Molecular phylogenetics reveals first record and invasion of Saccostrea species in the Caribbean Katrina M. Pagenkopp Lohan, Kristina M. Hill-Spanik, Mark E. Torchin, Ellen E. Strong, Robert C. Fleischer & Gregory M. Ruiz Marine Biology International Journal on Life in Oceans and Coastal Waters ISSN 0025-3162 Mar Biol DOI 10.1007/s00227-015-2637-5 1 23 Your article is protected by copyright and all rights are held exclusively by Springer-Verlag Berlin Heidelberg (outside the USA). This e- offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Mar Biol DOI 10.1007/s00227-015-2637-5 ORIGINAL PAPER Molecular phylogenetics reveals first record and invasion of Saccostrea species in the Caribbean Katrina M. Pagenkopp Lohan · Kristina M. Hill‑Spanik · Mark E. Torchin · Ellen E. Strong · Robert C. Fleischer · Gregory M. Ruiz Received: 29 August 2014 / Accepted: 17 February 2015 © Springer-Verlag Berlin Heidelberg (outside the USA) 2015 Abstract Taxonomic uncertainty often limits our ability data from the mitochondrial cytochrome oxidase I gene to resolve biogeographic patterns and discern biological were combined with morphological criteria to confirm the invasions. Within the bivalve mollusks, this uncertainty is identities of ten oyster species of Ostreidae, Isognomoni- particularly acute for oysters, as the high degree of pheno- dae, and Pteriidae, focusing on the Pacific and Caribbean typic plasticity of their shells creates taxonomic confusion. coasts of Panama, since tropical biota have received the The integration of molecular data with shell morphology least study. The results indicate that Crassostrea virginica, can differentiate species, providing new insights into bioge- previously only reported from this region along the Yucatan ography, invasions, and ecology of this functionally impor- Peninsula and coast of Venezuela, also occurs in the Carib- tant group. As an initial step in resolving the identities and bean waters of Panama. We also document the first record current geographic distributions of oyster species, sequence for a species of Saccostrea, a genus native to the Pacific, suggesting an invasion by an unknown non-native Saccos- trea species that is now widespread along the Caribbean Communicated by C. Riginos. from the Panama Canal west to Bocas del Toro. Sequences Electronic supplementary material The online version of this of the internal transcribed spacer region (ITS1) of the ribo- article (doi:10.1007/s00227-015-2637-5) contains supplementary somal gene complex (rDNA) did not reveal any hybridi- material, which is available to authorized users. zation. Considering the high connectivity of shipping and boating in Panama, Saccostrea sp. may have been intro- K. M. Pagenkopp Lohan · K. M. Hill-Spanik · R. C. Fleischer Center for Conservation and Evolutionary Genetics, Smithsonian duced to the Caribbean by either recreational or commer- Conservation Biology Institute, National Zoological Park, cial vessels, but the timing and potential ecological effects Washington, DC 20008, USA of this invasion remain unknown. K. M. Pagenkopp Lohan (*) · K. M. Hill-Spanik · G. M. Ruiz Marine Invasions Laboratory, Smithsonian Environmental Research Center, Edgewater, MD 21037, USA Introduction e-mail: [email protected] Oysters are ecosystem engineers, playing many vital roles Present Address: K. M. Hill-Spanik in coastal ecological processes, including nutrient cycling Grice Marine Lab, College of Charleston, Charleston, and benthic-pelagic coupling (Newell 2004), helping SC 29414, USA decrease eutrophication (Kemp et al. 2005), and creating habitat that serves as nurseries for commercially impor- M. E. Torchin Smithsonian Tropical Research Institute, Apartado, tant fish and crab species (Coen et al. 2007). Additionally, 0843-03092 Balboa, Ancon, Republic of Panama oysters serve as hosts for a variety of protistan parasites, some of which can cause massive host mortalities and alter E. E. Strong community structure and ecosystem dynamics (Villalba Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, et al. 2004; Burreson and Ford 2004; Carnegie and Cochen- DC 20560, USA nec-Laureau 2004). Although oysters contribute greatly to 1 3 Author's personal copy Mar Biol integral biotic processes in coastal waters, the geographic As the first step in a study to resolve the broad-scale bio- boundaries of many species are not well understood, and geography of oysters and their associated parasites across their taxonomy is often unclear. This is mainly due to their North and Central America, we used a combination of mor- phenotypically plastic shells, which can be greatly influ- phological criteria and sequences of the mitochondrial COI enced by environmental factors (Tack et al. 1992; Wilk and gene from multiple oyster species within the family Ostrei- Bieler 2009), making species difficult to identify and cir- dae and several families within the Pterioidea, focusing on cumscribe based on external shell characteristics alone. commonly occurring species from multiple locations along To add to the confusion, oyster species often have the Pacific and Caribbean coasts of Panama. Our goals widespread and overlapping geographic ranges, reflecting were to confirm the identities of the bivalve species col- both natural and anthropogenic mechanisms for disper- lected and to determine their distributions within Panama- sal. Many bivalve larvae have extended planktonic stages, nian waters. enabling them to disperse to distant locations (Scheltema 1986). Juvenile and adult oysters rafting on floating debris is another potential mechanism for the natural dispersal Materials and methods of some species, sometimes over vast distances (e.g., Ó Foighil et al. 1999; Donald et al. 2005). Bivalves can also Specimen collection be spread inadvertently via maritime traffic by attaching to the hulls of vessels (Gollasch 2002; Carlton 1992; David- We collected oysters from a variety of intertidal and subtidal son et al. 2009), as larvae in ballast water (Gollasch et al. habitats (primarily mangrove rhizophores, rocks, docks, pil- 2002; Briski et al. 2012), or associated with sea chests ings, etc.) from multiple locations on the Pacific and Carib- (Coutts et al. 2003; Coutts and Dodgshun 2007). Addition- bean coasts near the Panama Canal and at Bocas del Toro ally, many bivalve species have been moved deliberately (Fig. 1). Maps showing the distribution of three of the iden- for aquaculture purposes (McKindsey et al. 2007) and tified bivalves and all sampling locations were generated through the aquarium trade (Padilla and Williams 2004; using ArcGIS 10.2.2 for Desktop (Esri, Redlands, Califor- Weigle et al. 2005). All of these mechanisms aid in dis- nia). At the time of collection, physical data associated with persing bivalve species and modifying their distributions, the water were also collected (Electronic Supplementary which, when combined with the inherent difficulties of Material (ESM) Table S1). We placed oysters in coolers on morphological identification, can contribute to confusion in ice and, upon returning to the laboratory, kept the oysters understanding the geographic range of species or recogniz- at ~4 °C for no more than 72 h. Prior to the tissue sam- ing new invaders. pling, we removed epibionts and mud from the shells, then Increasingly, molecular markers are being used as an measured and shucked the oysters. Oysters were tentatively independent data source for species identification, to detect identified in the field to the lowest possible taxonomic level cryptic or unrecognized species, and to clarify persistent (genus or species), using standard bibliographic references taxonomic and biogeographic uncertainty. Molecular mark- [e.g., Romashko (1992), Tucker Abbott and Morris (1995), ers have been successfully used to differentiate members Kaplan (1982) and Coan and Valentich-Scott (2012)] and within the genus Crassostrea (e.g., Lam and Morton 2003; later compared with synoptic collections deposited in the Reece et al. 2008; Cordes et al. 2008; Sekino and Yamash- National Museum of Natural History. For molecular analy- ita 2013) and the genus Saccostrea (e.g., Lam and Morton ses, we sampled pieces of gill, mantle, and digestive gland 2006; Sekino and Yamashita 2013), and between other and preserved them in 95 % ethanol. The majority of shells closely related oyster genera (e.g., Klinbunga et al. 2005; were thoroughly cleaned, dried, labeled with unique identi- Liu et al. 2011), as well as aiding in resolving the distribu- fication numbers, and retained as vouchers. tions of other bivalve species. For example, sequence data confirmed the Trans-Atlantic geographic ranges of C. rhiz- DNA extraction, PCR amplification, and sequencing ophorae and C. gasar (Lapégue et al. 2002), as well as the identities and geographic ranges of multiple Crassostrea Following an overnight digestion with proteinase