Conservation Genetics

, Volume 15, Issue 4, pp 993–999

Discovery of the Roosevelt’s Barking Deer (Muntiacus rooseveltorum) in Vietnam

Authors

    • Department of Environmental Ecology, Faculty of Environmental ScienceHanoi University of Science, VNU
    • Centre for Natural Resources and Environmental Studies, VNU
  • Thanh V. Nguyen
    • Department of Genetics, Faculty of BiologyHanoi University of Science, VNU
  • Ha T. Duong
    • Department of Genetics, Faculty of BiologyHanoi University of Science, VNU
  • Ha M. Nguyen
    • Centre for Natural Resources and Environmental Studies, VNU
  • Long D. Dinh
    • Department of Genetics, Faculty of BiologyHanoi University of Science, VNU
    • Department of Fundamental SciencesVNU-School of Medicine and Pharmacy
  • Tuoc Do
    • Forest Inventory and Planning Institute
  • Hai D. Nguyen
    • Xuan Lien Nature Reserve
  • George Amato
    • Sackler Institute for Comparative GenomicsAmerican Museum of Natural History
Short Communication

DOI: 10.1007/s10592-014-0581-4

Cite this article as:
Le, M., Nguyen, T.V., Duong, H.T. et al. Conserv Genet (2014) 15: 993. doi:10.1007/s10592-014-0581-4

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.

Keywords

MuntjacMuntiacus rooseveltorumND4Cytochrome bG-fibrinogenConservation

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 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.
https://static-content.springer.com/image/art%3A10.1007%2Fs10592-014-0581-4/MediaObjects/10592_2014_581_Fig1_HTML.gif
Fig. 1

Known localities of the Roosevelt’s Barking Deer

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 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 NADH dehydrogenase subunit 4 (ND4), and a nuclear gene, the blood clotting protein, γ-fibrinogen.

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 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 γ-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).
Table 1

GenBank accession numbers, and associated samples that were used in this study

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

All sequences generated by this study have accession numbers: KJ425271–KJ425303

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 μl 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 μl (10 μl of mastermix, 5 μl of water, 2 μl of each primer at 10 pmol/μl and 2 μl of DNA or higher depending on the quantity of DNA in the final extraction solution). PCR condition was: 95 °C for 15 min 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 6 min. 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 1 % agarose gel (UltraPure™, Invitrogen). Gels were stained for 10 min in 1× TBE buffer at 2 pg/ml of ethidium-bromide and visualized under UV light. Successful amplifications were purified to eliminate PCR components using GeneJET™ 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 2.
Table 2

Primers used in this study

Segment

Primer

Length

Sequence

Reference

Cytb-1

L14724

450

5′-CGAAGCTTGATATGAAAAACCATCGTTG-3′

Irwin et al. (1991)

 

H15149

 

5′-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3′

H15149

Cytb-2

L15162

750

5′-GCAAGCTTCTACCATGAGGACAAATATC-3′

Irwin et al. (1991)

 

H15915R

 

5′-GGAATTCATCTCTCCGGTTTACAAGAC-3′

Irwin et al. (1991)

ND4

ND4-F

714

5′-CTCATRCCYCTGACCTGACTAT-3′

This study

 

ND4-R

 

5′-GCTATAAATTCGGTAAGTGGATT-3′

This study

16S

AR

567

5′-CGCCTGTTTATCAAAAACAT-3′

Palumbi et al. ( 1991)

 

BR

 

5′-CCGGTCTGAACTCAGATCACGT-3′

Palumbi et al. (1991)

G-fibrinogen

GL1

574

5′-AGAHAAYTGCTGCATCTTAGATG-3′

Gatesy (1997)

 

GR1

 

5′-TTCRTATTTCATAATTTCTTC-3′

Gatesy (1997)

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 tree-bisection 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 ≥70 % 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 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).
https://static-content.springer.com/image/art%3A10.1007%2Fs10592-014-0581-4/MediaObjects/10592_2014_581_Fig2_HTML.gif
Fig. 2

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)

The results of our phylogenetic analyses using two mitochondrial genes, cytochrome b and ND4, and a nuclear gene, γ-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 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 inter-specific 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 3 and James et al. 2008).
Table 3

Uncorrected (“p”) distance matrix showing percentage pairwise divergence calculated based on cytochrome b and ND4 genes

S. no.

Taxa

1

2

3

4

5

6

7

8

9

10

11

12

13

1.

M. vuquangensis (FJ705435)

            

2.

M. vuquangensis (AF042720)

2.2

           

3.

M. vuquangensis (Mu 1.1)

0.97

1.59

          

4.

M. vuquangensis (Mu 6.21)

0.96

1.67

0.09

         

5.

M. vuquangensis (Mu 6.9)

2.1

0.80

1.67

1.58

        

6.

M. vuquangensis (Mu 6.13)

2.03

0.71

1.59

1.49

0.06

       

7.

M. putaoensis (Mu 4.1)

5.89

5.77

5.78

5.49

5.78

5.77

      

8.

M. truongsonensis (Mu 1.9)

6.31

6.46

5.98

6.0

6.15

6.04

2.91

     

9.

M. truongsonensis (Mu 2.4)

6.04

6.29

5.71

5.82

5.88

5.77

2.91

0.38

    

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.

M. rooseveltorum (Mu 2.20)

5.72

6.18

5.4

5.72

5.50

5.49

2.59

3.08

2.91

0

  

12.

M. rooseveltorum (Mu 6.3)

5.66

6.18

5.34

5.72

5.50

5.44

2.49

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

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 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.

Copyright information

© Springer Science+Business Media Dordrecht 2014