852 SYSTEMATIC BIOLOGY VOL.54

Sy!(. Bwl. 54(51:852-859,2005Copyright © Society of Systomaiic BiiiiogISSN; lI>M-5l57prinl / 107iWi36X<mlineTXlIr 10.10Hl)/l()6.15150.'HK13.MB86

The Promise of DNA Barcoding for Taxonomy

PAUL D . N . HEBERT AND T. RYAN GREGORY

Department of Integrative Biology, Biodiversit]/ Institute of Ontario, Unwersiiy ofCuelph, GueJph. Ontario. NIG 21VI, Camda:E-mail: pbebert@iioguelpli.ca (P.D.N.H.)

DNA barcoding is a novel system (designed to providerapid, accurate, and automatable species identificationsby using short, standardized gene regions as internalspecies tags. As a consequence, it will make the Linnaeantaxonomic system more accessible, with benefits to ecol-ogists, conservationists, and the diversity of agenciescharged with the control of pests, invasive species, andfood safety. More broadly, DNA barcoding allows a dayto be envisioned when every curious mind, from pro-fessional biologists to schoolchildren, will have easy ac-cess to the names and biological attributes of any specieson the planet. In addition to assigning specimens toknown species, DNA barcoding will accelerate the paceof species discovery by allowing taxonomists to rapidlysort specimens and by highlighting divergent taxa thatmay represent new species. By augmenting their capa-bilities in these ways, DNA barcoding offers taxonomiststhe opportunity to greatly expand, and eventually com-plete, a global inventory of life's diversity.

Despite the potential benefits of DNA barcoding toboth the practitioners and users of taxonomy, it has beencontroversial in some scientific circles (Wheeler, 2004;Will and Rubinoff, 2004; Ebach and Holdredge, 2005; Willet al., 2005). A few have even characterized DNA barcod-ing as being "anti-taxonomy," arguing that its implemen-tation will signal the death of a system 250 years in themaking. We feel that this opposition stems from miscon-ceptions about the DNA barcoding effort. As such, wewelcome this opportunity to clarify both the rationaleand potential impacts of DNA barcoding. In respond-ing to this set of questions, we emphasize the multiplepositive impacts of this approach for taxonomy and bio-diversity science.

QUESTIONS

I. Given two billion US dollars (the amount acomprehensive program of DNA barcoding is estimated to

cost i\Nhitfield. 2003}), how ivould you spend this money tobenefit taxonomic and biodiversity research, and what wonki

be the legacy of these data?

This question ignores an inescapable reality; there isno prospect of a single $2 billion infusion of support forany biodiversity research program. Such a level of in-vestment may ultimately be achieved—but, if so, it willreflect a staged and geograpWcally dispersed process ofpositive funding decisions that will depend heavily onboth scientific progress and societal demand for species-level identifications. The small amount of funding so far

directed to DNA barcoding has yielded a rich harvestof scientific insights. This fact has led new organizationsto provide the support needed to explore the scalabil-ity of these results across the animal kingdom. The earlyand positive results from this second wave of investi-gations have now motivated larger research groups tocoalesce. In fact, the first alliances with a global reachhave been assembled to lead the development of bar-code sequence libraries for all birds and fishes. Segmentsof much more species-rich groups, such as plants andlepidopterans, are in the earlier stages of this process{www.barcoding.si.edu). These research groups may. inthe longer term, form the nuclear units needed for thebarcode initiative to move into the "big science" do-main where success depends upon the coupling of aclearly enunciated, socially significant research agendawith strong international research alliances.

It is also important to note that the quest for large-scale support for DNA barcoding is not being carried outat the expense of taxonomic funding. Indeed, it is clearthat any successful campaign to generate this supportwill result in a substantial infusion of funding for insti-tutions and individuals engaged in taxonomic research.Overall, the costs associated with DNA sequencing willrepresent a small component of DNA barcoding efforts;the majority of funding will be employed for global col-lection efforts, for the curation of resultant specimens,and for developing online databases containing detailedinformation about them. Moreover, it bears pointingout that the funding already acquired has come fromlarge foundations and government agencies and pro-grams with no tradition of supporting taxonomic re-search, and that in some cases DNA barcoding proposalshave competed directly with medical and comparativegenomics projects rather than any related to taxonomicresearch. Viewed from this perspective, any large-scaleDNA barcoding effort will represent a substantial boon,both financially and scientifically, to biodiversity and tax-onomic research. It will certainly leave a lasting legacy inthe form of a comprehensive, widely accessible systemfor the identification of species.

2. Globally, alpha taxonomic research (the discovery anddescription ofneiv species) is in crisis. Is DNA barcoding an

expedient solution to this problem or will it expediteits decline?

In our view the decline of alpha taxonomy is not aconsequence of the growing use of molecular methods.

2005 POINTS OF VIEW 853

as has sometimes been suggested (Wheeler, 2004). In fact,we expect DNA barcoding to aid the resurgence of tax-onomy. DNA barcoding programs will certainly directnew funding into the collection and cataloguing of spec-imens. They will, as well, aid taxonomic investigationsby helping to reveal cryptic species (Hebert et al., 2004a,2004b), by connecting sexes and life stages (Beskanskyet al., 2003), and by clarifying problems of synonymy thatnow consume much taxonomic effort (Alroy, 2002). Thenovelty and scientific promise of DNA barcoding willadditionally draw public interest to taxonomic and bio-diversity issues, encouraging young researchers to en-ter the discipline and both academic departments andbiomanagement agencies to hire them.

We are confident that DNA barcoding will play an in-creasingly important role as a taxonomic screening toolbecause of its ability to rapidly reveal the genetic dis-continuities that ordinarily separate distitict species (e.g.,Jarizen et a!., 2005; Smith et al., 2005). Its application inthis fashion will allow an inversion of standard taxo-nomic approaches that operate in an a priori fashion—-seeking the morphological discontinuities that signalreproductive isolation an:iong unsorted assemblages oforganisms. By contrast, DNA barcoding allows a moreefficient a posteriori approach where predefined, genet-ically divergent groups are examined for trait variation.In this sense, DNA barcoding will dearly be a powerfulenabler of alpha taxonomy.

3. Overlapping character variation betiveen and iL>ithi}ispecies is well documented for many character systems. Why

is this any more or less of a prohletnfor DNA harcoding?

Overlap in the variation of single characters is notproblematic for any taxonomic system, be it morpho-logical or molecular, so long as multiple characters areemployed for taxon diagnosis. One common misunder-standing of DNA barcoding is that it is based on a singlecharacter, namely "one DNA sequence." In fact, the 648-bp cytochrome c oxidase subunit 1 {coxl or COI ) generegion used as the DNA barcode standard for membersof the animal kingdom represents a complex compos-ite character involving hundreds of independently vary-ing components. Some of these component charactersare invariant and therefore not all 648 bp are informativewithin a given taxonomic assemblage, but most are vari-able. For example, we have found variation at 512 of the648 sites in a large set of lepidopteran barcodes (9715 se-quences from 2215 species and 1047 genera). This meansthat even within a single insect order, DNA barcodesintegrate the patterning of similarities and differencesamong hundreds of characters. In a certain sense, it islike the patterning generated by the scales on a moth'swing^—each scale is of almost no significance, but thecomposite character of wing coloration pattern is highlyinformative.

DNA barcoding using a single gene region doesnot assure complete taxonomic resolution, but it doespromise proximity. Based on past results for varied an-imal groups, DNA barcoding will deliver species-level

resolution in 95% to 97% of cases (Hebert et al., 2004b;Janzen et al., 2005; Ward et al., 2005). When it fails,it will narrow the options to a small number of con-generic taxa (which, in many cases, could be resolvedfully with additional genetic or other data). This impres-sive performance reflects two impoitant, and perhapsunexpected, observations: the rarity of mitochondrial se-quence sharing among species and the dearth of deepbarcode divergences within speciee. Constrained in-traspecific variation in diverse animal groups is a keyearly finding of the DNA barcode effort; one that meritsdeeper scientific investigation. Certainly, coxl shows farless variation within species than sonie early critics hadprojected would be the case (e.g.. Mallet and WiUmott,2003), and this may reflect the impact of selective sweepsrelated to the coevolution of nuclear and mitochondrialgenomes. Importantly, for the use of barcodes as species-level identifiers, barcode differences appear to accumu-late quickly, making it possible to distinguish all but theyoungest of sister species.

4. Many taxonomists already practice DNA barcodinginformalhf when delimiting and discovering species. Is thiszurong, and what data are sufficient to demonstrate that a

series of specimens represents a new species with traditionalor barcoding methodi?

We recognize both the general utility of genetic datain taxonomic studies and the strong concordance intaxonomic signals from different genes. However, weemphasize that there is no such thing as "informalDNA barcoding." A DNA barcode is not just any DNAsequence—it is a rigorously standardized sequence of aminimum length and quality from an agreed-upon gene,deposited in a major sequence database, and attached toa voucher specimen whose origins ar d current status arerecorded. In fact, it has already been established that onlythose coxl sequences that meet these strict criteria will bedesignated as DNA barcodes by the Mational Center forBiotechnology Information's GenBarik (NCBI, GenBank;www.ncbi.nlm.nih.gov/Genbank), the European Molec-ular Biology Laboratory (EMBL; www.embl.org), and theDNA Data Bank of Japan (DDBJ; www.ddbj.nig.ac.jp).

There is an important distinction between "describ-ing" and "delimiting" species, but a conflation of thetwo has created uneasiness about the use of DNA bar-codes as the foundation of future taxonomic descrip-tions. We emphasize that DNA barcoding seeks merely toaid indelimiting species—to highligh: genetically distinctgroups exhibiting levels of sequence divergence sugges-tive of species status. By contrast, DNA barcodes—bythemselves—are never sufficient to describe new species.At some stage, clearly divergent DNA barcodes, in com-bination with other information, will be used as the basisfor providing a new Lirmaean name (Smith et al., 2005)and, as with any taxonomic hypothesis, this would besubject to ongoing reevaluation. Fcir example, in a re-cent survey of North American birds, the threshold fordelineating probable new species was arbitrarily set at10 X the average within-species variation of the entire

854 SYSTEMATIC BIOLOGY VOL.54

barcode data set. This led to the revelation of four pre-sumptive new species (Hebert et al., 2004b), but decisionsregarding the formal recognition of these taxa are left,appropriately, to the ornithological community (notably,existing morphological and behavioral information sup-ports these new hypotheses), The synergy between DNAbarcoding and studies of morphological/ecological di-versity is further illustrated by the case of the skipperbutterfly, Astraptes fulgerafor, in which a combined mor-phological, natural history and barcoding approach re-vealed a complex of 10 species in one small area of CostaRica. Importantly, several of these species showed a rel-atively small barcode divergence, but a coupling of thisinformation with records on larval host plants and mor-phology illuminated the full diversity of the complex(Hebert etal., 2004a),

5. The proposed barcoding genes can fail to recover accuratespecies trees. Does this matter for DNA barcoding?

We emphasize that DNA barcodes do not aim to re-cover phylogenetic relationships; they seek instead toidentify known species and to aid the discovery of newones. Despite this fact, some opponents have claimedthat DNA barcoding fails as a taxonomic approach be-cause it does not always recover accurate species trees(e.g.. Will and Rubinoff, 2004). It is important in this re-gard to emphasize that current taxonomic placementsneed to be viewed as hypotheses not facts. Consider aprimary example offered by Will and Rubinoff (2004)in their critique of DNA barcoding: namely that DNAbarcodes suggest a very close affinity between the mothSinti/ra henrici and certain species o^ Acronicta (Hebertet al., 2003). Will and Rubinoff (2004:48) argue thatthis placement makes it "impossible [for DNA barcod-ing] to recover any taxonomic information below thesuprageneric level, not even genus membership." How-ever, rather than reflecting a failure of DNA barcod-ing, we believe this case illustrates the power of theapproach to illuminate taxonomic assignments in needof scrutiny. The traditional placement of S. henrici to adistinct genus reflects the fact that its adults have paleyellow forewings, showing striking divergence from thegrey/black forewings of Acronicta species. Yet larvalmorphology, adult forewing patterns, ecological niche,and genital anatomy all suggest that S. henrici has closeaffinities to Acronicta oblinata (D. Wagner, personal com-munication), a conclusion reinforced by DNA barcodes(Fig. 1). Its distinctive forewing coloration likely reflectsthe fact that larvae of S. henrici feed on grasses, asopposed to the tree-feeding habits of typical Acronictaspecies. A rapid shift in wing color has presumably beendriven by natural selection to aid substrate matching

FlCUKiL 1. A taxon identification tree generated through neighbor-joining analysis of K2P distances showing patterns of i:(),v7 sequencedivergence for 3] species of Acrouicta and 1 species of Siniyra. Speci-mens from different provinces (Canada) or states (USA) are shown indifferent colors.

Acronicta retanJata

Acronlca Interrupts

Acmnlcta connecta

Acronicta tiasa

Acronicta Innotata

Acronlca laettftca

Acronlca splnlgera

Acronicta grisea

Acronicta morula

Acronlca superans

Acranlcta fragllis

Acronicta lepuscullna

Acronicta Increta

— Acronicta exills

Acronicta tristis

Acronicta ovaa

Acronicta haesltata

Acronicta modica

Acronicta sp. 1Acronicta affllctaAcronicta tritonaAcronicta vinnvta

Acronicta amerkana

Acmnlcta dactyllna

Acronicta hastullferaAcronicta vulplna

Aavnicta funeralls

Acronicta ImpressaAcrcnicta impleta

Acronicta noctlvaga

Acronlca obllnia

Slmyra henrld

2005 POINTS OF VIEW 855

during adult life. Thus, this particular example revealsnot only the ability of DNA barcoding to refine existingtaxonomic hypotheses, but also to provide new insightsinto evolutionary trajectories (see Janzen et al., 2005, forother examples involving tropical iepidopterans).

6. Some species are not mitochondrially monophyletic,sharing polymorphisms with unrelated taxa. How will this

affect identifications using a barcoditig approach?

Although the horizontal exchange of mitochondria be-tween taxonomicnlly divergent organisms might theo-retically occur, evidence for it not been found amongthe thousands of animal species that have now beenbarcoded. Shared mitochondrial sequences (and hencebarcodes) have been observed, but only among closelyrelated species and presumably ns a result of ongoinghybridization. The taxonomic impacts of such sharingare far from catastrophic—they restrict identifications toa small coniplex of congeners. Shared barcodes do notrepresent a substantive taxonomic problem because theyare uncommon and their impacts are parochial.

It is worth emphasizing that critical tests of mitochon-drial sequence sharing between species are difficult toexecute. Many studies presume that discrepancies be-tween identifications made using morphological traitsand DNA barcodes signal flaws in the barcode data. Be-fore criHcaily accepting such conclusions, stronger vali-dation of the morphology-based assignments is needed.For example, Wahlberg et al. (2003) reported conflicts be-tween morphology and mitochondrial DNA divergencesin a tightly allied species complex of butterflies, but thisconclusion would have been far stronger if the morpho-logical assignments had been confirmed independentlyby several taxonomists (although these still might reflectan erroneous taxonomic hypothesis). It bears noting thatblind tests of DNA barcoding have been carried out onseveral occasions. Indeed, DNA barcoding has passeddouble-hWnd tests, in which the taxonomist providing thespecimens did not realize the full diversity of speciespresent in a sample (that is, until further examinationinspired by the barcoding results revealed key biolog-ical differences aniong them; e.g., Hebert et al., 2004a,2004b). Providing empirical demonstrations that DNAbarcodes are capable of consistent, accurate, and unam-biguous identifications is a key aspect of barcoding re-search, and the same should be expected of alternativeapproaches.

7. Should the completion of a DNA barcoding program everoccur, would this mark the beginnitig or etid of taxotiomic andbiodiversity research, and what will he the role of systematistsin a world where most identifications are done by "barcode"?

DNA barcoding will increase the scale and suc-cess of biodiversity science by greatly increasing ac-cess to species identifications. An automated DN A-basedsystem will free taxonomists from routine identifications,allowing them to direct their efforts to new collecfior\s,descriptions, and assessments of taxonomic relation-ships. Some DNA barcoding opponents have argued that

routine identifications are only a minor part of a tax-ononiist's work (Lipscomb et al., 2003; Wheeler, 2004;Will and Rubinoff, 2004), whereas others have lauded thepotenfial utility of automated identification systems, butonly if based on morphology (Gaston and O'Neill, 2004;Wheeler, 2004). We believe that species identifications area rate-limiting step for many ecological and biodiversityinvestigations, as well as for taxonomic research, andthat DNA barcoding will therefore both relieve a burdenon taxonomists and fill a current ni ed with importantbenefits to both taxonomy and biodiversity science.

In a barcoded world, taxonomists will retain their lead-ership role in the association, integration, and interpre-tation of knowledge about the character state variationthat delineates species and what thi: implies for higherlevel taxonomy. As noted earlier, their work on new as-semblages of life may often be expedited by using bar-code results to enable an a posteriori approach to speciesrecognition. Taxonomists will, of course, also continue toexploit other molecular and morphtilogical approachesto explore deeper taxonomic relationships.

8. Would the inevitable expansion of sequencingefforts that zvould come with a program of DNA

barcoding be concomitant with a decline in the qualityof taxonomic research?

It has been suggested that sequencing is too expensive,difficult, or time-consuming for taxonomists to carryout (e.g., Dunn, 2003; Mallet and Willmott, 2003; Seberget al., 2003). However, individual :axonomists are nomore required to perform their oun sequencing thanindividual photographers need to develop their ownphotographs. Barcoding has already moved to the "pho-tomat" stage, with tens of thousands of specimens beinganalyzed at low cost in high-volunie barcoding facili-ties (e.g., at the University of Guelph, Canada, and theSmithsonian Institution, USA). The direct "burden" ontaxonomic collaborators involves fueling the analyticaltrain by providing small fissue samples from identified,vouchered specimens to be barcodi 'd. As microfluidictechnologies mature over the next decade, one can expectthe development of affordable, user-friendly, compact —if not handheld—devices that integrate all stages fromDNA extraction through analysis of the barcode se-quence to gain an identification (e movement to the"Polaroid" stage in the analogy with photography). Al-though we might expect such instruments to becomestandard equipment for both taxonomic research and forthe broader community of organizations and individu-als that need rapid access to species identifications, thiscertainly does not imply that barccding will turn tax-onomists into molecular biologists.

We believe the tools provided by DNA barcoding willadd rigour to the generation and tesfing of taxonomic hy-potheses. Taxonomy has generally been executed usingdiscontinuities in analog (i.e., graded morphological)traits to infer species boundaries, an approach thathas generated a total of 1.7 million taxonomic hy-potheses over 250 years. DNA barcoding allows these

856 SYSTEMATIC BIOLOGY VOL.54

hypotheses to be tested using an independent digital (i.e.,DNA nucleotide-based) data stream. Although there hasbeen good correspondence between species recognizedthrough morphological approaches with designationsbased on barcodes, there are discordances. Tliese casesshould be welcomed as they will strengthen both taxo-nomic hypotheses and methods for analyzing barcodedifferences, and may lead to new discoveries regardingevolution and ecology. All of these benefits have beenevidenced in early barcoding efforts.

9. Assuming the technical problems of DNA barcodingcan be overcome, is it nozv, or xviU it ever be cost-effectiverelative to traditional methods to use DNA barcodes for

bioinventory ptirposes?

A major benefit of, and rationale for, DNA bar-coding lies in its cost-effectiveness for species iden-tification, especially in ambitious bioinventory andbiomonitoring programs (Smith eta!., 2005). As it stands,production-line systems for identifying even a smallgroup of thoroughly known species through morpho-logical approaches cost about $2 per specimen (e.g.,mosquito monitoring programs that deal with fewer than60 species; F. C. Hunter, personal communication). Whena team of taxonomic specialists targets a larger assem-blage of species in a specific geographic area, costs risesubstantially and the identification of single specimenscan cost $50 to $100 if all costs are internalized. Today,a DNA barcode can be generated for about $5 per spec-imen including labor and sequencing—and this cost isexpected to plummet. In time, DNA barcoding programshave the potential to become self-sufficient by charging asmall fee for identifications w^hile still maintaining openaccess for academic researchers.

Cost is only one criterion in evaluating the utility ofa taxonomic support system for biodiversity research.Speed, reliability, and accessibility are just as important,and we believe that DNA barcoding excels in these areas.By contrast, even smaller-scale biomonitoring programsbased on morphology currently face a major challenge indelivering results in a rapid, cost-effective fashion. Thiscan have dramatic economic consequences, as with thecurrent difficulty in identifying invasive species earlyenough to suppress an outbreak. Yet, the economic ben-efits of excluding even a single noxious invader, such asthe zebra mussel from North America, would have beensufficient after a decade to barcode most of the animalspecies on Earth.

10. Hypothesis-driven research is the foundation upon whichmost research agencies assign funding priorities,

yet taxonomy is discovery driven. Hozv would yourapproach to taxonomy convince these agencies of the

merits of taxonomic studies?

We agree that hypothesis-driven science dominatessmall-scale funding competitions and that taxonomyfares poorly because of its discovery-driven nature.On the other hand, every "big science" initiative—from subatomic physics to the human genome to space

exploration—has been discovery-driven, and this will bethe funding arena in which the global DNA barcodingprogram will operate if it rises to the challenge. As withmost other big science projects, DNA barcoding has expe-rienced claims that it is "not science," and that it threatensthe ability of smaller laboratories to carry out hypothesis-driven research. In past cases, such claims have alwaysproved shortsighted. The repeated observation is thatlarge-scale discovery science spins off hypotheses at afrenetic pace and reveals avenues of investigation thatcould never have been anticipated. In this sense, manyof the criticisms levied at DNA barcoding are remark-ably similar to those given a decade ago regarding thehuman genome project.

DNA barcoding has already been successful in at-tracting substantial funding from varied agencies andorganizations that have not been traditional funders oftaxonomy, but this has not been accomplished by selling"taxonomy" per se. Instead, the DNA barcoding initia-tive promotes the vision of a broadly accessible inventoryof life's diversity. It is only by emphasizing the benefitsto society and by sparking interest among the taxpay-ing public that support for a global biodiversity initiativewill be generated. Of course, that does not mean that tax-onomy is set to become a "high tech service industry" forother biologists, as some have suggested (e.g., Lipscombet al., 2003; Wheeler, 2004; Will and Rubinoff, 2004).A major goal of DNA barcodiiig is to enable the non-taxonomist majority of biologists—and indeed, anyone—to access taxonomic information directly while allowingprofessional taxonomists to focus on generating moresuch knowledge.

POSITION STATEMENT

Efforts to inventory eukaryotic diversity through mor-phological analyses have enjoyed much success. Thegeneration of nearly two million taxonomic hypothe-ses over the past 250 years is an impressive feat thathas provided a fomidational understanding of biolog-ical diversity, but many details await clarification. DNAbarcoding is positioned to aid the inventory of life byaccelerating species discovery, by testing current taxo-nomic hypotheses, and by making species identificationsmore easily available. These contributions will not bemade at the expense of core taxonomic values or fund-ing. DNA barcoding does not seek to abandon "mor-phological studies in favor of a narrow and whollymolecular identification system" (Will and Rubinoff ,2004: 47). Rather, it strives to build alliances betweenmolecular and morphological taxonomists {Hebert andBarrett, 2005). It seeks, as well, to preserve the Linnaeanprinciples by which species are named and classified.DNA barcodijig requires existing, morphologically de-rived species names for calibration, and it is thesenames that are recovered when barcoding is used foridentification.

It is generally accepted that the study of biodiver-sity is seriously imderfimded (Godfray, 2002). It is noteasy to attribute this to the theme of investigation, as

2005 POINTS OF VIEW 857

biodiversity science is important and attracts much pub-lic interest. However, this area of research does sufferfrom a culture of conflict. Rather than mounting grandcollaborations, the biodiversity community has a tradi-tion of polarization and infighting. DNA barcoding isno stranger to invectives—it has been tagged as "theo-retically vacuous technology" and as a "parlour trick"(Wheler, 2004; Will et al., 2005). The coupling of suchcomments with ad hominem attacks on DNA barcodingproponents brings little credit to the discipline.

Some critics charge that the DNA barcoding approachis fundamentally flawed, but the available data tell a verydifferent story: the success of DNA barcoding has sofar been startlingly impressive. As Smith (2005) notes,barcoding fared well in a test executed at the PEETconference. More importantly, a series of studies havenow investigated the effectiveness of DNA barcodingin species assemblages from varied geographic settings,and from numerous taxonomic groups with divergentlife history and evolutionary attributes. As a conse-quence of these sensitivity tests, barcode records are nowavailable for more than 13,000 animal species (and accu-mulating rapidly) and they reveal resolution that is noillusion (www.barcodinglife.org). In group after group,success in species identification exceeds 95% and thefew cases of compromised resolution involve the in-ability to discriminate a small group of closely alliedspecies (Hebert et al., 2004a, 2004b; Hebert et al., un-published). Typical results resemble those in Figure 1,which shows the patterning of DNA barcode divergencesfor 31 species of Acronicta, one of the most diverse lepi-dopteran genera in North America. In this case, there isno evidence of the sequence sharing between taxa thatwould be expected if hybridization were occurring orif species were too young to be discriminated. Instead,there is clean separation of species with barcode cohesionfor conspecifics even when they derive from disparatesites in eastern North America.

There is nothing exceptional about the barcode re-sults for Acronicta—studies on soil invertebrates fromthe arctic and on lepidopterans from the tropics showsimilar success in species resolution (Hogg and Hebert,2004; Janzen et al., 2005). This performance extendsinto marine settings: a barcode study which exam-ined more than 200 morphologically defined species ofAustralian fishes generated 100% success in their dis-crimination (Ward et al., 2005). Sensitivity tests across10-fold gradients in rates of mitochondrial evolution re-vealed high success in species identification from in-sect groups with both the lowest and highest rates ofevolution (Ball et al, 2005; Smith et al., 2005). Shifts innucleotide composition of the mitochondrial genomesimilarly fail to impact the resolution of DNA barcod-ing, as evidenced by success in groups, such as birds,with high G-fC composition and others, such as spiders,with extreme A+T bias (Hebert et al, 2004b; Barrett andHebert, 2005).

If these past studies are reflective of the general per-formance of barcodes across the animal kingdom, a com-prehensive coxl-based system will deliver taxonomic

resolution in excess of 99.99% when viewed from akingdom-level perspective. To grasp this, one need onlyimagine each of the 10,000 pits in Figure 2 as a reposi-tory for barcode data from a single species. Presumingthat there are 10 million animal species, the barcode li-brary for this kingdom could be represented by just 1000of these pages. The global avifauna, which consists ofabout 10,000 bird species, will occup}' only one of thesepages. Barcode records for the fishes of the world willoccupy three pages, while beetles will fill several hun-dred pages. Once all 10 million pits have been filled withbarcode data, the analysis of any new barcode sequencewill provide immediate transport to tlie correct page outof 1000, delivering 99.9% resolution. In fact, based ondata for North American birds, the barcode sequencewill provide perfect resolution by leading to an individ-ual species pit on the single bird page in 96% of cases.In the remaining cases, the newly gathered barcode willmatch sequences in two or three adjacent pits. In sum-mary, a short barcode will collapse the uncertainty inspecies identity from any one of 10 million species downto a single species in most cases, and to a small subset ofclosely allied species in other instances.

Funding is now in place to ensure that that the DNAbarcode library for animals will grow by at least 500,000records over the next 5 years, providing coverage forsome 50,000 species. Although this will be far from acomplete registry of species, it will allow DNA barcodesto function as an effective identification tool for thosetaxonomic groups with comprehensi ' e barcode records.For example, as barcode coverage for fishes, birds, andpest insects approaches completion, this will provideopen access to the identification of these species regard-less of life stage or condition. As this core of speciesrecords is joined by barcodes fron- other animals, aglobal identification system for this kingdom of life willrise.

Although we believe that the generality of barcodinghas now been demonstrated for the animal kingdom,there remains a need to both establish and evaluate bar-code protocols for the other kingdoms of life. The coreprinciples of barcode analysis (minin'alization and stan-dardization of sequence targets) are surely applicable tothese organisms, but the selection of gene regions andtests of their effectiveness remain in progress althoughearly results on plants (Kress et al., 2005) and protists(G. W. Saunders, personal communication) provide causefor optimism. Aside from its success in separating knownspecies, DNA barcoding will be a powerful aid in re-solving other taxonomic issues. Overlooked species havebeen regularly revealed, even in well-studied groupssuch as North American birds (Hebert et al, 2004b), but-terflies (Hebert et al, 2004a), and silk moths (Janzen et al.,2005). Its role in associating life stages (Beskansky et al.,2003) and genders (Janzen et al, 2005), and in clarify-ing synonymies, will also be of assistance in many othertaxonomic investigations.

The activation of any major science program demandsnot just a strong scientific rationale, but a demonstra-tion of societal relevance. DNA barcoding exhibits such

858 SYSTEMATIC BIOLOGY VOL.54

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Declined ilramiNcellv m law tsth esntury. bo racov-n 20m Currani populations aatta

FIGURE 2. A graphic representation of a matrix that would store barcode data on 10,000 species (e.g., nearly all known bird species). Onethousand such pages would house barcode records for 10 million species. In addition to providing a species epithet, such a system would act asa portal to all other collected information on a given species by linking into other comprehensive biological databases.

relevance by providing new access to identifications invaried contexts. Efforts to conser\'e life are currently con-strained by the need for an identification systeni, and webelieve that this need can only be met by DNA barcod-ing (see also Smith et al., 2005). Tlie ability of barcodes toidentify fragments of life has applications ranging fromthe resolution of cases of species substitution in the mar-ketplace (Marko et al., 2004) to the protection of food se-curity through, for example, screening animal feedstuffsfor ruminant waste. More generally, the ability of DNAbarcoding to deliver identifications quickly and cheaplyhas the potential to revolutionize humanity's relation-ship with biological diversity (Janzen, 2004).

If DNA barcoding proceeds on a large scale, it willgenerate important by-products for the scientific com-munity. All DNA extracts produced during the barcode

analysis of vouchered specimens will be stored, allowingfuture efforts to examine patterns of sequence diversityin other gene regions, and the collection programs in-stigated by DNA barcoding will expand the specimensavailable for morphological analysis. The barcode initia-tive will also create a Web-based system delivering notjust automated identifications, but also providing a por-tal to biological information for all species included inthe registry. Although DNA barcoding will not create the"encyclopedia of life," it will generate its index and tableof contents.

Because of both positive scientific results and its rec-ognized societal benefits, there is growing enthusiasmfor a large-scale DNA barcoding initiative. Two meet-ings at Cold Spring Harbor during 2003 clarified plansfor action (Stoeckle, 2003), and more meetings have

2005 POINTS OF VIEW 859

followed. The most recent, which was hosted by theNatural History Museum in London, attracted morethan 230 researchers (Marshall, 2005). The barcode move-ment also has a central organizing force: the Consortiumfor the Barcode of Life (CBOL), hosted by the Smith-sonian Institution in Washington, which was launchedin mid-2004. More than 80 organizations from 25 na-tions, including many prominent museums, have al-ready joined CBOL(www.barcoding.si.edu). The firstglobal barcode campaigns have been activated under itsauspices; they include plans to barcode all 10,000 speciesof birds and all 15,000 marine fishes by 2010. Clearly,work needs to expand beyond individual laboratoriesto tackle major projects such as these, and national bar-code networks are forming to establish specimen supplychains and to oversee core analytical facilities. The firstof these, the Canadian Barcode of Life Network, whichsaw activation in May 2005 (www.bolnet.ca), intends tobarcode at least 10,000 Canadian animal species over thenext 5 years.

We view these signs of growing s3Tiergy among thevarious sectors of the biodiversity community as ex-tremely hopeful. If developed to their full potential, his-tory may view the DNA barcoding enterprise as one thatnot only enhanced access to taxonomic information, butalso strengthened alliances among all those with interestsin the documentation, understanding, and preservationof biodiversity—an exciting prospect indeed.

ACKNOWLEDGEMENTS

We thank Mark Stoeckle and Dan Janzen for valuable revisionarysuggestions to earlier drafts of this article. We are also very grate-ful to the Gordon and Betty Moore Foundation, NSERC, CFi, andOIT for their support of DNA barcoding research at the University ofGuelph.

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First submitted 25 April 2005; reviews returned 9 June 2005;final acceptance 72 Jtily 2005

Associate Editor: Vhicent Savolainen