Restricted access
Research articles
Research articles
Complete DNA barcode reference library for a country's butterfly fauna reveals high performance for temperate Europe
Published:11 August 2010https://doi.org/10.1098/rspb.2010.1089
Abstract
DNA barcoding aims to accelerate species identification and discovery, but performance tests have shown marked differences in identification success. As a consequence, there remains a great need for comprehensive studies which objectively test the method in groups with a solid taxonomic framework. This study focuses on the 180 species of butterflies in Romania, accounting for about one third of the European butterfly fauna. This country includes five eco-regions, the highest of any in the European Union, and is a good representative for temperate areas. Morphology and DNA barcodes of more than 1300 specimens were carefully studied and compared. Our results indicate that 90 per cent of the species form barcode clusters allowing their reliable identification. The remaining cases involve nine closely related species pairs, some whose taxonomic status is controversial or that hybridize regularly. Interestingly, DNA barcoding was found to be the most effective identification tool, outperforming external morphology, and being slightly better than male genitalia. Romania is now the first country to have a comprehensive DNA barcode reference database for butterflies. Similar barcoding efforts based on comprehensive sampling of specific geographical regions can act as functional modules that will foster the early application of DNA barcoding while a global system is under development.
References
- 1
Brown W. M., George M.& Wilson A. C. . 1979 Rapid evolution of animal mitochondrial DNA. Proc. Natl Acad. Sci. USA 76, 1967–1971.doi:10.1073/pnas.76.4.1967 (doi:10.1073/pnas.76.4.1967). Crossref, PubMed, ISI, Google Scholar - 2
Hebert P. D. N., Cywinska A., Ball S. L.& deWaard J. R. . 2003 Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B 270, 313–321.doi:10.1098/rspb.2002.2218 (doi:10.1098/rspb.2002.2218). Link, ISI, Google Scholar - 3
Hebert P. D. N., Ratnasingham S.& deWaard J. R. . 2003 Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B (Suppl.) 270, S96–S99.doi:10.1098/rsbl.2003.0025 (doi:10.1098/rsbl.2003.0025). Link, ISI, Google Scholar - 4
Lahaye R., 2008 DNA barcoding the floras of biodiversity hotspots. Proc. Natl Acad. Sci. USA 105, 2923–2928.doi:10.1073/pnas.0709936105 (doi:10.1073/pnas.0709936105). Crossref, PubMed, ISI, Google Scholar - 5
Chase M. W.& Fay M. F. . 2009 Barcoding of plants and fungi. Science 325, 682–683.doi:10.1126/science.1176906 (doi:10.1126/science.1176906). Crossref, PubMed, ISI, Google Scholar - 6
Summerbell R. C., 2005 Microcoding: the second step in DNA barcoding. Phil. Trans. R. Soc. B 360, 1897–1903.doi:10.1098/rstb.2005.1721 (doi:10.1098/rstb.2005.1721). Link, ISI, Google Scholar - 7
Saunders G. W. . 2005 Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications. Phil. Trans. R. Soc. B 360, 1879–1888.doi:10.1098/rstb.2005.1719 (doi:10.1098/rstb.2005.1719). Link, ISI, Google Scholar - 8
Scicluna S. M., Tawari B.& Clark C. G. . 2006 DNA barcoding of Blastocystis. Protist 157, 77–85.doi:10.1016/j.protis.2005.12.001 (doi:10.1016/j.protis.2005.12.001). Crossref, PubMed, ISI, Google Scholar - 9
Sogin M. L., Morrison H. G., Huber J. A., Welch D. M., Huse S. M., Neal P. R., Arrieta J. M.& Herndl G. J. . 2006 Microbial diversity in the deep sea and the underexplored ‘rare biosphere.’. Proc. Natl Acad. Sci. USA 103, 12 115–12 120.doi:10.1073/pnas.0605127103 (doi:10.1073/pnas.0605127103). Crossref, ISI, Google Scholar - 10
Ebach M. C.& Holdrege C. . 2005 DNA barcoding is no substitute for taxonomy. Nature 434, 697.doi:10.1038/434697b (doi:10.1038/434697b). Crossref, PubMed, ISI, Google Scholar - 11
Will K. W., Mishler B. D.& Wheeler Q. . 2005 The perils of DNA barcoding and the need for integrative taxonomy. Syst. Biol. 54, 844–851.doi:10.1080/10635150500354878 (doi:10.1080/10635150500354878). Crossref, PubMed, ISI, Google Scholar - 12
Prendini L. . 2005 Comment on ‘Identifying spiders through DNA barcoding.’. Can. J. Zool. 83, 498–504.doi:10.1139/Z05-025 (doi:10.1139/Z05-025). Crossref, ISI, Google Scholar - 13
Cameron S., Rubinoff D.& Will K. . 2006 Who will actually use DNA barcoding and what will it cost? Syst. Biol. 55, 844–847.doi:10.1080/10635150600960079 (doi:10.1080/10635150600960079). Crossref, PubMed, ISI, Google Scholar - 14
Rubinoff D., Cameron S.& Will K. . 2006 A genomic perspective on the shortcomings of mitochondrial DNA for ‘Barcoding’ identification. J. Hered. 97, 581–594.doi:10.1093/jhered/esl036 (doi:10.1093/jhered/esl036). Crossref, PubMed, ISI, Google Scholar - 15
Hebert P. D. N., Stoeckle M. Y., Zemlak T. S.& Francis C. M. . 2004 Identification of birds through DNA barcodes. PloS Biol. 2, e312.doi:10.1371/journal.pbio.0020312 (doi:10.1371/journal.pbio.0020312). Crossref, PubMed, ISI, Google Scholar - 16
Clare E. L., Lim B. K., Engstrom M. D., Eger J. L.& Hebert P. D. N. . 2007 DNA barcoding of Neotropical bats: species identification and discovery within Guyana. Mol. Ecol. Notes 7, 184–190.doi:10.1111/j.1471-8286.2006.01657.x (doi:10.1111/j.1471-8286.2006.01657.x). Crossref, Google Scholar - 17
Borisenko A. V., Lim B. K., Ivanova N. V., Hanner R. H.& Hebert P. N. D. . 2008 DNA barcoding in surveys of small mammal communities: a field study in Suriname. Mol. Ecol. Resour. 8, 471–479.doi:10.1111/j.1471-8286.2007.01998.x (doi:10.1111/j.1471-8286.2007.01998.x). Crossref, PubMed, ISI, Google Scholar - 18
Hebert P. D. N., Penton E. H., Burns J. M., Janzen D. H.& Hallwachs W. . 2004 Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc. Natl Acad. Sci. USA 101, 14 812–14 817.doi:10.1073/pnas.0406166101 (doi:10.1073/pnas.0406166101). Crossref, ISI, Google Scholar - 19
Janzen D. H., Hajibabaei M., Burns J. M., Hallwachs W., Remigio E.& Hebert P. D. N. . 2005 Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding. Phil. Trans. R. Soc. B 360, 1835–1845.doi:10.1098/rstb.2005.1715 (doi:10.1098/rstb.2005.1715). Link, ISI, Google Scholar - 20
Smith A. M., Woodley N. E., Janzen D. H., Hallwachs W.& Hebert P. D. N. . 2006 DNA barcodes reveal cryptic host-specificity within the presumed polyphagous members of a genus of parasitoid flies (Diptera: Tachinidae). Proc. Natl Acad. Sci. USA 103, 3657–3662.doi:10.1073/pnas.0511318103 (doi:10.1073/pnas.0511318103). Crossref, PubMed, ISI, Google Scholar - 21
Smith A. M., Rodriguez J. J., Whitfield J. B., Deans A. R., Janzen D. H., Hallwachs W.& Hebert P. D. N. . 2008 Extreme diversity of tropical parasitoid wasps exposed by iterative integration of natural history, DNA barcoding, morphology, and collections. Proc. Natl Acad. Sci. USA 105, 12 359–12 364.doi:10.1073/pnas.0805319105 (doi:10.1073/pnas.0805319105). Crossref, ISI, Google Scholar - 22
Hajibabaei M., Janzen D. H., Burns J. M., Hallwachs W.& Hebert P. D. N. . 2006 DNA barcodes distinguish species of tropical Lepidoptera. Proc. Natl Acad. Sci. USA 103, 968–971.doi:10.1073/pnas.0510466103 (doi:10.1073/pnas.0510466103). Crossref, PubMed, ISI, Google Scholar - 23
Burns J. M., Janzen D. H., Hajibabaei M., Hallwachs W.& Hebert P. D. N. . 2008 DNA barcodes and cryptic species of skipper butterflies in the genus Perichares in Area de Conservación Guanacaste, Costa Rica. Proc. Natl Acad. Sci. USA 105, 6350–6355.doi:10.1073/pnas.0712181105 (doi:10.1073/pnas.0712181105). Crossref, PubMed, ISI, Google Scholar - 24
Lukhtanov V. A., Sourakov A., Zakharov E. V.& Hebert P. D. N. . 2009 DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification. Mol. Ecol. Resour. 9, 1302–1310.doi:10.1111/j.1755-0998.2009.02577.x (doi:10.1111/j.1755-0998.2009.02577.x). Crossref, PubMed, ISI, Google Scholar - 25
Rach J., DeSalle R., Sarkar I. N., Schierwater B.& Hadrys H. . 2008 Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata. Proc. R. Soc. B 275, 237–247.doi:10.1098/rspb.2007.1290 (doi:10.1098/rspb.2007.1290). Link, ISI, Google Scholar - 26
Meyer C. P.& Paulay G. . 2005 DNA barcoding: error rates based on comprehensive sampling. PLoS Biol. 3, e422.doi:10.1371/journal.pbio.0030422.g008 (doi:10.1371/journal.pbio.0030422.g008). Crossref, PubMed, ISI, Google Scholar - 27
Vences M., Thomas M., Bonett R. M.& Vieites D. R. . 2005 Deciphering amphibian diversity through DNA barcoding: chances and challenges. Phil. Trans. R. Soc. B 360, 1859–1868.doi:10.1098/rstb.2005.1717 (doi:10.1098/rstb.2005.1717). Link, ISI, Google Scholar - 28
Wiemers M.& Fiedler K. . 2007 Does the DNA barcoding gap exist?—a case study in blue butterflies (Lepidoptera: Lycaenidae). Front. Zool. 4, 8.doi:10.1186/1742-9994-4-8 (doi:10.1186/1742-9994-4-8). Crossref, PubMed, ISI, Google Scholar - 29
Elias M., Hill R. I., Willmott K. R., Dasmahapatra K. K., Brower A. V. Z., Mallet J.& Jiggins C. D. . 2007 Limited performance of DNA barcoding in a diverse community of tropical butterflies. Proc. R. Soc. B 274, 2881–2889.doi:10.1098/rspb.2007.1035 (doi:10.1098/rspb.2007.1035). Link, ISI, Google Scholar - 30
Whitworth T. L., Dawson R. D., Magalon H.& Baudry E. . 2007 DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae). Proc. R. Soc. B 274, 1731–1739.doi:10.1098/rspb.2007.0062 (doi:10.1098/rspb.2007.0062). Link, ISI, Google Scholar - 31
Shearer T. L.& Coffroth M. A. . 2008 Barcoding corals: limited by interspecific divergence, not intraspecific variation. Mol. Ecol. Resour. 8, 247–255.doi:10.1111/j.1471-8286.2007.01996.x (doi:10.1111/j.1471-8286.2007.01996.x). Crossref, PubMed, ISI, Google Scholar - 32
Spooner D. M. . 2009 DNA barcoding will frequently fail in complicated groups: an example in wild potatoes. Am. J. Botany 96, 1177–1189.doi:10.3732/ajb.0800246 (doi:10.3732/ajb.0800246). Crossref, PubMed, ISI, Google Scholar - 33
Dasmahapatra K. K., Elias M., Hill R. I., Hoffman J. I.& Mallet J. . 2009 Mitochondrial DNA barcoding detects some species that are real, and some that are not. Mol. Ecol. Resour. 10, 264–273.doi:10.1111/j.1755-0998.2009.02763.x (doi:10.1111/j.1755-0998.2009.02763.x). Crossref, PubMed, ISI, Google Scholar - 34
Barrett R. D. H.& Hebert P. D. N. . 2005 Identifying spiders through DNA barcodes. Can. J. Zool. 83, 481–491.doi:10.1139/Z05-024 (doi:10.1139/Z05-024). Crossref, ISI, Google Scholar - 35
Dincă V., Székely L., Kovács S., Kovács Z.& Vila R. . 2008 Pyrgus andromedae (Wallengren, 1853) (Hesperiidae) in the Romanian Carpathians. Nota lepid 31, 263–272. Google Scholar - 36
Dincă V., Cuvelier S., Zakharov E. V., Hebert P. D. N.& Vila R. In press. Biogeography, ecology and conservation of Erebia oeme in the Carpathians. Ann. Soc. Entomol. Fr.. ISI, Google Scholar - 37
Kudrna O. . 1977 A revision of the genus Hipparchia Fabricius. Faringdon, UK: E. W. Classey Ltd. Google Scholar - 38
Wakeham-Dawson A., Jakšić P., Holloway J. D.& Dennis R. . 2004 Multivariate analysis of male genital structures in the Hipparchia semele-muelleri-delattini complex (Nymphalidae, Satyrinae) from the Balkans: how many taxa? Nota lepid 27, 103–124. Google Scholar - 39
Alberti B. . 1943 Zur Frage der Hybridisation zwischen Colias erate Esp., hyale L. und edusa F. und ueber Umgrenzung der 3 Arten. Mitteilungen der Münchner Entomologischen Gesellschaft 33, 606–625. Google Scholar - 40
Porter A.& Geiger H. J. . 1995 Limitations to the inference of gene flow at regional geographic scales: an example from the Pieris napi group (Lepidoptera: Pieridae) in Europe. Biol. J. Linn. Soc. 54, 329–348. ISI, Google Scholar - 41
Descimon H.& Mallet J. . 2009 Bad species. Ecology of butterflies in Europe (eds, Settele J., Konvicka M., Shreeve T., Dennis R.& Van Dyck H. ), pp. 219–249. Cambridge, UK: Cambridge University Press. Google Scholar - 42
DeSalle R., Egan M. G.& Siddall M. . 2006 The unholy trinity: taxonomy, species delimitation and DNA barcoding. Phil. Trans. R. Soc. B 360, 1905–1916.doi:10.1098/rstb.2005.1722 (doi:10.1098/rstb.2005.1722). Link, ISI, Google Scholar - 43
Schlick-Steiner B. C., Seifert B., Stauffer C., Christian E., Crozier R. H.& Steiner F. M. . 2007 Without morphology, cryptic species stay in taxonomic crypsis following discovery. Trends Ecol. Evol. 22, 391–392. Crossref, PubMed, ISI, Google Scholar - 44
Mallet J. . 1995 A species definition for the modern synthesis. Trends Ecol. Evol. 10, 294–299.doi:10.1016/0169-5347(95)90031-4 (doi:10.1016/0169-5347(95)90031-4). Crossref, PubMed, ISI, Google Scholar - 45
Thomas J. A., Elmes G. W., Wardlaw J. C.& Woyciechowski M. . 1989 Host specificity among Maculinea butterflies in Myrmica ant nests. Oecologia 79, 452–457.doi:10.1007/BF00378660 (doi:10.1007/BF00378660). Crossref, PubMed, ISI, Google Scholar - 46
Als T. D., Vila R., Kandul N. P., Nash D. R., Yen S.-H., Hsu Y.-F., Mignault A. A., Boomsma J. J.& Pierce N. E. . 2004 The evolution of alternative parasitic life histories in large blue butterflies. Nature 432, 386–390.doi:10.1038/nature03020 (doi:10.1038/nature03020). Crossref, PubMed, ISI, Google Scholar - 47
Tartally A., Rákosy L., Vizauer T.-C., Goia M.& Varga Z. . 2008 Maculinea nausithous exploits Myrmica scabrinodis in Transylvania: unusual host ant species of a Myrmecophilous butterfly in an isolated region (Lepidoptera: Lycaenidae; Hymenoptera: Formicidae). Sociobiology 51, 373–380. ISI, Google Scholar - 48
- 49
Avise J. C. . 2000 Phylogeography: the history and formation of species. Cambridge, MA: Harvard University Press. Crossref, Google Scholar - 50
Funk D. J.& Omland K. E. . 2003 Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annu. Rev. Ecol. Syst. 34, 397–423. Crossref, Google Scholar - 51
Zakharov E. V., Lobo N. F., Nowak C.& Hellmann J. J. . 2009 Introgression as a likely cause of mtDNA paraphyly in two allopatric skippers (Lepidoptera: Hesperiidae). Heredity 102, 590–599.doi:10.1038/hdy.2009.26 (doi:10.1038/hdy.2009.26). Crossref, PubMed, ISI, Google Scholar - 52
Barber P.& Boyce S. L. . 2006 Estimating diversity of Indo-Pacific coral reef stomatopods through DNA barcoding of stomatopod larvae. Proc. R. Soc. B 273, 2053–2061.doi:10.1098/rspb.2006.3540 (doi:10.1098/rspb.2006.3540). Link, ISI, Google Scholar - 53
Karen W. E., Barnes D. K. A., Clark M. S.& Bowden D. A. . 2006 DNA barcoding: a molecular tool to identify Antarctic marine larvae. Deep Sea Res. Part II. Topical Studies in Oceanography 53, 1053–1060.doi:10.1016/j.dsr2.2006.02.013 (doi:10.1016/j.dsr2.2006.02.013). Crossref, ISI, Google Scholar - 54
Dincă V.& Vila R. . 2008 Improving the knowledge on Romanian Rhopalocera, including the rediscovery of Polyommatus amandus (Schneider, 1792) (Lycaenidae) and an application of DNA-based identification. Nota lepid 31, 3–23. Google Scholar - 55
Bickford D., Lohman D. J., Sodhi N. S., Ng P. K. L., Meier R., Winker K., Ingram K. K.& Das I. . 2006 Cryptic species as a window on diversity and conservation. Trends Ecol. Evol. 22, 148–155.doi:10.1016/j.tree.2006.11.004 (doi:10.1016/j.tree.2006.11.004). Crossref, PubMed, ISI, Google Scholar - 56
Pfenninger M.& Schwenk K. . 2007 Cryptic animal species are homogeneously distributed among taxa and biogeographical regions. BMC Evol. Biol. 7, 121.doi:10.1186/1471-2148-7-121 (doi:10.1186/1471-2148-7-121). Crossref, PubMed, ISI, Google Scholar - 57
Whinnett A., Zimmermann M., Willmott K. R., Herrera N., Mallarino R., Simpson F., Joron M., Lamas G.& Mallet J. . 2005 Strikingly variable divergence times inferred across an Amazonian butterfly ‘suture zone.’. Proc. R. Soc. B 272, 2525–2533.doi:10.1098/rspb.2005.3247 (doi:10.1098/rspb.2005.3247). Link, ISI, Google Scholar - 58
Hajibabaei M., Singer G. A. C., Hebert P. D. N.& Hickey D. A. . 2007 DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends Genet. 23, 167–172.doi:10.1016/j.tig.2007.02.001 (doi:10.1016/j.tig.2007.02.001). Crossref, PubMed, ISI, Google Scholar
