Barcoding the Fishes of North America

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Barcoding the Fishes of North America

• Philip A. Hastings
• Scripps Institution of Oceanography
• University of California San Diego

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• 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

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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

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• 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

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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
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CalCOFI component of the Pelagic Invertebrates
Collection
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• 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

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Barcoding efforts currently underway on North
America Fishes 2Establishing 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

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• 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

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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

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• Other Data Sources Relevant to Barcoding of North
  America Fishes
• Numerous phylogenetic and phylogeographic studies
• Dont 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

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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 200-300bp amplified via PCR
and sequenced
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• 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)

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• Comparison of CO1 Cytochrome b for Sebastes
• Research of John Hyde (SIO graduate student)
  working in Russ Vetters 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

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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
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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)

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Issues to be Addressed for Expanded Geographic
Coverage - FishBOL
Overlap of species ranges with other regions
few marine species are restricted to
geopolitical regions
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Latitudinal ranges of tropical eastern Pacific
chaenopsids (tube blennies)
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CO1 555 bp
Cyt b 555 bp
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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)

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Latitudinal ranges of tropical eastern Pacific
pomacentrids (damselfishes)