Mitochondrial DNA sequences suggest a revised taxonomy of Asian flapshell turtles (Lissemys SMITH, 1931) and the existence of previously overlooked taxa (Testudines: Trionychidae)

Abstract

We investigated relationships among Asian fl apshell turtles by using 2286 bp of mitochondrial DNA for phylogenetic reconstructions and relaxed molecular clock calculations. Currently three taxa are recognized, the unspotted species Lissemys scutata and L. punctata, with the unspotted subspecies L. p. punctata and the spotted subspecies L. p. andersoni. However, we found fi ve deeply divergent clades, two of which correspond to L. scutata (Myanmar; perhaps also adjacent Thailand and Yunnan, China) and L. p. andersoni (Indus, Ganges and Brahmaputra drainages; western Myanmar), respectively. Within L. p. punctata from peninsular India and Sri Lanka three distinct clades were identifi ed, two from peninsular India and one from Sri Lanka. The two clades from peninsular India are more closely related to L. p. andersoni than to fl apshell turtles from Sri Lanka. Due to a genetic divergence resembling L. scutata, we propose to separate Sri Lankan populations as the distinct species L. ceylonensis (Gray, 1856) from L. punctata. Furthermore, we suggest to restrict the name L. p. punctata (Lacepède, 1788) = L. p. punctata (Bonnaterre, 1789) to populations from southern peninsular India, whereas the name L. p. vittata (Peters, 1854) should be applied to unspotted fl apshell turtles from northern peninsular India. We classify all three taxa from the Indian subcontinent as subspecies because (1) there is morphological and genetic evidence that L. p. andersoni intergrades with L. p. vittata, and (2) the genetic divergence among L. p. punctata, L. p. andersoni and L. p. vittata resembles the degree of differentiation as observed between the latter two subspecies, whereas the differences between L. ceylonensis and L. scutata and among these species and the subspecies of L. punctata are about twice the values as observed among the subspecies of L. punctata. The formation of the subspecies of L. punctata was dated to have occurred between the uppermost Miocene and the Early Pleistocene (mean split ages of approx. 4.5 and 4.2 million years); the origin of L. ceylonensis and L. scutata, to a range between the Early Miocene and the Lower Pliocene (mean split ages of approx. 8 and 11 million years, respectively).

Phylogeography of the Asian softshell turtle Amyda cartilaginea (BODDAERT, 1770): evidence for a species complex

“…For the present study, 19 fresh blood or tissue samples of Amyda cartilaginea plus 14 tissue samples from historical museum specimens were used (Table S1). For fresh material, three mitochondrial and three nuclear DNA fragments were chosen, which have been successfully used in softshell turtles for phylogenetic and phylogeographic purposes (Weisrock & janzen 2000;enGstroM et al 2002enGstroM et al , 2004PraschaG et al 2007PraschaG et al , 2011McGauGh et al 2008;fritz et al 2010;lieBinG et al 2012;le et al 2014 ), namely, part of the 12S ribosomal RNA gene (12S rRNA or 12S), the complete cytochrome b gene (cyt b) plus adjacent DNA coding for tRNA-Thr, part of the 3' half of the NADH dehydrogenase subunit 4 gene (ND4) plus adjacent DNA coding for tRNAs, part of the gene coding for oocyte maturation factor Mos (Cmos), part of the gene coding for ornithine decarboxylase (ODC), and part of intron 1 of the orphan G protein-coupled receptor gene R35 (R35). For PCR and sequencing of the 12S, ND4, Cmos, ODC and R35 fragments of fresh samples, the same primer pairs were applied as in lieBinG et al (2012).…”

“…While this has allowed a pioneering assessment of the phylogenetic relationships (Meylan 1987), the rarity of material of many taxa hampered a detailed understanding of the species diversity. Thus, it is not surprising that several studies using molecular genetic approaches have gained a much refined understanding of trionychid taxonomy (Weisrock & janzen 2000;enGstroM et al 2002enGstroM et al , 2004PraschaG et al 2007PraschaG et al , 2011McGauGh et al 2008;fritz et al 2010;lieBinG et al 2012;le et al 2014) and resulted in the recognition of six additional species (Chitra chitra, C. vandijki, Lissemys ceylonensis, Pelodiscus axenaria, P. maackii, P. parviformis;enGstroM et al 2002;Mccord & Pritchard 2003;fritz et al 2010;PraschaG et al 2011; and two additional subspecies (Chitra chitra javanensis, Lissemys punctata vittata;enGstroM et al 2002;Mccord & Pritchard 2003;Pra-schaG et al 2011) compared to previous morphologybased assessments (WeBB 1982;Meylan 1987). However, molecular studies have not yet been conducted for the majority of trionychid species, so that the existence of further distinct, but currently unrecognized, taxa is most likely.…”

Molecular phylogeny of the softshell turtle genus Nilssonia revisited, with first records of N. formosa for China and wild-living N. nigricans for Bangladesh

“…Fifty-three Nilssonia samples were studied, representing the five currently recognized species Nilssonia formosa, N. gangetica, N. hurum, N. leithii and N. nigricans (see Appendix). Three mitochondrial genes were sequenced that have previously been shown to be useful for assessing the phylogenetic relationships of terminal chelonian taxa (e.g., le et al, 2006;Fritz et al, 2010Fritz et al, , 2012avarGas-raMírez et al, 2010;Wiens et al, 2010;PraschaG et al, 2011), viz. the partial 12S ribosomal RNA (12S rRNA) gene, the partial NADH dehydrogenase subunit 4 (ND4) gene, and the cytochrome b (cyt b) gene.…”

“…This suggests that each major river basin harbours a genetically distinct population of N. gangetica, which should be treated as a distinct management unit. In analogy to the widely used barcoding approach (heBert et al, 2003), uncorrected p distances of the mitochondrial cyt b gene have repeatedly been used as a yardstick for assessing the taxonomic status of turtles and tortoises (e.g., sPinks et al, 2004;varGas-raMírez et al, 2010;PraschaG et al, 2011;Fritz et al, 2012a, b;kindler et al, 2012) Pelodiscus maackii Russia: Primorsky Territory: Lake Khanka FM999003 FM999019 FM999011 HE801805 --HE801911…”