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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
FRPSULVLQJRIO;FRORXUOHVV*R7DT
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$WHPSODWHDQGO*R7DT
'1$SRO\PHUDVH XO 3&5ZDVFDUULHG
out using a thermocycler with 30 cycles
LQFOXVLYHRIRQHLQLWLDOGHQDWXUDWLRQDW&
DQG¿QDOH[WHQVLRQDW&IRUWKUHHDQG¿YH
PLQXWHVUHVSHFWLYHO\7KHRWKHUF\FOHV
FRQVLVWHGRIGHQDWXUDWLRQDW&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.
Lastly, many thanks to our colleagues (Mohd
)L]O 6LGT 0RKG 5DPML 5REHUWD &KD\D
Tawie Tingga and Noor Haliza Hasan) at
the Molecular Ecology Laboratory (MEL)
for their undying support and gracious
assistance. This research was supported
by a postgraduate scholarship (Zamalah) to
05$5DQG0R+()5*6
(25) grant awarded to MTA. This paper
DOVREHQH¿WHGIURPWKHFULWLFDOFRPPHQWV
E\'U/LP%RR/LDWDQG'U<X]LQH(VD
and the editorial comments by Ms Radina
0RKDPDG'HOLRIWKH&HQWUHRI/DQJXDJH
Studies, UNIMAS.
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Pertanika J. Trop. Agric. 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)