rssN (,127-4126 Vol )()(\t 2OOA
T h e J o u r n a l o f
WTLDLIFE
and PARKS
JounNlr- Op Wrorrre ANo Panrs, (2008) 25: 16-18
SneurNcn Vmnrrou Iu Mar,lvaN T,lpn (Tlpnus lxucus) INTeRREo Usnc Panrur SnqunNcns On Trm Cyrocrnovm
B SncunNr On Tun MrrocuouoRrAl DNA
. +JeffringJapning Rovie-Ryqn! _Cad Traelqttl, lVlarilyn-Jaoi E.s, Zainal-Zahar%aifiuddinr, Khairiah plohd Sharilf , Sivananttra"n Oia-eubilhvr.
Mohd. Farouk M. Y.a, Aniqa Akhtar Abdultdhl, &;eonien-idgf-_' '
rwildlife Genetic Resource Banking_Latroratory, Ex-situ conservation Division, Deparfnent of wildlife and National Parks (DWNP;, Peninsutar Maraysia, Ministry of Natural Resburces and
Environment Malaysia (NREI. KM t0 Jalan Cherjs. 56 t00 Kuala Lumpur. Malaysia 'Consultancy Divisio.". qY^Nl, NBE. r[M 10 Jalan Cheras, 56100 Kuila Lumpirr.
'Copenhagen Zoo. Roskildevej 38 Box 7 (OfficaSdr. Fasanvej 79), DK-2000 Fred,jriksberg, aDepartment of glemlsry }4alay_sia (KIMIA), Ministry of Science, Technology and Innovation
. Malaysia (MOSTIr. Jalan Sullan 4666t. peraling Jaya. Selangor Darul Ehiin. Malaysia
5Malaysian Agro-Biotechnoloey Insituti;fgtl,ryfilLrARbr.43400 serdane, S"i-eo,
xcorresponding aurhor email: jeffrine@ wildlife.gov.my
ABSTRACT comparison of 321.bp-long mtDNA cytochrome b sequences of wild and captive Malayan tapir (lapirus indicus) revealedlow variation among the individuals investigated. Phylogenetic anaJyses using,distance {neighbor-joiningt analysis- supponid the monophyleric sraius orth6 tra?tayan taprr. I wo hap totypes were identified ou t of | 3 Mal ayan tapir analyzed.
Keywords - sequence characterization, species-specific sites, phylogenetic analysis
AB_srRAK - Perbandingan 321 pasangan bes bagijujukan sitokrom b pada mitokondrial DNA ke atas Indlvrdu llar dan dalam kurungan Tapir Malaya menunjuklan variasi yang rendah. Analisa filogenetik menggunakan kaedah "disrance" menyokong starus monofiletik tapii Malaya. Dua ..hap-l,crype..
dikenalpasti daripada l3 individu Tapir Malaya yang dianalisa.
Kata kunci - pencirian sekuen, bes "species-specific,', analisa filogenetik
was done according to the manufacrurers' instructions. Sequences of olher families from the Order Perisiodacryla tFamily Equidae and Rhinocerotidae) were obtained from GenBantr:
Equas burchellii (DQ470805), Equus caballus (DQ297663). Equus przewalskii (DQ223534).
Rh i n oce ro s sondaicus (A1245'l 25 ), Dic e ro rh i nu s sumatrensis (N245723). Rhinoceros unicomis (X97336) and Ceratotherium simum (Y07726).
Cyl b sequence ol T. indicus tAFl4573q and't.
terrestris (AF056030) were also obtained from the GenBank.
This study compares a 321bp segment of the cytochrome b (c-yr D) of the mitochondrial DNA (mtDNA) gene to assess the sequence variation of the Malayan tapirs, All sequences were deposiled in the GenBank database (accession numbers EU224327 - EU224339). Samples were collected from l3 wild and captive-Malayan tapir (Table 1). Total genomic DNA from tisiue samples was isolated following a modified CTAB-based protocol (Grewe 2t al., 1993).
Blood samples were collected and preserved in Whatman FTA cards and DNA isolation
Rovrs-RvAN Er AL. / SeeusNcs VeruerloN IN MALAYAN TAPR Usrlc Cvrocsnolre B TABLE 1
Specimen information, GenBank accession numbers and haplotypes designation for the Tapirus rnclcus
No SamPles
Sex
Locality Origin GenBank Haplott?esAcc. No. Designation
Birth
Status 1 TAPOI
2 TAPO2 3 TAPO3 4 TAPO4 5 TAPO5 6 TAPO6 1 T APO'7 8 TAPO8 9 TAPO9 IO TAPIO I I T A P I I I2 TAPI2 1 3 T A P 1 3
I I 2 2 F
F M F M M F M F M
Melaka Zoo Melaka Zoo Melaka Zoo Melaka Zoo Melaka Zoo Terengganu Melaka Zoo Melaka Zoo Melaka Zoo Terengganu
Johor Perak
Selangor Captive Selangor Wild Singapore Captive
Selangor Wild
Terengganu Selangor Selangor Terengganu
Johor Perak
8U224327 EU224328 EU224329 8U224330 8U224331 8U224332 EV224333 EU224334 8U224335 8U224336 EU22433',7 EU224338 8U224339 wild
wia
wild wild wild
2 I I 2 I 2
Approximately, 350bp of the partial cytb fragment of the mtDNA was amplified in a GeneAmp@
PCR System 9700 (Applied Biosystems) by using two cyt b universal primers: CYTb I (5'- CCA TCC AAC ATC TCA GCA TGA TGA AA- 3') and CYTb 2 (5'-GCC CCT CAG AAT GAT ATT TGT CCT CA- 3') (Brodmann et al., 2001). The PCR amplification profile follows Ryan et. al. (2006). PCR products were visualized in a 1.0Vo agarose gel and positive products were directly purified using PCR Clean-Up System Kit (Promega). Cycle sequencing was performed on both strands with the Applied Blosystems 3130x1 Genetic Analyzer using the same primers as for the PCR amplification.
Alignments and standard genetic diversity indices were done according to Rya\ et. al.
(2006). Sequence characterization (variable sites, conserved sites, parsimony-informative sites, and nucleotide composition) was carried out using MEGA version 4 (Tamura et al., 2007). Analysis of the 321-bp partial cyt b gene on the T. indicus sequences revealed a low variation in their genetic diversity. Among the T. indicus sequences, two haplotypes were observed (Table l).
Haplotype and nucleotide diversity was O.582t0.092 and 0.0062610.00136, respectively.
Out of the 321bp (including outgroups), 222 positions were conserved (69.2V0) and 99 were variable (30.87o). Out of the latter, 77 positions (78.6Vo) were parsimoniously informative.
Among the Tapirus sequences alone, 44 variable sites ( 1 3 .77o) were obserued. Furthermore, among the T. indicus sequences, only six variable sites (1.9Vo) were observed with thrce parsimoniously
informative sites (data not shown).
Pair-wise distance analysis using the Kimura- two-parameters model (Kimura, 1980) was done to estimate genetic distances among the tapir sequences and all outgroups used as performed using MEGA version 4 (Tamura e/ al.,200'7).
Distance value ranges from 0 - 1.97o within the l. rncltcus sequences. lhe I. rndrcus sequences differed from T. tenestris with the average distance value of 14.87o. Compared to the outgroup sequences, distance value of 16.0% (Order Equidae) and 18.57o (Order Rhinocerotidae) were obtained.
The phylogenetic relationships of the Order Perissodactyla are summarized in Figure I (neighbor-joining; MEGA version 4). The reliability ofthe nodes in each ofthe analyses was assessed by 1000 bootstap iterations (Felsenstien,
1985). The Rhinocerotidae formed the basal clade of the Order Perissodactyla. Generally, topology of the phylogeny tree supports the monophyly of T. indicus. T. indicus further formed two groups supported by high bootsffap value (>987o for all methods). Sequence variations in the Malayan tapir are low, probably due to the short fragment ofthe gene used coupled by the conserved state of the cyt b gene itself (Martin and Palumbi, 1993).
Currently the analysis is extended to cover the complete cyt b gene and other highly variable genes (e.g. contol region) to provide a better view ol their genetic structure, The encouraging results had DromDted more research collaboration on (nucleai) microsatellite markers and other mtDNA sesments.
t 7
RovlE-RyAN ET AL. / Sequwce VerlenoN IN MeLeyeN Tnun UsrNc CyrocriRoras B ACKNOWLEDGEMENTS
We gratefully acknowledge rhe DWNp and NRE lor providing the funding. We are also gralelul lo the Chemisrry Deparrment TKIMIA) -for necessary facilities and equipments. Special thanksthe to rhe Director Ceneral of DWNP ana KIMIA lor the support and commitunent in this collaboration.
We also thank the Copenhagen Zoo lor providing funds lo conduct some of the laboratory analysisi We thank the Directors of Ex-Situ Conservition Division. Consultancy Division, and Institute for Biodiversity of DWNP l-or providing rhe suppon and guidance rhroughour rie proieit. We thank al\o the staff\ of Mela(a Zoo. taiping Zoo and T.
C. Arapaima for assisting in sample-s collection.
The authors declare thar ihe expeiiments comply with the current laws of Malavsia.
RrrrnrNcrs
Brodmann, P.D., Nicholas, G., Schaltenbmnd. p.
& IIg, E.C. (2001). Identifying unknown game specles: experience with nucleotide sequencing of the mitochondrial cytochrome b gene and a sub-sequent basic local alignment search tool search. European Food Reseatch and Technotogy, 212 491496 .
Fetsenstein, J. (1985). Confi dencelimits onphylogenies:
An approach using the bootsftap. Evotution,3t :'g3 7 9 1 .
Grewe, P.M., IGueger, C.C., Aquadro, C.F., Bermingham, E., Kincaid, H.L. & Miy, B. (1993).
Mitochondrial variation among lake trout (Salvelinus namaycush) strains stocked into Lake Ontorio.
Canadian Joumal of Fisheries and Aquatic Scicnces, 50:239'l-2403.
Kimura, M. (1980). A simple method for estimating evolutionary rate of base substitutions througi comparative studies of nucleotide segtJences. Joumal of Molecular Evolution, 16. lll-12}.
Martin, A.P. & Palumbi, S.R. (1993). horein Evoludon in Different Cellular Environment: Cytochrome b in Sharks and Mammals. Molecular Biology and Evo lutio n, l0(4\ : 87 3-89 t.
Ryan. J.R.J. & Esa. Y.B. (2006). ph)logenelic Analysis ol Hampala Fishes {Subfamily Cyprininae} in Malaysia Tnferred from Panial Mirochondrial Cyrochromi b DNA Sequences. ZaoloIical Science,23.. Zg3 g}l.
Tglnl'ra, K., Dudley, J., Nei, M. & Kumar, S. (2007).
MEC-A4t Molecular E\olurionary Genelics Analysis (MECA)sofiware \ersion 4.0. Molecular Biology and Evolut ion, 24(8)i 1596-1599.
Equus burchellii Equus caballus Equus przewalskii
us sumafrensrs Ceratotheium simum Rhinoceros sondaicus Rhinoceros unicornis
- 0.01 $b6fil,fol|6/stt6