Introduction:Polychaete annelids in the genus Hermodice , commonly known as bearded fireworms (Fig. 1), are facultative corallivores (Lewis, 2009) that inhabit reefs in the Atlantic Ocean, extending into the Gulf of Mexico (GoM) as well as the Caribbean and the Mediterranean Seas. Their destructive feeding behavior has been shown to stunt the branch growth of the hydrocoral Millepora complanata in the Caribbean (Whitman, 1988). Additionally, in the Mediterranean, bearded fireworms serve as a winter reservoir and vector for Vibrio shiloi, the pathogen responsible for the summer bleaching of Oculina patagonica (Rosenberg and Falkovitz, 2004; Sussman et al., 2003).

A recent study determined that bearded fireworms found in the Greater Caribbean are morphologically distinct from those in the Mediterranean and East Atlantic based on differences in branchial filament abundance (Fig. 1, right), as well as anal lobe morphology (Yaῆez-Rivera and Salazar-Vallejo, 2011). Hence, the genus Hermodice has been revised to include two species: H. carunculata in the Greater Caribbean and west Atlantic, and H. nigrolineata in the Mediterranean. However, no molecular data was used to support the delineation of amphi-Atlantic Hermodice species. Preliminary mitochondrial COI analysis (this study) indicated low levels of divergence between populations and revealed certain Mediterranean specimens that tend to cluster with a GoM phylogroup. Therefore, a broader genetic study including samples from additional locales as well as mitochondrial 16s rDNA sequence data has been conducted to determine whether the aforementioned delineation of amphi-Atlantic Hermodice populations can be supported by molecular evidence.

Results/ Discussion:Phylogenetic analyses supported the monophyly of amphi-Atlantic Hermodice (pp=0.99; boot=100) and resulted in a tree consisting of two clades (Fig. 3), one indicating a private lineage of only Mediterranean specimens (pp=1.0; boot=67%) and the other primarily containing samples from the Greater Caribbean (pp=1.0; boot=58%). A subclade of the Greater Caribbean population was also strongly supported (pp=1.0). These clades, however, showed weak bootstrap support and one of the Crete specimens clustered with samples from the Greater Caribbean for both combined and independent COI and 16S analyses.

Mean COI nucleotide distances ranged between 5.42 and 15.59 among GoM and Mediterranean populations. Genetic distances ranged from 0.7 to 1.1% within populations and from 0.8 to 3.0% between populations (Table 1). Analysis of Molecular Variance (AMOVA) for COI indicates that roughly 55% of variation occurred among the GoM and Mediterranean groups, whereas 44% of variation occurred within populations (Table 2).

Based on phylogenetic analyses as well as genetic distance calculations, the two amphi-Atlantic bearded fireworm populations do not constitute separate species (according to the Phylogenetic Species Concept). Therefore, the morphological distinction between Greater Caribbean and Mediterranean/ East Atlantic Hermodice populations is not corroborated by molecular evidence. Whereas other genetic studies of amphinomid polychaetes have revealed the presence of cryptic species complexes (Barroso et al., 2004), our study indicates high population connectivity across a wide geographic range for H. carunculata.

Variation d.f. Sum of Squares Variance Components Percentage of variation

Among groups 1 95.870 3.51027 Va 55.12

Among Populations Within Groups

3 10.213 0.06308 Vb 0.97

Within Populations

58 165.437 2.85235 Vc 43.91

Total 62 271.519 6.49571

Table 1: Mean genetic (COI) distances between (bottom left) and within (diagonal) populations calculated in MEGA. The mean number of (COI) nucleotide differences for the populations used in Arlequin analysis (top right) are also included.

Table 2: AMOVA results for COI showing variation among Mediterranean (Crete, Malta) and GoM (EFGB, WFGB, Sonnier) groups, among the populations within these groups, and within each population.

References:1. Barroso, R., Klautau, M., Sole Cava, A., & Paiva, P. (2010). Eurythoe complanata (polychaeta: Amphinomidae), the

‘cosmopolitan’ wreworm, consists of at least three cryptic species. Marine Biology, 157, 69-80. 2. Excoffier, L., Laval, G., Schneider, S. (2005). Evolutionary Bioinformatics Online, 1, 47.3. Huelsenbeck, J. P., Ronquist, F. (2002). University of California, San Diego, Computer program distributed by the

authors.4. Lewis, S., (2009). Master’s Thesis for George Mason University, 93.5. Maddison, W. P. and D.R. Maddison. (2011). Mesquite: a modular system for evolutionary analysis. Version 2.75 http://mesquiteproject.org Miller, M.A., Pfeiffer, W., and Schwartz, T. (2010) "Creating the CIPRES Science Gateway for inference of large

phylogenetic trees" in Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA pp 1 - 8.

6. Sussman, M. , Loya, Y. , Fine, M. & Rosenberg, E. (2003). The marine fireworm Hermodice carunculata is a winter reservoir and spring-summer vector for the coral-bleaching pathogen vibrio shiloi. Environmental Microbiology, 5(4), 250-255.

7. Swofford, D. L. (2002) PAUP: Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinaur Associates, Sunderland, Massachusetts.

8. Tamura, K., Dudley, J., Nei, M., Kumar, S. (2007). Molecular Biology and Evolution, 24, 1596.9. Whitman, J. D. (1988). Effects of predation by the fireworm Hermodice carunculata on milleporid hydrocorals. Bulletin of

Marine Science, 42(3), 446-458. 10. Yaῆez-Rivera, B. & Salazar-Vallejo, S. (2011). Revision of Hermodice Kinberg, 1857 (Polychaeta: Amphinomidae). Scientia

Marina, 75(2), 251-262.

Acknowledgements:We would like to thank the Research and Graduate Studies Office of TAMUG for funding this project as well as the captain and crew of the M/V Fling for diving support. Thanks to Patrick Schembri (Malta) and Sarah Faulwetter (Crete) for providing Mediterranean samples, Marissa Nuttall and Brett Gonzalez for GoM specimens, Pat Krug (Bahamas), Gonzalo Giribet (Belize, Florida Keys), Beatriz Yaῆez-Rivera (Yucatan, Mexico) and Alex Wolf (Curaçao). Ben Nguyen, Andrew Nguyenba and Laura Timm provided additional support with sequencing.

Joseph Ahrens, Elizabeth Borda, Alexandra Campbell, Anja SchulzeTexas A&M University at Galveston

High degree of connectivity among amphi-Atlantic populations of Hermodice carunculata (Amphinomidae, Annelida)

Materials and Methods:Collection: SCUBA divers hand collected live specimens from East Flower Garden Banks (EFGB), West Flower Garden Banks (WFGB), Sonnier Bank, and Panama City, FL to represent the GoM population. Caribbean specimens and sequence data were provided from Curaçao, the British Virgin Islands and Bahamas. Mediterranean samples were taken from Crete and Malta. All specimens were preserved in ethanol. Additional sequences for specimens from the Yucatan Peninsula (n=4), Brazil (GenBank), Panama and the Florida Keys (n=2) were also included in the phylogenetic analyses. (Fig. 2)

Processing: Using standard protocols, a fragment of the cytochrome c oxidase subunit I (COI) gene (~650bp) and 16srDNA (~450bp) was sequenced for 62 specimens. Sequences were aligned using Mesquite (Maddison and Maddison, 2011) and exported for analysis.

Analysis: Two phylogenetic analyses were performed: Bayesian inference in MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2002) as well as maximum parsimony using 500 bootstrap pseudoreplicates in PAUP*v. 4.0a122 (Swofford, 2002). Bayesian analysis was run on servers powered by CIPRES (Miller et al., 2010). Several amphinomid taxa were used as outgroups to determine support values for the midpoint root, but were not included in the consensus tree. Population genetic analyses for three GoM populations (EFGB, WFGB and Sonnier), Crete, and Malta were conducted using Arlequin 3.5.1.2 (Excoffier et al., 2006). Genetic distances (uncorrected) between populations were calculated in MEGA 4.1 (Tamura et al., 2007).

Topics for Future Research:An examination of the morphology of Mediterranean outliers will help to determine whether there is a genetic basis for the morphological criteria used by Yaῆez-Rivera and Salazar-Vallejo (2011).

Histological analysis of bearded fireworms undergoing posterior regeneration will provide insight into the developmental processes giving rise to the pygidium as well as important accessory internal and external morphological features like the anal lobe and botryoidal tissue.

Designing a short amplicon primer set and an unlabelled probe around an informative region of COI or 16s rDNA for use in high resolution melting analysis (HRMA) will allow for fast and inexpensive genotyping of Hermodice specimens from each of the two main clades.

Figure 1: Hermodice carunculata specimens collected from Sonnier Bank. Dorsal branchial filaments from H. carunculata (right, top) and H. nigrolineata (right, bottom) from Yaῆez-Rivera and Salazar-Vallejo (2011).

Malta Crete EFGB WFGB Sonnier Panama City Curaçao Virgin Islands Bahamas

Malta 0.008 6.24 15.59 13.25 15.22

Crete 0.009 0.010 12.80 11.01 12.48

EFGB 0.030 0.025 0.012 5.61 5.75

WFGB 0.025 0.021 0.012 0.009 5.42

Sonnier 0.028 0.023 0.010 0.010 0.009

Panama City 0.029 0.024 0.011 0.010 0.009 0.011

Curaçao 0.029 0.024 0.012 0.010 0.010 0.010 0.011

Virgin Islands 0.030 0.024 0.010 0.008 0.009 0.009 0.009 0.007

Bahamas 0.024 0.020 0.010 0.010 0.009 0.010 0.010 0.010 0.011

0.99/100

1/58

0.5

1

1/67

0.99/92

Curaçao

British Virgin Islands

Bahamas / Florida Keys

Panama City, FL (GoM)

Sonnier Bank (GoM)

Flower Gardens Banks (GoM)

Malta

Crete

Panama

Yucatan / Belize

Figure 2: World map displaying sample sites for this study. Points are color-coded according to the legend shown in Figure 3. Basemap taken from ArcGISOnline.

Figure 3: 50% majority rule consensus tree constructed using Bayesian inference. Scale bar indicates percent divergence among taxa. Posterior probability (BI)/ bootstrap values (MP) are shown for some clades. The subclade in the primarily Greater Caribbean clade received high posterior probability but was unsupported by bootstrap analysis. Note the Mediterranean (Crete) outlier within the Greater Caribbean clade.