The molecular phylogeny of the type-species of OodiniumChatton, 1912 (Dinoflagellata: Oodiniaceae), a highlydivergent parasitic dinoflagellate with non-dinokaryoticcharacters

Fernando Gomez • Alf Skovgaard

Received: 10 October 2014 / Accepted: 20 November 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Oodinium pouchetii (Lemmermann, 1899)

Chatton, 1912, the first described parasitic dinoflagel-

late, is the type of the Oodiniaceae Chatton, 1920. In

the taxonomical schemes, this family of metazoan

parasites includes Amyloodinium Brown & Hovasse,

1946 and Piscinoodinium Lom, 1981 that are respon-

sible of important damages in fish aquaculture.

Species of Oodinium Chatton, 1912 have unique

characteristics such as the possession of both non-

dinokaryotic and dinokaryotic nuclei within the life-

cycle, and the absence of the transversal (cingulum)

and longitudinal (sulcus) surface grooves in the

parasitic stage. We provide the first molecular data

for the genus Oodinium from specimens of O.

pouchetii infecting the chordate Oikopleura sp. (Tu-

nicata: Appendicularia) off the coasts of Brazil.

Although O. pouchetii lacks dinokaryotic characters

in the parasitic stage, the SSU rDNA phylogeny

revealed that it forms a distinct fast-evolved clade that

branches among the dinokaryotic dinoflagellates.

However, there is no clear relationship with other

dinoflagellates. Hence, the taxonomic affinity of the

family Oodiniaceae is unclear at the moment.

Introduction

A large number of dinoflagellates are known to

parasitise marine vertebrates and invertebrates (e.g.

Chatton, 1920; Cachon & Cachon, 1987; Shields,

1994). The first parasitic dinoflagellate to be described

was Oodinium pouchetii (Lemmermann, 1899) Chat-

ton, 1912 (= Gymnodinium pulvisculus (Ehrenberg,

1832) Stein, 1878 sensu Pouchet, 1885), an ectopar-

asite on the tail of the tunicate appendicularian

Oikopleura dioica Fol (see Pouchet, 1884, 1885).

The genus Oodinium Chatton, 1912 also includes

another parasite of appendicularians, O. fritillariae

Chatton, 1912, the parasite of annelids O. dogielii J.

Cachon & M. Cachon, 1971, the parasites of chaetog-

naths O. jordanii McLean & Nielsen, 1989 and O.

inlandicum Horiguchi & Ohtsuka, 2001, and an

undescribed species that parasitises ctenophores and

cnidarians (Chatton, 1912; Cachon & Cachon, 1971;

Electronic supplementary material The online version ofthis article (doi:10.1007/s11230-014-9538-8) contains supple-mentary material, which is available to authorized users.

F. Gomez (&)

Laboratory of Plankton Systems, Oceanographic Institute,

University of Sao Paulo, Praca do Oceanografico 191,

Cidade Universitaria, Butanta, Sao Paulo 05508-900,

Brazil

e-mail: fernando.gomez@fitoplancton.com

A. Skovgaard

Department of Veterinary Disease Biology, Faculty of

Health and Medical Sciences, University of Copenhagen,

Stigbøjlen 7, 1870 Frederiksberg C, Denmark

Present Address:

A. Skovgaard

Department of Environmental, Social and Spatial Change,

University of Roskilde, 4000 Roskilde, Denmark

123

Syst Parasitol (2015) 90:125–135

DOI 10.1007/s11230-014-9538-8

McLean & Nielsen, 1989; Mills & McLean, 1991;

Horiguchi & Ohtsuka, 2001). The oodinids that

parasitise fishes have been removed from the genus

Oodinium. Oodinium pillularis Schaperclaus, 1954

and O. limneticum Jacobs, 1946 have been transferred

to the genus Piscinoodinium Lom, 1981 and O.

ocellatum Brown, 1931 has been transferred to

Amyloodinium Brown & Hovasse, 1946, together with

the parasite of salps Amyloodinium amylaceum (Barg-

oni, 1894) Brown & Hovasse, 1946 (see Brown &

Hovasse, 1946; Lom, 1981). Oodinium cyprinodon-

tum Lawler, 1967 has been transferred to Crepidoodi-

nium Lom & Lawler, 1981, a genus that includes other

two species (Lom et al., 1993). Another parasite of

fishes, the type-species of the monotypic genus

Oodinioides Reichenbach-Klinke, 1970, is also clas-

sified within the family Oodiniaceae Chatton, 1920

(see Cachon & Cachon, 1987). Species of the genera

that infect fishes cause great damage in aquaculture

(Lauckner, 1984).

Historically, most species of parasitic dinoflagel-

lates have been classified as members of the orders

Blastodiniales or Syndiniales (Fensome et al., 1993).

Molecular phylogeny has demonstrated that Blastodi-

niales is an artificial assemblage with several of its

members now distributed among the dinokaryotic

dinoflagellates (Litaker et al., 1999; Saldarriaga et al.,

2001; Kuhn & Medlin, 2005; Levy et al., 2007;

Skovgaard et al., 2007; Gomez et al., 2009a; Coats

et al., 2010; Gomez & Skovgaard, 2014). Even genera

such as Amyloodinium and Piscinoodinium, classified

within the family Oodiniaceae, belong to different

phylogenetic clades. Amyloodinium ocellatum

branches with thecate dinoflagellates, the fish-killer

pfiesterids and cryptoperidiniopsoids, the parasites of

diatoms of the genus Paulsenella Chatton, 1920 and

the parasites of tintinnid ciliates of the genus Tintinn-

ophagus Coats, 2010 (see Litaker et al., 1999; Kuhn &

Medlin, 2005; Coats et al., 2010). Piscinoodinium spp.

branch with the so-called ‘thin-walled’ dinoflagellates

with mutualistic symbionts of reef-forming inverte-

brates and planktonic rhizarians (Pelagodinium Siano,

Montresor, Probert & de Vargas, 2010; Symbiodinium

Freudenthal, 1962), and free-living photosynthetic

species (Polarella Montresor, Procaccini & Stoecker,

1999 and Biecheleria Moestrup, Lindberg & Daugb-

jerg, 2009) (see Levy et al., 2007; Siano et al., 2010).

Whether one or both of the clades Amyloodinium-

pfiesterids or Piscinoodinium-‘thin-walled’ dinoflagellates

belongs to the family Oodiniaceae depends on the

phylogenetic position of the type Oodinium pouchetii.

However, up to date no sequence from the type-genus

of Oodiniaceae is available.

The heterotrophic dinoflagellates of the genus

Oodinium have been the subject of numerous cyto-

logical investigations that revealed the possession of

both non-dinokaryotic and dinokaryotic nuclei within

the life-cycle (Chatton, 1920; Hovasse, 1935; Cachon

& Cachon, 1971, 1977; McLean & Nielsen, 1989;

Horiguchi & Ohtsuka, 2001). During the early vege-

tative stage, the cell is ovoid and attached to its host by

a peduncle through which adsorption of metabolites

takes place (Cachon & Cachon, 1971). It lacks typical

dinoflagellate characters such as flagella and trans-

verse or longitudinal grooves. On the other hand, it has

organelles such as trichocysts and a pusule. At this

stage, the parasite and its nucleus grow tremendously

without any cellular division. The cell covering of the

trophont comprises thecal plates. However, assign-

ment of these plates into conventional Kofoid’s

tabulation system is difficult, due to the lack of

conventional reference points for the recognition of

plate series (Hovasse, 1935; Horiguchi & Ohtsuka,

2001). All these unique characters make Oodinium a

key genus for inferring dinoflagellate evolution.

During plankton surveys along the coasts of Brazil,

specimens of the appendicularian Oikopleura sp.

infected with Oodinium pouchetii were collected. This

study describes the life-cycle, with the first micrographs

of the dinospores, and provides the first molecular data

for O. pouchetii, the type-species of the family Oodi-

niaceae and the first described parasitic dinoflagellate.

Materials and methods

Sampling and isolation of materials

Specimens of O. pouchetii were isolated in the South

Atlantic Ocean at two locations off the coast of Sao

Paulo State, Brazil, at Sao Sebastiao Channel

(23�5004.0500S, 45�24028.820W) in July 2013 and off

Ubatuba (23�32020.1500S, 45�5058.9400W) in June

2014. The specimens were collected from the surface

using a phytoplankton net (20 lm mesh size). The live,

concentrated samples were examined in Utermohl

chambers at magnification of 920 with an inverted

microscope (Eclipse TS-100, Nikon, Tokyo) and

photographed with digital camera (Cyber-shot DSC-

126 Syst Parasitol (2015) 90:125–135

123

W300, Sony, Tokyo) mounted on the microscope

eyepiece. Trophonts that had detached before the first

division were micropipetted individually with a fine

capillary into a clean chamber and washed several

times in a series of drops of 0.2 lm-filtered and

sterilised seawater. Finally, trophonts of O. pouchetii

were placed in 0.2 ml Eppendorf tubes filled with

several drops of absolute ethanol. We used naturally

detached trophonts instead of cutting the attachment

peduncle of trophonts still attached to the host. In the

case of other ectoparasites that do not naturally

detached from the host (i.e. species of Ellobiopsis

Caullery, 1910), this manipulation increases the

probability of contamination with DNA from the

damaged host (Gomez et al., 2009a). Samples were

kept at room temperature and in darkness until

molecular analysis could be performed.

Life-cycle observations

After obtaining samples for DNA analysis, specimens

of O. pouchetii were used to investigate cell division,

and the morphology and behaviour of the dinospores.

Recently detached trophonts prior the first division

were individually placed in Utermohl chambers with

0.2 lm-filtered seawater, and periodically observed

under the microscope. Other recently detached troph-

onts were individually placed in 12-well tissue culture

plates with 0.2 lm-filtered seawater. In order to have

controlled environmental conditions, the plates were

placed in an incubator used for microalgae culturing,

at 23�C, 100 lmol photons m-2s-1 from cool-white

tubes and 12:12 h Light:Dark photoperiod. The cell

division and the swarmers were periodically photo-

graphed under light microscope.

PCR amplification and sequencing

The sample tubes containing specimens of O. pouch-

etii in ethanol were centrifuged and dried by placing

them overnight in a desiccator at room temperature.

Then 30 ll of sterile DNase-free water was added to

each sample tube and the samples were sonicated

through three 10-second pulses at an output setting of

1.0 (Coats et al., 2010) using a Virsonic 600 sonicator

(SP Scientific, Gardiner, NY) equipped with a micro-

tip. Ten microlitres of the crude cell lysate was used

for each polymerase chain reaction (PCR) amplifica-

tion. SSU rDNA was amplified using the primers

EukA and EukB (Medlin et al., 1988). A single PCR

was in one case (isolate #58) sufficient for obtaining a

PCR product suitable for sequencing. In two cases

(isolates #23 and #54), a nested PCR was performed

using 0.5 ll of the initial PCR product as template for a

second PCR with the newly-designed primers Ask1F

(5’-GAT TAA GCC ATG CAT GTC TCA G-3’) and

Ask2R (5’-GAA ACC TTG TTA CGA CTT CTC

C-3’). All PCR amplifications were performed in 25 ll

reaction volumes containing 1.25 unit of Biotaq

polymerase (Bioline Reagents Limited, London,

UK), buffer supplied with the polymerase, MgCl2 at

3.0 mM, dNTPs at 1.6 mM, and the forward and

reverse primers at 1.0 mM. The PCR was run in a

T100TM Thermal Cycler (Bio-Rad Laboratories, Her-

cules, CA) under the following conditions: initial

denaturation (94�C/2 min); 35 cycles of denaturation

(94�C/15 s), annealing (57�C/30 s), and extension

(72�C/2 min); final extension (72�C/7 min). Nested

PCR was run as touchdown PCR for 30 cycles: initial

denaturation (94�C/2 min); then 10 cycles of ‘touch-

down’ PCR with denaturation (94�C/15 s), 10 anneal-

ing steps (30 s) decreasing from 65 down to 56�C (1�C

decrease with each cycle), and extension (72�C/2

min); followed by 20 cycles of denaturation (94�C/15

sec), annealing (55�C/30 sec), and extension (72�C/2

min); and final extension (72�C/7 min). PCR products

were purified using Illustra GFX PCR DNA and Gel

Purification Kit (GE Healthcare, Little Chalfont, UK)

and sequenced bi-directionally with an ABI3730xl

sequencer (Macrogen Europe, Amsterdam, The Neth-

erlands) using the same primers as used for PCR and

additional internal primers [ND2F, ND7R, ND9R

(Ekelund et al., 2004); 528f (Elwood et al., 1985);

1209f (Giovannoni et al., 1988)]. Sequence reads were

aligned and assembled using the software ChromasPro

1.75 (Technelysium, Brisbane, Australia).

Phylogenetic analyses

Three phylogenetic analyses were performed based on

nearly complete SSU rDNA sequences. One analysis

(Alveolata tree) was based on an alignment of 73

sequences for species of the major alveolate groups

plus four cercozoan sequences serving as the out-

group. The second analysis (Dinokaryota tree) com-

prised sequences for dinokaryotes most similar to O.

pouchetii as identified through BLAST search (http://

blast.ncbi.nlm.nih.gov/Blast.cgi; Altschul et al.,

1997). Furthermore, sequences of a wide selection of

dinokaryotes were included, aiming at including spe-

cies of all mutualist symbiotic and parasitic

Syst Parasitol (2015) 90:125–135 127

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dinokaryote genera for which sequences were avail-

able. Two perkinsid and two syndinean sequences

were used as outgroups. The final matrix contained 64

sequences. The Dinokaryota tree analysis was repe-

ated with the addition of the two shorter O. pouchetii

sequences and three short sequences with highest

similarity to O. pouchetii according to a BLAST

search.

Sequences were aligned using Clustal X v2.1

(Larkin et al., 2007) and non-informative sites were

removed using Gblocks (Castresana, 2000) with

parameters set for less stringent conditions (minimum

number of sequences for a flanking position: 28;

minimum length of a block: 5; allow gaps in half

positions). Final alignments of the SSU rDNA

sequences spanned over 1,630 and 1,737 positions

(Alveolata and Dinokaryota trees, respectively).

Bayesian phylogenetic trees were constructed with

MrBayes v3.2 (Huelsenbeck & Ronquist, 2001).

MrBayes settings for the best-fit model (GTR?I?G)

were selected by AIC in MrModeltest 2.3 (Nylander,

2004). Four simultaneous Monte Carlo Markov chains

were run from random trees for a total of 2,000,000

generations in two parallel runs. A tree was sampled

every 100 generations, and the first 2,000 trees (burn-

in) were discarded before calculating posterior prob-

abilities and constructing Bayesian consensus trees.

The newly-generated sequences were deposited in

DDBJ/EMBL/GenBank under accession numbers

KM879217–KM879219.

Results

Life-cycle

Specimens of the appendicularian Oikopleura sp. were

observed with ectoparasitic trophonts of O. pouchetii.

The tails of the appendicularians contained up to

sixteen trophonts of different sizes (Fig. 1A–B).

Larger trophonts (up to 200 lm) showed dark brown

pigmentation and an oval shape (Fig. 1C–D); smaller

specimens were pyriform with yellowish pigmentation

(Fig. 1A–B; see video at http://youtu.be/DlCPkpK7

oSM). In younger specimens, the nucleus was distally

placed (opposite side of the attachment peduncle),

while the nucleus was centrally located in the larger

specimens. These large trophonts with dark brown

pigmentation were usually found detached from the

host at the bottom of the settling chambers; these were

isolated for PCR analysis. There was a high proportion

of detached mature trophonts on the bottom of the

settling chambers compared with those still attached to

the hosts. This may suggest that the manipulation due

to plankton net sampling and/or the stress or damage

of the captured hosts induce the final of the trophic life

when the parasite leaves its host by breaking its

peduncle attachment. The rupture of the stalk always

preceded sporogenesis. When the plankton sample

was allowed to settle for several hours, aggregations

of c.200 sporocysts were observed (Fig. 1Q–R).

Detached trophonts were individually placed in fil-

tered seawater in order to observe the different steps of

the sporogenesis process avoiding the influence of

other organisms and under controlled environmental

conditions. The divisions proceeded without inter-

ruption to form progressively smaller sporocysts that

eventually became liberated as flagellated dinospores

after 8–12 hours (Fig. 1F–W). The palintomic sporo-

genesis began with the retraction of the cell cytoplasm

(Fig. 1F, H). The cleavage of the trophont was longi-

tudinal (slightly oblique to the axis of the attachment

peduncle) (Fig. 1G, I). In the first generation, the two

daughter cells sometimes remained inside the theca

(Fig. 1K) and later they left behind an empty theca

(Fig. 1J). Further divisions always occurred outside

the theca. The nucleus divided before the cytoplasmic

furrow reached the nuclear level. The cell cytokinesis

began before the complete division of the nuclei

(Fig. 1L, M). Different steps of the development of the

first generation of daughter cells are shown in

Figs. 1G, I, K–O and the second generation of

daughter cells are illustrated in Fig. 1P–O. The dark

brown pigmentation of the adult trophont was pro-

gressively diluted along the successive divisions. This

suggested that this pigmented substance is a reserve

compound consumed during the sporogenesis. The

sporocyst generations followed one after the other

without any compensating growth until swarmers with

dinoflagellate characteristics appeared. The sporo-

genic divisions proceeded without pause and were

synchronic in the first generations. However, this

synchrony was lost in the last steps of the sporogenesis

(Fig. 1Q, S). This made it difficult to account the final

number of infective dinospores produced by each

trophont because the dinospores, swarmers, began to

swim when sporocysts were yet devoid of flagella. An

aggregation of c.200 immotile sporocysts was

128 Syst Parasitol (2015) 90:125–135

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observed after eight hours at a temperature of 23�C

(Fig. 1R). In the last step, these immotile sporocysts

divided again and formed a couple of dinospores each

that developed two flagella. The couple of dinospores

remained together, and began to move the flagella

(Fig. 1S, T). The transversal flagellum first vibrated

Fig. 1 Life-cycle stages of the ectoparasite Oodinium pouchetii from the coast of Brazil. Ectoparasitic phase of young trophonts

infecting Oikopleura sp. (A–E) and phase of multiplication (palintomic sporogenesis) (D–W). A, B, Young trophonts; C–E, Mature

trophont; F–H, Recently detached trophonts (note the retraction of the cytoplasm inside the theca and the large round nucleus); G, I, K,

L–O. First generation of daughter cells; G–I, The cleavage is longitudinal, slightly oblique to the axis of the attachment peduncle. The

daughter cells and the nuclei are oval; J, Empty theca; K–O, Division of the first generation; P, Note that cell division begins with

segmentation of the cell; P, Q, Second generation of daughter cells; R, Sporocysts of the penultimate generation; S, Sporocysts of the

penultimate generation (large) and sporocysts of the last generation that formed each a pair of swarmers. The inset shows the last

division; T–W, Recently formed dinospores, swarmers, forming couples; U, V, Note the longitudinal flagellum (lf), and the transversal

flagellum (tf) that vibrates outside the cingulum. Scale-bars: E–R, 20 lm; S–W, 10 lm

Syst Parasitol (2015) 90:125–135 129

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outside the cingulum, but eventually beat inside the

cingulum when the cells began to swim (Fig. 1U–W;

see video at http://youtu.be/DlCPkpK7oSM). The

swarmers began to disperse while other dinospores were

yet devoid of flagella. The small dinospores swam with

the typical dinoflagellate rotation, following a straight

trajectory at different levels in the settling chamber, and

with sudden accelerations. This feature made it difficult

to record the trajectories at high magnification. We

counted c.200 immotile sporocysts from each detached

trophont. Subsequently each immotile sporocyst divi-

ded into two swarmers. With this last division the total

number of swarmers from each detached trophont

would be 512, implying nine successive binary divisions

(29 = 512). The swarmers (c.9 lm long and c.7 lm wide)

showed a hemispherical contour of the hyposome and an

episome more conical with a round apex, and a well-

marked cingulum. The swarmers contained small yel-

low-greenish body inclusions, and a round nucleus

located in the centre of the cell (Fig. 1T–W).

Molecular phylogeny

Three partial SSU rDNA sequences (up to 1,713 bp)

for O. pouchetii were obtained: one sequence from

detached trophonts isolated at Sao Sebastiao Channel

on 10 July 2013 (isolate #23; KM879217), and two

sequences from detached trophonts isolated off Uba-

tuba on 6 June 2014 (isolate #54; KM879218) and on

13 June 2014 (isolate #58; KM878219). The three

sequences were identical. A BLAST search was

conducted on the new sequences to find related

sequences in the GenBank database. Initial BLAST

comparisons showed that, with the exception of the

partial environmental sequence (643 bp; EF539018,

retrieved from the western Pacific coast, which shared

99% identity with O. pouchetii), the closest identified

relatives in the database were dinokaryotes such as the

thecate parasites Blastodinium spp. (JX473665 and

DQ317538) and Duboscquodinium collinii Grasse,

1952 (HM483399), and free-living thecate species

[e.g. Peridinium umbonatum Stein, 1883 (GU001637)

and Scrippsiella trochoidea (Stein, 1883) Balech ex

Loeblich III, 1965 (HM483396)]. However, similari-

ties were low in all cases (84%).

We studied the phylogenetic position of O. pouch-

etii in three SSU rDNA phylogenetic trees. The first

tree contained diverse representatives of the alveolate

lineages, with ciliates, apicomplexans, perkinsids,

euduboscquellids (Marine Alveolate Group I), syn-

dineans (Marine Alveolate Group II) and dinokary-

otes. This tree (Supplementary file 1, Fig. S1) placed

unequivocally O. pouchetii within the dinokaryotic

lineage. Then, we studied the phylogenetic position of

O. pouchetii using a dataset including a variety of

dinoflagellate sequences, especially with representa-

tives of the parasitic dinokaryotes, including the

oodinioid dinoflagellates (species of Amyloodinium

and Piscinoodinium), other parasitic dinoflagellates,

and a diverse representation of the dinokaryotic

lineages, and rooted using perkinsid and syndinean

sequences as an outgroup (Fig. 2). We carried out an

additional analysis that also included shorter

sequences, especially partial SSU rDNA sequences

of environmental clones and other known dinoflagel-

lates (Supplementary file 1, Fig. S2). The clade of O.

pouchetii in the resulting tree is included as an inset in

Fig. 2.

In the Bayesian consensus tree (Fig. 2), the SSU

rDNA phylogeny revealed that sequences for Oodini-

um spp. formed a distinct clade among the dinokaryotic

dinoflagellates. The three sequences for O. pouchetii

formed a highly supported lineage (maximum poster-

ior probability value) with an environmental sequence

(EF539018) from the port of Hong Kong, Western

Pacific. In the tree containing short sequences (Sup-

plementary file 1, Fig. S2), this lineage branched with

strong support with an environmental sequence from

the sub-surface waters of the equatorial Pacific

(AJ402340), and two environmental sequences

(AY046653; AY046659) from the surface waters of

the South Pacific Ocean. The Oodinium clade branched

within the dinokaryotic dinoflagellates. However, it

was not possible to find any close genetically charac-

terised relatives of the family Oodiniaceae.

Discussion

The life-cycle of the trophont and the morphology of

the dinospores of O. pouchetii studied here coincide

with the original description of O. pouchetii. Pouchet

(1885, figure 27) illustrated a pair of recently formed

dinospores with long flagella and a cell contour similar

to those observed in the present specimens (Fig. 1T–

U). Chatton (1920) demonstrated that along the

growth of the Oodinium trophont, it progressively

losses the typical nuclear characteristic of the

130 Syst Parasitol (2015) 90:125–135

123

dinokaryotic dinoflagellates (condensed rod-like chro-

mosomes). Hovasse (1935) found that the nucleus of

swarmer cells possess a typical dinokaryon, and only

gradually acquire eukaryote-like organisation during

the feeding phase as ectoparasite. The characteristic

morphological features of a dinoflagellate do indeed

Fig. 2 Phylogenetic tree of the Dinokaryota based on phylogenetic analysis of 64 SSU rDNA sequences using Bayesian inference.

Perkinsozoa is used as outgroup. Parasitic taxa are highlighted. The species newly-sequenced in this study are in bold. Inset: The

Oodinium-clade from a corresponding tree (Supplementary file 1, Fig. S2) in which short sequences were included. Posterior

probabilities (when above 0.5) are given at nodes. The scale-bar represents the number of substitutions per site

Syst Parasitol (2015) 90:125–135 131

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disappear during the vegetative stage and can only be

seen again in the last stages of sporogenesis. Conse-

quently, O. pouchetii alternates between ultrastructure

features of basal dinoflagellates and dinokaryotes.

Also the trophonts of Noctiluca scintillans (Macart-

ney, 1810) Kofoid, 1920 do not retain the shared

characters of typical dinoflagellates such as a trans-

verse flagellum and permanently condensed chromo-

somes. The swarmers of species of Noctiluca Suriray,

1836 maintain the dinokaryotic characteristics includ-

ing two grooves, slightly differentiated flagella and

condensed chromosomes (Fukuda & Endoh, 2006).

Species of Noctiluca and other noctilucoids (species of

Spatulodinium J. Cachon & M. Cachon, 1968; Kofo-

idinium Pavillard, 1928) do not branch within the

dinokaryotic lineage (Gomez et al., 2010). Despite the

absence of dinokaryotic characters in the parasitic

phase, the molecular phylogeny reveals that O.

pouchetii is truly a member of the dinokaryotic

lineage.

The type of life-cycle has traditionally been used

for classification of the parasitic dinoflagellates into

families (Fensome et al., 1993). However, molecular

phylogeny has revealed that species of closely related

genera (i.e. Dissodinium Klebs, 1916 and Chytriodi-

nium Chatton, 1912) have different life-cycles

(Gomez et al., 2009a). The morphology of dinospores

seems to be more informative for the classification of

these parasites. However, it is not always easy to

observe the detailed morphology of the small swar-

mers when compared with the trophonts. For example,

before Skovgaard et al. (2007), the thecate swarmers

of Blastodinium Chatton, 1906 were usually referred

to as naked, gymnodinioid dinoflagellates (Chatton,

1920; Taylor, 2004). The swarmers of Amyloodinium

ocellatum were first described as naked gymnodinioid

dinoflagellate (Brown, 1934; Lom, 1981), but further

analysis with scanning electron microscopy revealed

that the swarmers were thecate (Landsberg et al.,

1994). Paulsenella vonstoschii Drebes & Schnepf

branches within a clade of thecate dinoflagellate.

However, the thecal plates have not yet been reported

(Kuhn & Medlin, 2005). Due to instrumental con-

straints, we were not able to discern the tabulation of

the dinospores of O. pouchetii. The thecal plates of the

trophonts of this species consist of four equatorial

series and are thin, with no visible ornamentation

(Hovasse, 1935). The assignment of these plates into

conventional Kofoid’s tabulation system is difficult,

due to the lack of conventional reference points (apical

pore plate, cingulum or sulcus) for the recognition of

plate series.

To date, Amyloodinium is still classified within the

family Oodiniaceae, but we have here shown that it is

distantly related to the type-species of the family.

Hence, O. pouchetii remains the only morphologically

identified member of Oodiniaceae with a sequence in

the GenBank database. Several environmental

sequences are related to O. pouchetii and thus may

represent potential unidentified members of the Oodi-

niaceae (Fig. 2; Supplementary file 1, Fig. S2).

Assuming that these environmental clones are not

artefacts and correspond to true organisms, unidenti-

fied relatives of Oodinium spp. are present in different

marine habitats. An environmental sequence from

coastal eutrophic waters unequivocally corresponded

to O. pouchetii (see Cheung et al., 2008). Sequences

for relatives of O. pouchetii were retrieved from

surface and subsurface waters of the oligotrophic

waters of the Pacific Ocean (Moon-van der Staay et al.,

2001; Lie et al., 2014). We do not know whether these

environmental clones branching with O. pouchetti are

parasitic or free-living organisms. Our results suggest

that there are dinoflagellates belonging to the family

Oodiniaceae that have not yet been morphologically

and ecologically characterised.

We must conclude that the parasite of fishes,

Amyloodinium and Piscinoodinium, should not be

placed in the family Oodiniaceae. In the classical

taxonomical schemes, Fensome et al. (1993) grouped

under the Oodiniaceae ectoparasites with suboval to

fusiform in outline trophonts, with a well-developed

peduncle or invasive organ that consists of a complex

rhizoid-like absorptive structure and with reproduc-

tion by palintomic sporogenesis. Another character of

this family is the possession of both non-dinokaryotic

and dinokaryotic nuclei within the life-cycle (Fen-

some et al., 1993). There is no evidence for the

presence of non-dinokaryotic nuclei in species of

Amyloodinium and Piscinoodinium. The thecate Am-

yloodinium ocellatum branched with other parasites

such as Paulsenella vonstoschii and Tintinnophagus

acutus Coats (as in Coats et al., 2010). The trophont of

O. pouchetii is also covered by thecal plates (Hovasse,

1935), and we can expect a relationship between these

taxa. Amyloodinium ocellatum can be distinguished

from O. pouchetii by the possession of rhizoid- and

root-like processes for attachment and by the

132 Syst Parasitol (2015) 90:125–135

123

production of starch grains. Species of the genus

Oodinium, on the other hand, are characterised by the

possession of a disk, rather than rhizoids, for attach-

ment and by the lack of starch grains (Brown &

Hovasse, 1946). Several of the congeneric species that

parasitise other metazoan groups show marked mor-

phological differences in comparison with the type-

species O. pouchetii (see McLean & Nielsen, 1989;

Mills & McLean, 1991; Horiguchi & Ohtsuka, 2001).

This raises questions of the correct generic affiliation

of these species. Other parasitic genera with some

morphological resemblance such as Cachonella Rose

& Cachon, 1952, Crepidoodinium, Protoodinium

Hovasse, 1935 or Oodinioides also lack molecular

information. Unfortunately, the strong bias in the

availability of sequences towards photosynthetic and

cultivable dinoflagellates hinders the advances in

dinoflagellate phylogeny. The percentage of parasitic

dinoflagellates for which at least one DNA sequence is

available is very low (7%; Gomez, 2014). Proper

taxon sampling is one of the greatest challenges to our

understanding of the phylogenetic relationships of the

parasitic dinoflagellates.

Analyses of environmental marine rDNA

sequences have revealed an extensive diversity of

ribotypes related to dinoflagellates, especially Syndi-

niales, widely distributed throughout the oceans

(Lopez-Garcıa et al., 2001; Guillou et al., 2008). The

closest relatives to the dinoflagellates, apicomplexans

and perkinsids, and nearly all the basal dinoflagellates

(Syndiniales, euduboscquellids and ellobiopsids), are

parasites. These basal dinoflagellates lack the charac-

teristics of dinokaryotic dinoflagellates such as the

condensed chromosomes in interphase. The propor-

tion of parasites is low (3%) among the core

dinoflagellates (Gomez, 2012). In our phylogenetic

analyses, we have included the available sequences for

other parasitic dinokaryotes. The parasite species of

the genera Amyloodinium, Tintinnophagus, Piscino-

odinium, Duboscquodinium Grasse, 1952, Chytriodi-

nium and Dissodinium branched with strong support in

different clades dominated by free-living dinoflagel-

lates (Litaker et al., 1999; Kuhn & Medlin, 2005; Levy

et al., 2007; Gomez et al., 2009a, b; Coats et al., 2010).

The phylogenetic position of Blastodinium remains

unclear with a weak relationship with peridinioid

clades (Skovgaard et al., 2007, 2012), and Blastodi-

nium is not always a monophyletic group in SSU

rDNA phylogenies (Coats et al., 2008; Skovgaard &

Salomonsen, 2009). With exception of Amyloodinium/

Paulsenella and Chytriodinium/Dissodinium, the

clades of parasitic dinokaryotes are not related to

each other.

Haplozoon Dogiel, 1906 and Oodinium have in

common that they comprise both fast-evolved dinok-

aryotes without any close known relatives (Fig. 2; see

also Saldarriaga et al., 2001; Rueckert & Leander,

2008). We observed in the SSU rDNA phylogeny that

the sequences for species of Amyloodinium, Tintinn-

ophagus, Piscinoodinium, Chytriodinium, Haplozoon

and Oodinium have long branches when compared

with the typical short-branched species of the main

clades of the core dinoflagellates. This has been

considered with caution because the branches of these

parasites are shorter in ingroup trees with a rich

taxonomic sampling of their relatives (Gomez &

Skovgaard, 2014). A longer branch usually is inter-

preted as either a longer time period since that taxon

split from the rest of the organisms in the tree or faster

evolutionary change in a lineage. We can only

hypothesise accelerated rates of evolutionary change

in parasitic dinoflagellates when compared with their

free-living counterparts.

Oodinium pouchetii is obviously highly derived not

only in relation to its morphology, but also to SSU

rDNA sequence. In this, as well as in many other

parasitic dinoflagellates, the detached trophont, its

sporogenesis stages and the small dinospores are only

recognised during routine phytoplankton analysis by

well-trained observers. Researchers focused on plank-

ton metazoans usually preserve the samples in formalin

and the parasites are unrecognisable due to fixation-

induced distortion (Skovgaard & Saiz, 2006). Conse-

quently, the abundance and role of Oodinium spp. in the

world oceans remains understudied.

Acknowledgements F.G. was supported by the Brazilian

Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico

(grant no. BJT 370646/2013-14). A.S. was supported through the

project IMPAQ - IMProvement of AQuaculture high quality fish fry

production, funded by the Danish Council for Strategic Research

(Grant No. 10-093522).

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