Abstract
We identified three distinct sympatric lineages of frogs among specimens previously considered a single species (Meris- togenys amoropalamus Matsui), based on 909 bp of mitochondrial DNA (12S rRNA and cytochrome b). To seek evidence of reproductive isolation between these lineages, we first analyzed a 249-bp fragment of the nuclear proopiomelanocortin (POMC) gene and found five haplotypes, of which two were limited to lineage 1 and three belonged to lineages 3 and 4. In a subsequent phylogenetic analysis of a 1313-bp fragment of nuclear POMC, Rag-1, and rhodopsin, lineage 1 was again distinct, while lineages 3 and 4 could not be differentiated. The results of the nuclear gene analyses suggest that lineage 1 is strongly isolated reproductively from lineages 3 and 4, which are not isolated from each other. This conclusion conforms to groupings based on larval morphology. These results indicate that frogs morphologically identified as M. amoropalamus should be split into two sympatric species, one of which contains two mitochondrial lineages that have presumably been retained via deep coalescence.References
Ballard, J.W.O. & Whitlock, M.C. (2004) The incomplete natural history of mitochondria. Molecular Ecology, 13, 729–744.
Dubois, A. (1992) Notes sur la classification des Ranidae (Amphibiens Anoures). Bulletin Mensuel de la Société Linnéenne de Lyon, 61, 305–352.
Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783–791.
Fouquet, A., Vences, M., Salducci, M.D., Meyer, A., Marty, C., Blanc, M. & Gilles, A. (2007) Revealing cryptic diversity using molecular phylogenetics and phylogeography in frogs of the Scinax ruber and Rhinella margaritifera species groups. Molecular Phylogenetics and Evolution, 43, 567-582.
Gonçalves, H., Martínez-Solano, I., Ferrand, N. & García-Parrs, M. (2007) Conflicting phylogenetic signal of nuclear vs mitochondrial DNA markers in midwife toads (Anura, Discoglossidae, Alytes): deep coalescence or ancestral hybridization? Molecular Phylogenetics and Evolution, 44, 494–500.
Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.
Hasegawa, M., Kishino, H. & Yano, T. (1985) Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution, 22, 160–174.
Hedges, S.B. (1992) The number of replications needed for accurate estimation of the bootstrap P value in phylogenetic studies. Molecular Biology and Evolution, 9, 366–369.
Holder, M.T., Anderson, J.A. & Holloway, A.K. (2001) Difficulties in detecting hybridization. Systematic Biology, 50, 978–982.
Huelsenbeck, J.P. & Ronquist, F.R. (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics, 17, 754–755.
Inger, R.F. & Stuebing, R.B. (2005) A field guide to the frogs of Borneo, Second Edition. Natural History Publications, Kota Kinabalu, Malaysia, 1–201.
Innan, H. & Nordborg, M. (2002) Recombination or mutational hot spots in human mtDNA? Molecular Biology and Evolution, 19, 1122–1127.
Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111–120.
Köhler, J., Vieites, D.R., Bonett, R.M., Hita García, F., Glaw, F., Steinke, D. & Vences, M. (2005) New amphibians and global conservation: a boost in species discoveries in a highly endangered vertebrate group. Bioscience, 55, 693-696.
Maddison, W.P. (1997) Gene trees in species trees. Systematic Biology, 46, 523–536.
Matsui, M. (1986) Three new species of Amolops from Borneo (Amphibia, Anura, Ranidae). Copeia, 1986, 623–630.
Matsui, M., Ito, H., Shimada, T., Ota, H., Saidapur, S.K., Khonsue, W., Tanaka-Ueno, T. & Wu, G.F. (2005) Taxonomic relationships within the pan-oriental narrow-mouth toad Microhyla ornata as revealed by mtDNA analysis (Amphibia, Anura, Microhylidae). Zoological Science, 22, 489–495.
Matsui, M., Maryati, M., Shimada, T. & Sudin A. (2007) Resurrection of Staurois parvus from S. tuberilinguis from Borneo (Amphibia, Ranidae). Zoological Science, 24, 101–106.
Matsui, M., Shimada, T., Liu, W.Z., Maryati, M., Khonsue, W. & Orlov, N. (2006) Phylogenetic relationships of the oriental torrent frogs of the genus Amolops and its allies (Amphibia, Anura, Ranidae). Molecular Phylogenetics and Evolution, 38, 659–666.
Nylander, J.A.A. (2002) MrModeltest v1.0b program distributed by the author. Available from http://www.ebc.uu.se/systzoo/staff/nylander.html (accessed 8 June 2008)
Palumbi, S.R., Martin, A., Romano, S., McMillan, W.O., Stice, L. & Grabowski, G. (1991) "The simple fool’s guide to PCR, version 2.0." Privately published, compiled by Palumbi S., Department of Zoology, University of Hawaii, Honolulu.
Rodriguez, F., Oliver, J.F., Marin, A. & Medina, J.R. (1990) The general stochastic model of nucleotide substitution. Journal of Theoretical Biology, 142, 485–501.
Roelants, K., Jiang, J. & Bossuyt, F. (2003) Endemic ranid (Amphibia: Anura) genera in southern mountain ranges of the Indian subcontinent represent ancient frog lineages: evidence from molecular data. Molecular Phylogenetics and Evolution, 31, 730–740.
Sheffler, I.E. (1999) Mitochondria. John Wiley and Sons. Inc., New York, 1–462p.
Shimada, T., Matsui, M., Sudin, A. & Maryati, M. (2007) Identity of larval Meristogenys from a single stream in Sabah, Malaysia (Amphibia: Ranidae). Zoological Journal of the Linnean Society, 151, 173–189.
Smith, S.A., Stephens, P.R. & Wiens, J.J. (2005) Replicate patterns of species richness, historical biogeography, and phylogeny in Holarctic treefrogs. Evolution, 59, 2433–2450.
Sota, T. & Vogler, A. (2001) Incongruence of mitochondrial and nuclear gene trees in the carabid beetles Ohomopterus. Systematic Biology, 50, 39–59.
Stephens, M., Smith, N.J. & Donnelly, P. (2001) A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics, 68, 978–989.
Swofford, D.L. (2002) PAUP*: Phylogenetic analysis using parsimony (*and other methods), Version 4. Sinauer Associates, Sunderland, MA, USA.
Tanaka-Ueno, T., Matsui, M., Chen, S.L., Takenaka, O. & Ota, H. (1998) Phylogenetic relationships of brown frogs from Taiwan and Japan assessed by mitochondrial cytochrome b gene sequences (Rana: Ranidae). Zoological Science, 15, 283–288.
Wiens, J., Fetzner, J.W.Jr., Parkinson, C.L. & Reeder, T.W. (2005) Hylid frog phylogeny and sampling strategies for speciose clades. Systematic Biology, 54, 719–748.
Yang, D. (1991) Phylogenetic systematics of the Amolops group of ranid frogs of Southeastern Asia and the Greater Sunda Islands. Fieldiana: Zoology, New Series, 63, 1–42.