DSpace at VNU: Discovery of the Roosevelt’s Barking Deer (Muntiacus rooseveltorum) in Vietnam

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 Roo-sevelt

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S H O R T C O M M U N I C A T I O N

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

Roo-sevelt’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 Muntjac Muntiacus rooseveltorum ND4 Cytochrome b G-fibrinogen 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 (Osgood1932), 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

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

M Le H M Nguyen

Centre for Natural Resources and Environmental Studies, VNU,

19 Le Thanh Tong Street, Hanoi, Vietnam

T V Nguyen H T Duong L D Dinh

Department of Genetics, Faculty of Biology, Hanoi University of

Science, VNU, 334 Nguyen Trai Road, Thanh Xuan District,

Hanoi, Vietnam

Present Address:

L D Dinh

Department of Fundamental Sciences, VNU-School of Medicine

and Pharmacy, 144 Xuan Thuy Road, Cau Giay District, Hanoi,

T Do Forest Inventory and Planning Institute, Vinh Quynh, Thanh Tri, Hanoi, Vietnam

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 DOI 10.1007/s10592-014-0581-4

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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

tru-ongsonensis, 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 mito-chondrial DNA from a partial 16S gene of these samples with that of the type specimen In addition, we recon-structed 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

Fig 1 Known localities of the

Roosevelt’s Barking Deer

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NADH dehydrogenase subunit 4 (ND4), and a nuclear

gene, the blood clotting protein, c-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

roosevelto-rum 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 distinc-tiveness and resolve the taxonomic status of M roose-veltorum 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 vuqu-angensis, and two outgroups, Panolia eldii and Elaphodus cephalophus, from GenBank were also added to the dataset (Table1)

Table 1 GenBank accession

numbers, and associated

samples that were used in this

study

All sequences generated by this

study have accession numbers:

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

KJ425275 KJ425295 – KJ425286 M 1.1 This study Muntiacus

vuquangensis

Muntiacus truongsonensis

truongsonensis

putaoensis

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

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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, 5 ll of

water, 2 ll of each primer at 10 pmol/ll and 2 ll 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 (UltraPureTM, Invitrogen) Gels were

stained for 10 min in 19 TBE buffer at 2 pg/ml of

ethi-dium-bromide and visualized under UV light Successful

amplifications were purified to eliminate PCR components

using GeneJETTM PCR Purification kit (Fermentas,

Can-ada) 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

Table2

Phylogenetic analyses

The sequences were aligned in BioEdit v7.1.3 (Hall1999)

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 imple-mented 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 algo-rithm, 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-replica-tions and simple taxon addition We regard bootstrap val-ues (BP) of C70 % as strong support and valval-ues of \70 %

as weak support (Hillis and Bull1993)

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 sam-pled 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 dis-carded from the final analyses using the burn-in function Two independent analyses were run simultaneously Both

Table 2 Primers used in this study

H15915R 50-GGAATTCATCTCTCCGGTTTACAAGAC-30 Irwin et al ( 1991 )

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runs were stabilized after 9,000 and 11,000 generations in

the analyses of the 16S and combined datasets,

respec-tively 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

diver-gence 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 vu-quangensis 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 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)

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mitochondrial-gene dataset (Amato et al 1999a), except

for the sister relationship between M truongsonensis and

M putaoensis Overall, our results indicate that each

spe-cies within the previously considered M rooseveltorum

species complex, including M putaoensis, M

roosevelto-rum, and M truongsonensis, is genetically distinct

Inter-specific genetic variation within the complex

ran-ges from 2.2 to 3.1 % based on two mitochondrial genes

(Table3) 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

ca-preolus (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

mito-chondrial DNA compared to other related taxa

Intra-spe-cific genetic distance within the three species of the

M rooseveltorum species complex based on cytochrome b

and ND4 is lower than 0.4 % (Table3 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

esca-lating 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 asso-ciate 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: Mun-tiacidae) from central Vietnam, and implications for conserva-tion Anim Conserv 1:61–68

Hall TA (1999) BioEdit: a user-friendly biological sequence align-ment 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 Cetartio-dactyla (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

Table 3 Uncorrected (‘‘p’’) distance matrix showing percentage pairwise divergence calculated based on cytochrome b and ND4 genes

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 –

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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

Publica-tions 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 4 Sinauer Associates, Massachusetts Timmins RJ, Duckworth JW, Long B (2008) Muntiacus roosevelto-rum 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 northeast-ern China roe deer Biochem Genet 45:93–102

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