Barcoding the Birds of North America Kevin C.R. Kerr University of Guelph Biodiversity Institute of Ontario Canada Collaborators: M.Y. Stoeckle, L.A. Weigt, C.J. Dove, C.M. Francis, P.D.N. Hebert

Introduction Preliminary birds study (Hebert et al. 2004) Specimens obtained from the Royal Ontario Museum: 260 species 130 with >2 replicates 437 sequences in total

Introduction The next steps are straightforward: Increase taxon density Increase replicates per species Illustrations © I. Lewington Map courtesy of “Birds of North American Online”

Analytical Chain Specimen Collection data Sample Photographs Extract Amplify Sequence Online barcode library

DNA sources Feathers were collected from banding stations across the country Tissues samples were loaned from museums; 96 well format matrix boxes facilitate the movement of tissue specimens

DNA sources Contributing Institutions: Royal Ontario Museum Burke Museum Canadian Wildlife Service Academy of Natural Sciences, Philadelphia American Museum of Natural History Field Museum Museum of Comparative Zoology Museum of Southwestern Biology Museum of Vertebrate Zoology National Museum of Natural History University of Alaska – Fairbanks University of Kansas

Methods The GenElute™ Mammalian Genomic DNA Purification Kit (Sigma-Aldrich) Silica-based membrane Individual tube format (2 ml tubes) Spin down on bench top centrifuge (allows 24 samples at a time)

Methods NucleoSpin® 96 Tissue (Machery-Nagel) Silica-based membrane 96-well plate format Requires larger high-speed centrifuge (2 plates at a time facilitates balancing) Supplemented in our lab with “Glass-Fibre” protocol (MUCH lower cost!)

Methods Biomek® NX automated workstation (Beckman Coulter)

Methods Our study provides evidence of the presence of pseudogenes in a few species (100-200bp) Not a problem with bidirectional sequencing Reverse Forward Contig Numt Numt 5’ 3’

Results Summary: 643 species (~93% coverage) 2,590 sequences 1-125 replicates per species Mean intraspecific distance = 0.23% (versus 0.27% from PLoS paper) Mean congeneric distance = 5.91% (versus 7.93% from PLoS paper) n = 24 n = 125

Species identification Example: Somateria complex Similar mtDNA sequences Barcodes are shared Horizontal transfer?

Species identification Pairs (14) = Species with unique barcodes Species with shared or overlapping barcodes = 6.4% Triplets (2) 93.6% White-headed gulls

Species “discovery” Example: Troglodytes troglodytes Of 405 new North American species inspected, 11 species harbour splits: Fulmarus glacialis Megascops kennicotti Toxostoma curvirostre* Psaltriparus minimus Poecile gambeli* Aphelocoma californica* Aphelocoma ultramarina Corvus corax* Troglodytes troglodytes* Thryomanes bewickii Catharus guttatus Eastern Western Outgroup 2% Divergence Western Eastern

Publication These findings were published last month in Molecular Ecology Notes (Kerr et al. 2007)

New results Summary: 654 species (up from 643) 3,412 sequences (up from 2,590) (plus impending sequences from collaborative projects on arctic geese and ptarmigan) n = 144 n > 300

Continued sampling Adequate geographical coverage is essential for uncovering deep genetic splits Range map of D. townsendi Range map of G. trichas Range map of C. americana

Continued sampling NJ tree for Certhia americana (eastern and western forms are 3.2% divergent)

Practical applications Unidentified hummingbird overwintering in Ontario Belongs to a complex of species difficult to identify DNA barcode was obtained from a single breast feather found in the snow, identifying this Rufous Hummingbird

Practical applications Barrow’s Goldeneye (B. islandica) is a rare species in eastern Canada DNA barcoding has been used to distinguish nest sites from its common cousin Bucephala islandica Bucephala clangula Barcodes are obtained from feathers used for nest lining © B. Gress

Future directions Number of Replicates per Species

Future directions Proportion of NA birds barcoded: 7% Remaining 93% Completed Map courtesy of “Birds of North American Online”, Image: © M. Danzenbaker

Acknowledgements Laboratory Collections Database Funding & Support The Hebert Lab Royal Ontario Museum Smithsonian Institution Burke Museum Smithsonian Institution Database Museum of Vertebrate Zoology BOLD Philadelphia Academy of Sciences Museum of Southwestern Biology American Museum of Natural History Funding & Support Louisiana State University Museum of Comparative Zoology The Moore Foundation Field Museum of Natural History Canadian Wildlife Service University of Kansas Federal Aviation Administration Bird banding stations