Biogeography of the Indonesian archipelago

17 Pages·2007·0.91 MB·English

BiologzralJoumal ofthe Linnpan Socieb (1998), 65: 329-345. With 7 figures
Article ID: bj980244
Biogeography of the Indonesian archipelago:
rnitochondrial DNA variation in the h i t b at,
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Eonycteris spelaea o
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SUSAN HISHEHI*, MICHAEL WESTERMAN' AND fro
LINCOLN H. SCHMITT' m
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La Tmbe Universig, Bundoora VIC 3083, Australia de
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Received 13 October 1997; acceptedf or publication 28 April 1998 p
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The fruit bat, Eonyctmk spelaea, occurs from India through the Philippines to the southeast n
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limit of its distribution in the Lesser Sunda islands of Indonesia. Mitochondria1 DNA (mtDNA) a
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variation was examined in Indonesian E. spelaea island populations by amplification of the /a
D-loop and digestion with restriction endonucleases. In addition, microgeographic variation rtic
was assessed by investigation of three cave populations within one island. A total of 24 le-a
genotypes, comprising two broad clades, was detected. The pattern of mtDNA variation b
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reflects the colonization history of E. spelaea with estimates of haplotype and sequence diversity tra
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highest in the older western populations and lowest at the eastern periphery of the species' t/6
distribution. These findings may also be associated with an environmental cline from west 5
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to east. There is also evidence that genetic distance between populations reflects geographic /3
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relationships, especially historical connectedness, as measured by Pleistocene sea-crossing 9
distances. At the microgeographic level, cave populations were heterogeneous and composed /26
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of diverse lineages suggesting restricted local interchange. 1
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0 1998 The Linnean Society of Iandon 5 b
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ADDITIONAL KEY WORDS:-enetic variation - Megachiroptera - phylogeography - gu
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population genetics - Pteropodidae. st o
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CONTENTS 05
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Introduction . . . . . . . . . . . . . . . . . . . . . . . 330 ay
Material and methods . . . . . . . . . . . . . . . . . . . 331 20
Results . . . . . . . . . . . . . . . . . . . . . . . . 333 19
Macrogeographic variation: island populations , . . . . . . . . . 333
Microgeographic variation: cave populations . . . . . . . . . . . 335
Discussion . . . . . . . . . . . . . . . . . . . . . . . 336
Variability and population structure . . . . . . . . . . . . . 336
Evidence of colonization history . . . . . . . . . . . . . . . 341
Historic and geographic correlates . . . . . . . . . . . . . . 341
Microgeographic differentiation: cave populations . . . . . . . . . 342
* Correspondence to S. Hisheh. Email: [email protected]
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0024-4066/98/110329+17 $30.00/0 0 1998 The Linnean Society of London
330 S. HISHEH ETA
Conclusion . . . . . . . . . . . . . . . . . . . . . 342
Acknowledgements . . . . . . . . . . . . . . . . . . . . 343
References . . . . . . . . . . . . . . . . . . . . . . . 343
INTRODUCTION
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Studies of biogeography have long focused on the Indonesian archipelago as an o
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intriguing and complex zone of faunal interchange. Its significance centres on its nlo
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position at the interface of two distinct biogeographic regions Asia and Australia d
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(Whitmore, 1987). This has led to much debate over whether this region is a d fro
zoogeographically transitional or unique zone (e.g. Mayr, 1976; Simpson, 1977). m
However, what remains clear is that these islands possess a number of features http
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periencing marked changes in the recent past (Mayr, 1976). The present land masses de
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were formed by the collision and subduction of the Australian and Pacific Plates ic
beneath the Asian Plate during the Pliocene. This gave rise to two island chains, .ou
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the Banda Arcs, of differing geological age and composition. The Inner Banda Arc .c
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islands (Lombok-Wetar-Banda) are principally volcanic, whereas the Outer Banda m
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Arc islands (Sumba-Tanimbar-Seram) are sedimentary in origin and represent io
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outliers of the Australian Plate (Veevers, 1991). During the Pleistocene, many of the n
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present islands were joined to form larger land masses due to lowered sea levels an
produced by glacial maxima (Morley & Flenley, 1987). Bali and Lombok are thought /artic
to have remained separate during the Pleistocene due to the depth of the Lombok le
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Strait, but many of the Inner Banda Arc islands were connected during this epoch b
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(Heaney, 1991). The major Outer Banda Arc islands (e.g. Sumba, Timor, Tanimbar tra
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and Kai) were not joined nor was there any connection between islands of different t/6
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geological origin (Audley-Charles, 1981 ). These geographic and geological histories /3
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may have considerable influence on patterns of differentiation of island fauna from 2
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the region. /2
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Second, Indonesia encompasses many fragmented insular populations (Mayr, 1
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1976) providing the opportunity to investigate patterns of variation in island 45
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populations as they become isolated from those of the mainland. In this way, the y
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study of island populations has contributed to the understanding of speciation and u
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vicariant events (Mayr, 1963). Further, the eastern Indonesian archipelago has a t o
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strongly linear orientation which allows the comparison of a series of populations 0
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each successively more distant from its colonizing source. M
Finally, a cliie in many environmental variables exists from west to east along ay
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the archipelago which may influence the distribution, habitat and genetics of its 0
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faunal elements (Mayr, 1976). In summary, Indonesia presents an interesting
evolutionary setting in which patterns of differentiation in its fauna may be influenced
by factors such as geological history, geographic features, climate and insularity.
The extent to which these have shaped genetic variation will shed light on the
prevailing evolutionary forces acting upon species in this dynamic region.
The present study, therefore, aims to assess the genetic variation within a
volant mammal, Eonycteris spehu, from the Indonesian archipelago in light of these
biogeographic factors. E. speha, the cave fruit bat, is classified in the suborder
Megachiroptera and the family Pteropodidae. It is a species of Indo-Malay origin
BIOGEOGRAPHY OF THE INDONESIAN ARCHIPELAGO 331
(Tate, 1946) and is common and widespread in southeast Asia (Start & Marshall,
1976). Its distribution ranges from India east to the Philippines, Sulawesi and the
Lesser Sundas (Koopman, 1989). Its presence in the Lesser Sundas (islands east of
Java) represents the southeastern limits of its distribution.
E. speha has been recorded from a variety of forest types and a range of altitudes.
It generally roosts in caves and is often found in large colonies. Its diet consists
primarily of fruit and nectar and observations suggest it is reliant on diverse food
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sources. E. spehu also appears to be a species of great dispersive ability and travels o
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considerable distances daily to feed (Payne, Francis & Phillipps, 1985; Start & nlo
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Marshall, 1976). Evidence indicates that individuals cluster in groups according to d
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sex and age when feeding and also within cave roosts (Bhat, Sreenivasan &Jacob, fro
1980; Start & Marshall, 1976). Kitchener, Gunnell & Maharadatunkamsi (1990) m
also found that there was a close clustering of juveniles and adult females at feeding http
localities. s://a
Here we investigate, at both macro- and microgeographic levels, genetic variation c
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in E. speha from eight Indonesian islands and three caves in Lombok island. Variation de
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is examined using restriction fragment length polymorphisms of mitochondrial DNA ic
(mtDNA). Restriction enzyme analysis is an appropriate and effective method for .ou
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assessing intra-specific genetic variation (Hillis & Moritz, 1990) and useful for .c
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microgeographic studies (Kessler & Avise, 1985). It becomes more so when combined
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with the technique of the polymerase chain reaction, PCR (White et al., 1989), io
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because of the ability to target specific DNA sequences for investigation. This study n
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utilizes this approach in assaying variation in the mitochondrial displacement loop n
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(D-loop) of E. speha. This sequence evolves rapidly within and between species rtic
(Moritz, Dowling & Brown, 1987) and is also a region for which universal and le
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mammalian primers are readily available (e.g. Kocher et al., 1989). mtDNA is a b
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sensitive indicator of historical demographic processes in addition to contemporary tra
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ones and also a marker for female dispersal patterns (e.g. Avise, 1994). t/6
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MATERIAL AND METHODS 12
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One hundred and four specimens of E. speha were collected from the following y
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Indonesian islands (Figure 1; sample size in parenthesis): Bali (12), Lombok (36), u
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Sumbawa (12), Sumba (7), Flores (lo), Adonara (lo), Alor (8) and Timor (9). The t o
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Lombok specimens were collected from three caves; one in southwest Lombok, cave 0
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1 (14) and two in east Lombok, caves 2 (6) and 3 (16). Mean latitude and longitude M
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of collection sites were recorded for each island population. Animals were mist- y
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netted and liver was removed and immediately placed in liquid nitrogen. Tissue 0
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samples were subsequently stored at -70°C. The carcass was preserved and
specimens lodged at the Western Australian Museum and Museum Zoologicum
Bogoriense.
Total genomic DNA was extracted from liver tissue according to Sambrook,
Frisch & Maniatis (1989). The tar et region was amplified by PCR using primers
comprising sequences of the tRNAf ro and tRNAh' genes which flank the D-loop of
the mammalian mitochondrial genome (Brown, 1983). Non-specific primers used
were mt15996L (Campbell et al., 1995) and mt607H (designed by M. Elphinstone).
Amplification was conducted in 50 pl volumes of 1 x reaction buffer, 100 pM
332 S. HISHEH ET AL
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Figure 1. Map of Indonesian region surveyed (islands from which E. spelaea has been sampled are .ou
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italicized). .c
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dNTPs, 2 mM MgC12, 0.2 pM primers, 1 unit Taq polymerase and 0.1 pg DNA linn
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template with denaturation at 93°C (1 min), annealing at 5OoC (1.5 min) and an
extension at 72OC (2min) for 35 cycles. The amplification product was digested /artic
with eight restriction endonucleases (EcoRI, SspI, Xbd, BdeI, MseI, RsaI, Suu3AI le
and Zp509I) and fragments separated on 5-1 0% polyacrylamide gels. Restriction -ab
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fragments were visualised by staining with ethidium bromide and fragment size was tra
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ascertained by comparison with appropriate size markers. t/6
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For each restriction enzyme, every morph was assigned an uppercase letter to /3
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represent its restriction fragment profile. Each haplotype was identified by an eight 2
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letter composite code according to its combination of morphs after Lansman et ul. /2
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(1 983). Heterogeneity of restriction enzyme morph frequency between populations 61
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was estimated by the contingency chi-square test and Fisher's exact test when 45
expected values were small. by
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Haplotype diversity, which is analagous to nuclear gene heterozygosity, was u
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calculated for each population according to Nei (1987) and corrected for small st o
sample size. GSb the proportion of genetic variation due to island population n 0
differentiation, was estimated according to Nei (1973). 5 M
An estimate of sequence divergence between haplotypes (p) was determined by ay
iteration according to Nei & Li (1979). Estimates of p were determined separately 20
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for restriction enzymes with 4- and 6-base recognition sequences and a mean value 9
was obtained, weighted by the product of the number of fragments and the length
of the recognition sequence. Bootstrap estimates were computed by resampling over
restriction enzymes. A tree of haplotype relationships was constructed from the
bootstrapped sequence divergence matrix using the neighbour-joining algorithm of
Saitou & Nei (1 988).
Sequence diversity within populations was estimated as the mean p between all
pairs of haplotypes (Nei & Jin, 1989). The net sequence diversity between pairs of
island populations was estimated as the average haplotype sequence diversity between
BIOGEOGRAPHY OF THE INDONESIAN ARCHIPELAGO 333
all possible inter-island pairs, corrected for average intra-island sequence diversity
according to Nei & Li (1979). The differentiation index (D) was estimated for each
island by averaging the sequence diversity between it and every other island. Principal
co-ordinate analyses were used to provide an ordination of distance matrices (Gower,
1966).
Two measures of contemporary geographic distances between islands were em-
ployed. Great-circle distance between populations was determined as the distance
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between the mean latitude and longitude of collection sites of each island. Minimum o
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sea-crossing distance was estimated by summing the shortest sea-crossing between nlo
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island pairs using, where possible, intervening islands as ‘stepping stones’. Pleistocene d
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minimum sea-crossing distances between islands were estimated based on the sea d fro
level being 120 m lower than the present and using the ‘stepping stone’ method as m
above. Correlations between genetic and geographic distance matrices were estimated http
and their significance tested according to Mantel (19 67) using randomization s
(Smouse, Long & Sokal, 1986). ://ac
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Genstat 5 (Genstat 5 Committee, 1993) and Minitab were used to conduct d
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statistical analyses and produce graphs. ic
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RESULTS io
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Macrogeographic variation: island populations an
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PCR amplification produced a DNA fragment 1360 basepairs in length and le
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digests of this region with eight restriction enzymes produced an average of 32 b
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fragments per individual. A total of 58 different fragments were identified across tra
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this species, of which 72% were variable. The frequency of the most common t/6
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restriction fragment morph ranged from 0.50 to 0.93. The geographic distribution /3
of EcoRI and RsaI morphs are shown in Figures 2A and 2B respectively. These /32
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examples indicate a tendency for variants to have marginalized distributions (eg. /2
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EcoRI morph B and RsaI morph C). 61
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Combining information from all restriction enzymes, a total of 24 haplotypes 45
were identified among eight island populations of E. speba and their geographic by
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distribution is shown in Table 1. The most common and widespread haplotype (1 0) u
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was found in 27 of 104 individuals. The remaining haplotypes were represented by t o
one to eleven individuals each, some of which were also localized to eastern or n 0
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western islands. M
Painvise sequence divergence (p) between the 24 haplotypes ranged from 0.3% ay
to 5.4% with a mean of 2.1% (Table 2). The neighbour-joining tree (Fig. 3) 20
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constructed from this distance matrix shows two broad clades of haplotypes. Clade 9
1 comprises the closely-networked cluster of haplotypes 1-19 (mean divergence
within = 1% YO) which are widespread throughout the sampled region and clade 2
comprises haplotypes 20-24 (mean divergence within = 1.27%) and is restricted to
western islands (Table 2). Mean sequence divergence between clades 1 and 2 was
3.56% reflecting that the two groups differ by more than one putative restriction
site for some enzymes.
Haplotype diversity and sequence diversity within island populations (Table 3)
indicated a tendency to decrease from west to east although a statistically significant
334 S. HISHEH ETAL.
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Figure 2. Distribution of (A) EcoRI and (B) RFaI morphs from eight island populations of E. spelaea. 0
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Inset: Morph distribution in cave populations from hmbok island. 9
association with longitude was not observed (Fig. 4). The island of Nor appeared
to be an outlier in this respect and when omitted, longitude and haplotype diversity
were correlated (r= -0.88, RO.01). Both measures of mtDNA diversity within
island populations are associated (r= 0.86, RO.01).
There was no indication that the number of localities sampled within each island
contributed to island population diversity measures (Table 3). Correlations of both
BIOGEOGRAPHY OF THE INDONESIAN ARCHIPELAGO 335
TABLE1. Geographic distribution of E.spelaea haplotypes in 8 island populations. Composite code
order: DdeI, EcoRI, MseI, RsaI, SauSAI, SspI, Xp5091, XbaI
Lombok
Haplotype Cave 1 Cave 2 Cave 3 Bali Sumbawa Sumba Flores Adonara Alor Tmor
1 AAABAAAB I 2
2 AAABAABA 1
3 AAABBAAB 1 2 1 2 1 1 D
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4 BAAABAAA 1 w
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5 BAABAAAA 1 2 2 lo
6 BAABAABA 1 ad
7 BAABAABB 2 ed
8 BAABABAA 1 fro
9 BAABABBB 1 3 1 2 m
1101 BBAAAABBBBAAAAAB 2 3 41 4 3 2 6 3 http
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12 BAABBABB 1 1 ://a
13 BAABBBAA 1 4 c
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14 BAABBBAB I d
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15 BAABBBBB 3 4 2 m
16 BAABCABB 8 3 ic.o
17 BAACABBB I u
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18 BAACCABB 1 7 .c
19 BAACCBBB 1 om
20 BACDCABA /b
2212 BBBBBCDDACAACCAA I iolinn
23 CBCDCACA ea
24 DBCDCACA 1 n/a
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sequence diversity and haplotype diversity with the number of localities sampled b
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were not significant; T= 0.14, P= 0.75 and r= 0.18, P= 0.66 respectively. trac
A large component of total variation was contributed by inter-island differentiation t/6
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(Gs,=0.24). A comparison of the levels of sequence divergence between island /3
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populations (Table 4) showed a range of 0 to 1.24% and indicated a strong 2
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geographic component to the variation. The arrangement of islands on the first axis /2
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of a principal coordinate analysis of the divergence matrix is strongly associated 1
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with longitude (Fig. 5), indicating that genetic relationships reflect the linear spatial 5
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separation of islands. This was supported by estimates of the differentiation index y
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(D) which ranged from 0.154 to 0.592 and showed that Timor, at the species’ u
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periphery, is the most differentiated population (Table 4). Further, haplotype diversity t o
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and D are significantly associated indicating that populations with low variability 0
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are also most genetically divergent (r= -0.82, R0.02; Fig. 6). M
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There was a trend for sequence divergence between island populations to be y
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associated with Pleistocene sea-crossing distance (Fig. 7) suggesting the influence of 0
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historical land connections on genetic variation, however, this did not quite reach
statistical significance (r= 0.45, P= 0.06). Genetic distance between populations was
less well described by contemporary sea-crossing (r= 0.30, P= 0.12) and not associated
with great-circle distance (r= -0.01 5, P= 0.47).
Microgeographic variation: cave populations
Within the island of Lombok, cave 1 was differentiated from the two eastern
caves for both EcoRI (R0.05) and &a1 (RO.01) morphs (Fig. 2A, B). In total, six
336 S. HISHEH ETA.
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Figure 3. Neighbour-joining tree of painvise sequence diversity estimates between 24 haplotypes of E. /3/3
spelaea. 29
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out of eight restriction enzymes showed statistically significant morph frequency 2
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differences between cave 1 and the other two caves. These findings are due to half 5 b
the individuals sampled from cave 1 possessing haplotypes 20-24 (clade 2) which y g
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were absent from the other two caves. This difference in clade frequency between e
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cave 1 and the other caves is statistically significant (p<O.OOl). t o
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All cave populations showed similar levels of haplotypic diversity, although cave 0
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1 had a substantially higher sequence diversity reflecting the presence of both clades M
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of haplotypes (Table 5). Comparing the magnitude of sequence diversity and linear y 2
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distance between the three caves, it appears that genetic and geographic distance 1
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are associated (Table 6), cave 1 being most divergent for both parameters.
DISCUSSION
biubili& and population structure
The mean mtDNA sequence divergence between E. spelaea haplotypes of 2.19'0
is concordant with levels reported in other conspecific mammalian studies using
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TABL2E. Percent sequence divergence (p) between 24 haplotypes of E. spelnea (bootstrapped estimates) ://a
1 ~ m5 cad
2 0.87 - 0 em
3 0.45 1.33 m ic
4 1.81 1.90 ~ 1.28 - !! .ou
56 01..8372 00..9403 11..8303 01..8374 -0. 42 - z! p.co
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7 0.90 0.85 1.38 1.76 0.85 0.43 - /b
8 1.64 1.54 2.10 1.51 0.66 1.07 1.63 - 0 io
9 1.48 1.63 1.95 2.52 1.63 1.21 0.59 0.80 - 1 lin
1101 01..9303 11..8308 00..4825 00..8329 00..4835 01..8279 01..8279 11..0683 21..0465 -0. 43 - -EI nean
1123 21..1308 21..0229 01..8663 11..2054 11..2098 01..8541 02..4016 02..0463 11..0204 00..8636 01..4211 -1. 61 2U /artic
1154 11..4995 22..0577 11..4041 21..0529 21..0663 21..0661 11..0405 01..8204 00..4817 11..2611 00..5999 00..5999 ~00 ..3778 -0. 41 - 2l le-ab
16 1.51 1.42 1.30 1.68 1.42 0.97 0.54 2.19 1.13 1.24 0.81 0.38 2.01 1.39 0.97 - stra
17 1.89 2.01 2.38 2.98 2.01 1.56 0.95 1.19 0.37 2.46 1.84 1.37 1.64 1.27 0.79 1.49 - c
18 1.94 1.83 1.71 2.13 1.83 1.34 0.92 2.59 1.49 1.63 1.20 0.74 2.39 1.77 1.32 0.34 1.11 > t/6
~ E 5
19 2.51 2.59 2.28 2.88 2.59 2.11 1.49 1.77 0.92 2.39 1.77 1.32 1.57 1.20 0.74 0.93 0.52 0.59 - /3
20 3.13 2.11 2.87 2.44 2.11 1.57 2.00 2.73 2.75 1.88 2.30 1.80 2.50 3.05 2.55 1.35 2.86 1.46 2.22 - 9 /3
2
2221 43..3324 23..9910 43..0855 33..3409 32..2125 32..2384 23..8839 23..5610 43..5699 22..9700 33..4255 33..3587 33..0286 44..2024 44..1364 33..5005 43..6799 33..1661 43..3983 21..5484 -0. 95 - F 9/26
23 4.34 3.91 4.05 4.28 3.91 4.04 4.59 4.25 5.33 3.62 4.17 4.29 3.96 4.92 5.04 3.71 5.44 3.81 4.57 1.99 1.52 0.47 - 8 61
24 4.47 4.04 4.17 3.99 3.63 3.76 4.31 3.97 5.06 3.36 3.91 4.03 3.71 4.67 4.78 3.48 5.16 3.58 4.34 1.81 1.33 0.34 0.34 - 24
1 2 3 4 5 6 7 8 9 1 0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 5 b
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338 S. HISHEH ETAL.
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Figure 4. Plot of haplotype diversity against longitude of eight island populations of E. spelaea. a
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TABL3E. Measures of mtDNA diversity in eight island populations of E. speluea p.c
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No. of m
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localities Mean Mean No. of Haplotype Sequence io
Island sampled latitude longitude n haplotypes diversity diversity lin
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Bali 8.28 114.66 12 0.86 1.2 an
Lombok 8.64 116.21 36 0.88 1.9 /a
Sumbawa 8.69 117.63 12 0.86 1.6 rtic
Sumba 9.66 119.52 7 0.62 0.8 le-a
Flores 8.54 121.20 10 0.36 0.3 b
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Adonara 8.33 123.15 10 0.57 0.7 tra
Alor 8.25 124.72 8 0.80 1.1 c
Timor 10.18 123.72 9 0.37 0.8 t/6
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TABL4E. Percent sequence divergence (lower triangle) and minimum Pleistocene sea-crossing distance 61
(km; upper triangle) between island populations of E. spelaea and island differentiation index (D) 24
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Bali Lombok Sumbawa Sumba Flores Adonara Alor Timor y
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Bali - 23 23 70 25.5 25.5 29.5 57.5 s
Lombok 0.099 - 0 47 2.5 2.5 6.5 34.5 t o
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Sumbawa 0.101 0 - 47 2.5 2.5 6.5 34.5 0
SFluomrebsa 00..08110 1 00..136676 00..220529 1-.0 94 44.5- 440. 5 484. 5 3762. 5 5 Ma
Adonara 0.140 0.108 0.096 0.3 13 0.271 - 4 32 y 2
Alor 0 0.074 0.034 0.078 0.502 0 - 28 0
1
Timor 0.900 0.474 0.385 1.243 0.177 0.414 0.552 9
~
~~ ~
D 0.294 0.184 0.154 0.444 0.497 0.192 0.177 0.592
restriction enzyme methods (Avise & Lansman, 1983). Estimates of nucleotide
substitution in E. speha fall within levels observed in two comparable studies in
bats. Pumo et al. (1988) observed up to &lo% divergence between haplotypes of
the fruit bat Artibeus jamaictmis which encompassed subspecific populations and