Identifying Canadian Freshwater Fishes through DNA Barcodes

Citation: Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E, et al. ( Identifying Canadian Freshwater Fishes through DNA Barcodes Nicolas Hubert 0 Robert Hanner 0 Erling Holm 0 Nicholas E. Mandrak 0 Eric Taylor 0 Mary Burridge 0 Douglas Watkinson 0 Pierre Dumont 0 Allen Curry 0 Paul Bentzen 0 Junbin Zhang 0 Julien April 0 Louis 0 Bernatchez 0 Hans Ellegren, University of Uppsala, Sweden 0 1 De partement de biologie , Pavillon Charles-Euge`ne-Marchand , Universite Laval , Sainte-Foy, Que bec , Canada , 2 Canadian Barcode of Life Network, Biodiversity Institute of Ontario, University of Guelph , Guelph, Ontario , Canada , 3 Department of Natural History , Royal Ontario Museum , Toronto, Ontario , Canada , 4 Great Lakes Laboratory for Fisheries and Aquatic Sciences, Fisheries and Oceans Canada , Burlington, Ontario , Canada , 5 Department of Zoology , Vancouver, British Columbia , Canada , 6 Fisheries and Oceans Canada , Central & Arctic Region, Freshwater Institute , Winnipeg, Manitoba , Canada , 7 Ministe`re des Ressources naturelles et de la faune du Que bec, Direction de l'ame nagement de la faune de Montre al, de Laval et de la Monte re gie , Longueuil, Que bec , Canada , 8 Fish and Wildlife Research Unit, University of New Brunswick , Fredericton, New Brunswick , Canada , 9 Department of Biology, Dalhousie University , Halifax, Nova Scotia , Canada Background: DNA barcoding aims to provide an efficient method for species-level identifications using an array of species specific molecular tags derived from the 59 region of the mitochondrial cytochrome c oxidase I (COI) gene. The efficiency of the method hinges on the degree of sequence divergence among species and species-level identifications are relatively straightforward when the average genetic distance among individuals within a species does not exceed the average genetic distance between sister species. Fishes constitute a highly diverse group of vertebrates that exhibit deep phenotypic changes during development. In this context, the identification of fish species is challenging and DNA barcoding provide new perspectives in ecology and systematics of fishes. Here we examined the degree to which DNA barcoding discriminate freshwater fish species from the well-known Canadian fauna, which currently encompasses nearly 200 species, some which are of high economic value like salmons and sturgeons. Methodology/Principal Findings: We bi-directionally sequenced the standard 652 bp ''barcode'' region of COI for 1360 individuals belonging to 190 of the 203 Canadian freshwater fish species (95%). Most species were represented by multiple individuals (7.6 on average), the majority of which were retained as voucher specimens. The average genetic distance was 27 fold higher between species than within species, as K2P distance estimates averaged 8.3% among congeners and only 0.3% among concpecifics. However, shared polymorphism between sister-species was detected in 15 species (8% of the cases). The distribution of K2P distance between individuals and species overlapped and identifications were only possible to species group using DNA barcodes in these cases. Conversely, deep hidden genetic divergence was revealed within two species, suggesting the presence of cryptic species. Conclusions/Significance: The present study evidenced that freshwater fish species can be efficiently identified through the use of DNA barcoding, especially the species complex of small-sized species, and that the present COI library can be used for subsequent applications in ecology and systematics. - Funding: This research was supported through funding to the Canadian Barcode of Life Network from NSERC, Genome Canada (through the Ontario Genomics Institute). Other sponsors listed at www.BOLNET.ca. Competing Interests: The authors have declared that no competing interests exist. DNA barcoding is designed to provide accurate, and automated species identifications through the use of molecular species tags based on short, standardised gene regions [1,2]. While humanity is facing increasing evidence of the erosion of Earths biodiversity, this approach is proving its effectiveness in characterising the complexity of the biodiversity realm at a pace unequalled by other characters [3]. The primary goals of DNA barcoding focus on the assembly of reference libraries of barcode sequences for known species in order to develop reliable, molecular tools for species identification in nature. Current results suggest that, in a large array of organisms, species are generally well delineated by a particular sequence or by a tight cluster of very similar sequences that allow unambiguous identifications [4,5,6,7,8,9,2,10,11,12]. Despite the great promise of DNA barcoding, it has been controversial in some scientific circles [13,14]. Yet, recent results illustrated some straightforward benefits from the use of a standardised molecular approach for identification [1,2]. First, intraspecific phenotypic variation often overlaps that of sister taxa in nature, which can lead to incorrect identifications if based on phenotype only [e.g. 15]. Second, DNA barcodes are effective whatever the life stages under scrutiny [e.g. 16, 17]. Third, cryptic variation and often spectacular levels of undetected taxonomic diversity have been frequently reported [e.g. 18, 19, 20]. Finally, DNA barcode libraries are fully available as they are deposited in a major sequence database, and attached to a voucher specimen whose origin and current location are recorded [2,3]. Once libraries are available, recent studies illustrate the vast array of applications that can be applied to them such as forensic engineering [21,22], ecology of cryptic communities [23], the tracking of invasive species [24,25] and identification of prey from predator stomach samples [e.g. 26]. With the aim of assigning specimens to known species based on molecular tags, a 648-bp segment of the 59 region of mitochondrial cytochrome c oxidase I (COI) gene forms the library of primary barcodes for the animal kingdom [1]. Mitochondrial DNA (mtDNA) presents several advantages that make it well suited for large scale molecular tagging. First, this genome is present in a large number of copies yielding substantial amounts of genomic DNA from a variety of extraction methods. Second, the high mutation rate and small effective population size make it often an informative genome about evolutionary patterns and processes [27,28]. For a barcoding approach to species identification to succeed, however, within-species DNA sequences need to be more similar to one another than to sequences in different species. Several processes such as pseudogenes ontogenesis, introgressive hybridisation, and retention of ancestral polymorphism pose potential difficulties in capturing species boundaries using mtDNA sequences [29,30,31,32]. The detection of mixed genealogy between closely related species has been previously estimated to occur in nea (...truncated)