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1-27-2006 Evolution of Endosymbiosis in (xylotrophic) Wood-Eating Bivalves Daniel L. Distel Principal Investigator; University of Maine, Orono

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Recommended Citation Distel, Daniel L., "Evolution of Endosymbiosis in (xylotrophic) Wood-Eating Bivalves" (2006). University of Maine Office of Research and Sponsored Programs: Grant Reports. 133. https://digitalcommons.library.umaine.edu/orsp_reports/133

This Open-Access Report is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in University of Maine Office of Research and Sponsored Programs: Grant Reports by an authorized administrator of DigitalCommons@UMaine. For more information, please contact [email protected]. Annual Report: 0129117

Annual Report for Period:06/2004 - 06/2005 Submitted on: 01/27/2006 Principal Investigator: Distel, Daniel L. Award ID: 0129117 Organization: University of Maine Title: Evolution of Endosymbiosis in (xylotrophic) Wood-Eating Bivalves

Project Participants Senior Personnel Name: Distel, Daniel Worked for more than 160 Hours: Yes Contribution to Project:

Post-doc

Graduate Student Name: Luyten, Yvette Worked for more than 160 Hours: Yes Contribution to Project: Graduate student participated in lab research with support from this grant. Name: Mamangkey, Gustaf Worked for more than 160 Hours: Yes Contribution to Project: Gustaf Mamangkey is a lecturer at the Tropical Marine Mollusc Programme, Faculty of Fisheries and Marine Sciences, Sam Ratulangi University,Jl. Kampus UNSRAT Bahu, Manado 95115,Indonesia. Gustaf has participated in this project as a scientific collaborator and is responsible for collecting all of the Indonesian specimens used in this study. Gustaf is an authority on the ecology and systematics of Indonesian Teridinidae. This year Gustaf has begun a Ph.D program at James Cook UniversityTownsville, Queensland 4811 AUSTRALIA. His participation in this project will contribute to his Ph.D training.

Undergraduate Student Name: Burgoyne, Adam Worked for more than 160 Hours: Yes Contribution to Project: Undergraduate student participated in lab research with support from this grant. Name: Willey, Jennifer Worked for more than 160 Hours: No Contribution to Project: Undergraduate student participated in lab research with support from this grant. Name: Chen, Tim Worked for more than 160 Hours: No Contribution to Project: Undergraduate student participated in lab research with support from this grant. Name: Eash, Angela Worked for more than 160 Hours: Yes Contribution to Project:

Page 1 of 8 Annual Report: 0129117 Undergraduate student participated in lab research with support from this grant. Name: Bosse, Ben Worked for more than 160 Hours: No Contribution to Project: Undergraduate student participated in lab research with support from this grant. Name: Amin, Mehwish Worked for more than 160 Hours: Yes Contribution to Project:

Technician, Programmer Name: Spenlinhauer, Tania Worked for more than 160 Hours: Yes Contribution to Project:

Name: Morrill, Wendy Worked for more than 160 Hours: Yes Contribution to Project:

Name: Cannone, Jamie Worked for more than 160 Hours: Yes Contribution to Project: Jamie Cannone is Dr. Gutell's research associate and has assisted in secondary structure inference and sequence alignment.

Other Participant Name: Gutell, Robin Worked for more than 160 Hours: Yes Contribution to Project: Dr. Gutell is a fculty member at the University of Texas and is a leading authority on RNA structure. Dr. Gutell is assisting in our research by inferring secondary structures for bivalve 18S and 28S rRNAs to assist in structural alignment of sequences.

Research Experience for Undergraduates

Organizational Partners

Other Collaborators or Contacts Gustaf Mamangkey, Faculty of Fisheries and Marine Sciences Sam Ratulangi University, Manado, 95115 INDONESIA, collected numerous valuable specimens for this project and will serve as a consultant in evaluating the data. This year Gustaf has begun a Ph.D program at James Cook UniversityTownsville, Queensland 4811 AUSTRALIA. His participation in this project will contribute to his Ph.D training. Dr. Robin Gutell, Faculty, University of Texas. Dr. Gutell and his research associate, Jamie Cannone were recruited as collaborators in this project to develop secondary structural models for the 18S and 28S ribosomal RNAs of specific bivalve taxa and to implement these models in creating and assessing the certainty of sequence alignments using structural features and compensatory substitutions as evidence for positional homology.

Page 2 of 8 Annual Report: 0129117 Activities and Findings

Research and Education Activities: (See PDF version submitted by PI at the end of the report) Some Relevant History: Funds for the current project were initially awarded to the University of Maine where I served as a faculty member until the spring of 2004. Shortly thereafter, I resigned from the University of Maine and accepted a position as Director of Genome Legacy Foundation (OGL). At this time, I requested and received a no-cost extension on this award, anticipating some inevitable disruption and delay in the progress of the project. I still retain a non-salary faculty position at the University of Maine, allowing me to continue to advise students. My graduate student has moved with me to the Ocean Genome Legacy Foundation but still retains her status as a full time student at the University of Maine, conducting her studies off campus. The Ocean Genome Legacy Foundation has requested ônew NSF awardee organizationö status and has completed all of the requirements of that request. As soon as the processing of this status change is complete, I will request transfer of this award to the Ocean Genome Legacy Foundation. In anticipation of the transfer, the project has been continued at OGL supported by existing OGL funds. Research and Education Activities The purpose of this investigation is to use molecular and morphological characters to explore the function, diversity, and evolution of wood boring, wood feeding, and symbiosis in wood-boring bivalves. We proposed to accomplished this by: 1) Producing a robust phylogeny for these morphologically diverse bivalves, and 2) Mapping on this phylogeny the occurrence of morphological, biochemical, and molecular traits associated with symbiosis, wood boring and wood feeding. The sampling strategy proposed includes a broad representation of the diversity of the families Teredinidae and Pholadidae and representative taxa within the parent Order Myoida. We proposed to perform phylogenetic analyses using two nuclear encoded (18S and 28S rRNA) and two mitochondrial (16S rRNA and COI) genes. The latter two biomarkers have been eliminated from the study for the following reasons: 1) we observed taxon-specific amplification failures using standard 'universal' PCR primers. These amplification failures are apparently due to variation in ôuniversalö primer regions. This is supported by the observation that all of the failed amplifications fall within a single phylogenetic lineage as identified by 18S and 28S phylogenies. Unfortunately this lineage includes the two primary taxa of interest in this study, the family Teredinidae, and the pholadid subfamily Xylophagainae. We note that the same result was observed for amplification of COI for members of the Xylophagainae by other investigators (pers. comm. V. Tunnicliffe), 2) Analyses to date indicate that 18S and 28S loci provide sufficient phylogenetic resolution to address the questions proposed here. An additional goal has been added to the project in the analysis stage. It was determined that the certainty of alignments for 28S genes, and therefore the certainty of resultant phylogenetic inferences, would be greatly improved by development of secondary structural models for bivalve 28S verified by in depth analysis of compensatory substitutions across the Class. To achieve this, a collaboration was initiated with the laboratory of Robin Gutell (University of Texas, Austin). To date we have completed a secondary structural model for Placopecten magellanicus 28S (the first available for a bivalve) and are in the process of producing additional structures for Mya arenaria 28S and 18S.

Page 3 of 8 Annual Report: 0129117 Please see attached pdf documents for a view of the 5' half of the P. magellanicus 2' structure). The complete structure can be viewed at http://www.rna.icmb.utexas.edu. Education To date, five undergraduate students and one graduate student have been trained in DNA sequencing, DNA cloning, sequence analysis, and phylogenetic analysis. We have also made progress toward implementing our K-12 outreach program. A collaboration has been established with the Ramey School, Ramey, Puerto Rico; a school serving the American military community in Puerto Rico. Science teacher Rex Roettger will work with me to develop research and educational materials for a high school research project to include eighteen 10th through 12th grade students. The students will conduct experiments to observe the growth of in their local waters and will record differences in infestation of wooden test panels placed in various depths and salinity regimes. The students will also contribute to and learn about this NSF funded project. They will preserve some of their specimens in ethanol for use in our NSF funded research and we will report to the students on how their efforts have contributed to the overall project.

Findings: (See PDF version submitted by PI at the end of the report) Results Please see attached pdf document for updated table describing sequencing progress to date. Findings Several important findings have been made: Results to date provide strong support for the following: 1) Wood feeding (xylotrophy) has arisen only once in bivalve evolution, rather than twice in two separate families as has been previously proposed. 2) All wood feeding bivalves share a single common ancestor that was likely a wood boring member of the family Pholadidae 3) The family Teredinidae (shipworms) appears to be a sister or daughter lineage of the subfamily Xylophagainae (deep-sea wood-borers) of the family Pholadidae. Thus, the of both families will likely require revision. The common ancestor of all wood feeders likely had the following features: a) Projections for mussel attachment on the internal surface of the valves (apophyses). This feature is found in non-wood-feeding pholads and Teredinidae but was lost in the deep-sea Xylophagainae. b) The intestine traversed the heart. The intestine traverses the heart in all non- wood-feeding pholads. This feature is retained in Xylophagainae, but the intestine is separated from the heart in all Teredinidae except . c) A single gill demibranch. The outer gill demibranch is retained in rock- and wood- boring pholadidae but is lost in all wood-feeding bivalves. d) A globular stomach. e) A caecum for storing wood particles during digestion. f) Reduced labial palps and crystaline style

Page 4 of 8 Annual Report: 0129117 g) Gill-born bacterial endosymbionts h) Protandrous hermaphroditism Several interesting evolutionary trends are also apparent. In Teredinidae: a) The ancestral teredinid had a globular stomach. The stomach becomes progressively more elongated in more recently divergent lineages. b) The heart was located in an anterior position. The heart migrates toward a posterior location in more recently divergent lineages. c) The pallets were composed of a single unit. In more recent lineages the pallets become segmented, but segmentation is secondarily lost in the most recently diverged lineages. d) In the ancestral teredinid, an extension of the rectum (the anal canal) served as a secondary storage site for wood particles. Recent research demonstrates that this organ, like the caecum, contains a brush border epithelium and is likely a secondary site for wood degradation and nutrient absorbtion. This feature is lost in more recently diverging lineages. e) The ancestral teredinid was a broadcast spawner. Internal fertilization and larvipary evolved only once in the most recently divergent Teredinidae. In xylophagainae: a) Apophyses are lost in all xylophagainae. Uncertain features: a) The formation of a calcareous tube lining the burrow appears in all Teredinidae and at least one genus of Xylophagainae (Xyloredo) but is absent in all non-wood-feeding Pholadidae. b) Pallets are present in all Teredinidae and at least one genus of Xylophagainae (Xylopholas) but are absent in all non-wood-feeding Pholadidae. c) The separation of the heart and intestine is observed in all Teredinidae except Kuphus, but is absent in all Pholadidae and Xylophgainae. The phylogenetic position of Kuphus, Xyloredo, and Xylopholas must be resolved before it is possible to determine which of these features is ancestral and which is derived and whether any have arisen independently in different lineages. We have obtained specimens of all three genera and are in the process of sequencing the appropriate genes. Three manuscripts are in preparation based on the described work: Distel, D.L., Morrill, W., Spenlinhauer,T., Luyten, Y., Cannone, ,J., and Gutell, R. (in preparation) ôA secondary structural model of the 28S rRNA from heterodont bivalves: Implications for sequence alignment and phylogeny of the bivalve order Myoida.ö Distel, D.L., Mamangkey, G.F., Burgoyne, A., Morrill, W., Spenlinhauer, T. Luyten, Y., and

Page 5 of 8 Annual Report: 0129117 Amin, M. (in preparation) Phylogenetic analysis of relationships among the bivalve families Pholadidae and Teredinidae based on large and small subunit ribosomal RNA sequences: Evidence for non-monophyly of Pholadidae. Mamangkey, G.F., Burgoyne, A., Morrill, W., Spenlinhauer, T. Luyten, Y., Amin, M. and Distel, D.L.(in preparation) Evolution of wood-boring, wood feeding and xylotrophic symbiosis in .

Training and Development: To date, six undergraduate students and one graduate student and one technician have been trained in DNA sequencing, DNA cloning, sequence analysis, and phylogenetic analysis. This project has also contributed to research experiences for to two high school students.

Outreach Activities: A collaboration has been established with the Ramey School, Ramey, Puerto Rico; a school serving the American military community in Puerto Rico. Science teacher Rex Roettger will work with me to develop research and educational materials for a high school research project to include eighteen 10th through 12th grade students. The students will conduct experiments to observe the growth of shipworms in their local waters and will record differences in infestation of wooden test panels placed in various depths and salinity regimes. The students will also contribute to and learn about this NSF funded project. They will preserve some of their specimens in ethanol for use in our NSF funded research and we will report to the students on how their efforts have contributed to the overall project. We have also recently embarked on a community outreach program with the Ipswich School district. The Ocean Genome Legacy Foundation is currently in the process of developing mentoring programs for K-12 teachers in the Ipswich School District. On January 23, 2006, OGLF, in conjunction with New England Biolabs, hosted 15 representatives from the Ipswich primary, middle and high schools, including teachers and administrators, for a three-hour development workshop to formulate a working plan for educational collaboration with the Ipswich school system. OGLF also maintains one part- time intern position for students from the Ipswich High School (position currently held by Sam Wilbur, sophomore).

Journal Publications

Books or Other One-time Publications

Web/Internet Site

URL(s): www.oglf.org Description: The Ocean Genome Resource and Ocean Genome Resource Database are respectively, a marine genome resource bank and an online catalog and database serving this bank. Both are under development by the Ocean Genome Legacy Foundation and are scheduled to become available to the public in the latter

Page 6 of 8 Annual Report: 0129117 half of 2006. All specimens and samples from this project are currently being accessioned into the Ocean Genome Resource and will be included in the online database which will be hosted on the OGL website (www.oglf.org). Subsamples of tissues, DNAs and amplification products from all accessioned specimens in the Ocean Genome Resource will be made available upon request for research purposes through the database via an on-line request form. Large and small subunit rRNA sequences obtained in this investigation will also be available through the Ocean Genome Resource Database via links to data files. These sequences will also be submitted to genbank where they will be connected to the samples in the OGR database via link-outs. Thus it will be possible to locate all samples or sequences from this investigation through searches of BLAST, Entrez, or the OGR database. Additionally, brief descriptions of all NSF funded research conducted at OGL will be available on the "Research" page at www.oglf.org.

Other Specific Products

Product Type: Physical collection (samples, etc.) Product Description: Ocean Genome Resource Database: Samples and specimens of bivalves tissues, DNAs and voucher specimes from 110 collections supported by this award have been deposited in the Ocean Genome Resource, a public access marine genome resource collection maintained and opperated by the Ocean Genome Legacy foundation. The samples are currently being accessioned. Sharing Information: The Ocean Genome Resource is scheduled to become available to the public in the latter half of 2006. All accessioned specimens will be included in an online database (currently under developpment) which will be hosted on the OGL website (www.oglf.org). Subsamples of tissues, DNAs and amplification products from all accessioned specimens can be requested via an on-line request form. Large and small subunit rRNA sequences obtained in this investigation will also be available through the Ocean Genome Resource Database via links to data files. These sequences will also be submitted to genbank where they will be connected to the samples in the OGR database via link-outs. Thus samples or sequences from this investigation can be located through searches of BLAST, Entrez, or the OGR database.

Contributions Contributions within Discipline: The work will provide new sequences and insights into the phylogeny of the Myoida which is currently under-represented in GenBank. We will also provide secondary structures for bivalve 18S and 28S rRNA molecules which will be widely useful within the discipline for use as sequence alignment aids. Currently, no published 28S and only one published 18S secondary structure is available for bivalves.

Contributions to Other Disciplines: The project is demonstrating a unique and distinct progression of evolutionary events triggered by the acquisition of symbionts by a metazoan lineage. Symbiosis has been recognized as a source of evolutionary innovation, however, no well documented and detailed progression of evolutionary adaptations has been observed in a single lineage to demonstrate the types of morphological and physiological changes that may be associated with establishment of a bacterial endosymbiosis. The evolution of bacteria- interactions is of general interest to the study of evolutionary biology, evolutionary medicine and infectious disease, developmental biology and immunology.

Contributions to Human Resource Development:

Page 7 of 8 Annual Report: 0129117 To date, six undergraduate students and one graduate student and one technician have been trained in DNA sequencing, DNA cloning, sequence analysis, and phylogenetic analysis. The work is also contributing to the professional development of one foriegn graduate student from a the developing nation of Indonesia and has contributed to research experiences for two high school students. Contributions to Resources for Research and Education: This project has contributed to the Ocean Genome Resource and database which will provide important physical resources (specimens), data and tools to the research and educational communiites. Contributions Beyond Science and Engineering:

Special Requirements

Special reporting requirements: None Change in Objectives or Scope: None Unobligated funds: less than 20 percent of current funds Animal, Human Subjects, Biohazards: None

Categories for which nothing is reported: Organizational Partners Any Journal Any Book Contributions: To Any Beyond Science and Engineering

Page 8 of 8 Secondary Structure: large subunit ribosomal RNA - 5' half

A U A G G U C A A A U G G G A C C G C G G G U U A A G A C U A CC C G G A G A U A G G G A U G U U C C C G C C A A A A C A G G C C G G G C U A C C A U U G U A G U A A G A A G C C A U A A C G G C U A A G G U C C C U A G C A G G G A G C C A A A U C G A U G A C G G C C U U C A G C U U G A C U A C U C U U U G C A C U G C G C G G C A U U A A A C G U G A C G U C C U A C A C A G A A G G U A G U G A U G U A U C U G C C G G C G C C A C G U A A G C G U U C G U A A G G C U G C A C G U U C G A C G U G C G U A U A G C A U A A A U G C U G G C A U G G U U A G C G A A U U C U U U C G G G C C G C A C G C A C G C G C G C G C C A U U C C U G G G C G A A A U G A G C G A A U U U A G C C G A G U A A U U A U C A U A C C C A A A A U A A C U C A U U A G A G C C G G G U G U U C G G C C G G A U C G G A G C U C G G A G A G G C C C G A C A C A G C C C C G G C C G U G C U U C G A G C C U U A U A A U C C G G A A U G C G U G G A U A G A U G U G C G A A G A A U A C C A A G U C G U G A U G G C C G C C U A G U G A G A C G 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G C A U C U U C G C C A U U G C G G U U C G G C C A G C U C A U U G C G U C G G G G A C G A C C G G U C G U G U U C G G G U G C C A G C C U G G C C A G C G C C G A U U C G G U G C U G C G A G A G U C C U G C U G G U C G G C A C A U G C U C A C G G G C C G U G C A U A A U U G C G G C C G C G U A G C Placopecten magellanicus A U U A C U A U U A A G U U G C A A G C C G UU G (AF342798) C G G A C G A U C G G U A G G C G U A G U U C C U G C C G G C G U 1.cellular organisms 2.Eukaryota A A U G C G G G A A G A U U C GA A U U G C U U C G 3.Fungi/Metazoa group 4.Metazoa U G G U C G G C 5' A G U U U 3' C G U G U G 5.Eumetazoa 6.Bilateria 7.Coelomata A G G C C G C G A U U A a C G 8.Protostomia 9. 10.Bivalvia U U G C A U A C G A U G U A U 11.Pteriomorphia 12.Pectinoida U A G C G C G C G C G C A U C U 13.Pectinoidea 14.Pectinidae G C C U C G A U G C G C 15.Placopecten U U C U October 2004 Citation and related information available at http://www.rna.icmb.utexas.edu Specimen name Source Collection site 18S bp of 28S bp of Status number sequence sequence completed completed 102 Scott Gallager Woods Hole Oceanog. 1782 3196 sequencing pedicellatus Inst. , MA, Culture, completed 106 Hiatella arctica R. McConnaughy La Jolla, CBM field 1810 1416 sequencing (formerly (SIO-UCSD) station, 15m completed Petricola carditoides) 107 Xylophaga Jack Merrill 12 miles east of 1840 3143 sequencing atlantica Southwest Harbor, ME, completed 100m 136 Mya arenaria Supermarket, Maine coast 1792 3106 sequencing Bangor, ME, completed 167 Cyrtopleura R. Mann, William Wachapreague, VA 1861 3120 sequencing costata and Mary, VA completed

169 Petricola R. Mann, William Wachapreague, VA 1799 1380 28S sequence pholidiformis and Mary, VA incomplete missing first 100 bp

172 Xylophaga Seth Tyler, U. of Dredged Wood, Friday 1837 1477 sequencing washingtona Wash. Harbor, WA completed 173 Unknown Seth Tyler, U. of Dredged Wood, Friday n n no pallets, no ID Wash. Harbor, WA 210 Martesia sp. Sherry Reed Ft. Pierce FL., 1610 n duplicate taxon (see (striata?) Australian stump 281) 212 Pholas dactylus Ben Totterdell, Rocky Shore, 1804 1454 sequencing Charmouth, Dorset, UK completed

216 Barnea truncata Roger Mann Wachapreague, VA 1824 1506 sequencing (VIMS) completed 217 sp. Dan Distel Tobago, driftwood 1828 1487 sequencing Bartschi completed 218 sp. Dan Distel Tobago, driftwood n 1503 duplicate taxon (see carinata 281) 220 Belfast Harbor, Collection panels, 1830 3149 duplicate taxon (see 9/22/00 Belfast pier 281) 229 Bankia gouldi Battelle Lab n n not yet dissected 230 Teredo navalis Batelle Station 10A n n duplicate taxon 236 Kuphus Donald Dan, Ft Zamboanga del Sur, 1795 1437 sequencing polythalamia Meyers FL Mindanao, Philippines completed 258 Xylophaga sp. R. Kilburn, Natal SE of Port Dunford,29 1777 1457 sequencing Museum, S. 02.2S: 32 19.6E, 760- completed Africa 800m 259 Bankia setacea Karen Ripley, Brown's Bay, WA, 1793 1490 sequencing WA from a rotted piling completed 260 reynii Praia Dura, Sao Praia Dura, Ubatuba, 1794 1462 sequencing Paulo, Brazil Brazil completed 261 Nausitora Praia Dura, Sao Praia Dura, Ubatuba, 1846 1427 sequencing fusticula Paulo, Brazil Brazil completed 263 Bankia carinata Dan Distel Lac Bay, Bonaire, 1791 1497 sequencing submerged wood completed 264 Spathoteredo Gustaf Manado Bay, Inodesia 1769 3180 sequencing obtusa Mamangkey, completed Manado 266 Bankia australis Gustaf Manado Bay, Inodesia 1821 1497 sequencing Mamangkey, completed Manado 267 Lyrodus massa Gustaf Manado Bay, Inodesia 1816 1467 sequencing Mamangkey, completed Manado 268 Teredothyra sp. Dan Distel Bachelor's Beach, 1819 1490 sequencing (dominicensus?) Bonaire, NA, sunken completed wood 3m 269 Teredo sp. Dan Distel Bachelor's Beach, n n not yet dissected Bonaire, NA, sunken wood 3m 270 Teredora sp. Dan Distel Lagoen, Bonaire, NA, n n not yet dissected malleolus? driftwood 271 Bankia sp. Dan Distel Lagoen, Bonaire, NA, n n not yet dissected zeteki? driftwood 273 Lyrodus sp. Dan Distel Bachelor's Beach, n n duplicate taxon pedicellatus? Bonaire, NA, sunken wood 3m 274 Bankia australis Gustaf West coast of Minahasa n n not yet dissected Mamangkey, Peninsula, Inodesia Manado 275 Spathoteredo Gustaf West coast of Minahasa n n duplicate taxon obtusa Mamangkey, Peninsula, Inodesia Manado 276 Nausitora Gustaf West coast of Minahasa 1818 1475 duplicate taxon dunlopei Mamangkey, Peninsula, Inodesia Manado 277 Lyrodus massa Gustaf West coast of Minahasa n n duplicate taxon Mamangkey, Peninsula, Inodesia Manado 278 Lyrodus Gustaf West coast of Minahasa n n duplicate taxon pedicellatus Mamangkey, Peninsula, Inodesia Manado 279 Dicyathifer Gustaf West coast of Minahasa n n not yet dissected manni Mamangkey, Peninsula,Inodesia Manado 280 Martesia striata Gustaf West coast of Minahasa n n duplicate taxon Mamangkey, Peninsula, Inodesia Manado 281 Martesia sp Gustaf West coast of Minahasa 1690 1503 sequencing Mamangkey, Peninsula, Inodesia completed Manado 282 Lyrodus sp. Dan Distel Banana River, FL, 1812 1173 sequencing mangrove wood completed 288 Xyloredo sp. Charles Fisher Gulf of Mexico, (27 1430 1430 sequencing 44.75 : 91 13.31) 540m, completed 289 Xylopholas sp. Charles Fisher Gulf of Mexico, (27 1800 1470 sequencing 44.75 : 91 13.31) 540m, completed oak 290 Hiatella arctica Gonzalo Girabet, Banyuls sur Mer, n n duplicate taxon Harvard U. France, dredge, 30-60m

291 Varicorbula Gonzalo Girabet, Florida, trawl FK-106 n 694 DNA extracted, but limulata Harvard U. degraded 297 Panope Taylor United,Inc Taylor shellfish farms 1762 1412 sequencing completed sequencing 328 Mya truncata Gulf of Maine,Inc Cobscook Bay, Maine 1823 3096 completed