3HUWDQLND-7URS$JULF6FL    TROPICAL AGRICULTURAL SCIENCE Journal homepage: http://www.pertanika.upm.edu.my/ Population Genetics of the Cave-dwelling Dusky Fruit Bat, Penthetor lucasi, Based on Four Populations in Malaysia Mohd Ridwan A. R.1, 2* and M. T. Abdullah2 1 Centre for Pre-University Studies, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia 2 Department of Zoology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia ABSTRACT The population genetics of P. lucasi was inferred using 1,061 base pairs (bp) of the Cytochrome b mitochondrial gene.$WRWDORILQGLYLGXDOVZHUHFODVVL¿HG a priori according to their localities, namely, Miri, Kuching, Sri Aman and Kelantan. Results showed that the populations of P. lucasi were separated into two haplogroups, namely, Haplogroup 1 (found in Miri and Kuching populations) and Haplogroup 2 (Miri, Kuching, Sri Aman and Kelantan populations). This separation was supported by bootstrap values LQWKHSK\ORJHQHWLFVDQDO\VHV LQWKHPD[LPXPOLNHOLKRRGDQGLQ%D\HVLDQ  A high level of genetic divergence was detected between two haplogroups (3.88%) and this separation could be related to historical events which include multiple colonisation DQG3OHLVWRFHQHUHIXJLDGXULQJWKH/DVW*ODFLDO0D[LPXPLFHDJHSHULRG+LJKJHQHWLF GLYHUJHQFHZLWKLQ0LUL  DQG.XFKLQJ  SRSXODWLRQVFRXOGEHGXHWRWKH presence of a species complex within the P. lucasi populations. The presence of haplotypes from both the populations in Haplogroup 1 and Haplogroup 2 might be due to the ability of WKLVSDUWLFXODUVSHFLHVRIEDWVWRSHUIRUPORQJGLVWDQFHÀLJKWIRUIRUDJLQJ$KLJKJHQHÀRZ EHWZHHQWKHVHSRSXODWLRQVVXJJHVWVDZLGHVSUHDGIHPDOHJHQHÀRZRIP. lucasi, judging from the distance of both localities. Meanwhile, the absence of a deep structure from the haplotype trees further proves that P. lucasi may have had a wide dispersal ability since the Pleistocene has allowed for genetic exchange to occur between the regions in Malaysia. ARTICLE INFO Article history: Received: 17 May 2010 Accepted: 6 July 2011 E-mail addresses: rahmanridwan@gmail.com (Mohd Ridwan A. R.), abdullahmt2@gmail.com (M. T. Abdullah) * Corresponding author ISSN: 1511-3701 © Universiti Putra Malaysia Press Keywords: Penthetor lucasi, population study, genetic GLYHUVLW\PLWRFKRQGULDO'1$ INTRODUCTION An understanding of a species population structure typically provides significant Mohd Ridwan A. R. and M. T. Abdullah LQIRUPDWLRQWRDGGUHVVTXHVWLRQVUHODWLQJ to both past and present evolutionary and behavioural processes of organism. Thus, WKHLQWURGXFWLRQRIPROHFXODUWHFKQLTXHVLV a great breakthrough in the pursuit of such understandings. This is especially true for studies in which traditional methods, such as the direct observation of individuals or populations, are greatly restricted (Burland & Worthington-Wilmer, 2001). Numerous studies on intraspecific phylogenetics and phylogeography of organisms have also positively impacted the current level of knowledge of species evolution and speciation. The use of genetic markers has led to the description and a better understanding on social life (Bryja et al., 7RGD\ studies on population genetics in bats have further revealed that phylogeographic variations are affected by various factors, such as seasonal migrations, geographical barriers, and past processes (Burland & :RUWKLQJWRQ:LOPHU  %U\MD et al.,  ,QWKH,QGR0DOD\DQUHJLRQVXFK studies have been conducted by various authors (e.g. Kitchener et al.DE Schmitt et al.+LVKHKet al $EGXOODK  0DKDGDWXQNDPVL et al., ,PHOGD7LQJJD 2WKHU than bats, population genetics studies on other taxa in this region have also been documented, including on birds (Rahman, 2000), fish (Esa et al., 2008) and frogs 5DPODK 7KHVHVWXGLHVKDYHXWLOLVHG various genetic markers, such as allozymes, 51$PW'1$DQGQXFOHDU'1$ 460 Isolation is one of the major factors facilitating evolutionary changes. A cave is a good example of habitat isolation, which is surrounded by mosaic habitat W\SHV+RZHYHUWKHSUHVHQFHRIJHQHÀRZ between populations over long distances will decrease differentiation, and it is assumed that genetic structuring is weak across the macrogeographical range in migratory bats (McCracken et al.,  :HEE  7LGHPDQQ  +LVKHK et al.,  Russell et al., 2005). In contrast, the nonmigratory ghost bat (Macroderma gigas) shows a clear genetic structuring among the populations in Australia (WorthingtonWilmer et al.,  The dusky fruit bat or Penthetor lucasi was selected for this study as it is known WROLYHVSHFL¿FDOO\QHDUWRWDOGDUNQHVVLQ isolated caves. This particular species has gone through several taxonomic reviews from Cynopterus (Ptenochirus) lucasi 7URXHVVDUW   WR Ptenochirus lucasi 7URXHVVDUW  DQGLVSUHVHQWO\SODFHG in the genus Penthetor $QGHUVHQ Maryanto, 2004). This bat is medium in size, with dark grey brown upperpart and pale buffy underpart. Sometimes, the specimens are observed to have a distinct dark shade at the centre of the head and paler near the eyes. It is widely distributed throughout the southern part of Thailand, Peninsular Malaysia, the Riau Archipelago, Borneo (Payne et al.,  &RUEHW  +LOO$EGXOODKet al.,)UDQFLV $EGXOODKet al., 2010) and Sumatra (Maryanto, 2004). A morphological Pertanika J. Trop. Agric. Sci. 35 (3) 460 - 484 (2012) 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia study on the species in Sarawak showed differences in the body and skull sizes (Sri Aman, Kuching and Miri populations). It was suggested that different ecological factors, such as breeding, crowding effect, foraging behaviour, resource availability and selective pressure, are the possible causes of the morphological variation among P. lucasi populations (Abd Rahman & Abdullah, 2010). This study aimed to examine the SK\ORJHQHWLFUHODWLRQVKLSVGLYHUVL¿FDWLRQ and genetic variation within the P. lucasi populations in Malaysia, inferring from the PW'1$&\WRFKURPHb (Cyt b) gene. It was hypothesised that P. lucasi had high site ¿GHOLW\IRUURRVWLQJ7KXVWKHUHZRXOGEH ORZJHQHÀRZDQGKLJKJHQHWLFGLYHUJHQFH among the isolated roosts in Malaysia. MATERIALS AND METHODS Samples Collection and DNA Extraction A total of 77 individuals of P. lucasi from four populations, namely Miri (33 individuals), Kuching (33 individuals), Sri $PDQ VL[LQGLYLGXDOV DQG.HODQWDQ ¿YH individuals), were used in this study (see Figure 1). The specimens were collected using mist nets and then euthanized using FKORURIRUPDQGSUHVHUYHGLQHWKDQRO prior to genetic analysis. Museum samples from the zoological collections at Universiti Malaysia Sarawak (Abdullah et al., 2010) DQGWKH'HSDUWPHQWRI:LOGOLIHDQG1DWLRQDO 3DUNRU':13 3DKDQJ ZHUHDOVRLQFOXGHG in this study. All the specimens used are listed in Appendix 1.'1$H[WUDFWLRQZDV done using the cetyltrimethylammonium EURPLGH &7$%  PHWKRG *UHZH et al.,   ZLWK WKH SUHVHQFH RI SURWHLQDVH .([WUDFWHG'1$ZDVYLVXDOL]HGRQ agarose gels containing ethidium bromide, UXQIRUDSSUR[LPDWHO\PLQXWHVDW9 and then photographed under ultraviolet 89 LOOXPLQDWLRQ7KHLVRODWHG'1$ZDV XVHGIRUIXUWKHUPW'1$DQDO\VHV Polymerase Chain Reaction (PCR) and DNA Sequencing Approximately 1061 base pairs (bp) of Cyt b were amplified following the standard protocol as described by Sambrook et al   $ SDLU RI &\W b primers ZHUH XVHG ¶&*$$*77*$7$7*$$ $$$&&$7&*77*¶ DQG NQRZQ DV L14724 (forward) (Irwin et al.,   DQG ¶$$&7*&$*7&$7&7&&**7 77$&$$*$&¶ NQRZQ DV + (reverse) (Irwin et al.,   $ WRWDO volume of 25 µl master mix was made FRPSULVLQJRI—O;FRORXUOHVV*R7DTŠ Flexi buffer, 1.5 µl of MgCl2 solution (25 mM), 0.5 µl of dNTP mix (10 mM), 1.0 µl of each forward and reverse primers (10 mM) 15.5 µl of deionised distilled water, —ORI'1$WHPSODWHDQG—O*R7DTŠ '1$SRO\PHUDVH X—O 3&5ZDVFDUULHG out using a thermocycler with 30 cycles LQFOXVLYHRIRQHLQLWLDOGHQDWXUDWLRQDWž& DQG¿QDOH[WHQVLRQDWž&IRUWKUHHDQG¿YH PLQXWHVUHVSHFWLYHO\7KHRWKHUF\FOHV FRQVLVWHGRIGHQDWXUDWLRQDWž&IRURQH minute, annealing at 40ºC for one minute and an extension at 72ºC for two minutes. $PSOL¿FDWLRQSURGXFWVZHUHWKHQYLVXDOLVHG using the agarose gel electrophoresis Pertanika J. Trop. Agric. Sci. 35 (3): 461 - 484 (2012) 461 Mohd Ridwan A. R. and M. T. Abdullah Fig. 1: Maps showing the type locality of P. lucasiVSHFLPHQVXVHGLQWKHPROHFXODUDQDO\VHV0LUL .XFKLQJ6UL$PDQ.HODQWDQ0DSZDVPRGL¿HGIURP'DOHW   PHWKRG'1$3XUL¿FDWLRQZDVGRQHXVLQJ WKH3URPHJD:L]DUG69*HODQG3&5&OHDQ 8S6\VWHP 3URPHJD&R 7KHSXUL¿HG VDPSOHVZHUHWKHQVHQWIRUVHTXHQFLQJDWD private laboratory using ABI prism TM Big dye TMWHUPLQDWRUF\FOHVHTXHQFLQJ5HDG\ Reaction Kit version 3.1 or using the ABI PRISM º  '1$ 6HTXHQFHU ZLWK WKH %LJ'\Hº7HUPLQDWRUY&\FOH6HTXHQFLQJ .LW7KHVHTXHQFLQJSURGXFWZDVUXQXVLQJ $%,;/FDSLOODU\'1$VHTXHQFHU  cm capillary). DOLJQPHQWVRI'1$VHTXHQFHVZHUHGRQH using CLUSTAL X (Thompson et al.,   VRIWZDUH 7KH SDLUZLVH GLVWDQFH between the populations were computed XVLQJWKH0ROHFXODU(YROXWLRQDU\*HQHWLF $QDO\VLV 0(*$  VRIWZDUH YHUVLRQ  (Kumar et al., 2004), with correction using a Kimura 2-parameter (K2P) model (Kimura,  7KHWLPHRIGLYHUJHQFHRIEDWVZDV estimated following Brown et al.   which was based on an evolutionary rate of Cyt b gene at 2% substitution rate per million years and calculated using Kimura-2 Sequence Alignment and Phylogenetic parameter distance matrix implemented in Analyses 0(*$YHUVLRQ (Kumar et al., 2004). A maximum likelihood (ML) tree was 7KH'1$VHTXHQFHUHVXOWVZHUHGLVSOD\HG using the CHROMAS version 1.45 constructed by using phylogenetics analysis VRIWZDUH 0F&DUWK\ 7KHPXOWLSOH using Parsimony (PAUP) version 4.0beta 462 Pertanika J. Trop. Agric. Sci. 35 (3) 462 - 484 (2012) 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia 6ZRIIRUG ZKHUHDVD%D\HVLDQWUHH was constructed using MrBayes version  +XHOVHQEHFN  5RQTXLVW   The Akaike Information Criterion (AIC) ZDVXVHGWRGHWHUPLQHWKHEHVW¿WPRGHO RI VHTXHQFH HYROXWLRQ LQ WKH VSHFLHV E\ using Modeltest 3.7 (Pasoda & Crandall,   7KH 0D[LPXP /LNHOLKRRG 0/  and Bayesian trees were constructed based RQ WKH *HQHUDO 7LPH UHYHUVLEOH *75  PRGHO 7DYDUH   DV GHWHUPLQHG E\ AIC. For ML, the heuristic search option was used in PAUP* with Tree-bisectionreconnection (TBR) branch swapping and UDQGRPDGGLWLRQDOVHTXHQFHUHSOLFDWHV The Bayesian analysis was performed with 2 745 000 generations implementing Metropolis-coupled Markov chain Monte Carlo (MCMC) with 100 generation and burn in=1000 for summary parameter values and trees. The trees were rooted with two outgroups, namely, Cynopterus brachyotis 7.$EG 5DKPDQ   DQG Rhinolophus philippinensis 7. Abd Rahman, 2010). To obtain a graphical representation of the Cyt b gene variation, minimum spanning networks (MSN) of haplotypes were constructed by allowing DOOWKHUHTXLUHGPXWDWLRQDOVWHSVWKDWZRXOG eventually link the different sub-networks. These haplotype networks were generated using the programme, Network 4.5.0.2 (Fluxus Technology 2004-2008). sites (S) and the mean number of nucleotide differences (K) were calculated using WKH 'QD63 YHUVLRQ  5R]DV et al., 2003). The Mantel test was conducted LQ$UOHTXLQ9HUVLRQ  ([RIILHU et al., 2005). Permutations of size 1000 were used to examine the effect of isolation-byGLVWDQFH ,%' E\WHVWLQJWKHFRUUHODWLRQ between geographical distance and genetic differentiation among the populations. The neutrality tests of Tajima’s, D (Tajima,  )XDQG/L¶VD* and F* (Fu & Li,  DQG)X¶VFs )X ZHUHXVHGWR test the hypothesis that all mutations are VHOHFWLYHO\QHXWUDO .LPXUD 7DMLPD D is based on the differences between the number of segregating sites and the average number of nucleotide differences 7DMLPD    )X DQG /L¶V D* and F* tests are based on molecular polymorphism GDWD )X /L )X¶VFs )X  assessment of the haplotype structure on the KDSORW\SHIUHTXHQF\GLVWULEXWLRQZDVXVHG as an additional neutrality test. The level of population subdivision (Fst) (Hudson et al.,  QXFOHRWLGHVXEGLYLVLRQ 1st) (Lynch &UHDVH DQGWKHQXPEHURIIHPDOH migrant (Nm) (Hudson et al.,   IRU GHWHUPLQLQJWKHJHQHÀRZZHUHFDOFXODWHG XVLQJ 'QD63 YHUVLRQ  5R]DV et al., 2003). The analysis of Molecular Variance (AMOVA) was used to estimate F-statistic ĭst  :HLU &RFNHUKDP YDOXHVLQ order to assess further differentiation among Population Genetic Analyses WKHSRSXODWLRQV7KHVLJQL¿FDQFHZDVWHVWHG Haplotype (h DQGQXFOHRWLGH ʌ GLYHUVLWLHV using 10 000 permutations, as performed 1HL 7DMLPD1HL QXFOHRWLGH XVLQJ WKH$UOHTXLQ9HUVLRQ  VRIWZDUH GLYHUJHQFH 'D WKHQXPEHURISRO\PRUSKLF ([FRI¿HUet al., 2005). Pertanika J. Trop. Agric. Sci. 35 (3): 463 - 484 (2012) 463 Mohd Ridwan A. R. and M. T. Abdullah RESULTS Analysis of Sequence A total of 1,061 bp of cyt b of 77 P. lucasi LQGLYLGXDOVZHUHVXFFHVVIXOO\VHTXHQFHG 2XW RI WKH WRWDO  ZHUH YDULDEOH VLWHV   FRPSULVLQJ  VLQJOHWRQ VLWHV  DQGSDUVLPRQ\LQIRUPDWLYHVLWHV (70.53%). On the average, the nucleotide composition consisted of adenosine (A) = WK\PLQH 7  F\WRVLQH &   DQGJXDQLQH *  7KHRYHUDOO IUHTXHQF\ GLVWULEXWLRQV RI QXFOHRWLGHV DW WKH¿UVWVHFRQGDQGWKLUGFRGRQSRVLWLRQV >YDOXHVLQSHUFHQWDJHV  $  42.6, T = 23.0, 41.2, 8.7, C = 27.0, 24.6, 44.3 DQG* @$OOWKHVHTXHQFHV ZHUHVXEPLWWHGWRWKH*HQ%DQNZLWKWKH DFFHVVLRQQXPEHUV*8*8 0/RIERRWVWUDSVVXSSRUWDQGLQ%33  with respect to the out-groups, C. brachyotis and R. philippinensis. Two clades were constructed from the phylogenetics trees, namely, Haplogroup 1 and Haplogroup 2. Haplogroup 1 comprised 31 haplotypes of P. lucasi from Miri and Kuching, while Haplogroup 2 consisted of 14 haplotypes of P. lucasi from Miri, Kuching, Sri Aman and Kelantan. Haplotype Network The phylogenetic structure among the samples from the four populations of P. lucasi was revealed by haplotype clustering on a minimum-spanning network (MSN) (Fig.4). Based on the unrooted network RI PW'1$ F\W b, the MSN showed a ‘star-like’ phylogeny in the P. lucasi populations in Malaysia. Furthermore, the Haplotypes Distribution of P. lucasi MSN topology pattern is similar to other Haplotype trees of P. lucasi were constructed haplotype trees (ML and Bayesian), which using the maximum likelihood (ML) and the LQFOXGHWZRJURXSVRIVHTXHQFHVIURPWKH Bayesian methods (see Fig.2 and Fig.3). populations of Miri-Kuching (Haplogroup *HQHUDOO\ ERWK WUHHV VKRZHG WKH VDPH 1) and Kuching-Miri-Sri-Aman-Kelantan grouping of P. lucasi, with only slight (Haplogroup 2), respectively. Within both differences in their topology. These trees sub-networks, most of the haplotypes were revealed the monophyly of P. lucasi  TABLE 1 Number of haplotypes and nucleotide diversity within each population of P. lucasi. Localities N Miri Kuching Sri Aman Kelantan 33 33 6 5 No. of haplotypes 26 17 3 4 Haplotype diversity ( h )† “ “ 0.733 ± 0.155 “ Nucleotide diversity ʌ ‚ 0.01584 ± 0.00321 0.01316 ± 0.00343 0.00082 ± 0.00023 0.00528 ± 0.00105 N=Number of individuals  (VWLPDWHGXVLQJ.LPXUDWZRSDUDPHWHUGLVWDQFH .LPXUD †Sites with gaps were completely excluded. 464 Pertanika J. Trop. Agric. Sci. 35 (3) 464 - 484 (2012) % Pairwise divergence*† 0.00 - 4.72   0.00 - 0.76 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia XQLTXH WR LQGLYLGXDOV   ZKLOH  haplotypes were associated with more than RQH LQGLYLGXDO  +DSORW\SH IUHTXHQFLHV were denoted by the proportional size of haplonodes. Thirty-seven mutational steps link the two haplogroups. Both the haplogroup sub-networks were rather complex with divergent branches marked with grey nodes, indicating hypothetical haplotypes (missing haplotypes). Within haplogroup 1, five haplotypes (namely, haplotypes 1, 10, 12, 13 and 25) were shared between Miri and Kuching populations, with a high IUHTXHQF\ VXJJHVWLQJ WKH IHPDOH JHQH flow. All the haplotypes from Miri and Kuching populations were divergent with the mutational step ranging from one to four. Within haplogroup 2, the Miri population GLYHUJHGE\RQHWR¿YHPXWDWLRQDOVWHSV The Kuching population was divergent with mutational steps ranging from one to three, while the Kelantan population diverged by one to four mutational steps. All Sri Aman haplotypes were divergent with a single mutational step. Nucleotide Divergence within and among the Populations $WRWDORIVHJUHJDWLQJVLWHVZHUHGHWHFWHG from 45 haplotypes that were distributed within and among the four populations of P. lucasi. From the total of 77 individuals, six haplotypes were shared between the SRSXODWLRQVQDPHO\++++ and H25 and all were shared between Miri and Kuching. The population from Miri VKRZHG WKH KLJKHVW IUHTXHQF\ RI XQLTXH haplotypes, with 26 haplotypes from a total of 33 individuals sampled (Table 1). The genetic divergence between the haplogroups is 3.88%. The genetic divergence within the population of P. lucasiUDQJHGIURPWR 7DEOH  whereas the divergence among population ranged from 0.003% to 0.14% (Table 2). The haplotype diversity (h) within WKH SRSXODWLRQ UDQJHG IURP  WR  TABLE 2 $QDO\VLVRIQXFOHRWLGHGLYHUVLW\ ʌ QHWQXFOHRWLGHGLYHUJHQFHDQGGLYHUJHQFHWLPHHVWLPDWHV DJH DPRQJ the four populations of P. lucasi. Localities 'LVWDQFH (KM) % Pair-wise divergence*† Nucleotide GLYHUVLW\ ʌ ‚ Net Nucleotide GLYHUJHQFH 'a) Miri-Kuching Miri–Sri Aman Miri-Kelantan Kuching-Sri Aman Kuching-Kelantan Sri Aman-Kelantan 516.5 420.8 1324.4 210.6  1178.2 0.003 0.13 0.14 0.14 0.14 0.01  0.02073 0.02061  0.01877 0.00463 -0.00220  0.02626 0.02878 0.02832 0.00327 Age of divergence (Kya)# 7.5 325 350 350 350 25 (VWLPDWHGXVLQJ.LPXUDWZRSDUDPHWHUGLVWDQFH .LPXUD  †Sites with gaps were completely excluded. Pertanika J. Trop. Agric. Sci. 35 (3): 465 - 484 (2012) 465 466 TABLE 3 6XPPDU\DQDO\VLVRIPW'1$F\WbVHTXHQFHVYDULDWLRQDPRQJWKHIRXUSRSXODWLRQVRIP. lucasi in Malaysia. N 33 H 26 S 73 Kuching 33 17 63 Sri Aman 6 3 2 Kelantan 5 4 12 Whole population 77 45  % sdiv 0.00  0.00  0.00  0.00  0.00  h† “ 0.011 “ 0.023 0.733 ± 0.155 “ 0.161 “ 0.006 ʌ‚ 0.01584 ± 0.00321 0.01316 ± 0.00343 0.00082 ± 0.00023 0.00528 ± 0.00105 “ 0.00177 K 16.80114 D  Fs -20.5431* D* -0.12175 F* -0.21304 r 0.0115  -0.37283 -23.0524*  0.31485 0.0220 0.86667 -0.05002 -7.09607* 0.06221  0.3467 5.60000  -1.16655   0.2300 20.83288 0.22450 -6.467 -1.06638 -0.65307 0.0081 1 QXPEHURIVHTXHQFH+ QXPEHURIKDSORW\SHV6 QXPEHURIVHJUHJDWLQJVLWHVVGLY SHUFHQWDJHRISDLUZLVHVHTXHQFHGLYHUJHQFH HVWLPDWHGE\.3GLVWDQFH .LPXUD h KDSORW\SHGLYHUVLW\ʌ QXFOHRWLGHGLYHUVLW\. DYHUDJHQXPEHURIQXFOHRWLGHGLIIHUHQFHVD 7DMLPD¶VVWDWLVWLFV 7DMLPD Fs = Fu’s statistics )X D* and F  )XDQG/L¶VVWDWLVWLFV )X /L U UDJJHGQHVVVWDWLVWLFV *P 6LJQL¿FDQFHZDVFDOFXODWHGXVLQJFRDOHVFHQWVLPXODWLRQLQ'QD63YHUVLRQ 5R]DVet al., 2003). † Sites with gap were completely excluded. Mohd Ridwan A. R. and M. T. Abdullah Pertanika J. Trop. Agric. Sci. 35 (3) 466 - 484 (2012) Population Miri 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia )LJ$PD[LPXPOLNHOLKRRGPDMRULW\UXOHFRQVHQVXVWUHHRIPW'1$F\Wb of P. lucasi. Bootstrap YDOXHVDERYHDUHLQGLFDWHGEHORZEUDQFK.&+.XFKLQJ.71.HODQWDQ050LUL6$6UL Aman. TABLE 4 Measures of geographical population differentiation in P. lucasi based on the analysis of molecular variance (AMOVA) Among groups Among population within groups Within population Variance component  Percentage % of variation 46.42 F-statistic ĭ ĭct = 0.46417 3.73415  18.77 34.81 ĭsc = 0.35030 ĭst = 0.65187 6LJQL¿FDQW P)  0.00000* 0.00000* 6LJQL¿FDQW3 Pertanika J. Trop. Agric. Sci. 35 (3): 467 - 484 (2012) 467 Mohd Ridwan A. R. and M. T. Abdullah TABLE 5 *HQHWLFGLIIHUHQWLDWLRQPDWUL[RIWKHSRSXODWLRQVFDOFXODWHGE\ĭst and P values is shown in parenthesis. Miri Kuching Sri Aman Kelantan Miri - 0.01525 (0.55856) 0.65238 (0.0000)* 0.6335 (0.0000)* Kuching Sri Aman Kelantan 0.70842 (0.0000)*  (0.0000)* 0.54475  - 6LJQL¿FDQWPZLWKSHUPXWDWLRQ )LJ$%D\HVLDQPDMRULW\UXOHFRQVHQVXVWUHHRIPW'1$F\WERIP. lucasi. The Bayesian posterior SUREDELOLWLHV %33 DUHLQGLFDWHGEHVLGHWKHWUHHEUDQFKQRGHV.&+.XFKLQJ.71.HODQWDQ05 0LUL6$6UL$PDQ 468 Pertanika J. Trop. Agric. Sci. 35 (3) 468 - 484 (2012) 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia Fig. 4: Haplotype mapping of 45 assigned haplo-nodes within the four populations of P. lucasi in Malaysia. All the nodes for the populations of Miri, Kuching, Sri Aman and Kelantan are represented by white, black, forward diagonal and diagonal cross, respectively. The grey nodes represent missing or unsampled haplotypes LQWKLVDQDO\VLV1RWHWKDWHDFKQRGHUHSUHVHQWVXQLTXHKDSORW\SHDQGQRGHVL]HVDUHSURSRUWLRQDOWRWKH KDSORW\SHIUHTXHQFLHVRIWKHJLYHQSRSXODWLRQ%ROGQXPEHUVLQGLFDWHGDWWKHQRGHEUDQFKHVDUHWKHQXPEHU of mutational steps to connect the nodes. Minimum-spanning network (MSN) was generated by Network SURJUDP )OX[XV7HFK  (Table 1). The intra-population nucleotide GLYHUVLW\ ʌ ZDVKLJKLQWKH0LULSRSXODWLRQ with 0.016. Among the populations, the nucleotide GLYHUVLW\ ʌ  UDQJHG IURP  WR  with an average nucleotide substitutions per site between populations (nucleotide GLYHUJHQFH'a UDQJLQJIURPWR A comparison between Miri and Sri Aman showed the highest nucleotide diversity with DQGDGLYHUJHQFH 'a) of 0.027, while the lowest nucleotide diversity of 0.004 was observed between Sri Aman and Kelantan, DORQJZLWKDGLYHUJHQFH 'a) value of 0.003 (Table 2). The Mantel analysis revealed a lack of significant relationship between nucleotide divergence and geographic GLVWDQFH FRUUHODWLRQFRHI¿FLHQWU  significant P =   DPRQJ WKH IRXU populations of P. lucasi. This indicated that the geographical distance was not a contributing factor in the nucleotide divergence within P. lucasi. Neutrality Test and Population Expansion The neutrality tests of Tajima’s D, Fu and Li’s, D* and F* and Fu’s F s, suggested that there were expansion events within all the P. lucasi populations. This was 3HUWDQLND-7URS$JULF6FL     Mohd Ridwan A. R. and M. T. Abdullah also supported by a ‘star-like’ shape of the network of P. lucasi. This ‘star-like’ pattern can be attributed to an expanding population 6ODWNLQ +XGVRQ5DKPDQ  Tajima’s D was positive for the total overall population, indicating a lack of recently derived haplotype (Table 3) (Fu & Li,  7KHQHJDWLYHYDOXHVRI)XDQG/L¶V D*(-1.06638), Fu and Li’s F*(-0.65307) and Fu’s Fs (-6.467) were observed for the total overall population, suggesting the presence of rare haplotypes or polymorphism in the population (Akey et al.  5DPODK  7KHDQDO\VLVIRUHDFKSRSXODWLRQ DOVRVKRZHGDKLJKO\VLJQL¿FDQWYDOXHRI Fu’s Fs for the Miri, Kuching and Sri Aman populations (Fs = -20.0525, P Fs = -23.5413, P   Fs   P = 0.000, respectively), indicating excess of the recent mutations, while the nonVLJQL¿FDQWYDOXHRI)XDQG/L¶VD* and F* (D* = -0.1218, P  F* = -0.2130, P D  P F*   P   D* = 0.0622, P = F  P = 0.58, respectively) indicated a demographic expansion for each of the populations. However, this was not observed for the Kelantan population. which are separated by the South China Sea. A high variation was observed among the JURXSV  EXWZDVQRWVLJQL¿FDQWO\ supported (P  %RWKWKHYDULDWLRQ among the population within the groups (18.77%) and the variation within (34.81%) WKHSRSXODWLRQVZHUHKLJKO\VLJQL¿FDQW P = 0.000). On other hand, the estimated ĭst values among the grouped populations VKRZHGDKLJKVLJQL¿FDQFHLQWKHSDLUZLVH differentiation (Table 5). The analysis between the populations revealed high levels of nucleotide (Nst) and population subdivision (Fst), with low level of migrant per generation (Nm) between the populations, and the exception between the Miri and Kuching populations. In particular, the P. lucasi of both the populations showed DKLJKJHQHÀRZ 1m  'HVSLWH the closer distance, both the populations in Kuching and Sri Aman showed low levels of migrant per generation (Nm = 0.30), LQGLFDWLQJORZIHPDOHJHQHÀRZ2YHUDOO WKHDQDO\VHVIURPWKHJHQHÀRZHVWLPDWRU gave a low level of female migrant per generation of P. lucasi in all the populations, except for the population from Miri. DISCUSSION Population Subdivision Genetic and Population History AMOVA was used to determine the extent of population differentiation in P. lucasi (Table 4). Population structuring was investigated by grouping the four populations into two broad geographical groups (namely, East and West Malaysia). The grouping was made based on the geographical distance between these two regions within Malaysia 2YHUDOOWKHDQDO\VLVRIESVHTXHQFHV of P. lucasi revealed low levels of nucleotide and haplotypes variation. The populations with low level of genetic diversity might have experienced a prolonged or severe demographic bottleneck in the recent times (Avise, 2000). A potential cause for such a bottleneck effect could be due to the 470 Pertanika J. Trop. Agric. Sci. 35 (3) 470 - 484 (2012) 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia multiple glaciations during Pleistocene HSRFK 5RTXHV  1HJUR  3LDJJLR et al.,   7KH ORZ OHYHOV RI JHQHWLF variation within P. lucasi populations also suggest that they might be recovering from catastrophic or stochastic events during their recent history (Ojeda, 2010). Meanwhile, climatic change and habitat loss may also contribute to reductions in genetic variability of the populations (Hadly et al., &KDQet al., 2005). A study by Chan et al. (2005) found that rodent species lost genetic variability as a response to major climatic changes and habitat changes during the Holocene. These conditions may also decrease the population size and range the species (Chan et al.,5RTXHV 1HJUR 3LDJJLRet al.,   Two haplogroups were observed for the P. lucasi populations, based on all the haplotype trees and network analyses with a high statistical support, suggesting that the isolation of the haplogroups was not a recent event (Piaggio et al.,  $KLJK genetic divergence was found between the two haplogroups (3.88%) in this study. The separation of the haplogroups might be explained in relation to the historical events (Ross et al.,5DPODK  High mutational steps (37 times) in MSN also suggest that the separation is an ancient event (William et al., 2005). A similar pattern of separation was also found in other WD[DLQFOXGLQJDQXUDQV 5DPODK DQG birds (Ramji, 2010). Although the historical glacial events appeared to have influenced the genetic structure of the P. lucasi, different patterns of colonisation events and refugia could exist between the haplogroups (William et al.,5REHUW 7KHGLYHUJHQFH between the haplogroups has a possibility of GDWLQJEDFNWR0\DZKLFKZDVZLWKLQ the Pleistocene epoch. The mammalian history was typically associated with the Pleistocene event, as it has been known as an important determinant for historical migration. Theoretically, the Sunda Shelf islands, namely, Borneo, Sumatra and Java, had repeatedly merged with Peninsular Malaysia to form a large landmass a number RIWLPHV 5XHGL )XPDJDOOL%LUGet al., 2005). The changing of the sea levels DQGWKHÀXFWXDWLQJWHPSHUDWXUHRIWKH0DOD\ Archipelago during Pleistocene had led to the repeated tropical rain forest isolation DQG IUDJPHQWDWLRQ ZKLFK FRQVHTXHQWO\ affected the forest-associated taxa (Ruedi )XPDJDOOL$QWKRQ\et al., 2007). It was hypothesised that some individuals of P. lucasi had migrated from their maternal roosts to establish new colonies. These colonies were expected to EHVXUURXQGHGE\DGHTXDWHIRRGUHVRXUFHV and secure places for shelter and breeding. As the colonies reached their carrying capacity, the initiator bats were forced to ¿QGPRUHIUDJPHQWHGKDELWDWVWRIRUPQHZ colonies. This stepping stone migration was repeated several times during the Pleistocene climate change period. Eventually, colonies with a common ancestor were assumed to be genetically mixed at intermediate refugia near the water bodies. The northern parts of Borneo (Miri and Sabah) were suggested DVWKHPDLQ4XDWHUQDU\UDLQIRUHVWUHIXJLD Pertanika J. Trop. Agric. Sci. 35 (3): 471 - 484 (2012) 471 Mohd Ridwan A. R. and M. T. Abdullah in Borneo, as described by many authors HJ$VKWRQ%UDQGRQ-RQHV &UDQEURRN0RUOH\+XQWet al., 2007). The discovery of pollens from Kalimantan also provided the evidence for the existence of the tropical rain forests GXULQJ/*0 $QVKDULet al., 2004). Furthermore, the reduction of moist rainforest, which was concentrated near water bodies, provided refugia for the animals (MacKinnon et al0RUOH\ 2000). The populations of P. lucasi were assumed to be isolated into these refugia over a long period of time. It was further speculated that P. lucasi colonised into the tropical rainforest during the interglacial dry period of Pleistocene maximum and dispersed during the cool wet period of 3OHLVWRFHQH PLQLPD *DWKRUQH+DUG\ et al., 2002), with the spread of the tropical rainforest. Therefore, repeated contraction and expansion of the rainforest during 4XDWHUQDU\ ZRXOG KDYH UHVXOWHG LQ WZR broad haplogroups in the northern and southwestern Borneo. It could be hypothesised that such occurrences might have affected the bats in terms of their movement and dispersal abilities. Based on the data obtained in the current study, it could be postulated that the age of divergence for all the populations of P. lucasi occurred between 7.5 - 350 kya. The late Pleistocene era dated back to 128 to 11 kya, while the Holocene era began 11 kya and has continued to the present (Cranbrook, 2000). Therefore, part of the divergence events of P. lucasi would have occurred from the 472 +RORFHQH WR WKH /DWH *ODFLDO 0D[LPXP /*0 RI3OHLVWRFHQHHSRFK The placement of haplotypes from Miri and Kuching in both Haplogroup 1 and Haplogroup 2 had led to the occurrence of a species complex which might be present within these populations. A high level of genetic divergence was detected between the haplotypes from all the P. lucasiSRSXODWLRQV  )DLVDO   also found a high divergence of 5% within the populations of P. lucasi from Borneo. The author has further suggested that a comprehensive genetic study is needed to verify the divergence. Meanwhile, recent reviews have also suggested that a criterion RI  VHTXHQFH GLYHUJHQFH LQ WKH &\W b gene is considered as an existence of the subspecies, whereas the values exceeding 10% are considered in bats as indicatives of species-level divergence (Bradley & Baker, %DNHU %UDGOH\ +RZHYHU WKHOHYHOVRIJHQHWLFGLYHUJHQFHDWPW'1$ PDUNHUVDORQHDUHQRWQHFHVVDULO\VXI¿FLHQW to identify the possible cryptic species 5XHGL 0F&UDFNHQ 0HDQZKLOH Ibanez et al. (2006) proposed species level UHFRJQLWLRQRQO\WRWKRVHPW'1$OLQHDJHVRI KLJKO\GLIIHUHQWLDWHGVSHFLHV ! ZKLFK also showed morphological differentiation and or ecological isolation. Nonetheless, the assumptions that are solely based on PW'1$ PDUNHUV KDYH EHHQ FULWLFLVHG because they reflect only an incomplete part of the natural history of the organisms (Ballard & Whitlock, 2003), or may be misled by the presence of pseudogenes Pertanika J. Trop. Agric. Sci. 35 (3) 472 - 484 (2012) 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia (Bensasson et al., 2001), and/or are affected E\WKHQDWXUDOOLPLWDWLRQVRIPW'1$PDUNHUV +XGVRQ 7XUHOOL 'XHWRWKHVH possible disadvantages, a cross-validation with independent nuclear markers is highly recommended (Zhang & Hewitt, 2003). According to Jayaraj (2008), the PLVFODVVL¿FDWLRQRIQHFWDULYRURXVEDWVLQWR different geographical clades in Malaysia might be due to their ability to perform ORQJGLVWDQFHÀLJKWIRUIRUDJLQJ7KHUHIRUH this kind of behaviour might explain the misclassification of P. lucasi haplotypes from Miri and Kuching present in both haplogroups. The Old World fruit bats can travel up to hundreds of kilometres, both within the mainland and across the ocean barriers (Shilton et al., 6RPHJRRG examples of the local species are Eonycteris spelaea and C. brachyotis, which can travel up to 50 km for foraging in a single night (Fukuda et al., 7KHKLJKPRELOLW\RI these species has made them very successful LQWHUPVRIGLVWULEXWLRQWKH\FDQEHIRXQG to inhabit various types of vegetations, from the lowland dipterocarp forest, peat swamp forest, kerangas, and up to montane forest (Payne et al.,)UDQFLV $VD megabat, P. lucasi is capable of travelling long distances and foraging in more places. This enables individuals to migrate from the north to the south of Sarawak, or vice versa. This is further demonstrated by the colonisation of bats in Krakatau Island, which proves that the bodies of water or oceans are not an effective barrier to impede the dispersion of the species of fruit bats :KLWWDNHU -RQHV7KRUQWRQet al.,   Population Partitioning and Gene Flow *HQHÀRZ 7KHOHYHORIJHQHÀRZLVH[SHFWHGWRGHFUHDVH with the increase of distance between two or more populations (Karuppudurai et al     &RQVHTXHQWO\ WKH QHDUHVW population is more similar at the neutral loci (Storz, 2002). This relationship refers to the isolation by distance, and assumes a stepping stone model of gene flow, which will SURYLGHDVXI¿FLHQWWLPHIRUWKHSRSXODWLRQ WRUHDFKDFRQGLWLRQRIHTXLOLEULXP .LPXUD :HLVV +RZHYHUWKHOHYHOVRIJHQH ÀRZDUHQRWRQO\GHSHQGHQWRQWKHGLVWDQFH between the populations, but also on the environment of the surrounding landscape between the populations (Storz, 2002). Thus, a high level of genetic variation within a population could result in a high level of JHQHÀRZVSHFL¿FDOO\IRUWKHSRSXODWLRQV in Miri and Kuching (Karuppudurai et al., 2007). This can be assumed since the sharing of haplotypes has been observed only (between) in the populations in Miri and Kuching, despite their notable distance from each other. This could have resulted from the continuous distribution of the P. lucasi population. In sedentary species, extrinsic barriers to gene flow and historical events may determine the extent of genetic partitioning among the populations (Karuppudurai et al., 2007). A barrier such as a developed area separating these localities has been suggested as a factor contributing to the failure of this particular species to be connected with each other and hence, impedes any gene ÀRZEHWZHHQWKHSRSXODWLRQV 6WRU]  Pertanika J. Trop. Agric. Sci. 35 (3): 473 - 484 (2012) 473 Mohd Ridwan A. R. and M. T. Abdullah Fluctuations in the world’s temperature and a series of lowering and rising of sea levels during the late Pleistocene might have somehow affected this particular species since it depended on the forest for food. These phenomena have also allowed for the formation of different types of forest (Campbell et al., 2006). According to Hudson et al.   D VLJQLILFDQW differentiation between the populations would be expected only if the Nm value was 6LPLODUUHVXOWVKDYHEHHQUHSRUWHG in P. poliochepalus and P. alecto (Webb & 7LGHPDQ Plecotus auritus (Burland et al., M. lyra (Rajan & Marimuthu, 2006) and C. sphinx (Karuppudurai, 2007). As for the populations of P. lucasi, only one population interaction showed a deviated value with its Nm!LHWKH0LUL.XFKLQJ SRSXODWLRQV7KHQRQVLJQL¿FDQWFRUUHODWLRQ between the geographical distance and the genetic diversity among the populations of P. lucasi in Malaysia has led to the rejection of genetic isolation by geographic distance. Therefore, factors other than the distance between the populations are responsible for the differentiation observed in the populations of P. lucasi. the populations in Haplogroup 1 and Haplogroup 2 is due to the ability of the dusky fruit bats to perform long-distance flights for foraging. A high gene flow was detected between these populations, suggesting continuous “stepping-stone” distributions of P. lucasi, despite the existing considerable distance between both localities. Meanwhile, the absence of a deep structure from the haplotype trees suggested that P. lucasi has a wide dispersal ability. The populations of P. lucasi were also expected to experience interpopulation JHQHWLFGLYHUJHQFHZKLFKFRXOGEHFODVVL¿HG LQWRGLIIHUHQWHYROXWLRQDU\VLJQL¿FDQWXQLWV (ESU) for management purposes. This study provided some useful insights into the phylogeoraphic relationships, genetic XQLTXHQHVV DQG SRSXODWLRQ VWUXFWXUH RI P. lucasi in Malaysia. However, further studies should be carried out using larger sample sizes per population and samples from other cave areas (e.g. Mulu in Sarawak, *RPDQWRQJDQG0DGDLLQ6DEDK ZLWKLQWKHLU geographical distribution for conservation management strategies of the populations of P. lucasi, which are highly dependent on the cave system for breeding and shelter, and the surrounding forested areas for CONCLUSION food resources. Additionally, information 7KH¿QGLQJVRIWKHFXUUHQWVWXG\LQGLFDWHG EDVHGRQWKHQXFOHDU'1$PDUNHUVDQGIDVW that the age of divergence for all the HYROYLQJPW'1$JHQHV PLFURVDWHOOLWHV LV populations of P. lucasi occurred between necessary to elucidate the complex status 350 – 7.5 kya. The divergence within the of P. lucasi. SRSXODWLRQVLQ0LUL  DQG.XFKLQJ (4.7%) could have led to the occurrence ACKNOWLEDGEMENTS of a species complex within P. lucasi. The authors would like to thank the Faculty The presence of the haplotypes from both of Resource Science and Technology, 474 Pertanika J. Trop. Agric. Sci. 35 (3) 474 - 484 (2012) 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia Universiti Malaysia Sarawak for various administrative and logistic aids throughout the course of this study. We would also like to express our appreciation to the Sarawak Forestry Corporation (SFC) and 6DUDZDN )RUHVWU\ 'HSDUWPHQW 6)'  IRU granting us the permission, with license QXPEHUXQGHUWKH6WDWH:LOG/LIH 3URWHFWLRQ5XOHVIRUUHVHDUFKSHUPLW QXPEHU13: ,,, 2XUKHDUWIHOW gratitude also goes to Mr. Haidar Ali and Mr. Saip Sulong for providing us with accommodation during our fieldwork at Niah NP and Wind Cave NR, and to the staff RIWKH=RRORJ\'HSDUWPHQWHVSHFLDOO\%HVDU Ketol and Huzal Irwan Husin, who assisted us during the conduct of this fieldwork. 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Sci. 35 (3) 480 - 484 (2012) 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia APPENDIX 1 List of samples of P. lucasi used in the genetic analyses. No Species Voucher/ Museum. No Locality Habitat *HQ%DQN Acc. No. 1 P. lucasi MZU/M/02120 Niah NP, Miri, Sarawak Limestone forest *8 2 P. lucasi MZU/M/02122 Niah NP, Miri, Sarawak Limestone forest *8 3 P. lucasi MZU/M/02123 Niah NP, Miri, Sarawak Limestone forest *8 4 P. lucasi MZU/M/02124 Niah NP, Miri, Sarawak Limestone forest *8 5 P. lucasi MZU/M/02125 Niah NP, Miri, Sarawak Limestone forest *8 6 P. lucasi MZU/M/02127 Niah NP, Miri, Sarawak Limestone forest *8 7 P. lucasi MZU/M/02128 Niah NP, Miri, Sarawak Limestone forest *8 8 P. lucasi MZU/M/02130 Niah NP, Miri, Sarawak Limestone forest *8  P. lucasi MZU/M/02131 Niah NP, Miri, Sarawak Limestone forest *8 Limestone forest *8 10 P. lucasi MZU/M/02133 Niah NP, Miri, Sarawak 11 P. lucasi MZU/M/02134 Niah NP, Miri, Sarawak Limestone forest *8 12 P. lucasi MZU/M/02135 Niah NP, Miri, Sarawak Limestone forest *8 13 P. lucasi MZU/M/02153 Niah NP, Miri, Sarawak Limestone forest *8 14 P. lucasi MZU/M/02154 Niah NP, Miri, Sarawak Limestone forest *8 15 P. lucasi MZU/M/02155 Niah NP, Miri, Sarawak Limestone forest *8 16 P. lucasi MZU/M/02156 Niah NP, Miri, Sarawak Limestone forest *8 17 P. lucasi MZU/M/02157 Niah NP, Miri, Sarawak Limestone forest *8 18 P. lucasi MZU/M/02163 Niah NP, Miri, Sarawak Limestone forest *8  P. lucasi 0=80 Niah NP, Miri, Sarawak Limestone forest *8 20 P. lucasi TK152463 Niah NP, Miri, Sarawak Limestone forest *8 21 P. lucasi TK152468 Niah NP, Miri, Sarawak Limestone forest *8 22 P. lucasi TK152470 Niah NP, Miri, Sarawak Limestone forest *8 23 P. lucasi TK152481 Niah NP, Miri, Sarawak Limestone forest *8 24 P. lucasi TK152482 Niah NP, Miri, Sarawak Limestone forest *8 25 P. lucasi TK152483 Niah NP, Miri, Sarawak Limestone forest *8 26 P. lucasi 7. Niah NP, Miri, Sarawak Limestone forest *8 27 P. lucasi 7. Niah NP, Miri, Sarawak Limestone forest *8 28 P. lucasi 7. Niah NP, Miri, Sarawak Limestone forest *8  P. lucasi 7. Niah NP, Miri, Sarawak Limestone forest *8 30 P. lucasi 7. Niah NP, Miri, Sarawak Limestone forest *8 31 P. lucasi 7. Niah NP, Miri, Sarawak Limestone forest *8 32 P. lucasi 7. Niah NP, Miri, Sarawak Limestone forest *8 Pertanika J. Trop. Agric. Sci. 35 (3): 481 - 484 (2012) 481 Mohd Ridwan A. R. and M. T. Abdullah 33 P. lucasi MZU/M/01685 Lambir NP, Miri, Sarawak Lowland 'LSWHURFDUS Forest *8 34 P. lucasi TK152883 Wind Cave NR, Kuching, Sarawak Limestone forest *8 35 P. lucasi TK152884 Wind Cave NR, Kuching, Sarawak Limestone forest *8 36 P. lucasi TK152885 Wind Cave NR, Kuching, Sarawak Limestone forest *8 37 P. lucasi TK152887 Wind Cave NR, Kuching, Sarawak Limestone forest *8 38 P. lucasi MZU/M/02173 Wind Cave NR, Kuching, Sarawak Limestone forest *8  P. lucasi MZU/M/02180 Wind Cave NR, Kuching, Sarawak Limestone forest *8 40 P. lucasi MZU/M/02207 Wind Cave NR, Kuching, Sarawak Limestone forest *8 41 P. lucasi 0=80 Wind Cave NR, Kuching, Sarawak Limestone forest *8 42 P. lucasi MZU/M/02210 Wind Cave NR, Kuching, Sarawak Limestone forest *8 43 P. lucasi MZU/M/02211 Wind Cave NR, Kuching, Sarawak Limestone forest *8 44 P. lucasi MZU/M/02212 Wind Cave NR, Kuching, Sarawak Limestone forest *8 45 P. lucasi MZU/M/02214 Wind Cave NR, Kuching, Sarawak Limestone forest *8 46 P. lucasi MZU/M/02216 Wind Cave NR, Kuching, Sarawak Limestone forest *8 47 P. lucasi MZU/M/02217 Wind Cave NR, Kuching, Sarawak Limestone forest *8 48 P. lucasi MZU/M/02226 Wind Cave NR, Kuching, Sarawak Secondary forest *8  P. lucasi MZU/M/02227 Wind Cave NR, Kuching, Sarawak Secondary forest *8 50 P. lucasi MZU/M/02232 Wind Cave NR, Kuching, Sarawak Secondary forest *8 51 P. lucasi 0=80 Wind Cave NR, Kuching, Sarawak Limestone forest *8 52 P. lucasi MZU/M/02233 Wind Cave NR, Kuching, Sarawak Limestone forest *8 482 Pertanika J. Trop. Agric. Sci. 35 (3) 482 - 484 (2012) 3RSXODWLRQ*HQHWLFVRIWKH&DYHGZHOOLQJ'XVN\)UXLW%DWPenthetor lucasi, Based on Four Populations in Malaysia 53 P. lucasi MZU/M/02235 Wind Cave NR, Kuching, Sarawak Limestone forest *8 54 P. lucasi MZU/M/02236 Wind Cave NR, Kuching, Sarawak Limestone forest *8 55 P. lucasi MZU/M/02234 Wind Cave NR, Kuching, Sarawak Limestone forest *8 56 P. lucasi MZU/M/02238 Wind Cave NR, Kuching, Sarawak Limestone forest *8 57 P. lucasi MZU/M/01716 Kubah NP, Kuching, Sarawak Mixed 'LSWHURFDUS Forest *8 58 P. lucasi 0=80 Padawan, Kuching, Sarawak Limestone forest *8  P. lucasi MZU/M/02240 Padawan, Kuching, Sarawak Limestone forest *8 60 P. lucasi MZU/M/02241 Padawan, Kuching, Sarawak Limestone forest *8 61 P. lucasi MZU/M/00568 Mount Penrissen, Kuching, Sarawak Montane forest *8 62 P. lucasi 0=80 Mount Penrissen, Kuching, Sarawak Montane forest *8 63 P. lucasi MZU/M/00570 Mount Penrissen, Kuching, Sarawak Montane forest *8 64 P. lucasi MZU/M/02242 Bako NP, Kuching, Sarawak Mixed 'LSWHURFDUS Forest *8 65 P. lucasi MZU/M/02243 Bako NP, Kuching, Sarawak Mixed 'LSWHURFDUS Forest *8 66 P. lucasi MZU/M/02244 Bako NP, Kuching, Sarawak Mixed 'LSWHURFDUS Forest *8 67 P. lucasi 0=80 Batang Ai NP, Sri Aman, Sarawak Lowland 'LSWHURFDUS Forest *8 68 P. lucasi 0=80 Batang Ai NP, Sri Aman, Sarawak Lowland 'LSWHURFDUS Forest *8  P. lucasi 0=80 Batang Ai NP, Sri Aman, Sarawak Lowland 'LSWHURFDUS Forest *8 70 P. lucasi 0=80 Batang Ai NP, Sri Aman, Sarawak Lowland 'LSWHURFDUS Forest *8 Pertanika J. Trop. Agric. Sci. 35 (3): 483 - 484 (2012) 483 Mohd Ridwan A. R. and M. T. Abdullah 71 P. lucasi 0=80 Batang Ai NP, Sri Aman, Sarawak Lowland 'LSWHURFDUS Forest *8 72 P. lucasi 0=80 Batang Ai NP, Sri Aman, Sarawak Lowland 'LSWHURFDUS Forest *8 73 P. lucasi ':13 *XD0XVDQJ.HODQWDQ NA *8 74 P. lucasi ':13 *XD0XVDQJ.HODQWDQ NA *8 75 P. lucasi ':13 *XD0XVDQJ.HODQWDQ NA *8 76 P. lucasi ':13 *XD0XVDQJ.HODQWDQ NA *8 77 P. lucasi ':13 *XD0XVDQJ.HODQWDQ NA *8 78 C.brachyotis TK152458 Mount Murud, Miri, Sarawak Montane forest *8  R. philippinensis 7. Niah NP, Miri, Sarawak Limestone forest *8 1$ 1RWDYDLODEOH13 1DWLRQDO3DUN15 1DWXUH5HVHUYH 484 Pertanika J. Trop. Agric. Sci. 35 (3) 484 - 484 (2012)