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 MarineAlaska – Mecklenberg et al., 2002Canada – Hart, 1973United States – Miller & Lea, 1973Mexico – Findley et al., 2005; Allen & Robertson, 1994

• East Coast MarineCanada – Scott & Scott, 1988United States - severalGulf of Mexico – McEachran & Fechhelm, 1998, 2006Caribbean – Böhlke & Chaplin, 1968

• FreshwaterMayden 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 HistoryUniversity of Kansas Natural History MuseumUniversity of British ColumbiaRoyal Ontario MuseumScripps Institution of OceanographyOthers

• New collecting efforts will be needed for some components of the fauna

Scripps Institution of OceanographyMarine Vertebrates Collection

Collecting Localities = 21,000Specimen Lots = 110,000

Specimens = approximately 2,000,000Species 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 samplesNext 200+ species will be readily obtainedFinal 250+ species will be a challenge to obtain

Many are rare in State waters Many are deep-sea species

• Collaboration neededCollecting in other regionsSequence 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

Specimen I D# Species Collection Date SequencedSIO 59-205 Sebastes cortezi 4/4/1959 1141bp cytochrome bSIO 68-90-8009 Sebastes cortezi 1/18/1968 1141bp cytochrome b, 386bp 12S rRNA, 586bp 16S rRNA, 624bp dloopSIO 68-90 Sebastes cortezi 1/18/1968 1141bp cytochrome bLACM 33538-1 Sebastes melanosema 6/21/1971 1141bp cytochrome b, 386bp 12S rRNA, 586bp 16S rRNA, 624bp dloopSIO 97-65 Sebastes notius 8/14/1996 1141bp cytochrome bSIO 65-410-53 Sebastes oblongus 1/ 21/ 1901 1141bp cytochrome b, 386bp 12S rRNA, 586bp 16S rRNA, 198bp dloopSIO 03-90 Sebastes pachycephalus 2/1/1999 1141bp cytochrome bCAS 27154 Sebastes rufinanus 9/8/1970 1141bp cytochrome b, 386bp 12S rRNA, 586bp 16S rRNA, 512bp dloopSIO68-89-53 Sebastes sinensis 1/18/1968 1141bp cytochrome bSIO 68-90-8012 Sebastes sinensis 1/18/1968 1141bp cytochrome b, 386bp 12S rRNA, 586bp 16S rRNA, 624bp dloopSIO 68-90-8044 Sebastes sinensis 1/18/1968 1141bp cytochrome b

DNA Sequence from Formalin Preserved Museum SpecimensJohn 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 200-300bp 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

• LengthsCO1 b: 555 bpCyt 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 UniversityPhil Hastings – SIO, University of California San DiegoRick Mayden – Saint Louis UniversityEd 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 Latitudinal ranges of tropical eastern Pacific tropical eastern Pacific chaenopsidschaenopsids(tube blennies)(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 Latitudinal ranges of tropical eastern Pacific tropical eastern Pacific pomacentridspomacentrids(damselfishes)(damselfishes)