DNA barcoding
Encyclopedia
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 classification but to identify an unknown sample in terms of a known 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.
Applications include, for example, identifying plant leaves even when flowers or fruit are not available, identifying insect larvae (which typically have fewer diagnostic characters than adults), identifying the diet of an animal based on stomach contents or faeces, and identifying products in commerce (for example, herbal supplements or wood).
Although several loci have been suggested, a common set of choices are:
cells contain mitochondria, and mitochondrial DNA (mtDNA) has a relatively fast mutation
rate, which results in significant variation in mtDNA sequences between species and, in principle, a comparatively small variance within species. A 658-bp
region of the mitochondrial cytochrome c oxidase
subunit I (COI) gene was proposed as a potential 'barcode'.
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., Wolbachia
), 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, or for one species to have more than one mtDNA sequence exhibited among different individuals).
As of 2009, databases of CO1 sequences included at least 620,000 specimens from over 58,000 species of animals.
for use in the DNA barcoding of flowering plant
s (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. Jesse Ausubel, a DNA barcode researcher not involved in that effort, suggested that standardizing on a sequence was the best way to produce a large database of plant sequences, and that time would tell whether this choice would be sufficiently good at distinguishing different plant species.
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 mtDNAvage ) 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 specimen
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”.
(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.
at the Area Conservacion 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
http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=650659 parasitoid
flies (Tachinidae
) that have been reared from caterpillar
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 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:
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
. 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 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 int data creation and analysis.
, 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., 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 vertebrate
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 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).
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.
Taxonomy
Taxonomy is the science of identifying and naming species, and arranging them into a classification. The field of taxonomy, sometimes referred to as "biological taxonomy", revolves around the description and use of taxonomic units, known as taxa...
method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular species
Species
In biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in many cases this definition is adequate, more precise or differing measures are...
. It differs from molecular phylogeny
Molecular phylogeny
Molecular phylogenetics is the analysis of hereditary molecular differences, mainly in DNA sequences, 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. 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.
Applications include, for example, identifying plant leaves even when flowers or fruit are not available, identifying insect larvae (which typically have fewer diagnostic characters than adults), identifying the diet of an animal based on stomach contents or faeces, and identifying products in commerce (for example, herbal supplements or wood).
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 sequencable 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 choices are:
- For animals and many other eukaryotes, the mitochondrial CO1 gene
- For land plants, the concatenation of the rbcL and matK chloroplast genes
Mitochondrial DNA
DNA barcoding is based on a relatively simple concept. Most eukaryoteEukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
cells contain mitochondria, and mitochondrial DNA (mtDNA) has a relatively fast mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
rate, which results in significant variation in mtDNA sequences between species and, in principle, a comparatively small variance within species. A 658-bp
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
region of the mitochondrial cytochrome c oxidase
Cytochrome c oxidase
The 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 proposed as a potential 'barcode'.
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., Wolbachia
Wolbachia
Wolbachia is a genus of bacteria which infects arthropod species, including a high proportion of insects , as well as some nematodes. It is one of the world's most common parasitic microbes and is possibly the most common reproductive parasite in the biosphere...
), horizontal gene transfer
Horizontal gene transfer
Horizontal 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...
(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).
As of 2009, databases of CO1 sequences included at least 620,000 specimens from over 58,000 species of animals.
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 genomeGenome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
for use in the DNA barcoding of flowering plant
Flowering plant
The flowering plants , also known as Angiospermae or Magnoliophyta, are the most diverse group of land plants. Angiosperms are seed-producing plants like the gymnosperms and can be distinguished from the gymnosperms by a series of synapomorphies...
s (or the larger group of land plants). One 2005 proposal was the nuclear internal transcribed spacer
Internal transcribed spacer
ITS 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, 28S rRNA and finally the 3'ETS...
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. Jesse Ausubel, a DNA barcode researcher not involved in that effort, suggested that standardizing on a sequence was the best way to produce a large database of plant sequences, and that time would tell whether this choice would be sufficiently good at distinguishing different plant species.
Vouchered specimens
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.Origin
The use of nucleotideNucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
sequence variations to investigate evolutionary relationships is not a new concept. Carl Woese
Carl Woese
Carl 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. He was also the originator of the RNA world hypothesis in 1977,...
used sequence differences in ribosomal RNA (rRNA) to discover archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
, 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 systematics
Systematics
Biological systematics is the study of the diversification of terrestrial life, 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. Hebert
Paul D.N. Hebert
Paul D. N. Hebert, FRSC is a Canadian biologist. He holds a Canada Research Chair in Molecular Biodiversity , and is a tenured full professor at the University of Guelph in Guelph, Ontario, Canada, as well as being a Fellow of the Royal Society of Canada, and the director of the Biodiversity...
from the University of Guelph
University of Guelph
The University of Guelph, also known as U of G, is a comprehensive public research university in Guelph, Ontario, Canada. It was established in 1964 after the amalgamation of Ontario Agricultural College, the Macdonald Institute, and the Ontario Veterinary College...
, Ontario
Ontario
Ontario is a province of Canada, located in east-central Canada. It is Canada's most populous province and second largest in total area. It is home to the nation's most populous city, Toronto, and the nation's capital, Ottawa....
, Canada
Canada
Canada is a North American country consisting of ten provinces and three territories. Located in the northern part of the continent, it extends 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 specimen
Specimen
A specimen is a portion/quantity of material for use in testing, examination, or study.BiologyA laboratory specimen is an individual animal, part of an animal, a plant, part of a plant, or a microorganism, used as a representative to study the properties of the whole population of that species or...
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
North America is a continent wholly within the Northern Hemisphere and almost wholly within the Western Hemisphere. It is also considered a northern subcontinent of the Americas...
(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
Polytypic
In zoology, polytypic refers to a taxonomic unit with more than one subgroup at the next lower level.-See also:*Linnaean taxonomy*monotypic*monotypic habitat...
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
Astraptes is the genus of flasher butterflies. They belong to the skipper butterfly subfamily Eudaminae, which was long included with the spread-winged skippers as a tribe.Species include:...
at the Area Conservacion de Guanacaste (ACG) in north-western Costa Rica
Costa Rica
Costa Rica , officially the Republic of Costa Rica is a multilingual, multiethnic and multicultural country in Central America, bordered by Nicaragua to the north, Panama to the southeast, the Pacific Ocean to the west and the Caribbean Sea to the east....
. This species was already known as a cryptic species complex
Cryptic species complex
In 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 whose morphology is very similar ....
, due to subtle morphological
Morphology (biology)
In biology, morphology is a branch of bioscience dealing with the study of the form and structure of organisms and their specific structural features....
differences, as well as an unusually large variety of caterpillar
Caterpillar
Caterpillars are the larval form of members of the order Lepidoptera . They are mostly herbivorous in food habit, although some species are insectivorous. Caterpillars are voracious feeders and many of them are considered to be 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 Belvosia
Belvosia
Belvosia is a genus of flies in the family Tachinidae.-Species:*B. albifrons Robineau-Desvoidy, 1830*B. aldrichi Townsend, 1931*B. analis Macquart, 1846*B. ansata Reinhard, 1951*B. argentifrons Aldrich, 1928...
http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=650659 parasitoid
Parasitoid
A parasitoid is an organism that spends a significant portion of its life history attached to or within a single host organism in a relationship that is in essence parasitic; unlike a true parasite, however, it ultimately sterilises or kills, and sometimes consumes, the host...
flies (Tachinidae
Tachinidae
Tachinidae 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...
) that have been reared from caterpillar
Caterpillar
Caterpillars are the larval form of members of the order Lepidoptera . They are mostly herbivorous in food habit, although some species are insectivorous. Caterpillars are voracious feeders and many of them are considered to be 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 Protocalliphora
Protocalliphora
Protocalliphora 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 Wolbachia
Wolbachia
Wolbachia is a genus of bacteria which infects arthropod species, including a high proportion of insects , as well as some nematodes. 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 cichlid
Cichlid
Cichlids are fishes from the family Cichlidae in the order Perciformes. Cichlids are members of a group known as the Labroidei along with the wrasses , damselfish , and surfperches . This family is both large and diverse. At least 1,300 species have been scientifically described, making it one of...
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 biotaBiota (ecology)
Biota are the total collection of organisms of a geographic region or a time period, from local geographic scales and instantaneous temporal scales all the way up to whole-planet and whole-timescale spatiotemporal scales. The biota of the Earth lives in the biosphere.-See...
. The COI gene of a group of extinct ratite
Ratite
A 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 the name from the Latin ratis...
birds, the moa
Moa
The moa were eleven 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 subfossil
Subfossil
Subfossil refers to remains whose fossilization process is not complete, either for lack of time or because the conditions 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 speciation
Speciation
Speciation 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.
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 FoundationGordon and Betty Moore Foundation
Gordon and Betty Moore Foundation seeks to develop outcome-based projects that will improve the quality of life for future generations. The private foundation focuses upon portfolios of large-scale initiatives and encourages collaboration so as to achieve the most significant and enduring outcomes...
, the Moorea Biocode Project is a 3-year project that brings together researchers from the Smithsonian Institution
Smithsonian Institution
The Smithsonian Institution is an educational and research institute and associated museum complex, administered and funded by the government of the United States and by funds from its endowment, contributions, and profits from its retail operations, concessions, licensing activities, and magazines...
, 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
Geneious
Geneious is suite of cross-platform bioinformatics software applications developed by Biomatters Ltd.- Features :Geneious comes in a Basic version that is free for academic use, and a commercial Pro version with added features. Geneious bundles various bioinformatics tools under one hood with an...
and two custom-developed plugins from the New Zealand
New Zealand
New Zealand is an island country in the south-western Pacific Ocean comprising two main landmasses and numerous smaller islands. The country is situated some east of Australia across the Tasman Sea, and roughly south of the Pacific island nations of New Caledonia, Fiji, and Tonga...
-based company, Biomatters
Biomatters
Biomatters Limited is the company behind the Geneious Pro software, a bioinformatics software development company headquartered in Auckland, New Zealand. Biomatters develops sequence analysis and bioinformatics software capable of scaling from a single user to the enterprise level of...
. 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 int data creation and analysis.
Criticisms
DNA barcoding has met with spirited reaction from scientists, especially systematistsSystematics
Biological systematics is the study of the diversification of terrestrial life, 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., Wolbachia
Wolbachia
Wolbachia is a genus of bacteria which infects arthropod species, including a high proportion of insects , as well as some nematodes. 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, this suggests that while mtDNA barcoding may work for vertebrate
Vertebrate
Vertebrates are animals that are members of the subphylum Vertebrata . Vertebrates are the largest group of chordates, with currently about 58,000 species described. Vertebrates include the jawless fishes, bony fishes, sharks and rays, amphibians, reptiles, mammals, and birds...
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 phenetics
Phenetics
In biology, phenetics, also known as 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. It is closely related to numerical taxonomy which is concerned with the use of...
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
Big Science
Big 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 taxonomy
Taxonomy
Taxonomy is the science of identifying and naming species, and arranging them into a classification. The field of taxonomy, sometimes referred to as "biological taxonomy", revolves around the description and use of taxonomic units, known as taxa...
, but barcoders respond that they compete for funding not with fields like taxonomy, but instead with other big science fields, such as medicine
Medicine
Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....
and genomics
Genomics
Genomics is a discipline in genetics concerning 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,...
. Barcoders also maintain that they are being dragged into long-standing debates over the definition of a species
Species problem
The species problem or species concept is a mixture of difficult, related questions that often come up when biologists identify species and when they define the word "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
Taxonomy
Taxonomy is the science of identifying and naming species, and arranging them into a classification. The field of taxonomy, sometimes referred to as "biological taxonomy", revolves around the description and use of taxonomic units, known as taxa...
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
Biodiversity
Biodiversity is the degree of variation of life forms within a given ecosystem, biome, or an entire planet. Biodiversity is a measure of the health of ecosystems. Biodiversity is in part a function of climate. In terrestrial habitats, tropical regions are typically rich whereas polar regions...
remains unknown, molecular barcoding can only hint at the existence of new taxa, but not delimit or describe them (DeSalle, 2006; Rubinoff, 2006).
DNA Barcoding Software
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 ProGeneious
Geneious is suite of cross-platform bioinformatics software applications developed by Biomatters Ltd.- Features :Geneious comes in a Basic version that is free for academic use, and a commercial Pro version with added features. Geneious bundles various bioinformatics tools under one hood with an...
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.
See also
- DNA taxonomy
- Consortium for the Barcode of LifeConsortium for the Barcode of LifeConsortium for the Barcode of Life runs the International Barcode of Life project, a collaborative effort which aims to use DNA barcoding to generate a unique genetic barcode for every species of life on earth...
- Identification (biology)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...
- Applied Food TechnologiesApplied Food TechnologiesApplied Food Technologies, Inc. is a privately held, woman-owned corporation in Alachua, Florida that develops diagnostics needed in the seafood industry and runs fee-for-service species identification and verification programs...
External links
- Barcode of Life Data Systems
- International Barcode of Life
- Consortium for the Barcode of Life
- Fish Barcode of Life Initiative (FISH-BOL)
- All Birds Barcoding Initiative (ABBI)
- Polar Flora and Fauna Barcoding website (Latest outpost in the Canadian Arctic in the field)
- The Barcode of Life Blog
- DNA Barcoding Community Network
- Guidelines for non COI gene selection