DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers
Abstract
DNA barcoding has been successfully implemented in the identification of previously described species, and in the process has revealed several cryptic species. It has been noted that such methods could also greatly assist in the discovery and delineation of undescribed species in poorly studied groups, although to date the feasibility of such an approach has not been examined explicitly. Here, we investigate the possibility of using short mitochondrial and nuclear DNA sequences to delimit putative species in groups lacking an existing taxonomic framework. We focussed on poorly known tropical water beetles (Coleoptera: Dytiscidae, Hydrophilidae) from Madagascar and dung beetles (Scarabaeidae) in the genus Canthon from the Neotropics. Mitochondrial DNA sequence variation proved to be highly structured, with >95% of the observed variation existing between discrete sets of very closely related genotypes. Sequence variation in nuclear 28S rRNA among the same individuals was lower by at least an order of magnitude, but 16 different genotypes were found in water beetles and 12 genotypes in Canthon, differing from each other by a minimum of two base pairs. The distribution of these 28S rRNA genotypes in individuals exactly matched the distribution of mtDNA clusters, suggesting that mtDNA patterns were not misleading because of introgression. Moreover, in a few cases where sequence information was available in GenBank for morphologically defined species of Canthon, these matched some of the DNA-based clusters. These findings demonstrate that clusters of close relatives can be identified readily in the sequence variation obtained in field collected samples, and that these clusters are likely to correspond to either previously described or unknown species. The results suggest that DNA-assisted taxonomy will not require more than a short fragment of mtDNA to provide a largely accurate picture of species boundaries in these groups. Applied on a large scale, this DNA-based approach could greatly improve the rate of species discovery in the large assemblages of insects that remain undescribed.
Footnotes
References
-
Avise J.C& Walker D.E . 1999 Species realities and numbers in sexual vertebrates: perspectives from an asexually transmitted genome. Proc. Natl Acad. Sci. 96, 992–995.doi:10.1073/pnas.96.3.992. . Crossref, PubMed, ISI, Google Scholar -
Belshaw R& Katzourakis A . 2005 BlastAlign: a program that uses blast to align problematic nucleotide sequences. Bioinformatics. 21, 122–123.doi:10.1093/bioinformatics/bth459. . Crossref, PubMed, ISI, Google Scholar -
Blaxter M.L . 2004 The promise of a DNA taxonomy. Phil. Trans. R. Soc. B. 359, 669–679.doi:10.1098/rstb.2003.1447. . Link, ISI, Google Scholar -
Bremer K . 1994 Branch support and tree stability. Cladistics. 10, 295–304.doi:10.1111/j.1096-0031.1994.tb00179.x. . Crossref, ISI, Google Scholar -
Cracraft J . 1983 Species concept and speciation analysis. Curr. Ornithol. 1, 159–187. Crossref, Google Scholar -
Davis J.I& Nixon K.C . 1992 Populations, genetic variation, and the delimitation of phylogenetic species. Syst. Biol. 41, 421–435. Crossref, ISI, Google Scholar -
DeSalle R, Egan M.G& Siddall M . 2005 The unholy trinity: taxonomy, species delimitation and DNA barcoding. Phil. Trans. R. Soc. B. 360, 1905–1916.doi:10.1098/rstb.2005.1722. . Link, ISI, Google Scholar -
Excoffier L, Smouse P& Quattro J . 1992 Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics. 131, 479–491. Crossref, PubMed, ISI, Google Scholar -
Floyd R, Abebe E, Papert A& Blaxter M . 2002 Molecular barcodes for soil nematode identification. Mol. Ecol. 11, 839–850.doi:10.1046/j.1365-294X.2002.01485.x. . Crossref, PubMed, ISI, Google Scholar -
Folmer O, Black M, Hoeh W, Lutz R& Vrijenhoek R . 1994 DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. PubMed, Google Scholar -
Godfray H.C.J . 2002 Challenges for taxonomy—the discipline will have to reinvent itself if it is to survive and flourish. Nature. 417, 17–19.doi:10.1038/417017a. . Crossref, PubMed, ISI, Google Scholar -
Gregory T.R& DeSalle R Comparative genomics in prokaryotes. The evolution of the genome& Gregory T.R . 2005pp. 585–675. Eds. San Diego:Elsevier. Crossref, Google Scholar -
Hebert P.D.N, Cywinska A, Ball S.L& DeWaard J.R Biological identifications through DNA barcodes. Proc. R. Soc. B. 270, 2003a 313–321.doi:10.1098/rspb.2002.2218. . Link, ISI, Google Scholar -
Hebert P.D.N, Ratnasingham S& DeWaard J.R Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. B. 270, Suppl. 1 2003b S96–S99.doi:10.1098/rspb.2002.2218. . Link, ISI, Google Scholar -
Hebert P.D.N, Penton E.H, Burns J.M, Janzen D.H& Hallwachs W Ten species in one: DNA barcoding reveals cryptic species in the Neotropical skipper butterfly Astraptes fulgerator. Proc. Natl Acad. Sci. 101, 2004a 14 812–14 817.doi:10.1073/pnas.0406166101. . Crossref, ISI, Google Scholar -
Hebert P.D.N, Stoeckle M.Y, Zemlak T.S& Francis C.M Identification of birds through DNA barcodes. PLOS Biol. 2, 2004b 1657–1663.doi:10.1371/journal.pbio.0020312. . Crossref, ISI, Google Scholar -
Hogg I.D& Hebert P.D.N . 2004 Biological identification of springtails (Hexapoda: Collembola) from the Canadian Arctic, using mitochondrial DNA barcodes. Can. J. Zool. 82, 749–754.doi:10.1139/z04-041. . Crossref, ISI, Google Scholar -
Hudson R.R& Coyne J.A . 2002 Mathematical consequences of the genealogical species concept. Evolution. 56, 1557–1565. Crossref, PubMed, ISI, Google Scholar -
Inward, D. G. 2003 The evolution of dung beetle assemblages. Ph.D. thesis, Imperial College, London. Google Scholar
-
Janzen D.H . 2004 Now is the time. Phil. Trans. R. Soc. B. 359, 731–732.doi:10.1098/rstb.2003.1444. . Link, ISI, Google Scholar -
Lipscomb D, Platnick N& Wheeler Q . 2003 The intellectual content of taxonomy: a comment on DNA taxonomy. Trends Ecol. Evol. 18, 65–68.doi:10.1016/S0169-5347(02)00060-5. . Crossref, ISI, Google Scholar -
Moritz C& Cicero C . 2004 DNA barcoding: promise and pitfalls. PLOS Biol. 2, 1529–1531.doi:10.1371/journal.pbio.0020354. . Crossref, ISI, Google Scholar -
Proudlove G& Wood P.J . 2003 The blind leading the blind: cryptic subterranean species and DNA taxonomy. Trends Ecol. Evol. 18, 272–273.doi:10.1016/S0169-5347(03)00095-8. . Crossref, ISI, Google Scholar -
Quicke D.J.L . 2004 The world of DNA barcoding and morphology—collision or synergism and what of the future. Systematist. 23, 8–12. Google Scholar -
Schneider S.D, Roessli D& Excoffier L ARLEQUIN version 2.000: a software for population genetics data analysis. 2000 Switzerland:Genetics and Biomery Laboratory, University of Geneva. Google Scholar -
Seberg O, Humphries C.J, Knapp S, Stevenson D.W, Petersen G, Scharff N& Andersen N.M . 2003 Shortcuts in systematics? A commentary on DNA-based taxonomy. Trends Ecol. Evol. 18, 63–65.doi:10.1016/S0169-5347(02)00059-9. . Crossref, ISI, Google Scholar -
Simon C, Frati F, Beckenbach A, Crespi B, Liu H& Flook P . 1994 Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87, 651–701. Crossref, ISI, Google Scholar -
Sites J.W& Marshall J.C . 2003 Delimiting species: a Renaissance issue in systematic biology. Trends Ecol. Evol. 18, 462–470.doi:10.1016/S0169-5347(03)00184-8. . Crossref, ISI, Google Scholar -
Smith M.A, Fisher B.L& Hebert P.D.N . 2005 DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar. Phil. Trans. R. Soc. B. 360, 1825–1834.doi:10.1098/rstb.2005.1714. . Link, ISI, Google Scholar -
Stoeckle M . 2003 Taxonomy, DNA, and the barcode of life. Bioscience. 53, 796–797. Crossref, ISI, Google Scholar -
Swofford D.L PAUP*: Phylogenetic analysis using parsimony. Version 4.0b. 2002 Sunderland, MA:Sinauer Associates. Google Scholar -
Tautz D, Arctander P, Minelli A, Thomas R.H& Vogler A.P . 2003 A plea for DNA taxonomy. Trends Ecol. Evol. 18, 70–74.doi:10.1016/S0169-5347(02)00041-1. . Crossref, ISI, Google Scholar -
Templeton A.R . 2001 Using phylogeographic analyses of gene trees to test species status and processes. Mol. Ecol. 10, 779–791.doi:10.1046/j.1365-294x.2001.01199.x. . Crossref, PubMed, ISI, Google Scholar -
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, doi:10.1098/rstb.2005.1717. . Link, ISI, Google Scholar -
Wheeler Q.D . 2004 Taxonomic triage and the poverty of phylogeny. Phil. Trans. R. Soc. B. 359, 571–583.doi:10.1098/rstb.2003.1452. . Link, ISI, Google Scholar -
Will K.W& Rubinoff D . 2004 Myth of the molecule: DNA barcodes for species cannot replace morphology for identification and classification. Cladistics. 20, 47–55.doi:10.1111/j.1096-0031.2003.00008.x. . Crossref, ISI, Google Scholar -
Wilson E.O . 2003 The encyclopedia of life. Trends Ecol. Evol. 18, 77–80.doi:10.1016/S0169-5347(02)00040-X. . Crossref, ISI, Google Scholar -
Wright S Evolution and the genetics of populations. 1978 Chicago:University of Chicago. Google Scholar
