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

Evolution of Protein Molecules

Astraptes fulgerator, first described in 1775, is a common and widely distributed neotropical skipper butterfly (Lepidoptera: Hesperiidae). We combine 25 years of natural history observations in northwestern Costa Rica with morphological study and DNA barcoding of museum specimens to show that A. fulgerator is a complex of at least 10 species in this region. Largely sympatric, these taxa have mostly different caterpillar food plants, mostly distinctive caterpillars, and somewhat different ecosystem preferences but only subtly differing adults with no genitalic divergence. Our results add to the evidence that cryptic species are prevalent in tropical regions, a critical issue in efforts to document global species richness. They also illustrate the value of DNA barcoding, especially when coupled with traditional taxonomic tools, in disclosing hidden diversity.

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Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator

Within uncharacterized groups, DNA barcodes, short DNA sequences that are present in a wide range of species, can be used to assign organisms into species. We propose an automatic procedure that sorts the sequences into hypothetical species based on the barcode gap, which can be observed whenever the divergence among organisms belonging to the same species is smaller than divergence among organisms from different species. We use a range of prior intraspecific divergence to infer from the data a model-based one-sided confidence limit for intraspecific divergence. The method, called Automatic Barcode Gap Discovery (ABGD), then detects the barcode gap as the first significant gap beyond this limit and uses it to partition the data. Inference of the limit and gap detection are then recursively applied to previously obtained groups to get finer partitions until there is no further partitioning. Using six published data sets of metazoans, we show that ABGD is computationally efficient and performs well for standard prior maximum intraspecific divergences (a few per cent of divergence for the five data sets), except for one data set where less than three sequences per species were sampled. We further explore the theoretical limitations of ABGD through simulation of explicit speciation and population genetics scenarios. Our results emphasize in particular the sensitivity of the method to the presence of recent speciation events, via (unrealistically) high rates of speciation or large numbers of species. In conclusion, ABGD is fast, simple method to split a sequence alignment data set into candidate species that should be complemented with other evidence in an integrative taxonomic approach.

ABGD, Automatic Barcode Gap Discovery for primary species delimitation

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

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A DNA barcode for land plants.

An evaluation of electrophoretic analysis as a potential tool for elucidating systematic relationships of Quercus utilized 478 trees representing 10 species from subgenus Erythrobalanus and 8 from subgenus Quercus. Leaf tissue was used to resolve 18 putative genetic loci representing 12 enzyme systems in each taxon. Subgeneric differentiation was apparent with intrasubgeneric genetic differentiation (mean modified Rogers' distance (D): Erythrobalanus 0.27; Quercus 0.44) being less than between subgenera (D = 0.68). Relationships among species within subgenera derived from electrophoretic data often compared well with published affinities. However, several discrepancies existed that might reflect disparities in rates of morphological, ecological, and allozymic divergence, or hybridization within subgenera, or both. Electrophoretic analysis of oak leaf tissue has the potential to yield valuable data for elucidating relationships among Quercus.

Electrophoretic evidence of relationships among Quercus (oaks) of eastern North America

This chapter is an overview of the techniques for DNA barcoding of fishes from field collection to DNA sequence analysis. Recommendations for modifications of field protocols and best tissue sampling practices are made. A variety of DNA extraction protocols is provided, including high-throughput robot-assisted methods. A pair of well-tested forward and reverse primers for PCR amplification and sequencing are presented. These primers have been successfully used for DNA barcode on a wide array of marine fish taxa and also work well in most freshwater and cartilaginous fishes. Recipes and cycling protocols for both PCR amplification and sequencing and cleanup methods for the reaction products are provided. A method for the consistent production of high-quality DNA barcodes from DNA sequence data is given and stringent guidelines for judging the quality of raw sequence data are laid out.

DNA Barcoding Fishes

Taxonomic identification of benthic macroinvertebrates is critical to protocols used to assess the biological integrity of aquatic ecosystems. The time, expense, and inherent error rate of species-level morphological identifications has necessitated use of genus- or family-level identifications in most large, statewide bioassessment programs. Use of coarse-scale taxonomy can obscure signal about biological condition, particularly if the range of species tolerances is large within genera or families. We hypothesized that integration of deoxyribonucleic acid (DNA) barcodes (partial cytochrome c oxidase subunit I sequences) into bioassessment protocols would provide greater discriminatory ability than genus-level identifications and that this increased specificity could lead to more sensitive assessments of water quality and habitat. Analysis of DNA barcodes from larval specimens of Ephemeroptera, Plecoptera, and Trichoptera (EPT) taxa collected as part of Maryland's Biological Stream Survey (MBSS) revealed , 2t o 33 as many DNA-barcode groups or molecular operational taxonomic units (mOTUs) as morphologically identified genera. As expected, geographic distributions for several mOTUs were tighter than for the parent genus, but few mOTUs showed closer associations with water-quality variables or physical-habitat features than did the genus in which they belonged. The need for improved protocols for the consistent generation of DNA barcodes is discussed.

https://bioone.org/journals/Freshwater-Science/volume-30/issue-1/10-012.1/Incorporation-of-DNA-barcoding-into-a-large-scale-biomonitoring-program/10.1899/10-012.1.pdf

Incorporation of DNA barcoding into a large-scale biomonitoring program: opportunities and pitfalls

Caribbean coral reef ecosystems are at the forefront of a global decline and are now facing a new threat: elimination of vulnerable species by the invasive lionfish (Pterois spp.). In addition to being threatened by habitat destruction and pollution, the critically endangered social wrasse (Halichoeres socialis), endemic to Belize’s inner barrier reef, has a combination of biological traits (small size, schooling, and hovering behavior) that makes it a target for the invasive lionfish. Based on stomach content analyses, this small fish comprises almost half of the lionfish diet at the inner barrier reef in Belize. The combination of lionfish predation, limited range, and ongoing habitat destruction makes the social wrasse the most threatened coral reef fish in the world. Other species with small range and similar traits occur elsewhere in the Caribbean and face similar risks.

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Invasive lionfish preying on critically endangered reef fish

Low levels of primary sequence similarity across insect taxa have led to the suggestion of conserved structural elements in the insect mitochondrial control region. Our aim was to determine whether previously described motifs and secondary structures exist in stoneflies (Plecoptera). Several motifs and structural elements previously described in Orthoptera and Diptera were found, including a conserved 'hairpin' structure that may play a role in the initiation of mtDNA replication. The repeat region had the highest percentage similarity, lowest A-T content and highest transition to transversion ratio, suggesting a unique evolutionary pattern for the repeats. Finally, we discuss the usefulness of the control region in population genetic and evolutionary studies.

Arrangement and structural conservation of the mitochondrial control region of two species of Plecoptera: utility of tandem repeat‐containing regions in studies of population genetics and evolutionary history

Lee A. Weigt

Papers

Electrophoretic evidence of relationships among Quercus (oaks) of eastern North America

DNA Barcoding Fishes

Incorporation of DNA barcoding into a large-scale biomonitoring program: opportunities and pitfalls

Invasive lionfish preying on critically endangered reef fish

Arrangement and structural conservation of the mitochondrial control region of two species of Plecoptera: utility of tandem repeat‐containing regions in studies of population genetics and evolutionary history