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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Biological identifications through DNA barcodes

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Molecular Evolutionary Genetics Analysis

The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-294X.2011.05403.x

Who is eating what: diet assessment using next generation sequencing.

The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. We first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.

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A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this ‘DNA barcoding’ initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the ‘DNA barcoding’ initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the ‘DNA barcoding’ initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings—Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of ‘DNA barcoding’.

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The unholy trinity: taxonomy, species delimitation and DNA barcoding

We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

/pdf/dna-primers-for-amplification-of-mitochondrial-cytochrome-c-46m2jyijnx.pdf

DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.

Vestimentiferan tubeworms inhabit sulfide-rich environments associated with deep-sea hydrothermal vents and cold-water seeps at continental margins. Twelve species have been described, and several more await formal descriptions. As a group, these worms are best known for their lack of a digestive system in adults and their dependence on endosymbiotic bacteria that supply nutrients derived from chemoautotrophism. The taxonomic status of Vestimentifera has been debated since their discovery. Furthermore, relationships within the Vestimentifera have been difficult to determine by morphological criteria. Several species display considerable phenotypic plasticity, further confounding efforts to establish evolutionary relationships. We used a fragment of the mitochondrial cytochrome c oxidase subunit I gene to examine evolutionary relationships among vent-endemic species (Riftia pachyptila, Oasisia alvinae, Ridgeia piscesae, Tevnia jerichonana) and seep-associated species(Escarpia laminata, E. spicata, Lamellibrachia barhami, L. columna, and an undescribed species) of these worms. The molecular data placed these vestimentiferan taxa within the phylum Pogonophora. The pogonophoran clade (including vestimentiferans) was then linked to the Annelida. Examination of sequence divergence suggests that extant vestimentiferans constitute a recent evolutionary radiation that diversified as a paraphyletic assemblage of seep-associated taxa (including the genera Lamellibrachia and Escarpia) and then gave rise to a clade of vent-endemic taxa (genera Riftia, Oasisia, Ridgeia and Tevnia).

/pdf/molecular-systematics-of-vestimentiferan-tubeworms-from-14r5ufwqaw.pdf

Molecular systematics of vestimentiferan tubeworms from hydrothermal vents and cold-water seeps

Vestimentiferan tube worms from deep-sea hydrothermal vents and cold-water seeps rely entirely on sulfur-oxidizing bacterial endosymbionts for nutriment. We examined host-symbiont co-evolution by comparing phylogenetic trees from symbiont 16S ribosomal DNA and host mitochondrial COI genes. The endosymbionts comprised two distinct clades, one associated with tube worms from basaltic vent habitats and the other associated with tube worms from sedimented seep-like environments. Within each symbiont clade, 16S rDNA sequences were nearly identical, suggesting that vent vestimentiferans share a single endosymbiont species that is distinct from the seep endosymbiont species. A third endosymbiont type, related to the seep species, was found in a tube worm collected from a whale carcass. Our results are consistent with a horizontal model of symbiont transmission.

Molecular Phylogenetics of Bacterial Endosymbionts and their Vestimentiferan Hosts

We report diagnostic differences in the nucleotide sequences of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from the zebra mussel (Dreissena polymorpha) and potentially co-occurring bivalves: the quagga mussel (Dreissena bugensis); the Asiatic clam (Corbicula fluminea), the dark false mussel (Mytilopsis leucophaeata), and the wedge clam (Rangia cuneata). The COI sequence of the deep-water "profunda" phenotype of the quagga mussel was nearly identical to that of shallow-water quagga mussels. Restriction fragment length polymorphisms (RFLPs) in this portion of COI produced species-specific differences in fragment numbers and sizes that could be used as diagnostic markers to distinguish the free-living larvae produced by these bivalves.

A diagnostic molecular marker for zebra mussels (Dreissena polymorpha) and potentially co-occurring bivalves: mitochondrial COI.

Allozyme data are presented for six discrete populations of the giant hydrothermal vent tube worm Riftia pachyptila Jones, 1981 collected throughout the species' known range along mid-ocean spreading ridges of the eastern Pacific Ocean Contrary to an earlier report, levels of genetic variation are relatively high in this species Estimates of gene flow based on F-statistics revealed that dispersal throughout the surveyed region is sufficiently high to counter random processes that would lead to losses of genetic diversity and significant population differentiation R pachyptila, like other species of tube worms, displays considerable morphologic variation among populations, but this diversity is not reflected in allozyme variation Vestimentifera, in general, appear to show extensive phenotypic plasticity In the light of the available genetic data, caution is warranted when making inferences about the taxonomic status of collections based on morphological variation alone A general decrease in estimated rates of gene flow between geographically more distant populations supports the hypothesis that dispersal in this species follows a stepping-stone model, with exchange between neighboring populations in great excess of long-distance dispersal High levels of gene flow have been recorded in a variety of vent fauna and may be a prerequisite for success of species found in the ephemeral habitats associated with regions of sea-floor hydrothermal activity

Michael B. Black

Papers

DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.

Molecular systematics of vestimentiferan tubeworms from hydrothermal vents and cold-water seeps

Molecular Phylogenetics of Bacterial Endosymbionts and their Vestimentiferan Hosts

A diagnostic molecular marker for zebra mussels (Dreissena polymorpha) and potentially co-occurring bivalves: mitochondrial COI.

Gene flow among vestimentiferan tube worm (Riftia pachyptila) populations from hydrothermal vents of the eastern Pacific