Identifying Canadian freshwater fishes through DNA barcodes

Abstract

Background: DNA barcoding aims to provide an efficient method for species-level identifications using an array of species specific molecular tags derived from the 5' 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.

Figure 1. Structure of the Freshwater Fish of Canada (FFC) database in BOLD, functionalities and connections with others public databases.

1, Project page with the list of the specimens analysed including a link to the specimen and sequence page; 2, Specimen page for an individual of Esox masquinongy including voucher information, taxonomy, collection location, collection site maps and specimen image; 3, Sequence page for the same individual of Esox masquinongy including specimens details, sequencing details including links to trace files, amino acid translation of sequence and trace viewer; 4, Taxon ID tree for the Canadian members of the Salmonid genus Oncorhynchus; 5, Connections with the GenBank.

Figure 2. Summary of the distribution of the genetic variability (K2P distances) at COI sequences for the 1360 individuals and 190 species analysed.

A. Distribution of the genetic distance within different taxonomic categories. B. Distribution of the genetic distances to the nearest-neighbour and mean intra-specific distance.