Eurythoe complanata: more cryptic species in the Pacific?Mirandia Johnson, Elizabeth Borda and Anja Schulze

Department of Marine Biology, Texas A&M University at GalvestonUndergraduate

QUESTIONS AND OBJECTIVES• Are there cryptic Eurythoe species in the Pacific?• What are the phylogenetic relationships among populations on opposite

sides of the Pacific Ocean?• To provide a phylogenetic molecular framework for characterizing species

diversity in the genus Eurythoe, in the absence of unambiguous morphological characters.

METHODSSampling• 29 Samples were included in this study representing: the Pacific coast of

Mexico (n=10), Gulf of California (GoC; n=2), Yucatan Peninsula (n=2), Caribbean coast of Panama (n=2), Sumatra, Indonesia (n=4), Sulawesi, Indonesia (n=4), American Samoa (n=1), Moorea (n=2), and from marine aquaria in St. Louis, MO (n=1) and Osnabrück, Germany (n=1). We also included representatives of E. complanata (n=5) from the Caribbean and E. complanata var. mexicana from GoC (n=6).

Amplification and Sequencing • Mitochondrial 16S rDNA and cytochrome c oxidase subunit 1 genes were

amplified and sequenced using standard protocols. • Sequences were edited in Sequencher v.4.10.1 and aligned using Muscle

(www.ebi.ac.uk/Tools/msa/muscle)Phylogenetic Analysis• Inter- and intraspecific COI pairwise genetic distances were calculated using

Mega 5: uncorrected and corrected with the K2P model of evolution.• Phylogenetic relationships were estimated under parsimony and maximum

likelihood assumptions. Parsimony analyzed with PAUP* and run with 1000 parsimony jackknife support values. Maximum likelihood analyses were run using RaxMLGUI with thorough bootstrap ( 10 reps; 1000 pseudo replicates).

ABSTRACTMitochondrial DNA data has shown that Eurythoe complanata is a cryptic species complex characterized by deeply divergent sympatric populations in the Atlantic and on either side of Panama, despite almost no morphological differences. Previous work examined populations primarily in the Caribbean and Atlantic Ocean, with little exploration of the phylogenetic relationships of Pacific populations. We aim to study these relationships using mitochondrial (COI and 16S rDNA) sequence data and include representatives from populations in the east Pacific (Mexico and the Gulf of California), Indo-Pacific (Indonesia, Moorea, and American Samoa), and from aquaria. The objectives of this study are to: 1) identify if there are cryptic species in the Pacific; 2) infer the phylogenetic relationships among amphi-Pacific populations; and 3) estimate the distributional patterns of animals that reproduce both sexually and asexually.

AcknowledgementsThis project is funded by NSF ATOL grant DEB 1036186 . Images: G. Rouse, C. Sanchez

Figure 3. Eurythoe specimen showing typical caruncle (B) and brachial filaments (C).

RESULTS AND DISCUSSIONTable 1. Average K2P corrected (blue; above diagonal) and uncorrected (green, below diagonal) COI pairwise distances (PD). A. Average interspecific and corrected intraspecific (along diagonal) PD. B. Average PD among Eurythoe representatives from select geographic regions.

ReferencesBarroso, R., M. Klautau, A. M. Sole-Cava, and P.C. Paiva (2010). "Eurythoe complanata (Polychaeta: Amphinomidae), the 'cosmopolitan' fireworm, consists of at least three cryptic species." Marine Biology 157(1): 69-80.Nygren, A., Pleijel, F. (2010) From one to ten in a single stroke – resolving the European Eumida sanguinea (Phyllodocidae, Annelida) species complex. Molecular Phylogenetics and Evolution 58(1): 132-141. Reish, D, and B Pernet. (2009) "Annelid Life Cycle Cultures." Annelids in Modern Biology. Ed. Daniel H. Shain. Hoboken, NJ: Wiley-Blackwell. 47-62.

INTRODUCTIONCryptic species• Cryptic species are morphologically similar species classified as a single

species . Morphology alone may severely underestimate the number of species (Nygren and Pleijel. 2010).

• A lack of defining characteristics has been correlated to wide geographic distributions (Barroso, et al. 2010).

Eurythoe • Eurythoe is represented by the circumtropical species, Eurythoe

complanata, which exhibits low morphological variation (Figure 3) across geographically disjunct populations (Barroso, et al. 2010).

• Barroso, et al. (2010) examined the morphology of over 200 samples from: the coast of Brazil; South Atlantic islands; Panama(Bocas del Toro, Taboga); Gold Coast and east coast of Africa; Red Sea; Sri Lanka; and the Azores. They found no clear morphological differences to distinguish these populations, but found molecular evidence for the existence of cryptic species.

• Eurythoe complanata can reproduce sexually and asexually (e.g. fragmentation and regeneration), but little is known about their planktonic larval stages (Reish and Pernet, 2009)

TABLE A1 2 3 4 5 6 7

1. E. complanata 0.3% 15.3% 21.7% 23.1% 20.6% 27.3% 27.2%

2. E. “mexicana” 12.4% 0.5% 24.7% 25.8% 27.6% 27.8% 27.8%

3. Eurythoe Pacific (A) 16.2% 17.9% -- 12.3% 14.2% 18.6% 21.2%

4. Eurythoe Pacific (B) 16.8% 18.4% 10% -- 8.7% 22.4% 22.3%

5. Eurythoe Pacific (C) 15.5% 19.2% 11.1% 7.4% 1.7% 22.1% 22.0%

6. Eurythoe Pacific (D) 18.9% 19.3% 14.4% 16.5% 16.3% -- 1.8%

7. Eurythoe Pacific (E) 18.8% 19.2% 15.9% 16.5% 16.3% 1.7% 0.4%

TABLE B 1 2 3 4 5 6 7 8 9 10

ASamoa

BSula

CSula

CSuma

DGoC

DGue

EAq. SL

EAq. Ger.

EBT

ESuma

1. A (Samoa) -- 12.3% 14.2% 13.4% 18.6% 18.6% 21.6% 21.0% 20.8% 21.1%

2. B (Sulawesi) 10.0% -- 7.6% 10.3% 22.4% 22.4% 23.0% 22.3% 22.1% 21.8%

3. C (Sulawesi) 11.3% 6.9% -- 1.8% 22.2% 22.2% 22.7% 22.1% 21.9% 21.6%

4. C (Sumatra) 10.7% 8.6% 2.1% -- 21.2% 21.2% 21.7% 21.1% 20.9% 20.6%

5. D (Gulf of Cali) 14.4% 16.5% 16.5% 15.8% -- 0.0% 2.1% 1.4% 1.4% 1.8%

6. D (Guerrero) 14.4% 16.5% 16.5% 15.8% 0.0% -- 2.1% 1.4% 1.4% 1.8%

7. E (Aq. St. Louis) 16.2% 16.8% 16.8% 16.2% 2.1% 2.1% -- 0.7% 0.3% 0.3%

8. E (Aq. Germany) 15.8% 16.5% 16.5% 15.8% 1.4% 1.4% 0.7% -- 0.0% 0.3%

9. E (Bocas del Toro) 15.8% 16.5% 16.5% 15.8% 1.5% 1.5% 0.5% 0.2% -- 0.0%

10. E (Sumatra) 16.0% 16.3% 16.3% 15.6% 1.9% 1.9% 0.5% 0.5% 0.3% --

Figure 1. Map showing the worldwide distribution of samples included in this study.

Highlights• The phylogeny supports the presence of at least five cryptic species in the Pacific. (Figure 2A;

Table 1) • Several clade representatives (B, C, and E) have broad geographic distributions, possibly assisted

by anthropogenic means and asexual reproductive modes (e.g. aquarium live rock).• Clade I appears to have west Pacific origins, possibly with recent introduction to the east Pacific.• Clade II is the most widespread, found in: Indonesia, the Yucatan peninsula, GoC, and western

coast of Mexico Caribbean side of Panama, and aquariums in Germany and Missouri.• High inter-clade genetic distances support distinct species. • Low intra-clade genetic distances were found among representatives from disjunct geographic

locations.

Future Studies• Continue collecting nuclear ITS sequence data, as an independent line of genetic evidence to

corroborate mitochondrial results. • Increase diversity by naming new species and revising old ones.