DNA Barcoding: an Exercise in Futility Or Utility?

SCIENTIFIC CORRESPONDENCE

DNA barcoding: An exercise in futility or utility?

Taxonomy, the science of naming and an ambitious programme of developing tid nuclear intergenic spacer trnH–psbA classifying organisms, is the foundation DNA barcodes for all species on the have been recommended as possible can- of all biology. Unfortunately, over the past planet, including those that are yet to be didate segments13–15. few decades, taxonomy is being comple- described. CBOL foresees many applica- DNA barcoding has invited several tely overshadowed by seemingly specta- tions of this technique, from fundamental criticisms. These range from skepticism cular and glamorous branches of biology. research on biodiversity to enforcement on the technique as an effective taxono- However, in the last few years some of of food laws, quarantine and phytosani- mic tool, to a more moderate view that it these subjects such as molecular biology tary laws and protection of wildlife11. could only complement the existing have rejuvenated taxonomy as a fashion- That the recent advances in sequencing approaches of conventional taxonomy. able science once again. The advent of technology have become even more Mallet and Willmott16 have argued that powerful DNA-based marker systems for rapid, accurate and inexpensive, means DNA barcodes based on a few specific identifying species has brought back the that the barcoding endeavour appears to genes may fail to distinguish closely charm of 18th century biology and prom- be both plausible and worthwhile. related species because of the persistence ises a less painstaking path for identifica- DNA barcoding, in principle, is a di- of ancestral polymorphism. It is also tion and discovery of new species on the agnostic technique that uses short DNA feared that DNA barcoding exercises ‘PCR-desk’ by ‘lay-taxonomists’ who sequence(s) for identification of species. may supplant genuine taxonomic projects need not necessarily be ‘naturalists’. The basic rationale of using the short and merely end up in spewing-out alter- DNA barcoding, a tool that obtains species- DNA sequence (universal molecular nate sets of data, without adding mean- specific DNA signature, is based on the yardstick), is that it allows to discrimi- ingful information on the taxa17. Some simple premise that sequence diversity nate among species of a taxon under the argue that DNA barcoding is gross over- within small stretches of the organism’s assumption that the sequences chosen simplification of the science of taxonomy genome can provide a ‘biological bar- have relatively lower ‘within-taxon’ and that DNA barcodes are no substitutes code’ to enable identification of any variation than that ‘between-taxa’. It in- to detailed understanding (morphologi- organism at the species level1,2. As a re- volves extraction of genomic DNA from cal, physiological and behavioural attri- sult, scientists are hoping that DNA bar- tissue samples collected from an individual butes) of taxa, practised in conventional coding will provide a ‘universal key’ that organism and using it for targetted ampli- taxonomy. Insisting that it as a manda- will allow identification of a species by fication of one or several regions (short- tory step in taxonomic studies has been running unknown DNA sequences through listed based on their information content) opposed on the grounds that this may a DNA barcode database. by polymerase chain reaction (PCR) and hinder the already slow process of de- The use of DNA barcoding is rela- sequencing the amplified products. The scribing new taxa. tively new (with the first publication resulting sequences are used as ‘barcodes’ In summary, while the above criti- appearing in 2003)1,3,4. Yet in this short for tagging the species. cisms may be valid among themselves, it span many investigators have reported The use of the technique can be traced has been grudgingly accepted that bar- robustness of this technique through a to the work of Carl Woese, who first codes or barcode-like techniques might flurry of papers. It is reported that with demonstrated the utility of rRNA genes after all be useful in specific circum- the use of a short sequence (~600 bp) of in inferring phylogenetic relationships stances and need not be viewed as a the cytochrome oxidase-I (COI) gene, the among microorganisms12. Currently this panacea for all taxonomic hurdles. The success rate of identification down to method is being proposed to be used for evidence so far however weighs in species level can be a remarkable – 98– non-microbes too, with appropriate genes. favour of DNA barcoding as a taxonomic 100% in many organisms, including For example, DNA barcodes based on a tool and the strength of the technique to birds5, fishes6 and butterflies7. Using this 5′, 648 bp fragment of the mitochondrial reveal cryptic species should make it a ‘barcode’, several cryptic species are COI gene, developed for a skipper but- valuable tool wherever conventional taxo- possible to be described within what had terfly species, Astraptes fulgerator, could nomy is found wanting18. It could also be previously been thought to be a single be used to differentiate a complex of at useful in analysing museum specimens species by conventional taxonomy8,9. least ten related species8. The effective- that cannot be easily subjected to con- These exciting developments have fu- ness of DNA barcodes based on COI was ventional taxonomic treatments and taxo- elled speculation that species could be also demonstrated in distinguishing 260 nomy of groups for which regular identified even without the expertise of species of North American birds5. More expertise is not available. conventional taxonomy10. In addition to recently, COI was used7 to discriminate Against this background, it is obviously providing rapid and accurate identification 521 species of Lepidoptera with a resolu- important to ask: should India commit of species, this technology also promises tion of 97.9%. While COI has generally itself to the barcoding of its organisms to uncover the phylogenetic affiliations been found and also accepted as the and if yes, to what purpose? Responses among different taxa. standard genomic region for barcoding to both the questions could be divided, This initial success in DNA barcoding animal species, for plants various because the concept of barcoding has led to the formation of the Consortium regions, including the chloroplast rbcL sown distrust between taxonomists and for the Barcode of Life (CBOL, http:// region, nuclear ITS (Internal Transcribed molecular systematists. In the Indian barcoding.si.edu). The Consortium has Spacer) region of rRNA genes and plas- context, the barcoding exercise should

CURRENT SCIENCE, VOL. 92, NO. 9, 10 MAY 2007 1213 SCIENTIFIC CORRESPONDENCE perhaps be weighed from at least two namely the Vector Control Research Further, we have also shown that a group important angles, namely (a) meeting the Centre, Pondicherry and St. Peter’s Col- of butterflies that are otherwise morpho- taxonomic challenges and providing a lege, Mumbai have been listed as mem- logically similar, are clearly differenti- robust identification of species and (b) bers of CBOL (http://barcoding.si.edu/). ated using the barcode (Figure 2). securing intellectual property rights We have recently initiated efforts to bar- In the Indian context, barcoding could (IPRs) for some of the country’s impor- code Indian butterflies in our laboratory, be useful in contributing to what might tant bioresources. especially those that might be economi- be called as ‘remote taxonomy’. Lack of Survey and description of Indian biota cally important, taxonomically intriguing type specimens and also access to large began as a strong programme almost 200 and endemic to the country. Specifically, collections often held elsewhere in inter- years ago. Despite this, we have not yet we have attempted to explore how the national museums has been among the completely described even a single group barcode of a select group of butterflies of most important constraints in practising of taxa, barring perhaps birds and butter- peninsular India places itself against the taxonomy in India. Of late, this con- flies; even mammals are being newly de- global barcodes available (Figure 1). straint is partly being overcome by the scribed. Further among those described, perhaps every group is burdened with taxonomic controversies of diverse kinds. Obviously these challenges range from subtle to serious, and require intervention that can successfully resolve the conflicts of classification. Besides the existing species lists, discovery of newer species especially from lower taxa, continuously demands expert taxonomic treatment. For example, in the recent past three new species of frogs, Philautus nerostagona from Wayanad District (Kerala) and Philautus anili sp. nov. and Philautus dubois sp. nov. from Wayanad and Ko- daikanal (Tamil Nadu) respectively19,20, were reported. Further, in a rather specta- cular discovery, a new family of frog, Nasikabatrachidae from the Western Ghats21 and a primate species Macaca munzala from the forests of Arunachal Pradesh22 were reported from the country. In fact, Aravind et al.23 showed that for a number of taxa, species discoveries are yet to attain an asymptote – a sugges- tion that there might be many more species waiting to be discovered. With three of the megadiversity hotspots in the country, it is not unlikely that we will have more discoveries than we can possi- bly address. Clearly, it would be to the advantage of taxonomy to complement existing taxonomic tools with DNA barcoding and aim at developing a robust identification scheme for specific groups24,25. Such a process can provide useful insights into the subtleties of an Figure 1. Consensus maximum parsimony tree of Indian and global Nymphalids. Species in box are from India. Numbers attached to species name indicate GenBank IDs. Idea malabarica, already identified taxonomic group (for Cupha erymanthis and Parantica aglea were collected from Sringeri, Chikamagalur District, 8 example, see Hebert et al. ). Further, DNA Karnataka, while the rest were collected from Botanical Garden, University of Agricultural Sci- barcoding information can also help in ences, Bangalore. DNA was extracted from thoracic region of each individual using the method 32 developing newer hypotheses relating of Vandewoestijne and Baguette . The extracted DNA was used for amplification of a 648 bp fragment of COI sequence using the primer pair: LepF (5′-ATTCAACCAATCATAAAGA- to the taxonomic position of species and TATTGG-3′) and LepR (5′-TAAACTTCTGGATGTCCAAAAAATCA-3′)8. The amplified thereby even resolve taxonomic quag- product was purified using Eppendorf gel cleanup kit (Perfectprep® Gel Cleanup) and mires. sequenced. Sequences were edited and aligned using CLUSTALW software. Kimura’s two- There have been several independent parameter model33 of base substitution was used to calculate genetic distances; consensus maxi- and concerted efforts to use DNA bar- mum parsimony trees were obtained using MEGA 3.1 software. DNA barcodes were able to clearly distinguish the various species. Butterfly species from different geographical areas coding in taxonomic studies in India. (whose sequences were obtained from GenBank) clustered with their respective members (spe- Currently only two institutions from India, cies/genus) from India, indicating that the DNA barcodes could be used to develop species IDs.

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Figure 2. Consensus maximum parsimony tree of blue tigers. Species in box are from India. Numbers attached to species name indicate the individual IDs. Individuals belonging to each species were collected from Botanical Garden, Uni- versity of Agricultural Sciences, Bangalore; Parantica aglea was collected from Sringeri, Chikamagalur District, Karnataka. DNA extraction, amplification, sequencing and analysis were done using the protocol mentioned in Figure 1. DNA barcodes were able to distinguish morphologically similar blue tigers, which is evident from the distinct cohesive clustering of individuals belonging to each species. As seen from the figure there was a clear differentiation of the blue tigers by the DNA barcode signatures, despite their apparent morphological similarities. Further, analysis based on the sequence data from GenBank (www.ncbi.nlm.nih.gov) for T. septentrionis (AF394182) from Malaysia and Parantica me- lusine (DQ175477) from Papua New Guinea, showed distinct clustering of the latter species with their respective species/genus from India. This result indicates that DNA barcodes across regions can be comparable to generate a larger taxonomic picture.

introduction of cyber-taxonomy or digi- from diluting the efficacy of the drug such as Trichogrammatidae (for their tal-taxonomy26,27. ‘Barcoding taxonomy’ that is eventually going to be extracted biocontrol uses) or orchids that might can further complement the efforts of from the herbal mix, to lowering the have immense commercial value. cyber-taxonomy for specific groups, where value of trade. It is estimated that about In conclusion, while the relative costs DNA technique-based interventions could 33 herbaceous species (depending on the and benefits of DNA barcoding techni- further resolve the group identities. geographical area of collection) can, in que continue to be debated, it is clear DNA barcoding information could theory, be in the mix of dry plant mate- that the tool can be effectively used in help in providing a correct species identi- rial. At our own laboratory, DNA analy- complementing conventional taxonomic fication tool, especially of those in which sis of raw materials collected from the studies and in securing IPRs for impor- biologically important properties or Bangalore market indicated at least four tant taxa. Viewed from this context, it molecules with IPR potential have been different species of Phyllanthus (Deepali will be important for the country to deve- identified. For example, the Phyllanthus et al., unpublished). Keeping in view lop skills and infrastructure to undertake (Euphorbiaceae) group of species is well India’s role in the raw drug trade glob- barcoding of at least some of the important known for its multifarious medicinal use ally, it is imperative that for such spe- taxa, both for conservation and commerce. ranging from antifungal to antiviral. In cific and complex groups, DNA barcodes

recent years, the group has drawn global are developed which can then provide 1. Hebert, P. D. N., Cywinska, A., Ball, attention due to its anti-hepatitis property unambiguous identification of species. S. L. and deWaard, J. R., Proc. R. Soc. 28,29 and the associated IPR claims . In This information would be useful in not London, Ser. B, 2003, 270, 313–321. India, it constitutes one of the most only providing diagnostics for rapid and 2. Marshall, E., Science, 2005, 307, 1037. important component in raw drug trade, easy identification of species in mixtures 3. Hebert, P. D. N., Ratnasingham, S. and wherein dry plant material is sold for in the raw drug trade, but also in drawing deWaard, J. R., Proc. R. Soc. London, export and domestic use. Unfortunately specific regulations to protect the natio- Ser. B (Suppl. 1), 2003, 270, S96–S99. because the Phyllanthus group has been nal markets. 4. Blaxter, M., Nature, 2004, 421, 122–124. historically plagued by taxonomic con- Besides the above-mentioned example, 5. Hebert, P. D. N., Stoeckle, M. Y., Zem- lak, T. S. and Francis, C. M., PLoS Biol., troversies30,31 and also because of the barcoding could potentially be useful in 2004, 2, e312. relative difficulty of identifying the spe- also identifying species in other groups 6. Ward, R. D., Zemlak, T. S., Innes, B. H., cies, it is common to find mixtures of with high potential of staking IPR claims, Last, P. R. and Hebert, P. D. N., Philos. species in the raw drug market. The such as medicinal leeches (for their anti- Trans. R. Soc. London, Ser. B, 2005, consequences of such mixtures can range coagulant property) or parasitoid wasps 360, 1847–1857.

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