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DNA Barcoding: An Introduction to Central Concepts and Controversies

DNA Barcoding: An Introduction to Central Concepts and Controversies

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An essay for the 2012 Undergraduate Awards (International Programme) Competition by Natasha Latysheva. Originally submitted for Ecology and Evolution at University of St. Andrews, with lecturer Dr Jeff Graves in the category of Life Sciences
An essay for the 2012 Undergraduate Awards (International Programme) Competition by Natasha Latysheva. Originally submitted for Ecology and Evolution at University of St. Andrews, with lecturer Dr Jeff Graves in the category of Life Sciences

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Published by: Undergraduate Awards on Aug 31, 2012
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10/27/2013

 
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DNA Barcoding: An Introduction to Central Concepts and Controversies
Although the concept of ‘genetic barcoding’ has been around since at least 1991 (Arnot 
et al.
, 1991), inthe last decade a group of innovative taxonomists, led by Paul Hebert, have championed the techniqueof DNA barcoding, bringing the ideas of molecular species identification to the forefront of bothtaxonomy and popular science.In essence, DNA barcoding seeks to match each biological species to a standardized genetic sequence.Using barcoding, researchers can identify unknown specimens by first sequencing their
sample’s
barcode (a preselected short stretch of DNA) and then matching it to a barcode stored within a massivedatabase of similar sequences, such as the popular and freely accessible GenBank. This simple but powerful technique has been largely integrated into the taxonomic repertoire, where it supplementstraditional morphological assessments. This essay will explore the fundamentals of DNA barcoding
 the theory, benefits, drawbacks and progress behind
taxonomy’s latest revolution
.
1.
 
Barcoding: the basics
DNA barcoding
1
requires a standardized DNA sequence to compare across taxa. The first gene proposedfor this role was a 648-base pair section of the thoroughly-studied mitochondrial gene, cytochrome-
c
oxidase subunit 1 (COI)
2
. However, the use of mtDNA can produce misleading results due to its unusualmaternal inheritance. Common circumstances such as hybridization, horizontal gene transfer, andmale-killing microorganisms have enormous potential to skew barcode analyses (e.g. otherwise-separate species might exhibit shared mtDNA, and the barcode would register only one species (Meier,2008)). Although researchers have been using molecular markers, such as allozymes and mtDNA, forcategorization purposes for years(Avise, 2004), the barcoding initiative is remarkable for its strivingtowards strict standardization, a comprehensive sequence database, and its ambitious plans formassive-scale international research cooperatives.The inherent simplicity of DNA barcoding technique sidesteps a number of the problems that havetraditionally plagued taxonomy. Perhaps most dramatically, barcoding allows the identification of manypreviously intractable subjects, including foodstuffs, soil, leather goods, and miniscule pieces of tissue.
1
 
The “barcode” analogy is misleading
- it connotes a static, unchangeable tag. Clearly, gene sequences are under the influenceof selection and are therefore
not 
static, much unlike the familiar barcodes plastered on commercial goods. Also, there is aslight degree of intraspecific variation in barcode sequences (so conspecifics may not have
identical 
barcode sequences).
2
Different barcodes may be more informative in different taxa. For example, therbcLandmatKchloroplast genes are utilised as barcodes in land plants (CBOL, 2009).
 
2
Species can also be identified at any life stage
be it eggs, seeds, larvae, or flowers. Also, theunambiguous nature of DNA barcodes contrasts attractively against the descriptive and oftentimessubjective gradations of morphology-based taxonomy, and barcoding-based species identification
isn’t 
hindered
by the fact that most extant species aren’t well characterized morphologically.
Finally,previously-perplexing taxonomic puzzles, such as those brought up by phenotypic convergence and
‘cryptic species’
, become more manageable with the use of barcoding.Practical applications of barcoding include roles in pinpointing of disease vectors, improvingmanagement and conservation of natural resources, and permitting more informed regulation of theillegal exploitation of organisms (as in the lamentable situation with bushmeat-hunting in Africa).Barcoding also
doesn’t rely on the expertise of a quickly
-dwindling supply of highly-experiencedtaxonomists. Barcoding a specimen is minimally costly, costing $2-5 for most labs and requiring a fewhours at most. Non-specialist researchers can rapidly sequence their sample, consult a barcodedatabase, and identify their organism.
2.
 
Main merits and concerns
Just how effective are DNA barcodes methods at matching unknown organisms to described taxa? Moreinterestingly, are DNA barcodes capable of distinguishing between closely-related sister species? Theanswer is an unsatisfying equivocation: it depends. Although all barcodes are sufficiently discriminativeto distinguish taxa at the genus level
3
(Hebert 
et al.
2003), species identifications
aren’t 
always sostraightforward.Consider a success first: Hebert 
et al.
’s canonical
study of COI sequences in 260 species of NorthAmerican birds (2004a). The study sought to find out how closely the species boundaries that barcodespredicted dovetailed with the preexisting (robust and well-resolved) taxonomic categorization.Barcoding proved to be a fruitful approach: each of the 260 species was found to have a different COIsequence, and COI sequence differences between closely related bird species were found to be 19-24times higher than the differences found within species. On the basis of these strongly supportiveresults, Hebert 
et al.
defined a
sequence threshold
of ten times the mean intraspecific variation for thegroup studied as the cut-off point that could be used to provisionally identify species.Compare this with another major study attempted to delimit cryptic butterfly species from theneotropics (Hebert 
et al.
, 2004b). The study concluded that 10 different species were present in the
 
3
Latin American area in question, but a later study pointed out major methodological flaws andchallenged this result, instead arguing that only three to 7 species were present (Brower, 2006). Suchconclusions have informed taxonomists that b
arcoding analyses aren’t as objective as
they might seemat first glance, and in extreme cases the results of barcoding studies are a function of the analyticalmethods used.Whitworth
et al.
’s 2007 study marked another
embarrassment for DNA barcoding proponents when it 
concluded that flies of the Calliphoridae family couldn’t be
discriminated using barcodes. In flies of thegenus Protocalliphora infected with endosymbiotic bacteria called
Wolbachia
, 60% of individual-to-species assignments were impossible to complete; any estimate of species diversity would haveundershot by 75%. These abysmal failures were likely due to widespread mitochondrial non-monophyly (resulting from
Wolbachia
-linked introgressive hybridization). Since
Wolbachia
infects upto 75% of insect species, this study suggests that mtDNA may be impotent as a tool for speciesidentification in insects.How do mtDNA barcodes fare when confronted with the formidable task of differentiating betweensister species? Johnson and Cicero (2004) also studied North American birds and concluded that mtDNA barcoding was woefully deficient at coping with the subtle genetic differences that characterizesister species: the average mtDNA sequence divergence in 39 pairs of sister species was 1.9%, withmost pairs falling below the 2.7% threshold set by Hebert 
et al.
for designating when there is sufficient mtDNA difference between species to count them as separate, bona fide species. Following thisthreshold, 29 of the 39 pairs of the morphologically-distinctive sister species would have beenmisclassified as the same species. Several other investigators have found mtDNA barcodes to beimprecise tools for judging species boundaries and for linking individuals to known taxa. Later, Meyerand Paulay (2005) have found that the lowest overall error for species identification was 4%. However,in incompletely sampled groups, error rates are substantial and species identification relies on the useof threshold between inter- and intra-specific divergences. It seems that DNA barcoding is not up to thetask under all circumstances.Animal mtDNA is already used to point out taxonomical errors: incongruities between the conclusionsof molecular and morphological studies can highlight potential errors in previous diversityassessments, which may prompt taxonomic revision. However, due to the substantial evidencepreviously references in this essay, it has been argued that mtDNA divergence is certainly
not 
suited tobe the
 primary 
tool for 1) inferring species boundaries and for 2) discovering biological diversity.mtDNA-based species boundaries investigations are hindered by a host of confounding phenomena,including the retention of ancestral polymorphism, male-biased gene flow, selection on any mtDNAnucleotide, introgression following hybridization, and paralogy following mtDNA infiltration into the

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