DNA barcoding is a taxonomic method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular species. It differs from molecular phylogeny in that the main goal is not to determine patterns of relationship but to identify an unknown sample in terms of a preexisting classification. Although barcodes are sometimes used in an effort to identify unknown species or assess whether species should be combined or separated, the utility of DNA barcoding for these purposes is subject to debate. The most commonly used barcode region, for animals, at least, is a segment of approximately 600 base pairs of the mitochondrial gene cytochrome oxidase I (COI).
Applications include, for example, identifying plant leaves even when flowers or fruit are not available, identifying insect larvae (which may have fewer diagnostic characters than adults and are frequently less well-known), identifying the diet of an animal, based on its stomach contents or faeces and identifying products in commerce (for example, herbal supplements or wood).
- 1 Choice of locus
- 2 Vouchered specimens
- 3 Origin
- 4 Case studies
- 5 Criticisms
- 6 DNA barcoding software
- 7 Current findings
- 8 See also
- 9 References
- 10 External links
Choice of locus
A desirable locus for DNA barcoding should be standardized (so that large databases of sequences for that locus can be developed), present in most of the taxa of interest and sequenceable without species-specific PCR primers, short enough to be easily sequenced with current technology, and provide a large variation between species yet a relatively small amount of variation within a species.
Although several loci have been suggested, a common set of standardized regions were selected by the respective communities:
- For animals and many other eukaryotes, the mitochondrial COI gene
DNA barcoding is based on a relatively simple concept. All eukaryote 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 (the Folmer 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 microorganisms, 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).
Identifying flowering plants
Kress et al. (2005) suggest that the use of the COI sequence "is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase I gene evolution in higher plants than in animals". A series of experiments was then conducted to find a more suitable region of the genome for use in the DNA barcoding of flowering plants (or the larger group of land plants). One 2005 proposal was the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer; other researchers advocated other regions such as matK.
In 2009, a collaboration of a large group of plant DNA barcode researchers proposed two chloroplast genes, rbcL and matK, taken together, as a barcode for plants. 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.
DNA sequence databases like GenBank contain many sequences that are not tied to vouchered specimens (for example, herbarium specimens, cultured cell lines, or sometimes images). This is problematic in the face of taxonomic issues such as whether several species should be split or combined, or whether past identifications were sound. Therefore, best practice for DNA barcoding is to sequence vouchered specimens.
The use of nucleotide sequence variations to investigate evolutionary relationships is not a new concept. Carl Woese used sequence differences in ribosomal RNA (rRNA) to discover archaea, which in turn led to the redrawing of the evolutionary tree, and molecular markers (e.g., allozymes, rDNA, and 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 specimens. This library would "provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing".
Identification of birds
In an effort to find a 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, the so-called "barcoding gap", was defined as 10 times the mean intraspecific variation for the group under study.
Identification of fishes
The Fish Barcode of Life Initiative (FISH-BOL), is a global effort to coordinate an assembly of a standardised DNA barcode library for all fish species, one that is derived from voucher specimens with authoritative taxonomic identifications. The benefits of barcoding fishes include facilitating species identification for all potential users, including taxonomists; highlighting specimens that represent a range expansion of known species; flagging previously unrecognized species; and perhaps most importantly, enabling identifications where traditional methods are not applicable.
Since its inception in 2005 FISH-BOL has been creating a valuable public resource in the form of an electronic database containing DNA barcodes for almost almost 10000 species, images, and geospatial coordinates of examined specimens. The database contains linkages to voucher specimens, information on species distributions, nomenclature, authoritative taxonomic information, collateral natural history information and literature citations. FISH-BOL thus complements and enhances existing information resources, including the Catalog of Fishes, FishBase and various genomics databases .
Delimiting cryptic species
The next major study into the efficacy of DNA barcoding was focused on the neotropical skipper butterfly, Astraptes fulgerator at the Area de Conservación de Guanacaste (ACG) in north-western Costa Rica. This species was already known as a cryptic species complex, due to subtle morphological differences, as well as an unusually large variety of caterpillar food plants. However, several years would have been required for taxonomists to completely delimit species. Hebert et al. (2004b) sequenced the COI gene of 484 specimens from the ACG. This sample included "at least 20 individuals reared from each species of food plant, extremes and intermediates of adult and caterpillar color variation, and representatives" from the three major ecosystems where Astraptes fulgerator is found. Hebert et al. (2004b) concluded that Astraptes fulgerator consists of 10 different species in north-western Costa Rica. These results, however, were subsequently challenged by Brower (2006), who pointed out numerous serious flaws in the analysis, and concluded that the original data could support no more than the possibility of three to seven cryptic taxa rather than ten cryptic species. This highlights that the results of DNA barcoding analyses can be dependent upon the choice of analytical methods used by the investigators, so the process of delimiting cryptic species using DNA barcodes can be as subjective as any other form of taxonomy.
A more recent example used DNA barcoding for the identification of cryptic species included in the ongoing long-term database of tropical caterpillar life generated by Dan Janzen and Winnie Hallwachs in Costa Rica at the ACG. In 2006 Smith et al. examined whether a COI DNA barcode could function as a tool for identification and discovery for the 20 morphospecies of Belvosia  parasitoid flies (Tachinidae) that have been reared from caterpillars in ACG. Barcoding not only discriminated among all 17 highly host-specific morphospecies of ACG Belvosia, but it also suggested that the species count could be as high as 32 by indicating that each of the three generalist species might actually be arrays of highly host-specific cryptic species.
In 2007 Smith et al. expanded on these results by barcoding 2,134 flies belonging to what appeared to be the 16 most generalist of the ACG tachinid morphospecies. They encountered 73 mitochondrial lineages separated by an average of 4% sequence divergence and, as these lineages are supported by collateral ecological information, and, where tested, by independent nuclear markers (28S and ITS1), the authors therefore viewed these lineages as provisional species. Each of the 16 initially apparent generalist species were categorized into one of four patterns: (i) a single generalist species, (ii) a pair of morphologically cryptic generalist species, (iii) a complex of specialist species plus a generalist, or (iv) a complex of specialists with no remaining generalist. In sum, there remained 9 generalist species classified among the 73 mitochondrial lineages analyzed.
However, also in 2007, Whitworth et al. reported that flies in the related family Calliphoridae could not be discriminated by barcoding. They investigated the performance of barcoding in the fly genus Protocalliphora, known to be infected with the endosymbiotic bacteria Wolbachia. Assignment of unknown individuals to species was impossible for 60% of the species, and if the technique had been applied, as in the previous study, to identify new species, it would have underestimated the species number in the genus by 75%. They attributed the failure of barcoding to the non-monophyly of many of the species at the mitochondrial level; in one case, individuals from four different species had identical barcodes. The authors went on to state:
The pattern of Wolbachia infection strongly suggests that the lack of within-species monophyly results from introgressive hybridization associated with Wolbachia infection. Given that Wolbachia is known to infect between 15 and 75% of insect species, we conclude that identification at the species level based on mitochondrial sequence might not be possible for many insects.
Mwabvu et al. (2013) observed a high level of divergence (19.09% for CO1, 520 base pairs) between two morphologically indistinguishable populations of Bicoxidens flavicollis millipedes in Zimbabwe, and suggested the presence of cryptic species in Bicoxidens flavicollis.
Marine biologists have also considered the value of the technique in identifying cryptic and polymorphic species and have suggested that the technique may be helpful when associations with voucher specimens are maintained, though cases of "shared barcodes" (e.g., non-unique) have been documented in cichlid fishes and cowries
Cataloguing ancient life
Lambert et al. (2005) examined the possibility of using DNA barcoding to assess the past diversity of the Earth's biota. The COI gene of a group of extinct ratite birds, the moa, were sequenced using 26 subfossil moa bones. As with Hebert's results, each species sequenced had a unique barcode and intraspecific COI sequence variance ranged from 0 to 1.24%. To determine new species, a standard sequence threshold of 2.7% COI sequence difference was set. This value is 10 times the average intraspecies difference of North American birds, which is inconsistent with Hebert's recommendation that the threshold value be based on the group under study. Using this value, the group detected six moa species. In addition, a further standard sequence threshold of 1.24% was also used. This value resulted in 10 moa species which corresponded with the previously known species with one exception. This exception suggested a possible complex of species which was previously unidentified. Given the slow rate of growth and reproduction of moa, it is probable that the interspecies variation is rather low. On the other hand, there is no set value of molecular difference at which populations can be assumed to have irrevocably started to undergo speciation. It is safe to say, however, that the 2.7% COI sequence difference initially used was far too high.
The Moorea Biocode Project
The Moorea Biocode Project is a barcoding initiative to create the first comprehensive inventory of all non-microbial life in a complex tropical ecosystem, the island of Moorea in Tahiti. Supported by a grant from the Gordon and Betty Moore Foundation, the Moorea Biocode Project is a 3-year project that brings together researchers from the Smithsonian Institution, UC Berkeley, France’s National Center for Scientific Research (CNRS), and other partners. The outcome of the project is a library of genetic markers and physical identifiers for every species of plant, animal and fungi on the island that will be provided as a publicly available database resource for ecologists and evolutionary biologists around the world.
The software back-end to the Moore Biocode Project is Geneious Pro and two custom-developed plugins from the New Zealand-based company, Biomatters. The Biocode LIMS and Genbank Submission plugins have been made freely available to the public and users of the free Geneious Basic software will be able to access and view the Biocode database upon completion of the project, while a commercial copy of Geneious Pro is required for researchers involved int data creation and analysis.
DNA barcoding has met with spirited reaction from scientists, especially systematists, ranging from enthusiastic endorsement to vociferous opposition. For example, many stress the fact that DNA barcoding does not provide reliable information above the species level, while others indicate that it is inapplicable at the species level, but may still have merit for higher-level groups. Others resent what they see as a gross oversimplification of the science of taxonomy. And, more practically, some suggest that recently diverged species might not be distinguishable on the basis of their COI sequences. Due to various phenomena, Funk & Omland (2003) found that some 23% of animal species are polyphyletic if their mtDNA data are accurate, indicating that using an mtDNA barcode to assign a species name to an animal will be ambiguous or erroneous some 23% of the time (see also Meyer & Paulay, 2005). Studies with insects suggest an equal or even greater error rate, due to the frequent lack of correlation between the mitochondrial genome and the nuclear genome or the lack of a barcoding gap (e.g., Hurst and Jiggins, 2005, Whitworth et al., 2007, Wiemers & Fiedler, 2007). Problems with mtDNA arising from male-killing 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 vertebrates, it may not be effective for the majority of known organisms.
Moritz and Cicero (2004) have questioned the efficacy of DNA barcoding by suggesting that other avian data is inconsistent with Hebert et al.'s interpretation, namely, Johnson and Cicero's (2004) finding that 74% of sister species comparisons fall below the 2.7% threshold suggested by Hebert et al. These criticisms are somewhat misleading considering that, of the 39 species comparisons reported by Johnson and Cicero, only 8 actually use COI data to arrive at their conclusions. Johnson and Cicero (2004) have also claimed to have detected bird species with identical DNA barcodes, however, these 'barcodes' refer to an unpublished 723-bp sequence of ND6 which has never been suggested as a likely candidate for DNA barcoding.
The DNA barcoding debate resembles the phenetics debate of decades gone by. It remains to be seen whether what is now touted as a revolution in taxonomy will eventually go the same way as phenetic approaches, of which was claimed exactly the same decades ago, but which were all but rejected when they failed to live up to overblown expectations. Controversy surrounding DNA barcoding stems not so much from the method itself, but rather from extravagant claims that it will supersede or radically transform traditional taxonomy. Other critics fear a "big science" initiative like barcoding will make funding even more scarce for already underfunded disciplines like taxonomy, but barcoders respond that they compete for funding not with fields like taxonomy, but instead with other big science fields, such as medicine and genomics. Barcoders also maintain that they are being dragged into long-standing debates over the definition of a species and that barcoding is less controversial when viewed primarily as a method of identification, not classification.
The current trend appears to be that DNA barcoding needs to be used alongside traditional taxonomic tools and alternative forms of molecular systematics so that problem cases can be identified and errors detected. Non-cryptic species can generally be resolved by either traditional or molecular taxonomy without ambiguity. However, more difficult cases will only yield to a combination of approaches. And finally, as most of the global biodiversity remains unknown, molecular barcoding can only hint at the existence of new taxa, but not delimit or describe them (DeSalle, 2006; Rubinoff, 2006).
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 Pro can be used for the sequence analysis components, and the two plugins made freely available through the Moorea Biocode Project, the Biocode LIMS and Genbank Submission plugins handle integration with the FIMS, the LIMS, workflow tracking and database submission.
The Barcode of Life Data Systems (BOLD) is a web based workbench and database supporting the acquisition, storage, analysis, and publication of DNA barcode records. By assembling molecular, morphological, and distributional data, it bridges a traditional bioinformatics chasm. BOLD is the most prominently used barcoding software and is freely available to any researcher with interests in DNA barcoding. By providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation in the global sequence databases. Because of its web-based delivery and flexible data security model, it is also well positioned to support projects that involve broad research alliances.
A recent study introduced a "barcode profiling" technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps. The method showed high reproducibility and achieved single-cell sensitivity. Other than profiling cancer cells, the method could also be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. 
- DNA taxonomy
- Consortium for the Barcode of Life
- Identification (biology)
- Applied Food Technologies
- DNA profiling
- Taxonomic impediment
- Paul DN Hebert et al. (2003). "Biological identifications through DNA barcodes". Proceedings of the Royal Society B 270: 313–321. doi:10.1098/rspb.2002.2218. PMC 1691236. PMID 12614582.
- Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH (June 2005). "Use of DNA barcodes to identify flowering plants". Proc. Natl. Acad. Sci. U.S.A. 102 (23): 8369–74. doi:10.1073/pnas.0503123102. PMC 1142120. PMID 15928076. Supporting Information
- Koch, H. 2010. Combining morphology and DNA barcoding resolves the taxonomy of Western Malagasy Liotrigona Moure, 1961. African Invertebrates 51 (2): 413-421.PDF fulltext
- Seberg O, Petersen G. (2009). "How Many Loci Does it Take to DNA Barcode a Crocus?". In Stout, Jane Catherine. PLoS ONE 4 (2): e4598. doi:10.1371/journal.pone.0004598. PMC 2643479. PMID 19240801.
- Eeva M Soininen et al. (2009). "Analysing diet of small herbivores: the efficiency of DNA barcoding coupled with high-throughput pyrosequencing for deciphering the composition of complex plant mixtures". Frontiers in Zoology 6: 16. doi:10.1186/1742-9994-6-16. PMC 2736939. PMID 19695081.
- CBOL Plant Working Group (August 4, 2009). "A DNA barcode for land plants". PNAS 106 (31): 12794–12797. doi:10.1073/pnas.0905845106. PMC 2722355. PMID 19666622.
- Kress WJ, Erickson DL (2008). "DNA barcodes: Genes, genomics, and bioinformatics". PNAS 105 (8): 2761–2762. doi:10.1073/pnas.0800476105. PMC 2268532. PMID 18287050.
- Renaud Lahaye et al. (2008-02-26). "DNA barcoding the floras of biodiversity hotspots". Proc Natl Acad Sci USA 105 (8): 2923–2928. doi:10.1073/pnas.0709936105. PMC 2268561. PMID 18258745.
- Fungal Barcoding Consortium (February 24, 2012). "Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi". PNAS 109 (16): 6241–6246. doi:10.1073/pnas.1117018109. PMC 3341068. PMID 22454494.
- Hebert PDN, Cywinska A, Ball SL, and deWaard JR (7 February 2003). "Biological identifications through DNA barcodes". Proceedings of the Royal Society B 270 (1512): 313–321. doi:10.1098/rspb.2002.2218. PMC 1691236. PMID 12614582.
- Ladoukakis ED, Zouros E (1 July 2001). "Direct evidence for homologous recombination in mussel (Mytilus galloprovincialis) mitochondrial DNA". Mol. Biol. Evol. 18 (7): 1168–75. doi:10.1093/oxfordjournals.molbev.a003904. PMID 11420358.
- Tsaousis AD, Martin DP, Ladoukakis ED, Posada D, Zouros E (April 2005). "Widespread recombination in published animal mtDNA sequences". Mol. Biol. Evol. 22 (4): 925–33. doi:10.1093/molbev/msi084. PMID 15647518.
- Melo-Ferreira J, Boursot P, Suchentrunk F, Ferrand N, Alves PC (July 2005). "Invasion from the cold past: extensive introgression of mountain hare (Lepus timidus) mitochondrial DNA into three other hare species in northern Iberia". Mol. Ecol. 14 (8): 2459–64. doi:10.1111/j.1365-294X.2005.02599.x. PMID 15969727.
- Johnstone RA, Hurst GDD (1996). "Maternally inherited male-killing microorganisms may confound interpretation of mitochondrial DNA variability". Biol. J. Linnaean Soc. 58 (4): 453–70. doi:10.1111/j.1095-8312.1996.tb01446.x.
- Hurst GD, Jiggins FM (August 2005). "Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts". Proceedings of the Royal Society B 272 (1572): 1525–34. doi:10.1098/rspb.2005.3056. PMC 1559843. PMID 16048766.
- Croucher PJP, Oxford GS, Searle JB (2004). "Mitochondrial differentiation, introgression and phylogeny of species in the Tegenaria atrica group (Araneae: Agelenidae)". Biological Journal of the Linnean Society 81: 79–89. doi:10.1111/j.1095-8312.2004.00280.x.
- Whitworth TL, Dawson RD, Magalon H, Baudry E (July 2007). "DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae)". Proceedings of the Royal Society B 274 (1619): 1731–9. doi:10.1098/rspb.2007.0062. PMC 2493573. PMID 17472911.
- Meier R (2008). "Ch. 7: DNA sequences in taxonomy: Opportunities and challenges". In Wheeler, Quentin. The new taxonomy. Boca Raton: CRC Press. ISBN 0-8493-9088-5.
- Jesse H. Ausubel (4 August 2009). "A botanical macroscope". Proceedings of the National Academy of Sciences 106 (31): 12569–70. doi:10.1073/pnas.0906757106. ISSN 0027-8424. PMC 2722277. PMID 19666620.
- Schander C, Willassen E (2005). "What can Biological Barcoding do for Marine Biology?" (PDF). Marine Biology Research 1 (1): 79–83. doi:10.1080/17451000510018962.
- Scott E. Miller (2007-03-20). "DNA barcoding and the renaissance of taxonomy". Proc Natl Acad Sci U S A. 104 (12): 4775–4776. doi:10.1073/pnas.0700466104. PMC 1829212. PMID 17363473.
- Hebert PD, Stoeckle MY, Zemlak TS, Francis CM (October 2004). "Identification of Birds through DNA Barcodes". PLoS Biol. 2 (10): e312. doi:10.1371/journal.pbio.0020312. PMC 518999. PMID 15455034. Supporting Information
- Ward RD, Hanner R, Hebert PDN (2009). "The campaign to DNA barcode all fishes, FISH-BOL.". Journal of Fish Biology 74: 329–356. doi:10.1111/j.1095-8649.2008.02080.x. PMID 20735564.
- Steinke D, Hanner R (2011). "The FISH-BOL collaborators' protocol". Mitochondrial DNA. 22 Suppl 1: 10–14. doi:10.3109/19401736.2010.536538. PMID 21261495.
- Becker S, Hanner R, Steinke D (2011). "Five years of FISH-BOL: brief status report". Mitochondrial DNA. 22 Suppl 1: 3–9. doi:10.3109/19401736.2010.535528. PMID 21271850.
- Hebert PD, Penton EH, Burns JM, Janzen DH, Hallwachs W (October 2004). "Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator". Proc. Natl. Acad. Sci. U.S.A. 101 (41): 14812–7. doi:10.1073/pnas.0406166101. PMC 522015. PMID 15465915. Supporting Information
- Brower AVZ (2006). "Problems with DNA barcodes for species delimitation: 'ten species' of Astraptes fulgerator reassessed (Lepidoptera: Hesperiidae)". Systematics and Biodiversity 4 (2): 127–32. doi:10.1017/S147720000500191X.
- "Database homepage for ACG caterpillar (Lepidoptera) rearing databases". Retrieved 2007-08-12.
- Smith MA, Woodley NE, Janzen DH, Hallwachs W, Hebert PD (2006). "DNA barcodes reveal cryptic host-specificity within the presumed polyphagous members of a genus of parasitoid flies (Diptera: Tachinidae)". Proc. Natl. Acad. Sci. U.S.A. 103 (10): 3657–62. doi:10.1073/pnas.0511318103. PMC 1383497. PMID 16505365.
- Smith MA, Wood DM, Janzen DH, Hallwachs W, Hebert PD (2007). "DNA barcodes affirm that 16 species of apparently generalist tropical parasitoid flies (Diptera, Tachinidae) are not all generalists". Proc. Natl. Acad. Sci. U.S.A. 104 (12): 4967–72. doi:10.1073/pnas.0700050104. PMC 1821123. PMID 17360352.
- Mwabvu, T.; Lamb, J.; Slotow, R.; Hamer, M.; Barraclough, D. 2013. Is millipede taxonomy based on gonopod morphology too inclusive? Observations on genetic variation and cryptic speciation in Bicoxidens flavicollis (Diplopoda: Spirostreptida: Spirostreptidae). African Invertebrates 54 (2): 349–356.Abstract & PDF
- Lambert DM, Baker A, Huynen L, Haddrath O, Hebert PD, Millar CD (2005). "Is a large-scale DNA-based inventory of ancient life possible?" (PDF fulltext). J. Hered. 96 (3): 279–84. doi:10.1093/jhered/esi035. PMID 15731217.
- LIMS Made Freely Available to DNA Barcoding Community - Bio-IT World
- Rubinoff D, Cameron S, Will K (2006). "A genomic perspective on the shortcomings of mitochondrial DNA for "barcoding" identification". J. Hered. 97 (6): 581–94. doi:10.1093/jhered/esl036. PMID 17135463.
- Kevin, C.R. Kerr, Mark Y. Stoeckle, Carla J. Dove, Lee A. Weigt, Charles M. Francis & Paul D. N. Hebert. 2006. Comprehensive DNA barcode coverage of North American birds. Molecular Ecology Notes. (OnlineEarly Articles). doi:10.1111/j.1471-8286.2006.01670.x Full text
- Funk DJ, Omland KE (2003). "Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA". Annu Rev Ecol Syst 34: 397–423. doi:10.1146/annurev.ecolsys.34.011802.132421.
- Meyer CP, Paulay G (December 2005). "DNA Barcoding: Error Rates Based on Comprehensive Sampling". PLoS Biol. 3 (12): e422. doi:10.1371/journal.pbio.0030422. PMC 1287506. PMID 16336051.
- Wiemers M, Fiedler K (2007). "Does the DNA barcoding gap exist? – a case study in blue butterflies (Lepidoptera: Lycaenidae)". Front. Zool. 4 (1): 8. doi:10.1186/1742-9994-4-8. PMC 1838910. PMID 17343734.
- "Numbers of Living Species in Australia and the World" (pdf) (Press release). A.D.Chapman, Australian Biological Resources Study. April 2007. Archived from the original on 2007-09-11.
- Moritz C, Cicero C (2004). "DNA Barcoding: Promise and Pitfalls" (PDF fulltext). PLoS Biol. 2 (10): 1529–31. doi:10.1371/journal.pbio.0020354. PMC 519004. PMID 15486587.
- Johnson NK, Cicero C (May 2004). "New mitochondrial DNA data affirm the importance of Pleistocene speciation in North American birds". Evolution 58 (5): 1122–30. doi:10.1554/03-283. PMID 15212392.
- Will KW, Mishler BD, Wheeler QD (2005). "The Perils of DNA Barcoding and the Need for Integrative Taxonomy" (PDF). Syst. Biol. 54 (5): 844–51. doi:10.1080/10635150500354878. PMID 16243769.
- Gregory TR (April 2005). "DNA barcoding does not compete with taxonomy" (PDF). Nature 434 (7037): 1067. doi:10.1038/4341067b. PMID 15858548.
- Desalle R (October 2006). "Species discovery versus species identification in DNA barcoding efforts: response to Rubinoff". Conserv. Biol. 20 (5): 1545–7. doi:10.1111/j.1523-1739.2006.00543.x. PMID 17002772.
- Rubinoff D (August 2006). "Utility of mitochondrial DNA barcodes in species conservation". Conserv. Biol. 20 (4): 1026–33. doi:10.1111/j.1523-1739.2006.00372.x. PMID 16922219.
- Rubinoff D (October 2006). "DNA barcoding evolves into the familiar". Conserv. Biol. 20 (5): 1548–9. doi:10.1111/j.1523-1739.2006.00542.x. PMID 17002773.
- Ullal AV, Peterson V, Agasti SS et al. (Jan 2014). "Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates.". Sci Transl Med. 6 (219): 219. doi:10.1126/scitranslmed.3007361. PMC 4063286. PMID 24431113.
- 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)
- Guidelines for non COI gene selection