DNA barcoding

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DNA barcoding is a taxonomic method which uses a short genetic marker in an organism's mitochondrial DNA to quickly and easily identify it as belonging to a particular species. It is based on a relatively simple concept: most eukaryote cells contain mitochondria and mitochondrial DNA (mtDNA) has a relatively fast mutation rate, which results in significant variance in mtDNA sequences between species and a comparatively small variance within species. A 648-bp region of the mitochondrial gene, known as cytochrome c oxidase I (COI), was initially proposed as a potential 'barcode'.

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[edit] Origin

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 archaebacteria, which in turn led to the redrawing of the evolutionary tree, and molecular markers (e.g., allozymes, rDNA, and mtDNA) 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, Professor 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”.

[edit] Case Studies

[edit] Identification of Birds

In an effort to find a correspondence 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 was defined as 10 times the mean intraspecific variation for the group under study.

[edit] Delimiting Cryptic Species

The next major study into the efficacy of DNA barcoding was focused on the neotropical skipper butterfly, Astraptes fulgerator. 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 north-western Costa Rica. 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 highlight the potential of DNA barcoding in the discovery of new species, when used in conjunction with traditionally collected data.

[edit] 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. Three criteria were set for the appropriate genetic loci:

  1. Significant species-level genetic variability and divergence
  2. An appropriately short sequence length so as to facilitate DNA extraction and amplification, and
  3. The presence of conserved flanking sites for developing universal primers.

At the conclusion of these experiments, Kress et al. (2005) proposed the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as a potential DNA barcode for flowering plants. These results suggest that DNA barcoding, rather than being a 'master key' may be a 'master keyring', with different kingdoms of life requiring different keys.

[edit] 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.

[edit] Criticisms

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. 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. Regarding the latter, exploratory studies have shown that about 96% of eukaryotic species surveyed can be detected with barcoding - though most of these would also be also resolvable with traditional means; the remaining 4% do, however, pose problems which can lead to error rates that are unacceptably high (up to 31% of false attributions) when relying on DNA barcoding alone. Other critics fear a "big science" initiative like barcoding will make funding even more scarce for already underfunded disciplines like taxonomy. Barcoders respond that they compete for funding not with fields like taxonomy, but instead with other big science fields, such as medicine and genomics.

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 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.

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. Another factor to consider is that because all mtDNA genes are maternally inherited, hybridization will lead to misleading results. And finally, as most of the global biodiversity remains unknown, molecular barcoding can only hint at the existence of new taxa, but not delimitate or describe them (DeSalle, 2006; Rubinoff, 2006a,b).

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Topics in phylogenetics
v • d • e
Relevant fields: phylogenetics | computational phylogenetics | molecular phylogeny | cladistics
Basic concepts: synapomorphy | phylogenetic tree | phylogenetic network | long branch attraction
Phylogeny inference methods: maximum parsimony | maximum likelihood | neighbour joining | UPGMA | Bayesian inference | Least Squares
Current topics: PhyloCode | DNA barcoding
List of evolutionary biology topics
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