This study aims to investigate the species diversity of rodents living in karst ecosystems of Thailand. A survey has been conducted throughout Thailand, 122 karsts sampled and 477 Murinae rodents live-trapped. Phylogenetic reconstructions were carried out using two mitochondrial markers (cytb, COI). A sequence-based species delimitation method completed by the analysis of the level of genetic divergence was then applied to define species boundaries within our dataset. The phylogenetic position of Niviventer hinpoon was also investigated and sequences obtained from the holotype specimen of this species were used to reliably identify samples of N. hinpoon. A total of 12 described Murinae species, corresponding to 17 deeply divergent genetic lineages, were encountered in limestone karsts of Thailand. Our study revealed an important genetic diversity within the traditionally recognized species Maxomys surifer (four highly divergent genetic lineages), Leopoldamys neilli (two highly divergent genetic lineages) and Berylmys bowersi (two highly divergent genetic lineages). These species could be considered as species complex and require further taxonomic work. This study also provides valuable information on the distribution of the two rodent species endemic to limestone karsts of Thailand, L. neilli and N. hinpoon. Leopoldamys neilli was the most abundant species encountered in Thai karsts during our survey. However, L. neilli specimens from western Thailand are genetically highly divergent from the remaining populations of L. neilli and could represent a separate species. Niviventer hinpoon, phylogenetically closely related to N. fulvescens, is much rarer and its distribution limited to central Thailand. Most of the other captured species are typically associated with forest ecosystems. This study suggests that limestone karsts play a key role in the preservation of the rodent species endemic to such habitat, but they would also provide refuges for the forest-dwelling Murinae rodents in deforested regions.

BackgroundHistorical biogeography and evolutionary processes of cave taxa have been widely studied in temperate regions. However, Southeast Asian cave ecosystems remain largely unexplored despite their high scientific interest. Here we studied the phylogeography of Leopoldamys neilli, a cave-dwelling murine rodent living in limestone karsts of Thailand, and compared the molecular signature of mitochondrial and nuclear markers.Methodology/Principal FindingsWe used a large sampling (n = 225) from 28 localities in Thailand and a combination of mitochondrial and nuclear markers with various evolutionary rates (two intronic regions and 12 microsatellites). The evolutionary history of L. neilli and the relative role of vicariance and dispersal were investigated using ancestral range reconstruction analysis and Approximate Bayesian computation (ABC).Both mitochondrial and nuclear markers support a large-scale population structure of four main groups (west, centre, north and northeast) and a strong finer structure within each of these groups. A deep genealogical divergence among geographically close lineages is observed and denotes a high population fragmentation. Our findings suggest that the current phylogeographic pattern of this species results from the fragmentation of a widespread ancestral population and that vicariance has played a significant role in the evolutionary history of L. neilli. These deep vicariant events that occurred during Plio-Pleistocene are related to the formation of the Central Plain of Thailand. Consequently, the western, central, northern and northeastern groups of populations were historically isolated and should be considered as four distinct Evolutionarily Significant Units (ESUs).Conclusions/SignificanceOur study confirms the benefit of using several independent genetic markers to obtain a comprehensive and reliable picture of L. neilli evolutionary history at different levels of resolution. The complex genetic structure of Leopoldamys neilli is supported by congruent mitochondrial and nuclear markers and has been influenced by the geological history of Thailand during Plio-Pleistocene.

Leopoldamys neilli is a Murinae rodent endemic to limestone karst of Thailand and the Lao PDR, but its ecology and the reasons of its endemism to karst are still totally unknown. The aim of this pilot study was to examine the plant composition of the diet of L. neilli at the level of order and family using DNA for molecular identification and to compare it with two other forest-dwelling Leopoldamys species, L. herberti and L. sabanus. A 202bp fragment of the rbcL gene was amplified and sequenced for twenty-three fecal samples of the three species using 454 pyrosequencing. We successfully identified a total of seventeen orders and twenty-one plant families, corresponding to thirty-three putative species, in the feces of these three Leopoldamys species. Solanaceae were the most common plants in the diet of L.neilli regardless of the region and sampling season, and they were also present in feces of both L. herberti and L. sabanus. The Araceae, Fabaceae, and Apocynaceae families were also identified in feces of L. neilli collected in various regions of Thailand and at different seasons. Plants of the Oleaceae family are consumed by both L. herberti and L. sabanus but were not found in the diet of L. neilli. Further improvements of the study, such as the use of additional genes, the creation of a reference collection, the microhistological examination of plant fragments to determine which parts of the plant are consumed, and the analysis of the animal diet of Leopoldamys are suggested to enhance the quality and accuracy of the results obtained.

Recently, Balakirev et al. (2013) presented a taxonomic revision of the genus Leopoldamys based on phylogenetic analyses. They identified five main Leopoldamys genetic lineages and suggested to rename several of them. According to these authors, the genetic lineage previously thought to belong to L. edwardsi (lineage L1) should be assigned to L. revertens while L. neilli (lineage L2) should be considered as a junior synonym of L. herberti. Using molecular and morphological data from a large sampling of Leopoldamys specimens, the aim of the present study was to investigate the taxonomic status of L. herberti and L. neilli. This study reveals that, contrary to Balakirev et al.'s statement, both genetic lineages L1 and L2 occur in Nakhon Ratchasima Province, close to the type locality of L. herberti. We also show that the external measurements and color pattern of L. herberti are highly similar to those of L1 specimens but are not consistent with the morphology of L2 specimens. Therefore these results strongly suggest that L. herberti should be assigned to the genetic lineage L1. Consequently L. neilli should not be considered as a junior synonym of L. herberti and this study confirms that the appropriate name of the genetic lineage L2 is L. neilli. Moreover, as our results show that L. herberti should be assigned to the lineage L1, this name has nomenclatural priority over L. revertens, the species name suggested by Balakirev et al. (2013) for this lineage.