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

2. 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

3. 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

4. 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

6. 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

7. CalCOFI component of the Pelagic Invertebrates Collection

8. 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

9. 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

10. 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

11. 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

12. Other Data Sources Relevant to Barcoding of North America Fishes
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

13. 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

14. 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)

15. 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: bp
Cyt b : truncated to 555 bp

16. 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

17. Barcoding Effort on North American Fishes – 3
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

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

19. Latitudinal ranges of tropical eastern Pacific chaenopsids
(tube blennies)

22. Cyt b 555 bp
CO1 555 bp

24. 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)

25. Latitudinal ranges of tropical eastern Pacific pomacentrids
(damselfishes)