BOLD: The Barcode of Life Data System: Barcoding

Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon ‘barcodes’. It was established previously that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. A new tools were developed recently to be complementary markers for (COI) DNA barcoding.

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Biological Identifications Through DNA Barcodes

The Barcode of Life Data System ( BOLD ) is an informatics workbench aiding the acquisition, storage, analysis and publication of DNA barcode records. By assembling molecular, morphological and distributional data, it bridges a traditional bioinformatics chasm. BOLD is freely available to any researcher with interests in DNA barcoding. By providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation in the global sequence databases. Because of its web-based delivery and flexible data security model, it is also well positioned to support projects that involve broad research alliances. This paper provides a brief introduction to the key elements of BOLD , discusses their functional capabilities, and concludes by examining computational resources and future prospects.

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BOLD : The Barcode of Life Data System (www.barcodinglife.org)

Because many animal species are undescribed, and because the identification of known species is often difficult, interim taxonomic nomenclature has often been used in biodiversity analysis. By assigning individuals to presumptive species, called operational taxonomic units (OTUs), these systems speed investigations into the patterning of biodiversity and enable studies that would otherwise be impossible. Although OTUs have conventionally been separated through their morphological divergence, DNA-based delineations are not only feasible, but have important advantages. OTU designation can be automated, data can be readily archived, and results can be easily compared among investigations. This study exploits these attributes to develop a persistent, species-level taxonomic registry for the animal kingdom based on the analysis of patterns of nucleotide variation in the barcode region of the cytochrome c oxidase I (COI) gene. It begins by examining the correspondence between groups of specimens identified to a species through prior taxonomic work and those inferred from the analysis of COI sequence variation using one new (RESL) and four established (ABGD, CROP, GMYC, jMOTU) algorithms. It subsequently describes the implementation, and structural attributes of the Barcode Index Number (BIN) system. Aside from a pragmatic role in biodiversity assessments, BINs will aid revisionary taxonomy by flagging possible cases of synonymy, and by collating geographical information, descriptive metadata, and images for specimens that are likely to belong to the same species, even if it is undescribed. More than 274,000 BIN web pages are now available, creating a biodiversity resource that is positioned for rapid growth.

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A DNA-Based Registry for All Animal Species: The Barcode Index Number (BIN) System

Although central to much biological research, the identification of species is often difficult. The use of DNA barcodes, short DNA sequences from a standardized region of the genome, has recently been proposed as a tool to facilitate species identification and discovery. However, the effectiveness of DNA barcoding for identifying specimens in species-rich tropical biotas is unknown. Here we show that cytochrome c oxidase I DNA barcodes effectively discriminate among species in three Lepidoptera families from Area de Conservacion Guanacaste in northwestern Costa Rica. We found that 97.9% of the 521 species recognized by prior taxonomic work possess distinctive cytochrome c oxidase I barcodes and that the few instances of interspecific sequence overlap involve very similar species. We also found two or more barcode clusters within each of 13 supposedly single species. Covariation between these clusters and morphological and/or ecological traits indicates overlooked species complexes. If these results are general, DNA barcoding will significantly aid species identification and discovery in tropical settings.

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DNA barcodes distinguish species of tropical Lepidoptera

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 neighbourjoining 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.

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DNA barcoding Australia's fish species

566 Abstract.-Yellowfin tuna, Thun­ nus albacares, were sampled from one region of the Atlantic Ocean. two re­ gions of the Indian Ocean, and six re­ gions of the Pacific Ocean. One of the Indian Ocean collections could not be allozymically analyzed; the remaining eight collections were examined for four polymorphic allozyme loci , with two restriction enzymes lBel I and Eco RI) that detect polymorphic restric­ tion sites in yellowfin tuna. Allele fre­ quencies at three of the allozyme loci were homogeneous across collections, whereas GPI-A* showed highly signifi­ cant differentiation (P<O.OOI). The GPI-A* data, taken together with the geographic location of the collections, suggested the existence ofat least four yellowfin tuna stocks: Atlantic Ocean, Indian Ocean, west-central Pacific Ocean, and east Pacific Ocean. Mito­ chondrial DNA differentiation was more limited, but spatial heterogene­ ity of the 24 observed haplotypes over the nine regions (P=O.048l and three oceans lP=O.009) was significant. The mtDNA data did not differentiate west­ central Pacific Ocean collections from east Pacific Ocean collections but did support the separation of Atlantic Ocean, Indian Ocean. and Pacific Ocean stocks.

Global population structure of yellowfin tuna, Thunnus albacares, inferred from allozyme and mitochondrial DNA variation

Five polymorphic microsatellite loci were examined in 1391 yellowfin tuna (Thunnusalbacares) from eight regions of the western (Coral Sea, eastern Australia, Fiji, Indonesia, Philippines and Solomon Islands) and eastern (California and Mexico ) Pacific Ocean. Across all samples, numbers of alleles per locus ranged from 7 to 30 (mean: 17.0), and observed heterozygosities per locus ranged from 0.223 to 0.955 (mean: 0.593). Temporal collections were available for three areas: no significant temporal heterogeneity was observed for the Coral Sea (1991/1992 and 1995/1996 collections) or eastern Australia (1994/1995, 1995/1996, 1996/1997 and 1997/1998), but there was slight but significant heterogeneity at one locus (cmrTa-161) between the two Philippines collections (1994/1995 and 1996/1997). Genotypes generally showed a good fit to Hardy–Weinberg expectations within populations; only cmrTa-208 in the pooled Coral Sea population gave a significant deviation after Bonferroni correction for 40 tests, with a small but significant excess of homozygotes. Four loci showed no evidence of population differentiation following contingency Chi-squared and FST analyses. The fifth locus, cmrTa-161, showed small but significant differentiation (FST=0.002, P<0.001). This heterogeneity was largely a result of the Philippines 1994/1995 and Fiji collections; there was no correlation with geographic distance. The average FST across all five loci was very low (FST=0.002), but it was significant (P<0.001). It is unclear whether this low but significant differentiation reflects noise in the dataset, perhaps arising from experimental error, or real population differentiation. The finding of very limited population heterogeneity accords with most of the earlier allozyme and mitochondrial DNA studies of yellowfin tuna in the Pacific Ocean.

Population structure of yellowfin tuna (Thunnus albacares) in the western Pacific Ocean, inferred from microsatellite loci

Samples of southern bluefin tuna, Thunnus maccoyii (Castelnau), taken from off the coasts of South Africa, Western Australia, South Australia and Tasmania from 1992 to 1994 were analysed for six polymorphic allozyme loci (ADA *, GDA *, GPI-A *, MPI *, PGDH * and PGM-1 *, n = 595 to 733 per locus) and for mitochondrial DNA variants revealed by three restriction enzymes (Bam HI, Bcl I and Eco RI) detecting polymorphic cut sites (n = 555). No significant spatial heterogeneity was detected. There were no sex-related differences in allele or mtDNA haplotype frequencies. Juveniles (30 to 35 cm and 46 to 54 cm) from what are thought to be two temporally-separated spawning peaks showed no significant genetic differentiation. There were also no significant differences in allele or haplotype frequencies between fish smaller than 70 cm and those larger than 70 cm. These data are consistent with the null hypothesis of a single unit stock of southern bluefin tuna, with a single spawning area. This is located to the south of Java and off the north-west coast of Australia.

Genetic population structure of southern bluefin tuna ( Thunnus maccoyii )

A genetic test was developed for the identification of the six species of billfish found in Australian waters (black marlin, Indo–Pacific blue marlin, striped marlin, Indo–Pacific sailfish, shortbill spearfish and broadbill swordfish). The test was based on the PCR–RFLP analysis of a 1400 bp region of the mitochondrial DNA molecule, the d-loop, using four restriction enzymes (Hinf I, Rsa I and Sau3A I andTaq I). A total of 33 composite haplotypes were observed among 160 fish; all were species-specific. Three of the species—black marlin, striped marlin and broadbill swordfish—showed sufficient intra-specific variation to be useful in population structure analyses.

B. H. Innes

Papers

DNA barcoding Australia's fish species

Global population structure of yellowfin tuna, Thunnus albacares, inferred from allozyme and mitochondrial DNA variation

Population structure of yellowfin tuna (Thunnus albacares) in the western Pacific Ocean, inferred from microsatellite loci

Genetic population structure of southern bluefin tuna ( Thunnus maccoyii )

PCR-based genetic identification of marlin and other billfish