Molecular systematics of Nycticebus coucang and its relationships to the other Malaysian primates based on cyt B gene sequences - Malaysian Agricultural Repository

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The Journal of

rssN 0121-812 Vol )orVI 2009-2010

WILDLIFE

ANd PARKS

Journal of Wldlife and Parks (2009 2010) 26 : 119-126

MOLECULAR SYSTEMATICS OF' Nycticebus coucqng AND ITS RELATIONSHIPS TO THE OTIIER MALAYSIAN PRIMATES BASED ON CYT B GENE SEQUENCES,

Badrul Munir Md-Zaint, Norlindawati Abd. Patehl, Ang Khai Chungt, Vun Vui Fuil, Za'inal Zlhari Zainuddin'?, Maklarin Lakinr, Ahmad Ampengr.4, Shukor M. No/ &

Mahani Mansor Clydet

tSchool of Environmentel qnd Nalural Resource Sciences, Faculty of Science and Technology,

Universiti Kebangsaan Malaysia, 43600 Bangu Selangor, Malaysia

'1Jctbqtan PERHILITAN, Km l0 Jalan Cheras, 56100 Kuala Lumpur, Malaysia 3 The Board ofTrustees of Sabah Parks, Kinabalu Park, Kota Kinabalu, Sabah, Malaysia

aSarawak Forestry Department, Kuching, Sarawak Malaysia

ABSTRACT

This research was conducted to portray the phylogenetic relationships of a genus of Malaysian primitive primate (Nycticebus) with several groups of Malaysian higher primates (Presbytis, Trachypithecus, Macaca, and Symphalangus). We sequenced 388 base pairs of the partial Cytochrome B mtDNA gene sequences. A single individual of larsias banc.lnus was selecled as an outgroup to root the tree. Data analyses were done using character-based approach (Maximum Parsimony, MP) and distance-based approach (Neighbour-joining, NJ). MP and NJ analysis show two major monophyletic clades betweerr Nyticebus coucang and other Malaysian primates. This study supports th€ separation between Prosimii and Anthropoidea primat€s.

Keywords: Nycticebus coucang, Molecular systematics, Malaysian primates, Cytochrome B, DNA sequences

INTRODUCTION

Although Malaysia is a relatively small country it is listed as one of the twelve world's mega diversity countries (Cox, 1997). The tropical rain forests of Malaysia provide natural habitats for a great variety ofspecies including primates. Bennett (1991) has listed out the entire Anthropoidea primates found throughout the country. Malaysia contains one species of great ape (Pongo pygmaeus), four lesser apes (Hylobates lar, H. agilis, H. muelleri and S. syndactylus), three cercopithecines (Macaca fascicularis, M. nemestrina and M. arctoides) and several colobine monkeys (iy'asalls larvatus, Presbytis spp and Trachypithecas spp). In addition, Malaysia also has two types olprosimians, Tarsias and. Nycticebus (Brandon-Jones et a/., 2004).

Cytochrome b mtDNA gene is well-known as a gene that evolves rapidly and can show variations within and between species (Irwin el a/., 1991). Caine' et al. Q006) emphasized that CytB gene is ideal for species identification because it shows limited variability within and much greater variation between species. This mitochonddal DNA gene is widely used to infer phylogenetic relationships for mammals (Irwin et aI.1991;Kocher et a/., 1989; Pesole etal., 1999) as well as in primate studies (Li et a1.,2001; Mortagnon et aL,2001;Md.-Zain et al.,2005a; Yod,er et al., 1996 Zhang & Ryder 1998a,b). Most of the phylogenetic relationship studies on Malaysian primates

120 Baclrul Munb Md-Zain, Norlinda ati Abd. Pateh, Ang Khai Chun, yun Vui Fui, Zainal Zahari Zainuddin, Maklarin Lakim, Ahnad Ampe g, Shukor M. Nor & Mahani Mansor Clyde

that used molecular data focused on the genus level, for examples in the genus P/erbytis (Md-Zain 2001), Trachypithecrr and Naralu (Md -Zain et a|.,2005b). Nevertheless, the molecular phylogeny relationships among the entire species of primates in Malaysia has never been carried out before.

In addition very limited inferences can be made on molecular systematics of Nycticebus as only a few sequences are available in Gene Bank. Thus, this study carries one objective, which is to determine phylogenetic relationships between primitive and higher primates in Malaysia.

MATERIALS AND METHODS Taxa sarnpled

All samples used in this research are listed in Table 1. Several institutions (PERHILITAN, Sabah Parks, and Sarawak Forestry Department) provided the necessary facilities and assistance for tissue sample collection.

DNA extraction, amplification and sequencing

The Cyt-b gene was choosen to reflect sequenc€ variation in mitochondrial DNA (mtDNA). Total DNA was extracted from tissue and blood using phenol chloroform extraction method (Hillis et ql., 1996), FTA@ Cards according to the Whatman@ Protocol for PCR and Gene All Tissue SV (Plus!) Mini Extraction Kit (Gene All). L14724 and Hl5l49 (Table 2) were used as primers to amplify the Cyt-b gene (Irwin et al. 1991; Kocher el al., 1989). The PCR components for the samples are shown in Table 3. Estimated fragment size of the CytB gene for all amplified samples, observed after electrophor€sis on 1.5% agarose gel is approximately 500bp (Figure l).

The PCR products were purified using QlAquick gel purification kit protocol (QIAGEN) and sent to company (1" Base, Kuala Lumpur) for sequencing.

Sequence Alignment and Data Analyses

Sequences were aligned using the ClustalW program, with minor adjustm€nts made manually.

Data matrix was analyzed by excluding 3l characters and u sing Tarsius bancarrJ as an outgroup.

Two primary methods were used to discern phylogenetic relationships: maximum parsimony (MP) and neighbor joining (NJ). These analyses were conducted using PAUP version 4.0. For unweighted MB trees were obtained by heuristic searches treating all nucleotide substitutions as unordered. Data were also subjected to bootstrap analysis with 2000 replications. Tree was also constructed using the NJ method by employing the Kimura 2 parameters distance option of PAUP.

Homoplasy was quantified using the consistency index (CI) and the homoplasy index (HI).

RESULTS AND DISCUSSION

The 357 remaining characters were examined. Results showed thal 23.53Yo were constant characters, 2.24yo chdracterc were parsimony uninformative and 74.23''/o were parsimony informative. Unweighted MP analysis produced a single bootstrap tree (length : 619, CI = 0.6559, ]JI:0.344I). Nycticebus, Symphalangas and Cercopithecidae were sorted into their own distinct monophyletic clade with 100% bootstrap support (Figure 2). NJ analysis yielded a single tree for which topology was slightly different from the MP tree (Figure 3). In the NJ 1lee, Nycticebus, and Cercopithecidae were sorted into their own distinct monophyletic clade with 100% bootstrap support while 99% bootstrap supported the Symphalangus clade. According the Hillis and Bull (1993), 70% or more is the minimum threshold in order to estimate phylogenies accurately.

Moleculor Slstematics Of Nycticebus coucang and l2l Its Relationships to the Other Malaysian Primates Based

On Cjt B Gene Sequences

Besides looking at tree topologies and bootstrap values, phylogenetic relationships between Nycticebus afid other Malaysian primates can also be derived from Kimura 2 Parameters distance matrix. Table 4 summarized the average percentage distance among Malaysian primates The average intra-specific genetic distance was 6.013 in the Nycticebus. The average inter-generic genetic distance between primitive primate (Nycticebus) and higher primates (Symphalangus, Trachypithecus atd Presbytis) was 91 .107

The topology of the tree is generally in agreement with the widely accepted classification of primates (Delson, 1980) that was based on fossil records and other molecular analyses (Arnason et al., 2000', Bro...uln et al., 1982; Ferris et al., 1981t Hayasaka et al., 1988) except the position of the Symphalangus. Basically genus Nycticebus is more closely related to family Cercopithecidae than to family Hylobatidae. However, CytB sequences shows that the Nycticebus is more closely related to genus Slmphalangus than to Macaque and leaf monkey.

The N. pygmaeus appears to be distantly related to other two types of lorises and it is more closely related to iy'. c. menagensis, This result strongly supports the study ofskull measurement by Groves (1998, 2001) that recognized N. pl,gmaeus as a separate species. N. c. menagensis is

closestto N. pygmaeas compared to other N. coucang utbspecies because ofa ring-species effect.

CytB gene sequences in this study showed interesting result between M c. coacazg samples from Peninsular Malaysia. Samples lrom Peninsular Malaysia together with M bengalensis werc from one clade, while the sample from Borneo together with N. coucang sample ftom Gene Bank and N.

c. menagensis were from another distinct clade. This result showed that the sample from Borneo (NcSP32) may be belongs to subspecies menqgensis that support Brandon-Jones et al. (2004) and Groves (2001). N. c. javanicus which is one ofM coacczg subspecies formed a separate branch.

This result may not support Brandon-IoBes et ql. (2004) that classified M c. jav.tnicus togelher with ,AI c. coucang, and N. c. menqgensis. However, this result is only based on one sample of-l{

c. javanicus from Gene Bank.

CONCLUSTON

This study has increased our understanding of phylogenetic relationships among Malaysian primates from the molecular perspectives of the CytB. Locus used in this study can be used to portray primate phylogenetic relationships as it was successfully used in other Asian colobines and cercopithecines. Even though this study showed each genus formed a distinct monophyletic clade, it also showed some conflicting result as the iy'.l,c/ic ebus clade is clos€r to Symphalangus than family Cercopithecidae. The species relationships amongNycticebus species however cannot be resolved. Thus, more data on nucleotide sequences ofboth mitochondrial and nuclear genes is required as well as larger number of samples.

ACKNOWLEDGEMENTS

We are deeply indebted to several institutions who provided us with necessary facilities and assistance for tissue sample collection including UKM, PERHILITAN, Zoo Taiping, Zoo Melaka, Sarawak Forestry Department, and Sabah Park. This research was made possible under grants IRPA 0802020019 EA30l lrom the Ministry of Science Technology and Innovation, Malaysia.

Badrul Munir Md-Zain, No indarati Abd Pateh, Ang Khai Chun, Vun rui Fui. Zainat Zahari Zainuddin, MaHsri Lakin' Ahmad Anpeng, Shukor M Nor & Mahani Mansor Clyde

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Bennett, E. L. 0991). Diurnal primates. In Kiew, R. (ed.). The State of Nature Conservation in Malaysia, pg. 150-172. Malayan Nature Society.

Brandon-Jones, D., Eudey, A. A., Geissmann, T, Groves, C. P, Melnick, D. J., Morales, J C, Shekelle, M., & Stewart, C.-B. (2004). Asian primate classification. Internqtionol Journal of Primatology 25 (l):94-164.

Brown, W. M., Prager, E. M., Wang, A. & Wilson, A. C. (1982). Mitochondrial DNA sequences of primates: tempo and mode ofevolution. Journal of Moleculat Ettolution 18:.225-239 Caine', L., Lima, G., Pontes, L., Abrantes, D, Pereira, M. & Pinheiro, M. F (2006)' Species

identification by cytochrome b gene: casework samples. ft ternational Congress Series 1288:145-147.

Cox, G. W (1997). Conservation biology: concepts and applications. 2'd edition. United State:

Wm. C. Brown Publishers.

Delson, E. (1980). Fossil Macaque, phyletic relationships and a scenario of development ln:

Lintlburg, D. G. (€ditor). The Macaques: studies in ecology, behavior and evolution, pg' 10-30. New York: Van Nostrand Reinhold.

Ferris. S. D., Wilson, A. C. & Brown, W. M. (1981). Evolutionary tree lor apes and humans based on cleavage maps ofmitochondrial DNA. Proc. Natl. lcad. Sci. USA 78:.2432-2436' Groves, C. (1998). Systematics ofTarsiers and Lorises Primates 39 (l):13-21.

Groves, C. (2001). Primate taxonomy. Smithsonian Instituition Press, Washington'

Hayasaka, K., Gojobori, T. & Hora, S. 0988). Molecular phylogeny and evolution of primate mitochondrial DNA. Molecular Biological Evolution 5(6): 626-644.

Hillis, D. M. & 8u11. J. J (1993). An empirical test of bootstrapping as a method for assessing confidence in phylogen€tic analysis. S.rrst. Biol 42:.182-192.

Hillis. D. M., Moritz, C., & Mable, B. K. 0996). Molecular systematics, Sinauer, Massachusetts.

Irwin. D. M.. Kocher, T. D. & Wilson, A. C. (1991). Evolution of the Cytochrome B gene of mammals. Journal Molecular Evolution 32:128-144.

Kocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V, Paabo, S., Villablanca, F. X , & Wilson, A. C. (1989). Dynamics ofmitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. lcad Sci I/'Sl 86: 6196-6200' Li, M., Liang B. & Tamate, H. B. (2001). Molecular phylogenetic relationships among Sichuan

Snub-nosed monkeys (Rhinopithecus roxellanae) inlerred from mitochondrial Cytochrome-b sequences' P r imat es 42(2): I 53 -160.

Md-Zain, B. M. (2001). Molecular systematics of the genus Presbytis. PhD thesis. New York:

Columbia University.

Md-Zain, B. M., Vun, V F., Zainal, Z. Z'Lakim, M., Ghumal, M. T & Mahani, M. C (2005a)' Genetic distance of Malaysian primates based on mitochondrial DNA cytochrome-b sequences. In Abd. Rahim, S., Surif, S., Abdullah, M. P, Samsudin, A. R , Mohd Rafek, A. G., Ratnam, W, Abd. Ghani, i., Md-Zain, B. M., Mohd, Said, M. N , Kee Alfian, A' A. & Ng, Y F. (eds). Proceeding of Second Regional Symposium on Environment and Natural Resources, pg. 39-41. Bangi: Universiti Kebangsaan Malaysia.

Md-Zain. B. M., Morales, J. C., Hassan, M. N., Jasmi, A. & Melnick, D. J. (2005b) Phylogenetic

position of the genus Trachypithecus as inlerred from Y-chromosome and autosomal

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Molec lar Systematics Of Nycticebus coucang an.J t23 hs Relationships to the Other Malalsian Primates Based

On Clt R Gene Sequences

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Table l: Sampling details No. Code/Accession

Number

Species

Locality

PmsBM23

P. m. siamensis

Ulu Besut, Terengganu

2 PmsBM24

P. m. siamensis

Ulu besut, Terengganu

3 PmrBM2T

P. m. robinsoni

Ulu Kenas, Perak

4 PmrBM3l

P. m. robinsoni

Ulu Kenas, Perak

5 PhBM67

P. hosei

Tawau Hill Park, Sabah

6 PhBMTO

P. hosei Lembah Danum, Sabah

7 PrBMl02

P. rubicunda Tawau Hill Park, Sabah

8 PIMK0l

P. rubicunda Tawau Hill Park, Sabah

9

TCBM0l T. crislatus Kota Kuala Muda, Kedah

10 TcBM03

T. cristatus Kota Kuala Muda, Kedah

11

TcAAO1

T. cristatus Samunsam, Sarawak

t 2 ToBM09

T. obscurus Changloon, Kedah

l 3 ToBMl0 T. obscurus Zoo Taiping, Perak

t4 ToL0l

T. obscurus Langkawi, Kedah

15 MnBMl02

Macacct nemestrina Zoo Melaka

16 MnBMl03

Macqca nemestrina Zoo Melaka

17 MaBM104

Macaca arctoides Zoo Melaka

l 8 MaZZ0|

Macaca arcloides

Unknown

19 MfMFO2

Macaca fascicularis Pulau Sapi, Sabah

20 MfSP5OL

Macaca fascicularis Tawau Hill Park, Sabah

22 HyZZ090 S. syndacrylus

Zoo Melaka

23 HyZZO95

S. syndactylus

Ipoh, Perak

HyZZ096

S. syndactylus Pahang

zo

TbA8011077

Tarsius bancanus Gene Bank

Badtal Munir Md-Zain, Norlindrwati Abd. Pateh, Ang Khai Chun, Vun l/ui Fui, Zainal Zahari Zainuddin, Maklarin Lakin, Ahnad Anpeng, Shukor M. Nor & Mahani Mansor Clyde

27 NcZZ098

N. c. coucang Selangor

2 8 NcZZ099

N. c. coucang Selangor

29 NcSP32

N. coucang Ranau, Sabah

3 0 NcAY687889

N. coucang Gene Bank

3 l NcmAY87836l

N. c. menagensis Gene Bank

NcjAY87836s

N. c. javanicus

Gene Bank 33 NbAY441477

Ny c t i c e b u s b e ngal ens is Gene Bank

NpAY6878892

N))cticebus pygmaeus Gene Bank

Table 2: Oligonucleotide primer pair and PCR conditions

Forward/Reverse Primer Sequences

Ll4'7 24: 5>CG A AGCTTGATATGAAAAACCATCGTTG-3) H1 5 149:5r-AAACTGCAGCCCCTCCGAATGATATTTGTCCTCA-3'

Phase Temperature (oC) Duration

Number ofCycles

Pre-denaturation 94 3 min

3 5

Denaturation

94 I min

Annealing 55 I min

Elongation

72 I min

Post-elongation

72

l0 min

Table 3 : PCR reagent mixture for amplification ofCytB gene.

Tissue sample FTA card Components

Concentrations Volume

(r.rl)

Concentrations

Volume

(rrl)

DNA Template

3.0-5.0

0r<

7a4 DNA Polymerase

5 U/pl 0 . 5 5 U/pl 0.5

M g C l , 50 mM 3.0 5 0 m M 3 . 0

dNTP 1 m M 1 . 0

l m M

1 . 0

PCR Bulfer 5X 5 . 0 5X 5 . 0

CytB L 20 pmol/pl 0.5 20 pmol/pl 0 . 5

CytB H 20 pmot/pl 0.5 20 pmol/pl 0 . 5

ddH,o

J 4 - f - J O . )

39.5

Total

50.0 50.0

Moleculqr $)stematics Of Nycticebus coucang and hs Relationships to the Other Malaysian Prinates Based On Clt B Gene Sequences

Table 4. Average percentage of genetic distance among and betweerr Presbytis, Trachypithecus, Macaca,

500 bp ---->

Figure I . Amplification product of Cyt b gene from Symphalangus and. N. coucang Symphalangus and Nl /ir ebrs using lhe Kimura 2 Paramelers

Presbytis Trachypithecus

Macaca Symphalangus Nycticebus

Presbytis 9.059 1 9 . 1 0 3 * 25.623*

Trachypithecus

21.046 3 . 8 0 1 21.192*

Macaca

27.822 22.145 9.160

Symphalangus 95.262 8 8 . 5 5 1 95.337 0 . 2 8 8

Nycticebus

119.940 116.024 121.7',l0 30.670 6.013

verage percentage iiorn Md-Zajn et

Lane I

Marker l0obp (invitrogen)

Lane 5 H. lar ( 22097 ) Lane 2

S. syndactylus (22090)

Lane 6 N. coucang (2'2098) Lane 3

S. syndactylus (22095)

Lane 7 N. coucang (22099)

Lane 4 S. syndactylus (22096)

Lane 8 N. coucang (SP(P)328)

Badnl Munir Md-Zain, Norlinda, ati Abd. Pateh, Ang Khai Chut1, Yltn l/ui Fui Zainal Zahari Zainuddin, Maklarin Lakm, Ahmad Ampeng, Shukor M. Nor & Mahani Mansor Clyde

>PmsB[r23

>PmsBlvl24

>PmrBM27

>PrnrBtvl3l

>PrBtul102

>PrlvlK01

>PhBtvl67

>PhBMTO

>TcBMDl

>TcB[403

>TcAAOl

>ToBlvl09

>ToBlvl10

>ToLOl

>lvlnB lvll 02

>lvlnB lvl1 03

>lvlaB lvl l 04

>lVaZZ01

>tvltMm2

>MISPsOL

>HyZZ090

>HYZZO96

>HYZZO95

>NEZO98

>NEZO99

>Nb4Y441477

>Nq4Y878365

>NcSP328

>NcAY6A788S

>Ncrn 4Y878361

>NpAY6B78B92

>TbA8011077

Figue 2. Phylogenetic r€lationships ofspecies studied based on maximum parsimony (bootsbap values shown in bmckets).

Molecular Systematics Of Nycticebus co cang and Its Relatianships to the Other Malatsian himates Based On Cyt B Gene Sequences

>PmsEirzil

>PmsBil24

>PtflSM27 :'PrfiEMS1

>pdM102

>F tlllDl rfhBME-/

>PhBIYPO

>TEBltml

>rt A,0t

>TcBMXl

>TqBM09

>lbBMt0

>Ts[D'l

>lrhBill (l2

>IuhB ltB

>{U!BM10{

*hal

>UIMffi

>ifiEiP50L

*toHlrr

>l{lzllE

>HYz.r!'j

>r@B

>AFZIE9

>l'lblY441il77 t{cEPl?8

>l.lcAYEC/889

>FlmAYgTKnl

>lqAYrrEs

>NpAY87W

>TbAE0t Irf7

Figure 3. Phylogenetic relationships ofspecies studied based on neighborjoining (bootstmp values shown in brackets).