Phylogeography of the Laotian Rock Rat (Diatomyidae: Laonastes): Implications for Lazarus Taxa Author(s): Minh Le, Ha M Nguyen, Ha T Duong, Thanh V Nguyen, Long D Dinh, Nghia X Nguyen, Luong D Nguyen, Tri H Dinh and Dang X Nguyen Source: Mammal Study, 40(2):109-114 Published By: Mammal Society of Japan DOI: http://dx.doi.org/10.3106/041.040.0206 URL: http://www.bioone.org/doi/full/10.3106/041.040.0206 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use Usage of BioOne content is strictly limited to personal, educational, and non-commercial use Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research Mammal Study 40: 109–114 (2015) © The Mammal Society of Japan Short communication Phylogeography of the Laotian rock rat (Diatomyidae: Laonastes): implications for Lazarus taxa Minh Le1,2,*, Ha M Nguyen2, Ha T Duong3, Thanh V Nguyen3, Long D Dinh3,**, Nghia X Nguyen4, Luong D Nguyen5, Tri H Dinh6 and Dang X Nguyen4 Department of Environmental Ecology, Faculty of Environmental Science, Hanoi University of Science, VNU, 334 Nguyen Trai Road, Thanh Xuan District, Hanoi, Vietnam Centre for Natural Resources and Environmental Studies, VNU, 19 Le Thanh Tong Street, Hanoi, Vietnam Department of Genetics, Faculty of Biology, Hanoi University of Science, VNU, 334 Nguyen Trai Road, Thanh Xuan District, Hanoi, Vietnam Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam Fauna and Flora International, 340 Nghi Tam Road, Tay Ho District, Hanoi, Vietnam Phong Nha-Ke Bang Conservation National Park, Quang Binh Province, Vietnam The Laotian rock rat, Laonastes aenigmamus, was first described in 2005 based on specimens collected in the vicinity of Khammouan Limestone National Biodiversity Conservation Area in the northern Annamite Range (Jenkins­et al 2005) The unique morphological charac­ ters and highly divergent mitochondrial DNA of the new species, which inhabits limestone habitat exclusively, confirm its distinctiveness among all living rodent families Jenkins et al (2005), as a result, considered the ­Laotian rock rat the only surviving member of the new monotypic rodent family Laonastidae Although sub­ sequent morphological study supported its phylogenetic position within the existing family Diatomyidae (Dawson et al 2006), this species has been demonstrated to be the last remaining species of an ancient clade as all of its rela­ tives went extinct since 11 million years ago (Dawson et al 2006; Huchon et al 2007) Historically, the family was widely distributed in Asia, from the western part of the continent, Pakistan and India, to the eastern region, Thailand, China, and Japan (Li 1974; Flynn et al 1986; Marivaux et al 2002), but until recently it was known only from a small area in Laos (Fig 1) (Jenkins et al 2005; Rivière-Dobigny et al 2011; Nicolas et al 2012) During our recent surveys conducted from 2012 to 2014 in two new sites, Phong Nha–Ke Bang National Park, a UNESCO World Heritage Site, located on the eastern side and in Hin Nam No National Biodiversity Conservation Area on the western side of the Annamite Range (Fig 1), a number of specimens of the Laotian rock rat were collected We sequenced the entire cytochrome b gene of the newly acquired samples, and analyzed new sequences with those derived from the previous studies to elucidate the phylogeographic pattern of the populations In addition, we estimated divergence time between major lineages of the Laotian rock rat using a relaxed molecular clock approach to provide insights into the evolution of this enigmatic species We discussed the ‘Lazarus effect’ (Flessa and Jablonski 1983), referring to a large temporal gap of fossil records of this family, and the importance of the Annamites in harboring ancient and unique lineages in the context of our results Materials and methods Eleven newly collected specimens of the Laotian rock rat were included in this study, nine from Phong Nha–Ke Bang National Park and two from Hin Nam No National Biodiversity Conservation Area, eastern Lao PDR (Fig 1) We sequenced the complete cytochrome b for all 11 samples using five primers (Irwin et al 1991) Additional 16 published sequences of the partial and complete cytochrome b from individuals of populations around Khammouan Limestone National Biodiversity Conser­ vation Area (Jenkins et al 2005; Huchon et al 2007; Rivière-Dobigny et al 2011) and from distinct clades G and H, whose samples were collected in Laos (Nicolas et al 2012), were also incorporated in the analyses Other haplotypes in Nicolas et al (2012) were much less diver­ *To whom correspondence should be addressed E-mail: le.duc.minh@hus.edu.vn **Present address: Department of Fundamental Sciences, VNU-School of Medicine and Pharmacy, 144 Xuan Thuy Road, Cau Giay District, Hanoi, Vietnam Mammal Study  40 (2015) 110 Fig. 1.  Locations of the Laotian rock rat’s samples included in this study G and H are localities of the samples belonging to clades G and H in Nicolas et al (2012) gent, and therefore not included in this study Four taxa were used to provide outgroup polarity (Fig 2) DNA was extracted using DNeasy Blood and Tissue kit (Qiagen, California) following the manufacturer’s instruction Extracted DNA was amplified by PCR mastermix (Fermentas, Canada) with 21 ml volume (10 ml of mastermix, ml of water, ml of each primer at 10 pmol/ml and ml of DNA) PCR condition was: 95°C for minutes to active HotStarTaq; with 40 cycles at 95°C for 30 s, 45° for 45 s, 72°C for 60 s; and the final extension at 72°C for minutes Successful amplifica­ tions were purified to eliminate PCR components using GeneJETTM PCR Purification kit (Fermentas, Canada) Purified PCR products were sent to Macrogen Inc (Seoul, South Korea) for sequencing The sequences were aligned in BioEdit v7.1.3 (Hall 1999) with default settings Data were analyzed using maximum parsimony (MP) and maximum likelihood (ML) as implemented in PAUP 4.0b10 (Swofford 2001) and Bayesian analysis as implemented in MrBayes 3.2.1 (Huelsenbeck and Ronquist 2001) Uncorrected pairwise divergences were calculated in PAUP 4.0b10 All settings were followed Le et al (2006), except that the number of generations in the Bayesian analyses was increased to 107 We selected the relaxed-clock method (Drummond et al 2006) to estimate divergence times The obtained data­ set was used as input for the computer program BEAST v1.7.2 (Drummond and Rambaut 2007) Priori criteria for the analysis were set by the program BEAUti v1.7.2 One calibration point, the split between Massoutiera + Ctenodactylus and Laonastes of 44.3 ± 3.5 million years ago (MYA) (Huchon et al 2007), was used to calibrate the phylogeny A general time-reversible (GTR) model using gamma + invariant sites with four gamma catego­ ries was employed along with the assumption of a relaxed molecular clock As for the priors, we used all default settings, except for the Tree Prior category that was set to Coalescent Constant Size This setting was selected based on the assumption that the populations have reached sta­ bility (Rivière-Dobigny et al 2011) The combined and non-partitioned dataset was used for a single run In addi­ tion, a random tree was employed as a starting tree The length chain was set to 107, and the Markov chain was sampled every 1,000 generations After the dataset with the above settings was analyzed in BEAST, the resulting likelihood profile was then examined by the program (Rambaut and Drummond 2009) to determine the burn-in cutoff point The final tree with calibration estimates was computed using the program TreeAnnotator v1.7.2 as recommended in the BEAST program manual Results Nine samples of the Laotian rock rat collected in Phong Le et al., Phylogeography of the Laotian rock rat111 Fig. 2.  The cladogram generated by the program BEAST Numbers above branches are MP and ML bootstrap values of important nodes, respec­ tively Numbers below branches are Bayesian single-model and mixed-model posterior probability values, respectively Asterisk indicates 100% value The MP analysis produced 225 most parsimonious trees (TL = 678, CI = 0.72, RI = 0.9) Of 1,140 aligned characters, 705 were constant, and 278 parsimony informative The 95% confidence interval values for each numbered node are presented in Table C: calibration point P + P + H: Pliocene + Pleistocene + Holocene Nha–Ke Bang National Park, Vietnam, and two samples collected in Hin Nam No National Biodiversity Conser­ vation Area, Lao PDR, were successfully sequenced For the ML analysis, a single tree was produced with the total number of attempted rearrangements of 4,211, and the score of the best tree recovered was 4181.43 In the single-model Bayesian analysis, lnL scores reached ­ ­equilibrium after 11,000 generations, while in the mixedmodel Bayesian analysis lnL attained stationarity after 12,000 generations in both runs Tree topologies obtained from MP, ML, and combined and partitioned Bayesian analyses are almost identical in resolving important nodes (Fig 2) Three major lineages A, B, and C were recovered by all analyses, although clade C was strongly supported only by the MP analysis (bootstrap value [BP] = 100%) In addition, clade A and B were placed as sister taxa by all analyses with significant support again originating from the MP analysis (BP = 80%) Within clade C, three main lineages were well sup­ ported statistically (BP > 70%, posterior probability [PP] ≥ 95%) Two samples Hin and Hin from the Hin Nam No National Biodiversity Conservation Area, were placed within the first lineage of clade C Within clade B, all analyses recovered four distinct groups with a high level of corroboration in all analyses Two samples from clade G were firmly clustered with samples Phong Nha and (Fig 2) Molecular analyses show that samples in clade A are significantly different from those in other clades by at least about 9% of pairwise divergence in cytochrome b data, while samples in clade C has the maximal pairwise divergence of 6.3% Samples within clade A have the highest pairwise divergence of 1.65% After 1,000 initial trees were discarded from the analy­ sis by the program Tracer v1.5, final divergence times were generated using the program TreeAnnotator v1.7.2 Age estimates and 95% confidence interval for important nodes specified in Fig are shown in Table The results show that the clades A, B, and C were separated about 12 MYA (95% Highest posterior density [HPD] = 7.92– 18.15) in the mid Miocene Clade A was split from clade B about 7.9 MYA (95% HPD = 3.84–11.58) at the end of the Miocene Lineages in clade C did not diversify until about MYA at the very end of the Miocene Diversifica­ tion within major lineages in clade A and C only occurred over the last MYA (Fig and Table 1) Mammal Study  40 (2015) 112 Table 1.  Time calibration for nodes in the phylogeny Node numbers are defined in Figure Node Age estimate (million years) 95% HPD (million years) 10 11 43.80 36.25 19.40 12.05 7.86 4.75 4.12 1.52 1.37 1.23 1.04 39.93–47.80 15.83–51.05 8.91–28.14 7.92–18.15 3.84–11.58 2.93–7.38 0.59–2.75 0.57–2.56 0.59–2.35 0.55–1.89 Discussion Our analyses revealed three deeply divergent and mono­ phyletic clades, i.e., clade B, consisting of samples col­ lected from Phong Nha–Ke Bang and two reportedly collected in Lao PDR, clade A, represented by a single sample from Laos, and clade C, including samples from the type locality in Khammouan Limestone National Bio­ diversity Conservation Area A minimal 9% divergence in the cytochrome b data between samples of the three clades A, B, and C, which consists of samples from the type locality, warrants specific status for the two geneti­ cally distinct populations, i.e., clades A and B (Bradley and Baker 2001) (Fig 2) However, morphological exam­ inations of all available specimens from clades B and C, and additional specimens of clade A, are needed to clarify this issue The phylogenetic results in this study are simi­ lar to those generated by Rivière-Dobigny et al (2011) in that three distinct lineages were resolved within clade C (Fig 2) The support level in this study, however, is generally higher for these clades Because the highly divergent lineages are distributed in a small area around Khammouan Limestone National Biodiversity Conservation Area, Rivière-Dobigny et al (2011) and Nicolas et al (2012) hypothesized that ­bar­riers between specific karsts, where each population inhabits, facilitate the genetic differentiation between them This hypothesis implies limited dispersal ability or highly specialized behaviors of rock rat In contrast, the long distance between the new locality of this species in Hin Nam No National Biodiversity ­Conservation Area and those surrounding Khammouan Limestone National Biodiversity Conservation Area, and between the locality in Phong Nha–Ke Bang National Park and the locality of clade G is intriguing (Fig 1), because the samples from the distant locations have close phylogenetic relationships, e.g., Hin and Hin with those from Khammouan within the first lineage in clade C, and Phong Nha samples with two from Laos in clade B (Fig 2) Since the samples from Hin Nam No are embedded in the clade consisting of Khammouan’s samples, the most parsimonious hypothesis suggests recent long-distance­dispersals of the population from the ancestral area in the western side of the country to Hin Nam No Similarly, the population in Laos (clade G in Nicolas et al 2012) could have dispersed from the original location in Phong Nha–Ke Bang recently Considering the deep divergences of the three clades A, B, and C, (Fig 2), we suggest that they have been isolated for a long period of time According to current calcula­ tion, clade A + B were split from clade C about 12 MYA (95% HPD = 7.92–18.15) in the mid Miocene In addi­ tion, it is thought that lineages in Vietnam and Laos have evolved into several distinct lineages over the last eight million years (Fig and Table 1) Nonetheless, as Nicolas et al (2012) generated somewhat different ­divergence times using the cytochrome b gene, it is recom­ mended that the estimates be reexamined in the future studies using more molecular markers, including nuclear genes According to the divergence time estimated in this study, at least two lineages of Laonastes must have once coexisted with the sister genus Diatomys, which was dis­ tributed widely in Asia While Diatomys disappeared from fossil records about 11 MYA (Dawson et al 2006), Laonastes has continued to evolve into multiple distinct lineages The Laotian rock rat was apparently able to avoid extinction episodes, which occurred in the mid Miocene During this time, the global climate experi­ enced the abrupt cooling after the period of warming beginning in the early Oligocene (Flower and Kennett 1994; Zachos et al 2001) The paleoclimatic pattern exerted profound changes in plant communities across Asia; humid and uniform vegetation was replaced by arid adapted species (Liu et al 2009; Lu et al 2010; Hui et al 2011; Miao et al 2012; Miao et al 2013) Turnover of herbivorous mammal communities in Asia significantly increased during this period as a result of habitat modi­ fication (Fortelius et al 2006; Liu et al 2009) As pri­ mary herbivores (Jenkins et al 2005; Nguyen et al 2012; Nguyen et al 2014), Laonastes and its diatomyid relatives might have gone through a period of serious Le et al., Phylogeography of the Laotian rock rat113 food shortage, which resulted in the mass extinction throughout much of their distribution range, during the mid Miocene Laonastes, a Lazarus taxon, could have survived through the extinction event because they resided in a refugium, the Annamites, in Southeast Asia It is hypoth­ esized that this region has been stable climatically and ecologically over a long period of time (Sterling et al 2006) Several important discoveries, including such an­ cient mammals as Saola (Vu et al 1993), Large-antlered Muntjac (Do et al 1994), and Annamite Striped Rabbit (Averianov et al 2000), support the hypothesis that the Annamites have served as a refugium for many taxa Our results once again highlight the importance of the Annamites in harboring unique and ancient evolutionary lineages The temporal gap in fossil records of diatomyids spanning over 11 million years perhaps results from the fact that this area has been insufficiently studied and/or fossils had been poorly preserved in the limestone forests Since the exact distribution range and population sta­ tus of the Laotian rock rat in Hin Nam No and Phong Nha–Ke Bang National Park are currently unknown, we recommend that more thorough surveys be conducted in both protected areas, particularly in Phong Nha–Ke Bang, to clarify these issues It is important to note the high level of hunting in Phong Nha–Ke Bang National Park, especially in the buffer zone, where this species is found The endangered Laotian rock rat along with other small mammal species has been frequently trapped for food by local people (Nguyen et al 2012) The highly distinct and threatened lineage distributed in the eastern side of the Annamites therefore requires a well-designed con­ servation plan to protect it from serious anthropogenic threats in the area Acknowledgments:  This study was supported by the National Foundation for Science and Technology Devel­ opment of Vietnam (NAFOSTED: Grant Nos 106.152010.30 to M Le and 106.15-2011.14 to D.X Nguyen) The Fauna and Flora International generously provided funding for research activities Comments from anony­ mous reviewers and the associate editor greatly improved the paper References Averianov, A O., Abramov, A V and Tikhonov, A N 2000 A New Species of Nesolagus (Lagomorpha, Leporidae) from Vietnam with Osteological Description Zoological Institute, St Petersburg, Russia, 22 pp Bradley, R D and Baker, R J 2001 A test of the genetic species concept: cytochrome b sequences and mammals Journal of Mammalogy 82: 960–973 Dawson, M R., Marivaux, L., Li, C.-K., Beard, C and Métais, G 2006 Laonastes and the “Lazarus effect” in recent mammals Science 311: 1456–1458 Do, T., Dung, V V., Dawson, S., Arctander, P and MacKinnon, J 1994 Introduction of a New 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Study 40: 109–114 (2015) © The Mammal Society of Japan Short communication Phylogeography of the Laotian rock rat (Diatomyidae: Laonastes): implications for Lazarus taxa Minh Le1,2,*, Ha M Nguyen2,... recommended in the BEAST program manual Results Nine samples of the Laotian rock rat collected in Phong Le et al., Phylogeography of the Laotian rock rat 111 Fig. 2.  The cladogram generated by the program... the previous studies to elucidate the phylogeographic pattern of the populations In addition, we estimated divergence time between major lineages of the Laotian rock rat using a relaxed molecular