DNA barcoding Australia's fish species

Abstract

Two hundred and seven species of fish, mostly Australian marine fish, were sequenced (barcoded) for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (cox1). Most species were represented by multiple specimens, and 754 sequences were generated. The GC content of the 143 species of teleosts was higher than the 61 species of sharks and rays (47.1% versus 42.2%), largely due to a higher GC content of codon position 3 in the former (41.1% versus 29.9%). Rays had higher GC than sharks (44.7% versus 41.0%), again largely due to higher GC in the 3rd codon position in the former (36.3% versus 26.8%). Average within-species, genus, family, order and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18% and 23.27%, respectively. All species could be differentiated by their cox1 sequence, although single individuals of each of two species had haplotypes characteristic of a congener. Although DNA barcoding aims to develop species identification systems, some phylogenetic signal was apparent in the data. In the neighbour-joining tree for all 754 sequences, four major clusters were apparent: chimaerids, rays, sharks and teleosts. Species within genera invariably clustered, and generally so did genera within families. Three taxonomic groups-dogfishes of the genus Squalus, flatheads of the family Platycephalidae, and tunas of the genus Thunnus-were examined more closely. The clades revealed after bootstrapping generally corresponded well with expectations. Individuals from operational taxonomic units designated as Squalus species B through F formed individual clades, supporting morphological evidence for each of these being separate species. We conclude that cox1 sequencing, or 'barcoding', can be used to identify fish species.

Figure 1

Distribution of K2P distances (percent) for coxI within different taxonomic categories. See also table 1. Note that cells with a frequency of less than 1% are not represented.

Figure 2

Variation in GC content for coxI among two groups of fishes. The first, second, third and fourth rows plot the GC content of sharks and rays codon position 1, sharks and rays codon position 3, teleosts codon position 1 and teleosts codon position 3, respectively. Codon position 2 is not shown as this shows very little variation within and among the two fish groups (table 3).

Figure 3

Neighbour-joining tree of 754 cox1 sequences from 207 fish species, using K2P distances. Multiple specimens of individual species are marked in blue. The three instances of deep intra-specific divergence are identified in orange. The three subgroups examined in more detail are identified.

Figure 4

K2P distance neighbour-joining tree of 61 cox1 sequences from 14 species of flathead (Platycephalidae, genera Platycephalus, Neoplatycephalus and Cymbacephalus). Bootstrap values greater than 50 shown. Specimen numbers for the Barcode of Life Database (BoLD, www.barcodinglife.org) given.

Figure 5

K2P distance neighbour-joining tree of 46 cox1 sequences from the eight species of tuna of the genus Thunnus. Bootstrap values greater than 50 shown. Specimen numbers for the Barcode of Life Database (BoLD, www.barcodinglife.org) given.

Figure 6

K2P distance neighbour-joining tree of 41 cox1 sequences from eight species of dogfish of the genus Squalus. Bootstrap values greater than 50 shown. Specimen numbers for the Barcode of Life Database (BoLD, www.barcodinglife.org) given.