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NEWS AND COMMENTARY

DNA barcoding plants in biodiversity hot spots...............................................................Progress and outstanding questionsPM Hollingsworth

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Heredity (2008) 101, 12; doi:10.1038/hdy.2008.16; published online 9 April 2008

DNA barcoding in animals is nowroutinely used for organismalidentification and has contributed

toward the discovery of new species.Although the approach has receivedstrong criticisms, a number of studieshave illustrated how sequencing just asingle organelle region (mitochondrialcytochrome oxidase 1, CO1) can serve asa powerful high-throughput tool for

biodiversity research (Hajibabaei et al.,2007). In plants, progress has beenhampered by slow substitution rates inmitochondrial DNA, and the search foran analogous region to animal CO1 hasfocused on chloroplast DNA. A numberof different chloroplast regions have

been proposed, but a consensus remainselusive (Pennisi, 2007; Ledford, 2008).The plant barcoding regions suggestedat the Second International Barcode ofLife Conference in Taipei (September2007) are summarised in Table 1.

A recent paper published by Lahayeet al. (2008) reports on the application of

DNA barcoding in plants and tacklestwo substantive issues. Firstly, theauthors provide new data to contributetoward this ongoing debate regardingthe most appropriate DNA regions for

barcoding in plants and secondly, theyapply one candidate barcoding regionto the flora of a global biodiversityhot spot.

To assess the comparative perfor-mance of different barcoding regions,71 specimens of 48 Costa Rican orchidspecies and 101 samples of 38 speciesfrom the Kruger National Park in SouthAfrica were examined with eight candi-

date barcoding regions. These includedrbcL, rpoC1, rpoB, trnH-psbA and matKthat feature in the preferred barcodesolutions of different research groupsdescribed above, with accD, nhdJand ycf5 that have been considered

previously as potential loci by theconsortium led by RBG Kew. TheatpF-H and psbK-I spacers very recentlyproposed by Kim et al. (Table 1) werenot included.

Of the regions Lahaye et al. (2008)analyzed, matK was their preferredoption. Their results support observa-tions from other groups that matK hasa rapid substitution rate compared to

other chloroplast coding regions (Chaseet al., 2007). Critically, however, theauthors report high levels of amplifica-tion success (100%) from a single primerpair, a result, which, to date, hasnot been obtained by other groups.This gene has a reputation for beingone of the more difficult chloroplastregions to routinely amplify and se-quence across divergent lineages, so thesuccess rate reported by Lahaye et al. isnotable. They used primers described

by Cuenoud et al. (2002) targeting aregion up to approximately 900bp inlength in the middle of the gene

(forward: 5

0

-CGATCTATTCATTCAATATTTC-30; reverse: 50-TCTAGCACACGAAAGTCGAAGT-30). Further testingof these primers on a broader sample setfor barcoding applications is nowneeded to assess whether the successrate of 100% is generalizable beyond thetaxa examined here.

The other region favored by Lahayeet al. was trnH-psbA. This is one ofthe most rapidly evolving chloroplastspacers, and the study by Kress andErickson (2007) also highlighted thepotential power of this intergenic spaceras a barcoding locus. Direct compara-

tive evaluation of these regions, withthe full set of other recently proposedcandidate barcoding loci in Table 1, isnow a priority to enable a standard

barcoding solution to be agreed inplants.

Resolving power of plantDNA barcodes

Using matK alone, or in combination

with trnH-psbA, Lahaye et al. reportedthat over 90% of species could bediscriminated (multiple individuals ofspecies resolved as monophyletic). Thisfigure is based on the 44 species fromthe Kruger National Park and CostaRica from which multiple accessionswere sampled. This is an encouraginglyhigh success rate for plant barcodingusing only organelle genes. However,this involves many comparisons inwhich just a single species was sampledfrom a genus or family, and wheremultiple congeneric species have beensampled, it does not necessarily include

the closest sister species. Although theability to distinguish among species in arestricted sample set has many potentialapplications, a desirable trait for DNA

barcoding is to be able to distinguishamong the different species within agenus. This is the performance measurethat perhaps most will be interested in.Re-examination of the data shows atotal of 17 genera from which multiplespecies (2 or 3) have been sampled.On the basis of the Unweighted PairGroup Method with Arithmetic mean(UPGMA) analysis of matK (Lahayeet al., Supplementary Figure S1), species

level discrimination was achieved in10/17 genera (reciprocal monophyly ofspecies where multiple conspecific in-dividuals were sampled, and non-zerolength branches between samples where

just single individuals represent spe-cies). In the seven other genera, therewere examples of non-monophyleticspecies topologies or identical se-quences shared between species.

The discriminatory abilities of matKwas followed up in the second part ofthe Lahaye et al. paper, which describesthe first published application of plantDNA barcoding for inventory work in a

floristic hot spot. The authors generatedand compiled matK sequences froman impressive data set of 1566 speci-mens representing 1084 orchid speciesfrom Mesoamerica. The sequences wereused to see if a barcode gap is presentin plants (a discontinuity betweenintra- and interspecific variation). Therewas, as expected, greater interspecificthan intraspecific sequence divergence.However, there were more than 500interspecific comparisons with zero differ-ences between species, and no cleardiscontinuity between intra- and interspe-cific divergences. The UPGMA tree ofthese data (Lahaye et al., Supplementary

Table 1 Plant barcoding regions proposed at the Second International Barcode of LifeConference

Research group Proposed plant barcode

Chase et al. (consortium led byRoyal Botanic Garden Kew, UK)

rpoC1+rpoB+matKorrpoC1+matK+trnH-psbA

Kim et al. (Korea University, Korea) matK+atpF-H+psbK-Ior matK+atpF-H+trnH-psbAKress and Erickson (Smithsonian

Institute, USA)

rbcL+trnH-psbA

Heredity (2008) 101, 12& 2008 Nature Publishing Group All rights reserved 0018-067X/08 $30.00

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Figure S2) also illustrates the highfrequency with which species cannot bedistinguished with matK, especially whenmultiple congeneric species are consid-ered. Species-level discrimination in thislarger data set is much lower than the 90%reported for the smaller data set described

above. The UPGMA tree is replete withexamples of identical sequences shared

between species (and genera), and a lackof reciprocal monophyly for species withmultiple accessions sampled.

Of course, in undertaking biodiver-sity inventory work in species-rich hotspots, there is no perfect taxonomy toserve as a baseline for performancemeasures. Lack of coincidence of matKsequence clusters with species bound-aries may reflect problems with DNA

barcoding in the group in question(either recent divergence or hybridiza-tion as biological causes, or contamina-

tion when carrying out large-scalemolecular surveys). However, it mayalso, in part, be attributable to thecurrent taxonomy needing updating.Lahaye et al. noted high levels ofdivergence among accessions of oneparticular orchid species. The divergentsequences coincided with morphologi-cal and geographical differences andrepresent an example of barcodingapproaches identifying potential crypticspecies warranting further taxonomicinvestigation.

Although both the final choice of a barcoding region and the percentageof plant species that will bedistinguishable by organelle barcodingremain to be determined, this studyprovides useful data toward thesetopics. The authors also report how

even genus-level resolution from DNA barcoding can have practical applica-tions. MatK sequences were ableto distinguish samples of the orchidgenus Phragmipedium, from 1500samples of other Mesoamerican orchids.All Phragmipedium species are listedon Convention on InternationalTrade in Endangered Species (CITES)Appendix 1 (trade completely forbid-den). Being able to distinguishthese, from orchid species forwhich trade is permissible with permits,provides a simple practical exampleof how the methodology could be

used by customs agencies to assessthe legitimacy of samples withoutneeding specialist knowledge of orchid

biology.PM Hollingsworth is at the Royal Botanic Garden,20 Inverleith Row, Edinburgh EH3 5LR, UK.

e-mail: P.Hollingsworth@rbge.org.uk

Chase MW, Cowan RS, Hollingsworth PM,van den Berg C, Madrinan S, Petersen G et al.(2007). A proposal for a standardised protocolto barcode all land plants. Taxon 56: 295299.

Cuenoud P, Savolainen V, Chatrou LW, Powell M,Grayer RJ, Chase MW (2002). Molecular

phylogenetics of Caryophyllales based onnuclear 18S rDNA and plastid rbcL, atpB,and matK DNA sequences. Am J Bot 89:132144.

Hajibabaei M, Singer GAC, Hebert PDN,Hickey DA (2007). DNA barcoding: how itcomplements taxonomy, molecular phyloge-netics and population genetics. Trends Gen et 23:

167172.Kress WJ, Erickson DL (2007). A two-locusglobal DNA barcode for land plants: thecoding rbcL gene complements the non-coding trnH-psbA spacer region. PLoS ONE2: e508.

Lahaye R, van der Bank M, Bogarin D, Warner J,Pupulin F, Gigot G et al. (2008). DNA barcod-ing the floras of biodiversity hotspots.Proc Natl Acad Sci USA 105: 29232928.http://www.pnas.org/cgi/content/abstract/105/8/2923?etoc.

Ledford H (2008). Botanical identities: DNA barcoding for plants comes a step closer.Nature 451: 616.

Pennisi E (2007). Taxonomy. Wanted: a barcode forplants. Science 318: 190191.

Editors suggested readingDasmahapatra KK, Mallet J (2006). DNA bar-

codes: recent successes and future prospects.Heredity 97: 254255.

Shiu S-H, Borevitz JO (2008). The next generationof microarray research: applications in evolu-tionary and ecological genomics. Heredity 100:141149.

Meirmans PG, Den Nijs HJCM, Van Tienderen PH(2006). Male sterility in triploid dandelions:asexual females vs asexual hermaphrodites.

Heredity 96: 4552.McGrath S, Hodkinson TR, Barth S (2007).

Extremely high cytoplasmic diversity in nat-ural and breeding populations of Lolium(Poaceae). Heredity 99: 531544.

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