Barcoding the Fishes of North America Philip A. Hastings Scripps Institution of Oceanography University of California San Diego

With the possible exception of Europe and selected regional faunas such as Japan, the North American ichthyofauna is the arguably the best known in the world Numerous relatively recent compilations of regional components of the fish fauna are available West Coast Marine Alaska – Mecklenberg et al., 2002 Canada – Hart, 1973 United States – Miller & Lea, 1973 Mexico – Findley et al., 2005; Allen & Robertson, 1994 East Coast Marine Canada – Scott & Scott, 1988 United States - several Gulf of Mexico – McEachran & Fechhelm, 1998, 2006 Caribbean – Böhlke & Chaplin, 1968 Freshwater Mayden et al., 1992 Miller et al., 2006 Several Guides, e.g., Page & Burr, 1999

North American Fishes The task of compiling fish diversity estimates for this region was recently simplified by release of the latest edition of Common and Scientific Names of Fishes from the United States, Canada, and Mexico (6th edition) by J. S. Nelson, E. J. Crossman, H. Espinosa-Pérez, L. T. Findley, C. R. Gilbert, R. N. Lea & J. D. Williams Earlier editions included marine and freshwater fishes of Canada and the continental United States (including Alaska) This edition expands coverage to include both marine and freshwaters of Mexico 3,700 species included Approximately 1,200 freshwater species Approximately 2,500 marine species (Does not generally include deep-sea species) We have a relatively good understanding of overall diversity, but new species continue to be described from this well-known region

Specimen availability for barcoding of North America Fishes Specimens in most large collections have been fixed in formalin, and thus generally unsuitable* A few collections with significant North American holdings have “announced” separate tissue collections American Museum of Natural History University of Kansas Natural History Museum University of British Columbia Royal Ontario Museum Scripps Institution of Oceanography Others New collecting efforts will be needed for some components of the fauna

Scripps Institution of Oceanography Marine Vertebrates Collection Collecting Localities = 21,000 Specimen Lots = 110,000 Specimens = approximately 2,000,000 Species Represented = 5,300 Tissue Collection = 700 species in ethanol

CalCOFI component of the Pelagic Invertebrates Collection

Barcoding efforts currently underway on North America Fishes - 1 Hebert Lab: Fishes of Canada West coast marine – ca 330 species East coast marine – ca 300 species (Bentzen & Kenchington) Freshwater – ca 180 species (Bernatchez) Funding source: Moore Foundation

Barcoding efforts currently underway on North America Fishes – 2 “Establishing a DNA Sequence Database for the Marine Fish Fauna of California” Ronald S. Burton (Marine Biology Research Division, SIO) Philip A. Hastings (Curator of Marine Vertebrates, SIO) Funding Source: California Sea Grant

Marine Fishes of California 875 species recorded from marine waters of the State and offshore waters (e.g., CalCOFI time series) Project involves collecting and assembling tissues and sequence data for Cytochrome b and 16s for all species Original proposal written before CO1 was widely advocated as the preferred sequence for the Barcode of Fishes Significantly more data on these sequences existed for fishes Proposal pending to California Sea Grant to fund collection of CO1 data from same specimens Provide a test of relative strengths of sequences in a regional fauna

Establishing a DNA Sequence Database for the Marine Fish Fauna of California: Progress to Date Cytochrome b and 16s sequences have been collected for nearly 400 species Data are posted on GenBank shortly after they become available Bottleneck now lies with collection of tissue samples Next 200+ species will be readily obtained Final 250+ species will be a challenge to obtain Many are rare in State waters Many are deep-sea species Collaboration needed Collecting in other regions Sequence sharing from other projects

Other Data Sources Relevant to Barcoding of North America Fishes 1.Numerous phylogenetic and phylogeographic studies Don’t generally use CO1 but tissues or DNA may be available 2. NMFS stock assessments: Sebastes, Salmonids, etc. Issues include: - Data collection for appropriate sequence - Archiving of associated voucher specimens in collections We are working with phylogeneticists to locate “vouchers,” long buried in their freezers - Catalog them into SIO collection - Provide post-publication link to voucher specimens 3. Formalin-fixed material in collections

DNA Sequence from Formalin Preserved Museum Specimens John R. Hyde, SIO & SWFSC, NMFS Tissue autoclaved in alkaline lysis buffer to break formalin induced crosslinks DNA purified on commercially available silica matrix columns DNA fragments from bp amplified via PCR and sequenced

Preliminary Results on Marine Fishes of California: Comparison of CO1 & Cytochrome b Most published tests of CO1 for discriminating fish species have been done on regional ichthyofaunas In many cases, these will not contain most closely related species (often allopatric) Need appropriate sequence data for a clade that includes closely related as well as more distantly related species (most stringent test for the effectiveness of a particular gene sequence to function as a bar code)

Comparison of CO1 & Cytochrome b for Sebastes Research of John Hyde (SIO graduate student) working in Russ Vetter’s lab (NMFS) 100 species in genus (66 in the North Pacific) Includes a full array of deep and very shallow (cryptic) lineages Taxa sampled: both genes for 70 species Lengths CO1 b: 555 bp Cyt b: 1100 bp Cyt b : truncated to 555 bp

CO1 versus Cytochrome b sequences for Sebastes (rockfishes): Preliminary Results 1. Both genes resolve most species (more individuals needed) 2. A few tip clades (species) are discriminated by truncated Cyt b but not by similar-length sequence of CO1 3. Neither sequence discriminates cryptic diversity recently identified using microsatellites Conclusion: both sequences perform adequately for discriminating most species except for very recently diverged ones

North America Fishes DNA Database NAFD(N)A Willy Bemis – Cornell University Phil Hastings – SIO, University of California San Diego Rick Mayden – Saint Louis University Ed Wiley – University of Kansas Intended Habitat Coverage: Freshwater and Marine Fishes Intended Geographic Coverage: All of North America including Mexico Target sequences: Multiple, including CO1 Goals include: Archiving voucher specimens, tissues and DNA isolates in established museum collections (SIO, University of Kansas, Cornell University, University of Alabama, others) Exploring the extent and basis of sequence variation for selected species Barcoding Effort on North American Fishes – 3

Issues to be Addressed for Expanded Geographic Coverage - FishBOL Overlap of species ranges with other regions – few marine species are restricted to geopolitical regions

Latitudinal ranges of tropical eastern Pacific chaenopsids (tube blennies)

CO1 555 bpCyt b 555 bp

Establishing a DNA Sequence Database for the Marine Fish Fauna of California Proposal is now pending with California Sea Grant to: 1. Collect and sequence additional individuals 2. Collect CO1 sequence data from same samples Provide a direct test of the effectiveness of Cytochrome b, 16s and CO1 sequence data for discriminating among species within a diverse ichthyofauna Providing multiple sequences in the archive maximizes the utility of barcoding by overcoming some possible complicating issues such as coalescence Could be especially important in some forensic applications (e.g., litigations for fisheries violations)

Latitudinal ranges of tropical eastern Pacific pomacentrids (damselfishes)