Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

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Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

With millions of species and their life-stage transformations, the animal kingdom provides a challenging target for taxonomy. Recent work has suggested that a DNA-based identification system, founded on the mitochondrial gene, cytochrome c oxidase subunit 1 (COI), can aid the resolution of this diversity. While past work has validated the ability of COI sequences to diagnose species in certain taxonomic groups, the present study extends these analyses across the animal kingdom. The results indicate that sequence divergences at COI regularly enable the discrimination of closely allied species in all animal phyla except the Cnidaria. This success in species diagnosis reflects both the high rates of sequence change at COI in most animal groups and constraints on intraspecific mitochondrial DNA divergence arising, at least in part, through selective sweeps mediated via interactions with the nuclear genome.

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Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species

BOLD: The Barcode of Life Data System: Barcoding

The evolution of marine microbes over billions of years predicts that the composition of microbial communities should be much greater than the published estimates of a few thousand distinct kinds of microbes per liter of seawater. By adopting a massively parallel tag sequencing strategy, we show that bacterial communities of deep water masses of the North Atlantic and diffuse flow hydrothermal vents are one to two orders of magnitude more complex than previously reported for any microbial environment. A relatively small number of different populations dominate all samples, but thousands of low-abundance populations account for most of the observed phylogenetic diversity. This "rare biosphere" is very ancient and may represent a nearly inexhaustible source of genomic innovation. Members of the rare biosphere are highly divergent from each other and, at different times in earth's history, may have had a profound impact on shaping planetary processes.

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Microbial diversity in the deep sea and the underexplored “rare biosphere”

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

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'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.

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Alina Cywinska

Papers

Biological identifications through DNA barcodes