DNA barcoding is a taxonomic method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular species. It differs from molecular phylogeny in that the main goal is not to determine patterns of relationship but to identify an unknown sample in terms of a preexisting classification. Although barcodes are sometimes used in an effort to identify unknown species or assess whether species should be combined or separated, the utility of DNA barcoding for these purposes is subject to debate. The most commonly used barcode region for animals and protists is a segment of approximately 600 base pairs of the mitochondrial gene cytochrome oxidase I (COI or COX1). This differs in the case of fungi, where part of Internal Transcribed Spacer 2 (ITS2) between rRNA genes is used, and again in plants, where multiple regions are used.
Applications include, for example, identifying plant leaves even when flowers or fruit are not available, identifying pollen collected on the bodies of pollinating animals, identifying insect larvae (which may have fewer diagnostic characters than adults and are frequently less well-known), identifying the diet of an animal, based on its stomach contents or faeces and identifying products in commerce (for example, herbal supplements, wood, or skins and other animal parts).
- 1 Choice of locus
- 2 Vouchered specimens
- 3 Origin
- 4 Case studies
- 5 Criticisms
- 6 Software
- 7 See also
- 8 References
- 9 External links
Choice of locus
A desirable locus for DNA barcoding should be standardized (so that large databases of sequences for that locus can be developed), present in most of the taxa of interest and sequenceable without species-specific PCR primers, short enough to be easily sequenced with current technology, and provide a large variation between species yet a relatively small amount of variation within a species.
Although several loci have been suggested, a common set of standardized regions were selected by the respective committees:
- For animals and many other eukaryotes, the mitochondrial COI gene
- For plants, the concatenation of the rbcL and matK chloroplast genes. These provide poor resolution for land plants, and a call was made for regions to be assessed that could complement rbcL and matK.
DNA barcoding is based on a relatively simple concept. All eukaryote cells contain mitochondria, and animal mitochondrial DNA (mtDNA) has a relatively fast mutation rate, resulting in the generation of diversity within and between populations over relatively short evolutionary timescales (thousands of generations). Typically, in animals, a single mtDNA genome is transmitted to offspring by each breeding female, and the genetic effective population size is proportional to the number of breeding females. This contrasts with the nuclear genome, which is around 100 000 times larger, where males and females each contribute two full genomes to the gene pool and effective size is therefore proportional to twice the total population size. This reduction in effective population size leads to more rapid sorting of mtDNA gene lineages within and among populations through time, due to variance in fecundity among individuals (the principle of coalescence). The combined effect of higher mutation rates and more rapid sorting of variation usually results in divergence of mtDNA sequences among species and a comparatively small variance within species. A 658-bp region (the Folmer region) of the mitochondrial cytochrome c oxidase subunit I (COI or COX1) gene was proposed as a potential 'barcode'.
Exceptions, where mtDNA fails as a test of species identity, can occur through occasional recombination (direct evidence for recombination in mtDNA is available in some bivalves such as Mytilus but it is suspected that it may be more widespread) and through occurrences of hybridization. Male-killing microorganisms, cytoplasmic incompatibility-inducing symbionts (e.g., Wolbachia), as well as heteroplasmy, where an individual carries two or more mtDNA sequences, may affect patterns of mtDNA diversity within species, although these do not necessarily result in bar-coding failure. Occasional horizontal gene transfer (such as via cellular symbionts), or other "reticulate" evolutionary phenomena in a lineage can lead to misleading results (i.e., it is possible for two different species to share mtDNA). In particular, mtDNA seems to be particularly prone to interspecific introgression  probably due to difference between sexes in mate-choice and dispersal. Additionally, some species may carry divergent mtDNA lineages segregating within populations, often due to historical geographic structure, where these divergent lineages do not reflect species boundaries.
Identifying flowering plants
Kress et al. (2005) suggest that the use of the COI sequence "is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase I gene evolution in higher plants than in animals". A series of experiments was then conducted to find a more suitable region of the genome for use in the DNA barcoding of flowering plants (or the larger group of land plants). One 2005 proposal was the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer; other researchers advocated other regions such as matK.
In 2009, a collaboration of a large group of plant DNA barcode researchers proposed two chloroplast genes, rbcL and matK, taken together, as a barcode for plants. Adding the nuclear internal transcribed spacer ITS2 region was proposed to provide better resolution between species. As of 2015, the search for better DNA barcodes for plants continues, with the proposal that the chloroplast region ycf1 may be suitable.
DNA sequence databases like GenBank contain many sequences that are not tied to vouchered specimens (for example, herbarium specimens, cultured cell lines, or sometimes images). This is problematic in the face of taxonomic issues such as whether several species should be split or combined, or whether past identifications were sound. Therefore, best practice for DNA barcoding is to sequence vouchered specimens.
The use of nucleotide sequence variations to investigate evolutionary relationships is not a new concept. Carl Woese used sequence differences in ribosomal RNA (rRNA) to discover archaea, which in turn led to the redrawing of the evolutionary tree, and molecular markers (e.g., allozymes, rDNA, and mtDNA sequences) have been successfully used in molecular systematics for decades. DNA barcoding provides a standardised method for this process via the use of a short DNA sequence from a particular region of the genome to provide a 'barcode' for identifying species. In 2003, Paul D.N. Hebert from the University of Guelph, Ontario, Canada, proposed the compilation of a public library of DNA barcodes that would be linked to named specimens. This library would "provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing".
Identification of birds
In an effort to find a relationship between traditional species boundaries established by taxonomy and those inferred by DNA barcoding, Hebert and co-workers sequenced DNA barcodes of 260 of the 667 bird species that breed in North America (Hebert et al. 2004a). They found that every single one of the 260 species had a different COI sequence. 130 species were represented by two or more specimens; in all of these species, COI sequences were either identical or were most similar to sequences of the same species. COI variations between species averaged 7.93%, whereas variation within species averaged 0.43%. In four cases there were deep intraspecific divergences, indicating possible new species. Three out of these four polytypic species are already split into two by some taxonomists. Hebert et al.'s (2004a) results reinforce these views and strengthen the case for DNA barcoding. Hebert et al. also proposed a standard sequence threshold to define new species, this threshold, the so-called "barcoding gap", was defined as 10 times the mean intraspecific variation for the group under study.
Identification of fish
The Fish Barcode of Life Initiative (FISH-BOL), is a global effort to coordinate an assembly of a standardised DNA barcode library for all fish species, one that is derived from voucher specimens with authoritative taxonomic identifications. The benefits of barcoding fishes include facilitating species identification for all potential users, including taxonomists; highlighting specimens that represent a range expansion of known species; flagging previously unrecognized species; and perhaps most importantly, enabling identifications where traditional methods are not applicable. An example is the possible identification of groupers causing Ciguatera fish poisoning from meal remnants.
Since its inception in 2005 FISH-BOL has been creating a valuable public resource in the form of an electronic database containing DNA barcodes for almost 10000 species, images, and geospatial coordinates of examined specimens. The database contains linkages to voucher specimens, information on species distributions, nomenclature, authoritative taxonomic information, collateral natural history information and literature citations. FISH-BOL thus complements and enhances existing information resources, including the Catalog of Fishes, FishBase and various genomics databases .
Delimiting cryptic species
The next major study into the efficacy of DNA barcoding was focused on the neotropical skipper butterfly, Astraptes fulgerator at the Area de Conservación de Guanacaste (ACG) in north-western Costa Rica. This species was already known as a cryptic species complex, due to subtle morphological differences, as well as an unusually large variety of caterpillar food plants. However, several years would have been required for taxonomists to completely delimit species. Hebert et al. (2004b) sequenced the COI gene of 484 specimens from the ACG. This sample included "at least 20 individuals reared from each species of food plant, extremes and intermediates of adult and caterpillar color variation, and representatives" from the three major ecosystems where Astraptes fulgerator is found. Hebert et al. (2004b) concluded that Astraptes fulgerator consists of 10 different species in north-western Costa Rica. These results, however, were subsequently challenged by Brower (2006), who pointed out numerous serious flaws in the analysis, and concluded that the original data could support no more than the possibility of three to seven cryptic taxa rather than ten cryptic species. This highlights that the results of DNA barcoding analyses can be dependent upon the choice of analytical methods used by the investigators, so the process of delimiting cryptic species using DNA barcodes can be as subjective as any other form of taxonomy.
A more recent example used DNA barcoding for the identification of cryptic species included in the ongoing long-term database of tropical caterpillar life generated by Dan Janzen and Winnie Hallwachs in Costa Rica at the ACG. In 2006 Smith et al. examined whether a COI DNA barcode could function as a tool for identification and discovery for the 20 morphospecies of Belvosia  parasitoid flies (Tachinidae) that have been reared from caterpillars in ACG. Barcoding not only discriminated among all 17 highly host-specific morphospecies of ACG Belvosia, but it also suggested that the species count could be as high as 32 by indicating that each of the three generalist species might actually be arrays of highly host-specific cryptic species.
In 2007 Smith et al. expanded on these results by barcoding 2,134 flies belonging to what appeared to be the 16 most generalist of the ACG tachinid morphospecies. They encountered 73 mitochondrial lineages separated by an average of 4% sequence divergence and, as these lineages are supported by collateral ecological information, and, where tested, by independent nuclear markers (28S and ITS1), the authors therefore viewed these lineages as provisional species. Each of the 16 initially apparent generalist species were categorized into one of four patterns: (i) a single generalist species, (ii) a pair of morphologically cryptic generalist species, (iii) a complex of specialist species plus a generalist, or (iv) a complex of specialists with no remaining generalist. In sum, there remained 9 generalist species classified among the 73 mitochondrial lineages analyzed.
However, also in 2007, Whitworth et al. reported that flies in the related family Calliphoridae could not be discriminated by barcoding. They investigated the performance of barcoding in the fly genus Protocalliphora, known to be infected with the endosymbiotic bacteria Wolbachia. Assignment of unknown individuals to species was impossible for 60% of the species, and if the technique had been applied, as in the previous study, to identify new species, it would have underestimated the species number in the genus by 75%. They attributed the failure of barcoding to the non-monophyly of many of the species at the mitochondrial level; in one case, individuals from four different species had identical barcodes. The authors went on to state:
The pattern of Wolbachia infection strongly suggests that the lack of within-species monophyly results from introgressive hybridization associated with Wolbachia infection. Given that Wolbachia is known to infect between 15 and 75% of insect species, we conclude that identification at the species level based on mitochondrial sequence might not be possible for many insects.
Mwabvu et al. (2013) observed a high level of divergence (19.09% for CO1, 520 base pairs) between two morphologically indistinguishable populations of Bicoxidens flavicollis millipedes in Zimbabwe, and suggested the presence of cryptic species in Bicoxidens flavicollis.
Marine biologists have also considered the value of the technique in identifying cryptic and polymorphic species and have suggested that the technique may be helpful when associations with voucher specimens are maintained, though cases of "shared barcodes" (e.g., non-unique) have been documented in cichlid fishes and cowries
Cataloguing ancient life
Lambert et al. (2005) examined the possibility of using DNA barcoding to assess the past diversity of the Earth's biota. The COI gene of a group of extinct ratite birds, the moa, were sequenced using 26 subfossil moa bones. As with Hebert's results, each species sequenced had a unique barcode and intraspecific COI sequence variance ranged from 0 to 1.24%. To determine new species, a standard sequence threshold of 2.7% COI sequence difference was set. This value is 10 times the average intraspecies difference of North American birds, which is inconsistent with Hebert's recommendation that the threshold value be based on the group under study. Using this value, the group detected six moa species. In addition, a further standard sequence threshold of 1.24% was also used. This value resulted in 10 moa species which corresponded with the previously known species with one exception. This exception suggested a possible complex of species which was previously unidentified. Given the slow rate of growth and reproduction of moa, it is probable that the interspecies variation is rather low. On the other hand, there is no set value of molecular difference at which populations can be assumed to have irrevocably started to undergo speciation. It is safe to say, however, that the 2.7% COI sequence difference initially used was far too high.
The Moorea Biocode Project
The Moorea Biocode Project is a barcoding initiative to create the first comprehensive inventory of all non-microbial life in a complex tropical ecosystem, the island of Moorea in Tahiti. Supported by a grant from the Gordon and Betty Moore Foundation, the Moorea Biocode Project is a 3-year project that brings together researchers from the Smithsonian Institution, UC Berkeley, France’s National Center for Scientific Research (CNRS), and other partners. The outcome of the project is a library of genetic markers and physical identifiers for every species of plant, animal and fungi on the island that will be provided as a publicly available database resource for ecologists and evolutionary biologists around the world.
The software back-end to the Moore Biocode Project is Geneious Pro and two custom-developed plugins from the New Zealand-based company, Biomatters. The Biocode LIMS and Genbank Submission plugins have been made freely available to the public and users of the free Geneious Basic software will be able to access and view the Biocode database upon completion of the project, while a commercial copy of Geneious Pro is required for researchers involved in data creation and analysis.
DNA barcoding has met with spirited reaction from scientists, especially systematists, ranging from enthusiastic endorsement to vociferous opposition. For example, many stress the fact that DNA barcoding does not provide reliable information above the species level, while others indicate that it is inapplicable at the species level, but may still have merit for higher-level groups. Others resent what they see as a gross oversimplification of the science of taxonomy. And, more practically, some suggest that recently diverged species might not be distinguishable on the basis of their COI sequences. Due to various phenomena, Funk & Omland (2003) found that some 23% of animal species are polyphyletic if their mtDNA data are accurate, indicating that using an mtDNA barcode to assign a species name to an animal will be ambiguous or erroneous some 23% of the time (see also Meyer & Paulay, 2005). Studies with insects suggest an equal or even greater error rate, due to the frequent lack of correlation between the mitochondrial genome and the nuclear genome or the lack of a barcoding gap (e.g., Hurst and Jiggins, 2005, Whitworth et al., 2007, Wiemers & Fiedler, 2007). Problems with mtDNA arising from male-killing microorganisms and cytoplasmic incompatibility-inducing symbionts (e.g., Wolbachia) are also particularly common among insects. Given that insects represent over 75% of all known organisms, this suggests that while mtDNA barcoding may work for vertebrates, it may not be effective for the majority of known organisms.
Moritz and Cicero (2004) have questioned the efficacy of DNA barcoding by suggesting that other avian data is inconsistent with Hebert et al.'s interpretation, namely, Johnson and Cicero's (2004) finding that 74% of sister species comparisons fall below the 2.7% threshold suggested by Hebert et al. These criticisms are somewhat misleading considering that, of the 39 species comparisons reported by Johnson and Cicero, only 8 actually use COI data to arrive at their conclusions. Johnson and Cicero (2004) have also claimed to have detected bird species with identical DNA barcodes, however, these 'barcodes' refer to an unpublished 723-bp sequence of ND6 which has never been suggested as a likely candidate for DNA barcoding.
The DNA barcoding debate resembles the phenetics debate of decades gone by. It remains to be seen whether what is now touted as a revolution in taxonomy will eventually go the same way as phenetic approaches, of which was claimed exactly the same decades ago, but which were all but rejected when they failed to live up to overblown expectations. Controversy surrounding DNA barcoding stems not so much from the method itself, but rather from extravagant claims that it will supersede or radically transform traditional taxonomy. Other critics fear a "big science" initiative like barcoding will make funding even more scarce for already underfunded disciplines like taxonomy, but barcoders respond that they compete for funding not with fields like taxonomy, but instead with other big science fields, such as medicine and genomics. Barcoders also maintain that they are being dragged into long-standing debates over the definition of a species and that barcoding is less controversial when viewed primarily as a method of identification, not classification.
The current trend appears to be that DNA barcoding needs to be used alongside traditional taxonomic tools and alternative forms of molecular systematics so that problem cases can be identified and errors detected. Non-cryptic species can generally be resolved by either traditional or molecular taxonomy without ambiguity. However, more difficult cases will only yield to a combination of approaches. And finally, as most of the global biodiversity remains unknown, molecular barcoding can only hint at the existence of new taxa, but not delimit or describe them (DeSalle, 2006; Rubinoff, 2006).
Software for DNA barcoding requires integration of a field information management system (FIMS), laboratory information management system (LIMS), sequence analysis tools, workflow tracking to connect field data and laboratory data, database submission tools and pipeline automation for scaling up to eco-system scale projects. Geneious Pro can be used for the sequence analysis components, and the two plugins made freely available through the Moorea Biocode Project, the Biocode LIMS and Genbank Submission plugins handle integration with the FIMS, the LIMS, workflow tracking and database submission.
The Barcode of Life Data Systems (BOLD) is a web based workbench and database supporting 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 the most prominently used barcoding software and 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.
- DNA taxonomy
- Pollen DNA barcoding
- Consortium for the Barcode of Life
- Identification (biology)
- Applied Food Technologies
- DNA profiling
- Taxonomic impediment
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