Barcoding Nemo: DNA-Based Identifications for the Ornamental Fish Trade

BACKGROUND:Trade in ornamental fishes represents, by far, the largest route for the importation of exotic vertebrates. There is growing pressure to regulate this trade with the goal of ensuring that species are sustainably harvested and that their point of origin is accurately reported. One important element of such regulation involves easy access to specimen identifications, a task that is currently difficult for all but specialists because of the large number of species involved. The present study represents an important first step in making identifications more accessible by assembling a DNA barcode reference sequence library for nearly half of the ornamental fish species imported into North America. METHODOLOGY/PRINCIPAL FINDINGS:Analysis of the cytochrome c oxidase subunit I (COI) gene from 391 species from 8 coral reef locations revealed that 98% of these species exhibit distinct barcode clusters, allowing their unambiguous identification. Most species showed little intra-specific variation (adjusted mean = 0.21%), but nine species included two or three lineages showing much more divergence (2.19-6.52%) and likely represent overlooked species complexes. By contrast, three genera contained a species pair or triad that lacked barcode divergence, cases that may reflect hybridization, young taxa or taxonomic over-splitting. CONCLUSIONS/SIGNIFICANCE:Although incomplete, this barcode library already provides a new species identification tool for the ornamental fish industry, opening a realm of applications linked to collection practices, regulatory control and conservation

Surviving historical Patagonian landscapes and climate: molecular insights from Galaxias maculatus

<p>Abstract</p> <p>Background</p> <p>The dynamic geological and climatic histories of temperate South America have played important roles in shaping the contemporary distributions and genetic diversity of endemic freshwater species. We use mitochondria and nuclear sequence variation to investigate the consequences of mountain barriers and Quaternary glacial cycles for patterns of genetic diversity in the diadromous fish <it>Galaxias maculatus </it>in Patagonia (~300 individuals from 36 locations).</p> <p>Results</p> <p>Contemporary populations of <it>G. maculatus</it>, east and west of the Andes in Patagonia, represent a single monophyletic lineage comprising several well supported groups. Mantel tests using control region data revealed a strong positive relationship when geographic distance was modeled according to a scenario of marine dispersal. (<it>r </it>= 0.69, <it>P = 0.055</it>). By contrast, direct distance between regions was poorly correlated with genetic distance (<it>r </it>= -0.05, <it>P </it>= 0.463). Hierarchical AMOVAs using mtDNA revealed that pooling samples according to historical (pre-LGM) oceanic drainage (Pacific vs. Atlantic) explained approximately four times more variance than pooling them into present-day drainage (15.6% vs. 3.7%). Further <it>post-hoc </it>AMOVA tests revealed additional genetic structure between populations east and west of the Chilean Coastal Cordillera (coastal vs. interior). Overall female effective population size appears to have remained relatively constant until roughly 0.5 Ma when population size rapidly increased several orders of magnitude [100× (60×-190×)] to reach contemporary levels. Maximum likelihood analysis of nuclear alleles revealed a poorly supported gene tree which was paraphyletic with respect to mitochondrial-defined haplogroups.</p> <p>Conclusions</p> <p>First diversifying in the central/north-west region of Patagonia, <it>G. maculatus </it>extended its range into Argentina via the southern coastal regions that join the Pacific and Atlantic oceans. More recent gene flow between northern populations involved the most ancient and most derived lineages, and was likely facilitated by drainage reversal(s) during one or more cooling events of the late Pleistocene. Overall female effective population size represents the end result of a widespread and several hundred-fold increase over approximately 0.5 Ma, spanning several climatic fluctuations of the Pleistocene. The minor influence of glacial cycles on the genetic structure and diversity of <it>G. maculatus </it>likely reflects the access to marine refugia during repeated bouts of global cooling. Evidence of genetic structure that was detected on a finer scale between lakes/rivers is most likely the result of both biological attributes (i.e., resident non-migratory behavior and/or landlocking and natal homing in diadromous populations), and the Coastal Cordillera as a dispersal barrier.</p

Identification of Birds through DNA Barcodes

Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10× average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens

Intraspecific Compared to Interspecific COI Distances (K2P) for Individual Species

<p>For each species in which this comparison was possible (<i>n</i> = 73), maximum intraspecific variation is compared to minimum interspecific congeneric difference. For illustration purposes shown here, 2.0% is chosen as a cutoff between usual values for intra- and interspecific variation. This divides the graph into four quadrants that represent different categories of species: (I) Intraspecific distance, <2%; interspecific distance, >2%: concordant with current taxonomy; (II) Intraspecific distance, >2%; interspecific distance, >2%: probable composite species (i.e., candidate for taxonomic split); (III) Intraspecific distance, <2%; interspecific distance, <2%: recent divergence, hybridization, or synonymy; (IV) Intraspecific distance, >2%; interspecific distance, <2%: probable misidentification of specimen.</p

Genetic Difference versus Geographic Distance

<p>For each same-species pair of specimens, the geographic distance between where specimens were collected is plotted against their COI divergence (K2P).</p

NJ Tree of COI Sequences from 260 Species of North American Birds

<p>Intraspecific divergences were sampled in 130 species; these are marked in blue. Four species showed deep intraspecific divergence: (a) <i>Sturnella magna,</i> (b) <i>Cistothorus palustris,</i> (c) <i>Vireo gilvus,</i> and (d) <i>Tringa solitaria.</i> Higher-order classifications in families (gray) and orders (gold) are highlighted, and are labeled on the left and right of the figure, respectively. Gold numerals indicate the two species that appear as paraphyletic lineages at the family level: (1) Oenanthe oenanthe and (2) <i>Hirundo rustica.</i></p

NJ Tree of COI Sequences from 30 Species in Family <i>Scolapacidae</i> (Sandpipers and Kin)

<p>The divergent pair of clustered sequences of Tringa solitaria is highlighted. An asterisk indicates a COI sequence from GenBank.</p

Comparison of Nucleotide Sequence Differences in COI among 260 Species of North American Birds

<p>Pairwise comparisons between 437 COI sequences are separated into three categories: differences between individuals in the same species, differences between individuals in the same genus (not including intraspecific differences), and differences between individuals in the same family (not including intraspecific or intrageneric differences).</p

DNA barcoding Australia's fish species

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