Discovery of the roosevelt’s barking deer muntiacus rooseveltorum in vietnam

Conserv Genet DOI 10.1007/s10592-014-0581-4 SHORT COMMUNICATION Discovery of the Roosevelt’s Barking Deer (Muntiacus rooseveltorum) in Vietnam Minh Le • Thanh V Nguyen • Ha T Duong Ha M Nguyen • Long D Dinh • Tuoc Do • Hai D Nguyen • George Amato • Received: 21 September 2013 / Accepted: 31 January 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Distribution and taxonomic status of the Roosevelt’s Barking Deer (Muntiacus rooseveltorum) have remained poorly understood after more than 80 years since its description All records of this species so far have been reported only from Lao PDR During recent surveys in central Vietnam, we found several specimens from local hunting trophies morphologically resembling this species Our molecular data, including both mitochondrial and nuclear genes, based on collected materials confirm for the first time that M rooseveltorum is distributed in Vietnam In addition, the phylogenetic analyses demonstrate that the Roosevelt’s Barking Deer represents a distinct evolutionary lineage closely related to the Truong Son Muntjac, in central Vietnam, and the Leaf Muntjac in Myanmar Given the rarity of this species and the escalating hunting and habitat loss in the region, it is important to conduct field research to assess its population status Such information is critically needed to design a conservation plan for this highly elusive and threatened taxon M Le (&) Department of Environmental Ecology, Faculty of Environmental Science, Hanoi University of Science, VNU, 334 Nguyen Trai Road, Thanh Xuan District, Hanoi, Vietnam e-mail: le.duc.minh@hus.edu.vn T Do Forest Inventory and Planning Institute, Vinh Quynh, Thanh Tri, Hanoi, Vietnam M Le Á H M Nguyen Centre for Natural Resources and Environmental Studies, VNU, 19 Le Thanh Tong Street, Hanoi, Vietnam T V Nguyen Á H T Duong Á L D Dinh Department of Genetics, Faculty of Biology, Hanoi University of Science, VNU, 334 Nguyen Trai Road, Thanh Xuan District, Hanoi, Vietnam Keywords Muntjac Á Muntiacus rooseveltorum Á ND4 Á Cytochrome b Á G-fibrinogen Á Conservation Introduction The Roosevelt’s Barking Deer (Muntiacus rooseveltorum) is one of the most poorly known mammal species in the world Since it was described as a new species in the early 20th century (Osgood 1932), no records have been reported until its rediscovery in Laos based on the molecular data derived from hunting trophies by the end of the century (Amato et al 1999b) However, after the type specimen was collected during the Kelley-Roosevelts and Delacour H D Nguyen Xuan Lien Nature Reserve, Cam Xuan Commune, Thuong Xuan District, Thanh Hoa, Vietnam G Amato Sackler Institute for Comparative Genomics, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024, USA Present Address: L D Dinh Department of Fundamental Sciences, VNU-School of Medicine and Pharmacy, 144 Xuan Thuy Road, Cau Giay District, Hanoi, Vietnam 123 Conserv Genet Asiatic expeditions, this species has never again been observed by scientists It has been recorded merely from three isolated localities in Laos based on the type and specimens from hunted animals (Amato et al 1999a, b; Timmins et al 2008) (Fig 1), although there was an unconfirmed report that it occurred in central Vietnam (Le et al 1999) This species is currently listed under the data deficient category in the IUCN Red list The taxonomic status of the Roosevelt’s Barking Deer is also controversial Morphologically, this species cannot be distinguished from other closely related taxa within the species complex, i.e., Muntiacus putaoensis and M truongsonensis, with a high level of confidence This is because these species were described based on few variable diagnostic characters from a limited number of specimens (Timmins et al 2008) So far, molecular studies have not been able to clarify the issues, as previous phylogenetic analyses employed only a small number of samples for Fig Known localities of the Roosevelt’s Barking Deer 123 each species, e.g., Amato et al (1999a) As a result, it is very challenging to study the distribution and population status of this potentially endangered taxon During our recent surveys in two central protected areas, Pu Hoat and Xuan Lien Nature Reserves, bordering with Lao PDR (Fig 1), we found several hunting trophies, which show morphological characters of the Roosevelt’s Barking Deer These characters include developed mental glands covered with long, stiff, and thick hairs on both sides of the jaw This species reportedly inhabits primary forests, which are restricted to isolated patches in the reserves To verify our finding, we compared the mitochondrial DNA from a partial 16S gene of these samples with that of the type specimen In addition, we reconstructed phylogenetic relationships of this species with other closely related taxa to shed light on current confusion surrounding its taxonomic status using other mitochondrial markers, including the complete cytochrome b and a partial Conserv Genet NADH dehydrogenase subunit (ND4), and a nuclear gene, the blood clotting protein, c-fibrinogen of M putaoensis, M truongsonensis, and M vuquangensis and two outgroup taxa available from GenBank were also included in the analyses To confirm the genetic distinctiveness and resolve the taxonomic status of M rooseveltorum with regard to other closely related taxa, we sequenced two mtDNA genes, the complete cytochrome b and partial ND4, and one nuclear gene, the c-fibrinogen, from four samples of M rooseveltorum, six samples of M vuquangensis, two samples of M truongsonensis, and one sample of M putaoensis Additional cytochrome b and ND4 sequences of the Giant Muntjac, Muntiacus vuquangensis, and two outgroups, Panolia eldii and Elaphodus cephalophus, from GenBank were also added to the dataset (Table 1) Materials and methods Taxonomic sampling and molecular data To genetically compare our collected samples with the type specimen, we sequenced the 16S gene for four samples of purported Muntiacus rooseveltorum from Pu Hoat and Xuan Lien Nature Reserves, as the only molecular data available for the type specimen of Muntiacus rooseveltorum is a 16S sequence (Amato et al 1999b) The sequences Table GenBank accession numbers, and associated samples that were used in this study All sequences generated by this study have accession numbers: KJ425271–KJ425303 Species names Cytb ND4 16S G-fib Sample number Reference Panolia eldii HM138200 HM138200 HM138200 – – Kong and Li (unpublished) Elaphodus cephalophus NC008749 NC008749 NC008749 – – Pang et al 2008 Muntiacus vuquangensis FJ705435 FJ705435 FJ705435 – – Hassanin et al 2012 Muntiacus vuquangensis - – AF108034 – – Amato et al 1999a Muntiacus vuquangensis AF042720 – – – – Giao et al 1998 Muntiacus vuquangensis KJ425275 KJ425295 – KJ425286 M 1.1 This study Muntiacus vuquangensis KJ425284 KJ425303 – KJ425294 M 6.13 This study Muntiacus vuquangensis KJ425283 KJ425302 – KJ425293 M 6.9 This study Muntiacus vuquangensis KJ425285 – – – M 6.21 This study Muntiacus truongsonensis - – AF108033 – – Amato et al 1999a Muntiacus truongsonensis KJ425276 KJ425296 – KJ425287 M 1.9 This study Muntiacus truongsonensis KJ425277 KJ425297 – KJ425288 M 2.4 This study Muntiacus putaoensis KJ425280 KJ425299 – KJ425290 M 4.1 This study Muntiacus putaoensis – – AF108032 – – Amato et al 1999a Muntiacus rooseveltorum – – AF108031 – – Amato et al 1999a Muntiacus rooseveltorum KJ425278 – KJ425271 – M 2.18 This study Muntiacus rooseveltorum KJ425279 KJ425298 KJ425272 KJ425289 M 2.20 This study Muntiacus rooseveltorum KJ425281 KJ425300 KJ425273 KJ425291 M 6.3 This study Muntiacus rooseveltorum KJ425282 KJ425301 KJ425274 KJ425292 M 6.4 This study 123 Conserv Genet For DNA extraction, bone samples were first cleaned with 10 % clorox in order to eliminate contaminated material on the sample surface Bone or dry tissue samples then were extracted following protocols specified in Le et al (2007) using DNeasy blood and tissue kit, Qiagen For the incubation step, the lysis usually took up to 72 h to let the bone to become completely digested During this step, the extraction was checked every 24 h to monitor the progress and added 20 ll increments of proteinase K A negative control was used in every extraction Extracted DNA from bones or old tissues was amplified by HotStarTaq mastermix (Qiagen, California) The PCR volume consisted of 21 ll (10 ll of mastermix, ll of water, ll of each primer at 10 pmol/ll and ll of DNA or higher depending on the quantity of DNA in the final extraction solution) PCR condition was: 95 °C for 15 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 In cases where PCR reactions did not work, the PCR product was used as template for the new PCR reactions Negative controls were used in all amplifications to check for possible contamination PCR products were subjected to electrophoresis through a % agarose gel (UltraPureTM, Invitrogen) Gels were stained for 10 in 19 TBE buffer at pg/ml of ethidium-bromide and visualized under UV light Successful amplifications 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 All primers used in this study, including newly designed ones, are shown in Table Phylogenetic analyses The sequences were aligned in BioEdit v7.1.3 (Hall 1999) with default settings Data were analyzed using three standard phylogenetic methods, 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) For MP analysis, heuristic analysis was conducted with 100 random taxon addition replicates using treebisection and reconnection (TBR) branch swapping algorithm, with no upper limit set for the maximum number of trees saved Bootstrap support (Felsenstein 1985) was calculated using 1,000 pseudo-replicates and 100 random taxon addition replicates All characters were equally weighted and unordered In addition, uncorrected pairwise distance was calculated for cytochrome b and ND4 in PAUP*4.0b10 For ML analysis, the optimal model for nucleotide evolution was determined using Modeltest 3.7 (Posada and Crandall 1998) The program selected GTR ? I and TrN ? G as the best-fit models for the 16S and combined analyses, respectively Analyses were conducted with stepwise-addition starting tree, heuristic searches with simple taxon addition, and the TBR branch swapping algorithm Support for the likelihood hypothesis was evaluated by bootstrap analysis with 100 pseudo-replications and simple taxon addition We regard bootstrap values (BP) of C70 % as strong support and values of \70 % as weak support (Hillis and Bull 1993) For Bayesian analyses, we used the optimal model determined by Modeltest with parameters estimated by MrBayes 3.2.1 Two simultaneous analyses with four Markov chains (one cold and three heated) were run of 10 million generations with a random starting tree and sampled every 1,000 generations Log-likelihood scores of sample points were plotted against generation time to determine stationarity of Markov chains Trees generated before log-likelihood scores reached stationarity were discarded from the final analyses using the burn-in function Two independent analyses were run simultaneously Both Table Primers used in this study Segment Primer Length Sequence Reference Cytb-1 L14724 450 50 -CGAAGCTTGATATGAAAAACCATCGTTG-30 Irwin et al (1991) 50 -AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-30 H15149 50 -GCAAGCTTCTACCATGAGGACAAATATC-30 Irwin et al (1991) 50 -GGAATTCATCTCTCCGGTTTACAAGAC-30 Irwin et al (1991) H15149 Cytb-2 L15162 750 H15915R ND4 ND4-F 714 ND4-R 16S AR 567 BR G-fibrinogen 123 GL1 GR1 574 -CTCATRCCYCTGACCTGACTAT-3 This study 50 -GCTATAAATTCGGTAAGTGGATT-30 This study 50 -CGCCTGTTTATCAAAAACAT-30 Palumbi et al ( 1991) 50 -CCGGTCTGAACTCAGATCACGT-30 Palumbi et al (1991) 50 -AGAHAAYTGCTGCATCTTAGATG-30 50 -TTCRTATTTCATAATTTCTTC-30 Gatesy (1997) Gatesy (1997) Conserv Genet runs were stabilized after 9,000 and 11,000 generations in the analyses of the 16S and combined datasets, respectively The posterior probability (PP) values for all clades in the final majority rule consensus tree are provided Results and discussion The analyses based on the 16S data show that four samples collected in Pu Hoat and Xuan Lien Nature Reserves were clustered with the sample of the type specimen (Fig 2a) with moderate support from MP and ML analyses (BP = 62 and 63 %, respectively) and with strong support from the Bayesian analysis (PP = 94 %) Genetic divergence based on the 16S gene fragments of five samples of M rooseveltorum is insignificant, with a maximum value of only 0.4 % between the type and sample Mu6.4 Moreover, three species, M putaoensis, M rooseveltorum, M truongsonensis, formed a clade independent of M vuquangensis with a high level of support from all three analyses (BP = 86 and 82, PP = 99) M putaoensis was strongly supported as the sister taxon to M truongsonensis only in the Bayesian analysis (PP = 93) The results of our phylogenetic analyses using two mitochondrial genes, cytochrome b and ND4, and a nuclear gene, c-fibrinogen (Fig 2a), corroborate very well to those generated from 16S data (Fig 2a) and from 12S, 16S, cytochrome b, and Dloop genes (Amato et al 1999a) The support level for all nodes in the cladogram based on the combined data (Fig 2b) is greatly improved compared to that for nodes in the cladogram based on the 16S data (Fig 2a) and for nodes in the cladogram based on the four Fig Both cladograms are the most parsimonious trees with branch length estimated by the MP analysis Numbers above branches are MP and ML bootstrap values, respectively Numbers below branches are Bayesian single-model posterior probability values Asterisk indicates 100 % value a Results based on the partial 16S dataset The MP analysis produced single most parsimonious tree (TL = 43, CI = 0.86, RI = 0.84) Of 567 aligned characters, 531 were constant, and 22 parsimony informative Red color coded terminal represents the type b Results based on combined mitochondrial and nuclear genes The MP analysis produced five most parsimonious trees (TL = 524, CI = 0.82, RI = 0.84) Of 2,431 aligned characters, 2,014 were constant, and 218 parsimony informative (Color figure online) 123 Conserv Genet Table Uncorrected (‘‘p’’) distance matrix showing percentage pairwise divergence calculated based on cytochrome b and ND4 genes S no Taxa M vuquangensis (FJ705435) – M vuquangensis (AF042720) 2.2 – M vuquangensis (Mu 1.1) 0.97 1.59 M vuquangensis (Mu 6.21) 0.96 1.67 0.09 – M vuquangensis (Mu 6.9) 2.1 0.80 1.67 1.58 M vuquangensis (Mu 6.13) 2.03 0.71 1.59 1.49 0.06 – M putaoensis (Mu 4.1) 5.89 5.77 5.78 5.49 5.78 5.77 M truongsonensis (Mu 1.9) 6.31 6.46 5.98 6.0 6.15 6.04 2.91 – M truongsonensis (Mu 2.4) 6.04 6.29 5.71 5.82 5.88 5.77 2.91 0.38 10 11 12 – – – – 10 M rooseveltorum (Mu 2.18) 6.18 6.17 5.57 5.71 5.66 5.57 2.21 3.08 3.09 – 11 12 M rooseveltorum (Mu 2.20) M rooseveltorum (Mu 6.3) 5.72 5.66 6.18 6.18 5.4 5.34 5.72 5.72 5.50 5.50 5.49 5.44 2.59 2.49 3.08 2.91 2.91 2.75 0 – 0.05 – 13 M rooseveltorum (Mu 6.4) 5.77 6.26 5.45 5.80 5.61 5.54 2.65 3.02 2.86 0.09 0.16 0.21 mitochondrial-gene dataset (Amato et al 1999a), except for the sister relationship between M truongsonensis and M putaoensis Overall, our results indicate that each species within the previously considered M rooseveltorum species complex, including M putaoensis, M rooseveltorum, and M truongsonensis, is genetically distinct Inter-specific genetic variation within the complex ranges from 2.2 to 3.1 % based on two mitochondrial genes (Table 3) This level of variation is somewhat lower than the value of about 5–6 % in a faster evolving gene, the control region, identified between Chinese ? Siberian and European roe deers, i.e., Capreolus pygargus and C capreolus (Randi et al 1998; Xiao et al 2007) Lower interspecific variation in this group of muntjac could indicate a recent radiation or a slower evolution rate of its mitochondrial DNA compared to other related taxa Intra-specific genetic distance within the three species of the M rooseveltorum species complex based on cytochrome b and ND4 is lower than 0.4 % (Table and James et al 2008) From the above results, it can be concluded that M rooseveltorum is present in two protected areas, Pu Hoat and Xuan Lien Nature Reserves, in central Vietnam Given the animals were hunted only two or three years ago, and many observations were reported recently by local people, it is likely that viable populations of this species still exist in the region However, hunting has been escalating in these protected areas (Le et al 1999; Osborn et al 2000; pers obs.) Le et al (1999) documented hundreds of traps being deployed by local people in Xuan Lien Nature Reserve Moreover, primary forests, the species’ natural habitat, currently covering\10 % of both reserves, are also disappearing quickly (Le et al 1999; Osborn et al 2000) It is therefore important that the population status of this species be surveyed immediately to design appropriate 123 13 – conservation measures to secure the survival of this elusive and rare species Acknowledgments The Nagao Natural Environment Foundation, Japan, the SeaWorld and Busch Gardens Conservation Fund, Vietnam National University, and the Alfred P Sloan Foundation generously provided funding for this project Eleanor Sterling and Martha Hurley supported the early field work M Le was supported by the National Foundation for Science and Technology Development of Vietnam (NAFOSTED: Grant No 106.15-2010.30) Comments from the associate editor and two anonymous reviewers helped improve the paper References Amato G, Egan MG, Rabinowitz A (1999a) A new species of muntjac, Muntiacus putaoensis (Artiodactyla: Cervidae) from northern Myanmar Animal Conserv 2:1–7 Amato G, Egan MG, Schaller GB, Baker RH, Rosenbaum HC, Robichaud WG, Desalle R (1999b) Rediscovery of Roosevelt’s Barking Deer (Muntiacus rooseveltorum) J Mammal 80:639–643 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap Evolution 39:783–791 Gatesy J (1997) More DNA support for a Cetacea/Hippopotamidae clade: the blood-clotting protein gene c-fibrinogen Mol Biol Evol 14:537–543 Giao PM, Tuoc D, Dung VV, Wikramanayake ED, Amato G, Arctander P, MacKinnon JR (1998) Description of Muntiacus truongsonensis, a new species of muntjac (Artiodactyla: Muntiacidae) from central Vietnam, and implications for conservation Anim Conserv 1:61–68 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT Nucleic Acids Symp 41:95–98 Hassanin A, Delsuc F, Ropiquet A, Hammer C, Jansen van Vuuren B, Matthee C, Ruiz-Garcia M, Catzeflis F, Areskoug V, Nguyen TT, Couloux A (2012) Pattern and timing of diversification of Cetartiodactyla (Mammalia, Laurasiatheria), as revealed by a comprehensive analysis of mitochondrial genomes C R Biol 335:32–50 Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis Syst Biol 42:182–192 Conserv Genet Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogeny Bioinformatics 17:754–755 Irwin DM, Kocher TD, Wilson AC (1991) Evolution of the cytochrome b gene of mammals J Mol Evol 32:128–144 James J, Ramakrishnan U, Datta A (2008) Molecular evidence for the occurrence of the leaf deer Muntiacus putaoensis in Arunachal Pradesh, north-east India Conserv Genet 9:927–931 Le TT, Le CV, Bui TD, Tran MH, Tran NQ, Nguyen SV, Monastyrskii AL, Eames JC (1999) A feasibility study for the establishment of Xuan Lien Nature Reserve, Thanh Hoa Province Birdlife International Vietnam Programme, Hanoi Le M, McCord WP, Iverson JB (2007) On the paraphyly of the genus Kachuga (Testudines: Geoemydidae) Mol Phylogenetics Evol 45:398–404 Osborn T, Fanning E, Grindley M (2000) Pu Hoat proposed nature reserve: biodiversity survey and conservation evaluation Society for Environmental Education, London Osgood W (1932) Mammals of the Kelley-Roosevelts and Delacour Asiatic expeditions Field Museum of Natural History Publications 312 Zoological Series 18:192–339 Palumbi SR, Martin AP, Romano S, McMillan WO, Stice L, Grabowski G (1991) The simple fools guide to PCR, version 2.0 Privately published Pang H, Liu W, Chen Y, Fang L, Zhang X, Cao X (2008) Identification of complete mitochondrial genome of the tufted deer Mitochondrial DNA 19:411–417 Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution Bioinformatics 14:817–818 Randi E, Pierpaoli M, Danilkin A (1998) Mitochondrial DNA polymorphism in populations of Siberian and European roe deer (Capreolus pygargus and C capreolus) Heredity 80:429–437 Swofford DL (2001) PAUP* Phylogenetic Analysis Using Parsimony (*and other methods), version Sinauer Associates, Massachusetts Timmins RJ, Duckworth JW, Long B (2008) Muntiacus rooseveltorum In: IUCN 2012 IUCN Red List of Threatened Species Version 2012.2 \www.iucnredlist.org[ Accessed 10 Sep 2013 Xiao CT, Zhang MH, Fu Y, Koh HS (2007) Mitochondrial DNA distinction of northeastern China roe deer, Siberian roe deer, and European roe deer, to clarify the taxonomic status of northeastern China roe deer Biochem Genet 45:93–102 123 ... (2007) Mitochondrial DNA distinction of northeastern China roe deer, Siberian roe deer, and European roe deer, to clarify the taxonomic status of northeastern China roe deer Biochem Genet 45:93–102... taxonomic status of the Roosevelt’s Barking Deer is also controversial Morphologically, this species cannot be distinguished from other closely related taxa within the species complex, i.e., Muntiacus. .. volume consisted of 21 ll (10 ll of mastermix, ll of water, ll of each primer at 10 pmol/ll and ll of DNA or higher depending on the quantity of DNA in the final extraction solution) PCR condition