Genetic Diversity and Gene Flow among Populations of Ophryotrocha

(Annelida: Polychaeta) on Vertebrate Bones at Different Depths

Elizabeth Tenney

Species of Ophryotrocha within the Family Dorvilleidae are well-studied polychaetes

found on whale-falls in all oceans at various shallow and deep water depths. The

opportunistic polychaete has also been discovered inhabiting experimentally deployed pig

and cow bones. However, while several studies have proposed phylogenies for Ophryotrocha

species, little is known about the genetic diversity and gene flow among Ophryotrocha

populations across different bone types, substrates, and depths. In the current study, we used

~ 600bp of the COI barcode to examine the genetic diversity and gene flow in populations of

Ophryotrocha discovered on whale, cow, and pig bones in the Mediterranean Sea. We found

that samples in our study formed two isolated branches. One branch contained all samples

collected from shallow depths (10 – 20 m), and one branch contained all samples collected

from 57 m. Pairwise distances ranged from 7.33% to 8.19% between the branches, neither of

which falls within previously recorded Ophryotrocha species lineages on the tree. Based on

this, we propose two cryptic species of Ophryotrocha. 21 haplotypes were present across the

38 samples found at shallow depths. These results provide evidence of gene flow among

shallow water populations of Ophryotrocha likely because of larval recruitment from large

intermixing populations. Future research could verify the presence of new species by

generating nuclear coding sequences. Substrate surrounding vertebrate bones should also be

sampled to examine the recruitment mechanisms of Ophryotrocha species in sampled

geographic locations.

Key Words: COI barcode, whale fall, Ophryotrocha, genetic diversity, gene flow,

phylogeny, haplotype, Mediterranean Sea

Introduction

Scientists have long speculated over the effect whale carcasses might have on their

ecological environment after death, believing that in time the carcass could host microbial

communities. Before the late 20th century, scientists had only gathered information

suggesting faunal communities inhabited them (Smith and Baco, 2003). In 1989, however, a

chance discovery of a whale carcass in the eastern Pacific Ocean spurred on several studies

with the purpose of investigating the invertebrate species that potentially thrive on naturally

or experimentally deployed whale carcasses (Smith et al., 1989). Since the initial discovery,

whale falls are now widely recognized as hosts of invertebrate faunal communities (Smith

and Baco, 2003).

Now, 21 whale-fall specific macrofaunal species have been discovered across the

world, with over 400 species recorded on whale fall in total (Smith and Baco, 2003). These

species inhabit the carcasses at different times during the four stages of biological and

chemical succession associated with whale falls, with different fauna at each stage: the initial

mobile-scavenger stage, the enrichment-opportunistic stage, the sulphophilic stage, and the

reef stage (Smith and Baco, 2003; Bennet et al., 1994). In the first stage, necrophages like

hagfish and sleeper sharks remove the soft tissue of the carcass; in the second, polychaetes,

crustaceans, and other opportunistic invertebrates begin to colonize the bones and

surrounding sediment. In the sulphophilic stage, the presence of chemoautotrophic sulphur-

based bacterial mats increases, and lastly, the reef stage represents the loss of organic matter

in the bones; here, suspension feeders colonize and deposit feeders feed on the substrate

(Smith and Baco, 2003).

After the scavenger species have removed the soft tissue from the whales or other

vertebrates, the invertebrate communities that begin to colonize are present for years;

therefore, these communities are an ideal stage to study. Polychaetes in particular are well

researched in whale fall literature (e.g., Dahlgren et al., 2004; Glover et al., 2005; Goffredi et

al., 2004; Fujiwara, 2006). There are also studies on other types of vertebrate bones, as

scientists have found that specific species of polychaetes colonize on different types of

experimentally deployed bones (Anderson, 2010; Jones et al., 2008; Taboada et al., 2014).

Cow and pigs bones are found to act as invertebrate community hosts, too, with species of

polychaetes largely found on these deployed mammal bones (Anderson, 2010; Jones et al.,

2008).

Ophryotrocha (Claparède & Mecznikow, 1869) is a model annelid typically found on

whale bones that is easy to keep in the laboratory, and therefore its evolution and speciation

have been widely studied (Heggoy et al., 2007). Discovered in 1869 and found to be within

the family Dorvelleidae, Ophryotrocha is a small marine worm with a relatively short life

span of under a year (Schleicherova et al., 2010). Ophryotrocha species can reproduce in a

variety of ways, including simultaneous hermaphrodism and gonochorism, and they breed

semi-continuously. After reproduction, a brood is produced and protected in a tube, waiting

to emerge (Shain, 2009). Once the larvae emerge, the adult species of Ophryotrocha die and

their offspring disperse through the water for weeks before settling as juveniles on various

substrates (Mercier et al., 2014).

Species in this genus are considered to be opportunistic (Fauchald, 1977). They are

commonly found in warm water habitats or polluted areas, though they are also present in

temperate and deep-sea locations. They are usually found at the sediment level, too, where

they act as grazers attempting to glean bacteria or detritus from the surface of the substrate

they are inhabiting – either sand, rock, or as has been discovered over the past decade,

submerged whale bones (Shain, 2009).

Species of Ophryotrocha are found worldwide, and the diversity of the genus has

increased significantly with the recent discovery of many new species from whale falls in

locations such as the Atlantic Ocean, the Mediterranean Sea, and even the Southern Ocean,

with species of Ophryotrocha present on the whale fall in these locations. While some

species have been described from single whale falls, others are known to have wide

distributions. For example, Ophryotrocha eutrophila has been found in the Mediterranean as

well as the Atlantic (Taboada et al., 2014; Wiklund et al., 2009). Scientists are frequently

discovering new species of Ophryotrocha in order to further understand the species and its

behaviour and habitat preferences (Wiklund et al., 2009; Taboada et al., 2014; Martin et al.,

1991; Cossu et al., 2014; Taboada et al., 2013; Amon et al., 2013).

Despite advances in documenting the diversity of Ophryotrocha species,

Ophryotrocha and its species diversity in varying geographic locations, substrates, and bone

types over time remain poorly known. Ophryotrocha species have primarily been studied on

deep sea whale fall, for example. However, it has been suggested that shallow water whale

falls are similar to other shallow water organically enriched substrates (Danise et al., 2014).

Deep water whale falls, on the other hand, have been found to be more similar to other deep

water habitats, like hydrothermal vents, indicating potential differences between species of

Ophryotrocha found on deep sea and shallow sea whale falls (Bennett et al., 1994).

Ophryotrocha may not be limited to specific mammal bones, either, and its presence on other

mammal or vertebrate bones could open up new research opportunities if studied more

thoroughly. Therefore, various water depth communities and different bone types may reveal

more about the nature of Ophryotrocha and its diversity.

To better understand the diversity of invertebrate populations inhabiting vertebrate

bones and to further differentiate between shallow water and deeper water communities, we

examined the diversity of Ophryotrocha on bones in the Mediterranean Sea. Dr. Sergi

Taboada (University of Barcelona) deployed cow, whale, and seagull bones on either sand or

rock substrates at depths of 10m, 20m, or 57m off of the Spanish Mediterranean coast.

Vertebrate bones were chosen based on bait fishing preferences in the area (Taboada et al.,

2014) Dr. Taboada sent his samples to us, where we then examined the gene flow and genetic

diversity of the species of Ophryotrocha found on those bones.

Given the dispersal mechanisms and larval stage in the Ophryotrocha life cycle, we

expected that there would be high genetic diversity and gene flow among Ophryotrocha

populations across bone types, substrates, geographic locations, and depths, due to the mixing

of Ophryotrocha species on similar substrates and consequent sexual reproduction (Taboada

et al., 2014). We also expected species of Ophryotrocha to vary in deep and shallow depths

because of the differing environments (Danise et al., 2014; Bennett et al., 1994). To test our

hypothesis, we used the COI barcode (Hebert et al., 2003) to identify the species of

Ophryotrocha found on the bones, and then we constructed a phylogenetic tree and haplotype

network to observe genetic diversity within and differences between species.

Methods

Sample Collection

Cow, pig, and whale bones were placed on rock substrate at a depth of 20m. Whale

bones and seagull bones were also deployed at 57m and 10m respectively off the shore of

Blanes Bay and Tossa de Mar (Figure 1). The bones were collected after 15 months (T2).

Ophryotrocha puerilis, O. eutrophila, and O. alborana as identified by morphology were

collected from the deployed vertebrate bones in Blanes Bay in the Spanish Mediterranean

coast. The three species of Ophryotrocha were extracted from the bones and 1mm tail snips

were removed and stored in ethanol.

DNA Amplification and Analysis

DNA was extracted using a QIAmp DNA Micro Kit #56304 (QIAGEN). A ~700bp

segment of the cytochrome oxidase I gene (COI) was then amplified using H and L universal

primers (Folmer et al., 1994) or Mega F (5’- TAYTCWACWAAYCAYAAAGAYATTGG -

3’) and Mega R (5’- TAKACTTCTGGRTGMCCAAARAAT -3’) primers. Primer were

provided by Kelly Pittenger and Samuel Davalos (Table 1). PCR was performed using the

following profile: 1µl DNA extract, 10x buffer, 50µM MgCl2, 25µM dNTP, 25µM forward

and reverse primers, and 0.5u Taq polymerase (Invitrogen). 25µl reactions were run at 95˚C

(5 min), 35 cycles of 95˚C (30 seconds), 45˚C or 48˚C (30 seconds), 72˚C (1 min), and a final

extending temperature of 72˚C (7 min).

Gel electrophoresis was performed using 1% agarose gel to verify COI amplification

and visualized through the fluorescence of ethidium bromide. Once COI amplification was

confirmed, the PCR amplifications were cleaned using ExoSAP-IT PCR Product Cleanup

protocol (Affymetrix/USB). DNA cycle sequencing was performed using 5µl or 10µl

reactions which contained ~50-300 ng of DNA, 5X Sequencing Buffer, Terminator-ready

reaction mix (TRR v.3 Life Technologies), and 1µM of either forward or reverse primers.

The reactions were run on the GeneAmp PCR thermocycler for 30 cycles of 96˚C (10

seconds), 50˚C or 49˚C (10 seconds), 60˚C (4 min), and held at 4˚C.

Sequences were analyzed using the ABI PRISM 3130A Genetic Analyzer (Life

Technologies). For all of the specimens, 610-658 bp of the COI gene were edited and aligned

using Sequencher 5.2.4 (GeneCodes). Species identity was examined from the edited

sequences using BLAST on the ‘somewhat similar’ setting.

Analysis

Sequencher alignments were exported as Fasta files, and all sequences were merged

with GenBank sequences using Mesquite 2.75. Sequence Matrix was used to format the

sequences for MEGA v.5.03. A neighbor-joining tree was generated using MEGA. Results

were visualized in FigTree v1.4.0 and a phylogram was generated. Population statistics and a

haplotype network were created using Arlequin 3.5.1.3. The haplotype network was

visualized in HapStar 0.7.

Results

We inferred the phylogenetic relationships of 43 specimens of Ophryotrocha from the

mitochondrial COI gene (Table 2). There were three main branches illustrated in our

neighbor-joining tree, with the species of Ophryotrocha we studied forming two separate

branches (Figure 2). Branch A consisted of all worms that were collected from the three bone

types in sand or rock substrate shallower than 20m (Opu_WRT2.1-2.11; 2.13-2.24;

Opu_CRT2.1-2.10; Opu_2013_BL1-5). Branch B consisted only of worms collected from

whale bones in sand at a 57m depth (Opu_2014_WTS1-5). The pairwise distances between A

and B ranges from 7.33% to 8.19%.

Branch B had greater nucleotide diversity than A, due to the greater phylogenetic

distance denoted by the longer branch length. Branch B had low haplotype diversity, with

two haplotypes displayed (Figure 3). Branch A had low nucleotide diversity, but high

haplotype diversity with 21 haplotypes across 38 specimens (Table 3; Figure 3). Species of

Opu_WRT had 12 haplotypes, species of Opu_CRT had 6 haplotypes, and species of

Opu_2013_BL had 5 haplotypes (Table 3). The specific values for haplotype and nucleotide

diversity for each locality of Branch A are illustrated in Table 3.

Discussion

This study presents the possible discovery of two cryptic species of Ophryotrocha.

Although the pairwise distance was lower than the average distance used for delineation of

Ophryotrocha species as presented by Dahlgren et al. (2001), there is no clear evidence of

gene flow between the two branches, A and B, in our analysis (Figure 2). Each branch forms

its own monophyletic group, and the delineation of the four most closely related species of

Ophryotrocha into their own branch offers evidence supporting discovery of new cryptic

species. These results represent an expansion of species diversity of Ophryotrocha found on

whale falls recently, as seen in recent research on new Ophryotrocha species by Wiklund et.

al. (2009), Taboada et al. (2013), Wiklund et al. (2012).

Within Branch A there are 21 haplotypes from a total of 38 specimens, with

haplotypes shared among each locality (Table 3). The haplotype diversity levels are

comparable to a study done by Schulze (2006) on the genetic diversity of Palolo worms in the

North Pacific and Caribbean Oceans (Schulze, 2006). Similarly, haplotype diversity was also

comparable across species of the ribbon worm Parborlasia corrugatus in the Southern

Ocean, indicating a similar trend among marine worms (Thornhill et al., 2008). Average

haplotype diversities in the specific studies for Palolo worms, Ribbon worms, and our

Ophryotrocha study are 0.864, 0.845, and 0.918 respectively (Schulze, 2006; Thornhill et al.,

2008). According to Wiens and Penkrot (2002), haplotypes that do not cluster and appeared

to be mixed among localities indicate evidence of gene flow. Therefore, Branch A seems to

represent a large population of the Mediterranean Sea with high levels of intermixing and

consequently gene flow among populations of Ophryotrocha. The gene flow may occur as a

result of worm recruitment from a large, mixed pool of larvae from the shallow depths of the

Mediterranean coast.

These results are not surprising, as Ophryotrocha is known for larval dispersal. The

adult species breed semi-continuously, and species may pseudocopulate for hours until a

brood is produced. The larvae emerge from the brood after 30-45 days (Shain, 2009). After

emerging, the larval species of Ophryotrocha disperse through the ocean for weeks before

settling to a suitable benthic substrate to inhabit (Mercier et al., 2014). The dispersal behavior

of Ophryotrocha is therefore evidence for potential gene flow, as intermixing among

populations can occur in a limited geographic range.

Our results also indicate differences in Ophryotrocha species dependent on deployed

bone depth, and there is no indication that bone type is linked to particular Ophryotrocha

species. Branch B was a monophyletic group found only on whale fall deployed at a depth of

57m, whereas Branch A consisted solely of worms collected from three different bone types

all in shallow water habitats (10m, 20m). These results indicate that variation in depth can

influence species diversity, illustrating differences in deeper water whale fall environments as

compared to shallow water whale fall environments. Similarly, Danise and Dominici (2014)

discovered variation in successional stages in deep and shallow water whale fall, suggesting

that different communities and species may inhabit whale fall at different depths (Danise and

Dominici, 2014).

Studies have also found that deep water whale falls are similar to other deep water

habitats like hydrothermal vents, and shallow water whale fall habitats mirror other shallow

water habitats (Danise et al., 2014; Bennet et al., 1994). This is likely a consequence of the

different environmental factors that are present at various depths. Therefore, the depth-

specific species diversity in our study may be specific to the general geographic location of

the whale fall and not the bone itself. Thus, species of Ophryotrocha may occur in benthic

habitats near whale fall and other vertebrate bones. Few studies focus on the presence of

Ophryotrocha on the substrate surrounding whale falls, but Smith and Baco (2003) noted that

polychaetes colonize the surrounding substrate before the bones (Smith and Baco, 2003).

This could indicate that Ophryotrocha are not dependent on bone habitat, as the dispersive

larvae colonize the general area first, and recruitment occurs on the substrate before the

populations of Ophryotrocha move to the bones; species of Ophryotrocha reproductively mix

and gene flow then occurs.

Future Directions

To further understand our results and assess whether we have discovered two new

species of Ophryotrocha, nuclear coding sequences should be generated for nuclear DNA

using either ITS1 or ITS2, as performed in other studies using polychaetes (Iannotta et al.,

2007; Boggemann, 2009). As the COI gene is a mitochondrial marker, and mitochondria are

passed on maternally, a nuclear marker will reveal whether our results reflect a maternal

lineage phenomenon or the general population dynamics at the genetic level.

To understand the dispersal mechanisms and the water depth variation of

Ophryotrocha species, the substrate surrounding the bones in deep and shallow water habitats

should be sampled for analysis and species classification. Many marine worms are commonly

found on various substrates in the Mediterranean, and Ophyrotrocha species may be similar.

For example, polychaetes have been associated with the sea grass Posidonia as well as the

rocky submerged coasts in the Mediterranean Sea (Iannotta et al., 2007; Giangrande et al.,

2003). If similar species of Ophryotrocha are found on the surrounding substrate of

vertebrate bones, this could indicate that the dispersal mechanisms of the larvae are not

reliant on bone habitat. Instead, they may disperse and settle in organically enriched

environments within a specific geographic area not limited to whale fall, denoting the

occurrence of larval recruitment across Ophryotrocha species.

Acknowledgements

We would like to thank Dr. Sergi Taboada (University of Barcelona) for all sample

collection contributions and guidance, as well as Dr. Damhnait McHugh and Dr. Nancy

Schult for their assistance and mentorship. We would also like to thank Colgate University

for the opportunity it provided for academic research. The work was funded by NSF-DEB

award number 1036537 to Dr. Damhnait McHugh.

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Figures

Figure 1. Geographic approximation of sample sites. Red denotes cow and whale bones on a rock

substrate at 20 m; orange denotes whale bones on a sand substrate at 57 m and yellow denotes seagull

bones at 10 m.

Mega Forward Primer 5’-TAYTCWACWAAYCAYAAAGAYATTGG -3’

Mega Reverse Primer 5’-TAKACTTCTGGRTGMCCAAARAATC -3’

Table 1. CO1 degenerate primers used to amplify the CO1 sequences in the samples.

Name Bone Type Substrate Depth (m)

Opu_WRT Whale Rock 20 Opu_CRT Cow Rock 20

Opu_2014_WTS Whale Sand 57 Opu_2013_BL Seagull Sand 10

Table 2. Abbreviation key delineating the terminology used in our phylogeny and results.

Figure 2. Neighbour-joining tree for Ophryotrocha specimens. ‘A’ represents one branch, and

‘B’ represents the second. Blue represents individuals found on whale bone and rock substrate at

20m; green represents cow and rock substrate at 20m; red represents seagull and sand substrate at

10m; pink represents whale bones on sand substrate at 57m.

A

B

Figure 3. CO1 haplotype network from branch A with 21 haplotypes total. Blue represents individuals

found on whale bone and rock substrate at 20m; green represents cow and rock substrate at 20m; red

represents seagull and sand substrate at 10mTwo or more colors represent shared haplotypes. Circles

containing numbered haplotypes are scaled to size according to the number of individuals possessing a

particular haplotype.

Locality N # of Haplotypes

Nucleotide

Diversity (π) Haplotype

Diversity (H) Mean # of pairwise distances

Opu_WRT 23 12 0.008659 +/- 0.004905

0.9091 +/- 0.0360

4.719368 +/- 2.397

Opu_CRT 10 6 0.009655 +/- 0.005726

0.8444 +/- 0.1209

5.600 +/- 2.936

Opu_2013_BL 5 5 0.010238 +/- 0.006913

1.0000 +/- 0.1265

5.600 +/-3.235

Table 3. Measures of intra-population variability for species of Ophryotrocha in the Mediterranean Sea

based on the CO1 gene. N is number of individuals per locality. Average nucleotide diversity: 0.00985;

average haplotype diversity: 0.918.

H18

H19

H8

H9

H20

H21

H10H3

H12

H1

H6

H15

H4

H5

H2

H14

H16H13

H11

H17

H7