Universidade Federal do Rio de Janeiro BÁSLAVI MARISBEL ...

Universidade Federal do Rio de Janeiro

BÁSLAVI MARISBEL CÓNDOR LUJÁN

BIODIVERSITY AND CONNECTIVITY OF CALCAREOUS SPONGES

(PORIFERA: CALCAREA) IN THE WESTERN TROPICAL ATLANTIC

Rio de Janeiro

2017

ii

Biodiversity and Connectivity of calcareous sponges (Porifera: Calcarea) in the Western

Tropical Atlantic

Báslavi Marisbel Cóndor Luján

Tese apresentada ao Programa de Pós-Graduação em

Biodiversidade e Biologia Evolutiva, Instituto de

Biologia, Universidade Federal do Rio de Janeiro,

como parte dos requisitos necessários à obtenção do

título de Doutor em Ciências Biológicas.

Orientadora: Dra. Michelle Klautau

Rio de Janeiro

Fevereiro/2017

iii


Tropical Atlantic



Banca examinadora:

Prof. Dra. Carla Zilberberg, IB/UFRJ

______________________________________________

Prof. Dr. Cristiano Valentim da Silva Lazoski, IB/UFRJ

_______________________________________________

Prof. Dr. Eduardo Carlos Meduna Hajdu, MN/UFRJ

_______________________________________________

Prof. Dr. Guilherme Ramos da Silva Muricy, MN/UFRJ

_______________________________________________

Prof. Dr. Marcelo Weksler, MN/UFRJ

_______________________________________________

Dr. Fernando Moraes, JBRJ (Suplente)

_______________________________________________

Prof. Dr. Paulo Cesar de Paiva, IB/UFRJ (Suplente)

_______________________________________________

Rio de Janeiro

Fevereiro/2017

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Trabalho realizado no Laboratório de Biologia de Porifera (LaBiPor)

Departamento de Zoologia, Instituto de Biologia

Universidade Federal do Rio de Janeiro – UFRJ

Orientadora: Profa. Dra. Michelle Klautau

Capa: Centro: Rede filogeográfica de Leucetta floridana (modificada). Sentido horário:

Leucetta floridana, Leucilla uter, Leucascus luteoatlanticus sp. nov., Ascandra torquata sp.

nov., Sycon conulosum sp. nov., Nicola tetela, triactina âncora de Amphoriscus hirstutus sp.

nov., triactina atrial de Grantessa tumida sp. nov., esqueleto de Leucandrilla pseudosagittata sp.

nov., esqueleto de Ernstia citrea. Créditos: A. Padua, E. Hajdu, F. Azevedo e T. Pérez.

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Ficha catalográfica

CÓNDOR LUJÁN, Báslavi Marisbel


Tropical Atlantic / Báslavi Marisbel Cóndor Luján. Rio de Janeiro: UFRJ, IB, 2017.

xiii, 259f. il; 29.7 (cm)

Orientador: Michelle Klautau.

Tese (Doutorado). UFRJ/IB/Programa de Pós-graduação em Biodiversidade e Biologia

Evolutiva, 2017.

Referências bibliográficas: 236-256f.

1. Esponjas Calcareas. 2. Mar do Caribe. 3. Costa do Brasil. 4. Conectividade genética. I.

Klautau, Michelle. II. Universidade Federal do Rio de Janeiro, Instituto de Biologia, Programa

de Pós-graduação em Biodiversidade e Biologia Evolutiva. III. Tese.

vi

Dedico esta tesis a mis queridos padres

quienes, estando lejos, están siempre a mi lado.

vii

Huk llactapi maypi llapan maskhay runakuna mamaqocha hatun yachaypi, Llapan maskhay

runakuna ruanku anchoveta challway, ñoqa huk punchay ruwayramuni qochayuyo sasa

llachayta, soqta huata llanqay tukukuy manan llakikamunichu (In Quechua by E. Pezo Zegarra)

Translation: In a country where almost all marine science is focused on the fishery of

Engraulins ringens (Peruvian anchoveta), I took the risky decision to study sponges. After six

carioca years, I do not regret.

viii

AGRADECIMENTOS

Esta tesis es el resultado de un maravilloso viaje a través de las primeras descripciones de las

esponjas del Mar del Caribe y del Brasil, colectas en lugares en los que nunca imaginé bucear,

interesantes diálogos con especialistas en Porifera y muchas horas dedicadas al microscopio, a

la amplificación de secuencias de ADN y a las simulaciones bayesianas. Todo esto sólo fue

posible gracias a la colaboración, apoyo e incentivo de muchísimas personas e instituciones.

Muchas gracias a todos y en especial a:

A minha orientadora Michelle Klautau por ter aceito a minha proposta de doutorado e ter me

mostrado o caminho para ser uma boa pesquisadora.

Aos membros do projeto MARRIO, em especial ao Eduardo Hajdu e ao Thierry Pérez por

terem organisado as maravilhosas expedições que geraram os resultados apresentados nesta tese.

Aos membros do LaBiPor, Andrézinho, Bernardo, Bárbara, Gabi, Fernanda, Mattheus,

Tayara, Taynara, Pedro e Raissa pela boa disposição para trabalhar em équipe e me auxiliar com

os pedidos de último momento pelo whatsapp (rsrs). Às meninas que alguma vez fizeram parte

do LabiPor, a Carol e Malena pelo trabalho de bancada e a Natalia e Vivi pela agradável

companhia.

Aos pesquisadores (alemḿ do LaBiPor) que dedicaram um tempo durante os mergulhos para

procurar e coletar Calcarea: A. Bispo, A. Ereskovsky, B. Thacker, C. Leal, C. Ruiz, C. Castello-

Branco, C. Diaz, G. Lôbo-Hajdu, H. Fortunato, J. García-Hernández, J. Vacelet, J. Carraro, L.

Van Bostal, M. Carvalho, O. Thomas, P. Chevaldonné, S. Chenesseau, S. Zea and S. Salani.

Aos pesquisadores espongólogos brasileiros e estrangeiros que conheci ao longo destes anos

(Simpósio de Porifera na Bahia, “La Martinique” Sponge Course, Pacotilles Expedition,

MARRIO Workshops). Todos eles, certamente, contribuiram na minha formação como

pesquisadora.

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Aos profesores do Museu Nacional e do Instituto de Biologia. Um especial “gracias” à

Ghennie Rodriguez pela orientação nas ańalises demográficas da Leucetta floridana.

Às agencias de fomento CAPES, CNPQ, CNRS, COFECUB, FAPERJ e Grupo Boticário de

Proteção à Natureza pelo financiamento das expedicçõs de coleta e os projetos do laboratório.

Ao programa PEC-PG e à CAPES pela concessão da bolsa de doutorado.

A mi mamá, papá y hermano por el apoyo incondicional y la confianza depositada en mi.

A Helmunt quien no sólo ha sabido darme su amor a lo largo de estos años, si no, también

me ha ayudado en momentos computacionales muy críticos.

Muchas Gracias! Muito obrigada!

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ABSTRACT

BIODIVERSITY AND CONNECTIVITY OF CALCAREOUS SPONGES (PORIFERA:

CALCAREA) IN THE WESTERN TROPICAL ATLANTIC


Supervisor: Dr. Michelle Klautau

Abstract of the thesis submitted to the Graduate Program Biodiversity and Evolutionary

Biology, Institute of Biology, Federal University of Rio de Janeiro – UFRJ, as part of the

requirements to obtain the title of Doctor in Biological Sciences.

The Western Tropical Atlantic (WTA) is a wide region that comprises the Tropical

Northwestern Atlantic (TNA), the North Brazil Shelf (NBS) and the Tropical Southwestern

Atlantic (TSA) provinces. Although it harbours a high diversity of marine species, sponges of

the class Calcarea have been poorly studied. This lack of knowledge hinders our understanding

of the biogeographical affinities of the calcareous sponges in the WTA and the role of the

Amazon River as an effective barrier to dispersal of these sponges. In the present study, the

diversity of the class Calcarea in the WTA was investigated using morphological and molecular

approaches and the population connectivity of a calcareous sponge, Leucetta floridana, was

assessed. With this contribution, the number of calcareous sponges from the WTA raised from

67 to 86, including the descriptions of 15 new species and four new records. Among them, 21

species are endemic to the TNA, three to the NBS and 22 to the TSA. The 14 species shared

between the TNA and TSA support a Caribbean-Brazilian affinity for calcareous sponges.

Genetic analyses evidenced five structured populations in L. floridana: one widespread

population maintaining gene flow between the TNA and TSA provinces and four other isolated

populations within the Caribbean Sea. The panmitic population suggested that the outflow of the

Amazon River in the Atlantic is not an effective barrier to the maintenance of gene flow among

trans-Amazonian populations of L. floridana.

Keywords: Caribbean Sea, NE Brazilian coast, phylogeography, demography, Amazon River.

Rio de Janeiro

February, 2017

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RESUMO

BIODIVERSIDADE E CONECTIVIDADE DE ESPONJAS CALCAREAS (PORIFERA,

CALCAREA) NO ATLÂNTICO TROPICAL OCIDENTAL



Resumo da Tese submetida ao Programa de Pós-Graduação em Biodiversidade e Biologia

Evolutiva, Instituto de Biologia, Universidade Federal do Rio de Janeiro – UFRJ, como parte

dos requisitos necessários à obtenção do título de Doutor em Ciências Biológicas.

O Atlântico Tropical Ocidental (WTA) é uma vasta região que compreende as províncias

Atlântico Noroeste Tropical (TNA), Plataforma Norte do Brasil (NBS) e Atlântico Tropical

Sudoeste (TSA). Embora abrigue uma grande diversidade de espécies marinhas, as esponjas da

classe Calcarea têm sido pouco estudadas. Essa falta de conhecimento dificulta nossa

compreensão das afinidades biogeográficas das esponjas Calcarea no WTA e do papel do Rio

Amazonas como uma barreira efetiva para a dispersão dessas esponjas. Neste estudo, foi

investigada a diversidade da classe Calcarea no WTA por meio de abordagens morfológicas e

moleculares e também foi avaliada a conectividade populacional da esponja Calcarea Leucetta

floridana. Com a presente contribuição, o número de esponjas da classe Calcarea no WTA

aumentou de 67 para 86 espécies, incluindo descrições de 15 espécies novas e 4 novos registros.

Entre elas, 21 espécies são endêmicas do TNA, três ocorrem só NBS e 22 são exclusivas do

TSA. As 14 espécies compartilhadas entre o TNA e o TSA sugerem uma afinidade Caribe-Brasil

para as esponjas calcareas. As análises genéticas evidenciaram cinco populações estruturadas

em L. floridana: uma população amplamente distribuida e mantendo fluxo gênico entre as

províncias TNA e TSA e quatro outras populações isoladas no Mar do Caribe. A população

panmítica sugeriu que a descarga do Rio Amazonas no Atlântico não é uma barreira efetiva à

manutenção do fluxo gênico entre populações trans-amazônicas de L. floridana.

Palavras-chave: Mar do Caribe, costa NE do Brasil, filogeografia, demographia, Rio

Amazonas.

Rio de Janeiro

Fevereiro de 2017

xii

INDEX

1. INTRODUCTION …............................................................................................................. 1

1.1 General introduction …........................................................................................................... 2

1.2 General Aim …........................................................................................................................ 5

1.3 Specific Aims …...................................................................................................................... 5

2. LITERATURE REVIEW …................................................................................................... 6

2.1. Geographic Scenario ….......................................................................................................... 7

2.1.1 The Western Tropical Atlantic …......................................................................................... 7

2.1.2 The Caribbean Sea …........................................................................................................... 8

2.1.3. The Tropical Brazilian Shelf and the Oceanic Islands ….................................................. 11

2.2. Connectivity within the Western Tropical Atlantic ….......................................................... 13

2.2.1 Estimating connectivity …................................................................................................. 13

2.2.2 General patterns of connectivity ….................................................................................... 14

2.2.3 Connectivity patterns in the Western Tropical Atlantic …................................................. 15

2.3. Porifera in the Western Tropical Atlantic …........................................................................ 16

2.3.1 Generalities of the Phylum Porifera ….............................................................................. 16

2.3.2 Generalities of the Class Calcarea …................................................................................. 17

2.3.3 Diversity of Calcarea in the Western Tropical Atlantic …................................................. 20

3. RESULTS ….......................................................................................................................... 21

3.1 Nicola gen. nov. with redescription of Nicola tetela (Borojevic & Peixinho, 1976) (Porifera:

Calcarea: Calcinea: Clathrinida) …............................................................................................. 22

3.2 Calcareous sponges (Porifera: Calcarea) from Curaçao including Brazilian shared species

and phylogenetic remarks …...................................................................................................... 34

3.3 New records of calcareous sponges (Porifera: Calcarea) from the Northeastern Brazilian

coast including three new species …......................................................................................... 132

3.4 Evolutionary history of the calcareous sponge Leucetta floridana in the Western Tropical

Atlantic ….................................................................................................................................. 176

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4. GENERAL DISCUSSION …............................................................................................. 215

5. CONCLUSIONS AND PERSPECTIVES ….................................................................... 233

6. REFERENCES …............................................................................................................... 236

APPENDIX ….......................................................................................................................... 257

Appendix 1 …........................................................................................................................... 257

Appendix 2 …........................................................................................................................... 258

Appendix 3 …........................................................................................................................... 259

Chapter 1

INTRODUCTION

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1.1 General Introduction

For many decades, the zoogeographical affinities within the Western Tropical Atlantic,

specifically between the Caribbean and the Brazilian coast, have been discussed and several

biogeographic divisions have been proposed. Both areas were once considered a single

biogeographic province, the Caribbean Province, which extended from the coast of Florida

(United States of America) down to Cabo Frio in Rio de Janeiro (Brazil) (Ekman, 1953). Some

years later, based on the Brazilian endemism of corals, hydrozoans, molluscs and fishes, Briggs

(1974) proposed a division in Caribbean, West Indian (Lesser Antilles) and Brazilian provinces.

The latter included also the Brazilian Oceanic Islands. The discovery of “Caribbean” reef fishes

under the Amazon freshwater plume (Collete & Rützler, 1977) made Helfman et al., (1997)

reconsider the presence of a unique province in this region. Palacio (1982) did not refuse this

but restricted the Tropical Province (Caribbean) down to the 23°C isotherm, between Espírito

Santo and Rio de Janeiro. Nonetheless, as knowledge on Brazilian endemic species increased,

the division in Caribbean and Brazilian provinces became more accepted (Briggs, 1995; Floeter

& Gasparini, 2000; Boschi, 2000).

In 2007, Spalding et al. introduced a new biogeographic classification, the Marine

Ecoregions of the World (MEOW) integrating previous biogeographic divisions,

geomorphological and oceanographic features and new data of species distribution and

dominant habitats. In that classification, the Western Tropical Atlantic (WTA) comprised three

biogeographic provinces: the Tropical Northwestern Atlantic (TNA), the North Brazil Shelf

(NBS) and the Tropical Southwestern Atlantic (TSA). More recently, modifications to those

provinces have been proposed by Floeter et al., (2008) and Briggs & Bowen (2012) based on

new studies on fish endemism and phylogeography.

The existence of different biogeographic provinces in the WTA relies on the possibility of

barriers that can prevent the dispersal of organisms within this broad area, such as the Amazon

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or Orinoco rivers. According to Rocha (2003), the freshwater and sediment outflow from the

Amazon river would act as a strong barrier to some reef fishes and might be responsible for the

endemism observed in the Brazilian coast. Nonetheless, the effectiveness of this barrier might

be restricted to shallow-water organisms and highly influenced by the sea-level fluctuations.

Therefore, larval dispersal (gene flow) between Caribbean and Brazilian populations of

widespread species would occur during interglacial periods and would be facilitated by the

sponge assemblages found underneath the Amazon river that would act as a connectivity

corridor.

Sponges are exclusively aquatic, sessile, filter-feeding, multicellular organisms. They are the

oldest extant metazoan group (Antcliffe et al., 2014) and present many adaptations to different

habitats, including marine and freshwater environments. They play an important ecological role

in marine ecosystems as they contribute to the recycling of nutrients from the water column into

the benthic communities (de Goeij et al., 2013). Moreover, they constitute one of the principal

marine invertebrate sources for the isolation of bioactive compounds of pharmaceutical interest

(Blunt, 2006, Perdicaris et al., 2013)

As all sponges have lecithotrophic larvae (Ereskovsky, 2010), low dispersal capabilities and

restricted geographic distribution ranges are expected. However, some studies revealed that

widespread species do occur along the WTA (Lazoski et al., 2001; Valderrama et al., 2009,

Zilberberg, 2006). These species constitute good models to evaluate the effectiveness of the

Amazon River as a real barrier to gene flow, nonetheless, up to date not such study was

performed. The knowledge on sponge connectivity within the WTA is fragmentary or restricted

to some species within one single biogeographic province (De Biasse et al., 2010, 2016;

Chaves-Fonnegra et al., 2015; López-Legentil & Pawlik, 2009; Richards et al., 2016; de Bakker

et al., 2016; Padua, 2012).

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Among the four extant classes of Porifera (Demospongiae, Homoscleromorpha,

Hexactinellida and Calcarea), Calcarea is the only with skeleton exclusively composed of

calcium carbonate). The knowledge on the diversity of calcareous sponges is somehow

restricted to geographical areas to which specialized taxonomists have access. For instance, the

Mediterranean Sea and the Caribbean Sea have comparable coastal extension (Coll et al., 2010;

Miloslavich et al, 2011); however, the number of calcareous sponges reported in the former is

ca. 65 whereas in the latter, it is only 23. This possibly reflects the lack of taxonomic effort in

the Caribbean Sea, a region considered a diversity hotspot for many other taxa.

Within this context, this study is intended to present the diversity and population

connectivity of calcareous sponges in the Western Tropical Atlantic, with a major emphasis on

the Caribbean Sea and the Northeastern Brazilian coast.

This thesis is organised in five chapters. The first chapter includes this general

introduction and the aims of this work. The second chapter sets the theoretical framework of this

study through a brief review on the characterization of the WTA, population connectivity and

calcareous sponges. The third chapter includes the results of this study and it is divided in four

articles. The first article proposes the new Calcinean genus Nicola based on the redescription of

the type material of “Guancha” tetela and the description of new specimens collected in

Curaçao. In the second and third articles, I described the calcareous sponges from Curaçao and

from the NE Brazilian coast, respectively, using morphological and molecular approaches. The

fourth article is the first study on population connectivity and demography of a calcareous

sponge (Leucetta floridana) in the WTA. The fourth chapter comprises a general discussion. The

fifth chapter presents the main conclusions and perspectives of this study.

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1.2 General Aim

To know the biodiversity and connectivity of the class Calcarea in the Western Tropical Atlantic

1.3 Specific Aims

1.3.1 To identify and describe the sponges of the class Calcarea from the Caribbean Sea.

1.3.2 To identify and describe the sponges of the class Calcarea from the Northeastern Brazilian

coast.

1.3.4 To assess the genetic structure of calcareous sponges in the Western Tropical Atlantic.

1.3.3 To test if the Amazon River is an effective barrier to connectivity among populations of

calcareous sponges.

Chapter 2

LITERATURE REVIEW

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2.1. Geographic Scenario

2.1.2 The Western Tropical Atlantic

The Atlantic Ocean emerged after the break-up of the supercontinent Pangaea in the late

Jurassic Period (163.5 -145 million years BP). Two important geological events occurred: (1)

the separation of the Pangaea in Laurasia and Gondwana, which resulted in the formation of the

Central Atlantic Ocean, and (2) the gradual south-to-north split of West Gondwana in South

America and Africa during the Early Cretaceous (145-100.5 million years BP) that originated

the South Atlantic Ocean (Seton et al., 2012). During the next million years, the Atlantic Ocean

continued spreading until its current configuration.

According to the latest marine biogeographic classification system (MEOW - Spalding et al.,

2007), the Tropical Atlantic Realm (TA, Figure 1A) comprises six biogeographic provinces

(Figure 1B): Tropical Northwestern Atlantic (TNA, n°12), North Brazil Shelf (NBS, n°13),

Tropical Southwestern Atlantic (TSA, n°14), St. Helena and Ascension Islands (n°15), West

African Transition (n°16) and Gulf of Guinea (n°17).

Figure 1. Biogeographical regionalization according to Spalding et al. (2007). A. Realms in theAtlantic Ocean. B. Biogeographic provinces in the Tropical Atlantic Realm. C. Ecoregions inthe Western Tropical Atlantic.

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The Western Tropical Atlantic (WTA) comprises the TNA, NBS and TSA biogeographic

provinces and includes 16 ecoregions (Figure 1C). The TNA comprises nine ecoregions:

Southern Gulf of Mexico (n°69), Western Caribbean (n°68), Southwestern Caribbean (n°67),

Southern Caribbean (n°66), Greater Antilles (n°65), Eastern Caribbean (n°64), Bahamiam

(n°63), Bermuda (n°62), and Floridian (n°63). The NBS includes the Guianian (n°71) and

Amazonia ecoregions (n°72). The TSA Province comprises São Pedro and São Paulo Islands

(n°73), Fernando de Noronha and Atoll das Rocas (n°74), Northeastern Brazil (n°75), Eastern

Brazil (n°76) and Trindade and Martin Vaz Islands (n°77). This study is focused on the TNA and

TSA provinces, specifically, the Caribbean Sea and the northeastern Brazilian coastal shelf.

2.1.2 The Caribbean Sea

The Caribbean Sea is a semienclosed basin within the TNA Province. It is bounded by Central

America on the west, Greater Antilles on the north, Lesser Antilles on the east and the northern

coast of South America on the south. It includes five ecoregions: Western Caribbean,

Southwestern Caribbean, Southern Caribbean, Greater Antilles and Eastern Caribbean. The

Caribbean basin comprises an area of approximately 2,754,000 km2, a volume of about 6.5x106

km3 and its coastline has an extension of more than 13,500 km (Miloslavich et al., 2011).

The geological age of the Caribbean Sea is related to the formation of the Caribbean Plate. In

the Middle Cretaceous (100.5–80 million years BP), a flood basalt event produced an oceanic

crust with a thickness of about 15-20 km (Burke, 1978). According to the model of Meschede &

Frisch (1998), this event took place somewhere between North and South America. An

alternative hypothesis (Pindell, 1994) suggested that the flood basalt occurred in the Pacific

Ocean, in the Galapagos hotspot, which afterwards, drifted eastwards (Figure 2).

9

Figure 2. Hypotheses to explain the origin of the Caribbean plate. A. Origin in an inter-American position. B. Pacific origin. Taken from Meschede & Frisch (1998).

The Caribbean Sea is divided into five deep water regions: the Yucatan Basin, the Cayman

Trough, the Colombian Basin, the Venezuelan Basin and the Grenada Basin, which are

separated by underwater ridges - the Cayman Ridge, the Nicaraguan Rise, the Beata Ridge and

the Aves Ridge, respectively (Draper et al., 1994, Figure 3). The average seafloor depth is

approximately 2,400 m and the deepest area (more than 7,500) is comprised between Cuba and

Jamaica, in the Cayman Trough (Matthews & Holcombe, 1985).

Figure 3. General geography and topography of the Caribbean Sea indicating the politicaldivisions and basins mentioned in the text. Source: Encyclopædia Britannica, Inc.

10

The present oceanographic configuration of the Caribbean Sea was established only after the

uplift of the Isthmus of Panama (2.8 million years BP - O'dea et al., 2016). Previously, the

Caribbean Sea experienced increased upwelling as it was connected to the Pacific Ocean (O'dea

et al., 2007). Nowadays, the Caribbean waters are clear, oligotrophic and with warm

temperatures ranging from 22 to 29°C (Kinder et al., 1985).

The Caribbean Sea receives the Atlantic inflow from the North Brazil current through several

passages located in the Lesser Antilles (Windward Islands Passages and Leeward Islands

Passages) and the Greater Antilles (Johns et al., 2002). The Caribbean current is formed mainly

by the masses of water that pass through the Windward Island Passages, specially through the

Grenada Passage located at the south of Grenade (near 11.51oN, Cherub, 2007). Within the

Caribbean basin, this current flows westward at 13–16°N and between Nicaragua and Jamaica it

turns northwest into the Gulf of Mexico through the Yucatan Channel. In the southernmost part

of the Caribbean Sea, one of the branches of the Caribbean Current forms the counterclockwise

Panama Gyre (Figure 4, Molinari et al., 1981).

Figure 4. Currents in the Caribbean Sea (as represented by the Mariano Global Surface VelocityAnalysis). Downloaded from http://oceancurrents.rsmas.miami.edu. Abbreviations: P=passage.

http://oceancurrents.rsmas.miami.edu/

11

Although the Caribbean waters are mainly clear, they are influenced by the freshwater

outflow from major rivers. The freshwater and sediments from the Amazon (Brazil) and

Orinoco (Venezuela) plumes enter through the North Brazil Current and the discharge of the

Magdalena river (Santa Marta, Colombia) directly interacts with the Caribbean current in the

Southwestern Caribbean (Cherub, 2007).

The Caribbean Sea is considered a global-scale hotspot for marine biodiversity (Roberts et

al., 2002). It encompasses a high diversity of flora and fauna distributed in different ecosystems

including coral reefs, mangroves and seagrasses (Miloslavich et al., 2010).

2.1.3 The Tropical Brazilian Shelf and the Oceanic Islands

The vast extension of the Brazilian coast (7,500 km) includes two major biogeographic realms,

the Tropical Atlantic (TA) and the Temperate South America (Spalding et al., 2007, Figure 1A).

In the present work only the TA was studied.

The Tropical Brazilian Shelf and the Oceanic Islands comprise seven ecoregions, one in the

NBS and six in the TSA (Figure 1C) . This area has been shaped by the Quaternary sea-level

changes over the past 5000 years, with the exposure of coastal sediments, river load, and coastal

drift (Dominguez et al., 1983).

The northern Brazilian coast, which corresponds to the Amazonian ecoregion of the NBS

(Figure 1C, n°72), is characterised by a combination of freshwater, estuarine and marine

ecosystems as a consequence of the outflow of the Amazon river. Benthic environments

comprise muddy-bank-shorelines (Anthony et al., 2010), rhodolith beds (Moura et al., 2016),

coral and sponge reefs (Colette & Rützler, 1976; Moura et al., 1999; 2016).

The northeastern and part of the southern Brazilian Coast (3°S - 20°S), which correspond to

the Northeastern Brazil (n°75), Eastern Brazil (n°76) and Trindade and Martin Vaz Islands

(n°77) ecoregions of the TSA, have an oligotrophic environment with little influence of river

runoff and only subjected to local upwelling (Soares et al., 2016). This area is characterised by a

12

variety of ecosystems, including mangrove forests, seagrass beds, coral reefs, sandy beaches,

rocky shores, lagoons and estuaries (Miloslavich et al., 2011; Moura et al., 2013).

The Brazilian oceanic islands include Fernando de Noronha Archipelago, Rocas Atoll (the

only atoll in the South Atlantic) and São Pedro and São Paulo Archipelago (ecoregions n° 73

and 74). These islands harbour tropical environments similar to those of the Caribbean Sea and

constitute very important hotspots of biodiversity (Soares et al., 2016). Mesophotic reefs and

rhodolith beds are also common within these islands (Magalhães et al., 2015; Amado-Filho et

al., 2016).

The entire coast of Brazil is under the influence of warm and temperate marine currents,

freshwater discharge from several rivers and upwelling events (Coelho-Souza et al., 2012;

Soares et al., 2016). In the Tropical Brazilian continental margin, the principal surface currents

are the Brazilian Current (BC) and the North Brazilian Current (NBC), originated from the

South Equatorial Current at about 5–6°S (Stramma, 1991; Silveira et al., 1994). The BC flows

to the south whereas the NBC runs to the north and northwest (Figure 5).

Figure 5. Surface currents in the Tropical Brazilian coast (as represented by the Mariano GlobalSurface Velocity Analysis): A. North Brazil Current (NBC) and B. Brazilian Current (BC).Downloaded from http://oceancurrents.rsmas.miami.

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2.2 Connectivity within the Western Tropical Atlantic

2.2.1 Estimating connectivity

Population connectivity is understood as the exchange of individuals among populations or

subpopulations (Gagnaire et al., 2015) and it can be divided into genetic and demographic

connectivity. Genetic connectivity refers to the degree to which gene flow affects evolutionary

processes within populations, whereas demographic connectivity refers to the degree to which

population growth and vital rates (survival, reproduction, emigration) are affected by dispersal

(Lowe & Allendorf, 2010).

The estimation of connectivity among marine populations is based on the assessment of

larval dispersal through indirect methods. These methods include the use of geochemical tags

from calcified structures (otoliths, statoliths and shells) or from artificial sources (fluorescent

compounds inserted in larvae (Thorrold et al., 2007), coupled biophysical models which

integrate oceanographic factors and larvae biological traits (e.g. Cowen et al., 2006; Paris et al.,

2007) and genetic approaches that allow the calculation of migration rates (e. g. Selkoe et al.

2010), among other parameters,

Molecular approaches include the amplification of neutral “frequency” or “sequence”

markers. Frequency markers such as microsatellites are adequate to answer questions in more

ecological timescales, whereas sequence markers such the cytochrome oxidase I (COI) aid to

resolve the history of divergence among populations. However, both types of markers present

some disadvantages. On the one hand, the isolation of microsatellites can be time-consuming

and consistent results may require large samplings and sophisticated analyses. In some cases, it

is even necessary to test the Mendelian condition of the loci before genotyping (Hellberg, 2009).

On the other hand, duplication or insertions from nuclear genome (pseudogenes) can alter

mtDNA patterns (Williams & Knowlton, 2001; Schizas, 2012).

14

Once chosen the adequate marker, further analyses to estimate connectivity comprise the

computation of indirect estimators (indexes) as the fixation index (FST) and the application of

direct methods such as parentage assignment or clustering analyses.

2.2.2 General patterns of connectivity

Marine population connectivity is driven by two main integrated components: (1) physical

processes and (2) biological traits (Cowen & Sponaugle, 2009). The former includes the

particular oceanographic processes occurring in the connectivity area (currents, tides, upwelling,

hurricanes), whereas, the latter corresponds to the life history of the target species including its

larval behaviour (mechanisms of defense against predators, vertical migrations, horizontal

swimming), chemical signals for settlement and adult spawning (Cowen et al., 2000; Cowen et

al, 2005). Other dispersal factors non-mediated by larvae such as adult rafting also influence

population connectivity (Kinlan et al., 2005; Cowen & Sponaugle, 2009).

In the last 20 years, several studies have notably contributed to the understanding of marine

population connectivity. Herein, I point out which I consider the most relevant conclusions:

1. Not all marine populations are open. Coastal marine populations were previously considered

open populations which would exchange individuals from very distant localities as a

consequence of the passive larval dispersal driven by water currents. Using Eulerian and

Lagrangian flow models, Cowen et al. (2000) found higher levels of larvae retention near the

natal localities than in distant areas. These results were also observed in further studies (Swearer

et al. 2002, Jones et al., 2005). Nowadays, studies suggest that populations range from fully

open to fully closed (Cowen & Sponaugle, 2009).

2. Larval dispersal capability may not be a good indicator of connectivity. Species with low

larval dispersal capabilities are believed to present structured populations or high rates of self-

recruitment (local replenishment); however, several studies on connectivity of reef fishes did not

evidence this. Bowen et al., (2006) found that Myripristis jacobus, a fish with short lifespan had

15

a population much less structured than Holocentrus ascensionis, a species with longer larval

duration. Almany et al. (2007) evidenced that fishes with different larval duration (orange

clownfish - Amphiprion percula and vagabond butterflyfish – Chaetodon vagabundus)

presented equal high levels of self-recruitment (60%). These studies indicated that larval

duration was a poor predictor of connectivity.

3. Genetic analyses should include oceanographic input. As traditional genetic analyses based

on the correlation between geographic distance (Eclidean distance) and genetic dissimilarity

failed to explain the connectivity patterns observed in several studies (e.g. Brandbury &

Bentzen, 2007), environmental information, such as ocean currents, started to be incorporated in

genetic calculations (White et al., 2010). That approach greatly improved the predictive value of

population genetics studies on small spatial scales (Selkoe et al., 2010). Within this context, two

adjacent localities are not necessarily connected nor two distant localities are genetically

isolated (White, 2010).

4. Still dealing with limited knowledge. Despite the considerable advances on marine

connectivity due to studies integrating different approaches, our knowledge is still fragmented

or restricted to specific taxa (mainly, fish and coral species). As pointed out by White (2010),

even with perfect simulations, the obtained results may refer to potential population

connectivity.

2.2.3 Connectivity patterns in the Western Tropical Atlantic

Our knowledge of population connectivity in the Tropical Western Atlantic is mainly based on

studies of reef fish connectivity using mitochondrial DNA sequences and with focus on the

Caribbean Sea and the Northeastern Brazilian coast.

Rocha et al. (2002) evaluated the role of the Amazon river as an effective barrier to the

dispersal of surgeonfishes (Acanthurus) and found levels of connectivity that correlated with

habitat preference. The species A. bahianus, which rarely settles outside shallow reefs, revealed

16

highly structured Brazilian and Caribbean populations, whereas A. chirurgus, collected on soft

sponge bottoms under the mouth of the Amazon River, presented genetically homogeneous

populations. This indicated that the Amazon outflow acted as a semi-permeable barrier. Rocha

(2003) suggested that the sponge assemblages discovered under the mouth of the Amazon river

(Colette & Rützler, 1976) would act as a corridor for species that require clear waters with

normal salinity but not for species strictly dependent on shallow reef habitats.

In the Caribbean Sea, Cowen et al. (2006) defined four broad regions of connectivity: (1) the

eastern Caribbean (Puerto Rico to Aruba); (2) the western Caribbean (Cuba to Nicaragua); (3)

Bahamas, Turks and Caicos Islands; and (4) the peripheral area of the Colombia-Panama Gyre.

Within these regions, population isolation and connectivity would be related to oceanographic

conditions such as the strong currents at the Mona Passage between Cuba and Puerto Rico and

the Colombia-Panama counter-gyre or to topographical features e.g. the shallow shelves that

remained exposed during the Pleistocene sea-level fluctuations.

2.3. Porifera in the Western Tropical Atlantic

2.3.1 Generalities of the Phylum Porifera

The phylum Porifera is composed of exclusively aquatic, sessile and multicellular organisms,

commonly known as sponges (Hooper et al., 2002). They are the oldest metazoan group still

extant and they present many ecological adaptations to different habitats including marine and

freshwater environments.

These animals are also characterised by the presence of a skeleton composed of spicules

(mineral aggregations of calcium carbonate or silica) or spongin (a protein from the family of

the collagen) and of an aquiferous system, principally used for filtering-feeding (Hooper et al.,

2002).1

1 Porifera also includes species without any type of skeleton (e.g. Hexadella and Oscarella spp.) or aquiferous system (carnivorous sponges).

17

Sponges present cells with high mobility and totipotency, which facilitate cellular

differentiation and transdifferentiation (Klautau, 2016). Except in species of the class

Homoscleromorpha, these cells do not form true tissues, however, the choanoderm (composed

of choancytes) and the pinacoderm (formed by pinacocytes), important components of the

aquiferous system, act as functional epithelia (Leys & Riesgo, 2012).

Sponges have short-lived lecithotrophic larvae (Ereskovsky, 2010) with a maximum lifespan

of two weeks (Fry, 1971; Maldonado, 2006). This may constrain their dispersion and

consequently, restrict their geographic distribution. Nonetheless, they present asexual processes

such as budding, gemmulation and fragmentation (Maldonado & Riesgo, 2008) that may

contribute to expand their distribution range.

According to the World Porifera Database (Van Soest et al., 2016), the phylum Porifera

currently comprises 8781 species distributed in four classes: Calcarea Bowerbank, 1862,

Demospongiae Sollas, 1885, Hexactinellida Schmidt, 1870 and Homoscleromorpha Gazave et

al, 2012. Among them, Calcarea is the only class that reunites the species whose skeleton is

exclusively composed of spicules made of calcium carbonate.

2.3.2 Generalities of the class Calcarea

The class Calcarea comprises marine sponges with a skeleton exclusively composed of calcium

carbonate, consisting of free, rarely linked or cemented spicules, to which a solid calcitic

skeleton can be added. They are viviparous and present blastula larvae (Manuel et al., 2002). In

this class, all the known types of aquiferous system in Porifera (asconoid, syconoid, sylleibid,

solenoid and leuconoid) are present (Figure 6, Cavalcanti & Klautau, 2011).

18

Figura 6. Aquiferous system of Porifera: asconoid (a), syconoid (b), sylleibid (c), leuconoid (d)and solenoide (e). Grey lines represent the choanoderm. Taken from Cavalcanti & Klautau(2011).

Calcarean sponges commonly reach small sizes (measured in mm or a few cm), however,

species of more than 20 cm in length were reported e.g. Leucandra multiformis, Leucetta

avocado, Pericharax heteroraphis, Sycon ciliatum (Poléjaeff, 1883; Koechlin, 1977; Van Soest

et al., 2012). Although most of the known species are white or beige; some yellow, red and pink

species have also been described e.g. Clathrina clathrus, C. rubra, Leucascus roseus. They can

be found in shallow tropical waters as well as in very deep waters in the Antarctic (Janussen &

Rapp, 2011; Rapp et al., 2011) and North Atlantic (Greenland, Rapp et al., 2013), in cryptic

environments protected of light such as overhangs, roofs of caves, crevices or underneath

boulders. Associated microorganisms have been found within the mesohyl of calcareous

sponges (Fromont et al., 2016) as well as benthic fauna (Padua et al., 2013) among the tubes or

atrium of these sponges.

The class Calcarea is divided into two monophyletic subclasses Calcinea and Calcaronea

Bidder, 1898(Figure 7). Calcaroneans are characterised by presenting sagittal spicules,

apinucleated choanocytes, amphiblastula larvae and diactines as the first spicules to be

produced, whereas calcineans have a skeleton mainly composed of regular spicules,

basinucleated choanocytes, calciblastula larvae and triactines as the first spicules to be secreted

(Manuel et al. 2002).

19

Figure 7. Calcaronea: A. Amphiblastula larvae (Lanna & Klautau, 2016), B. Apinucleatedchoanocyte (Borojevic et al., 2000), C. Sagittal spicules and diactines (Van Soest et al., 2012).Calcinea: D. Calciblastula larvae (Ereskovsky & Willenz, 2008). E. Basinucleated choanocytes(Borojevic et al, 1990). F. Regular spicules and diactines (Van Soest et al., 2012).

Phylogenetic relationships within these two subclasses remain unclear as many orders,

families and even genera are paraphyletic or polyphyletic (Voigt et al., 2012; Voigt & Wörheide,

2016). However, in the subclass Calcinea, certain skeletal traits have evidenced phylogenetic

signal in some genera, e.g. Clathrina and Borojevia (Rossi et al., 2011; Klautau et al., 2013).

The total number of described calcareous species (ca. 680) represents only about 8% of all

described extant sponges (Van Soest et al, 2012) and most of these records are restricted to areas

where the taxonomic effort on calcareous sponges has been intense (e. g. Australia, Japan and

Mediterranean Sea). Furthermore, the plasticity of a few morphological characters in some

Calcarean taxa difficults the species identification and sometimes require integrative approaches

(e.g. Valderrama et al., 2009; Imeseck et al., 2014; Azevedo et al., 2015; Klautau et al., 2016).

20

2.3.3 Diversity of Calcarea in the Western Tropical Atlantic

In general terms, the sponge diversity (including all the classes) is concentrated in the tropics

(Soares et al., 2016). However, the first attempt to elucidate the distribution patterns within the

class Calcarea (Van Soest et al., 2012) revealed very low values of diversity in that area. As

pointed out in the same study, those results did not reflect the real diversity but the need of

describing the sponge fauna from poorly explored areas.

The number of valid species known for the Western Tropical Atlantic, compiled from the

World Porifera Database (Van Soest et al., 2016; Van Soest, 2017), unpublished literature

(Azevedo, 2013) and submitted manuscripts (Azevedo et al., submitted) is 67 species. Some

species are shared between two or three biogeographic provinces and other species are endemic

to a province and in some cases, to an ecoregion.

Chapter 3

RESULTS

22

Nicola gen. nov. with redescription of Nicola tetela (Borojevic & Peixinho, 1976) (Porifera:

Calcarea: Calcinea: Clathrinida)

CÓNDOR-LUJÁN, B. & KLAUTAU, M.

Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av.

Carlos Chagas Filho, 373, 21941-902, Rio de Janeiro, RJ, Brasil. [email protected];

[email protected]

Corresponding author: [email protected]

Article published in ZOOTAXA 4103(3): 230-238

Abstract

Guancha tetela was originally described as a species having a peduncle and a skeleton

exclusively composed of sagittal triactines. Therefore, according to the most recent phylogeny

of Clathrinida, it should be placed in the genus Clathrina. This species was collected on the

Northeastern Brazilian coast in 1968 and it was not collected again until 2011 in Curaçao. In

this study, we reanalyzed the type material and the new specimens from Curaçao under a

morphological-molecular approach. Morphological analysis revealed the presence of tetractines

in the skeleton of all the studied specimens, including a slide of the holotype. In the molecular

phylogeny G. tetela grouped with genera containing tetractines, but as an independent new

lineage, different from all the other genera of Clathrinida. Based on these results, we propose

the erection of a new genus, Nicola gen. nov., to include species whose body is composed of

tubes without anastomosis nor branches but that run in parallel and coalesce at the apical and

basal regions. Moreover, the skeleton is exclusively composed of sagittal triactines and

tetractines.

Key words: Atlantic Ocean, Northeastern Brazilian Coast, Caribbean Sea, Curaçao, molecular

systematics, new genus, Guancha tetela.

Introduction

The genus Guancha was originally proposed by Miklucho-Maclay (1868) in order to describe

Guancha blanca, a species from the Canary Islands (Lanzarote) that presented a clathroid

cormus with a stalk. Although Miklucho-Maclay (1868) considered that the presence of a stalk

was sufficient to separate this species in a new genus, subsequent authors did not agree with this

point of view and placed G. blanca in different genera: Ascetta (Haeckel 1872; Vosmaer 1881;

Lendenfeld 1891), Leucosolenia (Lackschewitsch 1886; Vosmaer 1887; Breitfuss 1896, 1898;

23

Dendy & Row 1913; Arndt 1928; Hôzawa 1929; Burton 1930; Brøndsted 1931; Breitfuss 1932,

1935; Topsent 1936; Arndt 1941; Tanita 1943), and Clathrina (Minchin 1896; Jenkin 1908).

Only in 1976 the genus Guancha became accepted, with the description of G. tetela by

Borojevic and Peixinho (1976), and had its first diagnosis:

"Clathrinidae à cormus constitué d'un pédoncule et d'un corps clathroïde. Spicules

réguliers et parassagittaux, ou uniquement parasagittaux, orientés parallèlement dans les

parois des tubes, au moins dans la partie basale de l'éponge avec l'actine impaire basipète"

(Borojevic & Peixinho 1976) (Translation: Clathrinidae with a cormus composed of a stalk and

a clathroid body. Spicules are regular and parasagittal or only parasagittal, organised in parallel

in the tubes wall, at least at the basal part of the sponge with the unpaired actine basipetally

oriented).

Later on, in the Systema Porifera (Borojevic et al. 2002), the diagnosis proposed was:

"Clathrinidae with a cormus composed of a peduncle and a clathroid body. The peduncle

may be formed by true tubes with a normal choanoderm, or may be solid with a special

skeleton. The skeleton is composed of regular (equiangular and equiradiate) spicules to which

parasagittal spicules are added, at least in the peduncle. In some species only parasagittal

spicules are present. The unpaired actine of parasagittal spicules is always basipetally oriented."

Since the publication of the Systema Porifera and this new diagnosis of Guancha, four new

species were described within this genus: Guancha arnesenae Rapp, 2006, Guancha camura

Rapp, 2006, Guancha pellucida Rapp, 2006, and Guancha ramosa Azevedo et al., 2009. More

recently, however, molecular studies showed that Guancha was not a monophyletic genus, and

the authors proposed its synonymisation with Clathrina (Rossi et al. 2011; Klautau et al. 2013).

This synonymisation was confirmed when the type species of the genus (G. blanca) was

included in a molecular tree (Imešek et al. 2014). Currently, we follow what was proposed by

Klautau et al. (2013), that all Guancha species with only triactine spicules should be transferred

to Clathrina, and that Guancha species with triactines and tetractines should be assigned to any

of the other genera proposed or rediagnosed in the same article (namely Arthuria, Ascandra,

Borojevia, Brattegardia, and Ernstia).

According to this proposal, the species Guancha tetela, originally described as having a

skeleton exclusively composed of sagittal triactines, should be placed in Clathrina. However, a

more detailed revision of this species revealed the presence of tetractines in its skeleton, which

precludes its classification as a Clathrina. As G. tetela presents more triactines than tetractines,

it should then be considered Arthuria, however, the organisation of the cormus of G. tetela is

24

different of that of other Arthuria. Hence, molecular and detailed morphological analyses were

performed in the present work to verify the correct classification of G. tetela.

Material and Methods

The holotype of Guancha tetela is deposited in the Muséum Nationale d'Histoire Naturelle de

Paris under the number MNHN-LBIM-C-1975-1 and there are also two slides from the holotype

(one spicule slide and one section slide) deposited in the collection of the Museu Nacional do

Rio de Janeiro (MNRJ 40). In the present work we analysed the slides MNRJ 40. The holotype

of G. tetela was collected by dredging during a survey of the oceanographic vessel “Almirante

Saldanha” along the Northeastern Brazilian Continental Shelf. Recently (2011), five specimens

were collected from Curaçao by SCUBA diving and were deposited in the Porifera Collection of

the Biology Institute of the Universidade Federal do Rio de Janeiro (UFRJPOR 6714,

UFRJPOR 6723, UFRJPOR

6724, UFRJPOR 6746, and UFRJPOR 6767). Species names, voucher numbers, and GenBank

accession numbers of the DNA sequences used for a phylogenetic analysis are listed in Table 1.

Morphological analyses

For the preparation of spicule slides, fragments of the sponge were dissolved in sodium

hypochlorite (commercial bleach) in a test tube. After digestion, the spicules were washed five

times in distilled water and three times in absolute ethanol. They were then transferred to slides

and the ethanol was heat-evaporated. The mounting medium used was Entellan (Merck).

For the scanning electron microscopy (SEM), the spicules were placed on a cover-slip

mounted on a stub with double-sided carbon tape and sputter-coated with gold. The analysis was

performed in a JSM-6510 scanning electron microscope at the Institute of Biology of the

Universidade Federal de Rio de Janeiro.

For the preparation of the slides sections, small fragments of the sponge were stained with a

5% acid Fuchsin alcoholic solution for 10 min. The excess of Fuchsin was removed with

absolute ethanol for 5 min and the fragments were transferred to the slides, covered with some

drops of xylene and mounted with Entellan.

Spicules measurements were made using an ocular micrometer. The length and the width at

the base of each actine were measured for every spicule category. The results are presented in

tabular form, featuring length and width (minimum, mean, standard deviation [s], and

maximum), and sample size (n). Photographs were taken with a Zeiss AxioCam ERc5s coupled

to a ZEISS Stemi 2000C stereoscope and with a digital camera connected to a Zeiss Axioscop

microscope.

25

Molecular phylogenetic analyses

Genomic DNA was extracted with the guanidine/phenol-chloroform protocol (Sambrook et al.

1989) and stored at –20°C until amplification. The region comprising the partial 18S and 28S,

the spacers ITS1 and ITS2 and the 5.8S ribosomal DNA was amplified by PCR with the

following primers: 18S (5`-TCATTTAGAGGAAGTAAAAGTCG-3`) and 28S (5`-

GTTAGTTTCTTTTCCTC CGCTT -3`) (Lobo-Hajdu et al. 2004). Each PCR amplification

reaction mixture contained: 1X buffer (5X GoTaq R Green Reaction Buffer Flexi, PROMEGA),

0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM of each

primer, one unit of Taq DNA polymerase (Fermentas) and 1 µL of DNA, summing up to 15 µL

with Milli-Q water. PCR steps included one first cycle of 4 min at 94°C, 1 min at 50°C and 1

min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 50°C and one minute at 72°C, and a final

cycle of 6 min at 72°C. Forward and reverse strands were automatically sequenced in an ABI

3500 sequencer (Applied Biosystems). Sequences were aligned through the online version of

the program MAFFT v.7 (Katoh & Standley 2013) using the strategy Q-INS-i (Katoh & Toh

2008).

Phylogenetic analyses were performed under maximum likelihood (ML) and Bayesian

inference (BI). The ML analysis was conducted online on PhyML 3.0 (Guindon et al. 2010;

available at http://www.atgc-montpellier.fr/phyml). The model for the ML analysis was selected

using Modeltest 3.7 and the Akaike information criterion (AIC) (Posada & Crandall, 1998),

which indicated GTR (General Time Reversible). One thousand bootstrap pseudo-replicates

were performed (Felsenstein 1985). Bayesian inference reconstructions were obtained with

MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) under 106

generations and a burn-in of 1000 sampled trees, yielding a consensus tree of majority.

Table 1. Analyzed specimens with voucher numbers and GenBank accession numbers.

Species Voucher number Genbank accessionnumber

Ascaltis reticulum UFRJPOR6258 HQ588973Ascandra falcata UFRJPOR5856 HQ588962Ascandra contorta UFRJPOR6327 HQ588970Arthuria hirsuta ZMAPOR07061 KC843431Arthuria spiralatta MNRJ12860 KC985139Borojevia cerebrum UFRJPOR6322 HQ588964Borojevia brasiliensis UFRJPOR5214 HQ588978Brattegardia nanseni UFRJPOR6332 HQ588982Clathrina antofagastensis MNRJ 9289 HQ588985Clathrina blanca PMR-14307 KC479087

http://www.atgc-montpellier.fr/phyml

26

Clathrina clathrus UFRJPOR6315 HQ588974Clathrina lacunosa UFRJPOR6334 HQ588991Clathrina ramosa MNRJ 10313 HQ588990Ernstia tetractina UFRJPOR5183 HQ589000Ernstia sp. UFRJPOR6621 KC843433Guancha tetela UFRJPOR 6723 KU568492

Leucaltis clathria UFRJPOR 6944 KU568493Leucaltis nodusgordii QMG316050 AJ633857Leucettusa nuda MNRJ 10804 KC843453Leucascus simplex BMOO16283 KC843454Leucetta floridana PTL09.P100 KC843456Leucetta potiguar UFPEPOR547 EU781986

Results

Class Calcarea Bowerbank, 1864

Subclass Calcinea Bidder, 1898

Order Clathrinida Hartman, 1958 emend.

Nicola gen. nov.

Etymology: For Nicole Boury-Esnault in recognition of her dedicated work on the taxonomy of

sponges, including calcareous sponges.

Type species: Nicola tetela (Borojevic & Peixinho, 1976)

Diagnosis: Clathrinida with a globular to ovoid body composed of parallel tubes that coalesce at

the apical and basal regions. They do not anastomose nor ramify. The skeleton exclusively

contains sagittal spicules: triactines and tetractines. The paired actines are rudimentary and they

form a straight angle (180°s). The unpaired actine is always the longest actine. Diactines

including trichoxeas may be added. The aquiferous system is asconoid.

Nicola tetela comb. nov.

Synonyms: Guancha tetela, Borojevic & Peixinho 1976: 998

Material examined: Slide of the holotype (MNRJ 40), Station 1966, Northeastern Brazilian

Continental Shelf (Southern coast of Bahia State) (17°55'S, 37°30'W), collected by dredging by

the “Almirante Saldanha” (SAL) vessel, 17th August 1968, 47 m deep; UFRJPOR 6714,

UFRJPOR 6723, UFRJPOR 6724, Playa Kalki, Westpunt, Curaçao

(12°22'29.86 N,ʺ 69°09'30.63 W), collected by E. Hajdu and B. Cóndor-Luján, 21ʺ st August

2011, 5.6 m deep; UFRJPOR 6746, Sunset Waters, Soto, Curaçao

(12°16'01.58 N,ʺ 69°07'44.85 W), collected by E. Hajdu, 20ʺ th August 2011, 8.9 m deep;

27

UFRJPOR 6767, Sunset Waters, Soto, Curaçao (12°16'01.58 N,ʺ 69°07'44.85 W); collected byʺ

B. Cóndor-Luján; 22nd August 2011, 9–12 m deep.

Colour: bright orange in life and white in ethanol.

Description The specimens have a globular (Figure 1A) to ovoid body (Figure 1B), with apical

osculum and a peduncle at the base. The peduncle is formed by coalescent tubes with

choanoderm. Above the stalk, each tube is divided into two tubes which do not anastomose nor

ramify; instead, they run in parallel and then converge to form the osculum (Figure 1C). The

aquiferous system is asconoid, with choanocytes, intercalated by porocytes, covering the interior

of the tubes (Figure 1D). The surface is smooth and bright and the consistency is fragile.

The skeleton is composed of triactines and tetractines arranged in parallel, the triactines

being more abundant than the tetractines. The unpaired actine of the spicules is always

basipetally oriented (Figure 1E). The apical actine of the tetractines is projected into the lumen

of the tubes (Figure 1F).

Triactines are equally distributed all over the sponge body, whereas tetractines seem to be

more concentrated in the apical region, near the osculum (at least in UFRJPOR 6723). The size

of the spicules is very variable and although the unpaired actine is frequently much longer than

the paired ones (Figures 1G, H), it sometimes can be only a little longer (Figure 1I).

Spicules (Table 2, Figures 1G-K).

Triactines. Sagittal. Actines are straight, conical, with sharp tips. The unpaired actine presents a

constriction near its base. They present very variable size and are the most abundant spicules

(Figures 1G-I). Size: 75.0-440.0/5.0-10.0 µm (unpaired actine); 17.5-60.0/5.0-7.5 µm (paired

actine).

Tetractines. Sagittal. Actines are straight, conical with sharp tips. The unpaired actine has a

constriction near its base (Figures 1G, J). The apical actine is smooth and can be straight or

curved. It is longer and, generally, narrower than the paired actines (Figure 1K). They present

very variable size. Size: 80.0-370.0/5.0-10.0 µm (unpaired actine); 12.5-45.0/5.0-8.7 µm (paired

actine); 17.5-75.0/2.5-6.2 µm (apical actine).

Ecology: The Brazilian specimen was collected at 47 m deep in a calcareous-algae bottom,

whereas the specimens from Curaçao were found underneath broken corals in shallow waters

down to 12 m. No organisms were found on the surface or among the tubes of the studied

specimens.

28

Figure 1. Nicola tetela comb. nov. A-B: Live specimens: UFRJPOR 6714 (A) and UFRJPOR 6746 (B)(photos taken by E. Hajdu). C. Specimens after fixation (UFRJPOR 6714, 6723, 6724). D. Tangentialsection of the skeleton showing choanocytes and porocytes (pc). E. Detail of the apical region of the body.F. Apical actines projected into the lumen of a tube (arrow pointing to one apical actine). G. Spicules:Triactine (left) and tetractine (right). H – K: SEM images of spicules: H. Large triactine. I. Small triactine.J. Tetractine. I. Apical actine of a tetractine. All skeleton and spicule images were taken from thespecimen UFRJPOR 6723.

29

Table 2. Spicule measurements of Nicola tetela comb. nov., including the originalmeasurements of the holotype (MNRJ 40) by Borojevic & Peixinho (1976).

Specimen Spicule Actine Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max

UFRJPOR Triactine Unpaired 75.0 223.6 91.7 440.0 5.0 7.5 0.9 10 306723 Paired 27.5 31.3 4.3 47.5 5.0 6.4 1.0 7.5 30

Tetractine Unpaired 102.5 202.2 54.9 305.0 5.0 8.0 1.3 10 30Paired 25.0 31.4 3.2 37.5 5.0 6.1 1.2 8.7 30Apical 30.0 50.4 10.4 62.5 2.5 3.9 1.0 5 20

UFRJPOR Triactine Unpaired 75.0 231.6 95.3 435.0 5.0 6.5 1.2 8.7 306746 Paired 17.5 29.6 5.0 40.0 5.0 5.3 0.7 7.5 30

Tetractine Unpaired 80.0 215.0 110.5 370.0 5.0 6.7 1.0 7.5 6Paired 25.0 32.5 8.1 45.0 5.0 5.2 0.5 6.3 6Apical 35.0 58.5 15.5 75.0 2.5 3.3 1.1 5 5

UFRJPOR Triactine Unpaired 102.5 213.5 69.2 375.0 5.0 7.1 1.1 8.7 306767 Paired 20.0 33.4 6.7 50.0 5.0 6.3 1.2 7.5 30

Tetractine Unpaired 100.0 173.7 40.4 255.0 5.0 7.0 1.1 8.7 30Paired 12.5 29.8 7.1 40.0 5.0 5.6 1.0 7.5 30Apical 17.5 37.5 8.9 47.5 2.5 2.7 0.7 5 12

MNRJ 40 Triactine Unpaired 150.0 - - 400 7.0 - - 10 -(original) Paired 30.0 - - 60 - - - - -MNRJ 40(presentwork)

Triactine Unpaired 100.0 229.3 64.2 375.0 6.3 7.4 0.4 7.5 20Paired 32.5 36.9 3.0 42.5 5.0 6.4 1.0 7.5 20

Tetractine Unpaired 145.0 222.3 51.3 315.0 5.0 7.4 0.7 8.7 20Paired 30.0 35.0 5.0 45.0 5.0 7.1 0.8 8.7 20Apical 32.5 32.5 0.0 32.5 5.0 5.6 0.9 6.2 2

Remarks: Borojevic & Peixinho (1976) originally described the skeleton of this species as

being exclusively composed of triactines. Reanalysing the type material, we also found

tetractines, although those spicules were outnumbered by triactines (Figures 2A, B). Because of

the scarce quantity of tetractines in the holotype slide, they might have neglected them.

Moreover, Borojevic & Peixinho (1976) characterised the spicules as parasagittal, pointing out

the straight angle (90°s) formed by the unpaired and paired actines. We do recognize the

referred angle, however, we consider that it would be more correct to characterise these spicules

as sagittal, according to Boury-Esnault & Rützler (1997).

The molecular analysis produced the same tree topology with both phylogenetic methods

(ML and BI) and recovered the lineages found by Klautau et al. (2013) (Figure 3). Nicola tetela

comb. nov. did not cluster with any of the already known genera, not even Arthuria, confirming

that it is a new genus.

30

Figure 2. Photographs taken from the original slide of the holotype (MNRJ 40). A. Apicalregion. B. Basal region. In each photo a tetractine is indicated by an arrow.

Figure 3. Bayesian 50% majority rule consensus tree (106 trees sampled; burn-in =1000 trees)inferred from the ITS rDNA sequences under the GTR model. Bayesian posterior probabilities(BI) and bootstrap (ML) are given on the branches.

31

Discussion

The anastomosis of the cormus is an important taxonomic character in the order Clathrinida. For

example, all species of the genus Ascandra show a different anastomosis, with tubes free at least

at the apical region, while Arthuria, Borojevia, Brattegardia, Clathrina, and Ernstia have well

anastomosed tubes. In the new genus Nicola, tubes are not anastomosed but run in parallel and

are reconnected at the base and at the osculum. Another remarkable morphological character of

Nicola gen. nov. is its skeleton composed exclusively of sagittal spicules, which has not been

found in any other Calcinean genus. The triactines with reduced paired actines present in Nicola

gen. nov. are even similar to the “nail-spicules” found in the Calcaronean genera Kebira and

Grantiopsis (Calcaronea: Lelapiidae), most probably as a result of convergence. Our results

point that spicule shape and composition together with the organisation of the body are very

important characters for the taxonomy of Calcinea. Considering only spicule composition we

would expect to find a closer proximity of Nicola gen. nov. with Arthuria, however, we

observed in our phylogenetic tree that they are not sister genera. This means that the presence of

only sagittal spicules and the differentiated organisation of the cormus in Nicola gen. nov. are

strong characters that define well this new genus as separated from all the others known up to

date.

Acknowledgments

We are indebted to E. Hajdu and G. Lôbo-Hajdu for assistance and photographing during the

sample collections. Mark Vermeij and CARMABI are acknowledged for providing logistical

support in Curaçao. B. C. L. received scholarship from the Brazilian National Research Council

(CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). M.K.

is funded by fellowships and research grants from the CNPq, CAPES, and the Rio de Janeiro

State Research Foundation (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do

Rio de Janeiro - FAPERJ). This paper is part of the DSc. requirements of Báslavi Cóndor Luján

at the Biodiversity and Evolutionary Biology Program of the Federal University of Rio de

Janeiro.

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Calcareous sponges (Porifera: Calcarea) from Curaçao including Brazilian shared

species and phylogenetic remarks

Báslavi Cóndor-Luján1, Taynara Louzada1,2, Fernanda Azevedo1, André Padua1, Eduardo Hajdu3

& Michelle Klautau1

1Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av.

Carlos Chagas Filho, 373, CEP 21941-902, Rio de Janeiro, RJ, Brasil. 2 Universidade Federal do Estado do Rio de Janeiro, Instituto de Biociências, Av. Pasteur, 458,

Urca, CEP 22290-240, Rio de Janeiro, RJ, Brasil.3Universidade Federal do Rio de Janeiro, Museu Nacional, Departamento de Invertebrados,

Quinta da Boa Vista, São Cristóvão, CEP 20940-040, Rio de Janeiro, RJ, Brasil.

Corresponding author: M. Klautau, [email protected]

Journal: Journal of the Marine Biological Association of the United Kingdom.

ABSTRACT

This is the first inventory of calcareous sponges from Curaçao, Caribbean Sea. The

studied material comprised specimens sampled along the Curaçaoan Island as well as some

Brazilian specimens previously collected. They were analysed using morphological and

molecular (ITS and C-LSU) approaches. A total of 18 species were found and are described

here: 11 calcineans and seven calcaroneans. Eleven of these species are new to science; nine

are provisionally endemic to Curaçao, Amphoriscus micropilosus sp. nov., Arthuria vansoesti

sp. nov., Clathrina curaçaoensis sp. nov., Grantessa tumida sp. nov., Leucandra caribea sp. nov.,

Leucandrilla pseudosagittata sp. nov., Leucilla antillana sp. nov., Sycon conulosum sp. nov.,

Sycon magniapicalis sp. nov., and two are shared between Curaçao and Brazil, Ascandra

torquata sp. nov. and Clathrina aspera sp. nov. The formerly Brazilian endemic species C. lutea,

C. insularis, C. mutabilis and Borojevia tenuispinata have their distribution widen to the

Caribbean Sea. Clathrina cf. blanca, C. hondurensis and Leucetta floridana are new records for

Curaçaoan waters. The new phylogenetic affinities in Calcaronea as well as already reported

Calcinean relationships recovered in the molecular analyses are discussed.

Key words: Biodiversity, Calcinea, Calcaronea, Caribbean Sea, Southern Caribbean ecoregion,

Systematics, Western Tropical Atlantic.

mailto:[email protected]

35

INTRODUCTION

The Caribbean Sea is considered a global-scale hotspot of marine biodiversity (Roberts et al.,

2002). In its 2,754,000 km2 and over 13,500 km of coastline, it encompasses a high diversity of

flora and fauna distributed in different ecosystems including coral reefs, mangroves, seagrasses

and other environments (Miloslavich et al., 2010).

The sponges (Phylum Porifera) constitute one of the most diverse benthic faunal groups in

the subtidal habitats of coral reefs and mangroves (Diaz & Rützler, 2011), however, the studies

on sponge diversity are mostly restricted to the conspicuous species of the class Demospongiae.

As species of the class Calcarea are usually small and devoid of colour (Wörheide & Hooper,

1999; Rapp, 2006) and inhabit light protected or cryptic environments (e.g. overhangs, caves,

crevices), they are easily neglected in sponge fauna inventories. Furthermore, the plasticity of a

few morphological characters within some Calcarean taxa makes the identification of these

species difficult, sometimes requiring complementary approaches such as molecular analyses

(e.g. Valderrama et al., 2009; Imešek et al., 2014; Azevedo et al., 2015; Klautau et al., 2016).

The class Calcarea comprises species with skeleton composed exclusively of calcium

carbonate. It is divided in two monophyletic subclasses, Calcinea and Calcaronea Bidder, 1898.

Phylogenetic relationships within these two subclasses are still not well-understood as many

orders, families and even genera are polyphyletic (Voigt et al., 2012; Voigt & Wörheide, 2016).

However, certain skeletal traits have evidenced phylogenetic signal in some Calcinean genera

(Rossi et al., 2011; Klautau et al., 2013).

Up to date, only 24 calcareous sponges have been reported from the Caribbean Sea (Cóndor-

Luján & Klautau, 2016; Van Soest et al., 2016;) including representatives of both subclasses. In

a similar geographical range such as the Mediterranean Sea, whose basin comprises 2,969,000

km2 (Coll et al., 2010), the number of recorded calcareous species is more than doubled;

including ca. 65 species (Van Soest et al., 2016).

Among the Caribbean coral reefs, the Curaçaoan reefs are considered some of the healthiest

reefs, harbouring a great diversity of organisms including endemic species (Vermeij, 2012).

Although the sponge diversity within this island has been intensively assessed for many years,

very few calcareous species were reported for Curaçao. The pioneering study of Arndt (1927)

included the first record of the subclass Calcarea, Leucilla amphora Haeckel, 1872. Further

studies compelling shallow-water sponges (Van Soest, 1978, 1980, 1981, 1984; Hajdu & Van

Soest, 1992; Alvarez et al., 1998; De Weerdt, 2000), sponges from cryptic habitats (Van Soest,

2009) and deep-water sponges (Van Soest, 2014) did not report any other Calcarean species,

although one of them did mention the presence of calcareous sponges (Van Soest, 1981). More

36

recently, a former Brazilian endemic species, Nicola tetela (Borojevic & Peixinho, 1976) was

recorded for this island (Cóndor-Luján & Klautau, 2016). This very low number of records may

just reflect the lack of taxomomic expertise in this region, reinforcing the necessity of

faunistical studies on Calcarea.

The present work aimed to start filling the gap on the knowledge of Calcarea from the

Caribbean Sea through the description of the calcareous sponge fauna of Curaçao, integrating

morphological and molecular information. We also discuss the phylogenetic relationships within

Calcarea considering the species described here.

MATERIALS AND METHODS

Abbreviations

BMNH = The Natural History Museum, London, United Kingdom

GW = Gert Wörheide

IRB = Institut Ruđer Bošković, Zagreb, Croatia

MM = Michel Manuel

MNRJ = Museu Nacional do Rio de Janeiro, Brazil

PMJ = Phyletisches Museum Jena, Germany

PMR = Prirodoslovni Muzej Rijeka, Croatia

QM = Queensland Museum, Australia

SAM = South Australian Museum, Australia

UFRJPOR = Porifera collection of the Biology Institute of Universidade Federal do Rio de Ja-

neiro (UFRJ), Brazil

WAMZ = Zoological collection of the Western Australian Museum, Perth, Australia

ZMAPOR = Zoölogisch Museum, Instituut voor Systematiek en Populatiebiologie, Amsterdam,

The Netherlands

Study area: Curaçao

Curaçao is a volcanic island located in the Leeward Antilles Ridge, a boundary zone between

the Caribbean and the South American Tectonic Plates. It was originated in the Cretaceous

Period and received different Miocenic sediment depositions. Its current geology has been

shaped during the Quaternary (Hyppolyte & Mann, 2011).

It is localised in the southern Caribbean Basin, at 60 km north from Venezuela (Figure1A)

and it is included in the Southern Caribbean ecoregion of the Tropical Northwestern Atlantic

37

Province (TNA - Spalding et al., 2007). This island has a total area of 444 km2 and it is

surrounded by a fringing reef with 7.85 km2 situated 20-250 m from the coast (Vermeij, 2012).

Fig. 1. Study area. (A) location of Curaçao in the Caribbean Sea; (B) localities sampled along the coast of Curaçao.

Analysed Material

In this study, a total of 50 specimens were analysed. The material included specimens collected

in Curaçao in 2011 as well as Brazilian specimens already deposited in the Porifera Collection

of the Universidade Federal do Rio de Janeiro, Brazil (UFRJPOR) and that were conspecific

with the Curaçaoan specimens.

In Curaçao, eight localities along the western coast were surveyed (Figure 1B). The

collections were performed by SCUBA down to 20 m of depth. The specimens were

photographed in situ and carefully removed from the substrate with the aid of forceps and small

knives. At the CARMABI (Caribbean Research and Management of Biodiversity) Marine

Research Station, they were fixed in 96% ethanol.

Brazilian specimens were previously collected by SCUBA at depths ranging from four to 15

m, in localities within the southeastern coast (Rio de Janeiro State) and in a southern island

(Arvoredo Island in the Santa Catarina State).

All the samples are preserved in 96% ethanol and deposited in the UFRJPOR Collection.

Morphological analyses

The external morphology was examined through the observation of macroscopic characters on

the fixed specimens and complemented with information from the in situ pictures. The anatomy

was assessed through the analysis of the skeleton composition obtained from microscopy slides.

The preparation of section and spicule slides as well as the spicule measurements followed

38

standard procedures (Wörheide & Hooper, 1999; Klautau & Valentine, 2003; Cóndor-Luján &

Klautau, 2016). The spicule measurements are presented in tabular form, featuring length and

width (minimum, mean, standard deviation [SD] and maximum). Spicule measurements of the

species used for comparative purposes were obtained from the original descriptions or from

more detailed recent descriptions of the type material.

The species identifications followed the Systema Porifera (Hooper & Van Soest, 2002) and

additional literature (Klautau & Valentine, 2003; Klautau et al., 2013; Azevedo et al.,

submitted).

To illustrate the species descriptions, photographs were taken with a digital Canon camera

coupled to a Zeiss Axioscop microscope. Scanning electron microscopy (SEM) micrographs of

particular spicule ornamentations were taken at the Biology Institute of the UFRJ using a JSM-

6510 SEM microscope. The spicule preparation for SEM images followed Azevedo et al.

(2015).

Molecular analyses

The total genomic DNA was extracted using the guanidine/phenol-chloroform protocol

(Sambrook et al., 1989) or with a QIAamp DNA MiniKit (Qiagen) and stored at –20°C until

amplification. Two DNA regions were amplified. The C-region of the 28S (C-LSU) was

amplified using the primers fwd: 5'GAAAAGCACTTTGAAAAGAGA-3' (Voigt & Worheide,

2015) and rv: 5'-TCCGTGTTTCAAGACGGG-3' (Chombard et al., 1998) and the region

containing the partial genes 18S and 28S, the spacers ITS1 and ITS2 and the 5.8S ribosomal

DNA (named herein as ITS) was amplified with the primers: fwd: 5`-

TCATTTAGAGGAAGTAAAAGTCG-3` and rv: 5`-GTTAGTTTCTTTTCCTCCGCTT-3`)

(Lôbo-Hajdu et al., 2004). The ITS region was only amplified for calcinean species.

The PCR mixture included 1x buffer (5x GoTaq® Green Reaction Buffer Flexi,

PROMEGA), 0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM

of each primer, one unit of Taq DNA polymerase (Fermentas or PROMEGA) and 1 µL of DNA

in a volume of 15 µL. The PCR amplification comprised one first cycle of 4 min at 94°C, 1 min

at 50°C and 1 min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 48°C, 50°C or 52 °C and one

minute at 72°C, and a final cycle of 6 min at 72°C. Forward and reverse strands were

automatically sequenced in an ABI 3500 (Applied Biosystems) at the Biology Institute (UFRJ).

Sequences used in recent phylogenies (Klautau et al., 2013; Klautau et al., 2016; Voigt &

Wörheide, 2016) were retrieved from the Genbank database and are listed in Table 1 as well as

the ones generated in this study. These sequences were aligned through the MAFFT v.7 online

39

platform (Katoh & Standley, 2013) using the strategy Q-INS-i (Katoh & Toh, 2008) which

provides a better alignment as it considers the secondary structure of the amplified region. The

nucleotide substitution model that best fit the alignment was GTR+G+I for both DNA regions,

as indicated by the Bayesian Information Criterion in MEGA 6 (Nei & Kumar, 2000; Tamura et

al., 2013).

Phylogenetic reconstructions were performed under Maximum Likelihood (ML) and

Bayesian Inference (BI) approaches. The ML analyses were conducted on MEGA 6 using an

initial NJ tree (BIONJ) and a 1000 pseudo-replicates bootstrap. The BI reconstructions were

obtained with MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003)

under 3.5 x 106 generations and a burn-in of 3,500 sampled trees, yielding a consensus tree of

majority.

Table 1. Species used in the phylogenetic analyses with locality, voucher number and GenBank(GB) accession number. *Sequences generated in the present study.

Species Locality Voucher number GB accession numberCALCINEA LSU ITSAscandra contorta Mediterranean UFRJPOR6327 - HQ588970Ascandra coralicolla Norway UFRJPOR6329 - HQ588994Ascandra falcata Mediterranean UFRJPOR5856 - HQ588962Ascandra sp. Polynesia BMOO16290 - KC843446Ascandra spalatensis Adriatic Sea UFRJPOR7540 - KP740024Ascandra torquata sp. nov.* Brazil UFRJPOR6080 - This studyAscandra torquata sp. nov. Brazil UFRJPOR6084 - KC843448Ascandra torquata sp. nov.* Curaçao UFRJPOR6738 - This studyBorojevia aff. aspina Brazil UFRJPOR5245 - HQ588998Borojevia aff. aspina Brazil UFRJPOR5495 HQ589017 -Borojevia brasiliensis Brazil UFRJPOR5214 HQ589015 HQ588978Borojevia cerebrum Mediterranean UFRJPOR6322 HQ589008 HQ588964Borojevia croatica Adriatic Sea IRB-CLB6 - KP740023Borojevia sp. QMG313824 JQ272287 -Borojevia tenuispinata Brazil UFRJPOR6484 - KX548916Borojevia tenuispinata Brazil UFRJPOR6492 - KX548917Borojevia tenuispinata* Curaçao UFRJPOR6700 This study This studyBorojevia trispinata Brazil UFRJPOR6487 - KX548919Clathrina aspera* Brazil UFRJPOR5531(P) - This studyClathrina aspera* Brazil UFRJPOR6346 This studyClathrina aspera* Brazil UFRJPOR6472 This studyClathrina aspera* Brazil UFRJPOR6510 This studyClathrina aspera* Curaçao UFRJPOR 6758 This study -Clathrina aurea Brazil MNRJ 8998 HQ589005 HQ588968Clathrina clathrus Mediterranean UFRJPOR6315 HQ589009 HQ588974

Clathrina conifera Brazil UFRJPOR8991 HQ589010 HQ588959Clathrina coriacea Norway UFRJPOR6330 HQ589001 HQ588986

40

Clathrina curaçaoensis sp. nov.*

Curaçao UFRJPOR6734 This study This study

Clathrina cylindractina Brazil UFRJPOR 5206 HQ589007 HQ588979Clathrina fjordica Chile MNRJ8143 HQ588984Clathrina fjordica Chile MNRJ9964 HQ589016 -Clathrina helveola Australia QMG313680 JQ272291 HQ588988 Clathrina insularis Brazil UFRJPOR6527 - KX548920Clathrina insularis* Brazil UFRJPOR6530 - This studyClathrina insularis Brazil UFRJPOR6532 - KX548921Clathrina insularis* Brazil UFRJPOR6533 - This studyClathrina insularis Brazil UFRJPOR6536 - KX548922Clathrina insularis* Brazil UFRJPOR6537 - This studyClathrina insularis* Curaçao UFRJPOR6737 This study KC843435Clathrina lutea Brazil UFRJPOR5172 HQ589004 HQ588961Clathrina lutea Brazil UFRJPOR5173 - HQ588976Clathrina lutea Brazil UFRJPOR 6543 - KX548923Clathrina lutea Brazil UFRJPOR 6545 - KC843442Clathrina lutea* Curaçao UFRJPOR6761 - KC843445Clathrina lutea Virgin Islands ZMAPOR08344 - KC843444Clathrina lutea Florida, USA UFRJPOR5818 - KC843443Clathrina luteoculcitella Australia QMG313684 JQ272283 HQ588989Clathrina mutabilis* Brazil UFRJPOR6525 - This studyClathrina mutabilis Brazil UFRJPOR6526 - KX548925Clathrina mutabilis Brazil UFRJPOR6528 - KX548926Clathrina mutabilis* Brazil UFRJPOR6539 - This studyClathrina mutabilis* Brazil UFRJPOR6540 - This studyClathrina mutabilis* Curaçao UFRJPOR6704 - This studyClathrina mutabilis* Curaçao UFRJPOR6719 - This studyClathrina mutabilis Curaçao UFRJPOR6733 - KC843436Clathrina mutabilis* Curaçao UFRJPOR 6735 - This studyClathrina mutabilis* Curaçao UFRJPOR6740 - This studyClathrina mutabilis* Curaçao UFRJPOR6741 This study KC843437Clathrina mutabilis* Curaçao UFRJPOR6743 - This studyClathrina mutabilis* Curaçao UFRJPOR6744 - This studyClathrina mutabilis* Curaçao UFRJPOR6745 - This studyClathrina mutabilis* Curaçao UFRJPOR6747 - This studyClathrina sp. Polynesia UF:Porifera:1600 - KC843438Clathrina sp. Polynesia UFRJPOR6461 - KC843439Clathrina wistariensis Australia QMG313663 JQ272303 HQ588987Clathrina zelinhae* Brazil UFRJPOR 6627 This study -Leucetta chagosensis - BMOO16210 -- -Leucetta floridana Panama PTL09.P100 - KC843456Leucetta floridana* Curaçao UFRJPOR6726 - This studyLeucetta floridana* Curaçao UFRJPOR6765 - This studyLeucetta antarctica Antarctic MNRJ13798 - KC849700Leucetta microraphis Australia QMG313659 - AJ633874Leucetta pyriformis Antarctic MNRJ13843 - KC843457Leucetta potiguar Brazil UFPEPOR569 - EU781987Nicola tetela Curaçao UFRJPOR6723 KU568492 -

41

CALCARONEA LSUAmphoriscus micropilosus sp. nov. * Curaçao UFRJPOR6755(P) This study -Anamixilla torresi - - AY563636 -Aphroceras sp. SAM-PS0349 JQ272273 -Eilhardia schulzei QMG316071 JQ272256 -Grantessa tumida sp. nov.* Curaçao UFRJPOR6701(P) This study -Grantessa tumida sp. nov.* Curaçao UFRJPOR6695(P) This study -Grantessa aff. intusarticulata

GW979 JQ272278 -

Grantia compressa - - AY563538 -Grantiopsis heroni Australia QMG313670 JQ272261 -Grantiopsis cylindrica Australia GW973 JQ272261 -Leucandra aspera - - AY563535 -Leucandra falakra Adriatic Sea UFRJPOR8349 KT447560 -Leucandra nicolae QMG313672 JQ272268 -Leucandra sp. QMG316285 JQ272265 -Leucandra spinifera Adriatic Sea UFRJPOR8348 KT447561Leucandrilla pseudosagittata sp. nov.* Curaçao UFRJPOR6696 This study -L. pseudosagittata sp. nov.* Curaçao UFRJPOR6705 This study -Leucascandra caveolata QMG316057 JQ272259 -Leucilla antillana sp. nov.* Curaçao UFRJPOR6768 This study -Leuconia nivea - - AY563534 -Paraleucilla dalmatica Adriatic Sea UFRJPOR8346 KT447566 -Paraleucilla magna - GW824 KT447564 -Petrobiona massiliana - GW1729 JQ272307 -Sycettusa aff. hastifera Red Sea GW893 JQ272267 -Sycettusa cf. simplex Western India ZMAPOR11566 JQ272279 -Sycettusa sp. - MM-2004 AY563530 -Sycettusa tenuis Australia QMG313685 JQ272281 -Sycon ancora Adriatic Sea UFRJPOR8347 KT447568 -Sycon capricorn - QMG316187 JQ272272 -Sycon carteri Australia SAM-PS0143 JQ272260 -Sycon ciliatum - - AY563532 -Sycon conulosum sp. nov.* Curaçao UFRJPOR 6707 This study -Sycon cf. villosum - GW51115 KR052809 -Sycon magniapicalis sp. nov.*

Curaçao UFRJPOR6748 This study -

Sycon magniapicalis sp. nov.*

Curaçao UFRJPOR6763 This study -

Sycon raphanus - - AY563537 -Syconessa panicula Australia QMG313672 AM181007 -Synute pulchella - WAMZ1404 JQ272274 -Teichonopsis labyrinthica - SAMPS0228 JQ272264 -Utte aff. syconoides - QMG323233 JQ272269 -Utte aff. syconoides - QMG313694 JQ272271 -Ute ampullacea - QMG313669 JQ272266 -Vosmaeropsis sp. - MM-2004 AY026372 -

42

RESULTS

Taxonomy

Integrating traditional morphological examination with molecular phylogenetic analyses, we

recognised 18 species including 11 calcineans and seven calcaroneans. Within the subclass

Calcinea, the richest genus was Clathrina with seven species: C. aspera sp. nov., Clathrina cf.

blanca, C. curaçaoensis sp. nov., C. hondurensis, C. insularis, C. lutea, and C. mutabilis. In

Calcaronea, Sycon was the richest genera with two species: Sycon conulosum sp. nov and Sycon

magnapicalis sp. nov. The calcinean genera Ascandra and Borojevia as well as the calcaronean

Leucandrilla and Grantessa constitute not only new records for Curaçao, but also for the

Caribbean Sea.

Systematic Index

Class CALCAREA Bowerbank, 1862

Subclass CALCINEA Bidder, 1898

Order CLATHRINIDA Hartman, 1958

Genus Arthuria Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013

Arthuria vansoesti sp. nov.

Genus Ascandra Haeckel, 1872

Ascandra torquata sp. nov.

Genus Borojevia Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo, 2013

Borojevia tenuispinata Azevedo et al., submitted

Genus Clathrina Gray, 1867 sensu Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo,

2013

Clathrina aspera sp. nov.

Clathrina cf. blanca Miklucho-Maclay, 1868

Clathrina curaçaoensis sp. nov.

Clathrina hondurensis Klautau & Valentine, 2003

Clathrina insularis Azevedo et al., submitted

Clathrina lutea Azevedo et al., submitted

Clathrina mutabilis Azevedo et al., submitted

Genus Leucetta Haeckel, 1872

Leucetta floridana Haeckel, 1872

Subclass CALCARONEA Bidder, 1898

Order LEUCOSOLENIIDA Hartman, 1958

43

Family AMPHORISCIDAE Dendy, 1893

Genus Amphoriscus Haeckel, 1872

Amphoriscus micropilosus sp. nov.

Genus Leucilla Haeckel, 1872

Leucilla antillana sp. nov.

Family GRANTIIDAE Dendy, 1893

Genus Leucandra Haeckel, 1872

Leucandra caribea sp. nov.

Genus Leucandrilla Borojevic, Boury-Esnault & Vacelet, 2000

Leucandrilla pseudosagittata sp. nov.

Family HETEROPIIDAE Dendy, 1893

Genus Grantessa Lendenfeld, 1885

Grantessa tumida sp. nov.

Family SYCETTIDAE Dendy, 1892

Genus Sycon Risso, 1827

Sycon conulosum sp. nov.

Sycon magniapicalis sp. nov.

Description of taxa


TYPE SPECIES

Arthuria hirsuta (Klautau & Valentine, 2003).

DIAGNOSIS

"Calcinea in which the cormus comprises a typical clathroid body. A stalk may be present. The

skeleton contains regular (equiangular and equiradiate) triactines and tetractines. However,

tetractines are more rare. Diactines may be added. Asconoid aquiferous system" (Klautau et al.

2013).

Arthuria vansoesti sp. nov. (Figure 2, Table 2)

ETIMOLOGY

Named after Rob van Soest in recognition of his dedicated work on the taxonomy of the

sponges, including those from Curaçao.

44

TYPE LOCALITY

Daai Booi, St. Willibrordus, Curaçao.

TYPE MATERIAL

Holotype: UFRJPOR 6720 (specimen in ethanol and slides); Daai Booi, St. Willibrordus;

12°12'43.12"N, 69°05'8.42''W; 5.2 m deep; coll. B. Cóndor-Luján, 19 August 2011.

COLOUR

Yellow in life and white to beige in ethanol.

MORPHOLOGY AND ANATOMY

This species has a massive and smooth cormus. The holotype is 0.7 x 0.6 x 0.2 cm (Figures 2A-

B). The cormus is composed of irregular and loosely anastomosed tubes. A water-collecting tube

(2 x 1 mm) was present in the centre of the cormus of the holotype (arrow in Figure 2A). The

aquiferous system is asconoid. The skeleton has no special organization (Figure 2C) and it is

composed of abundant triactines (two shape categories) and rare tetractines.

SPICULES

Triactines I. Regular (equiangular and equiradiate). Most abundant spicules. Actines are

cylindrical, slightly undulated at the distal part and with rounded tips (Figure 2D). They

resemble the triactines of Clathrina aurea. Size: 72.5-87.5/3.8-5 µm.

Triactines II. Regular (equiangular and equiradiate). Rare. Actines are straight, slightly conical

with blunt to sharp tips (Figure 2E). Size: 52.5-82.5/2.5-5 µm.

Tetractines. Regular (equiangular and equiradiate). Basal actines are straight, cylindrical, with

rounded to blunt tips (Figure 2F). The apical actine is the shortest actine. It is straight and

smooth; however, some curved actines were found (Figure 2G). It has sharp or blunt tip. Size:

60-87.5/3.8-5 µm (basal actine) and 25/3.8-5 µm (apical actine).

ECOLOGY

This sponge was found in a cryptic habitat, underneath coral boulders.

GEOGRAPHIC DISTRIBUTION

Southern Caribbean (provisionally endemic to Curaçao, present study).

REMARKS

The genus Arthuria now comprises 11 valid species, A. africana (Klautau & Valentine, 2003),

A. alcatraziensis (Lanna et al., 2007); A. darwinii (Haeckel, 1870); A. dubia (Dendy, 1891); A.

hirsuta (Klautau & Valentine, 2003); A. spirallata Azevedo et al., 2015; A. sueziana (Klautau &

Valentine, 2003); A. tenuipilosa (Dendy, 1905); A. trindadensis Azevedo et al., submitted, A. tu-

buloreticulosa Van Soest & de Voogd, 2015 and A. vansoesti sp. nov. Most of them possess a

skeleton mainly composed of triactines with conical actines except for A. dubia, A. sueziana, A.

45

tubuloreticulata and A. vansoesti sp. nov. whose skeletons also include triactines with

cylindrical actines.

Arthuria vansoesti sp. nov. can be easily distinguished from A. dubia and A. sueziana

because they have different spicule width (Table 2), being thinner in the new species (2.5–5.0

µm) than in A. dubia (13.7 – 17.0 µm) and A. sueziana (8.0 – 11.8 µm). Besides, C. dubia has

granular cells which are absent in the new species. The species whose skeleton more resembles

A. vansoesti sp. nov. is the Indonesian A. tubuloreticulata, however, they present some

differences. A. tuboloreticulata is orange in live and its cormus has several oscula whereas the

new species is yellow in life and presents water-collecting tubes. Moreover, the spicules of the

Curaçaoan species have rounded tips and in the Indonesian species, they are sharp or blunt.

Table 2. Spicule measurements of Arthuria vansoesti sp. nov. (UFRJPOR 6720), A. dubia(BMNH 1891.9.19.2) taken from Klautau & Valentine (2003), A. sueziana (BMNH 1912.2.1.3)

and A. tuboreticulata (RMNH Por. 5547). H=holotype, L=lectotype.. B=basal and A=apicalactines.

Specimen Spicule ActineLength (µm) Width (µm) N

Min Mean SD Max Min Mean SD MaxUFRJPOR6720 (H)

Triactine I 72.5 79.8 4.9 87.5 3.8 4.6 0.6 5.0 30Triactine II 52.5 70.4 7.8 82.5 2.5 3.9 0.8 5.0 30Tetractine B 60.0 75.3 8.1 87.5 3.8 4.8 0.5 5.0 15

A 25.0 25.0 0.0 25.0 3.8 4.4 0.7 5.0 6BMNH 1891.9.19.2 (L)*

Diactine 77.5 256.0 97.0 418.2 - 13.7 5.0 - 30Triactine 92.5 151.5 14.0 170.0 - 15.5 1.8 - 30Tetractine B 120.0 140.3 9.5 155.0 - 16.0 1.0 - 9

A 100.0 110.0 6.8 117.5 - 9.8 0.5 -BMNH 1912.2.1.3 (H)

Trichoxea 250.0 - - - <0.3 - - -Triactine 75.0 91.3 10.9 137.5 - 10.3 1.5 - 30Tetractine B 70.0 86.0 6.1 97.5 - 9.4 1.4 - 30

A 50.0 56.3 4.5 62.5 - 5.0 0.0 - 4RMNH Por. 5547 (H)

Triactine 63 112.1 - 138 4 5.3 - 6.5 -Tetractine B 62 119.5 - 156 4 5.4 - 6 -

A 39 - - 132 3.5 - - 6.5 -

46

Fig. 2. Arthuria vansoesti sp. nov. (UFRJPOR 6720): (A) specimen in vivo; (B) specimen afterfixation; (C) tangential section of the body (cormus); (D) triactine I; (E) triactine II; (F)tetractine; (G) apical actine of tetractine.

47

Genus Ascandra Haeckel, 1872

TYPE SPECIES

Ascandra falcata Haeckel 1872

DIAGNOSIS

" Calcinea with loosely anastomosed tubes. Tubes are free, at least in the apical region. The

skeleton contains regular (equiangular and equiradiate) or sagittal triactines and tetractines. The

apical actine is very thin (needle-like) or very thick at the base. Diactines may be added.

Asconoid aquiferous system" (Klautau et al., 2016, emend).

Ascandra torquata sp. nov. (Figures 3 & 4, Table 3)

ETIMOLOGY

From the Latin torquere (= twist), for the twisted tip of the apical actine of the tetractines.

TYPE LOCALITY

Ponta do Vidal, Arvoredo Island, Reserva Biológica Marinha (REBIOMAR) do Arvoredo, Santa

Catarina, Brazil.

TYPE MATERIAL

Holotype (specimen in ethanol and slides) UFRJPOR 6084, Ponta do Vidal, REBIOMAR

Arvoredo, Santa Catarina, Brazil; 27°17’52.3’’S, 48°21’33.4’’W; 10-15 m deep; coll. F.

Azevedo, J. Carraro and A. Padua, 11 December 2009.

Paratypes (specimens in ethanol and slides) UFRJPOR 6080 and UFRJPOR 6082, Ponta do

Vidal, REBIOMAR Arvoredo, Santa Catarina, Brazil; 27°17'52.3"S, 48°21'33.4"W; 10-15 m

deep; coll. F. Azevedo, J. Carraro and A. Padua, 11 December 2009. UFRJPOR 6738, Playa

Jeremi, Soto, Curaçao; 12°19'43.73"N, 69°09'07.80"W; 15 m deep; coll. B. Cóndor-Luján and

E. Hajdu, 22 August 2011.

COLOUR

White in life and beige to light brown in ethanol.


The holotype (UFRJPOR 6084) measures 4.0 x 1.5 x 1.5 cm (Figure 3A). Cormus varying from

encrusting to massive, fragile in consistency and composed of irregular and loosely

anastomosed tubes at the base and free at the apical region. In fact, at the apical region, there is

no anastomosis as large vertical tubes arise and connect themselves culminating in oscula with

variable diameters (1-3 mm, Figure 3B). At the base, the surface is smooth while at the apical

region, it is very hispid due to diactines protruding the surface. Few diactines were also found in

the basal region of some specimens. The aquiferous system is asconoid. The skeleton has no

48

special organization and it is composed of abundant tetractines differentiated in two size

categories and of less frequent triactines and diactines (Figure 3C-D).

The specimen from Curaçao (UFRJPOR 6738, Figure 4A) is partitioned as shown in Figure 4B.

The largest fragment is 5 mm long and the free tubes can reach 5 mm high. In this specimen,

diactines were not observed (Figure 4C).

SPICULES (Table 3)

Diactines. Slightly sinuous with sharp tips (Figure 3E). Very variable size (length/width): 100.0-

395.0/5.0-17.5 µm.

Triactines. Regular (equiangular and equiradiate). Actines are conical, straight, with blunt to

sharp tips (Figures 3F-G and 4D). Subregular triactines were also found. Size (length/width):

45.0-197.5/7.5-15.0 µm.

Tetractines I. Regular (equiangular and equiradiate). The basal actines are slightly conical,

straight, with blunt to sharp tips (Figures 3H and 4E). The apical actine is smooth and conical.

Most apical actines are characteristically twisted, however, some straight ones with sharp tips

were also observed. Size (length/width): 57.0-225.0/5.0-22.5 µm (basal actine) and 50.0-

167.5/6.2-14.0 µm (apical actine).

Tetractines II. Regular (equiangular and equiradiate). Larger than tetractines I. The basal actines

are conical, straight, with blunt to sharp tips (Figure 3I and 4F). The apical actine is thinner than

the basal ones. It is smooth, straight, conical, with sharp tips. Some twisted apical actines were

also observed (Figure 3J). Size (length/width): 170.0-487.5/10.0-37.5 µm (basal actine) and

52.5-205.0/12.5-32.5 µm (apical actine).

ECOLOGY

Specimens inhabited sunlight protected environments. Brazilian specimens were collected in

vertical walls of large boulders while the Curaçaoan specimen was found underneath small

boulders. Amphipods, bryozoans, ophiuroids, polychaets and hydrozoans were found living in

association with the Brazilian specimens.


Disjunct distribution in the Western Atlantic: Southern Caribbean (Curaçao) and Southern Brazil

ecoregion of the Warm Temperate Southwestern Atlantic Province (Santa Catarina State).

REMARKS

Ascandra is currently composed of 15 species. Ascandra torquata sp. nov. can be easily distin-

guished from all them by the twisted apical actine of the tetractines which is absent in all the

other 14 known species. This new species is the third Ascandra registered for the Atlantic Ocean

49

- the other two are A. ascandroides (Borojević, 1971) from Brazil and A. corallicola (Rapp,

2006) from Norway.

A previous study evidenced that trichoxeas were not good taxonomic characters for

identifying Clathrina species as they could be present or not in C. mutabilis (Azevedo et al.,

submitted). The same seems to be valid for the diactines of A. torquata sp. nov. as they were

present in the specimens from Brazil but not in the one from Curaçao. Despite having this

morphological difference, the molecular analysis confirmed their conspecificity (Figure 31).

Therefore, at least for A. torquata sp. nov., diactines are not a reliable taxonomic character.

The clade of Ascandra was well supported in our ITS phylogenetic tree (pp=1, b=99, Figure

31), confirming the validity of this genus with the present diagnosis. In that tree, A. torquata sp.

nov. is a sister species of the type species of the genus A. falcata and of A corallicola. The ITS

sequence of A. torquata sp. nov. (UFRJPOR 6084) had already been published by Klautau et al.

(2013) as Clathrina sp. nov. 11.

Table 3. Spicule measurements of Ascandra torquata sp. nov. H=holotype; P=paratype.


UFRJPOR6084 (H)

Diactine - 100.0 208.9 90.7 382.5 7.5 12.0 3.0 17.5 30Triactine - 45.0 138.5 40.4 197.5 7.5 11.2 1.9 15 30Tetractine I basal 120.0 163.2 22.5 212.5 12.5 15.2 1.4 17.5 30

apical 72.5 112.0 30.8 167.5 7.5 8.8 1.7 14 30Tetractine II basal 195.0 256.1 38.8 337.5 10 26.6 6.6 37.5 22

apical 52.5 101.2 31.8 150.0 12.5 19.6 2.8 22.5 12UFRJPOR6080 (P)

Diactine - 115 245.5 53.5 335.0 5 10.8 2.4 15 30Triactine - 67.5 116.5 24.2 157.5 7.5 9.8 1.5 12.5 30Tetractine I basal 57.5 124.5 28.4 165.0 5 12.7 3.5 22.5 30

apical 62.5 112.2 26.5 162.5 7.5 9.6 1.8 12.5 25Tetractine II basal 207.5 360.7 81.6 487.5 21.2 32.1 6.3 32.1 30

apical 80.0 139.9 32.3 190.0 17.5 25.2 3.5 32.5 30UFRJPOR6082 (P)

Diactine - 142.5 255.8 73.1 395.0 5.0 14.3 2.8 15.0 17Triactine - 97.5 150.3 23.9 192.5 7.5 12.3 2.1 12.5 30Tetractine I basal 100.0 159.8 27.9 225.0 5.0 13.7 2.5 22.5 30

apical 65.0 118.5 19.9 150.0 7.5 10.5 2.2 12.5 30Tetractine II basal 170.0 272.5 47.7 375.0 21.3 29.6 3.8 45.0 30

apical 87.5 141.8 30.6 205.0 17.5 25.7 2.8 32.5 30UFRJPOR6738 (P)

Triactine - 75.0 116.4 20.0 150.0 8.8 11.6 1.6 15.0 30Tetractine I basal 85.0 122.5 22.0 192.5 10.0 12.4 2.1 17.5 30

apical 50.0 66.5 25.3 107.5 6.2 7.8 1.4 10.0 5Tetractine II basal 205.0 261.8 36.7 325.0 25 27.3 2.7 35 20

apical - 125.0 - - - 15.0 - - 1

50

Fig. 3. Holotype of Ascandra torquata sp. nov. (UFRJPOR 6084). (A-B) specimens afterfixation; (C-D) tangential section of the body indicating diactines (white arrows); (E) diactine;(F-G) triactines; (H) tetractine I; (I) tetractine II; (J); apical actines of tetractines I.

51

Fig. 4. Paratype of Ascandra torquata sp. nov. (UFRJPOR 6738). (A) specimen in vivo; (B)specimen after fixation; (C) tangential section of the body (cormus); (D) triactine; (E) tetractineI; (F) tetractine II.

52


TYPE SPECIES

Borojevia cerebrum (Haeckel, 1872)

DIAGNOSIS

"Calcinea in which the cormus comprises tightly anastomosed tubes. The skeleton contains

regular (equiangular and equiradiate) triactines, tetractines, and tripods. The apical actine of the

tetractines has spines. Aquiferous system asconoid" (Klautau et al., 2013).

Borojevia tenuispinata Azevedo et al., submitted (Figure 5, Table 4)

SYNONYMS

Borojevia tenuispinata: Azevedo et al., submitted

TYPE LOCALITY

Cabeço da Tartaruga, São Pedro e São Paulo Archipelago, Brazil.

MATERIAL EXAMINED

UFRJPOR 6700 and UFRJPOR 6708 (specimens in ethanol and slides); Daai Booi, St.

Willibrordus, Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5 m deep; coll. B. Cóndor-Luján, 18

August 2011.

COMPARATIVE MATERIAL EXAMINED

Borojevia tenuispinata. Holotype (specimen in ethanol and slides) UFRJPOR 6484; Cabeço da

Tartaruga, São Pedro e São Paulo Archipelago, Brazil; 0o54'57''N, 29o20'46''W; coll. G.

Rodríguez & F. Azevedo, 16 June 2011.

COLOUR

White in life and ethanol.


The largest specimen (UFRJPOR 6700) measures 0.7 x 0.4 x 0.3 mm. Cormus massive, rough

and compressible, composed of irregular and (mostly) tightly anastomosed tubes (Figure 5A).

The tubes in contact with the substrate are less tightly anastomosed than the most superficial

ones. The aquiferous system is asconoid. No water-collecting tubes were observed. The skeleton

has no special organization and it comprises triactines, tripods and tetractines (Figure 5B).

SPICULES

Triactines. Regular (equiangular and equiradiate). Actines are straight, conical, with blunt tips

(Figure 5C). They are the most abundant spicules. Size: 62.5-100/6.3-11.3µm.

53

Tripods. Regular (equiangular and equiradiate). Actines are straight, conical, with blunt tips.

They do not have the raised centre of typical tripods, instead they look like stout conical

triactines (Figure 5D). Size: 72.5-162.5/12.5-22.5 µm.

Tetractines. Regular (equiangular and equiradiate). The basal actines are straight, conical, with

blunt tips (Figure 5E). The apical actine is shorter and thinner than the basal ones and present

spines. The most common pattern of spine distribution observed consisted of spines arranged in

rows spreading from about two-thirds of the actine up to the tip (Figure 5F). Some few

tetractines with curved paired actines were also found. Size: 62.5-95.0/7.5-11.3 µm (basal

actine), 25-40/5-7.5 µm (apical actine).

ECOLOGY

This sponge was found in a cryptic habitat, underneath boulders.


São Pedro and São Paulo Islands ecoregion of the Tropical Southwestern Atlantic (São Pedro e

São Paulo Archipelago, Azevedo et al., submitted) and Southern Caribbean (Curaçao, this

study).

REMARKS

The genus Borojevia comprises eight species. Among them, six have a skeleton composition

(one category of tripods, triactines and tetractines) similar to the specimens from Curaçao.

These species are the Brazilians B. aspina (Klautau et al., 1994), B. brasiliensis (Solé-Cava et

al., 1991), B. tenuispinata and B. trispinata Azevedo et al., submitted, B. cerebrum (Haeckel,

1872) from the Mediterranean Sea and B. croatica Klautau et al., 2016 from the Adriatic Sea.

However, only B. brasiliensis, B. croatica and B. tenuispinata, have tetractines with apical

actines bearing a spine distribution pattern similar to the Curaçaoan specimens and share

comparable spicule size range (Table 4). Different from the species B. brasiliensis and B.

croatica, whose skeletons present more triactines than tetractines, in B. tenuispinata and in the

specimens from Curaçao, triactines and tetractines occur in the same proportion. The only

difference observed between the Caribbean specimens and the holotype of B. tenuispinata is

that the spicules of the former can attain larger sizes. This difference, however, can be perhaps

attributed to plasticity. Besides, in the ITS phylogenetic tree (Figure 31), the specimen

UFRJPOR 6700 clustered within the clade of B. tenuispinata (pp=0.98, b=100), indicating its

co-specificity.

54

Table 4. Spicule measurements of Borojevia tenuispinata from Curaçao (UFRJPOR6700 andUFRJPOR6708) and of the holotype (UFRJPOR 8464), B. brasiliensis (MNHN-LBIM.C.

1989.2) taken from Klautau & Valentine (2003) and B. croatica (UFRJPOR6865). H=holotype.


Min Mean SD Max Min Mean SD MaxUFRJPOR6700

Triactine 62.5 79.5 9.7 100.0 6.3 8.8 1.6 11.3 36Tripod 87.5 109.4 16.6 162.5 12.5 13.9 2.9 22.5 27Tetractine basal 67.5 78.8 7.1 92.5 7.5 8.5 1.0 10.0 30

apical 25.0 33.6 4.5 40.0 5.0 7.2 0.8 7.5 9UFRJPOR6708


apical 25.0 - - - - 7.5 - - 1UFRJPOR8464 (H)


apical 27.0 40.0 8.6 56.7 4.1 4.9 0.8 6.8 20MNHN-LBIM.C. 1989.2 (H)

Triactine 60.9 78.2 10.6 102.2 - 10.8 1.5 - 20Tripod 67 81 8.2 95.7 - 11 1.7 - 20Tetractine basal 56.5 75.3 10.0 91.3 - 10.4 1.3 - 20

apical 17.4 36.4 9.1 50.0 - 8-0 2.2 - 20UFRJPOR6865 (H)


apical - 20 . . . 5.0 - - 1

55

Fig. 5. Borojevia tenuispinata (UFRJPOR 6700): (A) specimen after fixation; (B) tangentialsection of the body (cormus); (C) triactine; (D) tripod; (E) tetractine; (F) detail of the spinedapical actine of a tetractine.

56

Genus Clathrina Gray, 1867

TYPE SPECIES

Clathrina clathrus (Schmidt, 1864)

DIAGNOSIS

"Calcinea in which the cormus comprises anastomosed tubes. A stalk may be present. The

skeleton ontains regular (equiangular and equiradiate) and/or parasagittal triactines, to which

diactines and tripods may be added. Asconoid aquiferous system" (Klautau et al., 2013).

Clathrina aspera sp. nov. (Figures 6 & 7, Table 5)

ETIMOLOGY

From the Latin asper (= rough), after the species rough surface.

TYPE LOCALITY

Water Factory, Willemstadt, Curaçao.

TYPE MATERIAL

Holotype: UFRJPOR 6758 (specimen in ethanol and slides); Water Factory, Willemstadt,

Curaçao; 12°06'30.88"N, 68°57'13.53"W; 13.2 m deep; coll. B. Cóndor-Luján and E. Hajdu, 23

August 2011.

Paratypes: UFRJPOR 5487 (specimen in ethanol and slides); Ilhas Botinas, Angra dos Reis, Rio

de Janeiro, Brazil; 23º03'19.36''S, 44º19'44.98''W; 1-3 m deep; coll. F. Azevedo & M. Klautau,

25 May 2007 and UFRJPOR 5531 (specimen in ethanol and slides); Praia do Bonfim, Angra dos

Reis, Rio de Janeiro, Brazil; 23°01'14.26''S, 44°19'48.18''W; 1-2 m deep; coll. M. Klautau, 27

May 2007.

ADDITIONAL ANALYSED MATERIAL

UFRJPOR 6345 (specimen in ethanol and slides); UFRJPOR 6472 (specimen in ethanol and

slides); Enseada dos Cardeiros, Arraial do Cabo, Rio de Janeiro, Brazil; 7m deep; coll. F.

Azevedo and G. Rodríguez; 22 May 2011 and UFRJPOR 6510 (specimen in ethanol and slides);

Praia do Forno, Arraial do Cabo, Rio de Janeiro, Brazil; 1.5 m deep; coll. E. Lanna and A.

Padua, April 2011.

COLOUR

White in life and beige to light brown in ethanol.


Cormus massive (Figure 6A) to almost spherical (Figure 7A), composed of thin, irregular to

regular and tightly anastomosed tubes finishing in large apical oscula. The holotype measures

0.8 x 0.6 x 0.2 cm (Figure 6B). It has rough surface due to the presence of large triactines on the

57

external tubes. Water-collecting tubes were not observed. The aquiferous system is asconoid.

The skeleton has no special organization and it is composed of two categories of triactines

(Figures 6C and 7B). The smaller triactines are the most abundant spicules whereas the large

triactines are less frequent and located on the surface. Near the oscular region, the triactines

become more sagittal.

SPICULES (Table 5)

Triactines I. Regular (equiangular and equiradiate). Actines are slightly conical to conical, with

blunt to sharp tips (Figures 6D, 6F and 7C-D). Some subregular triactines were also found

(Figure 7E). Size: 50.0-152.5/6.8-12.5 µm.

Triactines II: Regular (equiangular and equiradiate). Actines are conical and robust with sharp

tips (Figure 6E). Size: 127.5-325.0/17.5-37.5 µm.

ECOLOGY

The specimens inhabited shaded environments, underneath boulders or attached to an oyster

farming rope. They were found associated to macroalgae and to other invertebrates (octocorals,

bryozoans) or growing on oyster shells.


Southern Caribbean (Curaçao) and Eastern Brazil ecoregion of the Tropical Southwestern

Atlantic Province (Rio de Janeiro, Southern Brazilian Coast).

REMARKS

Within Clathrina, six species whose colour alive is white (or unknown) present two or more

spicule categories (Klautau & Valentine, 2003; Klautau et al. 2016; Azevedo et al., submitted);

however, only three have a cormus composed of tightly anastomosed tubes and large triactines

(including tripods) in the external tubes: C. clara Klautau & Valentine, 2003 from Christmas

Islands, C. laminoclathrata Carter 1886 from southern Australia and C. rotunda Klautau &

Valentine, 2003 from South Africa. Clathrina aspera sp. nov. can be easily distinguished from

those species as its skeleton comprises triactines whose actines are slightly conical to conical

and with blunt to sharp tips whereas the skeletons of the other three species only comprise

triactines whose actines are conical with sharp tips. Moreover, they differ in spicule size (Table

5) and external morphology.

Clathrina clara presents slightly thinner triactines II (21.8±3.5 µm) than those of C. aspera

sp. nov. (28.5±5.6 µm) and its cormus have water-collecting tubes which are absent in the new

Curaçaoan species. Clathrina laminoclathrata has three categories of triactines and even the

largest category (triactine III) is much thinner (18.0±3.0 µm). In this species, the large triactines

are present in the external tubes and can also be found at the base of the sponge (“basal lamina”

58

according to Carter, 1886) whereas in C. aspera sp. nov, they are restricted to the external tubes.

Clathrina rotunda has smaller triactines (triactines I: 52.6±3.6/5.8±0.7 µm and tripods:

59.0±9.8/9.6±2.4 µm) compared to C. aspera sp. nov. (triactine I: 115.4±12.9/9.7±1.1 µm and

triactine II: 227.5±50.8/28.5±5.6 µm – holotype measurements) and their actines are slightly

undulated whereas in the new Curaçaoan species, they are straight. Besides, water-collecting

tubes are also present in C. rotunda.

In the ITS phylogenetic tree, the sequences of C. aspera obtained from several specimens

(UFRJPOR 5531, UFRJPOR 6346, UFRJPOR 6472 and UFRJPOR 6510) clustered together.

However, as sequences of C. clara, C. laminoclathrata and C. rotunda were not available in the

Genbank databse, it was not possible to infer the phylogenetic affinities among them.

Fig. 6. Holotype of Clathrina aspera sp. nov. (UFRJPOR 6758): (A) specimen in vivo; (B)specimen after fixation; (C) tangential section of the body (cormus); (D and E) triactine I; (F)triactine II.

59

Fig. 7. Paratype of Clathrina aspera sp. nov. (UFRJPOR 5487): (A) specimen after fixation; (B)tangential section of the body (cormus) indicating triactines II (white arrows); (C-E) triactine I.

Table 5. Spicule measurements of Clathrina aspera sp. nov. (UFRJPOR 6758, UFRJPOR 5487and UFJPOR 5531), C clara (BMNH 1927.2.14.152), C. laminoclathrata (BMNH

1887.7.12.42) taken from Klautau & Valentine (2003) and C. rotunda (BMNH 1935.10.21.50).H=holotype, P=paratype, L=lectotype.

Specimen Spicule Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max

UFRJPOR 6758 (H)

Triactine I 75.0 115.4 12.9 152.5 7.5 9.7 1.1 12.5 40Triactine II 132.5 227.2 50.8 325.0 17.5 28.5 5.6 37.5 30

UFRJPOR5487 (P)

Triactine I 65.0 91.1 13.5 117.5 6.3 7.2 0.9 10 40Triactine II 232.5 242.5 9.0 250.0 27.5 31.7 3.8 35.0 3

UFRJPOR5531 (P)

Triactine I 57.5 95.0 15.7 127.5 6.3 7.6 1.2 10 40Triactine II 212.5 235.0 31.8 257.5 27.5 28.7 1.8 30 2

BMNH 1927.2.14.152 (H)

Triactine I 67.5 84.5 8.8 102.5 - 9.8 0.8 - 30Triactine II 102.5 164.5 34.3 245.0 - 21.8 3.5 - 30

BMNH 1887.7.12.42 (L)

Triactine I 50.0 72.0 15.0 113.0 - 8.0 2.0 - 30Triactine II 88.0 132.0 16.0 168.0 - 13.0 2.0 - 30Triactine III 125.0 188.0 31.0 235.0 18.0 3.0 30

BMNH 1935.10.21.50 (H)

Triactine I 45.6 52.6 3.6 57.6 - 5.8 0.7 - 20Tripod 45.6 59.0 9.8 79.6 - 9.6 2.4 - 20

60

Clathrina cf. blanca (Miklucho-Maclay, 1868) (Figure 8, Table 6)

SYNONYMS

Ascetta blanca, Haeckel, 1872: 38; Hansen, 1885: 20; Lendenfeld, 1891: 34; Arnesen, 1901: 9;

Mello-Leitão et al., 1961: 3.

Clathrina blanca: Minchin, 1896: 359; Jenkin, 1908: 438; Borojevic, 1971: 525; Ereskovsky,

1995: 730; Imešek et al., 2014: 23 Klautau et al., 2016: 37,38.

Clathrina cf. blanca: Rapp, 2015: 9.

Guancha blanca: Miklucho-Maclay, 1868: 221; Borojevic & Boury-Esnault, 1987: 14; Barthel

& Tendal, 1993: 84; Janussen et al., 2003: 17; Rapp, 2006: 352.

Leucosolenia blanca: Lackschewitsch, 1886: 300; Breitfuss, 1896: 426; Breitfuss, 1898a: 13;

Breitfuss, 1898: 105; Breitfuss, 1911: 224; Derjugin, 1915: 289; Breitfuss, 1927: 27; Arndt,

1928: 19; Hôzawa, 1929: 282; Brøndsted, 1931: 12; Breitfuss, 1930: 275; Breitfuss, 1932: 240;

Breitfuss, 1935: 7; Breitfuss, 1936: 5; Topsent, 1936: 9; Arndt, 1941: 45; Tanita, 1942b: 75.

TYPE LOCALITY

Lanzarote Islands, Canary Islands, Atlantic Ocean.

MATERIAL EXAMINED

UFRJPOR 6753 and UFRJPOR 6759 (specimens in ethanol and slides); Tug Boat, Caracasbaai,

Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 10 m deep; coll. B. Cóndor-Luján, 23

August 2011.

COLOUR

White in life and in ethanol.


This species has a globular clathroid body with an apical osculum and a stalk (Figure 8A). The

surface is smooth and the texture is soft. The consistency is compressible, even the stalk. In the

largest specimen (UFRJPOR 6759), the clathroid body measures 0.5 x 0.5 x 0.1 cm and the stalk

is 0.5 x 0.1 x 0.1 cm. The cormus is formed by irregular and tightly anastomosed tubes, which

converge at the centre of the sponge forming a single apical osculum. The stalk is formed by

true tubes with choanoderm (arrow in Figure 8A), except for the portion that attaches it to the

substrate which is solid. The aquiferous system is asconoid. The skeleton of the clathroid body

has no special organisation and it is composed of two categories of regular triactines (Figure

8B). The skeleton of the stalk is composed exclusively of parasagittal triactines, whose unpaired

actine is basipetally oriented. These spicules are more numerous and more closely disposed in

the median part of the stalk.

61

SPICULES

Triactines I of the clathroid body. Regular (equiangular and equiradiate) or subregular. Most

frequent spicules. Actines are cylindrical with blunt tips (Figure 8C). Some of them are slightly

undulated at the distal part. Size: 67.5-108.0/4.1-5.4 µm.

Triactines II of the clathroid body. Regular (equiangular and equiradiate). Actines are conical

and straight with sharp tips. Shorter and thicker than triactine I (Figure 8D). Size: 45.9-62.1/5.4-

8.1 µm.

Triactines of the stalk. Parasagittal (equiangular). The paired actines are slightly conical and

very short (sometimes they seem to be rudimentary). The unpaired actine is straight and

cylindrical to slightly conical with sharp tips (Figure 8F). Size: 40.5-67.5/5.4-8.1 µm (paired

actine) and 94.5-199.8/5.4-8.1 µm (unpaired actine).

ECOLOGY

This sponge was collected in a light protected environment, underneath coral boulder.


This species has an allegedly cosmopolitan distribution (Van Soest et al., 2016) including the

Caribbean Sea (Southen Caribbean, Curaçao, present study).

REMARKS

The external morphology of the specimens from Curaçao, a clathroid body attached to a stalk,

resembles the species formerly considered guanchas and now allocated in Clathrina (Klautau et

al., 2013). Among them, only two species have a clathroid body composed of tightly

anastomosed tubes, a stalk formed of true tubes and a skeleton composed of regular and

parasagittal triactines, as observed in the Curaçaoan specimens. These species are Clathrina

blanca (Miklucho-Maclay, 1868) from the Lanzarote Islands and C. pellucida (Rapp 2006) from

Norway.

Clathrina pellucida has a short stalk (less than 1/3 of the body) and a skeleton exclusively

composed of triactines with undulated actines whereas the Curaçaoan specimens have a longer

stalk (half the length of the body) and its skeleton also comprises triactines with straight actines.

Besides, the smaller triactines observed in the Curaçaoan specimens (Triactines II of the

clathroid body) are absent in C. pellucida.

Clathrina blanca is the species that most resembles the specimens from Curaçao. As spicule

sizes were not provided in the original description (Miklucho-Maclay, 1868), we used the

measurements provided by Haeckel (1872) for the parasagittal triactines (length/width): 50-

70/3-4 µm (paired actines) and 80-100/3-4 (unpaired actine). Compared to them, the analised

Curaçaoan specimens have thicker actines (5.4-8.1 µm). Compared to more detailed

62

descriptions of C. blanca reported from different localities, namely, Brazil (Borojevic, 1971),

Bay of Biscaye (Borojevic & Boury-Esnault, 1987), Norway (Rapp, 2006) and Adriactic Sea

(Imešek et al., 2014), we observed certain dissimilarities. In none of the referred descriptions,

spicules comparable to the small triactines observed in the specimens from Curaçao were

mentioned or illustrated. Moreover, the parasagittal triactines described for C. blanca sensu

Borojevic (1971) and C. blanca sensu Borojevic & Boury-Esnault (1987) are different to the

ones observed in our material (the paired actines are shorter in the Curaçaoan specimens). The

cormus of C. blanca sensu Imešek et al. (2014) presents several water-collecting tubes whereas

in our specimens, the tubes gather together in a single water-colllecting tube as also described

for C. blanca sensu Rapp (2006). As shown above, the specimens named after C. blanca show

a great range of morphological variability and it is possible that they constitute a species

complex, as already suggested by some authors (Muricy et al., 2011; Rapp, 2015).

The DNA amplification of the Curaçaoan specimens was not successful and its relationship

with the only available sequence of C. blanca from the Adriatic Sea (Imešek et al., 2014)

remains unknown. We name the specimens collected in Curaçao and analised in this study as

Clathrina cf. blanca until a molecular study considering the putative populations of Clathrina

“blanca” is done.

63

Fig. 8. Clathrina cf. blanca (UFRJPOR 6759): (A) specimen after fixation (arrow indicates thestalk); (B) tangential section of the clathroid body; (C) triactine I; (D) triactine II; (E) triactineof the stalk.

Table 6. Spicule measurements of Clathrina cf. blanca from Curaçao (UFRJPOR 6759).



Triactine I (body -

67.5 87.2 11.6 108.0 4.1 5.3 0.4 5.4 30

Triactine II (body) -

45.9 54.8 4.6 62.1 5.4 6.4 0.9 8.1 30

Triactine (stalk)

Unpaired 94.5 119.8 21.6 151.2 5.4 7.9 1.8 9.4 7Paired 40.5 51.8 5.7 62.1 8.1 8.1 0.0 8.1 10

Clathrina curaçaoensis sp. nov. (Figure 9, Table 7)

ETIMOLOGY

Named after the country of the type locality.

TYPE LOCALITY

Sunset Waters, Soto, Curaçao.

TYPE MATERIAL

Holotype: UFRJPOR 6734 (specimen in ethanol and slides); Sunset Waters, Soto, Curaçao;

12°16'01.58''N, 69°07'44.85''W; 3-10 m deep; coll. B. Cóndor-Luján, 20 August 2011.

COLOUR

Yellow in life and light beige in ethanol.


The analysed specimen has a massive cormus (0.7 x 0.4 x 0.3 mm) with a smooth and delicate

surface. The cormus is composed of irregular and loosely anastomosed tubes (Figure 9A). No

water-collecting tubes were observed. The aquiferous system is asconoid. The skeleton has no

special organization (Figure 9B) and it is exclusively composed of triactines. The tubes that

attach the sponge to the substrate are composed of parasagittal triactines (Figure 9C).

SPICULES (Table 7)

Triactines I. Regular (equiangular and equiradiate). Actines are slightly conical with blunt to

sharp tips (Figure 9D). Size: 87.5-130.0/7.5-10.0 µm.

Triactines II. Parasagittal (equiangular). Actines are slightly conical with blunt to sharp tips

(Figure 9E). Size: 57.5-80/7.5 µm (paired actine) and 99.9-137.7/8.1-8.8 µm (unpaired actine).

ECOLOGY

The specimen was found underneath boulders.

64



REMARKS

Within the genus Clathrina, seven species are yellow as C. curaçaoensis sp. nov.: C. aurea

Solé-Cava et al., 1991; C. chrysea Borojevic & Klautau, 2000, C. clathrus (Schmidt, 1864), C.

insularis Azevedo et al., submitted, C. lutea Azevedo et al., submitted, C. luteoculcitella

Wörheide & Hooper, 1999 and C. mutabilis Azevedo et al., submitted. However, compared to

C. curaçaoensis sp. nov, these species do not possess tubes differentiated for substrate

attachment supported by parasagittal triactines (triactines II) nor the same external morphology .

Among the non-yellow clathrinas with stalk (former “guanchas”), the species whose external

morphology most resembles that of C. curaçaoensis sp. nov. is C. arnesenae (Rapp, 2006).

Nonetheless, its skeleton does not comprise regular triactines as is the case of the new species;

instead, it is only composed of parasagittal triactines with cylindrical actines.

Regarding the triactines of the body of C. curaçaoensis sp. nov. (triactines I), they resemble

those of C. lutea. However, both species differ in their external morphology (as already

mentioned above). The cormus of C. lutea is formed by regular and tightly anastomosed tubes

while the cormus of C. curaçaoensis sp. nov. is composed of irregular and loosely anastomosed

tubes. Furthermore, in the ITS and C-LSU phylogenetic trees (Figures 31 and 32), C.

curaçaoensis sp. nov. (UFRJPOR 6734) did not cluster with C. lutea nor with any other yellow

clathrina. It appeared as a different lineage. Thus, based in morphological and DNA evidence,

we recognise it as a new species of Clathrina.

Table 7. Spicule measurements of Clathrina curaçaoensis sp. nov. (Holotype=UFRJPOR6734).

Spicule ActineLength (µm) Width (µm) N

Min Mean SD Max Min Mean SD MaxTriactine I 87.5 102.8 13.6 130.0 7.5 8.5 1.0 10.0 20Triactine II Unpaired 99.9 119.9 13.1 137.7 8.1 8.2 0.3 8.8 10

Paired 57.5 68.5 6.3 80.0 7.5 7.5 0 7.5 10

65

Fig. 9. Clathrina curaçaoensis sp. nov. (UFRJPOR 6734): (A) specimen after fixation; (B)tangential section of the body (cormus); (C) tangential section of an attachment tube; (D)triactine I; (E) triactine II (parasagittal).

Clathrina hondurensis Klautau & Valentine, 2003 (Figure 10, Table 8)

SYNONYMS

Clathrina hondurensis: Klautau & Valentine, 2003: 46;

Non Clathrina hondurensis: Rützler et al., 2014:101

Non Clathrina cf. hondurensis: Imešek et al., 2014:25; Klautau et al., 2016: 21.

TYPE LOCALITY

Turneffe, British Honduras, Caribbean Sea.

MATERIAL EXAMINED

UFRJPOR 6732 (specimens in ethanol and slides); Porto Mari, St. Willibrordus; 12°13'6.62"N,

69°05'13.26"W; 7.9 m deep; coll. B. Cóndor-Luján, 20 August 2011.

66


Clathrina hondurensis. Holotype (slide) BMNH 1938.3.28.4; Turneffe, British Honduras,

Caribbean Sea; coll. J.H. Borley, 20–22 March 1935.

COLOUR

White in life and light brown in ethanol.


The specimen is massive and it measures 1.4 x 1.0 x 0.2 cm (Figure 10A). The surface is

smooth and the consistency is compressible. The cormus is composed of irregular and tightly

anastomosed tubes (Figure 10B). No water-collecting tubes were observed. The aquiferous

system is asconoid. The skeleton has no special organization (Figure 10C) and it is composed of

triactines and rare trichoxeas (Figure 10C, arrow).

SPICULES (Table 8)

Trichoxeas. Straight and very slender (Figure 10D). Mostly broken. The size of the unique

whole trichoxea found is 325/2.5 µm.

Triactines. Regular (equiangular and equiradiate). Actines are conical with sharp tips (Figure

10E). Size: 100.0-212.5/13.6-25 µm.

ECOLOGY

This specimen was collected underneath boulders.


Southwestern Caribbean (Belize, Klautau & Valentine, 2003) and Southern Caribbean (Curaçao,

present study) ecoregions.

REMARKS

The external morphology and the shape of the triactines observed in the specimen from Curaçao

are similar to C. hondurensis. However, in the original description of that species, trichoxeas

were not reported and after the re-examination of the slides of the holotype, we did not find

them. As seen in other species, the presence of trichoxeas may not constitute a diagnostic

character, as they apparently constitute plastic characters. Regarding the spicule size, although

the triactines of the specimen from Curaçao can attain slightly larger sizes compared to the

specimen from Turneffe (Table 11), they are in the same size range. It is important to point out

that C. hondurensis was originally described based on one single specimen. Considering this,

the presence of trichoxeas and of slightly larger triactines in the specimen from Curaçao can be

attributed to intraspecific variation.

Rützler et al. (2014) recorded C. hondurensis from another Belizean locality (Belize barrier

reef near Carrie Bow), however, the skeleton of their specimen was composed of triactines

67

whose size range was 85-100 μm in length and 8-12 μm in width. Based on the original

description of C. hondurensis and the specimens from Curaçao, we believe that the specimen

reported by Rützler et al. (2014) does not correspond to C. hondurensis.

Recently, Klautau et al. (2016) considered the possibility of synonymy between C.

hondurensis and C. primordialis (Haeckel, 1872). In fact, both species present similar

morphology and their spicule size range do overlap, however, it is possible to recognize thicker

spicules in C. hondurensis (Table 6). As we could not successfully amplify any DNA region

from UFRJPOR 6732, we cannot affirm that the difference in width reflects plasticity and that

C. hondurensis and C. primordialis are synonyms. Therefore, we decided to maintain C.

hondurensis as a valid species and to identify the Curaçaoan specimen as C. hondurensis.

Fig. 10. Clathrina hondurensis (UFRJPOR 6732): (A) specimen in vivo; (B) specimen afterfixation; (C) tangential section of the body (cormus) skeleton; (D) detail of trichoxea indicatedby an arrow; (E) triactine.

68

Table 8. Spicule measurements of Clathrina hondurensis from Curaçao (UFRJPOR 6732) andof the holotype (BMNH 1938.3.28.4) and of C. primordialis taken from * Klautau & Valentine

(2003) and ** Haeckel (1872).

Specimen SpiculeLength (µm) Width (µm) N

Min Mean SD Max Min Mean SD MaxUFRJPOR 6732

Triactine 100.0 149.6 30.0 212.5 13.6 19.1 3.1 25 30Trichoxea 325.0 - - - 2.5 - - 1

BMNH 1938. 3.28.4

Triactine 120.0 142.9 15.5 175.0 12.5 16.8 2.4 20 20

BMNH 1938. 3.28.4*

Triactine 105.6 133.4 17.0 156.0 - 15.6 1.7 - 20

C. primordialis**

Triactine 100.0 - - 150.0 8 - - 12.0 -

Clathrina insularis Azevedo et al, submitted (Figure 11, Table 9)

SYNONYMS

Clathrina insularis: Azevedo et al, submitted

Clathrina sp. nov. 3 - UFRJPOR 6737a=UFRJPOR 6737: Klautau et al., 2013, 449 and 451.

TYPE LOCALITY

Cagarras, Fernando de Noronha Archipelago, Pernambuco, Brazil

MATERIAL EXAMINED

UFRJPOR 6737 (specimen in ethanol and slides); Playa Jeremi, Soto;

12°19'43.73''N,69°09'07.80''W; 14.9 m; coll. B. Cóndor-Luján, 22 August 2011.

ADDITIONAL MATERIAL RE-ANALYZED

Specimens in ethanol and slides: UFRJPOR 6533; Cagarras, Fernando de Noronha Archipelago,

Pernambuco, Brazil; 3°48'34.59''S, 32°23'27.91''W; 15 m; coll. F. Azevedo and G. Rodríguez,

27 June 2011. UFRJPOR 6530 and 6537; Ilha do Meio, Fernando de Noronha Archipelago,

Pernambuco, Brazil; 03°49'5.88''S,32°23'36.6''W;15 m ; coll. F. Azevedo and G. Rodríguez, 27

June 2011.

MATERIAL STUDIED FOR COMPARISON

Clathrina insularis. Holotype (specimen in ethanol and slides) UFRJPOR 6532; Cagarras,

Fernando de Noronha Archipelago, Pernambuco, Brazil; 3°48'34.59''S, 32°23'27.91''W; 15 m;

coll. F. Azevedo and G. Rodríguez, 27 June 2011.

COLOUR

Pale yellow in life and beige in ethanol.

69


The specimen is finely encrusting (0.4 x 0.4 x 0.1 mm, Figure 11A). The surface is smooth and

the texture is soft. The cormus is composed of irregular and loosely anastomosed tubes. Water-

collecting tubes are not present. The aquiferous system is asconoid. The skeleton has no

organization (Figure 11B) and it is composed of two categories of triactines.

SPICULES

Triactines I. Regular (equiangular and equiradiate). Actines are conical with sharp tips (Figure

9C). Size: 52.5-77.5/3.8-6.3 µm.

Triactines II. Regular (equiangular and equiradiate) or subregular (equiangular). Most abundant

spicules. Actines are cylindrical to slightly conical, distally undulated and with sharp tips

(Figure 9D-E). Size: 102.7-145.0/3.7-6.6 µm.

ECOLOGY

This sponge was found in a cryptic habitat, underneath boulder.


Fernando de Noronha and Atoll das Rocas ecoregion of the Tropical Southwestern Atlantic

Province (Fernando de Noronha Archipelago, Azevedo et al., submitted) and Southern

Caribbean (Curaçao, present study).

REMARKS

The external morphology and the skeleton composition of the Curaçaoan specimen match the

description of C. insularis. In the ITS phylogenetic tree (Figure 31), it clustered in the clade of

that species (pp=1, b=100), corroborating its morphological identification. The specimens

UFRJPOR 6530, UFRJPOR 6533 and UFRJPOR 6537 were already analysed in Azevedo et al.,

submitted, and herein, we provide their ITS sequences.

Table 9. Spicule measurements of Clathrina insularis (UFRJPOR 6737) from Curaçao and ofthe holotype (UFRJPOR 6532).


Min Mean SD Max Min Mean SD MaxUFRJPOR 6737


UFRJPOR 6532


70

Fig. 11. Clathrina insularis (UFRJPOR 6737): (A) specimen in vivo; (B) tangential section ofthe body (cormus), (C) triactine I, (D-E) triactine II.

Clathrina lutea Azevedo et al., submitted (Figure 12, Table 10)

SYNONYMS

Clathrina primordialis: Lehner & Van Soest, 1998: 97?

Clathrina sp. nov. 8 - UFRJPOR 6761 and ZMAPOR 08344: Klautau et al., 2013, 449 and 451.

TYPE LOCALITY

Pedra Lixa, Abrolhos Archipelago, Caravelas, Bahia, Brazil.

MATERIAL EXAMINED

UFRJPOR 6761 (specimen in ethanol and slides); Porto Mari, St. Willibrordus, Curaçao;

12°13'6.62"N, 69°05'13.26"W; 10.6 m deep; coll. Giselle Lôbo-Hajdu, 20 August 2011.


Clathrina lutea. Holotype (specimen in ethanol and slides) UFRJPOR 5173; Pedra Lixa,

Abrolhos Archipelago, Caravelas, Bahia, Brazil; 17°41'S, 38°59'W; 7 m deep; coll. C.

Zilberberg & L. Monteiro, 21 March 2005.

71

COLOUR

Dark yellow in life and white in ethanol.


This specimen has a rough massive cormus (1.0 x 0.4 x 0.1 cm, Figure 12A) composed of

regular and tightly anastomosed tubes (Figure 12B). Water-collecting tubes were observed. The

aquiferous system is asconoid. The skeleton has no organization and it is composed of one

category of triactines (Figure 12C). Some broken trichoxeas were found in the spicule slide.

SPICULES

Triactines. Regular. Actines are cylindrical to slightly conical, slightly undulated and with blunt

tips (Figure 12D). Size: 75.0-97.5/7.5-10 µm.

ECOLOGY

This sponge was collected in a light protected environment, inside a small crevice.


Within the Tropical Southwestern Atlantic includes the Floridian (Florida, United States of

America: UFRJPOR 5818, Klautau et al., 2013), Eastern Caribbean (Virgin Islands: ZMAPOR

08344, Klautau et al., 2013) and Southern Caribbean (Curaçao, present study) ecoregions. In the

Tropical Northwestern Atlantic includes Eastern Brazil (Abrolhos Archipelago) and Fernando de

Noronha and Atoll das Rocas (Rocas Atoll) ecoregions (Azevedo et al., submitted).

REMARKS

Compared to the type material of C. lutea, our specimen has slightly larger triactines and actines

are not very undulated. Besides, some broken trichoxeas were found in the spicule slide of the

Curaçaoan material whereas in the Brazilian specimens they were not observed. Despite these

slight differences, the Curaçaoan specimen grouped in the clade of C. lutea (pp=1, b=100,

Figure 31), which included Brazilian and Caribbean specimens.

In 1998, Lehner & Van Soest reported C. primordialis to Jamaica. After revising the

description of that record, including the in vivo figure (Figure 23, page 97), we rather consider it

as C. lutea based principally on its external morphology.

Table 10. Spicule measurements of Clathrina lutea from Curaçao (UFRJPOR 6761) and of theholotype (UFRJPOR 5173).


Min Mean SD Max Min Mean SD MaxUFRJPOR 6761 Triactine 75.0 88.8 5.6 97.5 7.5 9.4 0.7 10.0 30UFRJPOR 5173 Triactine 69.3 78.5 3.8 84.0 6.5 7.5 0.4 8.3 30

72

Fig. 12. Clathrina lutea (UFRJPOR 6761): (A) specimen in vivo (photo taken by G. Lôbo-Hajdu); (B) specimen after fixation; (C) tangential section of the body (cormus); (D) triactine.

Clathrina mutabilis Azevedo et al., submitted (Figure 13, Table 11)

SYNONYMS

Clathrina mutabilis: Azevedo et al., submitted

Clathrina sp. nov. 4 - UFRJPOR 6733 and UFRJPOR 6741: Klautau et al., 2013, p: 449 and

451.

TYPE LOCALITY

Cagarras, Fernando de Noronha Archipelago, Pernambuco, Brazil.

MATERIAL EXAMINED

Specimens in ethanol and slides: UFRJPOR 6704 and UFRJPOR 6719; Playa Kalki, Westpunt,

Curaçao; 12°22'29.86"N, 69°09'30.63"W; 6.7 m deep; coll. E. Hajdu and B. Cóndor-Luján, 21

August 2011; UFRJPOR 6717; Water Factory, Willemstadt, Curaçao; 12°06'30.88"N,

73

68°57'13.53"W; coll. B. Cóndor-Luján, 19 August 2011; UFRJPOR 6733 and UFRJPOR 6735,

Porto Mari, St. Willibrordus, Curaçao; 12°13'6.62"N, 69°05'13.26"W; 7.9 m deep; coll. B.

Cóndor-Luján, 20 August 2011; UFRJPOR 6740; Sunset Waters, Soto, Curaçao; 12°16'01.58"N,

69°07'44.85"W, 9-12 m deep; coll. B. Cóndor-Luján, 22 August 2011; UFRJPOR 6741,

UFRJPOR 6743 and UFRJPOR 6744; Sunset Waters, Soto, Curaçao; 12°16'01.58"N,

69°07'44.85"W, 8.9 m, 9.8 m and 7.2 m deep, respectively; coll. B. Cóndor-Luján, 22 August

2011. Slides: UFRJPOR 6699; Hook’s Hut, Willemstadt, Curaçao; 12°07'18.94"N,

68°58'11.46"W; < 10 m deep; coll. B. Cóndor Luján and G. Lôbo-Hajdu, 17 August 2011;

UFRJPOR 6712; Daai Booi, St. Willibrordus, Curaçao; 12°12'43.12"N, 69°05'8.42"W; 4.9 m

deep; coll. B. Cóndor-Luján, deep, 18 August 2011; UFRJPOR 6736 (specimens in ethanol and

slides); Playa Jeremi, Soto, Curaçao; 12°19'43.73 N, 69°09'07.80 W; 4.9 m deep; coll. B.ʺ ʺ

Cóndor-Luján, 22 August 2011; UFRJPOR 6747; Sunset Waters, Soto, Curaçao; 12°16'01.58"N,

69°07'44.85"W, 4.9 m deep; coll. B. Cóndor-Luján, 22 August 2011; UFRJPOR 6750; Tug Boat,

Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W, coll. B. Cóndor-Luján, 23

August 2011.


Clathrina mutabilis. Holotype (specimen in ethanol and slides) UFRJPOR 6526; Cagarras,

Fernando de Noronha Archipelago, Pernambuco, Brazil; 3°48'34.59''S, 32°23'27.91''W; 15 m;

coll. F. Azevedo and G. Rodríguez, 27 June 2011.

COLOUR

Pale yellow in life and white to beige in ethanol.


The specimens have a massive cormus composed of irregular and loosely anastomosed tubes

(Figure 11A) which form water-collecting tubes. When tubes are contracted, this sponge has a

more encrusting growth form (UFRJPOR 6743, Figure 11B). The largest fragment deposited in

our collection belongs to the specimen UFRJPOR 6735 (Figure 11C) which measures 0.8 x 0.6

x 0.2 cm. The aquiferous system is asconoid. The skeleton has no organization (Figure 11D) and

it is composed of two categories of triactines.

SPICULES (Table 11)

Triactines I. Regular (equiangular and equiradiate) but some subregular spicules (equiangular

but not equiradiate) were also found. Actines are conical, straight, with sharp tips (Figure 13E).

Smaller than triactines II. Size: 60-112.5/7.5-10 µm.

74

Triactines II. Regular (equiangular and equiradiate), subregular (equiangular but not

equiradiate) (Figure 13F) or parasagittal (Figure 13G). Frequent. Actines are slightly conical to

cylindrical, slightly undulated, with blunt tips. Size: 100.0-195.0/5-11.3 µm.

ECOLOGY

The specimens were found underneath boulders and broken corals.


Fernando de Noronha and Atoll das Rocas (Fernando de Noronha Archipelago , Azevedo et al.,

submitted) and Southern Caribbean (Curaçao , present study) ecoregions. Clathrina mutabilis

was found in all Curaçaoan sampled localities.

REMARKS

In the ITS phylogenetic tree, these specimens clustered within the clade of C. mutabilis

(pp=0.81, b=91%, Figure 31) and they match its morphological description. However, some

slight differences were found. Compared to the holotype, the skeleton of the Curaçaoan

specimens has more parasagittal triactines II and does not present trichoxeas (or at least, they

were not observed). This may be the result of plasticity. It is important to point out that in the

analised specimens, it was possible to observe water-collecting tubes, and thus, we complement

the original description of this species providing one more diagnostic character.

Table 11. Spicule measurements of Clathrina mutabilis from Curaçao (UFRJPOR 6741,UFRJPOR 6704 and UFRJPOR 6745) and of the holotype (UFRJPOR 6526). U=unpaired and

P=paired actines.



Triactine I 100.0 123.8 10.4 145.0 7.5 8.9 0.9 10 30ParasagittalTriactine I

U 125.0 166.0 17.3 195.0 5 8.2 1.1 10 30P 100.0 116.1 11.2 132.5 5 7.5 1.1 10 30

Triactine II 67.5 82.4 8.5 95.0 6.3 8.0 0.8 8.8 30UFRJPOR6704

Triactine I 107.5 135.5 15.9 162.5 7.5 10.3 1.0 11.3 20ParasagittalTriactine I

U 122.5 142.4 15.6 177.5 7.5 8.3 1.0 10 30P 75.0 98.7 12.8 127.5 6.3 7.6 0.5 8.8 30

Triactine II 60.0 95.5 12.6 112.5 7.5 8.8 1.2 10 20UFRJPOR6745

Triactine I 100.0 126.0 18.1 160.0 7.5 9.0 1.1 10 20ParasagittalTriactine I

U 150.0 169.5 13.1 195.0 7.5 9.1 1.0 10 30P 97.5 121.0 12.9 140.0 7.5 8.3 0.9 10 30

Triactine II 60.0 81.6 13.4 102.5 7.5 8.6 1.1 10 20UFRJPOR6526


75

Fig. 13. Clathrina mutabilis: (A) specimen UFRJPOR 6704 in vivo; (B) specimen UFRJPOR6743 in vivo; (C) specimen UFRJPOR 6735 after fixation; (D) tangential section of the body(cormus); (E) triactine I; (F) triactine II; (G) parasagittal triactine II. Skeleton images were takenfrom slides of the specimen UFRJPOR 6741.

76

Genus Leucetta Haeckel, 1872

TYPE SPECIES

Leucetta primigenia Haeckel, 1872.

DIAGNOSIS

"Leucettidae with a homogeneous organisation of the wall and a typical leuconoid aquiferous

system. There is neither a clear distinction between the cortex and the choanoskeleton, nor the

presence of a distinct layer of subcortical inhalant cavities. The atrium is frequently reduced to a

system of exhalant canals that open directly into the osculum" (Borojevic et al., 2002).

Leucetta floridana Haeckel, 1872 (Figure 14, Table 12)

SYNONYMS

Amphoriscus floridanus: Haeckel, 1872: 144.

Dyssycus floridanus: Haeckel, 1872: 144.

Leucaltis floridana: Haeckel, 1872: 144.

Leucaltis impura: Haeckel, 1872: 144.

Leucaltis pura: Haeckel, 1872: 144.

Leucetta aff. floridana: Lehnert & van Soest, 1998: 99.

Leucetta floridana: de Laubenfels, 1950: 146; Moraes et al., 2006: 167; Muricy et al., 2008:

132; Valderrama et al., 2009: 9; Lanna et al., 2009: 7; Muricy et al., 2011: 36-37; Rützler et al.,

2014: 102; Azevedo et al, submitted.

Leucetta microraphis: Tanita, 1942: 111; Borojevic & Peixinho, 1976: 1003.

Leucetta sp. : Moraes et al., 2003: 17.

Leucilla floridana: Jenkin, 1908: 453.

Lipostomella floridana: Haeckel, 1872: 144.

TYPE LOCALITY

Coast of Florida, United States of America.

MATERIAL EXAMINED

UFRJPOR 6726 (specimen in ethanol and slides); Water Factory, Willemstadt; 12°06'30.88"N,

68°57'13.53"W; 17.8 m deep; coll. E. Hajdu, 19 August 2011; UFRJPOR 6757 (specimen in

ethanol and slides); Tug Boat, Caracasbaai, Willemstadt; 12°04'08.20"N, 68°51'44.40"W; 6.2

m deep; coll. B. Cóndor-Luján, 23 August 2011; UFRJPOR 6765 (specimen in ethanol); Hook’s

Hut, Willemstadt, Curaçao; 12°07'18.94"N, 68°58'11.46"W; 13.3 m deep; coll. E. Hajdu, 18

August 2011.

77

COLOUR

White to light blue in life and grayish white to brown in ethanol.


This species has a massive growth form (Figure 14A). The largest specimen measures 1.6 x 2.6

cm (Figure 14B). The body is ridged and hispid. The three analised specimens presented a

single apical osculum (largest diameter=0.5 cm). In the specimen UFRJPOR 6757, the osculum

is particularly elongated and bears a very delicate margin. The atrial cavity is wide and also very

hispid. The aquiferous system is leuconoid. The consistency is very rough and incompressible.

SKELETON

The skeleton is typical of the genus. It does not have a special organization and it is composed

of two size categories of triactines (I and II) and tetractines (I and II). The cortex and atrial wall

are thin whereas the choanosome is thick. Triactines II and tetractines II, which are the largest

spicules, are found in in the cortex and in the choanosome, tangentially disposed. Tetractines II

are rare. Triactines I and tetractines I are spread in the choanosome and in the atrium. The apical

actine of tetractines I penetrates the exhalant canals and the atrial cavity. Near the atrium,

triactines I and tetractines I become sagittal.

SPICULES

Triactines I. Regular. Actines are conical, straight, with blunt tips (Figure 14C). Frequent.

Sagittal triactines I were also observed. Size: 87.5-175.0/10.0-22.5 µm.

Triactines II. Regular. Actines are conical, straight, with blunt tips (Figure 14D). Very variable

size: 378.4-2378.4/54.1-389.2 µm.

Tetractines I. Regular. Actines are slightly conical, straight, with blunt to sharp tips (Figure

14E). The apical actine is smooth, thinner than the basal actines and has a sharp tip. Sagittal

tetractines I were also observed. Size: 102.5-200.0/11.2-20.0 µm (basal actine) and 25-50/7.5-10

µm (apical actine).

Tetractines II. Regular. Rare. Actines are conical, straight, with blunt tips (Figure 14F). Very

variable size: 464.9-2162.2/108.1-270.3 µm.

ECOLOGY

This species was found underneath boulders close to some incrusting and massive (cf. Clathria)

demospongias. No associated organism was found on the surface of the analised specimens.


Widespread in the Western Atlantic: Tropical Southwestern Atlantic Province including the

Floridian (Haeckel, 1872), Bermuda (Bermudas, de Laubenfels, 1950), Greater Antilles

(Jamaica, Lehner & van Soest, 1998) and the Southern Caribbean (Urabá and San Andrés,

78

Valderrama et al., 2009 and Curaçao, present study) ecoregions, North Brazil Shelf Province

(Pará, Borojevic & Peixinho, 1976) and Tropical Northwestern Atlantic Province comprising the

Northeastern Brazil, Eastern Brazil, Fernando de Noronha and Atoll das Rocas ecoregions

(Borojevic & Peixinho, 1976; Valderrama et al., 2009).

REMARKS

Our specimens match the original description of L. floridana as well as the redescription

provided by Valderrama et al. (2009). Haeckel's measurements are provided here: Triactines and

tetractines I: 150-250/10-15 µm and triactines and tetractines II: 700-1500/100-150 µm and

those of Valderrama et al., (2009) are presented in Table 12. Leucetta floridana is not only one

of the few species of Calcarea already reported for the Caribbean Sea but it is also one of the

most widespread species along the Western Tropical Atlantic.

Table 12. Spicule measurements of Leucetta floridana from Curaçao (UFRJPOR 6726 andUFRJPOR 6757) and of its redescription (UFRJPOR 5360. Valderrama et al., 2009).



Triactine I - 121.5 143.6 14.6 172.8 10.8 13.9 1.6 16.2 20Triactine II - 972.0 1431.3 354.9 2378.4 129.6 214.5 52.5 345.6 21Tetractine I B 105.0 139.1 23.3 200.0 12.5 15.2 2.2 20.0 20

A 25.0 33.4 8.4 45 7.5 8.1 1.2 10 8Tetractine II B 675.7 1228.2 293.6 1621.6 108.1 171.8 49.7 216.2 9

UFRJPOR6757

Triactine I - 87.5 126.7 22.2 175.0 10.0 15.5 3.2 22.5 30Triactine II - 378.4 1383.3 673.9 2378.4 54.05 207.7 98.3 389.2 24Tetractine I B 102.5 138.3 19.6 200.0 11.2 15.3 2.8 20.0 30

A 40.0 44.4 4.3 50.0 10 10.0 0.0 10 4Tetractine II B 464.9 1294.9 568.7 2162.2 108.1 172.0 59.1 270.3 9

UFRJPOR5360

Triactine I - 105.6 143.3 28.7 217.8 9.9 17.1 4.9 33,0 30Triactine II - 257.4 696.2 279.7 1181.5 33.0 102.1 46.2 194.6 30Tetractine I - 105.6 137.4 24.1 224.4 9.9 15.4 3.6 26.4 30Tetractine II - 278.0 665.5 301.0 1042.5 48.7 102.5 51.3 180.7 8

79

Fig. 14. Leucetta floridana (UFRJPOR 6757): (A) specimen in vivo; (B) specimen afterfixation; (C) triactine I; (D) triactine II; (E) tetractine I; (F) tetractine II.

Genus Amphoriscus Haeckel, 1872TYPE SPECIES

Amphoriscus chrysalis (Schmidt, 1864)

DIAGNOSIS

"Amphoriscidae with syconoid organization of the aquiferous system. Scattered spicules in the

choanosome are always absent" (Borojevic et al., 2002).

80

Amphoriscus micropilosus sp. nov. (Figures 15 & 16, Table 13)

ETIMOLOGY

From the Latin pilosus (= hairy), for the presence of microdiactines crossing the cortex.

TYPE LOCALITY

Sunset Waters, Soto, Curaçao.

TYPE MATERIAL

Holotype: UFRJPOR 6739 (specimens in ethanol and slides); Sunset Waters, Soto, Curaçao;

12°16'01.58"N, 69°07'44.85"W; 13.1 m deep; coll. B. Cóndor-Luján, 22 August 2011.

Paratypes: UFRJPOR 6755 and UFRJPOR 6756 (specimens in ethanol and slides); Tug Boat,

Caracasbaai, Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 8.6 m deep; coll. B.

Cóndor-Luján, 23 August 2011.

COLOUR

White in life and in ethanol. Transparent and bright.


This sponge has a very variable external morphology (Figure 15A-F), which can be tubular

(Figures 15A and 15D) to flatened sac-shaped (Figures 15C and 15F) but always with an apical

osculum. The surface is smooth, although microdiactines protrude through the surface. The

consistency is rough. The largest specimen (UFRJPOR 6756) measures 1.2 x 0.4 cm and it

presents a short peduncle (arrow in Figure 15D). The osculum has a margin sustained by T-

shaped triactines and it is surrounded by short trichoxeas. The aquiferous system is syconoid.

SKELETON

The skeleton is typical of the genus (Figure 15G). The cortical skeleton is composed of

perpendicular microdiactines spread through the surface (Figure 15H) or organized in tufts (only

in UFRJPOR 6739, Figure 15I), triactines and the paired actines of the subcortical tetractines.

The triactines are distributed tangentially to the surface (arrow in Figure 15J). The choanosomal

skeleton is inarticulated, composed of the apical actines of the subcortical tetractines and by rare

subatrial triactines. The apical actine of the tetractines crosses the choanosome and occasionally

reaches the atrium (black arrow in Figure 15K). The unpaired actine of the subatrial triactines

points toward the cortex (white arrow in Figure 15K). The atrial skeleton is exclusively

composed of tetractines with their apical actine projected into the atrial cavity (asterisk in Figure

15K).

SPICULES

Microdiactines. Fusiform, straight, with sharp tips. Size: 27.0-94.5/1.1-1.4 µm

81

Cortical triactines. Sagittal. Actines are smooth, conical, with sharp tips (Figure 16A).

Sometimes the paired actines are curved. Size: 102.6-310.5/8.1-18.9 µm (paired actines), 89.1-

310.5/6.7-18.9 µm (unpaired actine).

Subcortical tetractines. Sagittal. Actines are straight, smooth, conical, with sharp tips (Figure

16B). The apical actine is very large. They are the largest spicules in this species. Size: 172.8-

572.4/21.6-64.8 µm (paired actine), 183.6-540/30-64.8 µm (unpaired actine), 162.0-

1036.8/21.6-64.8 µm (apical actine).

Subatrial triactines. Sagittal. Actines are conical, smooth, straigth, with sharp tips (Figure 16C).

The paired actines are shorter than the unpaired ones and some are slightly curved. Sometimes,

one paired actine is longer than the other one. Size: 81.0-240.3/8.1-21.6 µm (paired actine),

91.8-610/8.1-18.9 µm (unpaired actine).

Atrial tetractines. Sagittal. Actines are conical, straight, smooth and have sharp tips. The

unpaired actine is slightly longer than the paired ones (Figure 16D). The apical actine is the

shortest actine. Size (length/width): 100.0-264.6/10-24.3 µm (paired actine), 83.7-297/10-21.6

µm (unpaired actine), 27-115/8.1-13.5 µm (apical).

ECOLOGY

Specimens were found underneath broken coral boulders. No associated organism was found.

Some balls of sediment were found inside the atrial cavity of the holotype.


Southern Caribbean (provisionally endemic to Curaçao present study).

REMARKS

The genus Amphoriscus comprises 16 species (Van Soest et al., 2016, Van Soest, 2017). Among

them, four species have been reported for the Caribbean Sea: Amphoriscus oviparus (Haeckel,

1872), A. perforatus (Haeckel, 1872), A. testiparus (Haeckel, 1872), and A. urna Haeckel, 1872,

however, none of them present a skeleton composition similar to A. micropilosus sp. nov.

Different from the new species, whose subatrial skeleton is exclusively composed of triactines,

all the referred species have subatrial tetractines and lack cortical microdiactines. Moreover, A.

perforatus presents atrial triactines, which are absent in A. micropilosus sp. nov., and A. urna

lacks the cortical triactines present in the Curaçaoan species.

Considering the species recorded out of the Caribbean Sea, the species that most resembles

the new species is A. elongatus (Poléjaeff, 1883), originally described from the Indian Ocean.

Nonetheless, these two species differ in spicule size (Table 13), mainly in the width of the apical

actine of the atrial tetractines. which is thinner in the new species (8.1-13.5 µm) than in A.

elongatus (16-20 µm). Besides, in A. elongatus the radial tubes “meet in threes, in fours, or in

82

larger numbers around the same shallow invagination of the gastric cavity” (Poléjaeff, 1883),

which was not observed in the new species.

83

Fig. 15. Amphoriscus micropilosus sp. nov.: (A-C) UFRJPOR 6756, UFRJPOR 6739 andUFRJPOR 6755 in vivo; (D-F) UFRJPOR 6756, UFRJPOR 6739 and UFRJPOR 6755 afterfixation; (G) cross section of the skeleton; (H) trichoxeas along the surface in UFRJPOR 6755;(I) tufts of trichoxeas in UFRJPOR 6739; (J) cortical skeleton showing triactines (arrow); (K)choanosomal and atrial skeletons and the apical actine of cortical tetractines (black arrow),unpaired actine of a subatrial triactine (white arrow) and apical actine of an atrial tetractine (*).Abbreviations: cx=cortex; at=atrium. All photos were taken from the holotype slide (UFRJPOR6739) except when indicated.

Fig. 16. Spicules of Amphoriscus micropilosus sp. nov. (Holotype=UFRJPOR 6739): (A)cortical triactine; (B) subcortical tetractine; (C) subatrial triactine; (D) atrial tetractine.

Table 13. Spicule measurements of Amphoriscus micropilosus sp. nov. (UFRJPOR 6739,UFRJPOR 6755 and UFRJPOR 6756) and of A. elongatus . H=holotype. P=paired, U=unpaired,

A=apical and B=basal actines.


Min Mean SD Max Min Mean SD Max

UFRJPOR6739 (H)

Micro-diactine - 51.3 75.6 16.8 94.5 1.1 1.3 0.1 1.4 7Cortical Triactine

P 102.6 204.0 51.7 310.5 8.1 14.4 2.8 18.9 14U 110.7 229.5 54.5 283.5 6.7 14.1 3.0 18.9 15

SubcorticalTetractine

P 356.4 475.2 71.1 572.4 32.4 52.9 9.2 64.8 20U 216.0 392.0 127.2 540.0 48.6 54.5 4.7 64.8 10A 162.0 449.6 158.8 658.8 21.6 49.7 12.4 64.8 19

Subatrial Triactine

P 81.0 177.5 46.7 240.3 8.1 13.7 3.7 21.6 20U 91.8 186.3 50.5 243.0 8.1 12.4 2.6 16.2 20

84

Atrial tetractine

P 167.4 205.2 29.8 264.6 12.2 17.1 3.6 24.3 9U 140.4 228.6 45.2 297.0 12.2 15.6 3.0 21.6 9A 51.3 65.1 11.4 81.0 8.1 11.5 1.9 13.5 8

UFRJPOR6755

Micro-diactine - 27.0 51.3 22.7 81.0 1.1 1.2 0.2 1.4 4Cortical triactine

P 113.4 174.2 32.3 240.3 8.1 14.0 2.6 18.9 20U 89.1 221.1 64.9 310.5 8.1 14.4 3.2 18.9 20


P 172.8 223.2 62.6 367.2 21.6 30.5 7.5 43.2 14U 183.6 229.0 54.8 324.0 32.4 40.0 9.0 54.0 5A 291.6 491.8 217.8 1036.8 21.6 35.7 11.4 54.0 13

Subatrial triactine

P 86.4 165.4 41.8 232.2 8.1 14.0 2.5 18.9 20U 135.0 242.2 61.6 351.0 9.5 13.7 2.4 18.9 20

Atrial tetractine

P 113.4 173.3 34.1 229.5 10.8 14.0 1.7 16.2 20U 83.7 200.0 56.6 283.5 10.8 15.6 2.5 21.6 19A 27.0 49.7 16.2 78.3 8.1 11.1 1.8 13.5 19

UFRJPOR6756

Micro-diactine - 45.0 61.7 16.7 87.5 1.0 1.2 0.1 1.2 6Cortical triactine

P 107.5 157.3 23.6 212.5 10 12.0 1.5 17.5 27U 157.5 195.4 23.0 230.0 10.0 12.2 0.8 12.5 26


P 250.0 363.9 67.9 510.0 25.0 39.3 5.9 50 23U 250.0 279.0 23.3 315.0 30.0 37.0 4.5 40.0 5A 325.0 555.0 110.4 740.0 30.0 42.3 7.0 55.0 30

Subatrial triactine

P 125.0 160.8 23.5 207.5 12.5 12.9 0.9 15 9U 255.0 346.3 110.2 610.0 10.0 13.8 2.4 18.8 15

Atrial tetractine

P 100.0 142.3 25.4 212.5 10.0 11.9 1.7 15 27U 125.0 184.7 43.4 250.0 10.0 12.4 1.8 17.5 22A 55.0 78.8 17.8 115.0 10.0 10.3 0.8 12.5 10

A.elongatus

Micro-diactine - - 100 - - - 2.5 - - -Cortical triactine

P - - - 250 - 15 - - -U - - - 450 - 15 - - -

Cortical tetractine

B - 600 - - - 70 - - -A 600 - - - - - - -

Atrial tetractine

P - 250 - - 16 - - 20 -U - - - 450 16 - - 20 -


TYPE SPECIES

Leucilla amphora Haeckel, 1872

DIAGNOSIS

"Amphoriscidae with sylleibid or leuconoid organization. The choanoskeleton is formed

primarily by the apical actines of giant cortical tetractines and the unpaired actines of subatrial

triactines or tetractines. It may contain dispersed spicules, but a typical articulated

choanoskeleton is always absent" (Borojevic et al., 2002, emend).

85

Leucilla antillana sp. nov. (Figures 17 & 18, Table 14)

ETIMOLOGY

From its distribution in Curaçao located in the Leeward Antilles Ridge.

TYPE LOCALITY

Water Factory, Willemstadt, Curaçao.

TYPE MATERIAL

Holotype: UFRJPOR 6768 (specimen in ethanol and slides); Water Factory, Willemstadt,

Curaçao; 12°06'30.88"N, 68°57'13.53"W; 9.9 m deep; coll. B. Cóndor-Luján, 23 August 2011.

COLOUR



This species has an irregular tubular shape being wider at the base (Figure 17A). It measures 1.0

x 0.8 x 0.1 cm. The surface is slightly hispid due to some protruding spicules. Its texture is

rough. The osculum is apical and has a delicate crown of trichoxeas (arrown in the Figure 17B).

The aquiferous system is leuconoid.

SKELETON

The skeleton is characteristic of the genus (Figure 17C). The cortical skeleton is exclusively

formed by tetractines (Figure 17D) with the basal actines tangentially disposed on the surface.

The choanosomal skeleton is inarticulated, composed of the apical actine of the cortical

tetractines (arrow in Figure 16E), which occasionally crosses the atrial skeleton, and of the

unpaired actine of the subatrial triactines (white arrow in Figure 16F). The subatrial triactines do

not form a continuous layer, instead, they are irregularly scattered at this region. The atrial

skeleton is composed of tetractines with the apical actine projected into the atrium (black arrow

in Figure 16F).

SPICULES (Table 14)

Cortical tetractines. Sagittal. Actines are conical with sharp tips. The paired actines are

frequently curved. The apical actine is straight and it is the longest actine (Figure 17A). Some

undulated apical actines were also observed. Size: 350-485/35-60 μm (paired actine), 75-

200/35-60 μm (unpaired actine) and 285-550/35-55 μm (apical actine).

Subatrial triactines. Sagittal. Actines are conical with sharp tips. The paired actines are straight

and smaller than the unpaired one (Figure 17B-C). Some slightly curved paired actines were

also observed. Very variable size: 175-460/15-60 μm (paired actine) and 225-490/15-55 μm

(unpaired actine).

86

Atrial tetractines. Sagittal. Actines are conical with very sharp tips. The unpaired actine is

slightly longer than the paired ones (Figure 17D). The apical actine is the thinnest and shortest

actine. Size: 120-270/10-12.5 μm (paired actine), 185-355/10-12.5 μm (unpaired actine) and 15-

50/5-10 μm (apical actine).

ECOLOGY

This species was found underneath coral boulders.



REMARKS

The genus Leucilla comprises 13 valid species distributed in all oceans (Van Soest et al., 2016).

Among these species, none of them has the same skeleton composition as Leucilla antillana sp.

nov. The species that most resemble our new species are Leucilla uter Poléjaeff, 1883 (type

locality: Bermudas) and L. sacculata Carter, 1890 (type locality: Fernando de Noronha

Archipelago, Brazil). However, their skeletons include subatrial tetractines, which are absent in

L. antillana sp. nov. Besides, the size range of the other spicule categories does not match our

new species (Table 14). The apical actine of the cortical tetractines of L. uter is almost twice

longer (400--1200 μm) than that of L. antillana sp. nov. (285-550 μm) and its atrial tetractines

are thicker as well (20 μm against 10-12.5 μm). Comparing to L. sacculata, L. antillana sp. nov.

has thinner spicules (84.7 μm against 15-60 μm). Leucilla sacculata and L. uter also differ from

L. antillana sp. nov. in the ornamentation of the osculum. In L. antillana sp. nov., the osculum is

surrounded by a crown of trichoxeas whereas in L. sacculata it is surrounded by a crown of

microdiactines and in L. uter it is naked. However, this last difference should be taken with

caution as the presence of a crown of trichoxeas is not consistent among individuals of the same

species within some calcaronean genera.

87

Fig. 17. Leucilla antillana sp. nov. (UFRJPOR 6768): (A) specimen in vivo; (B) specimen afterfixation with the crown of trichoxea (arrow); (C) cross section of the skeleton; (D) tangentialsection of the cortex; (E) choanoskeleton with the apical actine of a tetractine (arrow); (F) atrialskeleton with a subatrial triactine (white arrow) and an apical actine of an atrial tetractine (blackarrow). Abbreviations: ct=cortex, at=atrium.

88

Fig. 18. Spicules of Leucilla antillana sp. nov. (UFRJPOR 6768): (A) cortical tetractine; (B)large subatrial triactine; (C) small subatrial triactine; (D) atrial tetractine.

Table 14. Spicule measurements of Leucilla antillana sp. nov. (H=holotype=UFRJPOR 6768),L. uter and L. sacculata. P=paired, U=unpaired, A=apical and B=basal actines.

Species Spicule ActineLength (µm) Width (µm) N


Cortical Tetractine

P 305.0 404.4 62.0 485.0 35 50.1 6.4 60.0 17U 75.0 131.0 45.3 200.0 35 48.8 7.4 60.0 10A 285.0 405.0 64.6 550.0 35 49.2 7.1 55.0 19

Subatrial Triactine

P 175.0 276.0 91.3 460 15.0 32.8 13.1 60.0 20U 225.0 389.4 71.2 490.0 15.0 34.4 13.4 55.0 18

Atrial Tetractine

P 120.0 224.0 34.8 270.0 10 10.3 0.8 12.5 20U 185.0 265.0 41.9 355.0 10 10.5 1.0 12.5 20A 15.0 30.0 8.9 50.0 5 8.0 1.7 10.0 20

Leucilla uter

Micro-diactine - - 400 - - - 2.5 - - -Cortical Tetractine

B 400.0 - - 600.0 - - - 50 -A 400.0 - - 1200.

0- - - 50 -

Subatrial Triactine

U - - - 600.0 30 - - 50 -P - - - 420.0 21 - - 35 -

89

Atrial Tetractine

P - - - 400.0 - 20 - - -U 250.0 - - 350.0 - 20 - - -A - - - 200.0 - 20 - - -

Leucilla sacculata

Micro-diactine - - 84.7 - - - - - - -Cortical Tetractine

- - 564.4 - - - - - 84.7 -

Subatrial Triactine

- - 564.4 - - - - - 84.7 -

Subatrial Tetractine

- - 564.4 - - - - - 84.7 -

Genus Leucandra Haeckel, 1872

TYPE SPECIES

Leucandra egedii (Schmidt, 1870)

DIAGNOSIS

"Grantiidae with sylleibid or leuconoid organization. Longitudinal large diactines, if present, are

not restricted to the cortex, but lie obliquely across the external part of the sponge wall and

protrude from the surface of the sponge" (Borojevic et al., 2002).

Leucandra caribea sp. nov. (Figures 19-21, Table 17)

ETIMOLOGY

Named after its distribution in the Caribbean Sea.

TYPE LOCALITY

Tug Boat, Caracasbaai, Willemstadt, Curaçao.

TYPE MATERIAL

Holotype: UFRJPOR 6754 (specimen in ethanol and slides); Tug Boat, Caracasbaai,

Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 13.9 m deep; coll. B. Cóndor-Luján, 23

August 2011.

COLOUR

White in life and beige in ethanol.


This species has a sac-shaped external morphology: it is wide at the base and becomes narrower

near the apical osculum (Figure 19A). It measures 0.7 cm length and 0.3 cm width (Figure 19B).

This sponge is quite smooth and compressible. Near the suboscular region, scattered short

trichoxeas protrude through the surface (Figure 19C). Although some trichoxeas do protrude the

90

surface, it is not very hispid. The osculum is supported by triactines, tetractines and has a

discrete crown of trichoxeas (Figures 19D and 19E). The aquiferous system is leuconoid with

subspherical choanocytary chambers ranging from 28 to 34 µm (Figure 19F).

SKELETON

The skeleton is typical of the genus (Figure 20A). As mentioned before, the oscular margin has

a differentiated skeleton. It is composed of T-shaped triactines and tetractines tangentially

disposed and short trichoxeas perpendicular to the cortex. The cortical skeleton is composed of

tangential triactines (Figure 20B). The unpaired actine can point either to the surface or to the

choanosome. The choanosomal skeleton does not have a special organization and it is formed by

triactines of variable size (as shown in Figures 18F and 19A). Several exhalant choanosomal

canals with a diameter varying from 140 to 300 µm were observed within this region. They are

surrounded by tetractines with the apical actine projected inside them (Figure 20C). Some

triactines lining the canals were also found (Figure 20D). No subatrial skeleton was observed.

The atrial skeleton is formed by triactines (Figures 20E) and rare tetractines (Figure 20F). The

apical actine of the tetractines protrudes into the atrial cavity (arrow in Figure 19F).

SPICULES

Cortical triactines. Subregular or sagittal. Actines are slightly conical with sharp tips. The

paired actines are frequently curved and slightly longer than the unpaired one which is always

straight (Figure 21A). Compared to the atrial triactines, the cortical triactines are thicker. Size:

110-340/7.5-16.3 μm (paired actine) and 105-325/7.5-16.3 μm (unpaired actine).

Choanosomal triactines. Regular or subregular. Actines are conical with sharp tips (Figure

21B). They are largest spicules in L. caribea sp. nov. Very variable size: 370-960/25-75 μm.

Triactines and tetractines of the canals. Sagittal. Actines are conical with sharp tips. The paired

actines are curved, following the shape of the choanosomal canals (Figure 21C). The apical

actine of the tetractines is smooth and it is thinner than the basal ones (as shown in Figures 20C-

D). The size of the triactines is similar to that of the tetractines. Size of tetractines: 112.5-

220.0/7.5-12.5 μm (paired), 62.5-210.0/7.5-12.5 μm (unpaired) and 45.0-110.0/5.0-10.0 μm

(apical).

Atrial triactines (shown in Figure 20E): Sagittal. Actines are conical with sharp tips. Compared

to the cortical triactines, the angle formed by the paired actines of the atrial triactines is more

open. Size: 132.3-253.8/8.1-13.5 μm (paired actine) and 164.7-253.8/5.4-13.5 μm (unpaired

actine).

91

Atrial tetractines. Sagittal. Actines are conical with sharp tips (Figure 21D). The apical actine is

smooth. Size: 150-262.5/7.5-15.0 μm (paired actine), 162.0-297.0/8.1-16.2 μm (unpaired actine)

and 35.0-132.5/7.5-12.5 μm (apical actine).

ECOLOGY

This specimen was found underneath a coral boulder.



REMARKS

Among the species of Leucandra reported from the Caribbean Sea, namely L. crustacea

(Haeckel, 1872), L. barbata (Duchassaing & Michelotti, 1864), L. curva (Schuffner, 1877), L.

multiformis Poléjaeff, 1883, L. rudifera Poléjaeff, 1883, and L. typica (Poléjaeff, 1883) (Van

Soest et al., 2016), only L. typica possess a similar skeleton composition as that of L. caribea

sp. nov. The skeletons of the other Caribbean species include cortical tetractines (L. crustacea

and L. curva), diactines (L. barbata, L. multiformis and L. rudifera) and atrial grapnel spicules

(also in L. rudifera), all of which are not present in L. caribea sp. nov.

Leucandra caribea sp. nov. can be differentiated from L. typica as the former possess an

atrial skeleton mainly composed of triactines and few tetractines whereas in the latter triactines

and tetractines are in the same proportion (or at least, Poléjaeff did not indicate the opposite).

Besides, in the new species, the choanosomal canals are lined by tetractines and triactines and in

L. typica, they are only lined by tetractines. In L. typica, trichoxeas (<300/1 μm) are scattered in

the choanosome and spindle-shaped microdiactines (100/4 μm) are concentrated in the

suboscular region whereas in L. caribea sp. nov., trichoxeas (>100/1.2) were found only in the

suboscular region.

Within the other species of Leucandra with similar skeleton composition and external

morphology, L. falakra Klautau et al., 2016 from the Adriactic Sea is the one that most

resembles L. caribea sp. nov. Nonetheless, they have some important differences. The

choanosomal skeleton of the Curaçaoan species is composed of one single type of triactine

(370-960/25-75) while in the Adriatic species, it is composed of small (paired actine: 94.5-

180.9/8.1-13.5 μm and unpaired actine: 70-143.1/8.1-16.2) and giant triactines (342-

1047.6/48.6-118.8 μm). In addition, although almost all the spicule categories have similar size

(Table 17), the unpaired actine of the atrial tetractines is shorter in L. falakra (59.4-126.9 µm)

than in the new species (162-297 µm).

92

Table 17. Spicule measurements of Leucandra caribea sp. nov. (H=holotype: UFRJPOR 6754)and Leucandra falakra (H=holotype: UFRJPOR 8349). P=paired, U=unpaired and A=apical

actines.



Trichoxea (crown)

- - - - >105 1.1 1.2 0.2 1.6 15

Trichoxea (cortex)

- - - - >108 1.1 1.2 0.2 1.6 10

Cortical triactine

P 110-.0 218.4 65.2 340.0 7.5 11.6 2.4 16.3 25U 105.0 219.3 63.7 325.0 7.5 12.6 2.4 16.3 25

Choanosomaltriactine

- 370.0 576.3 188.8 960.0 25.0 41.3 10.8 75.0 24

Canal tetractine

P 112.5 179.3 32.0 220.0 7.5 9.8 1.8 12.5 10U 62.5 140.6 60.4 210.0 7.5 10.0 2.0 12.5 4A 45.0 70.9 19.0 110.0 5.0 7.9 1.3 10.0 14

Atrial triactine

P 132.3 209.4 41.8 253.8 8.1 11.2 1.9 13.5 12U 164.7 210.6 35.0 253.8 5.4 10.1 3.4 13.5 6

Atrial tetractine

P 150.0 206.9 28.7 262.5 7.5 11.6 1.9 15.0 20U 162.0 237.6 35.1 297.0 8.1 12.9 2.5 16.2 20A 35.0 69.0 28.3 132.5 7.5 9.6 1.2 12.5 20

UFRJPOR8349

Cortical triactine

P 94.5 136.4 24.0 180.9 8.1 11.1 1.9 13.5 20U 70.2 106.0 18.8 143.1 8.1 11.4 2.4 16.2 20

Cortical and choanosomal triactine

- 342.0 624.5 192.3 1047.6

48.6 81.5 20.6 118.8

23

Choanosomaltriactine

P 162.0 214.2 39.8 288.9 13.5 18.3 4.0 27.0 20U 108.0 189.7 58.9 351.0 13.5 19.8 4.1 29.7 20

Canal tetractine

P 99.9 154.0 26.4 199.8 8.1 12.4 2.4 16.2 19U 45.9 143.0 56.5 288.9 9.5 12.4 1.9 16.2 19A 50 80.6 24.4 137.5 7.5 9.6 1.5 12.5 20

Atrial triactine

P 140.4 222.7 33.7 294.3 9.5 15.1 2.5 20.3 30U 78.3 111.2 24.4 159.3 8.1 12.3 1.7 16.2 30

Atrial tetractine

P 145.8 191.4 26.0 256.5 10.8 14.9 2.6 18.9 30U 59.4 92.0 22.1 126.9 10.8 13.1 1.7 16.2 16A 67.5 110.3 30.3 162.0 8.1 11.9 2.8 16.2 15

93

Fig. 19. Leucandra caribea sp. nov.: (A) specimen in vivo; (B) specimen after fixation; (C)detail of trichoxeas (arrow) along the surface; (D) tangential section of the oscular region; (E)detail of the oscular trichoxeas (arrow); (F) cross section of the skeleton. Abbreviations:c=choanosomal canals, cc=choanocytary chambers.

94

Fig. 20. Skeleton of Leucandra caribea sp. nov.: (A) cross section of the skeleton; (B) cortex;(C) tetractine of a choanosomal canal; (D) triactine of a choanosomal canal; (E) atrial triactine;(F) atrial tetractine with the apical actine projected into the atrial cavity (arrow). Abbreviations:cx=cortex, c=choanosomal canal, ct=cortical triactines, at=atrium.

95

Fig. 21. Spicules of Leucandra caribea sp. nov: (A) cortical triactine; (B) choanosomaltriactine; (C) tetractine of choanosomal canals; (D) atrial tetractine.

Genus Leucandrilla Bojorevic, Boury-Esnault & Vacelet, 2000

TYPE SPECIES

Leucilla wasinensis Jenkin, 1908

DIAGNOSIS

"Grantiidae with leuconoid organization. In addition to triactines the cortex contains tetractines,

with the apical actines turned into the choanoderm. The articulated choanoskeleton is supported

by subatrial triactine spicules, and numerous rows of choanosomal triactines and/or tetractines,

with apical actines of cortical tetractines in the distal region" (Borojevic et al., 2002).

Leucandrilla pseudosagittata sp. nov. (Figures 22-24, Table 18)

ETIMOLOGY

Derived from the presence of subcortical tetractines with pseudosagittal shape.

96

TYPE MATERIAL


Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 15.2 m deep; coll. E. Hajdu, 23 August

2011.

Paratypes: UFRJPOR 6696 (specimen in ethanol and slides); Sunset Waters, Soto, Curaçao;

12°07'18.94"N, 68°58'11.46"W; <10 m deep; coll. B. Cóndor-Luján and G. Lôbo-Hajdu, 17

August 2011 and UFRJPOR 6705 (specimen in ethanol and slides); Water Factory, Willemstadt,

Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5 m deep; coll. B. Cóndor-Luján, 18 August 2011.

TYPE LOCALITY


COLOUR



This species has a cylindrical massive body (Figure 22A-E). The holotype is the largest

specimen and it measures 3.8 cm x 2.0 cm (Figures 22A and 22C). In this specimen, the

choanosomal wall is thinner at the apical region (0.2 cm) and thicker at the basal region (0.6

cm).The surface is slightly rough and the consistency is hard. The osculum is apical and its

diameter is 1.2 mm. The atrial cavity is not hispid and measures 0.4 cm. The aquiferous system

is leuconoid with spherical choanocytary chambers.

SKELETON

In the three analised specimens the oscular margin is composed of T-shaped spicules including

triactines and rare tetractines (Figure 23A). The skeleton is not typical of the genus (Figure 23B)

as it presents rare tetractines with pseudosagittal shape. The cortical skeleton is composed of

triactines (Figure 23B and 23C) tangentially disposed on the surface. The subcortical skeleton is

formed by large sagittal tetractines. Some of them have pseudosagittal shape (white arrow in

Figure 23D). The longer paired actine of the pseudosagittal tetractines and the apical actine of

the sagittal tetractines cross the choanosome and sometimes reach the atrial skeleton. The

choanosomal skeleton has no special organization and some subcortical tetractines invade this

region. The choanosomal canals (average diameter: 108-238 µm) are surrounded by small

tetractines whose apical actine is projected into them (Figure 23E). The poorly developed

subatrial skeleton is formed only by tetractines (black arrow in Figure 23D). The atrial skeleton

is exclusively composed of tetractines with the apical actine projected into the atrial cavity

(Figure 23F).

97

SPICULES

Cortical triactines. Sagittal. Actines are conical with blunt to sharp tips. Some paired actines are

slightly curved (Figure 24A). Very variable size: 97.2-421.2/8.1-21.6 µm (paired actine), 75.6-


Subcortical tetractines I. Sagittal. Actines are conical with sharp tips. The paired actines are

slightly curved (Figure 24B). The apical actine is generally longer than the paired ones. Size:

248.4-1188.0/27.0-75.6 µm (paired actine), 162.0-564.0/27.0-64.8 µm (unpaired actine) and

345.6-1122.0/21.6-75.6 µm (apical actine).

Subcortical tetractines II (indicated in Figure 24D). Pseudosagittal. Rare. Actines are conical

with sharp tips. Size: 270.0-529.2/37.8-54.0 µm (shorter paired actine), 237.6-1134.0/32.4-75.6

µm (longer paired actine), 270.0-1036.8/32.4-75.6 µm (unpaired actine) and 162.0-540.0/21.6-

64.8 µm (apical actine).

Canal tetractines. Sagittal. Actines are conical with sharp tips. The paired actines are curved

following the shape of the canals and they are longer than the other actines (Figure 24C). Size:

118.8-264.6/8.1-17.6 µm (paired actine), 70.2-213.3/8.1-17.6 µm (unpaired actine) and 40.5-

132.3/8.1-13.5 µm (apical actine).

Subatrial tetractines. Sagittal. Actines are conical with sharp tips (Figure 24D). The apical

actine is shorter than the basal ones. Size: 205.2-993.6/21.6-75.6 µm (paired), 205.2-766.8/21.6-

70.2 µm (unpaired) and 129.6-432.0/16.2-54.0 µm (apical actine).

Atrial tetractines. Sagittal. Actines are conical with blunt to sharp tips (Figure 24E). Sometimes

the paired actines are slightly curved. The apical actine is conical, shorter than the basal ones

and has sharp tips. Size: 67.5-391.5/8.1-21.6 µm (paired actine), 59.4-270.0/8.1-21.6 µm

(unpaired actine) and 21.6-108.0/6.4-13.5 µm (apical actine).

ECOLOGY

The three specimens were collected in light protected habitats. The holotype and one paratype

(UFRJPOR 6696) were collected underneath boulders. A polychaete was found inside the

holotype.



REMARKS

Although the skeleton of the analysed specimens from Curaçao presents rare subcortical

tetractines with pseudosagittal shape, which would suggest their allocation in one of the genera

of the family Heteropiidae, we placed them in the genus Leucandrilla following Borojevic et

al., 2000: "In any calcaronean sponge with a strong cortex, some subcortical spicules may be in

98

the position and have the shape of pseudosagittal spicules, due to the restriction of their growth

by the rigidity of the cortical skeleton. They should not be interpreted as an indication that the

sponge belongs to the family Heteropiidae."

Three species are considered within the genus Leucandrilla: L. intermedia (Row, 1909)

from the Red Sea, L. lanceolata (Row & Hôzawa, 1931) from Southwestern Australia and L.

wasinensis (Jenkin, 1908) from East Africa. Leucandrilla pseudosagittata sp. nov. can be easily

differentiated from them by the absence of diactines and choanosomal triactines and by the

presence of pseudosagittal subcortical tetractines.

The two sequences of L. pseudosagittata sp. nov. formed a monophyletic clade (pp=1,

bb=100) within the large clade LEUCII (which did not include any Heteropiidae species, see

Phylogenetic section). In both BI and ML phylogenetic trees, L. pseudosagittata nested with

other Grantiidae and Amphoriscidae species but with low support. In the BI tree (Figure 30), it

grouped with Amphoriscus micropilosus sp. nov., Leucilla antillana sp. nov., Sycon conulosum

sp. nov. and S. megapicalis sp. nov. (pp=0.57) and in the ML tree (data not shown), it clustered

with Leucandra nicolae, Paraleucilla magna and P. dalmatica (b=44.7).

Fig. 21. Leucandrilla pseudosagittata sp. nov.: (A-B) Specimens in vivo; (C-E) specimens afterfixation.

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Fig. 22. Skeleton composition of Leucandrilla pseudosagittata sp. nov. (paratype UFRJPOR6705): (A) osculum; (B) cross section of the skeleton; (C) cortex; (D) choanoskeletonindicating the subcortical tetractine (white arrow) and the subatrial tetractine (black arrow); (E)choanosomal canals (c); (F) atrial skeleton. Abbreviations: ct=cortex, at=atrium.

100

Fig. 23. Spicules of Leucandrilla pseudosagittata sp. nov. (paratype UFRJPOR 6705): (A)cortical triactine; (B) subcortical tetractine; (C) tetractine of a canal; (D) subatrial tetractine; (E)atrial tetractine.

Table 18. Spicule measurements of Leucandrilla pseudosagittata sp. nov. (UFRJPOR 6696,UFRJPOR 6705 and UFRJPOR 6752). H=holotype. P=paired, U=unpaired and A=apical

actines.

Specimen Spicule ActineLength Width N


Cortical Triactine

P 97.2 273.2 77.4 399.6 8.1 14.9 3.6 21.6 20U 97.2 207.4 70.3 345.6 5.4 14.1 4.3 21.6 20

SubcorticalTetractine I

P 248.4 543.8 161.5 810.0 27.0 43.5 10.3 64.8 20U 162.0 271.1 90.0 399.6 32.4 37.8 5.7 43.2 10A 367.2 541.8 104.2 702.0 21.6 38.4 10.9 64.8 17

SubcorticalTetractine II

P + 356.4 574.4 154.7 896.4 32.4 53.0 11.3 64.8 11P - 313.2 402.8 71.8 529.2 37.8 46.4 5.8 54.0 10U 324.0 479.5 105.7 648.0 32.4 49.7 8.0 59.4 10A 162.0 261.4 78.7 378.0 21.6 32.9 6.5 43.4 10

Canal Tetractine

P 118.8 200.7 39.9 256.5 8.1 12.6 2.8 17.6 20U 97.2 135.8 25.4 189.0 8.1 12.4 2.6 17.6 20A 54.0 89.4 23.1 124.2 8.1 10.2 1.8 13.5 20

Subatrial P 205.2 522.7 172.1 993.6 21.6 44.8 12.3 75.6 20

101

Tetractines U 205.2 388.3 113.8 637.2 21.6 35.4 7.9 48.6 20A 129.6 276.9 87.8 432.0 16.2 33.2 8.2 43.2 14

Atrial Tetractine

P 67.5 201.1 57.5 297.0 8.1 13.1 3.2 18.9 19U 59.4 187.5 54.3 240.3 8.1 12.6 3.4 18.9 11A 32.4 62.9 23.0 105.3 6.4 9.3 2.1 14.9 11

UFRJPOR 6705

Cortical Triactine

P 129.6 307.3 85.0 410.4 9.5 13.8 3.2 21.6 21U 129.6 261.4 65.3 367.2 9.5 14.0 3.4 21.6 12


P 259.2 639.4 211.0 972.0 27.0 54.3 13.5 70.0 20U 162.0 350.1 103.3 464.4 27.0 39.6 11.4 64.8 12A 345.6 511.2 147.7 864.0 37.8 51.3 8.7 70.2 20


P + 453.6 680.4 162.5 1134.0

43.2 58.4 7.9 70.2 16

P - 237.6 460.1 145.5 648.0 43.2 52.4 3.6 54.0 10U 270.0 583.2 145.9 756.0 48.6 53.3 4.0 64.8 15A 270.0 359.1 78.3 486.0 43.2 52.0 7.0 64.8 8

Canal Tetractine

P 143.1 210.7 32.1 264.6 8.1 12.5 2.5 17.6 20U 97.2 143.3 29.1 205.2 9.5 13.1 2.3 17.6 18A 62.1 86.4 16.7 124.2 8.1 9.6 1.5 13.5 20

Subatrial Tetractines

P 259.2 489.8 207.9 864.0 32.4 48.3 13.2 70.2 20U 216.0 405.0 131.6 669.6 32.4 48.3 11.8 70.2 20A 129.6 279.0 99.5 432.0 21.6 35.1 9.4 54.0 12

Atrial Tetractine

P 135.0 230.0 69.4 391.5 8.1 13.8 3.5 21.6 20U 94.5 164.4 43.8 264.6 9.5 13.4 3.1 21.6 20A 27.0 46.4 14.0 64.8 8.1 9.5 2.3 13.5 19

UFRJPOR 6752

Cortical Triactine

P 118.8 276.5 89.3 421.2 10.8 15.0 2.7 20.3 20U 75.6 242.5 84.9 421.2 12.2 15.1 2.3 18.9 20


P 313.2 689.6 222.4 1188.0

32.4 52.9 11.0 75.6 20

U 194.4 360.7 134.3 594.0 32.4 36.7 7.1 54.0 10A 367.2 693.6 232.2 1122.

032.4 51.3 11.0 75.6 20


P + 237.6 539.2 197.1 810.0 32.4 45.5 11.2 75.6 14P - 270.0 459.0 121.5 594.0 37.8 47.0 6.3 54.0 10U 313.2 548.1 258.1 1036.

832.4 48.6 12.6 75.6 12

A 216.0 346.8 93.1 540.0 32.4 39.0 5.2 43.2 9Canal Tetractine

P 118.8 186.4 46.5 245.7 8.1 12.0 2.4 16.2 20U 70.2 128.5 41.2 213.3 8.1 10.9 2.6 16.2 20A 40.5 81.7 23.5 132.3 8.1 10.2 1.7 13.5 20


P 280.8 518.4 191.1 853.2 32.4 46.5 10.7 59.4 13U 302.4 502.7 167.1 766.8 32.4 44.7 9.4 54.0 11A 216.0 332.6 66.1 432.0 27.0 40.0 10.6 54.0 10

Atrial Tetractine

P 153.9 205.9 48.0 332.1 9.5 13.8 3.0 21.6 29U 126.9 202.8 40.9 270.0 9.5 13.8 3.1 21.6 24A 21.6 60.5 23.0 108.0 6.7 9.8 1.9 14.9 30

102

Genus Grantessa Lendenfeld, 1885TYPE SPECIES

Grantessa sacca Lendenfeld, 1885

DIAGNOSIS

"Heteropiidae with syconoid organization and an articulated choanoskeleton. A thin cortex is

formed by triactines but lacks longitudinal large diactines. The distal part of the radial tubes is

frequently decorated by tufts of radially arranged diactines, indicating a close relationship to the

genus Syconessa" (Borojevic et al., 2002).

Grantessa tumida sp. nov. (Figures 24 & 25, Table 19)

ETIMOLOGY

From the Latin tumidus (=swollen), for the presence of subatrial and atrial spicules with distally

swollen unpaired actines.

TYPE LOCALITY

Sunset Waters, Soto, Curaçao

TYPE MATERIAL

Holotype: UFRJPOR 6766 (specimen in ethanol and slides); Sunset Waters, Soto, Curaçao;

12°16'01.58"N, 69°07'44.85"W; 13.1 m deep; coll. B. Cóndor-Luján, 23 August 2011.

Paratypes: UFRJPOR 6701 (specimen in ethanol and slides); Daai Booi, St. Willibrordus,

Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5 m deep; coll. B. Cóndor-Luján, 18 August 2011

and UFRJPOR 6695 (specimen in ethanol and slides); Hook’s Hut, Willemstadt, Curaçao;

12°07'18.94"N, 68°58'11.46"W; 6.3 m deep; coll. B. Cóndor-Luján & G. Lôbo-Hajdu, 17

August 2011.

COLOUR

Beige to light brownish in life and beige in ethanol.


This species has a tubular to sac-shaped body with an apical osculum surrounded by a crown of

trichoxeas. The holotype (UFRJPOR 6766) is the largest specimen. It measures 0.5 x 0.2 cm

(Figure 24A). The surface is smooth to the touch although diactines protrude through the

surface. The consistency is compressible. The aquiferous system is syconoid.

SKELETON

The osculum is surrounded by a crown of trichoxeas supported by T-shaped triactines and rare

tetractines (Figure 24B). The skeleton is typical of the genus. The cortical skeleton is composed

of diactines and triactines (Figure 24C). The diactines are perpendicularly arranged in tufts and

103

do not penetrate the choanosome. The triactines are tangentially disposed with the paired actines

laying tangentially to the subcortical region (black arrow in Figure 24C). The subcortical

skeleton is composed of pseudosagittal triactines with the longest paired actine (actine 1)

penetrating the choanosome (white arrow in Figure 24C). The tubar skeleton is articulated

(Figure 24D), composed of several rows of triactines with the unpaired actine pointing to the

cortex. The subatrial skeleton is composed of triactines with the unpaired actine pointing to the

cortex (Figure 24E). The atrial skeleton is composed of triactines and tetractines with the

unpaired actine adjacent to the atrium (in the common position of the paired actines). Some

atrial tetractines with the unpaired actine disposed in the traditional position were also found.

The apical actine of the tetractines penetrates the atrial cavity (Figure 24F).

SPICULES

Diactines. Fusiform, straight, with tips usually sharp (Figures 25A-B). Size: 108.0-637.2/5.4-

16.2 µm.

Cortical triactines. Sagittal. Actines are conical with sharp tips. The paired actines are less

straight than the unpaired one (Figure 25C). Size: 48.6-102.6/4.1-8.1 µm (paired actine), 51.3-


Subcortical triactines. Pseudosagittal. Actines are conical with sharp tips. One of the paired

actines is shorter and more curved than the other. The unpaired actine is straight (Figure 25D).

Size: 67.5-145.8/4.1-5.4 µm (paired actine 1), 51.3-113.4/2.7-5.4 µm (paired actine 2), 54.0-


Tubar triactines I. Sagittal. Actines are conical with sharp tips. One of the paired actines is

shorter and more curved than the other. The unpaired actine is straight and usually slightly

longer than the paired ones (Figure 25E). Size: 62.1-140.4/5.4-8.1 µm (paired actine), 21.6-

72.9/4.1-8.1 µm (short paired actine), 99.9-202.5/5.4-8.1µm (unpaired actine).

Tubar triactines II. Sagittal. Actines are conical with sharp tips. The paired actines have almost

the same size and are slightly curved. The unpaired actine is straight and generally longer than

the paired ones (Figure 25F). Size: 48.6-121.5/5.4-8.1 µm (paired actine), 72.9-205.5/5.4-8.1

µm (unpaired actine).

Subatrial triactine. Sagittal. Actines are slightly conical with sharp tips. The unpaired actine is

straight and longer than the paired ones. The paired actines are inwardly curved (Figure 25G).

Some paired actines with different lengths in the same spicule were also found. Size: 40.5-

108.8/4.1-6.8 µm (paired actine), 35.1-243.0/4.1-8.1 µm (unpaired actine).

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Atrial triactine. Sagittal. Actines are conical with sharp tips. The unpaired actine is elongated

and swollen at the distal part (Figures 25H-I). Size: 89.1-162.0/4.1-8.1µm (paired actine),

159.3-283.5/5.4-8.1 µm (unpaired actine).

Atrial tetractine I: Sagittal. Actines are conical with sharp tips. The unpaired actine is longer

than the paired ones (Figure 25J). Some apical actines are curved. Size: 78.3-148.5/4.1-5.4 µm

(paired actine), 108.0-280.8/5.4-8.1 µm (unpaired actine), 13.5-108.0/4.1-6.8 µm (apical actine).

Atrial tetractine II. Sagittal. Actines are conical with sharp tips. The unpaired actine is elongated

in tetractines II and, unlike the unpaired actine of the tetractines I, it is swollen at the distal part

(Figure 25H). The apical actine is curved and it is the shortest actine of this species. Size: 94.5-

151.2/4.1-8.1 µm (paired actine), 162.0-283.5/5.1-8.1 µm (unpaired actine), 13.5-43.2/4.1-5.4

µm (apical actine).

ECOLOGY

The specimens were found underneath coral boulders. One of the paratypes (UFRJPOR 6695)

was covered with sediment when collected.



REMARKS

Among the 28 valid species of the genus Grantessa, only G. ramosa sensu Borojevic (1967)

from South Africa and G. tenhoveni Van Soest & de Voogd, 2015 from Indonesia present atrial

spicules with swollen unpaired actine. Grantessa ramosa sensu Borojevic (1967) is very similar

to the Curaçaoan species, however, they present some dissimilarities. In G. tumida sp. nov. all

the spicules (but the diactines) are thinner (Table 19) and the apical actine of the atrial

tetractines is straight whereas in G. ramosa sensu Borojevic (1967) it is curved. Differently

from G. tenhoveni, the skeleton of our new species does not bear subatrial tetractines nor atrial

tetractines with a very long apical actine (660-960 µm) as described for the Indonensian

species.

It is important to point out here that G. ramosa sensu Borojevic (1967) differs greatly from

the original description of G. ramosa (Haeckel, 1872), also from South Africa, in spicule size

and shape (Table 22). Based on the written descriptions and drawings, they probably belong to

two different species.

The C-LSU sequences of G. tumida sp. nov. (UFRJPOR6701 and UFRJPOR6695) grouped

together in the same cluster (pp=1, b=100) within the large clade LEUCI (which included

species of the Grantiidae and Heteropiidae families). Nonetheless, G. tumida sp. nov. did not

group with the other Grantessa species, G. aff. intusarticulata (Figure 30).

105

Fig. 24. Grantessa tumida sp. nov. (Holotype=UFRJPOR 6766): (A) specimen after fixation;(B) osculum; (C) distal part of the skeleton indicating a cortical (black arrow) andpseudosagittal subcortical triactine (white arrow); (D) tubar skeleton; (E) subatrial skeleton; (F)atrial skeleton.

106

Fig. 25. Spicules of Grantessa tumida sp. nov (Holotype=UFRJPOR 6766): (A, B) diactine; (C)cortical triactine; (D) subcortical triactine; (E) tubar triactine I; (F) tubar triactine II; (G)subatrial triactine; (H) atrial triactine; (I) detail of the swollen tip of the atrial triactine; (J) atrialtetractine II.

107

Table 19. Spicule measurements of Grantessa tumida sp. nov. (UFRJPOR 6695, UFRJPOR6701 and UFRJPOR 6766), G. ramosa (Haeckel, 1872) and G. ramosa sensu Borojevic (1967).

H=holotype, P=Paired, U=unpaired, A=apical, CP=cortical paired and IP=internal pairedactines.

Specimen Spicule Actine Length (µm) Width (µm) N

Min Mean S Max Min Mean S Max

UFRJPOR6695

Diactine 108.0 250.6 99.6 432.0 5.4 9.7 2.8 16.2 20Cortical triactine

P 48.6 62.1 9.8 81.0 5.4 5.4 0.0 5.4 20U 51.3 93.9 21.7 121.5 5.4 5.6 0.5 6.8 20

Pseudosagittal Triactine

P1 67.5 106.7 27.3 140.4 4.1 5.1 0.6 5.4 15P2 51.3 73.7 17.9 108.0 4.1 5.1 0.6 5.4 13U 54.0 93.4 24.5 124.2 4.1 5.1 0.6 5.4 15

Tubar Triactine 1

P1 72.9 110.0 22.2 140.4 5.4 6.4 1.1 8.1 20P2 29.7 50.1 14.3 72.9 5.4 5.8 0.6 6.8 20U 99.9 138.4 21.4 164.7 5.4 6.0 0.8 8.1 20

Tubar Triactine

P 51.3 91.8 20.8 121.5 5.4 6.4 1.1 8.1 20U 99.9 153.4 35.3 207.9 5.4 6.0 0.8 8.1 20

Subatrial Triactine

P 43.2 83.5 21.8 108.0 4.1 5.2 0.9 6.8 15U 70.2 156.2 60.8 229.5 4.1 5.8 0.9 6.8 14

Atrial triactine

P 94.5 119.8 17.4 148.5 4.1 5.1 0.6 5.4 14U 159.3 223.3 32.9 283.5 5.4 6.1 1.0 8.1 20

Atrial Tetractine I

P 78.3 117.5 22.7 148.5 4.1 5.1 0.6 5.4 12U 121.5 196.9 53.9 270.0 5.4 6.4 1.2 8.1 12A 24.3 56.2 17.8 86.4 4.1 4.9 0.7 5.4 10

Atrial Tetractine II

P 94.5 119.2 14.0 148.5 4.1 5.3 0.6 6.8 14U 162.0 222.3 29.5 270.0 5.1 5.6 0.5 6.8 18A 24.3 30.2 5.2 37.8 4.1 5.0 0.7 5.4 6

UFRJPOR6701

Diactine - 108.0 360.7 110.0 615.6 5.4 10.8 3.5 16.2 20Cortical triactine

P 56.7 78.4 10.7 102.6 5.4 5.5 0.6 8.1 20U 86.4 117.5 15.8 140.4 5.4 6.0 0.8 8.1 20


P1 78.3 125.1 17.5 145.8 5.4 5.4 0.0 5.4 20P2 56.7 92.2 15.6 108.0 4.1 5.2 0.5 5.4 20U 78.3 105.3 13.9 129.6 4.1 5.3 0.8 8.1 20

Tubar Triactine 1

P1 62.1 89.4 9.1 105.3 5.4 6.2 1.2 8.1 20P2 27.0 45.6 12.3 72.9 4.1 5.6 1.6 8.1 20U 105.3 129.9 20.5 194.4 5.4 6.1 0.9 8.1 20

Tubar Triactine 2

P 78.3 88.0 7.9 105.3 5.4 6.1 1.1 8.1 20U 72.9 129.2 27.7 189.0 5.4 6.2 1.2 8.1 20

Subatrial Triactine

P 48.6 74.9 11.8 89.1 4.1 5.1 0.6 5.4 16U 35.1 151.6 59.2 243.0 4.1 5.6 1.0 8.1 16

Atrial triactine

P 89.1 120.4 19.8 162.0 4.1 5.2 0.5 5.4 20U 162.0 223.4 27.2 270.0 5.4 6.3 1.2 8.1 20

Atrial Tetractine I

P 83.7 115.0 25.9 145.8 4.1 5.1 0.6 5.4 10U 129.6 179.6 49.3 280.8 5.4 5.9 1.0 8.1 8A 21.6 46.5 24.4 86.4 4.1 5.1 0.6 5.4 9

Atrial P 94.5 125.3 18.6 151.2 4.1 5.1 0.6 5.4 10

108

Tetractine II

U 189.0 230.6 28.3 270.0 5.4 6.3 1.3 8.1 12A 13.5 29.2 11.7 43.2 4.1 5.1 0.6 5.4 5

UFRJPOR6766 (H)

Diactine 194.4 381.2 138.2 637.2 5.4 9.7 2.8 16.2 20Cortical Triactine

P 51.3 79.4 12.7 102.6 4.1 5.3 0.4 5.4 20U 81.0 128.9 22.3 175.5 4.1 5.5 0.8 8.1 20


P1 78.3 110.7 18.1 137.7 4.1 5.3 0.3 5.4 15P2 62.1 84.6 14.2 113.4 2.7 5.0 0.8 5.4 15U 56.7 101.8 26.9 126.9 4.1 5.3 0.4 5.4 10

Tubar Triactine 1

P1 62.1 95.9 15.2 113.4 5.4 6.3 1.2 8.1 20P2 21.6 50.2 11.5 72.9 5.4 5.6 1.5 8.1 20U 124.2 162.9 21.2 202.5 5.4 5.9 0.7 6.8 20

Tubar Triactine 2

P 48.6 95.6 13.3 118.8 5.4 6.0 1.0 8.1 20U 83.7 145.9 27.9 205.2 5.4 5.9 0.8 8.1 20

Subatrial Triactine

P 40.5 80.3 17.3 99.9 4.1 5.3 0.6 6.8 16U 67.5 162.0 47.6 224.1 4.1 5.7 1.0 6.8 16

Atrial triactine

P 97.2 123.1 13.2 148.5 4.1 5.7 1.1 8.1 20U 189.0 233.1 26.7 283.5 5.4 6.1 1.0 8.1 20

Atrial Tetractine I

P 97.2 118.8 17.5 137.7 4.1 5.1 0.7 5.4 4U 108.0 160.7 53.4 234.9 5.4 6.4 1.3 8.1 4A 13.5 83.4 27.6 108.0 5.4 5.7 0.6 6.8 10

Atrial Tetractine II

P 94.5 121.5 16.9 148.5 5.4 6.2 1.1 8.1 10U 189.0 240.5 28.7 283.5 5.4 6.1 1.1 8.1 11A 13.5 21.2 8.4 35.1 5.4 5.4 0.0 5.4 6

Grantessaramosa (Haeckel, 1872)

Diactine - 60.0 - - 80.0 - - - -Tubar Triactine

P 40.0 - - 80.0 6.0 - - 8.0U 80.0 - - 120.0 6.0 - - 8.0

Subatrial Triactine

P 40.0 - - 80.0 6.0 - - 8.0U 160.0 - - 200.0 6.0 - - 8.0

Atrial Tetractine

P 50.0 - - 80.0 6.0 - - 8.0U 100.0 - - 120.0 6.0 - - 8.0A 20.0 - - 30.0 - 8.0 - -

Grantessaramosa sensu Borojevic (1967)

Diactine - 150.0 - - 380.0 6.0. - - 12.0Cortical Triactine*

U 70.0 - - 150.0 8.0 - - 12.0

Subcortical Triactine

CP 80.0 - - 180.0 15.0 - - 18.0IP 150.0 - - 240.0 15.0 - - 18.0U 100.0 - - 200.0 14.0 - - 16.0

Tubar Triactine

U 150.0 - - 230.0 14.0 - - 18.0

Subatrial Triactine

P 80.0 - - 120.0 9.0 - - 11.0U 180.0 - - 250.0 12.0 - - 14.0

AtrialTriactine

P 60.0 - - 180.0 8.0 - - 10.0U 200.0 - - 400.0 8.0 - - 10.0

Atrial Tetractine

P 60.0 - - 180.0 - 25.0 - -U <200 - - <400 - 25.0 - -A - - - 140.0 - 25.0 - -

* Paired actines slightly longer than unpaired actine.

109

Genus Sycon Risso, 1827

TYPE SPECIES

Sycon humboldti Risso, 1827

DIAGNOSIS

"Sycettidae with radial tubes partially or fully coalescent; distal cones are decorated by tufts of

diactines. The inhalant canals are generally well defined between the radial tubes and are often

closed at the distal end by a membrane that is perforated by an ostium, devoid of a skeleton.

There is no continuous cortex covering the distal ends of the radial tubes. Skeleton of the atrium

and of the tubes composed of triactines and/or tetractines" (Borojevic et al., 2002).

Sycon conulosum sp. nov. (Figures 26 & 27, Tables 20 & 21)

ETIMOLOGY

Derived from the conulose appearance of the surface.

TYPE LOCALITY

Daai Booi, St. Willibrordus, Curaçao.

TYPE MATERIAL

Holotype: UFRJPOR 6707 (specimen in ethanol and slides); Daai Booi, St. Willibrordus,

Curaçao; 12°12'43.12"N, 69°05'8.42"W; 3-5m; coll. B. Cóndor-Luján, 18 August 2011.

COLOUR



This species has a ficiform shape (Figure 26A). It measures 0.9 cm long and 0.7 cm wide. The

surface is hispid with tufts of diactines protruding through the body. It has a conulose

appearance because of the distal cones, which are very separated. The osculum (diameter=0.9

mm) is apical with no crown and it is supported by sagittal triactines (Figura 26B-C). The

atrium measures 2.4 mm. The radial tubes are fully coalescent (Figure 26D). The aquiferous

system is syconoid.

SKELETON

The skeleton is typical of the genus. The skeleton of the distal cones is composed of short

diactines, rare trichoxeas and triactines (white arrow in Figure 26E). The tubar skeleton is

articulated, composed of several rows of triactines with the unpaired actine pointing towards the

distal cones (black arrow in Figure 26E). The subatrial skeleton is composed of triactines whose

unpaired actine points to the surface (arrow in Figure 26F). The atrial skeleton is composed of

110

tangential triactines (black arrow in Figure 26G) and fewer tetractines (white arrow in Figure

26G). The apical actine of the tetractines penetrates the atrial cavity.

SPICULES (Table 20)

Diactines. Fusiform with tips usually sharp (Figure 27A). Size: 100-275/6.3-11.3 µm.

Triactines of the distal cones. Sagittal. Actines are conical with sharp tips. The paired actines are

frequently curved (Figure 27B). In the distal part of the cone, the unpaired actine is very long.

Size: 50-102.5/5-10 µm (paired actine) and 65-175/5-8.8 µm (unpaired actine).

Tubar triactines I. Sagittal. Actines are conical with sharp tips. One of the paired actines is

shorter and more curved than the other. The unpaired actine is straight and it is generally slightly

longer than the paired ones (Figure 27C, left). Size: 32.5-60/5-10 µm (short paired actine), 67.5-

95/5-11.3 µm (long paired actine) and 82.5-125/5-10 µm (unpaired actine).

Tubar triactines II. Sagittal. Actines are conical with sharp tips. The paired actines are slightly

curved and have equal sizes. The unpaired actine is straight and generally longer than the paired

ones (Figure 27C, right). Size: 40-80/3.8-10 (paired actine) and 62.5-155/3.8-10 µm (unpaired

actine).

Subatrial triactines. Sagittal. Actines are slightly conical with sharp tips. The paired actines are

inwardly curved. Some paired actines have different lengths. The unpaired actine is straight and

longer than the paired ones (Figure 27D). Size: 42.5-80/5-8.8 µm (paired actine) and 88.5-

172.5/5-10 µm (unpaired actine).

Atrial triactines. Sagittal. Actines are conical, straight and have sharp tips. The unpaired actine

is usually longer than the paired ones (Figure 27E). Size: 80-130/7.1-14.3 µm (paired actine)

and 80-172.5/5-10 µm (unpaired actine).

Atrial tetractines. Sagittal. Actines are conical, straight with sharp tips (Figure 27F). The apical

actine is the shortest actine of this species. Size: 62.5-90/7.5-11.3 µm (paired actines), 92.5-

130/7.5-10 µm (unpaired actine) and 15-40/5-10 µm (apical actine).

ECOLOGY

This species was found in a light protected environment. No associated organisms were found.



REMARKS

Within the 88 accepted species of Sycon (Van Soest et al., 2016), 15 have the same skeleton

composition as the analised specimen from Curaçao: S. abyssale Borojevic & Graat-Kleeton,

1965, S. ampulla (Haeckel, 1870), S. arcticum (Haeckel, 1870), S. barbadensis (Schuffner,

1877), S. brasiliense Borojevic, 1971, S. boreal (Schuffner, 1877), S. dunstervillia (Haeckel,

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1872), S. formosum (Haeckel, 1870), S. humboldt Risso, 1827, S. raphanus Schmidt, 1862), S.

scaldiense (Van Koolwijk, 1982), S. schmidti (Haeckel, 1872), S. setosum Schmidt, 1862, , S.

tuba Lendenfeld, 1891 and S. villosum (Haeckel, 1870). Sycon conulosum sp. nov. can be easily

differentiated from S. abyssale, S. brasiliense, S. dunstervilla and S. formosum because of the

shape of their diactines. In the new species, diactines are fusiform whereas in S. abyssale and S.

brasiliense, one of the tips is lanceolated and in S. dunstervilla and S. formosum, diactines have

distally swollen tips (originally described as “keulen” or “kolb”). The remaining 11 species

differ from the new species in spicule size (Table 21).

112

Fig. 26. Sycon conulosum sp. nov. (UFRJPOR 6707): (A) specimen after fixation; (B) osculum;(C) detail of the oscular T-shaped triactines; (D) cross section of the skeleton; (E) skeleton witha tuft of diactines, a triactine of the distal cone (white arrow) and a tubar triactine (black arrow);(F) subatrial skeleton indicating a subatrial triactine (arrow); (G) atrial skeleton indicating atrialtriactine (black arrow) and atrial tetractine (white arrow). Abbreviations: at=atrium.

Fig. 27. Spicules of Sycon conulosum sp. nov. (UFRJPOR 6707): (A) diactine of the distal cone;(B) triactine of the distal cone; (C) tubar triactine I (left) and tubar triactine II (right); (D)subatrial triactine; (E) atrial triactine; (F) atrial tetractine.

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Table 20. Spicule measurements of Sycon conulosum sp. nov. (UFRJPOR 6707).


Min Mean SD Max Min Mean SD MaxDiactine 100.0 216.5 48.0 275.0 6.3 8.4 1.3 11.3 20Triactine(distal cone)

Paired 50.0 77.4 16.4 112.5 5.0 7.2 1.6 10.0 20Unpaired 65.0 124.1 28.9 175.0 5.0 6.4 1.3 8.8 20

Tubar Triactine 1

Paired (>) 67.5 78.3 7.7 95.0 5.0 8.1 1.6 11.3 20Paired (<) 32.5 48.6 6.5 60.0 5.0 8.3 2.2 10.0 20Unpaired 82.5 64.6 1.6 125.0 5.0 8.3 1.6 10.0 20

Tubar Triactine 2

Paired 40.0 63.5 10.5 80.0 3.8 7.4 1.7 10.0 20Unpaired 62.5 106.4 21.2 155.0 3.8 7.3 1.7 10.0 20

Subatrial Triactine

Paired 42.5 65.1 9.9 80.0 5.0 5.8 1.2 8.8 20Unpaired 82.5 135.1 20.2 172.5 5.0 7.1 1.6 10.0 20

Atrial Triactine

Paired 80.0 103.0 11.8 130.0 7.1 10.0 2.2 14.3 14Unpaired 80.0 126.6 23.7 172.5 5.0 7.9 1.9 10.0 14

Atrial Tetractine

Paired 62.5 77.5 11.4 90.0 7.5 9.1 1.9 11.3 4Unpaired 92.5 111.3 20.3 130.0 7.5 8.8 1.4 10.0 4Apical 15.0 27.9 9.6 40.0 5.0 7.3 1.7 10.0 12

Table 21. Original measurements of Sycon ampulla, S. arcticum, S. barbadense, S. boreale, S.humboldti, S. raphanus, S. scaldiense, S. schmidti, S. setosum, S. tuba and S. villosum. Values in

micrometers (µm). *Taken from Haeckel (1872).

Species - typelocality

Distal cone Tubar triactine

Subatrialtriactine

Atrial triactine

Atrial tetractine

S. ampulla – Atlantic coast of South America

Diactine: 100-500/5

Paired: 50-80/5Unpaired: 100-150/5

- 60-80/5 Unpaired: 60-80/5Apical: 40-60/5(rarely 100)

S. arcticum– Arctic Ocean

Diactine: 1000-3000/10-40Trichoxea: 100-300/1-5Triactine:smaller than tubar

Paired: 100-200/10Unpaired: 200-300/10

- Paired: 50-150/10Unpaired: 200-250/10

Paired: 50-150/10Unpaired: 200-250/10Apical: 20-40/10

S. barbadense– Barbados

Diactine: 400/9 Unpaired: ≤ 150

- ≤ 200/9 Paired: ≤ 150/9Unpaired: ≤ 200/9Apical: ≤ 80/13

Sycon boreale - Norway

Diactine: 500/9 Paired: 500Unpaired: 180

- Paired: shorterthan unpaired.Unpaired:180/6.8

Apical: 50/13

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S. humboldti* – Western Mediterranean

Diactine I:500–2000/20-40Diactines II: 200-400/5-20Triactine:Paired: 30-60/ 10-15Unpaired: 150-250/20-30

80-120/8-12 - Paired: 50-120/8Unpaired: 50-200/5-8

Paired: 50-20/8Unpaired: 50-200/5-8Apical: 100-120

S. raphanus* – Adriatic Sea

Diactine: 1000–3000/20 -24

Paired: 100-180/10 – 12Unpaired: 150 – 250/10 – 12

?/5-8 (Subregular to sagittal)150 – 250/8 – 10

Subregular to sagittalApical: 60 – 120

S. scaldiense –Netherlands

Diactine: 500–700/5-10Triactine: 30-60/5-10

80-150/9-11 Paired:80-200/5-10Unpaired:180-350/5-10

Paired: 100 - 280/5-10Unpaired: 200– 300/5-10

Paired:100-280/5-10Unpaired: 200–300/5-10Apical: 180–350/5-10

S. schmidti –Adriatic Sea

Diactine I:100 – 300/10Diactine II: 100–500/20 - 30

Paired: 100-150/10Unpaired: 200-300/10

- Subregular200-400/10-15

SubregularBasal:200-400/10-15Apical:40-50/12-16

S. setosum* –Adriatic Sea

Diactine:1000-3000/20Diactine: 100-200/1

Paired: 80-160/5-8Unpaired:120-200/5-8

- Subregular100-115/5

SubregularApical: 300-600/5

S. tuba– Adriatic Sea

Diactine: 300/10

Paired: 280/7Unpaired: 220/7

- Paired: 280/7Unpaired: 320/7

Paired: 280/7Unpaired: 320/7Apical: 200-260/7

S. villosum– North Atlantic

Diactine:1000-3000/10-40Triactine:100-200/5-8

100-300/30 SagittalPaired: 100/30Unpaired:300/10-30

SagittalPaired: 100/30Unpaired: 300/10-30

Paired: 100-150/5-8Unpaired: 300-400/5-8Apical: 500-800/≤10

Sycon magniapicalis sp. nov. (Figures 28 & 29, Table 22)

ETIMOLOGY

From the Latin magna (=large), for the long apical actine of the atrial tetractine.

TYPE LOCALITY


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TYPE MATERIAL


Willemstadt, Curaçao; 12°04'08.20"N, 68°51'44.40"W; 14.9 m deep; coll. B. Cóndor-Luján, 23

August 2011.

Paratype: UFRJPOR 6763 (specimen in ethanol and slides); Water Factory, Willemstadt,

Curaçao; 12°06'30.88"N, 68°57'13.53"W; 8.4 m deep; coll. E. Hajdu, 23 August 2011.

COLOUR

White to beige in life and yellowish in ethanol.


This species has a globular body with an apical osculum (Figure 28A). The consistency is hard,

although compressible. The surface is very hispid with long diactines and trichoxeas protruding

through the surface. The holotype measures 6.5 cm length x 2.5 cm width (Figure 28B). The

osculum has a neck with a delicate crown of trichoxeas (arrows in Figure 28B-C). The radial

tubes are fully coalescent. The atrial cavity is hispid and the aquiferous system is syconoid.

SKELETON

The osculum has a neck composed of T-shaped triactines and tetractines and a crown of

trichoxeas (Figure 28D). The triactines and tetractines are arranged parallel to each other

(Figure 28E). In the paratype, the crown is more conspicuous as shown in Figure 28C. The

skeleton of the body is typical of the genus (Figure 28F). The distal cones have diactines,

triactines and rare trichoxeas tangentially disposed (Figure 28G). The diactines comprise two

size categories. In the larger diactines, the proximal part crosses the choanosome and

occasionally, reaches the atrium. The tubar skeleton is articulated, exclusively formed by

triactines with the unpaired actine pointing to the distal cones (Figure 28H). The subatrial

skeleton is composed of tetractines (white arrow in Figure 28I) and rare triactines (black arrow

in Figure 28I) with the unpaired actine pointing to the surface. The atrial skeleton is formed by

tetractines. The basal actines lay tangentially and the very long apical actine is projected into the

atrium (Figure 28I).

SPICULES

Trichoxeas. Straight with tips always broken. Ticker than usually. Size: >1690.2/1.4-5.4 μm.

Diactines I. Fusiform and straight. The proximal tip is lanceolated while the distal tip is usually

abruptly sharp (Figure 29A). Size: 100.0-864.0/5.4-21.6 μm.

Diactines II. Fusiform and straight. The proximal tip is sharp and the distal tip was always

found broken. They are the largest diactines in this species. Size: > 2970.0/23.0-32.4 μm.

116

Triactines of the distal cones. Sagittal. Actines are conical with sharp tips. Some paired actines

can be slightly longer than the unpaired one (Figure 29B). Size: 54.0-153.9/5.4-10.8 μm (paired

actine) and 40.5-175.5/5.4-10.8 μm (unpaired actine).

Tubar triactines. Sagittal. Actines are conical with sharp tips (Figure 29C). Some paired actines

are curved. Size: 54.0-135.0/5.4-10.8 μm (paired actine) and 35.1-189.0/ 5.4-10.8 μm (unpaired

actine).

Subatrial triactines. Sagittal. Actines are conical with sharp tips (Figure 29D). The unpaired

actine is straight and longer than the paired ones. The paired actines are inwardly curved. In

some triactines, one paired actine can be longer than the other. Size: 67.5-126.9/2.7-6.8 μm

(paired actine), 27.0-99.9/2.7- 6.8 μm (smaller paired actine) and 67.5-245.7/4.1- 8.1 μm

(unpaired actine).

Subatrial tetractines. Sagittal. Actines are conical with sharp tips. The unpaired actine is straight

and longer than the basal actines. It can be slightly conical. The paired actines are inwardly

curved (Figure 29E). The apical actine is the shortest actine in this species Size: 40.0-143.1/4.1-

8.1 µm (paired actine), 143.1-237.6/5.4-10.8 μm (unpaired actine) and 10.8-75.6/2.7-6.8 μm

(apical actine).

Atrial tetractines. Sagittal. The basal actines are conical with sharp tips (Figure 29F). The apical

actine is slightly conical to cylindrical, straight, smooth and very long. It can be straight or

curved. Size: 110.7-237.6/5.4-13.5 μm (paired actine), 29.7-151.2/5.4-16.2 μm (unpaired actine)

and 124.2 - 496.0/5.4-16.2 μm (apical actine).

ECOLOGY

Specimens were collected underneath coral boulders, partially covered with sediment.



REMARKS

Surprisingly, Sycon plumosum Tanita, 1943 from Palau (Carolinas Islands) is the only species of

Sycon that presents the same skeleton composition as that of the specimens from Curaçao.

However, they greatly differ in spicule size as almost all the spicule categories are smaller in S.

magniapicalis sp. nov. (only the atrial tetractines have similar size in both species).

Additionally, S. plumosum lacks the oscular neck observed in both Curaçaoan specimens.

In the C-LSU phylogenetic tree (Figure 30), the sequences of S. magniapicalis sp. nov.

(UFRJPOR6748 and UFRJPOR6763) grouped together with high support (pp=1, b=99.9),

confirming their co-specificity. Nonetheless, they did not cluster with the other species of Sycon

(S. ancora, S. capricorn, S. cf. vilosum, S. ciliatum, S. conulosum sp. nov. and S. raphanus).

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Only in the BI tree, S. magniapicalis sp. nov. appeared as a sister taxon (pp=0.99) of the clade

formed by Sycon conulosum sp. nov. + L. antillana (pp=1, b=99.9).

Fig. 28. Sycon magniapicalis sp. nov.: (A) specimen in vivo; (B) specimen after fixation; (C)paratype after fixation; (D) skeleton of the osculum indicating the neck (asterisk) and the crown;(E) detail of the oscular T-shaped triactine (white arrow) and tetractine (black arrow); (F) cross

118

section of the skeleton; (G) distal cones with diactines II (black arrow) and triactines (whitearrow); (H) tubar skeleton; (I) subatrial skeleton with tetractine (white arrow) and triactine(black arrow); (J) atrial skeleton with tetractines. Abbreviations: at=atrium. All picturescorrespond to the holotype except when indicated.

Fig. 29. Spicules of Sycon magniapicalis sp. nov. (UFRJPOR 6748): (A) diactine I; (B) triactineof the distal cone; (C) tubar triactine; (D) subatrial triactine; (E) subatrial tetractine; (F) atrialtetractine.

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Table 22. Spicule measurements of Sycon magniapicalis sp. nov. (UFRJPOR6748 andUFRJPOR6763) and of S. plumosum from Tanita, 1943. P=paired, U=unpaired and A=apical.



Trichoxea 864.0 - - - 1.3 3.6 1.2 5..4 20Diactine I 100.0 285.7 121.9 486.0 5.4 12.1 3.6 18.9 16Diactine II >2835 - - - 23.0 26.4 1.3 27.0 12Triactine (cone)

P 64.8 110.6 17.9 145.8 5.4 9.7 1.6 10.8 20U 40.5 97.7 34.5 175.5 5.4 8.8 1.8 10.8 20

Tubar Triactine

P 54.0 94.0 26.9 135.0 5.4 7.2 1.6 10.8 19U 35.1 65.5 25.2 118.8 5.4 6.3 1.2 8.1 20

Subatrial Triactine

P 67.5 88.2 16.6 126.9 4.1 5.5 0.7 6.8 20P- 29.7 53.2 16.8 78.3 4.1 5.4 0.6 6.8 13U 121.5 184.4 29.6 245.7 5.4 6.1 0.9 8.1 20


P 75.6 106.0 20.2 143.1 4.1 5.5 1.0 8.1 20U 143.1 194.5 23.5 237.6 5.4 7.4 1.1 8.1 20A 16.2 28.9 15.1 75.6 2.7 4.7 0.9 6.8 19

Atrial Tetractine

P 116.1 157.0 19.4 183.6 5.4 8.3 1.3 10.8 15U 29.7 67.5 34.3 151.2 5.4 7.5 1.5 10.8 13A 221.4 313.6 84.4 496.0 5.4 8.8 1.7 10.8 11

UFRJPOR6763

Trichoxea - 216.0 671.7 386.0 1690.2 1.4 3.2 2.0 5.4 20Diactine I - 100.0 432.3 178.0 864.0 10.8 15.3 3.0 21.6 22Diactine II - >2970 - - - 24.3 27.4 1.7 32.4 17Triactine (cone)

P 54.0 89.1 24.3 153.9 5.4 6.8 1.2 8.1 20U 40.5 64.4 21.5 121.5 5.4 5.9 1.0 8.1 20

Tubar Triactine

P 54.0 104.1 23.2 129.6 5.4 8.5 1.3 10.8 20U 94.5 156.6 25.7 189.0 5.4 8.8 1.5 10.8 20

Subatrial Triactine

P + 72.9 91.0 13.0 113.4 2.7 4.9 1.0 6.8 20P - 27.0 57.8 22.0 99.9 2.7 4.8 0.9 5.4 20U 67.5 169.0 37.6 234.9 4.1 6.1 1.3 8.1 20


P 75.6 103.8 14.6 132.3 5.4 5.7 0.8 8.1 20U 148.5 191.3 24.9 232.2 5.4 7.1 1.5 10.8 20A 10.8 20.9 9.6 51.3 2.7 4.2 1.2 5.4 20

Atrial Tetractine

P 110.7 148.4 25.4 237.6 6.8 9.7 1.8 13.5 20U 32.4 63.5 25.9 113.4 5.4 9.3 2.6 16.2 20A 124.2 227.0 56.5 345.0 8.1 10.8 2.5 16.2 19

Sycon plumosum

Diactine - 800 - - 3000 30.0 - - 35 -Triactine (cone)

P 200 - - 240 - 16 - - -U 140 - - 200 16 - - -

Tubar Triactine

P 170 - - 240 15 - - 18 -U 270 - - 360 15 - - 18 -

Subatrial Triactine

P 120 - - 180 8 - - 10 -U 250 - - 360 8 - - 10 -


P 120 - - 180 8 - - 10 -U 250 - - 360 8 - - 10 -A 70 - - 100 8 - - 10 -

Atrial Tetractine

P 170 - - 200 12 - - 16 -U 200 - - 280 12 - - 16 -A 130 - - 350 12 - - 16 -

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Phylogenetic analyses

Calcaronean phylogeny

C-LSU sequences of six new Calcaronean species, Amphoriscus micropilosus sp. nov. (n=1),

Grantessa tumida sp. nov. (n=2), Leucilla antillana sp. nov. (n=1), Leucandrilla

pseudosagittata sp. nov. (n=2), Sycon conulosum sp. nov. (n=1) and Sycon magniapicalis sp.

nov. (n=2) were produced and provided herein.

The aligned C-LSU sequences had a total length of 458 bp including gaps. BI and ML

approaches recovered similar tree topologies including monophyletic Calcaronean ( pp=1,

b=79) and Calcinean with high BI posterior probability (pp:1) and ML bootstrap (b=100)

support. The complete C-LSU phylogenetic tree obtained by BI is shown in Figure

S1(Supplementary Material).

Within the Calcaronean cluster, we recovered two major clades (Figure 30). One of them

included all Heteropiidae and some Grantiidae species and it was previously referred as LEUC I

(pp=0.89, b=73) by Voigt et al, (2012). The other clade BAE + LEUC II (pp=1, b=90) nested

the Baerida species (BAE: Eilhardia schulzei, Leuconia nivea and Petrobiona massiliana) and

almost all the other Leucosolenida species but Leucosolenia sp. (LEUC II).

In the LEUC I clade, Grantessa tumida sp. nov. did not cluster with G. aff. intusarticulata,

instead it clustered with the Grantiidae species, Synute pulchella (pp=0.93, b=58), suggesting

the non-monophyly of the genus Grantessa. Interestingly, G. tumida sp. nov. as well as S.

pulchella do not present tetractines in the tubar skeleton whereas the specimens identified as G.

aff intusarticulata do have this spicule category.

Sycon was recovered again as a polyphyletic genus (Voigt et al., 2012; Klautau et al., 2016).

The sequences of the new species, S. conulosum sp. nov. and S. magniapicalis sp. nov., as well

as S. ancora, S. carteri, S. cf. ciliatum and S. raphanus appeared in different clades within the

clade BAE + LEUC II whereas S. capricorn and S. carteri nested in the LEUC I clade (pp=0.89.

b=73).

The Amphoriscidae species (A. micropilosus sp. nov., L. antillana sp. nov., Paraleucilla

dalmatica and P. magna) did not cluster together. Contrary to expected, L. antillana sp. nov.

evidenced a high affinity (pp=1, b= 99.9) with S. conulosum sp. nov. These two species are very

different, however, their skeletons do not have choanosomal tetractines (tubar or subatrial).

Two relationships found by Klautau et al. (2016) using the whole 28S LSU were also

recovered in our phylogeny: (1) the genus Paraleucilla (including P. dalmatica and P. magna)

formed a highly supported clade with Leucandra nicolae in the BI analyses (pp=1) and (2)

Leucandra spinifera and L. aspera clustered together as sister species (pp=0.97, b=54).

121

Calcinean phylogeny

We provided ITS and C-LSU sequences for three new species, Ascandra torquata sp. nov. (2

ITS), Clathrina aspera sp. nov. (4 ITS and 1 C-LSU) and C. curaçaoensis sp. nov. (1 ITS and 1

C-LSU), and for five already known calcinean species, Borojevia tenuispinata (1 ITS and 1 C-

LSU), C. lutea (1 ITS), C. insularis (1 ITS and 1 C-LSU), C. mutabilis (8 ITS and 1 C-LSU), C.

zelinhae (1 C-LSU) and Leucetta floridana (2 ITS).

The aligned ITS sequences had a total length of 1200 bp including gaps. BI and ML

approaches recovered similar tree topologies. The BI phylogenetic tree is shown in Figure 31.

The genera Ascandra (pp=1, b=99), Borojevia (pp=0.7, b=65) and Clathrina (pp=1, b=100)

were recovered as monophyletic clades including the type species of each genus and the

sequences of the new species described herein.

The Calcinean C-LSU clade recovered in the general Bayesian tree is shown in Figure 32.

Two important phylogenetic relationships were recovered in both, the ITS and C-LSU

phylogenies: (1) the yellow C. aurea, C. clathrus, C. curaçaoensis sp. nov, C. lutea and C.

luteoculcitella clustered together in one clade (ITS: pp=1, b=55; C-LSU: pp=0.86, b=65)

whereas the other yellow clathrinas C. mutabilis and C. insularis, appeared in separate clades

and (2) Clathrina aspera sp. nov. clustered together with the geographically distant Australian

species C. helveola and C. wistariensis (ITS and C-LSU: pp=1, b=100).

Although several clathrinas from the Tropical Atlantic Ocean were included in the ITS

phylogenetic analysis, C. mutabilis appeared closer to a clathrina from the French Polynesia

(pp=1, bb=100). Another interesting grouping was observed within the clade of L. floridana.

The Curaçaoan specimens evidenced a higher affinity with the Brazilian specimen than with the

Panamanian one despite their geographic distance.

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Figure 30. Calcaronean cluster from the Bayesian phylogenetic tree inferred from the C-LSUsequences. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap).BAE (Baerida), LEUC I (Leucosolenida I) and LEUC II (Leucosolenida II) refer to clades foundby Voigt et al, 2012. *Sequences generated in this study.

123

Figure 31. Bayesian phylogenetic tree inferred from the ITS sequences of the Calcineanspecies. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap).*Sequences generated in this study.

124

Figure 32. Calcinean cluster from the Bayesian phylogenetic tree inferred from the C-LSUsequences. Posterior probabilities and bootstrap values are given on the branches (pp/bootstrap).*Sequences generated in this study.

DISCUSSION

Biodiversity and Distribution

With the present work we increased from two to 20 the number of known species of

calcareous sponges from Curaçao. We found a total of 12 representants of the subclass Calcinea

and eight of the subclass Calcaronea. With the addition of these Curaçaoan records, the number

of calcareous sponges reported for the Caribbean Sea rose from 24 to 40 species, an increase of

66.7% in our knowledge of the biodiversity of Caribbean calcareous sponges.

The finding of nine provisionally Curaçaoan endemic species (45%; 9/20) - Amphoriscus

micropilosus sp. nov., Arthuria vansoesti sp. nov., Clathrina curaçaoensis sp. nov., Grantessa

tumida sp. nov., Leucandra caribea sp. nov., Leucandrilla pseudosagittata sp. nov. Leucilla

antillana sp. nov., Sycon conulosum sp. nov. and Sycon magniapicalis sp. nov. - may indicate a

high endemism of Calcarea in this island, however, it is also possible that it is just showing how

understudied this class is in the Caribbean Sea.

Considering the geographic distribution of calcareous sponges species, only six species

were previously reported occurring in both, the Brazilian Coast and the Caribbean Sea, Leucetta

125

floridana, Leucaltis clathria, Leucandra barbata, Leucandra rudifera, Nicola tetela and Sycon

ampulla (Muricy et al., 2011; Cóndor-Luján & Klautau, 2016). In this study, we expanded the

geographical distribution of four former Brazilian endemic species (B. tenuispinata, C.

insularis, C. lutea and C. mutabilis, Azevedo et al., submitted) to the Caribbean Sea (Curaçao)

and reported two new species (Ascandra torquata sp. nov. and Clathrina aspera sp. nov.)

present in both regions. Therefore, 12 species are currently shared between the Brazilian coast

and the Caribbean Sea. This result suggests a Brazilian-Caribbean sponge affinity for Calcarea

and questions the role of the Amazon and Orinoco Rivers as effective barriers to the dispersal

between these two regions.

Taxonomy and Phylogeny

Important phylogenetic traits with systematic implications can be discussed based on our

results. The phylogenetic importance of the skeleton composition in Calcinean systematics has

already been demonstrated (Rossi et al., 2011; Klautau et al., 2013), nonetheless, in Calcaronea

this has not been tested so far. In this study, we found that the presence of subatrial tetractines

clustered Leucilla antillana sp. nov. with Sycon conulosum sp. nov. and the absence of tubar

tetractines separated Grantessa tumida sp. nov. from Grantessa aff. intusarticulata. Whether

choanosomal tetractines (subatrial or tubar) do have phylogenetical signal across other species

within Calcaronea should be analysed in further studies with a larger number of species.

The close affinity between Heteropiidae and Grantiidae with large longitudinal diactines

was firstly observed in a 28S-phylogeny (Voigt et al., 2012) and it was recently recovered with

the shorter C-LSU sequences (Voigt & Worheide, 2016). This relationship was based on two

highly supported clades: (1) Sycettusa spp. + Grantessa aff. intusarticulata and Aphroceras sp.

and (2) Vosmaeropsis sp. + Sycettusa spp. and Sycon ciliatum. Herein, we recovered that

relationship with a third pair of species: G. tumida sp. nov. and Synute pulchella.

Azevedo et al., (submitted) suggested that trichoxeas were not reliable characters to

differentiate species in Clathrina (as specimens of C. mutabilis with and without trichoxeas

clustered together). We not only recovered the same pattern in another Clathrina species

(Clathrina lutea) but also in Ascandra as diactines can be present or not in A. torquata sp. nov.

Perhaps in the future, not only trichoxeas but also diactines will not be considered as diagnostic

characters for all Calcinean sponges.

The obtained ITS and C-LSU phylogenies suggested again that the yellow colour appeared

more than once in Calcinea (Rossi et al., 2011; Klautau et al., 2013) and that affinities found

between Atlantic and Indo-Pacific species (C. aspera sp. nov. and C. wistariensis; C. mutabilis

and Clathrina sp. from French Polynesia) should receive attention in further studies.

126

ACKNOWLEDGEMENTS

We would like to thank Giselle Lôbo-Hajdu for assistance during the sample collections. Mark

Vermeij and CARMABI are acknowledged for providing logistical support in Curaçao. We are

thankful to Marcelo Soares for the assistance with the SEM procedures.

FINANCIAL SUPPORT

This study was funded by the Brazilian National Research Council (CNPq), Coordination for

the Improvement of Higher Education Personnel (CAPES), Foundation Grupo Boticário de

Proteção à Natureza and Rio de Janeiro State Research Foundation (FAPERJ). B. C. L. received

a scholarship from CAPES. T. L. received a scholarship from FAPERJ. F.A. and A.P. are granted

with post-doc scholarships from FAPERJ and CAPES. E.H. and M.K. have fellowships from

CNPq.

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SUPPLEMENTARY MATERIAL

Figure S1. Bayesian phylogenetic tree inferred from the C-LSU sequences of the Calcaroneanand Calcinean species. Posterior probabilities and bootstrap values are given on the branches(pp/bootstrap). BAE (Baerida), LEUC I (Leucosolenida I) and LEUC II (Leucosolenida II) referto clades found by Voigt et al, 2012. *Sequences generated in this study.

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New records of calcareous sponges (Porifera: Calcarea) from the Northeastern Brazilian

coast including three new species

BÁSLAVI CÓNDOR-LUJÁN1, FERNANDA AZEVEDO1, ANDRÉ PADUA1, EDUARDO

HAJDU2 & MICHELLE KLAUTAU1*

1Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av.

Carlos Chagas Filho, 373, Rio de Janeiro, RJ, Brasil, 21941-902. E-mail:

[email protected]; [email protected]; [email protected];

[email protected] Federal do Rio de Janeiro, Museu Nacional, Departamento de Invertebrados,

Quinta da Boa Vista, São Cristóvão, Rio de Janeiro, RJ, Brasil, 20940-040. E-mail:

[email protected].

*Corresponding author: [email protected]

Journal to be submitted: ZOOTAXA

ABSTRACT

Despite the efforts done in order to know the diversity of calcareous sponges along the Brazilian

coast, several areas still remain unexplored or poorly investigated. In this study, we examined

the material collected in recent expeditions along the NE Brazilian coast using morphological

and molecular approaches. Sampled localities included three protected areas: Área de Proteção

Ambiental dos Recifes de Corais de Maracajaú in Rio Grande do Norte State, Fernando de

Noronha Archipelago in Pernambuco State and Abrolhos National Marine Park in Bahia State

and other localities in Ceará State and Rio Grande do Norte. Collections were performed by

snorkeling and SCUBA down to 20 m deep. A total of 14 species were identified including three

new species: Amphoriscus hirsutus sp. nov., Grantia grandisapicalis sp. nov. and Leucascus

luteoatlanticus sp. nov. Arthuria vansoesti Borojevia brasiliensis, Clathrina aspera and C.

luteoculcitella constitute new records for the NE Brazil. Clathrina mutabilis, Ernstia citrea and

E. rocasensis are recorded for the first time from Rio Grande do Norte, Bahia and Pernambuco,

respectively. Clathrina aurea, C. lutea, Leucascus roseus and Leucilla uter are recorded from

new localities within the Abrolhos Marine National Park.

Keywords: Brazilian continental shelf, Fernando de Noronha and Atoll das Rocas, NE Brazil,

Eastern Brazil, Calcaronea, Calcinea.

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Introduction

The Northeastern Brazilian coast comprises a large area of more than 3,300 km, representing

almost 45% of the Brazilian coast. It is located in the Tropical Southwestern Atlantic Province

and covers three entire marine ecoregions, São Pedro and São Paulo Islands, Fernando de

Noronha and Atoll das Rocas and Northeastern Brazil, and part of the Eastern Brazil ecoregion

(Spalding et al. 2007). It also includes nine Brazilian political-administrative divisions (States):

Maranhão, Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Alagoas, Sergipe and

Bahia. This vast region encompasses a variety of ecosystems, coral and algal reefs, estuaries,

mangroves, rocky shores, sandy beaches, seagrasses, tide pools and coastal and oceanic islands

(Moraes 2011; Longo & Amado-Filho 2014; Araújo & Amaral 2016), housing a great diversity

of marine taxa, including sponges (Muricy et al. 2011). However, due to its huge dimension and

habitat heterogeneity, several areas are still unexplored or badly known concerning its marine

biodiversity, especially for some faunistic groups, such as sponges of the class Calcarea.

The class Calcarea (Porifera) is composed of sponges whose skeleton is built exclusively of

calcium carbonate. They are divided into two monophyletic subclasses: Calcaronea and

Calcinea. Calcaroneans are characterised by presenting sagittal spicules, apinucleated

choanocytes and amphiblastula larvae and diactines are the first spicules to be produced,

whereas calcineans have a skeleton mainly composed of regular spicules, basinucleated

choanocytes, calciblastula larvae and triactines are the first spicules to be secreted (Manuel et al.

2002). Within the subclasses of Calcarea, molecular phylogenetic studies have demostrated that

high taxonomical categories such as orders, families and even some calcaronean genera are

polyphyletic (Voigt et al. 2012; Voigt & Wörheide 2015; Klautau et al. 2016), whereas other

studies have evidenced monophyletic genera in Calcinea (Rossi et al. 2011; Klautau et al.

2013).

According to the catalogue of Brazilian Porifera, among the 47 species of calcareous sponges

recorded from the Brazilian coast, 26 species (55.3%) were reported from the Northeastern

region (Muricy et al. 2011). This might suggest that the diversity of Calcarea within this region

is well investigated, however, recent studies in this area revealed new species to science

(Cavalcanti et al. 2014, 2015; Azevedo et al. submitted), which shows how the diversity of this

class is still poorly known.

The first records of calcareous sponges from the NE Brazil were published in the 19th

century: Amphoriscus synaptum (Schmidt in Haeckel, 1872) and Leucilla sacculata (Carter,

1890). Almost one century later, Borojevic & Peixinho (1976) reported 24 species including

four new species to science at that time. In the last decade, the knowledge of calcareous sponges

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from the NE Brazil increased by studies focusing on specific localities such as the Potiguar

Basin (Rio Grande do Norte, Lanna et al. 2009), Bahia State (Hajdu et al. 2011) and oceanic

and mid-shelf islands (Azevedo et al. submitted), or in genera as Paraleucilla and Vosmaeropsis

(Cavalcanti et al. 2014, 2015, respectively). Despite this considerable increase of descriptions in

the last years, the diversity and distribution of calcareous sponges in the NE Brazil is still

underestimated.

In the present work, using morphological and molecular approaches, we studied material

collected in recent expeditions conducted along the NE Brazilian coast including three Protected

Areas (Corais de Maracajaú, Abrolhos Marine National Park and Fernando de Noronha

Archipelago) and two other localities in Ceará and Rio Grande do Norte States.

Material and Methods

Analysed material

In this study, we examined the calcareous sponges collected in three expeditions carried out in

the NE Brazilian Coast during 2014 and 2016: ExpoCERN (Expedição Poriferos do Ceará e

Rio Grande do Norte, April 2014), Abrolhos Expedition (December 2015) and TAXPOmol

Biodescoberta 2016 Expedition (April 2016). As some localities are within protected areas,

namely, Abrolhos Marine National Park, Fernando de Noronha and Área de Proteção Ambiental

dos Recifes de Corais de Maracajaú (APARC- Maracajaú), these expeditions received the

adequate support and permissions from the ICMBio (Instituto Chico Mendes de Conservação da

Biodiversidade). The sampled localities where calcarean specimens were found are detailed in

Table 1 and Figure 1.

The collections were conducted by snorkeling and SCUBA diving down to 20 m deep. The

specimens were photographed in situ and fixed in 96% ethanol. All the samples are preserved in

96% ethanol and deposited in the Porifera Collection of the Biology Institute of the

Universidade Federal do Rio de Janeiro, Brazil (UFRJPOR) or in the Museu Nacional do Rio de

Janeiro (MNRJ/UFRJ).

Morphological procedures

The external morphology and internal anatomy were assessed through the observation of the

fixed specimens and the examination of microscopy slides. Sections and spicule slides

preparations followed standard protocols (Wörheide & Hooper 1999; Klautau & Valentine

2003). The spicule measurements including length and width (minimum, mean, standard

deviation [SD] and maximum) are presented in tabular form. The species identifications

followed the Systema Porifera (Hooper &Van Soest 2002) and additional appropriate literature

135

for calcineans (Klautau & Valentine 2003; Klautau et al. 2013; Cavalcanti et al. 2013). To

illustrate the species descriptions, photographs were taken with a digital AxioCam MRC5

coupled to a Zeiss Imager A2 microscope. Additionally, spicules were placed on a cover-slip,

mounted on a stub with double-sided carbon tape and sputter-coated with gold for scanning

electron microscopy. Microphotographs were taken with a JSM-6510 SEM at the Institute of

Biology of the Universidade Federal de Rio de Janeiro (Brazil).

Table 1. Sampled localities, ecoregions sensu Spalding et al. (2007) and geographiccoordinates. Brazilian States: BA=Bahia, CE=Ceará, PE=Pernambuco, RN=Rio Grande doNorte.

L. Locality Ecoregion Geographic Coordinates1 Porto do Pecém, São Gonçalo do

Amarante, CENortheastern Brazil 03º32.106'S, 38º47.893'W

2 Ressurreta, Fernando de Noronha Archipelago, PE

Fernando de Noronha andAtoll das Rocas

03°48.817'S, 32°23.483'W

3 Farol Tereza Pança, Área de Proteção Ambiental dos Recifes de Corais de Maracajaú, Maracajaú, RN

Northeastern Brazil 05º24.133'S, 35º17.849'W

4 Batente das Agulhas, Natal, RN Northeastern Brazil 05°33.841'S, 35°04.367'W5 Parcel das Paredes, Abrolhos Marine

National Park, BAEastern Brazil 17°54.319'S, 38°56.658'W

6 Mato Verde, Ilha Santa Bárbara, Abrolhos Marine National Park, BA

Eastern Brazil 17°57.847'S, 38°41.979'W

7 Portinho Sul, Ilha Santa Bárbara, Abrolhos Marine National Park, BA


8 Ilha Guarita, Abrolhos Marine National Park, BA


9 Chapeirão 1, Abrolhos Marine National Park, BA


10 Chapeirão 2, Abrolhos Marine National Park, BA


Molecular procedures

The total genomic DNA was extracted with the guanidine/phenol-chloroform protocol

(Sambrook et al. 1989) or with the QIAamp_DNA MiniKit (Qiagen) and stored at –20°C. For

calcinean species, the region containing the partial 18S and 28S, the spacers ITS1 and ITS2 and

the 5.8S ribosomal DNA (ITS) was amplified with the primers: fwd: 5`-

TCATTTAGAGGAAGTAAAA GTCG-3` and rv: 5`-GTTAGTTTCTTTTCCTCCGCTT-3`)

(Lôbo-Hajdu et al. 2004). For calcaroneans, the C-region of the 28S (C-LSU) was amplified

using the primers fwd: 5'GAAAAGCACTTTGAAAAGAGA-3' (Voigt & Wörheide 2015) and

rv: 5'-TCCGTGTTTCAAGACGGG-3' (Chombard et al. 1998).

136

Figure 1. Map of South America highlighting the Northeastern Brazilian coast and the sampledlocalities in the present study. (A) Ceará State; (B) Fernando de Noronha Archipelago; (C) RioGrande do Norte State, and (D) Abrolhos Marine National Park. The numbers refers to thesampled localities in Table 1.

The PCR mixture included 1x buffer (5x GoTaq® Green Reaction Buffer Flexi,

PROMEGA), 0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM

of each primer, one unit of Taq DNA polymerase (Fermentas or PROMEGA) and 1 µL of DNA

in a volume of 15 µL. The PCR amplification comprised one first cycle of 4 min at 94°C, 1 min

at 50°C and 1 min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 50°C and one minute at 72°C,

and a final cycle of 6 min at 72°C. Forward and reverse strands were sequenced in an ABI 3500

(Applied Biosystems). The new sequences and the ones retrieved from the GenBank database

are listed in Table 2. The set of sequences was aligned through the MAFFT v.7 online platform

(Katoh & Standley 2013) using the strategy Q-INS-i (Katoh & Toh 2008). The nucleotide

substitution models that best fit the alignment were GTR+G for C-LSU and TN93+G for ITS

sequences, as indicated by the Bayesian Information Criterion in MEGA 6 (Nei & Kumar 2000;

Tamura et al. 2013).

Phylogenetic reconstructions were obtained using Bayesian Inference (BI) and Maximum

Likelihood (ML) methods. The BI reconstructions were conducted in MrBayes 3.1.2

(Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) under 106 generations and a

burn-in of 1000 sampled trees, yielding a consensus tree of majority. As the TN93 model is not

implemented in Mr.Bayes, we used the GTR model. The ML analyses were performed on

137

MEGA 6 using an initial NJ tree (BIONJ) and a bootstrap of 1000 pseudo-replicates, yielding a

mid-point rooted tree. BI posterior probabilities (pp) values as well as ML bootstrap values (b)

are indicated on the branches of the inferred trees. In order to know the genetic intraspecific

variability within the sequenced species, we calculated the uncorrected p distance considering

complete deletion in MEGA 6.

Table 2. Species used in the phylogenetic analyses with locality, voucher number and GenBank(GB) accession number for the ITS (Calcinea) and C-LSU (mostly Calcaronea) regions.*Sequences generated in the present study. H=holotype, P=paratype.

Species Locality Voucher number GenBank NumberCALCARONEAAmphoriscus micropilosus Curaçao UFRJPOR6755 (H) Curaçao paperAmphoriscus hirsutus sp. nov.* NE Brazil UFRJPOR7570 (H) This studyAnamixilla torresi - - AY563636Aphroceras sp. - SAM-PS0349 JQ272273Grantessa tumida Curaçao UFRJPOR6701 (P) Curaçao paperGrantessa aff. intusarticulata - GW979 JQ272278Grantia compressa - - AY563538Grantia grandisapicalis sp. nov.* NE Brazil UFRJPOR7567 (H) This studyLeucandra aspera - - AY563535Leucandra falakra Adriatic Sea UFRJPOR8349 (H) KT447560Leucandra nicolae - - JQ272268Leucandra sp. - QMG316285 JQ272265Leucandra spinifera Adriatic Sea UFRJPOR8348 (H) KT447561Leucandrilla pseudosagittata Curaçao UFRJPOR 6705 (P) Curaçao paperLeucascandra caveolata - QMG316057 JQ272259Leucilla antillana Curaçao UFRJPOR 6768 (H) Curaçao paperLeuconia nivea - - AY563534Paraleucilla dalmatica Adriatic Sea UFRJPOR8346 (H) KT447566Paraleucilla magna Adriatic Sea IRB-P14 KT447564Sycettusa aff. hastifera Red Sea GW 893 JQ272282Sycettusa cf. simplex Western India ZMAPOR11566 JQ272279Sycettusa tenuis Australia QMG313685 JQ272281Sycettusa sp. - - AY563530Sycon ancora Adriatic Sea UFRJPOR8347 (P) KT447568Sycon carteri Australia SAM-PS0143 JQ272260Sycon ciliatum - - AY563532Sycon conulosum Curaçao UFRJPOR6707 (H) Curaçao paperSycon cf. villosum - GW51115 KR052809Sycon magniapicalis Curaçao UFRJPOR 6748 (H) Curaçao paperSycon raphanus - - AY563537Syconessa panicula Australia QM G313672 AM181007Utte aff. syconoides - QMG323233 JQ272269Utte aff. syconoides - QMG313694 JQ272271Ute ampullacea - QMG313669 JQ272266Vosmaeropsis sp. - MM-2004 AY026372

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CALCINEAAscandra falcata Mediterranean UFRJPOR5856 HQ588962Ascaltis reticulum Mediterranean UFRJPOR 6260 HQ588977Borojevia aff. aspina Brazil UFRJPOR 5245 HQ588998Borojevia brasiliensis SE Brazil UFRJPOR5214 HQ588978Borojevia brasiliensis SE Brazil UFRJPOR5230 HQ588999Borojevia brasiliensis SE Brazil UFRJPOR 5406 KX548909Borojevia brasiliensis* NE Brazil UFRJPOR 7384 This studyBorojevia cerebrum Mediterranean UFRJPOR6322 HQ588964Borojevia croatica Adriatic Sea IRB-CLB6 KP740023Borojevia tenuispinata Brazil UFRJPOR6484 (H) KX548916Borojevia trispinata Brazil UFRJPOR6419 (H) KX548918Clathrina antofagastensis Chile MNRJ 9289 HQ588985Clathrina aphrodita Peru MNRJ 12994 KC985138Clathrina aspera SE Brazil UFRJPOR 5531 (P) Curaçao paperClathrina aspera Curaçao UFRJPOR 6758 (H) Curaçao paperClathrina aspera* NE Brazil UFRJPOR 7390 This studyClathrina aspera* NE Brazil UFRJPOR 7391 This studyClathrina aurea SE Brazil MNRJ 8998 HQ588968Clathrina aurea* NE Brazil UFRJPOR7544 This studyClathrina aurea* NE Brazil UFRJPOR7571 This studyClathrina aurea* NE Brazil UFRJPOR7574 This studyClathrina aurea* NE Brazil UFRJPOR7584 This studyClathrina blanca Adriatic Sea PMR-14307 KC479087Clathrina clathrus Mediterranean UFRJPOR6315 HQ589009 (C-LSU)

HQ588974 (ITS)Clathrina conifera Brazil MNRJ8991 HQ588959Clathrina coriacea Norway UFRJPOR6330 HQ588986 Clathrina curaçaoensis Curaçao UFRJPOR6734 Curaçao paperClathrina cylindractina Brazil UFRJPOR5206 HQ588979Clathrina fjordica Chile MNRJ 8143 HQ588984Clathrina hispanica Mediterranean UFRJPOR 6305 KC843432Clathrina insularis Brazil UFRJPOR6532 (H) KX548921Clathrina insularis Brazil UFRJPOR6536 (P) KX548922Clathrina helveola Australia QMG313680 HQ588988 Clathrina lacunosa Norway UFRJPOR 6334 HQ588991Clathrina lutea NE Brazil UFRJPOR5172 HQ588961Clathrina lutea NE Brazil UFRJPOR5173 (H) HQ588976Clathrina lutea NE Brazil UFRJPOR 6543 KX548923Clathrina lutea NE Brazil UFRJPOR 6545 KC843442Clathrina lutea* NE Brazil UFRJPOR 7549 This studyClathrina lutea* NE Brazil UFRJPOR 7561 This studyClathrina lutea* NE Brazil UFRJPOR 7579 This studyClathrina lutea* NEBrazil UFRJPOR 7591 This studyClathrina lutea* NE Brazil UFRJPOR 7592 This studyClathrina lutea* NE Brazil MNRJ 18900 This studyClathrina luteoculcitella Australia QMG313684 HQ588989Clathrina luteoculcitella Indonesia ZMAPOR08657 KX548906Clathrina luteoculcitella* NE Brazil UFRJPOR 7551 This study

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Clathrina luteoculcitella* NE Brazil UFRJPOR 7569 This studyClathrina mutabilis NE Brazil UFRJPOR6526 (H) KX548925Clathrina mutabilis NE Brazil UFRJPOR 6528 (P) KX548926Clathrina mutabilis* NE Brazil UFRJPOR 7386 This studyClathrina mutabilis Curaçao UFRJPOR 6741 KC843437Clathrina nuroensis Peru MNRJ 13032 KC985136Clathrina peruana Peru MNRJ 12839 (H) KC985135Clathrina primordialis Adriatic Sea UFRJPOR 6863 KP740016Clathrina ramosa Chile MNRJ 10313 HQ588990Clathrina rubra Adriatic Sea PMR 14306 KC479088Clathrina wistariensis Australia QMG313663 HQ588987Ernstia tetractina Brazil UFRJPOR 5183 HQ589000Leucascus luteoatlanticus sp. nov.* NE Brazil UFRJPOR 7582 (H) This studyLeucascus simplex Polynesia BMOO16283 KC843454

RESULTS

Taxonomy

From a total of 69 specimens analysed, we identified three calcaronean and 11 calcinean species

including three new species to science: Amphoriscus hirsutus sp. nov., Grantia grandisapicalis

sp. nov. and Leucascus luteoatlanticus sp. nov. Among the already known species, four are

reported for the first time from the NE Brazil: Borojevia brasiliensis (Solé- Cava et al., 1991),

Clathrina aspera Cóndor-Luján et al., in press, C. luteoculcitella Worheide & Hooper, 1999 and

Arthuria vansoesti Cóndor-Luján et al., in press.

Seven species already recorded from the NE were also found in this study. Clathrina

mutablis Azevedo et al., submitted was originally described from Fernando de Noronha

Archipelago and herein, we expand its distribution to Maracajaú. Ernstia citrea Azevedo et al.,

submitted, was only recorded from Rocas Atoll (Rio Grande do Norte) and now it was found in

the Abrolhos Marine National Park. Ernstia rocasensis Azevedo et al., submitted, previously

reported from Rocas Atoll and Abrolhos Archipelago, is being reported from Fernando de

Noronha Archipelago now. Clathrina aurea Solé- Cava et al., 1991, C. lutea Azevedo et al.,

submitted, Leucascus roseus Lanna et al., 2007 and Leucilla uter Poléjaeff, 1883 were found in

new localities within the Abrolhos Marine National Park.

In the next section, the new records from the NE Brazil as well as the allegedly cosmopolitan

Leucilla uter are broadly described and illustrated. Calcineans already reported from the NE

region and found in the studied material are listed in Table 11.

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Description of species

Class CALCAREA Bowerbank, 1862

Subclass CALCARONEA Bidder, 1898

Order LEUCOSOLENIIDA Hartman, 1958

Family AMPHORISCIDAE Dendy, 1893

Genus Amphoriscus Haeckel, 1872

Amphoriscus hirsutus sp. nov. (Figures 2-3, Table 3)

Etimology. From the Latin hirsutus (= full of bristles), for the presence of diactines protruding

along the body.

Type locality. Ilha Guarita, Abrolhos National Marine Park, Bahia State.

Type material. Holotype (ethanol): UFRJPOR 7570; Ilha Guarita, Abrolhos Marine National

Park; 10.3 m deep, collected by A. Padua & F. Azevedo, 4/XII/2014.

Colour. White in life and yellowish in ethanol.

Description. This sponge has a friable, tubular body with an apical osculum (Figure 2A). The

holotype measures 22 x 6 mm. The osculum has a margin composed of sagittal tetractines and it

is surrounded by a crown of trichoxeas (Figure 2B). The surface is very hispid due to diactines

and anchor-like triactines which protrude through the sponge surface. The diactines are

distributed along the body (arrows in Figure 2B) and the anchor-like triactines are present at the

basal region of the sponge (arrowheads in Figure 2B-C). The aquiferous system is syconoid.

Skeleton. The skeleton is typical of the genus (Figure 2D). Diactines cross perpendicularly the

cortex (arrow in Figure 2D), which is composed of tetractines. The basal actines of the cortical

tetractines lay tangentially to the surface (black arrow in Figure 2E). The apical actine of the

tetractines and the diactines cross the choanosome, eventually reaching the atrium. The

choanosomal skeleton is inarticulated and it is formed by the apical actines of the cortical

tetractines (white arrow in Figures 2E-F) and the unpaired actine of subatrial triactines (black

arrows in Figure 2F). Subatrial-like triactines (arrowhead in Figure 2F) and tetractines (arrow in

Figure 2G) were found scattered in this region. The atrial skeleton is composed of tetractines

with the apical actine projected into the atrial cavity (Figure 2H). The anchor-like triactines are

organised in tufts (Figure 2I) with the unpaired actine located in the choanosome and the paired

actines protruding the sponge surface.

Spicules

Diactines (Figure 3A). One tip is sharp and the other, which protrudes the cortex, is lanceolated.

Very variable size: 462.5-2150.0/12.5-20.0 µm.

141

Cortical tetractines (Figure 3B). Sagittal. Actines are conical, straight, smooth and have sharp

tips. The paired actines can be curved. The apical actine is the largest actine and can be slightly

undulated. Size: 150.0-165.0/12.5-17.5 µm (unpaired actine), 200-350/7.5-20.0 µm (paired

actine) and 150-450/10-20 µm (apical actine).

Choanosomal tetractines (Figure 3C). Very rare. Sagittal. Subatrial-like. Actines are slightly

conical and with sharp tips. The paired actines are curved and can have different length.

Subatrial triactines (Figure 3D-F). Sagittal. Actines are conical, straight to slightly curved and

with sharp tips. The paired actines can have different length. Size: 315.0–565.0/10–15 µm

(unpaired actine) and 165.0–365.0/7.5–12.5 µm (paired actine).

Atrial tetractines (Figure 3G). Sagittal. Actines are slightly conical, straight, smooth and with

sharp tips. The paired actines can be curved. The apical actine is the shortest one. Size: 80-

285/7.5-12.5 µm (unpaired actine), 165.0-400.0/5-12.5 µm (paired actine) and 50-150/5-8.7 µm

(apical actine).

Anchor-like triactines (Figures 3H-I). Sagittal. The unpaired actine is very long. It is curved at

the base and very straight from the median region to the distal part (Figure 3H). The paired

actines are rudimentary and centripetally curved (Figure 3I). Size: 1650->1750/10 µm (unpaired

actine) and 25.0–40/6.2–7.5 µm.

Ecology. This specimen was found covered with sediment, surrounded by algae.

Geographic Distribution. Provisionally endemic to the NE Brazil (present study).

Remarks. Amphoriscus hirsutus sp. nov. is the second Amphoriscus species bearing anchor-like

triactines. The other species is A. ancora Van Soest, 2017 from the Guyana Shelf. Although both

species have similar skeletons, they present relevant differences. In A. ancora, diactines and

anchor-like triactines are restricted to the base of the sponge, whereas in A. hirsutus sp. nov. the

diactines are present along the whole body and the anchor-like triactines are present not only in

the base but also in adjacent (basal) regions. The new Brazilian species present triactines and

tetractines scattered in the choanosomal skeleton which are absent in A. ancora. Besides, the

size (mean length/width) of the atrial tetractines of A. hirsutus (paired actine: 293.2/8.6 µm,

unpaired actine: 190.0/9.4 µm and apical actine: 88.4/7.2 µm) exceeds that of A. anconra

(paired actine: 99.0/7.7 µm, unpaired actine: 87/8.6 µm and apical actine: 26/5.3 µm).

Additionaly, the osculum of A. hirsutus presents a crown of trichoxeas whereas in A. ancora, it

is naked, however, as this is a plastic character, it must be treated with caution.

Another species of Amphoriscus recorded from the Brazilian coast is Amphoriscus synaptum

(Schmidt in Haeckel, 1872), however, it can be easily differentiated from A. hirsutus sp. nov. by

the presence of anchor-like tetractines in the former.

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Not considering the anchor-like spicules, the species that more resemble A. hirsutus sp. nov.

in skeleton composition is A. buccichii Ebner, 1887 from the Adriatic Sea, however, the

skeleton of the latter also comprises small subcortical tetractines (length: 80-120 µm) which are

absent in the new species. Moreover, they differ in spicule size. A. buccichii has smaller

diactines (60-200/3-5 µm) and thicker cortical tetractines (width: 30–40 µm) compared to A.

hirsutus sp. nov. (size of diactines: 462.5-2150/12-20 µm and width of cortical tetractines: 7.5-2

µm). For additional comparisons, all the measurements of A. ancora and A. buccichii are

presented in Table 3.

Table 3. Measurements of Amphoriscus hirsutus sp. nov. (UFRJPOR 7570), A. ancora and A.buccichii. *Taken from the original descriptions. H=holotype. P=paired, U=unpaired andA=apical actines.

Species Spicule Actine Length (µm) Width (µm) NMin Mean SD Max Min Mean SD Max

UFRJPOR7570(H)

Diactine 462.5 2150.0 510.3 2150.0 12.5 16.9 2.6 20.0 14Cortical Tetractine

P 200.0 280.4 46.1 350.0 7.5 12.6 3.4 20-0 25U 150.0 157.5 10.6 165.0 12.5 15.0 3.5 17.5 2A 150.0 324.8 85.6 500.0 10.0 14.0 2.9 20.0 26

Subatrial Triactine

P 165.0 266.8 51.6 365.0 7.5 10.1 7.5 12.5 22U 315.0 443.8 70.9 565.0 10.0 11.8 1.7 15.0 21

AtrialTetractine

P 165.0 293.2 64.0 400.0 5.0 8.6 1.6 12.5 24U 80.0 190.0 65.9 285.0 7.5 9.4 1.8 12.5 17A 50.0 88.4 31.4 150.0 5.0 7.2 1.0 8.7 20

Anchor-liketriactine

P 25.0 32.1 5.8 40.0 6.2 7.3 0.5 7.5 7U 1650.0 - - >1750.0 - 10 - -

A.ancora*

Diactine 600.0 1017.0 - 1230.0 3.0 4.2 - 6.5 -Cortical Tetractine

P 135.0 239.0 - 320.0 8.0 15.4 - 21.0 -U 151.0 216.0 - 330..0 12.0 16.1 - 21.0 -A 258.0 311.0 - 366.0 8.0 17.8 - 20.0 -

Subatrial Triactine

P 129.0 218.0 - 324.0 7.0 8.6 - 10.5 -U 201.0 367.0 - 468.0 7.0 10.1 - 12.0 -

AtrialTetractine

P 57.0 99.0 - 165.0 3.0 7.7 - 11.0 -U 42.0 87.0 - 186.0 3.0 8.6 - 12.0 -A 9.0 26.0 - 60.0 3.0 5.3 - 15.0 -

Anchor-liketriactine

P 18.0 24.8.0 - 29.0 5.0 6.8 - 9.0 -U 96.0 386.0 - 660 7.0 8.6 - 10.0 -

A.buccichii*

Diactine 60.0 - - 200 3.0 - - 5.0 -Cortical Tetractine

P 360.0 - - 420 30.0 - - 40.0 -U 360.0 - - 540 30.0 - - 40.0 -A 300 - - 420 30.0 - - 40.0 -

Subatrial Triactine

P 100 - - 120 6.0 - - 7.0 -U 200 - - 260 6.0 - - 7.0 -

AtrialTetractine

P 150 - - 200 7.0. - - 10.0 -U 300 - - 400 7.0 - - 10.0 -A 100 - - 150 6.0 - - 12.0 -

143

Figure 2. Amphoriscus hirsutus sp. nov. (UFRJPOR 7570). A. Specimen in vivo. B. Specimenafter fixation showing diactines (arrows) and anchor-like triactine (arrowhead). C. Anchor-liketriactine. D. Cross section of skeleton with diactines (black arrow). D. Cortex with the pairedactines (black arrow) and apical actine (white arrow) of a tetractine. F. Choanosome showing anapical actine of a cortical tetractine (white arrow), unpaired actines of subatrial triactines (black

144

arrows) and a choanosomal triactine (arrowhead). G. Choanosome with subatrial-like tetractine.H. Atrial skeleton. I. Tuft of anchor-like triactines. Abbreviations: cx=cortex; at=atrium.

Figure 3. Spicules of Amphoriscus hirsutus sp. nov. (UFRJPOR 7570). A. Diactines. B. Corticaltetractines. C. Choanosomal subatrial-like tetractine. D-F. Subatrial triactines. G. Atrialtetractine. H. Anchor-like tetractine. I. Paired actines of the anchor-like tetractine.

145


Leucilla uter Poléjaeff, 1883 (Figures 4-5, Table 4)

Synonyms.

Amphoriscus chrysalis Burton, 1963: 545.

Leucilla australiensis, Borojevic, 1967: 221; Borojevic & Peixinho, 1976: 1031

Leucilla uter Poléjaeff, 1883: 53; Dendy & Row, 1913: 784; Borojevic & Boury-Esnault, 1987:

35; Muricy & Silva, 1999: 160, Muricy et al., 2011: 25

Leucilla? uter, Muricy et al., 1991: 1187.

Type locality. Poléjaeff described specimens from Bermudas and Philippines, however the

lectotype deposited in the Natural History Museum (London) is from Bermudas.

Type specimens. Lectotype (ethanol and dried) BMNH 1884.4.22.21, paralectotypes (slides):

BMNH.4.22.30 and BMNH 1884.4.22.31.

Material examined. UFRJPOR 7545 (ethanol), Mato Verde, Ilha Santa Bárbara, Abrolhos

Marine National Park, 7.1 m deep, collected by A. Padua & F. Azevedo, 3/XII/2014.

Colour. Bright white in life and in ethanol.

Description. The analysed specimen has a sac-shaped body with an apical osculum (Figure

4A). It measures 9.4 x 3.3 mm (Figure 4B). The osculum has a crown of trichoxeas (2.6 x 1.7

mm) supported by sagittal T-shaped tetractines. In the apical part of the body, diactines protrude

through the surface (arrow in Figure 4B). The surface is rough and the consistency is friable.

The aquiferous system is leuconoid.

Skeleton. The skeleton is typical of the genus (Figure 4C). The cortical skeleton is formed by

tetractines and very rare triactines (arrow in Figure 4D). The basal actines of the tetractines are

tangentially disposed and the apical actine penetrates the choanosome and can reach the atrium.

Some diactines were also observed in this region. The choanosomal skeleton is inarticulated,

formed by the apical actine of the cortical tetractines (white arrow in Figure 4E) and by the

unpaired actine of the subatrial triactines (black arrow in Figure 4E) and rare subatrial

tetractines. Rare tetractines and triactines were found scattered in the choanosome, with their

unpaired actine pointing to the surface (as shown in Figure 4C). The atrial skeleton is

exclusively composed of tetractines with the apical actine projected into the atrium (Figure 4F).

Spicules

Diactines (Figure 5A). Fusiform (sharp tips). Size (length/width): >1250/6-10 μm.

Cortical triactines (Figures 5B). Sagittal. Very rare. Actines are straight, slightly conical with

sharp tips. The paired actines can be slightly curved and longer than the unpaired one. Size:

200.0-205.0/10.0-11.2 μm (paired actine) and 122.5/10.0-12.5 μm (unpaired actine).

146

Cortical tetractines (Figure 5C-D). Sagittal. Actines are conical with sharp tips. The paired

actines are frequently inwardly curved. The apical actine is the longest actine. Size: 225.0-

530.0/22.5-40.0 μm (paired actine), 265.0-500.0/30-42.5 μm (unpaired actine) and 255-

1000/22.5-40.0 μm (apical actine).

Choanosomal triactines (Figure 5E) and tetractines (Figure 5F-G). Sagittal. Rare. Actines are

conical with sharp tips. Different from the cortical tetractines, the actines are always straight.

Size of triactines: 250.0/22.5 μm (paired actine) and 237.5/22.5-25 μm (unpaired actine). Size of

tetractines: 335-400/15-40 μm (paired actine), 227.5-425.0/27.5-37.5 μm (unpaired actine) and

87.5/25.0 μm (apical actine).

Subatrial triactines (Figure 5H). Sagittal. Actines are conical with sharp tips. The paired

actines are smaller than the unpaired one and can be slightly curved. Very variable size: 75-

320.0/7.5-22.5 μm (paired actine) and 150-540/7.5-20.0 μm (unpaired actine).

Subatrial tetractines (Figure 5I). Sagittal. Rare. Actines are conical with sharp tips, similar to

subatrial triactines. The apical actine is shorter and thinner than the basal ones. Size: 112.5-

285.0/7.5-15 μm (paired actine), 175.0-350.0/7.5-20 (unpaired actine) and 50.0-87.5/7.5 μm

(apical actine).

Atrial tetractines (Figure 5J). Sagittal. Actines are conical with very sharp tips. The paired

actines are longer than the unpaired one. The apical actine is the thinnest and shortest actine.

Size: 115.0-285.0/5.0-12.5 μm (paired actines), 137.5-187.5/7.5-12.5 μm (unpaired actine) and

62.5-150.0/7.4-10.0 μm (apical actine).

Ecology. This specimen was collected underneath boulders.

Geographic distribution. Leucilla uter is an allegedly cosmopolitan species. It was originally

described from Bermudas and Philippines (Poléjaeff, 1883) and then, recorded from Brazil

where it is widely distributed, from Alagoas to Rio de Janeiro States (Borojevic & Peixinho

1976; Muricy et al. 2011).

Remarks. The analysed specimen in the present study mostly matches the original description

of Leucilla uter (Poléjaeff 1833) as well as the description of its lectotype (Borojevic & Boury-

Esnault, 1987). The only difference is the presence of rare diactines and cortical triactines in our

specimen. As the validity of those spicules as diagnostic characters within Leucilla species has

been questioned (Borojevic & Boury-Esnault, 1987), we decided not to differentiate the studied

specimen as a new species and to identify it as L. uter until an update revision of this genus is

done.

It is worth to mention that the revised specimen slightly differs from the specimens

misidentified as L. australiensis by Borojevic & Peixinho (1976) and re-identified as L. uter in

147

Muricy et al. (2011) in the size of the cortical triactines and of the atrial tetractines. The

unpaired actines of both spicule categories are larger in the specimens described by Borojevic &

Peixinho (cortical triactine=200-400/10-16 μm and atrial tetractine=270-400/10-12 μm) than in

our specimen (cortical triactine=122.5/10-12.5 μm and atrial tetractine=137.5-187.5/7.5-12.5

μm).

This is the first record of L. uter occurring in very shallow waters (<10 m). Previously, it was

reported from depths ranging from 10 to 61 m (Muricy et al. 2011).

Figure 4. Leucilla uter (UFRJPOR 7545). A. Specimen in vivo. B. Specimen after fixation. C.Cross section of the skeleton. D. Cortex with cortical triactines (arrow). E. Inarticulated

148

choanosomal skeleton with the apical actine of a cortical tetractine (white arrow) and theunpaired actine of a subatrial triactine (arrow). F. Atrial skeleton. Abbreviations: cx=cortex;at=atrium.

Figure 5. Spicules of Leucilla uter (UFRJPOR 7545). A. Diactines. B. Cortical triactine. C-D.Cortical tetractines. E. Choanosomal triactine. F-G Choanosomal tetractines. H. Subatrialtriactine. I. Subatrial tetractine. J. Atrial tetractine.

149

Table 4. Measurements of Leucilla uter of the specimen UFRJPOR 7545, a Brazilian specimenby Borojevic & Peixinho (1976) and of the type specimen by Poléjaeff (1893).


UFRJPOR 7545

Trichoxea 2250 - - - - 2.5 - - 2Diactine 1250 - - - 6 8 - 10 3Corticaltriactine

Paired 200.0 201.7 2.9 205.0 10.0 10.4 0.7 11.2 3Unpaired - 122.5 - - 10.0 11.2 1.8 12.5 2

Cortical tetractine

Paired 225.0 368.5 85.4 530 22.5 32.5 6.1 40.0 20Unpaired 265.0 381.7 66.5 500.0 30.0 36.1 4.3 42.5 20Apical 255.0 535.0 171.3 1000 22.5 31.3 5.7 40.0 26

ChoanosomalTriactine

Paired - 250.0 0.0 - - 22.5 0.0 2Unpaired - 237.5 0.0 - 22.5 23.8 1.8 25.0 2

ChoanosomalTetractine

Paired 335.0 365.7 35.4 400.0 15.0 30.7 8.7 40.0 7Unpaired 227.5 370.4 71.8 425 27.5 33.8 4.9 37.5 6Apical - 87.5 - - - 25 - - 1

Subatrial Triactine

Paired 75.0 186.9 77.7 320.0 7.5 11.6 4.8 22.5 20Unpaired 150.0 370.8 124.5 540.0 7.5 13.9 4.9 20.0 20

SubatrialTetractine

Paired 112.5 222.3 50.4 285.0 7.5 10.1 2.5 15.0 13Unpaired 175.0 275.9 45.7 350.0 7.5 10.8 3.2 20.0 14Apical 50.0 73.1 17.2 87.5 - 7.5 0.0 - 4

AtrialTetractine

Paired 115.0 204.8 50.6 285.0 5.0 7.9 1.7 12.5 15Unpaired 137.5 158.1 21.2 187.5 7.5 10.3 2.5 12.5 4Apical 62.5 95.2 22.3 150.0 5.0 7.4 1.1 10.0 21

Leucilla uter sensu Borojevic &Peixinho, 1976

Corticaltriactine

Paired 170.0 - - 400.0 10.0 - - 16.0 -Unpaired 200.0 - - 400.0 10.0 - - 16.0 -

CorticalTetractine

Basal 300.0 - - 700.0 20.0 - - 60.0 -Apical 180.0 - - 600.0 20.0 - - 60.0 -

ChoanosomalTriactine

300.0 - - 600.0 30.0 - - 50.0 -

ChoanosomalTetractine

Basal 300.0 - - 600.0 30.0 - - 50.0 -Apical - - 200.0 - - - - -

Subatrialtriactine

Paired 170.0 - - 250.0 10.0 - - 16.0 -Unpaired 300.0 - - 400.0 - 16.0 - - -

Atrialtetractine

Paired 200.0 - - 350.0 10.0 - - 12.0 -Unpaired 270.0 - - 400.0 10.0 - - 12.0 -Apical 40.0 - - 80.0 - - - - -

Originaldescription

Diactine - - 400.0 - 2.5 - - -Corticaltetractine

Basal 400.0 - - 600.0 - - - 50.0 -Apical 400.0 - - 1200 - - - 50.0 -

Choanosomaltetractine

Basal 400.0 - - 600.0 - - - 50.0 -Apical 400.0 - - 1200 - - - 50.0 -

Subatrialtriactine

Unpaired - - - 600.0 30.0 - - 50.0 -Paired - - - 420.0 21.0 - - 35.0 -

Subatrialtetractine

Unpaired - - - 600.0 30.0 - - 50.0 -Paired - - - 420.0 21.0 - - 35.0 -

Atrial tetractine*

Paired - - - 400.0 12.5 20.0 - - -Unpaired 250.0 - - 350.0 12.5 20.0 - - -Apical - - - 200.0 12.5 20.0 - - -

150

Family Grantiidae Dendy, 1893

Genus Grantia Fleming, 1828

Grantia grandisapicalis sp. nov. (Figure 6 and 7, Table 5)

Etimology. From the Latin grandis (= large) for the long apical actine of the atrial tetractines.

Type locality. Chapeirão 2, Abrolhos Marine National Park, Bahia State, Brazil.

Type material. Holotype (ethanol): UFRJPOR 7567, Chapeirão 2, Abrolhos Marine National

Park, 15.2 m deep, collected by A. Padua & F. Azevedo, 04/XII/2014.

Colour. White in life and beige in ethanol.

Description. This sponge has a globular body with an apical osculum (Figures 6A-B). The

holotype measures 14 x 6 mm. The surface is very hispid because of diactines protruding

through the cortex (arrow in Figure 6B). The osculum (diameter=2.5 mm) is sustained by a

margin composed of T-shaped triactines and tetractines and it is surrounded by a crown of

trichoxeas (arrowhead in Figure 6B). The atrium is hispid due to the long apical actines of the

atrial tetractines. The aquiferous system is syconoid with elongated chambers arranged side by

side.

Skeleton. The skeleton is typical of the genus (Figure 6C). The cortical skeleton is composed of

perpendicular diactines (white arrow in Figure 6D) and tangential triactines (black arrow in

Figure 6D). Diactines do not penetrate the choanosome. The tubar skeleton is articulated,

composed of several rows of triactines and tetractines (in less proportion) with the unpaired

actine pointing to the cortex (Figure 6E). The subatrial skeleton is composed of triactines with

the unpaired actine pointing to the choanosome. The atrial skeleton is exclusively composed of

tetractines with the apical actine projected into the atrial cavity (Figure 6F).

Spicules

Trichoxea of the crown (Figure 7A). Slender. Size: More than 500 μm long.

Diactines I (Figure 7B). Fusiform and small. Size: 300-580/22.5-30.0 μm.

Diactines II (Figure 7C). Fusiform and large. Very variable size: 1000.0 to > 3125.0/35.0-55.0

μm.

Cortical triactines (Figures 7D-F). Sagittal. Actines are conical, straight and have sharp tips.

The paired actines are frequently longer than the unpaired one and can be slightly curved. Size:

75.0-142.5/7.5-11.3 μm (paired actine) and 47.5-107.5/7.5-12.5 μm (unpaired actine).

Tubar triactines (Figures 7G-I). Sagittal. Actines are conical, straight with sharp tips (Figure

7G). The paired actines have different sizes (Figure 7H) and one of them can be curved (Figure

7I). Size: 32.5-67.5/5-10 μm (paired actine 1), 62.5-120.0/5-7.5 μm (paired actine 2) and 119.4-

140/5-8.8 μm (unpaired actine).

151

Tubar tetractines (Figures 7J-K). Sagittal. Actines are conical, straight with sharp tips (Figure

7J). Some paired actines have different sizes and one of them can be curved. The unpaired

actine can be longer than the paired ones (Figure 7K). Size: 67.5-142.5/5.0-7.5 μm (paired

actines), 80.0-197.5/6.2-8.7 μm (unpaired actine) and 17.5-35.0/5-7.5 μm (apical actine).

Subatrial triactines (Figure 7L). Sagittal. Actines are slightly conical, straight with sharp tips.

The paired actines can be curved. Size: 50-110/5-7.5 μm (paired actines) and 127.5-207.5/7.5

μm (unpaired actine).

Atrial tetractines (Figures 7M-N). Sagittal. Actines are conical with sharp tips (Figure 7M).

The apical actine is long, thick and distally curved (Figure 7N). Size: 80.0-175.0/5-8.7 μm

(paired actines), 80.0-112.5/5-6.2 μm (unpaired actine) and 100-172.5/5-8.7 μm (apical actine).

Ecology. The surface of the analysed specimen was found covered with sediment.

Geographic distribution: This species is provisionally endemic to the NE Brazil (present

study).

Remarks. The genus Grantia comprises 40 valid species (Van Soest et al. 2016). Among them,

only G. aculeata Urban, 1908 from the Mediterranean Sea, G. infrequens Carter, 1886 from

Australia, G. intermedia Thacker, 1908 from Cape Verde and G. kempfi Borojevic & Peixinho,

1976 from Brazil present triactines and tetractines in the tubar skeleton. Grantia kempfi is the

species that more resembles G. grandisapicalis sp. nov., however, they can be distinguished by

the choanocytary chambers which are ramified near the atrium in the former and elongated in

the latter. Besides, the skeleton of G. kempfi comprises comprises subatrial tetractines and atrial

triactines, which are absent in the new species. The other species of Grantia reported from

Brazil is G. atlantica Ridley, 1881 (Muricy et al. 2011), which does not present subatrial

triactines nor tubar tetractines (Borojevic & Peixinho 1976).

152

Figure 6. Grantia grandisapicalis sp. nov. (UFRJPOR 7567). A. Specimen in vivo. B.Specimen after fixation indicating crown (arrowhead) and diactines (arrow). C. Cross section ofthe skeleton. D. Cortical skeleton with diactine (white arrow) and cortical triactine (blackarrow). E. Tubar skeleton with triactine (white arrow) and tetractine (black arrow). F. Atrialskeleton. Abbreviations: cx=cortex; at=atrium.

153

Figure 7. Spicules of Grantia grandisapicalis sp. nov. (UFRJPOR 7567). A. Broken trichoxea.B. Diactine I. C. Broken diactine II. D-F. Cortical triactines. G-I. Tubar triactines. J-K. Tubartetractines. L. Subatrial triactine. M. Atrial tetractine. N. Apical actine of an atrial tetractine.

Table 5. Spicule measurements of Grantia grandisapicalis sp. nov. (UFRJPOR 7567).


Min Mean SD Max Min Mean SD MaxDiactine I - 300.0 440.0 140.0 580.0 22.5 27.5 4.3 30.0 3

Diactine II - 1000.0 >3125.0 35 42.0 6.9 55.0 7

Corticaltriactine

Paired 75.0 112.1 18.0 142.5 7.5 9.0 1.2 11.3 21Unpaired 47.5 74.5 14.2 107.5 7.5 10.3 1.4 12.5 20

Tubartriactine

Paired - 32.5 48.4 9.8 67.5 5.0 6.2 1.3 10 20Paired+ 62.5 87.7 14.1 120.0 5.0 6.4 1.2 7.5 21Unpaired 119.4 90.0 14.3 140.0 5.0 7.3 1.1 8.8 20

154

Tubartetractine

Paired 67.5 94.6 17.8 142.5 5.0 7.0 1.0 7.5 30

Unpaired 80.0 141.6 28.8 197.5 6.2 7.7 0.5 8.7 30

Apical 17.5 26.4 5.0 35.0 5.0 5.4 0.8 7.5 16

Subatrialtriactine

Paired 50.0 80.5 19.3 110.0 5.0 5.9 1.2 7.5 10

Unpaired 127.5 160.8 27.3 207.5 7.5 7.5 0.0 7.5 10Atrialtetractine

Paired 80.0 125.0 29.5 175.0 5.0 6.6 1.1 8.7 20Unpaired 80.0 99.4 13.9 112.5 5.0 5.6 0.7 6.2 4Apical 100 132.2 18.1 172.5 5.0 7.2 0.9 8.7 23

Subclass Calcinea Bidder, 1898

Family Clathrinidae Minchin, 1900


Arthuria vansoesti Cóndor-Luján, Azevedo, Padua, Hajdu, Klautau, in press (Figure 8, Table 6)

Synonyms

Arthuria vansoesti Cóndor-Luján et al., in press

Type locality. Daai Booi, St. Willibrordus, Curaçao, Caribbean Sea.

Type specimens. Holotype (ethanol): UFRJPOR 6720 (holotype); Daai Booi, St. Willibrordus;

12°12'43.12"N, 69°05'8.42W; 5.2 m deep; collected by B. Cóndor-Luján, 19/VIII/2011.

Material examined. UFRJPOR 7557 (ethanol), Chapeirão2, Abrolhos Marine National Park;

15.2 m deep; collected by F. Azevedo & A. Padua, 4/XII/2014.

Colour. Light yellow in life and beige (or yellowish white) in ethanol.

Description. The analysed specimen has a massive cormus (6 x 5 x 3 mm) formed by irregular

and loosely anastomosed tubes and presents water-collecting tubes (Figures 8A-B). The

aquiferous system is asconoid. No granular cells were observed. The skeleton has no special

organization and it is composed of abundant triactines (distinguished in two shape categories)

and rare tetractines.

Spicules (Table 6)

Triactines I (Figure 8C). Regular (equiangular and equiradiate). Very frequent. Actines are

cylindrical, slightly undulated at the distal part and with rounded tips. Size: 70.0-102.5/2.5-5.0

μm.

Triactines II (Figure 8D). Regular (equiangular and equiradiate). Actines are straight, slightly

conical with blunt to sharp tips. Size: 52.5-92.5/2.5-5.0 μm

Tetractines (Figures 8E-F). Regular (equiangular and equiradiate). Rare. Basal actines are

cylindrical, slightly undulated at the distal part and with rounded to blunt tips (Figure 11E). The

155

apical actine is straight, smooth and bear sharp tips (Figure 11F). Size: 80.0-85.0/3.8-5 μm

(basal actine) and 25.0-67.5/2.5-5.0 μm (apical actine).

Geographic distribution. Curaçao (Cóndor-Luján et al. in press) and Abrolhos Marine

National Park (present study)

Remarks: This species was originally described from the Caribbean Sea (Cóndor-Luján et al.

in press) and we are expanding its distribution to the NE Brazil. The other Arthuria occurring in

the NE is A. trindadensis Azevedo et al., submitted. The new species can be easily differentiated

from A. trindadensis by the in vivo colour, which is yellow in the former and white in the latter.

Besides, the skeleton of A. trindadensis comprises triactines whose actines only have sharp tips

whereas A. vansoesti has triactines with rounded (Triactines I) and sharp (Triactines II) tips.

Table 6. Spicule measurements of Arthuria vansoesti from Abrolhos (UFRJPOR 7557) and ofthe holotype . *Taken from Cóndor-Luján et al. in press.


UFRJPOR 7557

Triactine I - 70.0 85.6 7.4 102.5 2.5 3.5 0.7 5.0 25Triactine II - 52.5 76.5 9.9 92.5 2.5 3.4 0.9 5.0 20Tetractine basal 80.0 81.7 2.9 85.0 3.8 4.6 0.7 5.0 3

apical 25.0 46.3 30.0 67.5 2.5 3.8 1.8 5.0 2Holotype* Triactine I 72.5 79.8 4.9 87.5 3.8 4.6 0.6 5.0 30

Triactine II 52.5 70.4 7.8 82.5 2.5 3.9 0.8 5.0 30Tetractine basal 60.0 75.3 8.1 87.5 3.8 4.8 0.5 5.0 15

apical 25.0 25.0 0.0 25.0 3.8 4.4 0.7 5.0 6

156

Figure 8. Arthuria vansoesti (UFRJPOR 7557). A. Specimen in vivo. B. Specimen afterfixation. C. Triactine I. D. Triactine II. E. Tetractine. F. Apical actine of a tetractine.


Borojevia brasiliensis (Solé-Cava, Klautau, Boury-Esnault, Borojevic & Thorpe, 1991) (Figure

9, Table 7)

Synonyms

Clathrina cerebrum Borojevic 1971: 526

Clathrina brasiliensis Solé-Cava et al. 1991: 382; Klautau et al. 1994: 372; Muricy & Silva

1999: 160; Klautau & Borojevic 2001: 403; Klautau & Valentine 2003: 11; Muricy et al. 2011:

33.

Borojevia brasiliensis Klautau et al. 2013: 459

Type locality. Arraial do Cabo, Brazil

Type specimens. Holotype (ethanol): MNHN-LBIM.C. 1989.2, Arraial do Cabo (Enseada), Rio

de Janeiro, Brazil. Collected by G. Muricy, 16/XII/ 1986.

157

Material examined. UFRJPOR 7384 (ethanol), Porto do Pecém, São Gonçalo do Amarante,

Ceará State, collected by E. Hajdu & S. Salani, 2/IV/2014.

Colour. White in life and ethanol.

Description. This species has a massive, rough and slightly compressible cormus (17 x 6 x 3

mm) composed of irregular and tightly anastomosed tubes (Figures 9A-B). The aquiferous

system is asconoid and the oscula are spread through the cormus. No cells with granules were

observed. The skeleton has no special organization and it comprises tripods that are in fact large

triactines, triactines and tetractines. Triactines are more abundant than tetractines.

Spicules (Table 7)

Tripods. (Figures 9C-D). Equiangular, subregular or parasagittal. Actines are straight, slightly

conical with blunt tips. They do not have the centre raised as in typical tripods. Size: 121.5-

153.9/9.4-12.1 µm.

Triactines.. Regular (equiangular and equiradiate). Very abundant. Actines are straight, conical

with blunt tips (Figure 9E). Sagittal triactines with curved paired actines were also observed

(Figure 8F). Size: 62.1-72.6/8.1-10.8 µm.

Tetractines.. Regular (equiangular and equiradiate). Actines are straight, conical with blunt tips

(Figure 9G-H). Sagittal tetractines with curved paired actines were also observed (Figure 8I).

The apical actine is shorter and thinner than the basal ones and has spines. Spines are located

near the tip of the actine (Figure 9J). Some tetractines with no spines were also observed. Size:

62.1-83.7/8.1-10.8 µm (basal actine) and 37.5-65.0/5.0-7.5 µm (apical actine).

Geographic distribution. SE Brazil (Klautau & Valentine 2003; Muricy et al. 2011) and NE

Brazil (this study).

Remarks. Four Borojevia species are known from Brazilian waters: B. aspina (Klautau, Solé-

Cava, & Borojevic, 1994), B. brasiliensis, B. tenuispinata and B. trispinata Azevedo et al.,

submitted. Among these, our specimen more resembles B. brasiliensis as it presents a skeleton

with more triactines than tetractines and similar spines distribution along the apical actine of the

tetractines, however, the tripods are different. Compared to previous descriptions of B.

brasiliensis (Solé-Cava et al. 1991; Klautau & Borojevic 2001; Klautau & Valentine 2003) the

specimen from Ceará have tripods with longer and less conical actines (Table 7). Nonetheless,

as the variability in the shape of tripods in Borojevia has been assigned to polymorphism or

plasticity (Klautau et al. 2016), the observed difference in the analysed specimen does not

constitute a diagnostic character. Therefore, we identified UFRJPOR 7384 as B. brasiliensis.

Furthermore, in the phylogenetic tree, it clustered within the B. brasiliensis clade with a high

support (pp=0.98, b=95, Figure 15), corroborating the morphological identification.

158

Figure 9. Borojevia brasiliensis (UFRJPOR 7384). A. Specimen in vivo. B. Specimen afterfixation. C-D. Tripods. E-F. Triactines. G-I. Tetractines. J. Apical actine of a tetractine.

Table 7. Spicule measurements of Borojevia brasiliensis from Ceará (UFRJPOR 7384) and of the holotype. *Taken from Klautau & Valentine (2003).


UFRJPOR7384

Tripod 121.5 138.0 9.4 153.9 9.4 11.2 0.8 12.1 20Triactine 62.1 68.8 3.8 72.6 8.1 8.7 0.8 10.8 20Tetractine basal 62.1 71.4 4.8 83.7 8.1 9.2 0.9 10.8 20

apical 37.5 50.0 7.0 65.0 5.0 6.7 0.8 7.5 20Holotype* Tripod 67 81 8.2 95.7 - 11 1.7 - 20

Triactine 60.9 78.2 10.6 102.2 - 10.8 1.5 - 20Tetractine basal 56.5 75.3 10.0 91.3 - 10.4 1.3 - 20

apical 17.4 36.4 9.1 50.0 - 8.0 2.2 - 20

159

Genus Clathrina Gray, 1867 sensu Klautau, Azevedo, Cóndor-Luján, Rapp, Collins & Russo,

2013

Clathrina aspera Cóndor-Luján, Azevedo, Padua, Hajdu, Klautau, in press (Figure 10, Table 8).

Synonyms

Clathrina aspera Cóndor-Luján et al., in press.

Type locality. Water Factory, Willemstadt, Curaçao, Caribbean Sea.

Type specimens. Holotype (ethanol): UFRJPOR 6758, Water Factory, Willemstadt, Curaçao,

12°06'30.88"N, 68°57'13.53"W, 13.2 m deep, collected by B. Cóndor-Luján and E. Hajdu,

23/VIII/2011. Paratypes (ethanol): UFRJPOR 5487, Ilhas Botinas, Angra dos Reis, Rio de

Janeiro, Brazil; 23º03'19.36''S, 44º19'44.98''W; 1-3 m deep; collected by F. Azevedo and M.

Klautau, 25/V/2007 and UFRJPOR 5531; Praia do Bonfim, Angra dos Reis, Rio de Janeiro,

Brazil; 23°01'14.26''S, 44°19'48.18''W; 1-2 m deep; collected by M. Klautau, 27/V/2007.

Material examined. UFRJPOR 7390 and UFRJPOR 7391 (ethanol), Farol da Praia de

Maracajaú, Área de Proteção Ambiental dos Recifes de Corais de Maracajaú (APARC-

Maracajaú), Rio Grande do Norte State, collected by B. Cóndor-Luján, 2 m deep, 8/IV/2014.

Colour. White in life and in ethanol.

Description. The largest specimen (UFRJPOR 7390) measures 13 x 11 x 4 mm. The cormus is

massive and composed of irregular and tightly anastomosed tubes (Figures 10A-B). Because of

the presence of large triactines located on the external tubes, the surface is rough. The

aquiferous system is asconoid with oscula widespread on the surface. No water-collecting tubes

were observed. The skeleton has no special organization and it comprises two categories of

triactines.

Spicules.

Triactines I (Figure 10C). Regular (equiangular and equiradiate). Large and tripod-like. Actines

are straight, conical with sharp tip. Size: 225.0-390.0/20.0-35.0 µm.

Triactines II (Figure 10D). Regular (equiangular and equiradiate). Actines are straght,

cylindrical to slightly conical with sharp tips. Size: 45.0-125.0/5.0-8.7 µm.

Ecology. This sponge was found underneath boulders. Adjacent individuals to UFRJPOR 7391

were eaten by fish (field observation).

Geographic distribution. Brazil, including the NE (this study) and SE regions, and Curaçao

(Cóndor-Luján et al. in press).

Remarks. The present record of C. aspera from Rio Grande do Norte contributes to the

understanding of its distribution and genetic connectivity in the Western Tropical Atlantic as it

160

was previously reported from two very distant localities, Curaçao (Caribbean Sea) and Rio de

Janeiro (Cóndor-Luján et al. in press). In both ITS and C-LSU phylogenetic reconstructions

(Figures 14 and 15), the NE specimens clustered with sequences of the type material of

C.aspera (UFRJPOR5531 and UFRJPOR6758) with high support (ITS: pp=0.99, b=99 and C-

LSU: pp=1, b=91) in concordance with the morphological identification.

Figure 10. Clathrina aspera (UFRJPOR 7390). A. Specimen in vivo. B. Specimen afterfixation. C. Triactine I. D. Triactines II.

Table 8. Spicule measurements of Clathrina aspera from Rio Grande do Norte (UFRJPOR 7390 and UFRJPOR 7391) and from the holotype. *Taken from Cóndor-Luján et al. in press


UFRJPOR Triactine I - 245.0 - - - 20 - - 17390 Triactine II 50.0 78.9 24.0 112.5 5.0 7.0 0.8 8.7 40

UFRJPOR Triactine I 225.0 317.5 50.0 390.0 20.0 25.6 3.6 35.0 127391 Triactine II 45.0 78.6 24.5 125.0 5.0 6.7 1.0 7.5 40

Holotype* Triactine I 132.5 227.2 50.8 325.0 17.5 28.5 5.6 37.5 30Triactine II 75.0 115.4 12.9 152.5 7.5 9.7 1.1 12.5 40

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Clathrina luteoculcitella Wörheide & Hooper, 1999 (Figure 11, Table 9)

Synonyms

Clathrina luteoculcitella Wörheide & Hooper 1999: 868; Klautau & Valentine 2003: 30,

Klautau et al. 2013: 12.

Clathrina aff. luteoculcitella: Van Soest & de Voogd 2015:13.

Type locality. Great Barrier Reef, Australia

Type specimen. Holotype (ethanol): QMG 313684, ‘The Patch’, at the N end of the channel

between Heron Island and Wistari Reef, Great Barrier Reef, 23°26.6'S, 151°53.4'E, 25 m deep.

Material examined. UFRJPOR 7551 (ethanol); Chapeirão1, Parque Nacional Marinho dos

Abrolhos; 16.2 m; collected by A. Padua & F. Azevedo, 04/XII/2014; UFRJPOR 7569 (ethanol);

Chapeirão2, Parque Nacional Marinho dos Abrolhos; 15.2 m; collected by A. Padua & F.

Azevedo, 04/XII/2014.

Colour. Beige in life and white in ethanol.

Description. The largest specimen measures 21 x 13 x 5 mm. The cormus is massive, rough and

slightly compressible. It is composed of thin, regular and tightly anastomosed tubes which form

folds (giving an apparent spherical shape, Figure 11A). The aquiferous system is asconoid. The

oscula are spread along the surface (Figure 11B), however, in UFRJPOR 7551, structures

similar to water-collecting tubes (laterally located) were observed. The skeleton has no special

organization and it comprises one single category of triactines and very rare diactines. Diactines

were only found in UFRJPOR 7551 and most of them were located tangentially to the surface

(only one diactine perpendicularly inserted was observed).

Spicules

Triactines (Figure 11C). Regular (equiangular and equiradiate). Actines are slightly conical to

conical, distally undulated and with sharp tips. Size: 59.5-90.0/5.4-10 µm.

Diactines (Figure 11D). Very slender, trichoxea-like. Most of them were broken but it was

possible to distinguish two tips: one sharp tip and another lanceolated (wider). Size: >162.5/2.5

µm.

Ecology. The specimen UFRJPOR 7551 was found in a crevice, on a red calcareous algae. Balls

of sediment were found inside the tubes of UFRJPOR 7569.

Geographic distribution: Australia (Wörheide & Hooper 1999), Indonesia (Van Soest & de

Voogd 2016) and NE Brazil (present study).

Remarks. The analysed specimens resemble the Brazilian endemic species Clathrina

angraensis Azevedo et al., 2007 in external morphology and spicule size (48-100/6.8±10 µm),

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however, the cormus of the latter does not form folds and its triactines are less conical than

those observed in the specimens from Abrolhos.

The in vivo colour of the studied specimens is beige, nonetheless, in the ITS phylogenetic

analysis, they grouped (pp=1, b=99, Figure 15) with the yellow C. luteoculcitella from

Australia, indicating its conspecificity. Different from the Australian specimens whose skeleton

comprise abundant diactines perpendicularly disposed to the surface of the cormus (Wörheide &

Hooper 1999; Klautau & Valentine 2003), in the Brazilian specimens, (thinner) diactines were

rarely found and, when observed, they were tangential to the surface. This indicates that

diactines may constitute plastic morphological characters in Clathrina, as recently suggested by

Azevedo et al. submitted. Clathrina luteoculcitella constitutes the first confirmed record of an

Indo-Pacific species present in the Western Atlantic Ocean. This disjunct distribution is puzzling

and should receive attention in further studies as it is possible that this species was introduced in

the Brazilian coast by anthropogenic means (M. Klautau, pers. Obs.).

Figure 11. Clathrina luteoculcitella (UFRJPOR 7551). A. Specimen in vivo. B. Specimen afterfixation. C. Triactines. D. Broken diactine.

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Table 9. Spicule measurements of Clathrina luteoculcitella from Abrolhos (UFRJPOR 7551and UFRJPOR 7569) and from the original description (Wörheide & Hooper 1999).


UFRJPOR 7551 Diactine >87.5 - - >162.5 2.5 2.5 0 2.5 6Triactine 72.5 81.4 5.0 90.0 7.5 7.8 0.7 10 20

UFRJPOR 7569Original description

Triactine 59.5 75.3 8.6 86.5 5.4 6.3 1.2 8.1 20Diactine 90.0 164.4 - 220.0 2.0 3.1 - 6.0Triactine 68.0 77.7 - 84.0 8.0 9.4 - 12.0

Family Leucascidae Dendy, 1892

Genus Leucascus Dendy, 1892

Leucascus luteoatlanticus sp. nov. (Figures 12 and 13, Table 10)

Etimology. From the Latin lutea (=yellow) for the yellow colour of the cormus and the type

locality in the Atlantic Ocean.

Type locality. Parcel das Paredes, Abrolhos Marine National Park, Bahia State, Brazil

Type material. Holotype: UFRJPOR 7582; Parcel das Paredes, Abrolhos Marine National Park,

Bahia State, Brazil, 14. 6 m deep; collected by A. Padua & F. Azevedo, 5/XII/2014.

Material used for comparison: Holotype of L. flavus (ethanol): ZMAPOR 13145, Sulawesi,

Bone Baku, Indonesia, station BB/NV/120597, collected by N. J. de Voogd, 16/V/1997.

Colour. Yellow in life and beige in ethanol.

Description: The analysed specimen measured 18 x 10 x 3 mm. This species has a rough, firm

and massive cormus composed of regular and tightly anastomosed tubes (Figure 12A). It is

possible to recognize thin and delicate cortical and atrial membranes delimiting the cormus and

the atrium, respectively. The cortical membrane is perforated by inhalant apertures. The

aquiferous system is solenoid. The oscula are located on the top of elevations and do not have

ornamentation (Figure 12B).

Skeleton. The skeleton comprises triactines and tetractines (Figure 12C). In the cortex, there

are more triactines than tetractines (Figure 12D), whereas in the tubes and in the atrium,

triactines and tetractines seem to be present in equal proportions (Figure 12F). Most

choanocytary tubes are hispid because of the apical actine projected inside of them (Figure

12E).

Spicules

Triactines (Figure 13A). Regular (equiangular and equiradiate). Actines are slightly conical to

conical, straight with sharp tips. Size: 80.0-125.0/7.5-11.3 µm.

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Tetractines (Figures 13B-C). Regular (equiangular and equiradiate). Actines are slightly

conical, straight with sharp tips (Figure 13B). The apical actine is slightly conical, thinner than

the basal ones and bear spines. The spines are distributed along the apical actine (Figure 13C).

Size: 75-105.0/7.5-10 µm (basal actines) and 40.0-85.0/5-7,5 µm (apical actine).

Ecology. A crustacean and a polychaeta were found inside the atrium.

Geographic distribution. Provisionally endemic to the NE Brazil (present study).

Remarks. The specimen analysed resembles Leucascus flavus Cavalcanti et al., 2013 from

Indonesia. Both species are yellow in vivo, present a cortical skeleton mainly composed of

triactines and the skeleton of their choanocytary tubes have triactines and tetractines in equal

proportions. However, they can be differentiated by their atrial skeletons which present more

tetractines than triactines in L. flavus, whereas in the Brazilian specimen, triactines and

tetractines are present in similar proportions. As this character (proportion of spicules in the

atrial skeleton) has been validated as a diagnostic character in the revision of Leucascus

(Cavalcanti et al. 2013), we propose to name the analysed specimen herein as a new species, L.

luteoatlanticus sp. nov. Additionally, as Van Soest & de Voogd (2015) provided in situ pictures

of L. flavus, it was possible to recognize a wider atrium in L. flavus when compared to L.

luteoatlanticus sp. nov.

Before this study, only two species of Leucascus were reported for the Brazilian coast, L.

roseus Lanna et al., 2007 and L. albus Cavalcanti et al., 2013. Different from L. luteoatlanticus

sp. nov., which is yellow in vivo and have spicules with conical actines, L. roseus is pink and

possesses spicules with cylindrical actines whereas L. albus is white and its skeleton bears

microdiactines.

Table 10. Spicule measurements of Leucascus luteoatlanticus sp. nov. (UFRJPOR 7582) and ofthe holotype of L. flavus. *Taken from Cavalcanti et al. (2013).


UFRJPOR 7582

Triactine 80.0 100.3 11.9 125.0 7.5 9l4 1.1 11.3 20Tetractine basal 75.0 92.4 9,2 105.0 7.5 8l4 0.9 10 20

apical 40.0 61.9 13.6 85.0 5.0 5l5 1.0 7.5 16Leucascus flavus*

Triactine 70.0 100.9 9.5 120.0 7.5 9l7 1.1 12.5 30Tetractine basal 75.0 98,7 7.4 115.0 7.5 8l9 1.3 10.0 30

apical 41.3 49.6 5.3 60.7 3.6 4l2 0.6 4.9 30

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Figure 12. Leucascus luteoatlanticus sp. nov. (UFRJPOR 7582). A. Specimen in vivo. B.Specimen after fixation. C. Cross section of the skeleton. D. Cortical membrane. E. Atrialmembrane. F. Choanocytary tube with projected apical actines (arrow). Abbreviations:cx=cortex; ct=choanocytary tube.

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Figure 13. Spicules of Leucascus luteoatlanticus sp. nov. (UFRJPOR 7582). A. Triactines. B.Tetractine. C. Apical actine of a tetractine covered with spines.

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Table 11. New locality records of calcareous sponges from the NE coast. Species - Type Locality

Material examined

New Record Locality, depth, collectors, collection date

Clathrina aurea -Arraial do Cabo, Rio de Janeiro, Brazil.

UFRJPOR 7544 L6, 7.1 m, coll. A Padua & F. Azevedo, 03/XII/2014.UFRJPOR 7548 L9, 16.2 m, coll. A. Padua & F. Azevedo, 04/XII/2014.UFRJPOR 7552UFRJPOR 7553UFRJPOR 7554

L9, 16.2 m, coll. A. Padua & F. Azevedo, 04/XII/2014.

UFRJPOR 7572 L5, 11.7 m, coll. A. Padua & F. Azevedo, XII/2014.UFRJPOR 7571UFRJPOR 7574UFRJPOR 7576

L5, 14.3 m, coll. A. Padua & F. Azevedo, XII/2014.

UFRJPOR 7580UFRJPOR 7584UFRJPOR 7585

L5, 14.6 m, coll. A. Padua & F. Azevedo, XII/2014.

MNRJ 18965 L10, 13.1 m, coll. A. Bispo & J. Carraro, 04/XII/2014.Clathrina lutea - Pedra Lixa, Abrolhos Archipelago, Bahia, Brazil.

UFRJPOR 7549UFRJPOR 7550UFRJPOR 7555

L9, 16.2 m, coll. A Padua & F. Azevedo, 4/XII/2016.

UFRJPOR 7561 UFRJPOR 7563 UFRJPOR 7564 UFRJPOR 7565UFRJPOR 7566 UFRJPOR 7568


UFRJPOR 7577UFRJPOR 7578

L5, 14.3 m, coll. T. Pérez & O. Thomas, 05/XII/2014.

UFRJPOR 7579 UFRJPOR 7583UFRJPOR 7589UFRJPOR 7590

L5, 14.6 m, coll. A. Padua & F. Azevedo, 05/ XII /2014.

UFRJPOR 7591 L10, 9-15 m, coll. A. Bispo & J. Carraro, 04/ XII /2014.UFRJPOR 7592UFRJPOR 7593 UFRJPOR 7594UFRJPOR 7595 UFRJPOR 7596UFRJPOR 7597UFRJPOR 7598UFRJPOR 7599UFRJPOR 7600UFRJPOR 7601UFRJPOR 7602

Abrolhos Marine National Park, T. Pérez & O. Thomas, 04/XII/2014.

MNRJ 18844 L10, 11.6 m, coll. E. Hajdu, 04/XII/2014.MNRJ 18900 L5, 5 – 16 m, coll. A. Bispo & J. Carraro, , 04/XII/2014.

Clathrina mutabilis - Fernando de Noronha Archipelago, Brazil

UFRJOR 7386 L4, 20 m, coll. B. Cóndor-Luján, 07/IV/2014.UFRJPOR 7388 L3, 2 m, coll. B. Cóndor-Luján, 08/IV/2014.

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Ernstia citrea - Rocas Atoll, Rio Grande do Norte, Brazil

UFRJPOR 7547 L7, 7.1 m, coll. A. Padua & F. Azevedo, 03/XII/2014.UFRJPOR 7556UFRJPOR 7560

L10, 15.2, coll. A. Padua & F. Azevedo, 04/XII/2014.

UFRJPOR 7573 L5, 14.3 m, coll. A. Padua & F. Azevedo, 05/XII/2014.

Ernstia rocansensis - Rocas Atoll, Rio Grande do Norte, Brazil

UFRJPOR 8547 L2, 4 m, coll. A. Bispo & S. Salani, 23/IV/2016.

Leucascus roseus - Alcatrazes Archipelago, São Sebastião, São Paulo, Brazil.

UFRJPOR 7558UFRJPOR 7559


UFRJPOR 7575 L5, 14.3 m, coll. A. Padua & F. Azevedo, 04/XII/2014.UFRJPOR 7581 L5, 14.6 m, coll. A. Padua & F. Azevedo, 04/XII/2014.UFRJPOR 7586UFRJPOR 7587UFRJPOR 7588


MNRJ18884 MNRJ 18885

L10, 10 – 15 m, coll. A. Bispo & J. Carraro, 04/XII/2014.

Molecular analyses

In this study, 19 DNA sequences were generated. We provided sequences for the new species

described herein: C-LSU for Amphoriscus hirsutus sp. nov. and Grantia grandisapicalis and

ITS for Leucascus luteoatlanticus sp. nov. Furthermore, new ITS sequences were generated for

Clathrina aspera, C. aurea, C. lutea, C. luteoculcitella, C. mutabilis and Borojevia brasiliensis.

An additional C-LSU sequence of C. aspera was presented as well.

The C-LSU sequences produced an alignment of 457 bp including gaps. Both phylogenetetic

methods (BI and ML) recovered the same tree topology (BI tree is shown in Figure 14). The

phylogenetic tree yielded similar results to those previously obtained (Voigt & Wörheide 2016;

Cóndor-Luján et al. in press). With the addition of the new sequences, Amphoriscus and

Grantia appeared as non monophyletic genera. Grantita grandispicalis sp. nov. nested within

the clade of Heteropiidae and Grantiidae, instead of clustering with Grantia compressa, which is

the type species of the genus. Interestingly, the skeleton of G. grandisapicalis sp. nov. comprises

tubar tetractines whereas that of G. compressa does not. Furthermore, A. hirsutus sp. nov. whose

choanosomal skeleton is mainly composed of triactines clustered with Sycon conulosum +

Leucilla antillana (pp=0.99, b=90) whose tubar and subatrial skeletons (respectively) are

exclusively composed of triactines (Cóndor-Luján et al. in press).

The intraspecific variation (estimated by the uncorrected p distance) of the ITS Calcinean

sequences ranged between 0 and 1.4%. Borojevia brasiliensis, C. lutea and C. aspera showed

169

no intraspecific variation (0%), The values ranged from 0 to 0.5% in C. mutabilis and from 0-

0.7% in C. luteoculcitella. The highest variation was observed in C. aurea (0-1.4%).

Figure 14. Bayesian phylogenetic tree inferred from the C-LSU sequences of the studiedspecies. Bayesian posterior probabilities and bootstrap values (pp/b) are given on the branches.* Sequences generated in this study.

170

The alignment of the ITS sequences had a total length of 1349 bp including gaps. Both the

BI and ML methods yielded similar tree topologies (ML tree is shown in Figure 15). The

phylogenetic tree (1) recovered the monophyletic genera Clathrina (pp=0.96, b=71) and

Borojevia (pp=1 b=100) obtained in previous studies using a similar set of sequences (Klautau

et al. 2013, 2016; Azevedo et al. submitted; Cóndor-Luján et al. in press) and (2) revealed a

close affinity between Leucascus (L. simplex + L. luteoatlanticus sp. nov.) and Ascaltis (A.

reticulum) supported by high values (pp=0.1, b=99).

DISCUSSION

With the results of the present study, the list of calcareous sponges from the NE Brazil increased

from 42 (Azevedo et al. submitted; Van Soest et al. 2016) to 49 species. Among these, 42.9%

(18) are endemic from this region, 23.8% (10) also occur in the Caribbean Sea and 19% (8)

have an amphi-Atlantic distribution (being present in South Africa or in the Mediterranean Sea).

Whether this apparent high endemism in the NE Brazil reflects a true distribution pattern for

Calcarea remains uncertain as formerly NE Brazilian endemic species have been recently

recorded from other areas, e.g. Grantia kempfi in the Guyana Shelf (Van Soest 2017) and

Borojevia tenuispinata, Clathrina mutabilis, C. insularis and Nicola tetela from Curaçao

(Cóndor-Luján & Klautau 2016; Cóndor-Luján et al. in press). Further samplings from adjacent

localities are necessary to validate this observed endemic pattern.

The new records provided herein bring new insights to understand the distribution patterns of

certain Brazilian calcareous sponges and highlight interesting affinities among the northeastern

localities. The new record of Ernstia rocasensis from Fernando de Noronha suggests an affinity

among three isolated areas: Rocas Atoll, Abrolhos and Fernando de Noronha Archipelagos.

Apart from this species, Leucetta floridana is the only species previously reported from these

localities (although it has a broader distribution in the Western Tropical Atlantic, Valderrama et

al. 2009; Muricy et al. 2011). The occurrence of C. mutabilis in Maracajaú reinforces the

calcarean affinity between the Brazilian continental shelf and the oceanic islands, supported by

the previous records of C. aurea, Leucaltis clathria, Leucascus roseus, and Leucetta floridana

(Muricy et al. 2011). The finding of C. aspera in the NE Brazil (Maracajaú) constitutes an

important intermediate record as previously, the known distribution of this species was disjunct:

Curaçao and Rio de Janeiro (Cóndor-Luján et al. in press) and therefore, we point out the

importance of continuing to preserve this protected area.

The new molecular affinities found in this study suggested an apparent phylogenetic signal

in the skeleton composition (choanosomal triactines) of some calcaronean species, as already

171

pointed out (Cóndor-Luján et al. in press). However, more species with different skeleton

characteristics should be included in the trees to verify this hypothesis. Besides, it is necessary

that more detailed morphological descriptions are made to allow mapping the characters on

phylogenetic trees.

ACKNOWLEDGEMENTS

We are indebted to Marcelo Soares and Ana Luisa Pires Moreira for providing technical support

with collecting licenses, Olivier Thomas, Thierry Pérez, Mariana Carvalho, André Bispo, Sula

Salani, Cristiana Castello-Branco, Camille Leal, João Carraro and Humberto Fortunato for field

assistance and sponge collection, Carol Leite for some slides preparations and the staff of the

Laboratório de Protistologia of Prof. Inácio da Silva Neto for providing assistance with

microscopy images. The dive centres Atlantis Divers, Mar de Noronha Divers and Natal Divers

are acknowledged for proving technical diving assistance. We thank the Brazilian government

agencies that authorized the sampling licenses in MPAs; Instituto Chico Mendes de

Conservação da Biodiversidade (ICMBio), Instituto de Desenvolvimento Sustentável e Meio

Ambiente do Rio Grande do Norte (IDEMA), Reserva Biológica Marinha do Atol das Rocas,

Parque Nacional Marinho de Fernando de Noronha, and the Brazilian Navy (Terminal Portuário

do Pecém). This work was funded by the Brazilian National Research Council (CNPq),

Coordination for the Improvement of Higher Education Personnel (CAPES), Foundation Grupo

Boticário de Proteção à Natureza and Rio de Janeiro State Research Foundation (FAPERJ).

B.C.L. received a scholarship from CAPES, F.A. has a post-doc fellowship by CAPES and A.P.

has a post-doc fellowship by FAPERJ. E.H. and M.K. have fellowships by CNPq.

172

Figure 15. Maximum Likelihood phylogenetic tree inferred from the ITS sequences of theCalcinean species. Bayesian posterior probabilities and bootstrap values (pp/b) are given on thebranches. * Sequences generated in this study. FN: Fernando de Noronha. RN: Rio Grande doNorte.

173

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Evolutionary history of the calcareous sponge Leucetta floridana in the Western Tropical

Atlantic

Cóndor-Luján, B. 1; García-Hernández, J.2; Schizas, N.2; Pérez, T.3; Zea, S4. Klautau, M. 1

1 Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av.

Carlos Chagas Filho 373, CCS, Bloco A, A0-100, 21941-902, Rio de Janeiro, RJ, Brasil.2 Caribbean Laboratory of Marine Genomics, University of Puerto Rico-Mayagüez, P.O. Box

9000, Mayagüez, PR, 00681, United States of America. 3Institut Méditerranéen de Biodiversité et d’Ecologie marine et continentale, CNRS, Aix

Marseille Univ, IRD, Avignon Univ. Station Marine d’Endoume, rue de la Batterie des Lions,

13007 Marseille, França.4Centro de Estudios en Ciencias del Mar – CECIMAR, Universidad Nacional de Colombia,

Sede Caribe; c/o INVEMAR, Calle 25 2-55, Rodadero Sur - Playa Salguero, Santa Marta,

Colombia. *Corresponding author: [email protected]

Running title: Evolutionary history of Leucetta floridana

ABSTRACT

Sponges have short-lived lecithotrophic larvae, which constrain their dispersion and

consequently restrict their geographic distribution. However, some calcareous species as

Leucetta floridana are widespread in the Western Tropical Atlantic (WTA). In order to elucidate

the historical processes that originated its current distribution and to evaluate the role of the

Amazon River as a barrier to the gene flow between Caribbean and Brazilian sponge

populations, several genetic analyses using ITS sequences were performed. A total of 162

specimens from different localities (Puerto Rico, Lesser Antilles, Panama, Colombia, Curaçao

and Brazil) was studied. Genetic variation was assessed through sequence-type and nucleotidic

diversity indexes. Phylogenetic trees and a MJ network were constructed to explore the

genealogical relationships among individuals. To determine population structure, FST

comparisons and AMOVA were performed. Neutrality tests, mismatch distributions and

Bayesian skyline plots were conducted to infer demography patterns. The phylogenetic trees as

well as the networks showed high structured populations consistent with FST and AMOVA

results: four populations restricted to the Caribbean Sea and one population widespread in the

WTA. The presence of a large widespread population in the WTA rejects the role of the Amazon

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River as an effective barrier to gene flow between Caribbean and Brazilian localities.

Demography analyses indicated an event of population expansion in the Caribbean Sea during

the Last Maximum Glaciation (20 millions year BP). The patterns of population connectivity

and demography within the WTA are discussed and a hypothetical evolutionary scenario for L.

floridana is presented.

Key words: Amazon River, Brazilian Coast, Caribbean Sea, Greater and Lesser Antilles,

Population connectivity, Porifera

INTRODUCTION

In the marine environment, organisms with short-lived larvae and consequent low dispersal

capabilities, such as sponges, are not expected to attain widespread geographic distributions.

Some morphological and molecular studies on sponges have rejected the putative

cosmopolitanism of some species (Solé-Cava et al., 1991; Boury-Esnault et al., 1992; Hajdu &

van Soest, 1992; Klautau et al., 1994; Muricy et al., 1996; Klautau et al., 1999; Klautau &

Valentine, 2003; Valderrama et al., 2009), while others revealed that species with widespread

distribution do occur in the Western Tropical Atlantic Ocean (WTA) (Lazoski et al., 2001;

Valderrama et al., 2009).

The occurrence of species with this wide distribution represents a good opportunity to assess

the species dispersal across biogeographic barriers such as the Amazon River. Up to date, no

study on the effectiveness of the Amazon River as a barrier to gene flow was performed within

Porifera. Some recent studies evaluated the genetic connectivity of sponges only within the

Tropical Northwestern Atlantic (TNA), e.g. Callyspongia vaginalis (De Biasse et al., 2010,

2016), Cliona delitrix (Chaves-Fonnegra et al., 2015) and Xestospongia muta (López-Legentil

& Pawlik, 2009; Richards et al., 2016; de Bakker et al., 2016), or exclusively in the Tropical

Southwestern Atlantic (TSA) (Padua et al., in prep.: Clathrina aurea) but none considered

populations along the entire Western Tropical Atlantic.

Different biochemical and molecular approaches have been used to unravel the genetic

structure of sponge populations (isozymes, nuclear and mitochondrial DNA regions). In

calcareous sponges, as the traditional mitochondrial DNA marker (COI) was not appropriate for

phylogeography inferences or population structure studies (Worheide et al., 2000), nuclear

intron regions (ITS and ATPSb-Iii) were employed (Wörheide et al., 2002; Bentlage and

Wörheide, 2007, Wörheide et al., 2008). Among them, the ITS proved to be the most suitable

one (Wörheide et al., 2008). Recently, the mitochondrial gene cox3 was proposed as an

alternative phylogeography marker since it evidenced a variability similar to that of nuclear

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intron data (Voigt et al., 2012). However, due to the substantial variation at both intra- and inter-

specific levels found in mitochondrial markers (Lavrov et al., 2013), the use of cox3 remains

uncertain. Therefore, for the class Calcarea, the ITS region is still a useful marker at population

level.

The calcareous sponge Leucetta floridana (Calcarea: Calcinea: Leucettidae) has a patchy

distribution within reefs and inhabits environments protected from light such as small crevices,

overhangs, and vertical slopes (Valderrama et al., 2009). It has been found from -2 to -90 m of

depth (Muricy et al., 2011). As a calcinean species, it is assumed to have a lecithotrophic larvae

(calciblastula) (Borojevic et al., 1990; Maldonado & Berquist, 2002), however, the reproduction

of this species has not been investigated yet.

Leucetta floridana is one of the most widespread calcareous sponges in the TWA and thus,

constitute a good model for evaluating population connectivity within the Western Tropical

Atlantic. In this study, we assessed the genetic structure of this species at phylogeographic,

population and demographic levels in order to (1) understand the historical processes that

culminated in its current geographic distribution and to (2) evaluate the role of the Amazon

River as a barrier to the gene flow between Caribbean and Brazilian populations of sponges.

MATERIALS AND METHODS

Analysed material

The analysed material comprised 162 specimens preliminary identified as Leucetta floridana

from 18 localities within the Tropical Northwestern Atlantic (TNA, n=147) and the Tropical

Southwestern Atlantic (TSA, n=15), at depths varying from -2 to -70 m.

Localities in the TNA included four ecoregions, Greater Antilles - Puerto Rico (n=12),

Eastern Caribbean – Anguilla (n=1), Saint Martin (n=52), Antigua (n=12), Les Saintes (n=8),

Guadeloupe (n=1), Martinique (n=23), and Saba (n=3), Southwestern Caribbean - San Andrés

(n=4), Panama (n=19), Urabá (n=3) and Santa Marta (n=7) and Southern Caribbean - Curaçao

(n=2), whereas in the TSA comprised three ecoregions, Northeastern Brazil – Ceará (n=5) and

Rio Grande do Norte (RN, n=4), Fernando de Noronha and Atoll das Rocas – Fernando de

Noronha Archipelago (n=4) and Rocas Atoll (n=1) and Eastern Brazil – Abrolhos Archipelago

(n=1) (Table 1, Figure 1).

Most of the Caribbean specimens (131 specimens) were collected during several campaigns:

Expedition PaCoTilles (Patterns of Connectivity among the Lesser Antilles, 2015, n=87), Santa

Marta Expedition (2014, n=7), Martinique Expedition (2013, n=10), Puerto Rico Expedition

(2015, n=12) and PorToL Project (2012, n=15). After collection, specimens were fixed and

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stored in ethanol 93 or 96% until DNA extraction. All voucher specimens are deposited in the

Porifera Collection of the Universidade Federal of Rio de Janeiro (UFRJPOR) in Brazil,

Universidad de Puerto Rico or INVEMAR (Instituto de Investigaciones Marinas y Costeras) in

Colombia.

The other 31 specimens were already deposited in the Porifera Collection of the UFRJPOR

and were analysed in previous studies (Valderrama et al., 2009; Klautau et al., 2013; Cóndor-

Luján et al., in press). Detailed information of any specimen can be accessed in Table S1 of the

Supplementary Material.

Additional sequences of Leucetta species were retrieved from Genbank (Table 2) for

comparative phylogenetic analyses described in the corresponding section.

Figure 1. Map indicating the localities considered in this study. Coloured circles representdifferent ecoregions according to the MEOW (Spalding et al., 2007). Blue: Eastern Caribbean,green: Southwestern Caribbean, yellow: Southern Caribbean, red: Northeast Brazil, orange:Fernando de Noronha and Atoll das Rocas, and pink= Eastern Brazil. The Amazon River isindicated.

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Table 1. Locality, geographic coordinates, depth, and Genbank accession numbers of thespecimens of Leucetta floridana analysed in this study. CE=Ceará, PE=Pernambuco, RN=RioGrande do Norte, FN=Fernando de Noronha Archipelago. *Sequences generated in the presentstudy.

Locality Geographic Coordinates Depth (m) Genbank Number

Greater Antilles Ecoregion – Puerto RicoCayoTurrumote 17°56'20.13''N, 67° 2'43.95''W 12.9 - 17.4 N=2* Cayo Mario 17°57'16.89''N, 67° 3'53.42''W 09.0 - 17.7 N=4* Cayo Conserva 17°55'56.91''N, 67° 5'35.01''W 12.6 - 15.6 N=2* Veril – Fallen Rock 17°54'5.04''N, 66°55'27.59''W 31.5 N=1*Pinnacles 17°56'3.96''N, 67° 1'49.80''W 9.9 - 17.1 N=2*Veril - El Hoyo 17°52.529'N, 67° 2.671'W 31.5 N=1*Eastern Caribbean Ecoregion – Lesser AntillesLittle Scrub 2, Anguilla 18°17.903'N, 62°57.294'W 23.5 N=1* Trou David, Terres Basses, SaintMartin

18°04.402'N, 63°07.149'W 6.1 N=4*

Rocher Créole, Saint Martin 18°07.038'N, 63°03.419'W 8.0 – 10.0 N=1* Les Arches1, Saint Martin 18°07.588'N, 62°58.248'W 10 - 20 N=10* Basse Espagnoles, Saint Martin 18°07.821'N, 63°00.270'W 6 - 10 N=16* Chico2, Saint Martin 18°06.501'N, 62°59.005'W < 22 N=11*Les Arches2, Saint Martin 18°07'32.81''N, 62°58'21.49''W < 16 N=7*Southeastern Coast, Saba 17°37.066'N, 63°13.580'W < 27 N=3*Nanton Point, Saint Paul, Antigua and Barbuda

16°59'51.60''N, 61°45'37.22''W < 20 N=9*

Five Islands, Antigua and Barbuda

17°04.990'N, 61°54.840'W 4 N=1*

Diamond Bank, Saint Paul, Antigua and Barbuda

17°12.000'N, 61°52.800'W 7 – 8 N=2*

Cave Cathédrale, Guadeloupe 16°27.740' N, 61°31.837'W 13.7 N=1*Cave 1, Les Saintes, Guadeloupe

15°52.984'N, 61°34.25'W 8 - 10.7 N=9*

Grottes des couleurs, Pointe Burgos, Grande Anse, Anses d'Arlet, Martinique

14°29.787'N, 61°05.351'W <10 KX355573KX355574N=7*

Anses d´Arlet, Martinique 14°30.377'N, 61°05.850'W? <10 KX355575N=1*

Anse de Fortune, Anses d'Arlet, Martinique

14°30.377'N, 61°05.850'W 3 – 6 N=9*

Southwestern Caribbean EcoregionBocas del Toro, Panama 09º20.914'N, 082º09.394'W? < 17 EU78189

EU78190EU78191N=8*

Las Cuevas, Bocas del Toro, Panamá

09º20.914'N, 082º09.394'W <20? N=7*

Cayo Zapatilla, Bocas del Toro, Panamá

09º14.881'N, 82º01.952'W <20? N=2*

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La Piscinita, San Andrés, Colombia

12°33'N, 81°43'W? 2- 5 EU781972 EU781973 EU781974

West View, Leward-reef, San Andrés, Colombia

12°33'N, 81°43'W? 5 EU781971

Bajo Agua Viva, Urabá, Colombia

07°53''N, 76°38'W? 15 EU781968 EU781969EU781970

El Morro, Santa Marta Bay, Colombia

11°14'57.50"N, 74°13'54.50"W 11 - 21 N=4*

Punta Venado, Ensenada de Taganga, Santa Marta, Colombia

11°16'25.00"N, 74°12'24.00"W 18 – 18.6 N=2*

Punta Gaira, Gaira Bay, Santa Marta, Colombia

11°13'08.00"N, 74°14'30.00"W 14 N=1*

Southern Caribbean Ecoregion - CuraçaoWater Factory, Willemstadt 12°06'30.88"N, 68°57'13.53"W 17.8 N=1Hook’s Hut, Willemstadt 12°07'18.94"N, 68°58'11.46"W 13.3 N=1Northeastern Brazil Ecoregion- BrazilPotiguar Basin, RN 04º37'31.7"S, 36º46'00.7"W 70 EU781985

N=2*Risca das Bicudas, RN 04º57'00.9"S, 36º07'49.7"W 10 EU781978Urca do Tubarão, RN 04º50'52.7"S, 36º27'02.1"W N=1*Station 30 - CENPES, CE 2º52'S, 39 º10"W 33 EU781980

EU781982EU781983EU781984N=1*

Fernando de Noronha and Atoll das Rocas Ecoregion - BrazilBarretinha, Rocas Atoll, RN 3°51'36'S, 33°49'04''W 12 EU781975Sela Gineta Island, FN, PE 3°48'49''S, 32°23'29''W 7 EU781976Ressurreta, FN, PE 3°48'49''S, 32°23'29''W 4 – 7.3 EU781977

N=2*Eastern Brazil EcoregionAbrolhos Marine National Park, Bahia - BrazilParcel das Paredes 17°58'S, 38°40'W 8 EU781979

Table 2. Species names, voucher numbers, locality and Genbank accession numbers of thesequences used in the phylogenetic analysis.

Species Voucher Number Locality Genbank NumberLeucetta antarctica MNRJ 13798 Antarctic KC849700Leucetta chagosensis BMOO1612 French Polynesia KC843455Leucetta microraphis QMG313659 Australia AJ633874Leucetta pyriformis MNRJ13843 Antarctic KC843457Leucetta potiguar UFPEPOR 547 Brazil EU781986Leucetta potiguar MNRJ 8474 Brazil EU781981Leucetta potiguar UFPEPOR 569 Brazil EU781987Leucetta potiguar UFPEPOR 588 Brazil EU781988

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DNA extraction, amplification, sequencing and alignment

The DNA of 136 specimens was extracted following the guanidine/phenol-chloroform protocol

(Sambrook et al., 1993) and stored at –20°C until amplification. The region comprising the

partial 18S and 28S, the spacers ITS1 and ITS2 and the 5.8S ribosomal DNA was amplified by

PCR with the primers: 18S (5`-TCATTTAGAGGAAGTAAAAGTCG-3`) and 8S (5`-

GTTAGTTTCTTTTCCTCCGCTT-3`) (Lôbo-Hajdu et al., 2004). Each PCR amplification

reaction mixture contained: 1X buffer (5X GoTaq R Green Reaction Buffer Flexi, PROMEGA),

0.2 mM dNTP, 2.5 mM MgCl2, 0.5 µg/µL bovine serum albumin (BSA), 0.33 µM of each

primer, one unit of Taq DNA polymerase (Fermentas) and 1 µL of DNA, summing up to 15 µL

with Milli-Q water. PCR steps included one first cycle of 4 min at 94°C, 1 min at 50°C and 1

min at 72°C, 35 cycles of 1 min at 92°C, 1 min at 50°C and 1 min at 72°C, and a final cycle of 6

min at 72°C. Successful amplification was visualized in an 1% agarose electrophoresis gel and

purified with the GE Kit. Forward and reverse strands were automatically sequenced in an ABI

3500 (Applied Biosystems).

The electropherograms of the sequences generated in this study as well as the 25 sequences

of L. floridana retrieved from the Genbank database were visually edited using Chromas Lite.

Electropherograms with dubious base attribution (double-peaks) were not considered in any

further analyses and are not presented here. All sequences generated in this work were deposited

in the GenBank database (www.ncbi.nlm.nih.gov).

Phylogenetic analyses

In order to verify the identity of the specimens attributed to L. floridana, phylogenetic

reconstructions, including sequences from other Leucetta species (Table 2), were performed

under maximum-likelihood (ML) and Bayesian Inference (BI).

Sequences were aligned through the MAFFT v.7 online version (Katoh & Standley, 2013)

using the method Q which considers the secondary structure of the amplified region (Katoh et

al., 2005) and consequently, guarantees a better alignment. The nucleotide substitution model

that best fit the alignment was determined using jModelTest2 (Darriba et al., 2012; Guindo &

Gascuel, 2003) and corresponded to GTR + G.

The ML analyses were conducted on MEGA 6 (Tamura et al., 2013) using an initial NJ tree

(BIONJ) and 1000 bootstrap pseudo-replicates. The BI reconstructions were obtained with

MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) under 106

generations and a burn-in of 1 000 sampled trees, yielding a consensus tree of majority.

http://www.ncbi.nlm.nih.gov/

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The genetic divergence among all the species and the genetic variability among individuals

were assessed by the calculus of the uncorrected p distance, considering complete deletion in

MEGA 6.

Phylogeography analyses

Considering only the individuals of L. floridana, sequences were re-aligned as described in the

above section. Gaps were considered as informative characters. The ITS sequence-types

(referred herein as ST) were calculated by DNASP v. 5.10.01 (Librados & Rozas, 2009). The

nucleotide (π) and the ITS sequence-type (STh) diversities were determined using ARLEQUIN

v. 3.5.1 (Schneider et al., 2000).

To assess the evolutionary and geographical relationships, two analyses were considered: (1)

a tree including the STs within each ecoregion and additional Leucetta sequences, rooted on the

most distant lineage(s) and (2) an unrooted tree (network) considering only the specimens of L.

floridana. Phylogenetic analyses for the rooted tree followed the procedures detailed in the

above section (ML and BI approaches). Networks were calculated using the Median Joining

algorithm (Bandelt et al., 1999) within the program NETWORK v. 4.6 (Forster et al., 2007). ST-

groups (STG) were defined by an analysis of molecular variance (AMOVA) as it maximizes

variation among the groups and minimizes variation within the groups.

Population structure

The population structure across different geographical partitions (provinces, ecoregions and

localities) was assessed through pairwise FST comparisons (Weir & Cockerham, 1984) and an

analysis of molecular variance (AMOVA) as implemented in ARLEQUIN. The statistical

significance of estimates was assessed by 10 000 permutations.

Demographic structure

In order to test for population expansion across geographical (provinces, ecoregions and

localities) and phylogenetic (clades and STGs) groups, Tajima´s D (D) and Fu´s Fs (Fs)

neutrality tests were performed. Moreover, the SSD and raggedness index (r) values based on

mismatch distribution were calculated. All calculations considered 104 simulations and were

computed in ARLEQUIN.

To explore the historical demography of L. floridana, Bayesian skyline analyses (Drummond

et al., 2005) were performed in BEAST 1.8 (Drummond et al., 2012) for each structured

population (n≥10) as suggested by the AMOVA and network results. The nucleotide substitution

model was determined using the Bayesian Information Criterion (BIC) implemented in MEGA

6. The chosen model was Jukes Cantor but as it is not implemented in BEAUti 1.8 (Drummond

et al., 2012), we used the closest model with the lowest BIC: the HKY model. As the ITS

184

substitution rate within Calcarea is unknown, we used the 1.0% per MY estimated for the

demospongiae Prosuberites laughlini (Worheide et al., 2004) which was also employed to

estimate phylogeography patterns in other Leucetta species (Worheide et al., 2008). The

analyses assumed a strict molecular clock model and the numbers of groups were set to six

(previous exploratory tests with the same sequence set showed no difference in the Bayesian

Skyline Plot when using six or 10 groups). A MCMC analysis of 108 generations was run with

10% burn-in. The results were summarized in piecewise-constant Bayesian Skyline Plots using

TRACER 1.5 (Rambaut & Drummond, 2009).

RESULTS

Genetic Diversity

P distance

The length of the alignment including all the Leucetta sequences was 764 pb, including 136

variables sites, 74 parsimony-informative sites and 61 singletons. All specimens preliminarily

identified as L. floridana clustered in a clade supported by high ML bootstrap and BI posterior

probability (b=99, p=1) values, corroborating the initial identification (Figure 2).

The genetic divergences among the species of Leucetta were as follows: L. floridana–L.

potiguar: 2.5-3.4%, L. floridana–L. microraphis: 4.7-5.3%, L. floridana–L. chagosensis: 7.1-

7.7% , L. floridana–L. antarctica: 8.0-8.6%, and L. floridana–L. pyriformis: 8.5-9.1%. Leucetta

potiguar, a species restricted to the TSA Province, appeared as the sister taxon of L. floridana as

already reported in previous studies (Valderrama et al., 2009; Klautau et al., 2013).

The length of the alignment considering only L. floridana individuals was 733 pb with 708

invariable sites, 23 variable sites and two sites with alignment gaps. The intraspecific variability

inferred by the p-distance ranged from 0 to 1.8%. Interestingly, this same range value (0-1.8%)

was found (1) between Caribbean and Brazilian individuals and (2) among Caribbean

individuals. Among Brazilian individuals, the range varied between 0 and 0.1%.

Sequence-type richness

A total of 24 STs were found when considering gaps as informative characters. Among them,

two STs were shared by the TNA and TSA (ST1 and ST2), 16 were exclusive to the TNA and

one to the TSA (Table 3). ST1 was the most frequent ST, found in 98 individuals (60.5%) and

present in all the studied ecoregions except the Southern Caribbean (Curaçao). ST2 was

restricted to the Southwestern Caribbean and Fernando de Noronha and Atoll das Rocas

ecoregions. Besides ST1 and ST2, other five STs were shared among different ecoregions and

185

localities (ST12, ST15, ST18, ST19 and ST14). Seventeen STs were private to one locality or

island (ST3, ST5, ST6, ST7, ST8, ST9, ST10, ST11, ST13, ST14, ST16, ST17, ST20, ST21,

ST22, ST23, ST24).

The ecoregion with more STs was Eastern Caribbean (K=13) followed by Southwestern

Caribbean (K=10, Table 3). Greater Antilles, Northeastern Brazil and Fernando de Noronha and

Atoll das Rocas presented the same ST contribution (K=2) whereas the Southern Caribbean and

Eastern Brazil presented only one ST. Within the Eastern Caribbean, the island with more STs

was Saint Martin (K=8) followed by Martinique with five STs. Both localities presented the

same number of private STs (P=4). In the Southwestern Caribbean, the localities with more STs

were Panama and San Andrés, both with four STs. Within the Brazilian localities, Fernando de

Noronha and Ceará were the most diverse localities presenting two STS.

Sequence-type (STd) and nucleotidic (π) diversities

The overall STd was 0.62±0.04 and the total π was 0.003±0.002. Similar values were obtained

for the TNA whereas slightly lower values were observed for the TSA (Table 3). Among the

ecoregions, the Southwestern Caribbean presented the highest values: STd=0.83±0.04 and

π=0.0078±0.0039. Within this ecoregion, San Andrés had the highest values: STd=1±0.18 and

π=0.0075±0.0054, being followed by Panama with STd=0.71± 0.07 and π =0.005±0.003.

Greater Antilles (Puerto Rico) presented the lowest values in STd=0.17 ± 0.133 and in π=

0.0002 ± 0.0004.

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Figure 2. Maximum Likelihood phylogenetic tree inferred from the ITS sequences of Leucettafloridana and other Leucetta species. ML bootstrap and BI posterior probability values areindicated on the branches.

Table 3. Genetic Diversity. N=number of sequences, S/T=Segregating/Total number of sites, K=number of STs, P=number of private STs, STd= ST diversity, π=nucleotide diversity, SD=standard deviation. RN=Rio Grande do Norte State, FN=Fernando de Noronha Archipelago.

Ecoregion Locality N S/N K P STd (SD) π(SD)Tropical Northern Atlantic 147 24/733 23 16 0.638 (0.045) 0.0038 (0.0022)Greater Antilles

Puerto Rico 12 1/732 2 1 0.167 (0.134) 0.0002 (0.0004)Eastern Caribbean 100 19/733 13 8 0.558 (0.056) 0.0022 (0.0014)

Anguilla 1 0/732 1 0 - -Saint Martin 52 16/733 8 3 0.3167 (0.084) 0.0015 (0.0011)Saba 3 0/732 1 0 0 0Antigua 12 13/732 4 2 0.454 ( 0.17) 0.003 (0.002)Guadeloupe 1 0/732 1 0 - -Les Saintes 8 1/732 2 0 0.25 (0.18) 0.0003 (0.0005)Martinique 23 4/733 5 3 0.613 (0.104) 0.0016 (0.0012)

Southwestern Caribbean 33 14/732 10 7 0.835 (0.042) 0.007 (0.0039)San Andrés 4 10/731 4 3 1.0 (0.177) 0.0075 (0.0055)Panama 19 9/732 4 2 0.713 (0.074) 0.0055 (0.0032)Urabá 3 1/732 2 1 0.667 (0.314) 0.0009 (0.0011)Santa Marta 7 8/732 2 1 0.476 (0.171) 0.0052 (0.0034)

Southern CaribbeanCuraçao 2 0/732 1 0 0 0

Tropical Southern Atlantic 15 2/732 3 1 0.457 (0.141) 0.0007 (0.0007)Northeastern Brazil 9 1/732 2 1 0.389 (0.164) 0.0005 (0.0006)

Ceará 5 1/732 2 1 0.6 ( 0.175) 0.0008 (0.0009)RN 4 0/732 1 0 0 0

FN and Atoll das Rocas 5 1/732 2 0 0.6 (0.175) 0.0008 (0.0009)FN 4 1/732 2 0 0.5 (0.265) 0.0007 (0.0008)Rocas Atoll 1 0/731 1 0 - -

Eastern BrazilAbrolhos 1 0/732 1 0 - -

Clade 1= STG1+STG2 144 15/733 17 - 0.527 (0.0497) 0.0014 (0.0011)

Clade 2= STG3 9 2/733 3 - 0.556 (0.165) 0.0008 (0.0008)

Clade 3 9 4/732 4 - 0.694 (0.147) 0.0022 (0.0016)

STG1 139 12/733 15 - 0.492 (0.051) 0.0012 (0.0009)

STG2 5 1/733 2 - 0.4 (0.237) 0.0005 (0.0007)

STG4 6 1/732 2 - 0.333 (0.215) 0.0004 (0.0006)

STG5 3 1/732 2 - 0.667 (0.314) 0.0009 (0.0011)

All 162 25/733 24 - 0.624 (0.044) 0.0035 (0.0021)

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Phylogenetic and phylogeographic inferences

The ML phylogenetic tree obtained from the STs of L. floridana observed within each

ecoregion and the other leucettas is shown in Figure 3A. In both ML and Bayesian analysis, the

clade of L. floridana was subdivided into three clusters with high values of ML bootstrap (b)

and Bayesian posterior probability (pp): clade 1 (b=91 and pp=0.99, in light green), clade 2

(b=94 and pp=0.99, in green) and clade 3 (b=97 and pp=0.96, dark green).

Figure 3. A. Maximum-Likelihood phylogenetic tree inferred from the ITS sequences ofLeucetta floridana and other Leucetta species indicating the sequence-type (STs) found withineach ecoregion. Bootstrap and posterior probability values (ML/BI) are indicated on thebranches. B. Parsimony Median-Joining tree obtained from the ITS sequences of Leucettafloridana. The size of the circles is proportional to the frequency of the STs. Colours representdifferent localities. Numbers between STs indicate mutational steps and when not indicated, itrefers to one-mutational step.

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Clade 1 included representatives from all studied ecoregions in the TSA (Greater Antilles,

Eastern Caribbean, Southwestern Caribbean and Southern Caribbean, in black) and in the TNA

(Northeastern Brazil, Fernando de Noronha and Atoll das Rocas and Eastern Brazil, in red) and

from depths varying from -4 to -70 m. This clade clustered 144 individuals grouped in 17 STs.

Clades 2 and 3 comprised exclusively Caribbean specimens. Clade 2 included nine individuals

of the Southwestern Caribbean representing three STs whereas Clade 3 grouped nine specimens

from four STs.

The parsimony MJ network yielded additional information to the phylogenetic ML and IB

trees, unraveling five ST-groups (Figure 3B) supported by the AMOVA analyses (Tables S2 and

S3, Supplementary Material). Clade 1 was divided into STG1 and STG2, Clade 2 corresponded

to STG3 and Clade 3 was separated in STG4 and STG5. (Reference to Supplementary Material:

STG1 = STGA+ STGB + STGC + STGD, STG2 = STGE, STG3 = STGF, STG4 = STGG and

STG5=STGH). STG1 and STG2 are separated by a three-mutational-step distance. STG1 has a

star-like shape in which rare STs are one-mutational-step close to the common central ST1,

indicating a population expansion pattern. STG4 and STG5 are situated in the opposite side of

the network and are separated by two-mutational steps. SGT3 has apparent central position,

however, it is composed of few individuals (n=9). Determined STGs have certain geographic

correspondence: STG1 is distributed along the TSA and TNA. STG2, STG3, and STG4 are

restricted to the Southwestern Caribbean whereas STG5 occurs only in the Eastern Caribbean.

Population Structure

AMOVA

In the analyses of molecular variance (AMOVA), two hypothetical scenarios of population

structure yielded the maximum variation among groups (ɸCT=0.549). The first hypothetical

scenario (H13) comprised eight groups: (1) Puerto Rico + Anguilla + Saint Martin + Saba +

Antigua + Les Saintes + Guadeloupe, (2) Martinique, (3) San Andrés, (4) Panama, (5) Uraba,

(6) Santa Marta, (7) Curaçao, and (8) Brazil. The second scenario (H19) split the Brazilian

group into (1) Coastal Brazil (Rio Grande do Norte + Ceará + Abrolhos) and (2) Insular Brazil

(Rocas Atoll + Fernando de Noronha). Interestingly, in both hypothesis, the only highly

structured locality within the Eastern Caribbean was Martinique, whereas all localities in the

Southwestern Caribbean represented separated populations. Additionally, Curaçao is well

differentiated from the others despite its low sample size. All tested groups and the F-statistics

obtained are presented in Table S4.

FST Comparisons

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Significant FST indexes ranged from moderate (0.14<FST<0.25) to high (0.25≤FST≤1) values

(Table 4). High values were observed when comparing localities within the Southwestern

Caribbean (except for Panama) with the other Caribbean (0.47≤FST≤1) and Brazilian localities

(0.35≤FST≤0.96). Despite the small number of sequences analysed from Curaçao, high FST values

were observed between this island and other Caribbean localities (Les Saintes and Puerto Rico).

Within the Lesser Antilles, high pairwise FST values (0.47≤<FST<≤0.57) were obtained between

Martinique and some of the other islands (Saint Martin, Saba, Antigua and Les Saintes). Among

ecoregions, pairwise FST comparisons yielded significant high values in Greater Antilles x

Southwestern Caribbean (FST=0.86) and Southern Caribbean x Northeastern Brazil (FST=0.73)

whereas the lowest value was found in Fernando de Noronha and Atoll das Rocas x

Southwestern Caribbean (FST=0.19) (Table 5).

When sequences were grouped according to the clades recovered in the phylogenetic

analyses, all pairwise comparisons were significant (Table 6). FST values ranged from 0.82

(Clade 2 x Clade 3) to 0.88 (Clade 1 x Clade 3). When the same set of sequences was grouped

considering the ST groups, the FST values obtained were even higher (0.85≤FST≤ 0.96) in almost

all cases except in STG1 x STG2 (FST=0.779).

Demography Structure

Sequence-type (ST) and nucleotidic (π) diversity

According to Grant and Bowen (1998), historical processes can be inferred for species or

populations based on haplotipic and nucleotidic diversity indexes. Herein, we suggest some

demographic events in L. floridana based on the observed ST (sequence-type) and π

(nucleotidic) diversity indexes.

The overall values of STd>0.5 and π <0.005 suggest the occurrence of a recent bottle-neck

event within the populations of L. floridana. In the TNA, this must have been followed by a

rapid population growth. Within the Eastern Caribbean, the values observed in Martinique

(STd=0.61±0.1 and π=0.002 ±0.001) indicate a rapid growth within this island whereas Antigua,

Saint Martin and Les Saintes may represent founder populations (STd<0.5 and π <0.005). The

high values observed in the Southwestern Caribbean (STd>0.5 and π >0.005) suggest the

presence of a large stable population or a secondary contact among different populations in this

area or at least in Panama and San Andrés. Differently, Santa Marta, given its diversity indexes

(H<0.5 and π >0.005) may have suffered an ancient bottleneck or may represent divergent

populations geographically subdivided. In the TNA, the low values of STd and π of the

Brazilian population may indicate that they have also experienced a recent bottleneck or

represent founder populations.

Table 4. Pairwise FST values among sampled localities or islands. Caribbean localities are PR: Puerto Rico, AG: Anguilla, SN: Saint Martin, SAB: Saba,AN: Antigua, GU: Guadeloupe, LS: Les Saintes, MT: Martinique, PAN: Panama, SAN: San Andrés, URA: Uraba, SM: Santa Marta and CUR: Curaçao.Brazilian localities: CE: Ceará, RN: Rio Grande do Norte, RAT: Rocas Atoll and ABR: Abrolhos Archipelago. Significant values are in bold.

PR AG SN SAB AN GU LS MT PAN SAN URA SM CUR CE RN FN RAT ABRAG -1SN -0.02 -0.96SAB -0.19 0 -0.18AN 0 -1 -0.02 -0.19GU -1 0 -0.96 0 -1LS 0.01 -1 -0.04 -0.17 -0.03 -1MT 0.61 0.38 0.52 0.51 0.47 0.38 0.57PAN 0.25 -0.36 0.31 0.09 0.14 -0.36 0.21 0.48SAN 0.57 -0.38 0.53 0.23 0.29 -0.37 0.45 0.60 0.16URA 0.97 0.91 0.85 0.95 0.75 0.91 0.95 0.88 0.39 0.53SM 0.73 0.333 0.71 0.54 0.51 0.33 0.67 0.75 0.34 0.29 0.52CUR 0.86 1 0.37 1.0 0.20 1 0.81 0.22 0.21 0.18 0.95 1.0CE 0.31 -0.5 0.05 0.12 0.001 -0.5 0.23 0.53 0.18 0.36 0.92 0.6 0.67RN -0.13 0 -0.13 0 -0.13 0 -0.11 0.56 0.13 0.31 0.96 0.58 1 0.19FN 0.11 -1 -0.06 -0.09 -0.09 -1 0.05 0.53 0.14 0.2 0.92 0.57 0.71 0.15 0RAT 0.85 1 0.28 1 -0.04 1 0.78 0.59 -0.02 -0.83 0.91 0.43 1 0.57 1 0.33ABR -1 0 -0.96 0 -1 .0 -1 0.38 -0.36 -0.37 0.91 0.33 1 -0.5 0 -1 1 0

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Table 5. Pairwise FST values among ecoregions and provinces. Ecoregions within the TNA(Tropical Northwestern Atlantic): GA= Greater Antilles, EC=Eastern Caribbean,SWC=Southwestern Caribbean and SC=Southern Caribbean. Ecoregions within the TSA(Tropical Southwestern Atlantic): NB=Northeastern Brazil, FA=Fernando de Noronha and Atolldas Rocas and EB=Eastern Brazil. Significant values (p<0.05) are in bold.

GA EC SWC SC NB FA EB TSAEC 0.025

SWC 0.863 0.367SC 0.263 0.138 0.196NB 0.106 0.036 0.243 0.732FA 0.310 0.057 0.192 0.674 0.231EB -1.0 -0.797 -0.258 1.0 -0.75 -0.5

TNA - - - - - - - 0.035

Table 6. Pairwise FST values among Clades and ST-Groups (STG). All values were significant.

Clade 1 = STG1+STG2

Clade2= STG3

Clade 3= ST4+STG5

STG1 STG2 STG4

Clade 2 = STG3 0.871Clade 3 0.883 0.824STG1 - 0.892 0.901STG2 - 0.928 0.87 0.779STG4 0.884 0.908 - 0.904 0.956STG5 0.907 0.922 - 0.923 0.955 0.847

Neutrality Tests

The values obtained in the neutrality tests considering localities, ecoregions, clades and ST-

groups are shown in Table 7. In the overall sample, both tests indicated a deviation from

neutrality (Tajima's D=-1.21 and Fu's Fs=-9.56), however, Tajima's D test was not significant

(p=0.09). Significant Tajima's D and Fu´s Fs values were obtained for the Eastern Caribbean

ecoregion (D=-1.78, p=0.012 and Fs=-4.26, p= 0.047), Clade 1 (D=-1.73, p=0.013 and Fs=-

11.96, p=0.0) and STG1 (D=-1.97, p=0.002 and Fs=-13.32, p=0) indicating population

expansion within these groups (Aris-Brosou & Excoffier, 1996).

Congruent significant tests were not observed among the other groups tested. Tajima's D

values were significant for Saint Martin (D=-2.04, p=0.004) and Antigua (D=-2.1, p=0.002)

whereas the Fu´s Fs test was significant for the TNA (Fs=-8.068, p=0.012).

Mismatch Distribution

Most geographical partitions analysed herein evidenced a multimodal mismatch distribution,

except for Martinique which had an approximate unimodal distribution (Figures 4A-F). Among

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the genetic groups tested, unimodal-like mismatch distributions were observed for Clade1 and

STG1 (Figures 4G-H). These unimodal distributions (Poisson-like) are the signature of

demographic or spatial expansions (Rogers and Harpending 1992; Bunje and Wirth, 2008). In

none of the cases, the SSD and the Harpending's raggedness index were significant (Figure 4,

Table S5) and consequently, it was not possible to reject the sudden expansion model.

As expected due to the low variability found in Puerto Rico, Northeast Brazil and Fernando de

Noronha and Rocas Atoll, Clade 2, Clade 3, STG3, STG4 and STG5, no clear distribution

pattern was observed for them.

Table 7. Demography statistics of Leucetta floridana. TNA=Tropical Northern Atlantic,TSA=Tropical Southern Atlantic, RN=Rio Grande do Norte, p=p value.

Province/Ecoregion Locality Tajima´s D (p) Fu´s FS (p)TNA Caribbean Sea -1.0404 (0.1416) -8.0681 (0.0119)Greater Antilles Puerto Rico -1.1405 (0.1683) -0.4757 (0.1334)Eastern Caribbean Lesser Antilles -1.7835 (0.0124) -4.2641 (0.0472)

Saint Martin -2.0454 (0.0037) -2.4497 (0.09)Antigua -2.1039 (0.0023) 1.11937 (0.7496)Les Saintes -1.0548 (0.2096) -0.1820 (0.2007)Les Saintes + GuadeloupeMartinique -0.0204 (0.4933) -0.4473 (0.3819)

Southwestern Caribbean 1.6576 (0.9596) 0.7867 (0.6623)San Andrés 0.0834 (0.6645) -0.3992 (0.2231)Panama 1.9623 (0.9809) 4.3061 (0.9612)Urabá 0.0 (1.0) 0.2007 (0.3965)Santa Marta 0.8755 (0.8333) 5.1781 (0.9822)

TSA Brazil -0.3988 (0.2884) -0.4474 (0.2619)Northeastern Brazil Ceará + RN 0.15647 (0.7512) 0.47744 (0.3977)

Ceará 1.2247 (0.9435) 0.6261 (0.5025)Fernando de Noronha (FN) and Atoll das Rocas

FN Archipelago + Rocas Atoll

0.0 (1.0) 0.6261 (0.4986)

FN Archipelago 0.0 (1.0) 0.1718 (0.3388)Clade 1= STG1+STG2 -1.7347 (0.0133) -11.9645 (0.0)

Clade 2= STG3 0.15647 (0.7615) -0.5321 (0.139)Clade 3 0.9867 (0.8481) -0.0611 (0.45050)STG1 (1 A) -1.6636 (0.0177) -11.0736 (0.0001)STG2 (1 B) 0.0 (1.0) 0.0902 (0.2959)STG4 (3 A) 0.0 (1.0) -0.0027 (0.2612)STG5 (3 B) 0.0 (0.9873) 0.2007 (0.3867)All -1.2085 (0.099) -9.5358 (0.0056)

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Bayesian Skyline Plot

Bayesian Skyline Plots (BSP) evidenced a population expansion pattern when considered (1) the

complete set of L. floridana sequences (Figure 5A), (2) STG1+STG2 = Clade 2 (Figure 5B), (3)

STG1 (Figure 5C) and (4) AMOVA-G1: Puerto Rico + Anguilla + Saint Martin + Saba +

Antigua + Les Saintes + Guadeloupe (Figure 5D). In the other groups tested, the BSP did not

detect any demographic event probably due to the small number of individuals per group

(n<10).

DISCUSSION

Genetic Structure

Although the AMOVA results indicated some genetic structure when geographical partitions

were considered, the highest FST values were observed among clades (inferred by ML and BI

methods) and ST-Groups (obtained in the MJ network). Therefore, we recognize the occurrence

of five structured populations of L. floridana within the Tropical Atlantic Ocean: four

populations restricted to the Caribbean Sea and a widespread population in the Western Tropical

Atlantic.

Caribbean restricted populations

Cowen et al. (2006) defined four broad regions of connectivity within the Caribbean: (1) the

eastern Caribbean (Puerto Rico to Aruba); (2) the western Caribbean (Cuba to Nicaragua); (3)

the Bahamas and the Turks and Caicos Islands; and (4) the peripheral area of the Colombia-

Panama Gyre. They also considered smaller areas of isolation within each region. In this study,

the four Caribbean-restricted populations of L. floridana matched two of the referred

connectivity regions and evidenced isolation patterns within them.

In the peripheral area of the Colombian-Panama Gyre, which roughly coincides with the

Southwestern ecoregion (Spalding et al., 2007), we found three structured populations: SGT3 in

the area of direct influence of the Colombian-Panama Gyre (Panama and Urabá), STG4 in

distant localities (San Andrés and Santa Marta) and STG2 in closer localities (Panama and San

Andrés)

Isolation of STG3 from the Caribbean Sea (FST= 0.89 – 0.93) and connectivity among the

individuals within this population can be explained by the cyclonic circulation of the

Colombian-Panama Countercurrent (counter-clockwise to the Central Caribbean Current) which

may act as a larval retention current.

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Figure 4. Mismatch distribution of Leucetta floridana for (A) Antigua, (B) Saint Martin, (C)Panama, (D) Martinique, (E) Southwestern Caribbean, (F) Eastern Caribbean, (G) Clade 1 and(H) STG1. Bars show the observed frequency distribution for the number of pairwisedifferences among all individuals sampled. The solid lines show the expected distribution undereach population expansion model. SSD: Sum of Squared Deviations, r: Harpending's raggednessindex.

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Figure 5. Bayesian Skyline Plots for (A) all ITS sequences, (B) Clade1= ST-Group1 + ST-Group2 of Leucetta floridana, (C) ST-Group1 and (D) AMOVA Group 1: Puerto Rico +Anguilla + Saint Martin + Saba + Antigua + Les Saintes + Guadeloupe of Leucetta floridana.The maximum time is the upper 95% HPD of the root height. The median estimate (black solidline) and 95% HPD limits (blue line) are indicated.

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As the coast of Santa Marta is directly influenced by the Central Caribbean Current (Diaz et

al., 2001), this can explain why individuals from this locality are isolated from Urabá and

Panama, presenting a private ST (ST6). However, they are somewhat connected with some

individuals from San Andrés (FST<0.25 but not significant), as observed in STG4. This can be

attributed to the presence of the Colombian-Panama Countercurrent in Santa Marta during

raining periods (Diaz et al., 2001). This latitudinal connectivity between San Andrés and Santa

Marta (12.6°N and 11.2°N, respectively) has already been reported in other marine taxa e.g. the

reef fish Stegastes partitus (Ospina-Guerrero et al., 2008).

Different to STG4, STG2 reunites individuals from longitudinally close (82.1 and 81.7°W)

but latitudinally distant (9.3 and 12.6°N) localities (Panama and San Andrés) that may be

warranting the gene flow through the Colombian-Panama Countercurrent. This scenario has also

been suggested for another Caribbean sponge (Cliona delitrix, Chaves-Fonnegra et al., 2015)

Besides the barriers cited above, the whole genetic structure observed within this large area

(peripheral area of the Colombia-Panama Gyre) suggests the presence of a strong barrier

between San Andrés and Urabá as no STG including both localities was found (FST>0.5 but not

significant). An alternative explanation to this could relay on past geological events that

occurred within this area isolating populations.

The last structured population is located in the Eastern Caribbean connectivity region of

Cowen et al., (2006). STG5 included individuals from two islands located in the northern part of

the Lesser Antilles arc (Saint Martin and Antigua) and no individuals from the southern islands

(Martinique nor Guadeloupe). Whether this corresponds to a split of the Eastern Antilles

population in northern and southern populations remains uncertain based on our results,

however, this should receive more attention in further studies compelling the Lesser Antilles.

A Western Tropical Atlantic widespread population

The occurrence of a large population of L. floridana with intense genetic exchange between

Caribbean and Brazilian localities (STG1) does not only reject the role of the Amazon river as

an effective barrier to gene flow in the Western Tropical Atlantic but also suggests the presence

of biological and/or physical mechanisms that would facilitate the observed connectivity

pattern.

In a broad sense, the population connectivity observed in L. floridana may be mediated by

(1) the North Brazilian Current enabling the flow of migrants from the Brazilian coast to the

Caribbean Sea and (2) the sponge assemblages under the mouth of the Amazon river acting as a

connectivity corridor (as proposed by Rocha et al., 2003 for fish species).

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Within this context, it is suggested that the larva of L. floridana can pass underneath the

Amazon freshwater outflow (-30 m; Curtin, 1986). Although knowledge on the ultrastructure

and behaviour of calciblastula larva is scarce, we know that this larva is hollow, oval and

surrounded by ciliated cells (Amano & Hori, 2001; Ereskovsky & Willenz, 2008), which may

be responsible for its motility. In some Demospongiae, the cilia are used for larval rotation along

the longitudinal axis, apparently allowing the larvae to re-adjust its depth while drifting

(Maldonado et al., 2003). These ciliated cells may play the same role in L. floridana, facilitating

its sinking under the Amazon mouth. Alternatively, the larva of L. floridana can undergo a

passive mechanism, being dragged to the bottom by masses of water with more salinity

(Maldonado, 2006).

Similar patterns of high genetic connectivity along the Western Tropical Atlantic had been

attributed to efficient larval dispersal in other marine taxa (e.g. Rocha et al., 2002; Rodriguez et

al., 2013). In Porifera, this is paradoxal since sponge larvae are lecithotrophic (Ereskovsky,

2010) and remain in the water column for minutes to a few days (usually <2 weeks -

Maldonado, 2006; Padua et al., 2013), which most probably restrict their dispersal and

colonization capabilities.

Some sponge larvae exhibit complex mechanisms that contribute to expand their pelagic

lifespan and consequently increase their dispersal capability such as (1) the incorporation of

dissolved compounds by pinocytotic activity in Tedania ignis (Jaeckle, 1995), (2) the capacity to

phagocyte and digest bacteria and small (<4 μm) unicellular organisms by the ciliated cells of

Halichondria parenchymella (Ivanova, 1999) or (3) just being unpalatable to predators (fish),

observed in some Caribbean sponges (Lindquist & Hay, 1996). Whether this occurs in L.

floridana or in any other calcareous sponge is unknown but should not be discarded.

Another mechanism that improve the dispersal capacity of some sponge larvae is the

fragmentation of reproductive sponges. A fragment containing larvae can travel for a long time

before the larvae are released and this may contribute to maximise the dispersal capacity

(Maldonado & Uriz, 1999).

Demographic history

Our results evidenced a population expansion pattern for L. floridana, specifically within the

widespread population STG1. Diversity indexes, neutrality tests and mismatch distributions

indicated that this occurred in the Caribbean Sea. Complementary information provided by the

bayesian skyline plot situated the onset of this event ca. 20.000 years BP. This estimated date

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coincides with the final period of the Pleistocene: the Last Glaciation Maximum (LGM, 30.000

– 19.000 years BP, Lambeck et al., 2001).

During the Pleistocene (2.6 million –11.700 years BP), the glacial-interglacial cycles

changed the sea level, superficial sea temperatures, current patterns, upwelling intensity and

coastal habitats (Rohling et al., 1998; Lambeck et al., 2002). Global sea levels were 115–130 m

lower than today (Fairbank, 1989; Lambeck et al., 2002) and tropical sea-surface temperature

oscillated between 1 °C and 3 °C (Herbert et al., 2010). During the LGM, the area of coastal

habitats was reduced by approximately 92% in the Gulf of Mexico and Caribbean Sea

(Bellwood & Wainwright, 2002). Under those conditions, some species became extinct and

several others remained as fragmented populations that nowadays are highly genetically

structured (Ludt & Rocha, 2015).

It is feasible that the shallow overhangs and vertical slopes inhabited by L. floridana during

the Pleistocene ended up being exposed due to the low sea levels and consequently, some

populations were decimated. We suggest that L. floridana suffered a bottle-neck event during

the late Pleistocene and that its populations remained isolated for a long period of time, which is

reflected in the high genetic population structure observed in this study. However, after the

LGM, one of the isolated population (STG1) expanded within the Caribbean.

In the Western Tropical Atlantic, demographic events have been reported as a result of the

fluctuating conditions during the Pleistocene. In the Tropical Northwestern Atlantic, population

bottlenecks have been evidenced in invertebrates including gastropods (Johnston et al., 2012),

decapods shrimps (Cook et al., 2010) and blenny fish (Eytan & Hellberg, 2010) whereas

population expansion was observed in penaeid shrimps (McMillen-Jackson & Bert, 2003),

spiny lobster (Naro-Maciel et al., 2011) and West Indian manatee (Vianna et al. 2006). Although

most of these events have been recorded in the TNA, population expansion has also been

reported in the Tropical Southwestern Atlantic (e.g. Rodriguez-Rey et al., 2013).

Within sponges, genetic structure due to interglacial-glacial cycles have been observed in

the demosponges Hymeniacidon flavia and H. sinapium (Hoshino et al., 2008) from the

Northwest Pacific and the Indo-Pacific calcarean Leucetta chagosensis (Wörheide et al., 2002;

2008) as well as population expansion of Pericharax heteroraphis (Bentlage & Worheide, 2007)

within the Great Barrier Reef in Australia.

Evolutionary history of L. floridana

Based on the phylogenetic, genetic structure and the demographic results obtained in this study

and additional (palaeo)geological information, we propose the following evolutionary scenario

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for L. floridana. Ancestral populations of this species were broadly distributed in the Western

Tropical Atlantic. Once the outflow of the Amazon River started to be deposited in the Atlantic

in the late Miocene (10.4 million years BP - Hoorn, 1996), populations were isolated and L.

floridana must have been restricted to the Tropical Northwestern Atlantic.

During the Pleistocene, important changes in marine taxa occurred (Lundt & Rocha, 2015).

In L. floridana, we suggest both genetic isolation and connection due to glacial-interglacial

cycles. The glacial periods isolated some populations, as evidenced by the high genetic structure

observed within the Caribbean-restricted ST-groups (STG2, STG3, STG4 and STG5).

According to the demographic bayesian analyses, these ST-groups share a more ancient history

when compared to STG1.

During the most recent interglacial periods, the distribution of one population of L. floridana

(STG1) was expanded to the northeastern Brazilian coast and maintained connectivity

(evidenced by the common ST1) through the sponge corridor, as suggested for other trans-

Amazonian species (Rocha, 2003). This southward dispersal from the Caribbean to Brazilian

localities has been also observed in reef fish (Lima et al., 2005; Rocha et al., 2008) and

although it conflicts with today's current patterns, it must respond to past oceanographic

conditions (Iturralde-Vinent & MacPhee, 1999). According to Bowen et al., 2001, populations

can disperse and potentially coalesce after periods of isolation. Therefore, even if trans-

Amazonian individuals of L. floridana underwent restricted genetic flow for some period of

time as a result of the fluctuating conditions of the Pleistocene, they were able to restore

connectivity later. This must have been facilitated by the large population size of this

widespread species. By the end of the last glaciation maximum, the widespread population

(STG1) suffered a bottleneck and a posterior sudden expansion event in the Lesser Antilles

(most probably, in Martinique). Whether the Lesser Antilles acted as a Pleistocean refugia is

uncertain as not palaeogeological information is available, however, the presence of many

private ST may support this (Maggs et al., 2008).

Acknowledgements

The authors thank Adeline Pouget-Cuvelier, Amandine Vaslet, Julien Chalifour and Philippe

(Filipo) Thélamon for providing relevant information on sampling localities in the Lesser

Antilles and also Alexander Ereskovsky, Bob Thacker?, César Ruiz, Cristina Diaz, Eduardo

Hajdu, Fernanda Azevedo, Jean Vacelet, Laurent Van Bostal, Pedro Leocorny, Pierre

Chevaldonné and Sandrine Chenesseau for collecting L. floridana in the Caribbean Sea.

Ghennie Rodriguez is acknowledged for providing invaluable support for the demography

200

analyses. This work was partly supported by the Associated International Laboratory

‘MARRIO’. B.C.L. received a scholarship from the Brazilian Coordination for the Improvement

of Higher Education Personnel (CAPES). J.E.G.H. received grants from the American Museum

of National History Lerner-Gray Fund for Marine Research (sponge collection) and the Sea-

Grant Puerto Rico (DNA sequencing). M.K. is funded by fellowships and research grants from

the Brazilian National Research Council (CNPq), CAPES, and the Rio de Janeiro State

Research Foundation (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio

de Janeiro - FAPERJ). T.P. is funded by grants from The French National Center for Scientific

Research (CNRS).

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SUPPLEMENTARY MATERIAL

Table S1. Voucher number, locality, geographic coordinates and Genbank accession number of the specimens of Leucetta floridana analysed in this study.Abbreviations: Brazilian States: BA=Bahia, CE=Ceará, RN= Rio Grande do Norte, PE=Pernambuco. SM-MNR=Saint Martin Marine Natural Reserve.

Voucher Number Sample Locality Geographic Coordinates Depth (m) Genbank Number

Tropical Northwestern AtlanticGreater Antilles ecoregion: Puerto Rico (n=12)PR52 Cayo Mario 17°57'16.89''N, 67°03'53.42''W 9.0 This studyPR81 Cayo Mario 17°57'16.89''N, 67°03'53.42''W 10.8 This studyPR97 Veril – Fallen Rock (Guanica) 17°54'05.04''N, 66°55'27.59''W 31.5 This studyPR99 Cayo Conserva 17°55'56.91''N, 67°05'35.01''W 12.6 This studyPR105 Cayo Conserva 17°55'56.91''N, 67°05'35.01''W 15.6 This studyPR112 Pinnacles 17°56'03.96''N, 67°01'49.80''W 9.9 This studyPR117 Veril - El Hoyo 17°52.0529'N, 67°02.671'W 31.5 This studyPR120 Pinnacles 17°56'03.96''N, 67°1'49.80''W 17.1 This studyPR136 Turromote 17°56'20.13''N, 67°2'43.95''W 17.4 This studyPR141 Turromote 17°56'20.13''N, 67°2'43.95''W 12.9 This studyPR147 Cayo Mario 17°57'16.89''N, 67°3'53.42''W 13.2 This studyPR153 Cayo Mario 17°57'16.89''N, 67°3'53.42''W 17.7 This studyEastern Caribbean ecoregion: Lesser Antilles (n=100)Anguilla (n=1) This studyUFRJPOR 8278 Little Scrub2 18°17.903'N, 62°57.294'W 23.5 This studySaint Martin (n=52)UFRJPOR 7789 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W <20? This studyUFRJPOR 7968 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7970 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7971 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7972 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7974 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W < 22 This studyUFRJPOR 7985 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 7986 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 7990 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 7992 Les Arches 2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 7993 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 8000 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This studyUFRJPOR 8001 Les Arches2, Tintamare Island, SM-MNR 18°07'32.81''N, 62°58'21.49''W < 16 This study

UFRJPOR 8022 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This studyUFRJPOR 8025 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This studyUFRJPOR 8028 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This studyUFRJPOR 8029 Trou David, Les Terres Basses 18°04.402'N, 63°07.149'W 6.1 This studyUFRJPOR 8030 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 9.8 This studyUFRJPOR 8031 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 9.8 This studyUFRJPOR 8098 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 8 - 10 This studyUFRJPOR 8142 Rocher Créole, SM-MNR 18°07.038'N, 63°03.419'W 8 - 10 This studyUFRJPOR 8150 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8151 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8152 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8153 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8155 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8157 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8158 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8181 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8182 Les Arches1, Tintamare Island, SM-MNR 18°07.588'N, 62°58.248'W 10 – 20? This studyUFRJPOR 8192 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This studyUFRJPOR 8193 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This studyUFRJPOR 8194 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This studyUFRJPOR 8195 Basses Espagnoles 18°07.821'N, 63°00.270'W 10 This studyUFRJPOR 8196 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8197 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8198 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8199 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8216 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8217 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8218 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8219 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8221 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8222 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8223 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8224 Basses Espagnoles 18°07.821'N, 63°00.270'W 6 - 10 This studyUFRJPOR 8231 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 20 This studyUFRJPOR 8232 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13.8 This studyUFRJPOR 8234 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This studyUFRJPOR 8235 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This study

UFRJPOR 8237 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This studyUFRJPOR 8238 Chico2, Tintamare Island, SM-MNR 18°06.501'N, 62°59.005'W 13 - 20 This studySaba (n=3)UFRJPOR 8040 SA1, Southeastern Coast 17°37.066'N, 63°13.580'W < 27 This studyUFRJPOR 8041 SA1, Southeastern Coast 17°37.066'N, 63°13.580'W < 27 This studyUFRJPOR 8042 SA1, Southeastern Coast 17°37.066'N, 63°13.580'W < 27 This studyAntigua and Barbuda (n=12)UFRJPOR 7936 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7938 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7939 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7940 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7942 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7943 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7949 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7950 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 7952 Nanton Point, Saint Paul 16°59'51.60''N, 61°45'37.22''W < 20 This studyUFRJPOR 8093 Five Islands 17°04.990'N, 61° 54.840'W 4 This studyUFRJPOR 8185 Diamond_Bank, Saint Paul 17°12.000'N, 61°52.800'W 7 - 8 This studyUFRJPOR 8186 Diamond Bank, Saint Paul 17°12.000'N, 61°52.800'W 7 - 8 This studyGuadeloupe (n=9)UFRJPOR 7655 Cave1, Les Saintes 15°52.984'N, 61°34.25'W < 11 This studyUFRJPOR 7659 Cave1, Les Saintes 15°52.984'N, 61°34.25'W < 11 This studyUFRJPOR 7648 Cave1, Les Saintes 15°52.984'N, 61°34.25'W < 11 This studyUFRJPOR 7827 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8087 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8558 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8091 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8092 Cave1, Les Saintes 15°52.984'N, 61°34.25'W 8 - 10 This studyUFRJPOR 8340 Cave Cathédrale, ? 16°27.740'N, 61°31.837'W 13.7 This studyMartinique (n=23)UFRJPOR 7403 Grottes des couleurs, Pointe Burgos, Grande

Anse, Les Anses d’Arlet14°29.787'N, 61°05.351'W <10 m This study

UFRJPOR 7404 Pointe Burgos, Grande Anse, Anses d'Arlet <10 m This studyUFRJPOR 7405 Grottes des couleurs, Pointe Burgos, Grande


UFRJPOR 7406 Grottes des couleurs, Pointe Burgos, Grande Anse, Les Anses d’Arlet

14°29.787'N, 61°05.351'W <10 m This study


14°29.787'N, 61°05.351'W <10 m This study


14°29.787'N, 61°05.351'W <10 m This study


14°29.787'N, 61°05.351'W <10 m This study


14°29.787'N, 61°05.351'W <10 m KX355573


14°29.787'N, 61°05.351'W <10 m KX355574

UFRJPOR 7412 Les Anses d’Arlet <10 m KX355575UFRJPOR 7423 Grottes des couleurs, Pointe Burgos, Grande


UFRPOR 7424 Grottes des couleurs, Pointe Burgos, Grande Anse, Les Anses d’Arlet

14°29.787'N, 61°05.351'W <10 m This study

SP2 Les Anses d’Arlet <10 m This studyUFRJPOR 7777 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7778 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7845 Pointe Burgos, Grande Anse, Les Anses

d’Arlet14°29.787'N, 61°05.351'W? This study

UFRJPOR 7859 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 3? This studyUFRJPOR 7861 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 3 This studyUFRJPOR 7862 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 5 This studyUFRJPOR 7863 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7865 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7871 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studyUFRJPOR 7877 Anse de Fortune, Les Anses d’Arlet 14°30.377'N, 61°05.850'W 6 This studySouthwestern Caribbean ecoregion (n=33)Panamá (n=19)UFRJPOR 6389 Bocas del Toro 09º20.914'N, 82º9.394'W? <20? This studyPC BT 12 Bocas del Toro 09º20.914'N, 82º9.394'W? <20? EU781989PC BT 22 Bocas del Toro 09º20.914'N, 82º9.394'W? <20? EU781990PC BT 23 Bocas del Toro 09º20.914'N, 82º9.394'W <20? EU781991UFRJPOR 6948 Las Cuevas, Bocas del Toro 09º20.914'N, 82º9.394'W <20? This studyUFRJPOR 6951 Las Cuevas, Bocas del Toro 09º20.914'N, 82º9.394'W <20? This studyUFRJPOR 6953 Las Cuevas, Bocas del Toro 09º20.914'N, 82º9.394' W <20? This studyUFRJPOR 6954 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This studyUFRJPOR 6956 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This study

UFRJPOR 6959 Bocas del Toro <20? This studyUFRJPOR 6960 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This studyUFRJPOR 6961 Las Cuevas, Bocas del Toro 09º20.914'N, 82º09.394'W <20? This studyUFRJPOR 6975 Bocas del Toro <20? This studyUFRJPOR 6976 Bocas del Toro 17 This studyUFRJPOR 6977 =MNRJ 15748B

Bocas del Toro <20? This study

UFRJPOR 6978 =MNRJ 15754B

Bocas del Toro <20? This study

UFRJPOR 6988 Bocas del Toro <20? This studyUFRJPOR 6989 Cayo Zapatilla, Bocas del Toro 09º14.881'N, 82º01.952'W <20? This studyUFRJPOR 6989A Cayo Zapatilla, Bocas del Toro 09º14.881'N, 82º01.952'W <20? This studyColombia (n=14)SM01 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 21 This studySM02 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 19 This studySM03 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 20 This studySM04 El Morro, Santa Marta Bay, Santa Marta 11°14'57.50"N, 74°13'54.50"W 11 This studySM06 Punta Venado, Ensenada de Taganga, Santa

Marta11°16'25.00"N, 74°12'24.00"W 18 This study

SM07 Punta Venado, Ensenada de Taganga, Santa Marta

11°16'25.00"N, 74°12'24.00"W 18.6 This study

SM12 Punta Gaira, Gaira Bay, Santa Marta 11°13'08.00"N, 74°14'30.00"W 14 This study UFRJPOR 5357 Bajo Agua Viva, Urabá 07°53''N, 76°38'W? 15 EU781970 UFRJPOR 5359 Bajo Agua Viva, Urabá 07°53''N, 76°38'W? 15 EU781969UFRJPOR 5360 “Bajo Agua Viva”, Urabá 07°53''N, 76°38'W? 15 EU781968UFRJPOR 5363 “West View”, Leward-reef, San Andrés 12°33'N, 81°43'W? 5 EU781971UFRJPOR 5364 La Piscinita, San Andrés 12°33'N, 81°43'W? 2 - 5 EU781972UFRJPOR 5366 La Piscinita, San Andrés 12°33'N, 81°43'W? 2 - 5 EU781973UFRJPOR 5367 La Piscinita, San Andrés 12°33'N, 81°43'W? 2 - 5 EU781974Southern Caribbean ecoregion: Curaçao (n=2)UFRJPOR 6726 Water Factory, Willemstadt 12°06'30.88"N, 68°57'13.53"W 17.8 Curaçao paperUFRJPOR 6765 Hook’s Hut, Willemstadt 12°07'18.94"N, 68°58'11.46"W 13.3 Curaçao paperTropical Southwestern AtlanticNortheastern Brazil ecoregion: Brazil (n=9)BPOTPOR 200 Potiguar Basin, RN 04º37'31.7"S, 36º46'00.7"W 70 This studyBPOTPOR 202 Potiguar Basin, RN 04º37'31.7"S, 36º46'00.7"W 70 EU781985BPOTPOR 610 Risca das Bicudas, RGN 04º57'00.9"S, 36º07'49.7"W 8-10? EU781978BPOTPOR 634 Urca do Tubarao, RGN 04º50'52.7”S, 36º27'02.1"W 8 This study

MNRJ 8440 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781983MNRJ 8445 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781984MNRJ 8465 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781982MNRJ 8481 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 This studyMNRJ 8488 Station 30 - CENPES, CE 02º52'S, 39º10'W 33 EU781980Fernando de Noronha and Atoll das Rocas ecoregion: Brazil (n=5)MNRJ 7725 Barretinha, Rocas Atoll, RGN 03°51'36''S, 33°49'04''W 12 EU781975MNRJ 8609 Sela Gineta Island, Fernando de Noronha

Archipelago, PE03°48'49''S, 32°23'29''W 7 EU781976

MNRJ 8602 Ressurreta, Fernando de Noronha Archipelago, PE

03°48'49''S, 32°23'29''W 4 EU781977

UFRJPOR 6480 Ressurreta, Fernando de Noronha Archipelago, PE

03°48'49''S, 32°23'29''W 7.3 This study

UFRJPOR 6481 Ressurreta, Fernando de Noronha Archipelago, PE

03°48'49''S, 32°23'29''W 7.3 This study

Eastern Brazil ecoregion (n=1): NE BrazilUFRJPOR_4703 Parcel das Paredes, Abrolhos Archipelago,

BA17°58'S, 38°40'W 8 EU781979

211

Table S2. ST-Groups and its corresponding STs as suggested by the network topology of Leucetta floridana. (Number of sequences within each STG and ST)

STGA (110) STGB (1) STGC (4)

STGD (24) STGE (5) STGF (9) STGG (6) STGH (3)

ST1(98) ST21 (1) ST2 (3) ST12 (3) ST7 (1) ST4 (6) ST6 (5) ST17 (1)

ST3 (2) ST8 (1) ST14 (14) ST10 (4) ST5 (1) ST9 (1) ST18 (2)

ST13 (2) ST15 (4) ST11 (2)

ST19 (4) ST16 (3)

ST20 (1)

ST22 (1)

ST23 (1)

ST24 (1)

Table S3. AMOVA. Hypotheses of ST-groups (STG-Tests) for Leucetta floridana.with the obtained F.statistics: CT=variation among hypotheticalɸSTGs, SC= variation within STs among hypothetical groups and ST= variation within STs. All values were significant (10 000 permutations). Theɸ ɸchosen hypothese is indicated in bold. (Reference to principal text: STG1=STGA+STGB+STGC+STGD, STG2=STGE, STG3=STGF, STG4=STGG andSTG5=STGH).

STGS within Clade 1 Clade2 Clade3 ɸCT ɸSC ɸST

STG-Test1 STGA STGB STGC STGD STGE STGF STGG STGH 0.679 0.943 0.982STG-Test2 STGA STGB STGC STGD STGE STGF STGG STGH 0.759 0.931 0.983STG-Test3 STGA STGB STGC STGD STGE STGF STGG STGH 0.767 0.929 0.983STG-Test4 STGA STGB STGC STGD STGE STGF STGG STGH 0.757 0.922 0.981STG-Test5 STGA STGB STGC STGD STGE STGF STGG STGH 0.768 0.919 0.981STG-Test6 STGA STGB STGC STGD STGE STGF STGG STGH 0.667 0.939 0.980STG-Test7 STGA STGB STGC STGD STGE STGF STGG STGH 0.689 0.910 0.972STG-Test8 STGA STGB STGC STGD STGE STGF STGG STGH 0.752 0.882 0.971STG-Test9 STGA STGB STGC STGD STGE STGF STGG STGH 0.729 0.922 0.979STG-Test10 STGA STGB STGC STGD STGE STGF STGG STGH 0.724 0.894 0.971STG-Test11 STGA STGB STGC STGD STGE STGF STGG STGH 0.749 0.883 0.970STG-Test12 STGA STGB STGC STGD STGE STGF STGG STGH 0.692 0.903 0.970STG-Test13 STGA STGB STGC STGD STGE STGF STGG STGH 0.704 0.926 0.978STG-Test14 STGA STGB STGC STGD STGE STGF STGG STGH 0.719 0.895 0.970STG-Test15 STGA STGB STGC STGD STGE STGF STGG STGH 0.757 0.886 0.972

Table S4. AMOVA. Hypotheses of population structure (H) and the obtained F-statistics: ɸCT=variation among hypothetical groups, ɸSC= variation withinlocalities among hypothetical groups and ɸST= variation within localities. Significant values are indicated in bold (10 000 permutations). Ecoregions: SC=Southern Caribbean, NE Brazil=Northeastern Brazil, FN and RA=Fernando de Noronha and Atoll das Rocas and EB=Eastern Brazil. Localities:PR=Puerto Rico, AG=Anguilla, SN=Saint Martin, SB=Saba, AN=Antigua, SNT=Les Saintes, GU=Guadeloupe, MT=Martinique, SAN=San Andrés,URA=Uraba, SM=Santa Marta, CUR=Curaçao, RN=Rio Grande do Norte, CE=Ceará, AT= Rocas Atoll, FN=Fernando de Noronha Archipelago,ABR=Abrolhos Archipelago.

Tropical Northern Atlantic Tropical Southern Atlantic F- statisticsGreater Antilles

Eastern Caribbean SouthwesternCaribbean

SC NE Brazil FN andRA

EB ɸCT ɸSC ɸST

H1 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR -0.054 0.433 0.402H2 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR -0.061 0.433 0.398H3 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.251 0.318 0.489H4 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.170 0.347 0.458H5 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.319 0.271 0.503H6 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.317 0.271 0.502H7 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.270 0.305 0.492H8 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.262 0.308 0.490H9 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.377 0.223 0.516H10 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.399 0.201 0.520H11 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.388 0.209 0.516H12 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.378 0.218 0.514H13 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.5488 -0.1148 0.4970H14 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.421 0.081 0.468H15 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.436 0.061 0.471H16 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.532 -0.121 0.475H17 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.538 -0.148 0.470H18 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.532 -0.170 0.452H19 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.5494 -0.1192 0.496H20 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.541 -0.157 0.469H21 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.429 0.096 0.483H22 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.423 0.102 0.482H23 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.376 0.130 0.457H24 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.341 0.204 0.476H25 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.362 0.177 0.476H26 PR AG SN SB AN SNT GU MT SAN PAN URA SM CUR RN CE AT FN ABR 0.354 0.184 0.474

214

Table S5. Mismatch distribution values. SSD=sum of squared deviation, r=Harpending's raggedness index.

SSD SSD P value r r p value

Groups

Antigua 0.0280 0.18 0.1717 0.5357

Les Saintes 0.2791 0.071 0.3125 0.4099

Les Saintes + Guadaloupe 0.3067 0.0889 0.3580 0.2832

Saint Martin 0.0059 0.4314 0.2760 0.5558

Panama 0.1336 0.0666 0.2870 0.0246

San Andrés 0.1243 0.3188 0.2778 0.7281

Santa Marta 0.4535 0 0.7279 0.9399

Uraba 0.0898 0.6033 0.5555 0.8964

Puerto Rico 0.0229 0.2384 0.4722 0.4358

Fernando de Noronha 0.02194 0.6456 0.25 0.9243

Ceará 0.0542 0.5037 0.4 0.3706

Eastern Caribbean 0.4050 0.0003 0.1161 0.9996

Southwestern Caribbean 0.0515 0.0201 0.0829 0.054

Northeast Brazil 0.0062 0.452 0.2006 0.4564

FN and Rocas Atoll 0.0542 0.5042 0.4 0.3863

Tropical Southwestern Atlantic 0.0116 0.3149 0.1619 0.4790

Tropical Northwestern Atlantic 0.4707 0.0001 0.0677 1.0

Clade 1 0.3638 0.0 0.0931 1.0

Clade 2 = STG3 0.0275 0.2272 0.2037 0.3274

Clade 3 0.0330 0.487 0.1088 0.6829

STG1 0.3337 0 0.1130 0.9996

STG2 0.0072 0.7642 0.2 0.9411

STG4 0.003 0.745 0.2222 0.9268

STG5 0.0898 0.5988 0.5556 0.8925

All 0.4539 0.0001 0.0629 1.0

Chapter 4

GENERAL DISCUSSION

216

This study contributed to the knowledge of the biodiversity of calcareous sponges from the

Western Tropical Atlantic (WTA). Nineteen species, including 15 new species and four new

records, were added to the 67 previously known species from this area (Van Soest et al., 2016;

Van Soest, 2017; Azevedo et al., submitted), summing up a total of 86 calcareous sponges in the

WTA (Table 1).

Compared to regions with coastline extensions similar to the study area (ca. 29 000 km) such

as Australia and Japan, whose coastlines cover 25,670 and 29,751 km, respectively (CIA, 2013),

the richness of Calcarea is, unexpectedly, higher in the WTA. Sixty-seven species have been

reported from Australia (Leocorny et al., 2016; Van Soest et al., 2016) and 79 from Japan (Van

Soest et al., 2016). This is very surprising as the calcareous sponges from those Indo-Pacific

regions have been thoroughly studied (e.g. Japan: Hôzawa, 1916, 1918, 1929, 1933, 1940;

Hôzawa & Tanita, 1941; Tanita, 1942, 1943 and Australia: Lendenfeld, 1885; Carter, 1886;

Dendy, 1892, 1893; Dendy & Row, 1913; Dendy & Frederick, 1924; Row & Hôzawa, 1931;

Wörheide & Hooper, 1999, 2003).

Considering the biogeographical provinces within the study area, the major contribution was

to the TNA (Tropical Northwestern Atlantic), as the previous number of reported species from

this province was 23 and it was raised to 45. Twelve new species (Amphoriscus micropilosus sp.

nov., Arthuria vansoesti sp. nov., Ascandra torquata sp. nov., Clathrina aspera sp. nov., C.

curaçaoensis sp. nov., Ernstia sp. nov. , Leucandra caribea sp. nov., Leucandrilla

pseudosagittata sp. nov., Leucilla antillana sp. nov., Grantessa tumida sp. nov., Sycon

conulosum sp. nov., Sycon magniapicalis sp. nov.) and nine new records (Arthuria hirsuta,

Borojevia tenuispinata, Clathrina aurea, C. blanca, C. cylindractina, C. insularis, C. lutea, C.

mutabilis, and Nicola tetela) were reported herein.

The new 21 records from the TNA have certainly changed the distribution of the species

richness within the ecoregions of this province. Before this study, the Greater Antilles presented

217

the highest number (8) of species (Haeckel, 1870, 1872; Lehnert & Van Soest, 1998) and it was

followed by Bermuda, with six species (Poléjaeff, 1883; de Laubenfels, 1950). Now, the richest

ecoregion is the Southern Caribbean, with 23 species (Arndt, 1927; Haeckel, 1872; this study),

followed by the Eastern Caribbean, with 13 species (Duchassaing & Michelotti, 1864;

Schuffner, 1877; this study) and the Southwestern Caribbean, with nine species (Rozemeijer &

Dulfer, 1987; Valderrama et al., 2009; Klautau et al., 2013; this study). Additionally, all these

new records are from localities that once had very few or no valid records of Calcarea.

Previously, three species were recorded from Colombia (Rozemeijer & Dulfer, 1987;

Valderrama et al., 2009), two from Panama (Valderrama et al., 2009; Klautau et al., 2013), one

from Curaçao (Arndt, 1927) and none from Martinique. Nowadays, those numbers have

increased and there are five, seven, eight and 20 calcareous sponges from Colombia, Panama,

Curaçao and Martinique, respectively.

In the Tropical Southwestern Atlantic (TSA), the richness of calcareous sponges has

increased from 48 (Muricy et al., 2011; Cavalcanti et al., 2014, 2015; Azevedo et al., submitted)

to 54 species. Although known species were found again in the Abrolhos Archipelago (e.g. C.

lutea, C. aurea, L. roseus), three new species (Amphoriscus hirsutus sp. nov., Leucascus

luteoatlanticus sp. nov. and Grantia grandisapicalis sp. nov.) and two new records (C.

luteoculcitella and Ernstia vansoesti) were described from there. This evidenced that sampling

in unexplored sites within “known” localities can still reveal novelties in species richness and

distribution. Furthermore, the six records raised the number of valid species from the Eastern

Brazil ecoregion from 25 to 30, and now it is the richest ecoregion of the TSA (Borojevic, 1971;

Borojevic & Peixinho, 1976; Cavalcanti et al., 2014, 2015; Azevedo et al., submitted; this

study). Nonetheless, in some areas or localities within this ecoregion, such as the littoral of

Espírito Santo State, no Calcarea has ever been recorded (Muricy et al., 2011).

218

In the North Brazilian Shelf (NBS), only 11 species have been reported so far (Borojevic &

Peixinho, 1976; Cavalcanti et al., 2013; Van Soest, 2017). This apparent poor diversity of

Calcarea may be an artefact of the lack of investigation on sponge diversity in this province.

Future samplings in this region may provide important insights for understanding former

disjunct species distribution in the Caribbean Sea and NE Brazilian coast.

Comparative information on Porifera2 richness within the WTA is available across some

ecoregions. Considering the Southern Caribbean (SC) and the Eastern Brazil (EB),

Demospongiae is accounted as the richest class with 201 (88.2%, Van Soest et al., 2012; 2014;

Alvizu et al., 2013) and 121 (79.6%, Van Soest et al., 2016) species, respectively. Within

Calcarea, 23 species were recorded from the SC (10.1%) and 30 from the EB (19.7%, this

study). Homoscleromorpha has four species from the SC (1.7%) and one single record from the

EB (0.7%, Domingos et al., 2016). Interestingly, in these “well-sampled” ecoregions, the

observed percentages are close to the ones obtained for global diversity of Porifera:

Demospongiae, 83%; Calcarea, 8% and Homoscleromorpha: 1% (Van Soest et al., 2012).

Despite the limited knowledge on calcareous sponges within certain localities in the WTA, it

is possible to elucidate some general distribution patterns. Likewise Demospongiae (Van Soest

& Hajdu, 1997) and Homoscleromorpha species (Domingos et al., 2016), most of the calcareous

sponges (65.1%=56 species) presented herein are endemic from the WTA. Among the non-WTA

endemic species, 13 species (15.3%) are amphi-Atlantic3, eight (9.4%) are restricted to the

2 Hexactinellida and certain Demospongiae recorded at more than 200 m (published in VanSoest et al., 2014; Hestetum et al., 2016 for the SC) were not considered as the depthboundary for MEOW is 200 m deep (Spalding et al., 2007).

3 Five from the West African Transition (Tropical Atlantic realm; Borojevic & Peixinho,1872; Thacker, 1908), three from the Temperate Southern Africa realm (Klautau &Valentine, Urban 1908; Brøndsted, 1931) and five from the Temperate Northern Atlanticrealm (Topsent, 1892, Miklucho-Maclay, 1868; Borojevic & Boury-Esnault, 1987, Sarà &Gaino, 1971; Klautau et al., 2016).

219

Western Atlantic4, four are Atlanto-Pacific5 and the other four are cosmopolitan6. Although the

amphi-Atlantic distribution of certain calcareous sponges proved to be the result of

overconservative systematics (Solé-Cava et al., 1991; Klautau et al., 1994); the presence of

Arthuria hirsuta, originally described from South Africa (Klautau & Valentine, 2003), was

confirmed in the Caribbean (La Martinique, Pérez et al., submitted, Appendix: Table 1). In this

study, the occurrence of the Australian Clathrina luteoculcitella in NE Brazil was corroborated

by molecular methods and whether or not this correspond to a natural distribution should be

investigated. Unpublished results from the Pacotilles campaign revealed the presence of this

species also in Bequia (Eastern Caribbean), which would give some cues for the understanding

these unexpected distribution patterns. The records from the WTA of the cosmopolitan species

should be revised, except for those of Vosmaeropsis sericata as its type locality is included in

the WTA (Cavalcanti et al., 2015).

A higher number of Calcaroneans was recorded from the WTA (46 spp.) compared to

Calcineans (39 spp.); however, the proportion of trans-Amazonian species is higher in Calcinea

(33.3%=13 spp.7) than in Calcaronea (15.2%=7 spp.8). It is possible that there is some kind of

bias in this result, but the possibility that the calciblastula larva (from Calcinea) can disperse

better than the amphiblastula (from Calcaronea) underneath the Amazon plume should not be

discarded. However, this hypothesis requires further investigation.

4 Seven that can also be found in SE or S Brazil (Warm Temperate Southwestern Atlantic;Muricy et al., 2011; Azevedo et al., submitted) and one also recorded in the Northern Gulfof Mexico (WTNA; Haeckel, 1872).

5 Three with records in the Indo-Pacific (Wörheide & Hooper, 1999; Van Soestet al., 2015;Poléjaeff, 1883; Kelly et al., 2009) and one in the SE Pacific (Azevedo et al., 2015).

6 As the type locality of Vosmaeropsis levis is unclear, its distribution was not consideredherein.

7 Arthuria vansoesti, Ascandra torquata, Borojevia tenuispinata, Clathrina aspera, C. aurea,C. cylindractina, C. insularis, C. lutea, C. luteoculcitella (considering material fromPacotilles), C. mutabilis, Leucaltis clathria, Leucetta floridana and Nicola tetela.

8 Grantia kempfi, Leucandra barbata, L. rudifera, L. serrata, Leucilla uter, Sycettusa flamma,and Vosmaeropsis complanatispinifera.

220

A biogeographic province is a large geographic area characterised by the presence of

endemic species and delimited by particular geomorphological, hydrogeographic and

geochemical factors (Spalding et al., 2007). The >10% endemism criterion proposed by Briggs

(1974) has been widely employed for defining biogeographic provinces within the Tropical

Atlantic (e.g. Floeter & Soares-Gomez, 1999; Floeter & Gasparini, 2000; Briggs & Bowen,

2012). In this study, the percentage of endemic species in both the TNA and TSA surpassed the

10%, being 46.7% (21 out of 45 species) in the former and 40.7% (22 out of 54 species) in the

latter. Therefore, it is possible to say that the existence of the TNA and TSA provinces is

supported by the observed high endemism of calcareous sponges within these areas.

Nonetheless, there are several species that occur in more than one province. Among the 86

species from the WTA, 16 (18.6%) species are shared between the TNA, NBS and/or TSA.

Leucaltis clathria and Leucetta floridana have been recorded from the three provinces

(Borojevic & Peixinho, 1976; Haeckel, 1872; Klautau et al., 2013; Valderrama et al., 2009) and

they are considered species with a wide distribution. Grantia kempfi, Sycettusa flamma and

Vosmaeropsis complanatispinifera are shared between the NBS and the TSA (Borojevic &

Peixinho, 1976; Cavalcanti et al., 2015; Van Soest, 2017). Eleven species, Arthuria vansoesti,

Borojevia tenuispinata, Clathrina aspera, C. aurea, C. insularis, C. lutea, C. mutabilis,

Leucandra barbata, L. rudifera, Leucilla uter and Nicola tetela were recorded from the TNA

and TSA (Azevedo et al., submitted; Cóndor-Luján & Klautau, 2016, Muricy et al., 2011; this

study) but not in the NBS.

The occurrence of 13 species in the TNA and TSA provinces suggests a Caribbean-Brazilian

affinity within the class Calcarea. Although this affinity constitutes a novelty for Calcarea, it

was already observed in Demospongiae some decades ago (Hechtel, 1976; Colette & Rützler,

1977) and indicated in more recent studies (Moraes, 2011; Muricy et al., 2011; Moura et al.,

2016; Soares et al., 2016).

221

Hechtel (1976) reported numerous Demospongiae from both the West Indies and Brazil and

suggested that larvae of those species would cross the Orinoco and Amazon barriers,

considering the assumption of hard substrate availability close to the river's discharge which

would permit sponge settlement. One year later, Colette & Rützler (1977) not only reported

“Caribbean” sponges underneath the mouth of the Amazon River but also mentioned that there

was no apparent effect of freshwater and that there was abundant hard substrata on the bottom

of the Amazon mouth. Recently, Moura et al. (2016) reported the existence of a calcium

carbonate system underneath the Amazon river plume, which would act as a connectivity

corridor for wide depth–ranging reef-associated species, such as sponges.

The presence of calcareous species in both provinces also suggests a possible connectivity

between the TNA and TSA populations, which could be explained by a stepping-stone model of

population structure in the North Brazil Shelf Province (NBS) (in the Venezuelan Southeastern

Coast, Guyanas or in the Northern Brazilian Coast) facilitating the gene flow between these two

provinces. Therefore, it is quite probable to find several of the 13 shared species in the NBS (up

to date, only Leucetta floridana and Leucaltis clathria had been reported from the three

provinces).

In this study, L. floridana, one of the most widespread species in the WTA, was used as a

model to assess connectivity between TNA and TSA populations. The results evidenced five

structured populations: one widespread population maintaining gene flow between the TNA and

TSA provinces and four other isolated populations within the Caribbean Sea. The finding of a

panmitic widespread population in the WTA was really unexpected as it rejects the hypothesis

that the discharge of the Amazon River is an effective barrier to the maintenance of gene flow

among trans-Amazonian populations of calcareous sponges. Interestingly, a similar genetic

222

pattern was observed in the Demospongiae Chondrilla aff. nucula collected in the Caribbean

and in the Brazilian coast (Boury-Esnault et al., 20169).

Cryptic species are not unusual in Calcarea because of the plasticity of some characters in

certain genera and species. Studies assessing the genetic identity of former Brazilian

populations of Clathrina clathrus, C. primordialis and Borojevia cerebrum revealed that they

were cryptic species and a posterior discovery of diagnostic morphological characters justified

the erection of new species: C. aurea, C. cylindractina, C. conifera, and B. brasiliensis (Klautau

et al., 1994; Solé-Cava et al., 1991). In L. floridana, the observed genetic variability (0-1.8%)

is within the expected intraspecific range estimated for other leucettas (Valderrama et al., 2009)

and other Calcinean species (Rossi et al., 2011). Moreover, the high morphological variability of

L. floridana did not match the structured populations found in this study. Individuals with

different morphotypes (based on the shape of the oscula) and/or colour-morphs (light blue, pink

and white) were found in the same population or ST-group. Likewise, several individuals

collected in different habitats (completely protected from light, such as underneath boulders or

inside crevices, and semi-exposed to light, such as vertical slopes), depths (- 2 and - 40 m) or

localities (adjacent or distant) grouped in the same population. Therefore, there is no evidence to

support an ongoing cryptic speciation in L. floridana.

The high intraspecific variation within Leucetta floridana is comparable to that of other two

species with similar distribution in the WTA: C. lutea with 0-1.6% and C. mutabilis with 0-2%

(values taken from Chapter 3.4). An exploratory ITS sequence-type network of these two

Clathrina species (Appendix: Figures 2 and 3) showed that each species presented an ITS

sequence-type shared by Caribbean and Brazilian individuals (Brazil-Saint Martin and Brazil-

Curaçao-La Martinique-Panama, respectively). Furthermore, genetic analyses10 with C. lutea

sequences evidenced a similar phylogeographic and population pattern as those of L. floridana.

9 Results recently presented by N. Boury-Esnault in the II Workshopt ABC/CNRS (LIA MARRIO).

10 Results I recently presented in the II Workshop ABC/CNRS (LIA MARRIO).

223

According to Avise (2000), phylogeographic breaks shared among taxa with varying life

histories provide a rational basis for determining where the effective gene flow is restricted or

totally absent and thus, facilitate the detection of physical features that may act as important

barriers. Consequently, further studies should be addressed to verify the congruence of the

phylogeographic and population patterns of C. lutea and L. floridana as they might reveal

important traits for understanding connectivity and species evolutionary history in the WTA.

Leucetta floridana and L. potiguar are morphologically very similar species but present

strong diagnostic morphological characters (Lanna et al., 2009). Different from L. floridana, L.

potiguar is a NE Brazilian endemic species. In all known phylogenetic trees (Valderrama et al.,

2009; Klautau et al., 2013; Azevedo et al., submitted; this study), they appear as sister species.

Given their current geographic distribution and their phylogenetic position (Figure 2 of Chapter

3.4 of this study), it is possible to infer that the ancestral species of these pair of eucettas had a

widespread distribution in the WTA (including the Caribbean Sea and the NE Brazilian coast).

In this study, the genetic divergence estimated by the uncorrected p distance between L.

floridana and L. potiguar varied from 2.5 to 3.4%. If considered the mutation rate of 1% per

MY calculated for Demospongiae (Wörheide et al., 2004), it is possible to infer that the

cladogenetic event occurred ~4 MY BP, during the Pliocene (~5.3 – 2.6 MY BP). Interestingly,

the Amazon River became fully established at ~7 MY BP (Figuereido et al., 2009) and during

the early Pliocene, it started to drain water and sediments on a large scale to the Atlantic Ocean

(Latrubesse et al., 2010). As the discharge of freshwater and sediments from the Amazon River

has been recognised as responsible for the formation of pairs of sister species of reef fishes in

the WTA (Rocha, 2003 and references therein), this can also be suggested for this pair of sisters

species of Leucetta.

Table 1. List of recorded species from the Western Tropical Atlantic. Abbreviations: TL=type locality. Brazilian States - AL: Alagoas, BA: Bahia, CE:Ceará, ES: Espírito Santo, MA: Maranhão, PB: Paraíba, PA: Pará, PE: Pernambuco, RN: Rio Grande do Norte, RJ: Rio de Janeiro, SC: Santa Catarina,SP: São Paulo. FN: Fernando de Noronha. Provinces in the Tropical Atlantic (TA) – NBS: North Brazil Shelf, TNA: Tropical Northwestern Atlantic,TSA: Tropical Southwestern Atlantic, WAT: West African Transition. Other provinces - WTNA: Warm Temperate Northwest Atlantic, WTSA: WarmTemperate Southwestern Atlantic, WTSEP: Warm Temperate Southeastern Pacific. Realms - CIP: Central Indo-Pacific, TAA: Temperate Australasia,TNA: Temperate Northern Atlantic, TNP: Temperate Northern Pacific, TSAfrica: Temperate Southern Africa, TSAmerica: Temperate South America. E:Eastern, N: Northern, NE: Northeastern, S: Southern, SE: Southeastern, SW: Southwestern, W:Western. USA: United States of America. ** Indicates new species and * new records obtained in this study.

Species Distribution Ecoregion, Province, Realm

References

Subclass Calcaronea

1 Amphoriscus ancora Van Soest, 2017 Guyana Shelf (TL) Guianan, NBS Van Soest, 2017

2 Amphoriscus hirsutus sp. nov.** Abrolhos Archipelago (BA), NE Brazil(TL)

Eastern Brazil, TSA This study

3 Amphoriscus micropilosus sp. nov.** Curaçao (TL) S Caribbean, TNA This study

4 Amphoriscus synapta (Schmidt in Haeckel, 1872)

BA, NE Brazil (TL) NE or E Brazil, TSA Haeckel, 1872

5 Amphoriscus testiparus (Haeckel, 1872) Cuba (TL) Greater Antilles, TNA Haeckel, 1872

6 Amphoriscus urna Haeckel, 1872 Venezuela (TL) S Caribbean, TNA Haeckel, 1872

7 Grantessa anisactina Borojevic & Peixinho, 1976

Paraíba, NE Brazil (TL) E Brazil, TSA Borojevic & Peixinho, 1976

8 Grantessa tumida sp. nov. ** Curaçao (TL) S Caribbean, TNA This study

9 Grantia atlantica Ridley, 1881 Vitória Bank (ES), SE Brazil E Brazil, TSA Ridley, 1881

10 Grantia grandisapicalis sp. nov. ** Abrolhos Archipelago (BA), NE Brazil(TL)

E Brazil, TSA This study

11 Grantia kempfi Borojevic & Peixinho, 1976 Guyana Shelf Guianan, NBS Van Soest, 2017

AP, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976

PE (TL), NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Off Abrolhos, BA, NE Brazil E Brazil, TSA Borojevic & Peixinho, 1976

12 Leucandra amorpha Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883

13 Leucandra armata (Urban, 1908) Off Marajó Island, PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976

CE, PE, AL, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Off Abrolhos, BA, NE Brazil and ES, SE Brazil Brazil

E Brazil, TSA Borojevic & Peixinho, 1976

Francis Bay (TL), South Africa Natal, Agulhas, TSAfrica (realm)

Brøndsted, 1931; Urban, 1908, 1909

14 Leucandra barbata (Duchassaing & Michelotti, 1864)

Dry Tortugas Floridian, TNA de Laubenfels, 1936

Virgin Islands (TL) E Caribbean, TNA Duchassaing & Michelotti, 1864

Colombia SW Caribbean, TNA Rozemeijer & Dulfer, 1987

PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Off Abrolhos, BA, NE Brazil and ES, SE Brazil

E Brazil, TSA Borojevic & Peixinho, 1976

15 Leucandra caribea sp. nov.** Curaçao (TL) S Caribbean, TNA This study

16 Leucandra crassior Ridley, 1881 PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Vitória Bank (TL), ES, SE Brazil E Brazil, TSA Ridley, 1881

17 Leucandra crosslandi Thacker, 1908 PB, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Boa Vista Island, Cape Verde (TL) Cape Verde, WAT Thacker, 1908

18 Leucandra crustacea (Haeckel, 1872) Bermuda Bermuda, TNA De Laubenfels, 1950

Venezuela (TL) S Caribbean, TNA Haeckel, 1872

19 Leucandra curva (Schuffner, 1877) Barbados E Caribbean, TNA Schuffner, 1877

20 Leucandra hentschelii Brøndsted, 1931 NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Simonstown, South Africa (TL) Agulhas Bank, Agulhas,TSAfrica (realm)

Brøndsted, 1931

21 Leucandra multiformis Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883

22 Leucandra rudifera Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883

Trindade Island Trindade and Martin Vaz Islands, TSA

Moraes et al.., 2006

Arraial do Cabo, RJ, SE Brazil SE Brazil, WTSA, TSAmerica

Muricy et al., 1991, Muricy & Silva, 1999.

Cape Verde Cape Verde, WAT Thacker, 1908

23 Leucandra serrata Azevedo & Klautau, 2007 RN, NE Brazil NE Brazil, TSA Lanna et al., 2009

RJ, SE Brazil (TL) SE Brazil, WTSA, TSAmerica

Azevedo & Klautau, 2007

24 Leucandra typica (Poléjaeff, 1883) Bemuda (TL) Bermuda, TNA Poléjaeff, 1883

25 Leucandrilla pseudosagittata sp. nov.** Curaçao (TL) S Caribbean, TNA This study

26 Leucilla amphora Haeckel, 1872 Puerto Rico (TL) Greater Antilles, TNA Haeckel, 1872

Curaçao S Caribbean Arndt, 1927

Senegal, West Africa Sahelian Upwelling, WAT

Borojevic & Boury-Esnault, 1987

27 Leucilla antillana sp. nov.** Curaçao (TL) S Caribbean, TNA This study

La Martinique* E Caribbean, TNA Pérez et al., submitted: Leucilla sp. nov.

28 Leucilla sacculata (Carter, 1890) FN (TL), NE Brazil FN and Atoll das Rocas, TSA

Carter, 1890

29 Leucilla uter Poléjaeff, 1883 Bermuda (TL) Bermuda, TNA Poléjaeff, 1883

Curaçao S Caribbean, TNA Arnd, 1927

CE, RN, PB, PE, AL, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Abrolhos Archipelago*, Off Abrolhos and Off Belmonte, BA, NE Brazil and ES, SE Brazil

E Brazil, TSA Borojevic & Peixinho, 1976, this study

Arraial do Cabo, RJ, SE Brazil SE Brazil, WTSA, TSAmerica

Muricy et al., 1991; Muricy & Silva, 1999; Borojevic & Peixinho, 1976.

Philippines E Philippines, Western Coral Triangle, CIP

Poléjaeff, 1883

30 Leucosolenia horrida (Schmidt in Haeckel, 1872)

Florida. USA (TL) Floridian, TNA Haeckel, 1872

31 Leucosolenia salpinix Van Soest, 2017 Guyana Shelf (TL) Guianan, NBS Van Soest, 2017

32 Paraleucilla incomposita Cavalcanti et al., 2014

Arraial d'Ajuda (TL), BA, NE Brazil E Brazil, TSA Cavalcanti, et al., 2014

33 Paraleucilla oca Cavalcanti et al., 2014 Itaparica Island (TL), BA, NE Brazil E Brazil, TSA Cavalcanti, et al., 2014

34 Paraleucilla solangeae Cavalcanti et al., 2014

Guarajuba, BA, NE Brazil (TL) NE Brazil, TSA Cavalcanti, et al., 2014

35 Paraleucilla sphaerica Lanna et al., 2009 Potiguar Basin, NE Brazil (TL) NE Brazil, TSA Lanna et al., 2009

36 Sycettusa flamma (Poléjaeff, 1883) AP, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976

Off Bahia (TL), NE Brazil NE or E Brazil, TNA Poléjaeff, 1883

37 Sycon ampulla (Haeckel, 1872) Venezuela (TL?) S Caribbean, TNA Haeckel, 1972

RJ, SE Brazil and Florianópolis, SC, S Brazil (TL?)

SE Brazil, WTSA, TSAmerica

Haeckel, 1972; Mello-Leitão et al., 1961; Muricy & Silva, 1999.

Azores, Portugal Azores Canaries Madeira, Lusitanian, TNA (realm)

Topsent, 1892

38 Sycon barbadense (Schuffner, 1877) Barbados (TL) E Caribbean, TNA Schuffner, 1877

39 Sycon brasiliense Borojevic, 1971 Cabo São Tomé, RJ, SE Brazil (TL) E Brazil, TSA Borojevic, 1971

40 Sycon conulosum sp. nov.** Curaçao (TL) S Caribbean, TNA This study

41 Sycon formosum (Haeckel, 1870) Cuba (TL) Greater Antilles, TNA Haeckel, 1870

42 Sycon frustulosum Borojevic & Peixinho, 1976

PE (TL), NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

43 Sycon magniapicalis sp. nov.** Curaçao (TL) S Caribbean, TNA This study

44 Sycon vigilans Sarà & Gaino, 1971 Punta Manara (TL), Italy Mediterranean Sea, TNA (realm)

Sarà & Gaino, 1971

PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Urca, RJ (SE) and SC (S), Brazil SE Brazil, WTSA Borojevic &Peixinho, 1976; Mothes & Lerner, 1994.

45 Vosmaeropsis complanatispinifera Cavalcantiet al., 2015

AP, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976

RN (TL), NE Brazil E Brazil, TSA Borojevic & Peixinho, 1976; Cavalcanti et al., 2015

46 Vosmaeropsis levis Hozawa, 1940 Mexico (TL) Atlantic or Pacific Atlantic?

TNA? Hôzawa, 1940

Cabo de São Tomé, RJ E Brazil, TSA Borojevic, 1971

47 Vosmaeropsis sericata Ridley, 1881Cosmpolitan

Vitória Bank, SE Brazil (TL) E Brazil, TSA Ridley 1881

Cape Verde Cape Verde, WAT Thacker, 1908

Chile WTSEP, TSAmerica Breitfuss, 1898

SUBCLASS CALCINEA

48 Arthuria hirsuta Klautau & Valentine, 2003* Stil Bay (TL), Cape Town, South Africa

Agulhas Bank, Agulhas,TSAfrica (realm)

Klautau & Valentine, 2003

La Martinique* E Caribbean, TNA Pérez et al., submitted

49 Arthuria trindadensis Azevedo et al., submitted

Trindade Island Trindade and Martin Vaz Islands, TSA

Azevedo et al., submitted

50 Arthuria vansoesti sp. nov.**

Curaçao (TL) S Caribbean, TNA This study

La Martinique* E Caribbean, TNA Pérez et al., submitted: Arthuria sp. nov.

Abrolhos Archipelago, BA, NE Brazil*


51 Ascaltis agassizii Haeckel 1872 Florida (TL), United States of America Floridian, TNA Haeckel, 1872

Gulf of Mexico TNA? or TA? (realms) Haeckel, 1872

52 Ascalits panis (Haeckel, 1870) Florida, United States of America Floridian, WTNA Haeckel, 1870

Carrier Bow Cay, Belize W Caribbean, TNA Rutzler et al., 2014

Cape Verde, West Africa Cape Verde, WAT Thacker, 1908

53 Ascaltis poterium (Haeckel, 1872) PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Cosmopolitan Australia (TL) CIP? or TAA? (realms) Haeckel, 1872

Arctic Artcic (realm) Breitfuss, 1932

Chile TSA (realm) Ridley, 1881; Breitfuss, 1898

54 Ascaltis reticulum (Schmidt, 1862) PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976

Cosmopolitan PE, NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

ES, SE Brazil E Brazil, TSA Borojevic & Peixinho, 1976

Banyuls-sur-Mer (TL) and Adriatic Sea, Mediterranean Sea

Mediterranean Sea, TNA (realm)

Schmidt, 1862; Klautau et al., 2016 (more references in it)

Arctic Artcic (realm) Breitfuss, 1932

Japan TNP (realm) Hôzawa, 1940; Tanita, 1943

55 Ascandra ascandroides (Borojevic, 1971) PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976

Cabo São Tomé (TL), RJ, SE Brazil E Brazil, TSA Borojevic, 1971

Arraial do Cabo, RJ (SE) and SC (S) SE Brazil, WTSA Klautau & Borojevic, 2001; Mothes & Lerner, 1994

Bay of Biscay S European Atlantic Shelf, Lusitanian

Borojevic & Boury-Esnault, 1987

56 Ascandra atlantica (Thacker, 1908) NE Brazil NE Brazil, TSA Borojevic & Peixinho, 1976

Vitoria, SE Brazil E Brazil, TSA Borojevic & Peixinho, 1976

Boa Vista (TL), Cape Verde Cape Verde, WAT Thacker, 1908Klautau & Valentine

57 Ascandra torquata sp. nov.** Bocas del Toro, Panama* SW Caribbean, TNA Personal observation

Santa Marta, Colombia* SW Caribbean, TNA Personal observation

Curaçao S Caribbean, TNA This study

Arvoredo Archipelago (TL), SC, S Brazil

S Brazil, WTSA, TSAmerica

This study

58 Borojevia brasiliensis (Solé-Cava et al., 1991)

CE, NE Brazil* NE Brazil, TSA This study

Cabo de São Tomé, RJ, SE Brazil E Brazil, TSA Borojevic, 1971

Arraial do Cabo, RJ, SE Brazil (TL) SE Brazil, WTSA, TSAmerica

Solé-Cava et al., 1991Muricy et al., 2011 (and references in it)

59 Borojevia tenuispinata Azevedo et al, submitted

Curaçao* S Caribbean, TNA This study*

São Pedro e São Paulo Archipelago (TL), NE Brazil

São Pedro e São Paulo Islands, TSA

Azevedo et al, submitted

60 Borojevia trispinata Azevedo et al, submitted São Pedro e São Paulo Archipelago (TL), NE Brazil

São Pedro e São Paulo Islands, TSA

Azevedo et al, submitted

Arraial do Cabo, RJ, SE Brazil SE Brazil, WTSA Azevedo et al, submitted

61 Clathrina aspera sp. nov.** Curaçao (TL) S Caribbean, TNA This study

RN, NE Brazil NE Brazil, TSA This study

RJ, SE Brazil SE Brazil, WTSA This study*

62 Clathrina aurea (Solé-Cava et al., 1991) Bocas del Toro, Panama* SW Caribbean, TNA Personal observation

Martinique* Eastern Caribbean, TNA

Pérez et al., submitted*

FN, NE Brazil FN Archipelago and Atoll das Rocas, TSA

Muricy et al., 2001 (more references in it)

Potiguar Basin, NE Brazil NE Brazil, TSA Lanna et al.., 2009

Abrolhos Archipelago*, BA, NE Brazil E Brazil, TSA Azevedo et al., in press; this study

RJ (TL) and SP, SE Brazil SE Brazil, WTSA Solé-Cava et al., 1999; Muricy et al., 2011 (references in it)

S Peru WTSEP, TSAmerica Azevedo et al., 2015

63 Clathrina blanca Miklucho-Maclay, 1868*Cosmopolitan

Curaçao* S Caribbean, TNA This study

SP, SE Brazil and SC, S Brazil SE Brazil, WTSA Borojevic, 1971

Canary Islands (TL) and Norway Lusitanian and N European Sea, TNA (realm)

Miklucho-Maclay, 1868; Rapp,2006 (references in it)

Antarctic Southern Ocean (realm) Brøndsted, 1931

Japan TNP (realm) Hôzawa, 1929; Tanita, 1943

64 Clathrina conifera (Klautau & Borojevic, 2001)

NE Brazil NE Brazil, TSA Borojevic & Pexinho, 1976; Klautau & Valentine, 2003

Arraial do Cabo, RJ (TL), SE brazil SE Brazil, WTSA, TSAmerica

Borojevic & Pexinho, 1976; Klautau & Valentine, 2003

Adriatic Sea Mediterranean Sea, TNA (realm)

Klautau et al., 2006

65 Clathrina curaçaoensis sp. nov.** Curaçao (TL) S Caribbean, TNA This study

66 Bocas del Toro, Panama* SW Caribbean, TNA Personal observation

Clathrina cylindractina (Klautau et al., 1994)*

Arraial do Cabo, RJ, SE Brazil (TP) SE Brazil, WTSA Klautau et al., 1994

67 Clathrina hondurensis Klautau & Valentine, 2003

Belize (TP) W Caribbean, TNA Klautau & Valentine, 2003

Carrie Bow Cay, Belize W Caribbean, TNA Rützler et al., 2014

Bocas del Toro, Panama* SW Caribbean, TNA Personal observation


68 Clathrina insularis Azevedo et al., submitted Curaçao* S Caribbean, TNA This study

Martinique* E Caribbean, TNA Pérez et al., submitted: Clathrina sp. nov. 1

FN (TL), NE Brazil FN and Atoll das Rocas, TSA


69 Clathrina luteoculcitella Wörheide & Hooper, 1999*

Abrolhos, NE Brazil* Eastern Brazil, TSA This study

Great Barrier Reef (TL), Australia NE Australian Shelf, CIP

Wörheide & Hooper, 1999

70 Clathrina lutea Azevedo et al., submitted Florida, USA Floridian, TNA Klautau et al., 2013; Zea et al.,2014

Little San Salvador, Bahamas Bahamian, TNA Zea et al., 2014

Jamaica* Greater Antilles, TNA Lehner & Van Soest, 1998 (C. primordialis ), this study


Virgin Islands E Caribbean, TNA Klautau et al., 2013

Abrolhos Archipelago (TL) E Brazil, TSA Azevedo et al.., submitted; thisstudy*

71 Clathrina mutabilis Azevedo et al., submitted Panamá and Colombia* SW, TNA Personal observation*

Curaçao* S Caribbean, TNA This study*

Martinique* E Caribbean, TNA Pérez et al., submitted: Clathrina sp. nov. 2*

FN Archipelago, NE Brazil (TL) FN and Atoll das Rocas, TSA


RN, NE Brazil* NE Brazil, TSA This study

72 Ernstia citrea Azevedo et al., submitted Rocas Atoll, NE Brazil FN and Atoll das Rocas, TSA


Abrolhos Archipelago, NE Brazil* Eastern Brazil, TSA This study

73 Ernstia multispiculata Azevedo et al., submitted

Rocas Atoll, NE Brazil FN and Atoll das Rocas, TSA


74 Ernstia santipauli Azevedo et al., submitted São Pedro e São Paulo Archipelago (TL), NE Brazil

São Pedro and São Paulo Islands


75 Ernstia sp. nov. ** La Martinique (TL) E Caribbean, TNA Pérez et al., submitted; Fontana et al., in prep.

76 Ernstia rocasensis Azevedo et al., submitted Rocas Atoll (TL) and FN Archipelago, NE Brazil

FN and Atoll das Rocas, TSA

Azevedo et al., submitted; this study

Abrolhos Archipelago, BA, NE Brazil* E Brazil, TSA Azevedo et al., submitted; this study

77 Ernstia sulphurea Miklucho-Maclay, 1868 FN Archipelago, NE Brazil FN and Atoll das Rocas, TSA


Trindade Island, SE Brazil Trindade and Martin Vaz Islands, TSA


Lanzarote Beach, Canary Islands (TL) Azores Canaries Madeira, Lusitanian

Miklucho-Maclay, 1868

78 Leucaltis clathria Haeckel, 1872 Florida, USA (TL) Floridian, TNA Haeckel, 1872

Bocas del Toro, Panama SW Caribbean, TNA Klautau et al., 2013;Zea et al., 2014; personal observation*

Guyana Shelf Guianian, NBS Van Soest, 2017

PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976;

CE, RG, AL, SE, NE BraziL NE Brazil, TSA Borojevic & Peixinho, 1976; Lanna et al., 2009

ES, SE Brazil E Brazil, TSA Borojevic & Peixinho, 1976.

79 Leucascus luteoatlanticus sp. nov.** Abrolhos Archipelago (TL), BA, NE BraziL


80 Leucascus roseus Lanna et al., 2007 Potiguar Basin, RN, NE Brazil NE Brazil, TSA Lanna et al., 2009

Abrolhos Archipelago, BA, NE Brazil Eastern Brazil, TSA Cavalcanti et al., 2013; this study*

RJ and SP (TL), SE Brazil Southeastern Brazil, WTSA

Cavalcanti et al., 2013; Lanna et al., 2007;

81 Leucascus aff. simplex sensu Cavalcanti et al., 2013

PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976; Cavalcanti et al., 2013

82 Leucetta floridana (Haeckel, 1872) Bermuda Bermuda, TNA de Laubenfels, 1950

Bahamas Bahamian, TNA Zea et al., 2014

Florida (TL) Floridian, TNA Haeckel, 1872

Jamaica and Cayman Islands Greater Antilles, TNA Lehner & van Soest, 1998; Milaslovich et al., 2011

Panama and Colombia (Santa Marta*) SW Caribbean, TNA Klautau et al., 2013; this study (Santa Marta)*; Valderrama et al., 2009; Zea et al., 2014


Lesser Antilles* including St. Eustatius E Caribbean, TNA García-Hernández et al., 2016; Pérez et al., submitted; this study;

MA and PA, N Brazil Amazonia, NBS Borojevic & Peixinho, 1976

MA, CE, RN, PA, PE and AL, NE Brazil

NE Brazil, TSA Borojevic & Peixinho, 1976; Lanna et al, 2009; Valderrama et al., 2009

FN Archipelago and Rocas Atoll FN and Atoll das Rocas, TSA

Borojevic & Peixinho, 1976; Moraes et al., 2006; Valderrama et al., 2009

Abrolhos, BA, NE Brazil and ES, SE Brazil.

E Brazil, TSA Borojevic & Peixinho, 1976; Valderrama et al., 2009

83 Leucetta imberbis (Duchassaing & Michelotti, 1864)

Virgin Islands (TP) E Caribbean, TNA Duchassaing & Michelotti, 1864

Santa Marta, Colombia SW Caribbean, TNA Rozemeijer & Dulfer, 1987

Jamaica Greater Antilles, TNA Lehner & Van Soest, 1998

84 Leucetta potiguar Lanna et al., 2009 Potiguar Basin (TL), RN and CE, NE Brazil

NE Brazil, TSA Lanna et al., 2009; Valderramaet al., 2009

85 Leucettusa corticata (Haeckel, 1872) Cuba Greater Antilles, TNA Haeckel, 1872

New Zealand S New Zealand?, TAA Kelly et al., 2009

86 Nicola tetela (Borojevic & Peixinho, 1976) Curaçao* S Caribbean, TNA This study

Sint Eustatius E Caribbean, TNA García-Hernández et al., 2016

Abrolhos (TL), BA, NE Brazil E Brazil, TSA Borojevic & Peixinho, 1976

Chapter 5

CONCLUSIONS AND PERSPECTIVES

234

This study certainly contributed to our comprehension of the marine biodiversity of the Western

Tropical Atlantic, filling in the gap of knowledge on calcareous sponges from poorly studied areas

such as the Caribbean Sea. The 86 species now reported from the WTA represents approximately

12% of all known Calcarea (ca. 730 species; Van Soest et al., 2012; Rapp, 2013; Cavalcanti et al.,

2013, 2014, 2015 ; Azevedo et al., 2015, submitted; Van Soest & Voogd, 2015; Klautau et al., 2016;

Leocorny et al., 2016; Van Soest, 2017; Azevedo et al., submitted). However, the richness of this

region is still underestimated as new species are still being discovered from new localities in the

Caribbean Sea (Lesser Antilles, Pacotilles campaign, pers. obs.) and in the NE Brazil (Bahia, F.

Cavalcanti, pers. comm.). Besides, new species of the subclass Calcaronea from Colombia and

Panama (not included in this study) are being described.

As suggested for other marine taxa (Ludt & Rocha 2005), the current distribution of calcareous

sponges may be related to the major changes occurred during the Pleistocene in the WTA. It would

be interesting to compare phylogeographic patterns across different genera in Calcarea (e.g.

Clathrina and Leucetta) to see if they corroborate this hypothesis.

As mentioned before, the systematics of calcareous sponges is considered particularly difficult

because of the plasticity of some of the characters (Van Soest et al., 2012). In this study, molecular

approaches played an important role in providing useful information for the identification of species

whose morphological characters were very plastic (e.g., diactines in Ascandra torquata) and for the

allocation of certain species in its correct genus (e.g. Nicola tetela). In the latter case, I should point

out the importance of revising the type material and not to limit to the examination of the original

description.

The Amazon River outflow may constitute a semi-permeable barrier to calcareous sponges. It

would allow the dispersal of some species such as Leucetta floridana, Clathrina aurea, C. insularis,

C. lutea, and C. mutabilis (whose occurrences in the TNA and TSA were corroborated by

morphological and molecular methods) but also, restrict the dispersal of other species (the TNA and

235

TSA endemic species). At least, for one calcareous species, L. floridana, the discharge of freshwater

and sediments from the rivers of the TWA (Orinoco and Amazonas) does not constitute an effective

barrier to gene flow. Studies using other molecular markers are recommended to give more support

to this finding.

In order to fully understand marine connectivity, it is also very important to perform studies on

the reproduction of the target species (e.g. larval behaviour) as it provides quite useful information

to explain the observed genetic patterns. Non-physical barriers such as chemical defenses and adult

ecology should be considered as well. As most of the mentioned information is scarce or even

inexistent in Calcarea, it is crucial to begin investigating these aspects if a more precise

understanding of calcareous sponges connectivity in the Western Tropical Atlantic is desired.

Chapter 6

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APPENDIX 1

Calcareous sponge species recorded in Martinique . C= Cave (dark, semi-dark caves, tunnels, overhangs), M= Mangrove, R= Reef (hard bottoms ingeneral, including coral reefs). Species marked by an * indicate new records for the Eastern Caribbean. Modified from Pérez et al., submitted.

Order Family Species External traits Genbank Accesion Numbers

Habitat

Clathrinida Clathrinidae

Arthuria hirsuta (Klautau & Valentine, 2003) *

White, massive to semi-spherical clathroid body. Hispid.

KX355564 C

Arthuria sp. nov. Yellow, clathrate with water-collecting tube. Soft.

C

Clathrina aurea Solé-Cava, Klautau, Boury-Esnault, Borojevic & Thorpe, 1991*

Light yellow, clathrate with several oscula.

KX355565 -KX355567

C

Clathrina sp. nov. 1* Light yellow, clathrate with water-collecting tube. Soft.

KX355568 -KX355571

C

Clathrina sp. nov. 2* Light yellow, clathrate and thin encrusting. Soft.

KX355572 C

Clathrina sp. White clathrate. CErnstia sp. nov. Lemon yellow clathrate. Soft. C

Leucosolenida Amphoriscidae Leucilla sp. nov. Bright white, tubular with apical osculum. Delicate.

C

Leucilla sp. 1 CLeucilla sp. 2 CAmphoriscus sp. C

Grantiidae Leucandra sp. CLeucettidae Leucetta floridana (Haeckel,

1872)*White, massive with apical osculum. Rough.

KX355573 -KX355575

C, R

Sycettidae Sycon sp. Beige, globular with apical osculum surrounded by crown.

M

APPENDIX 2

Median-Joinig network of Clathrina lutea. ST2 is shared between Caribbean (Saint Martin)and Brazilian specimens of C. lutea. Taken from presentation at II Workshop MARRIO.

APPENDIX 3

Preliminary Median-Joining network of Clathrina mutabilis. ST1 is shared betweenCaribbean (Curaçao, Martinique and Panama) and Brazilian specimens of C. mutabilis.

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