DNA barcoding is a
taxonomicTaxonomy is the practice and science of classification. The word finds its roots in the Greek , taxis and , nomos...
method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular
speciesIn biology, a species is:* a taxonomic rank or* a unit at that rank ....
. It differs from
molecular phylogenyMolecular phylogenetics, also known as molecular systematics, is the use of the structure of molecules to gain information on an organism's evolutionary relationships. The result of a molecular phylogenetic analysis is expressed in a phylogenetic tree....
in that the main goal is not to determine classification but to identify an unknown sample in terms of a known classification.
Applications include, for example, identifying plant leaves even when flowers or fruit are not available, identifing the diet of an animal based on stomach contents or feces, and identifying products in commerce (for example, herbal supplements or wood).
Mitochondrial DNA
It is based on a relatively simple concept: most
eukaryoteA eukaryote is an organism whose cells contain complex structures enclosed within membranes. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear envelope, within which the genetic material is carried...
cells contain mitochondria and mitochondrial DNA (mtDNA) has a relatively fast
mutationIn biology, a mutation is a randomly derived change to the nucleotide sequence of the genetic material of an organism.Mutations can be caused by copying errors in the genetic material during cell division, or by exposure to mutagens , or can be induced by the organism itself, by cellular processes...
rate, which results in significant variation in mtDNA sequences between species and, in principle, a comparatively small variance within species. However, because all mtDNA genes are maternally inherited (direct evidence for recombination in mtDNA is available in some bivalves such as
Mytilus but it is suspected that it may be more widespread), any occurrences of hybridization, male-killing microoroganisms, cytoplasmic incompatibility-inducing symbionts (e.g.,
WolbachiaWolbachia is a genus of inherited bacteria which infects arthropod species, including a high proportion of insects. It is one of the world's most common parasitic microbes and is possibly the most common reproductive parasite in the biosphere...
),
horizontal gene transferHorizontal gene transfer , also Lateral gene transfer , is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism. By contrast, vertical transfer occurs when an organism receives genetic material from its ancestor, e.g...
(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, or for one species to have more than one mtDNA sequence exhibited among different individuals). A 648-
bpIn molecular biology, two nucleotides on opposite complementary DNA or RNA strands that are connected via hydrogen bonds are called a base pair . In the canonical Watson-Crick base pairing, adenine forms a base pair with thymine , as does guanine with cytosine in DNA. In RNA, thymine is replaced...
region of the mitochondrial
cytochrome c oxidaseThe enzyme cytochrome c oxidase or Complex IV is a large transmembrane protein complex found in bacteria and the mitochondrion.It is the last enzyme in the respiratory electron transport chain of mitochondria located in the mitochondrial membrane...
subunit I (COI) gene was initially proposed as a potential 'barcode'.
Origin
The use of
nucleotideNucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides play central roles in metabolism...
sequence variations to investigate evolutionary relationships is not a new concept.
Carl WoeseCarl Richard Woese is an American microbiologist and physicist. Woese is famous for defining the Archaea in 1977 by phylogenetic taxonomy of 16S ribosomal RNA, a technique pioneered by Woese and which is now standard practice...
used sequence differences in ribosomal RNA (rRNA) to discover
archaeaThe Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon . They have no cell nucleus or any other organelles within their cells...
, which in turn led to the redrawing of the evolutionary tree, and molecular markers (e.g., allozymes, rDNA, and mtDNAvage ) have been successfully used in molecular
systematicsBiological systematics is the study of the diversification of life on the planet Earth, both past and present, and the relationships among living things through time. Relationships are visualized as evolutionary trees...
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. HebertPaul D.N. Hébert, FRSC is a Canadian biologist at the University of Guelph in Guelph, Ontario, Canada where he is a tenured full professor. He is also a Canada Research Chair in Molecular Biodiversity and a Fellow of the Royal Society of Canada....
from the
University of GuelphThe University of Guelph, also known as U of G, is a medium-sized university located in Guelph, Ontario, established in 1964. While the U of G offers degrees in many different disciplines, the university is best known for its focus on life sciences, based in part on a long-standing history of...
,
OntarioOntario is a province located in east-central Canada, the largest by population and second largest, after Quebec, in total area. Ontario is bordered by the Canadian provinces of Manitoba to the west and Quebec to the east, and 5 U.S...
,
CanadaCanada is a country occupying most of northern North America, extending from the Atlantic Ocean in the east to the Pacific Ocean in the west and northward into the Arctic Ocean...
, proposed the compilation of a public library of DNA barcodes that would be linked to named
specimenIn biology, a specimen is an individual animal, part of an animal, plant, part of a plant, or microorganism used as a representative to study the properties of the whole population of that species or subspecies...
s. 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 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 AmericaNorth America is the northern continent of the Americas, situated in the Earth's northern hemisphere and in the western hemisphere. It is bordered on the north by the Arctic Ocean, on the east by the North Atlantic Ocean, on the southeast by the Caribbean Sea, and on the west by the North Pacific...
(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
polytypicIn zoology, polytypic refers to a taxonomic group with more than one subgroup at the next lower taxonomic level.-See also:*Linnaean taxonomy*monotypic...
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.
Delimiting cryptic species
The next major study into the efficacy of DNA barcoding was focused on the neotropical skipper butterfly, Astraptes fulgeratorAstraptes is the genus of flasher butterflies, which belong to the spread-wing skipper subfamily.Species include:* Astraptes alardus – Frosted Flasher* Astraptes alector – Gilbert's Flasher...
at the Area Conservacion de Guanacaste (ACG) in north-western Costa RicaCosta Rica, officially the Republic of Costa Rica is a country in Central America, bordered by Nicaragua to the north, Panama to the east and south, the Pacific Ocean to the west and south and the Caribbean Sea to the east.Costa Rica, which translates literally as "Rich Coast", constitutionally...
. This species was already known as a cryptic species complexIn biology, a cryptic species complex is a group of species which satisfy the biological definition of species, that is, they are reproductively isolated from each other, but their morphology is very similar ....
, due to subtle morphologicalIn biology morphology is the form, structure and configuration of an organism.This includes aspects of the outward appearance as well as the form and structure of the internal parts like bones and organs...
differences, as well as an unusually large variety of caterpillarCaterpillars are the larval form of a member of the order Lepidoptera . They are mostly phytophagous in food habit, with some species being entomophagous. Caterpillars are voracious feeders and many of them are considered pests in agriculture...
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 BelvosiaBelvosia is a genus of tachinid flies in the family Tachinidae. The type species originally was placed in Latreillia Robineau-Desvoidy, 1830, but that name was found to be a junior homonym of Latreillia Roux, 1830, a genus of crabs....
http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=650659 parasitoidA parasitoid is an organism that spends a significant portion of its life history attached to or within a single host organism which it ultimately kills in the process. Thus they are similar to typical parasites except in the certain fate of the host. In a typical parasitic relationship, the...
flies (TachinidaeTachinidae is a large and rather variable family of true flies within the insect order Diptera, with more than 8,200 known species and many more to be discovered. There are over 1300 species in North America. Insects in this family are commonly called tachina flies or simply tachinids.They occur in...
) that have been reared from caterpillarCaterpillars are the larval form of a member of the order Lepidoptera . They are mostly phytophagous in food habit, with some species being entomophagous. Caterpillars are voracious feeders and many of them are considered pests in agriculture...
s 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 ProtocalliphoraProtocalliphora or Bird blowflies are a blow fly genus containing many species which are obligate parasites of birds. The larvae suck the blood of nestlings and are found in the nests of birds...
, known to be infected with the endosymbiotic bacteria WolbachiaWolbachia is a genus of inherited bacteria which infects arthropod species, including a high proportion of insects. It is one of the world's most common parasitic microbes and is possibly the most common reproductive parasite in the biosphere...
. 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:
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
cichlidCichlids are fish from the family Cichlidae in the order Perciformes. The family Cichlidae, a major family of perciform fish, is both large and diverse. There are at least 1300 scientifically described species, making it one of the largest vertebrate families. Numerous new species are discovered...
fishes and cowries.
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
genomeIn modern molecular biology the genome refers to all of its hereditary information encoded in DNA .The genome includes both the genes and the non-coding sequences of the DNA. The term was adapted in 1920 by Hans Winkler, Professor of Botany at the University of Hamburg, Germany...
for use in the DNA barcoding of
flowering plantThe flowering plants or angiosperms are the most diverse group of land plants. The flowering plants and the gymnosperms are the only extant groups of seed plants...
s. Three criteria were set for the appropriate genetic
lociIn the fields of genetics and evolutionary computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
:
- Significant species-level genetic variability and divergence
- An appropriately short sequence length so as to facilitate DNA extraction and amplification, and
- 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 spacerITS refers to a piece of non-functional RNA situated between structural ribosomal RNAs on a common precursor transcript. Read from 5' to 3', this polycistronic rRNA precursor transcript contains the 5' external transcribed sequence , 18S rRNA, ITS1, 5.8S rRNA, ITS2, 26S rRNA and finally the 3'ETS...
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.
Cataloguing ancient life
Lambert et al. (2005) examined the possibility of using DNA barcoding to assess the past diversity of the earth's
biotaBiota may refer to:* Biota , the plant and animal life of a region* Biota , a superdomain in taxonomy* Biota , an evergreen coniferous tree, Platycladus orientalis* Biota , an avant-prog band from Colorado, USA...
. The COI gene of a group of extinct
ratiteA ratite is any of a diverse group of large, flightless birds of Gondwanan origin, most of them now extinct. Unlike other flightless birds, the ratites have no keel on their sternum - hence their name, which comes from the Latin for raft...
birds, the
moaThe moa were ten species of flightless birds endemic to New Zealand. The two largest species, Dinornis robustus and Dinornis novaezelandiae, reached about in height with neck outstretched, and weighed about ....
, were sequenced using 26
subfossilSubfossil refers to remains whose fossilization process is not complete, either for lack of time or because the condition in which they were buried were not optimal for fossilization....
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
speciationSpeciation is the evolutionary process by which new biological species arise. The biologist Orator F. Cook seems to have been the first to coin the term 'speciation' for the splitting of lineages or 'cladogenesis,' as opposed to 'anagenesis' or 'phyletic evolution' occurring within lineages...
. It is safe to say, however, that the 2.7% COI sequence difference initially used was far too high.
Criticisms
DNA barcoding has met with spirited reaction from scientists, especially
systematistsBiological systematics is the study of the diversification of life on the planet Earth, both past and present, and the relationships among living things through time. Relationships are visualized as evolutionary trees...
, 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 microoroganisms and cytoplasmic incompatibility-inducing symbionts (e.g., WolbachiaWolbachia is a genus of inherited bacteria which infects arthropod species, including a high proportion of insects. It is one of the world's most common parasitic microbes and is possibly the most common reproductive parasite in the biosphere...
) are also particularly common among insects. Given that insects represent over 75% of all known organisms
http://www.environment.gov.au/biodiversity/abrs/publications/other/species-numbers/02-exec-summary.html#allspecies, this suggests that while mtDNA barcoding may work for
vertebrateVertebrates are members of the subphylum Vertebrata, chordates with backbones or spinal columns. About 58,000 species of vertebrates have been described. Vertebrata is the largest subphylum of chordates, and contains many familiar groups of large land animals. Vertebrates comprise cyclostomes, bony...
s, 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
pheneticsIn biology, phenetics, also known as numerical taxonomy or taximetrics, is an attempt to classify organisms based on overall similarity, usually in morphology or other observable traits, regardless of their phylogeny or evolutionary relation....
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 scienceBig Science is a term used by scientists and historians of science to describe a series of changes in science which occurred in industrial nations during and after World War II, as scientific progress increasingly came to rely on large-scale projects usually funded by national governments or groups...
" initiative like barcoding will make funding even more scarce for already underfunded disciplines like
taxonomyTaxonomy is the practice and science of classification. The word finds its roots in the Greek , taxis and , nomos...
, but barcoders respond that they compete for funding not with fields like taxonomy, but instead with other big science fields, such as
medicineMedicine is the art and science of healing. It encompasses a range of health care practices evolved to maintain and restore health by the prevention and treatment of illness....
and
genomicsGenomics is the study of the genomes of organisms. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. The field also includes studies of intragenomic phenomena such as heterosis, epistasis, pleiotropy and other...
.
The current trend appears to be that DNA barcoding needs to be used alongside traditional
taxonomicTaxonomy is the practice and science of classification. The word finds its roots in the Greek , taxis and , nomos...
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
biodiversityBiodiversity is the variation of life forms within a given ecosystem, biome, or for the entire Earth. Biodiversity is often used as a measure of the health of biological systems...
remains unknown, molecular barcoding can only hint at the existence of new taxa, but not delimit or describe them (DeSalle, 2006; Rubinoff, 2006).
See also
- DNA taxonomy
- Consortium for the Barcode of Life
Consortium for the Barcode of Life is an international collaborative effort which aims to develop a mechanism capable of generating a unique genetic barcode for every species of life on earth....
- Identification (biology)
Identification in biology is the process of assigning a pre-existing individual or class name to an individual organism. Identification of organisms to individual names may be based on individualistic natural body features Identification in biology is the process of assigning a pre-existing...
External links