*Division of Veterinary and Biomedical Sciences, Murdoch University, Perth, Western Australia;
Department of Virology, Biomedical Primate Research Center, Rijswijk, the Netherlands;
Division of Quarantine, Ministry of Agriculture and Natural Resources, Balikpapan, Indonesia; and
§Max-Planck-Institute for Evolutionary Anthropology, Leipzig, Germany
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Abstract |
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Introduction |
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Today, orangutans exist in increasingly fragmented and isolated populations. While the Sumatran orangutan is primarily found in northern Sumatra, the Bornean is distributed in Central, West, and East Kalimantan, Sarawak, and Sabah. They are, however, not found in Brunei and South Kalimantan (Rijksen and Meijaard 1999
). The determination of the intrasubspecific variation between isolated Bornean populations has been stated to be essential for both the management of orangutan reintroduction projects and the planning of conservation strategies to preserve the remaining wild populations (De Boer 1982
; Courtenay, Groves, and Andrews 1988
; Janczewski, Goldman, and O'Brien 1990
; The World Conservation Union/Species Survival Commission 1993
; Uchida 1996
; Xu and Arnason 1996
). Studies of morphological features have indicated that the extent of interpopulation differentiation within Borneo may approach that between Borneo and Sumatra (Groves, Westwood, and Shea 1992
; Uchida 1998
). By using discriminant analyses of orangutan skulls from different localities, Groves , Westwood, and Shea (1992)
concluded that there were three distinct populations of orangutans: those in Sumatra, those in southwestern Borneo, and those in the remainder of Borneo. Avoiding the confounding effects of age-related size differences of skulls by examining tooth morphology, Uchida found significant differences between two Bornean populations from Northwest and Southwest Kalimantan that were as great in magnitude as those from the Borneo-Sumatra comparison (Uchida 1998
).
Analysis of molecular variation is increasingly employed in evaluation of animal populations for purposes of taxonomic clarification, genetic variability assessment, and identification of origin of confiscated illegal pets (Morin, Moore, and Woodruff 1992
; Morin et al. 1993
; Zhi et al. 1996
; Warren et al. 2000
). Only two studies have used samples of Bornean orangutans of known origin for the assessment of genetic variability (Zhi et al. 1996
; Warren et al. 2000
). The study of Zhi et al. (1996)
compiled a total of 33 individuals from four areas in Borneo, as well as 6 Sumatran individuals from two locations. The analytical methods used, nuclear minisatellite loci analysis, mtDNA restriction fragment length polymorphisms, and the analysis of mitochondrial 16S rRNA sequences, revealed a separation between Bornean and Sumatran orangutans at approximately 1.5 MYA and considerable diversity within the Bornean and Sumatran subspecies. However, the methods used did not detect geographically defined genetic variation within Borneo. Recent microsatellite DNA studies provide evidence that east and west Bornean populations, while subject to genetic drift, have similar genetic backgrounds (Warren et al. 2000).
Sequence analysis of the most variable segment of the control region of the rapidly evolving mtDNA molecule has long been the method of choice for analysis of population level diversity in humans and great apes (Vigilant et al. 1991
; Morin et al. 1993
; Woodruff 1993
; Garner and Ryder 1996
; Gagneux et al. 1999
). Assessment of molecular variation within Borneo requires the use of a reasonable number of samples of known origin analyzed with a highly informative genetic locus. This study presents the results of the first comprehensive analysis of mtDNA control region sequences from 41 Bornean orangutans from six locations, as well as sequences from five Sumatran orangutans.
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Materials and Methods |
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Analysis of DNA Sequences
Sequence analysis was conducted using MacVector, version 6.0, and AssemblyLIGN software packages (Oxford Molecular Ltd., U.K.), and aligned sequences were manually edited using the sequence alignment editor Se- Al, version 1.0a1 (Rambaut 1995
). Estimates of Wright's fixation index FST (Wright 1951
; Cockerham and Weir 1984
) were computed using ARLEQUIN, version 1.1 (Schneider 1997
). Molecular variance among groups and populations was calculated using AMOVA, which is part of the ARLEQUIN software. The interpopulation distances were calculated using the Iwave program (Harpending et al. 1993
). Maximum-likelihood tree reconstruction was performed using PUZZLE, version 4.0.2 (Strimmer and von Haeseler 1999
). Phylogenetic analysis of the sequences was performed with the PHYLIP package, version 3.572 (Felsenstein 1995
). Trees were visualized using TREEVIEW, version 1.5.3, software (Page 1998
). Most recent common ancestor (MRCA) estimations were performed using FLUCTUATE (Kuhner 1998
).
Nucleotide Sequences
The nucleotide sequences of the orangutan mitochondrial DNA control regions described in this article have been deposited in the EMBL/GenBank data libraries (accession numbers AJ391095AJ391103 and AJ391105AJ391141.
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Results |
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Population pairwise FST values and percentages of sequence divergence were calculated for the groups of individuals from the six Bornean regions and those with a Sumatran origin (table 2 ). Results indicate that the six regions sampled in Borneo represent four significantly differentiated (P = 0.05) populations, with the individuals collected from the geographically adjacent regions of Sarawak and Northwest Kalimantan and those collected from Southwest and Central Kalimantan, respectively, not being genetically distinct. At the more stringent 1% significance level, the individuals from Sabah were also undifferentiated from the other two northerly populations from Northwest Kalimantan and Sarawak.
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AMOVA analysis was used to further substantiate the apportionment of the genetic diversity within Borneo (table 3 ). The first analysis classified the Bornean sequences into six populations (SARA, NK, SAB, EK, SK, and CK) and considered these six populations one group for comparison with the single population of the Sumatran group. The analysis indicated that most of the variation (70.8%) distinguished the Bornean and Sumatran groups. Since the six Sumatran individuals did not constitute a geographically defined population and an understanding of variation within Borneo was of interest, the analysis was performed with the same six Bornean populations and using the three Bornean groups that were defined by the population pairwise FST analysis (CK/SK, EK, and SARA/SAB/NK). This analysis showed that most of the diversity (46.4%) was found within populations but that a considerable proportion of the diversity distinguished populations within groups (26%), as well as the groups themselves (27.6%).
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Maximum-likelihood (ML) tree reconstruction using the quartet-puzzling method was also applied to the data (Strimmer and von Haeseler 1997
). The transition/transversion ratio was estimated from the data using a Tamura- Nei model of substitution (Tamura and Nei 1993
), and the gamma-distribution parameter alpha, which describes the extent of rate heterogeneity among sites, was estimated assuming gamma-distributed rates (table 4
). Comparison of these parameters for the control region segment in bonoboos, chimpanzees, and humans revealed similar levels of rate heterogeneity but a two- to threefold reduced transition-transversion ratio in orangutans. ML analysis produced trees consistent in their branching patterns with the ones obtained from the NJ analyses (data not shown). ML trees were constructed with and without the assumption of a molecular clock. Results indicated that all data sets analyzed (all orangutans plus the chimpanzee outgroup, all orangutans, and only Bornean orangutans) failed the clock test at the 5% significance level. This result was likely an effect of analyzing a large number of lineages that were differentiated by a relatively small number of mutations, and the ML approach may not be the best way to test for constancy of evolutionary rates in large mtDNA data sets (A. von Haeseler, personal communication). The program FLUCTUATE (Kuhner 1998
) was then used to estimate the time to the MRCA of the mtDNA segment studied. The Bornean orangutan female effective population size (Ne) was estimated to be 22,000, and the age of the MRCA was estimated to be 860,000 years ago. The combined data set of Bornean and Sumatran orangutans led to an estimate of 28,000 for the female Ne and an MRCA of 1.1 MYA. These estimates were derived using the assumptions of a mutation rate for the sequence of 0.33 changes per site per million years, a mutation rate based on human data (Ward et al. 1991
), a generation time of 20 years, a constant population size, and the ML estimator of
. The estimates have a large variance. To put the values in perspective, the same assumptions were used to derive estimates for the ages of the MRCAs of west African chimpanzees (61 individuals; MRCA 420,000 years ago) and bonobos (31 individuals; MRCA 340,000 years ago) (Gagneux et al. 1999
). These values suggest that the genetic depth of Bornean orangutans is twice as deep as that of western chimpanzees, and 2.5 times as deep as that of bonobos.
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Discussion |
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Our phylogenetic analyses confirm the findings of wide genetic variation between Bornean and Sumatran orangutans. Despite the identification of distinct geographic clusters of Bornean orangutans, no particular cluster was clearly more related to the Sumatran apes than any other Bornean population. These data contrast with findings by Groves, Westwood, and Shea (1992)
that suggested that the southwest population (SK) was more related to the Sumatran population than any other orangutan population in Borneo. However, it should be noted that only the skulls from Southwest Kalimantan, Northwest Kalimantan, Sarawak, and Sabah were studied, and individuals from Central and East Kalimantan were not included.
Our observations may have implications for the interpretation of the proposed migration routes of the Bornean orangutan ancient ancestors from Sumatra to Borneo across the Sunda landmass (fig. 3 ) (Rijksen and Meijaard 1999
). Natural geographic barriers may have forced the isolated colonization of at least four different regions of Borneo. This notion is now supported by the more recent genetic diversification of orangutans observed in the four different regions of Borneo (table 3
). Estimations of the MRCA suggested that Sumatran and Bornean populations diverged approximately 1.1 MYA. Furthermore, our data suggest that at least four distinct Bornean subpopulations diverged 860,000 years ago. This is in agreement with previous estimates of divergence dates for Bornean and Sumatran orangutans, which ranged from 0.6 to 3.4 Myr (Bruce and Ayala 1979
; Janczewski, Goldman, and O'Brien 1990
; Ruvolo et al. 1994
; Zhi et al. 1996
). Thus, the Bornean and Sumatran orangutans were reproductively isolated long before the islands were geographically isolated by rising seas in the Late Pleistocene. This isolation could have been due to geographical barriers such as ancient river systems or due to behavioral barriers (Zhi et al. 1996
). Indeed, differences in social interactions have been recorded in field studies of Sumatran and Bornean orangutans (Galdikas 1978
; Rijksen 1978
; Peters 1995).
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The results of this study are consistent with behavioral research that indicates that female orangutans have smaller ranges than males and generally stay in specific geographic locations (Rijksen and Meijaard 1999
). In fact, geographic boundaries formed by rivers and mountains prevent isolated orangutan populations from traversing such terrain. Recent microsatellite studies revealed no differentiation between East and West Kalimantan (Warren et al. 2000). However, it is important to note that microsatellite analyses provide data reflecting a more historical time point than the mtDNA, which is less conservative and has a rapid mutation rate. Current data suggest that historically, the orangutan populations within Borneo were large enough within specific regions to enable gene flow and prevent a genetic bottleneck. However, the mtDNA does indicate that the four distinct subpopulations within Borneo represent reproductively isolated populations that show significant genetic diversity.
The phylogenetic analyses enabled the determination and confirmed the probable place of origin for most of the confiscated individuals that were used in this study. There was also evidence that there may be even greater genetic variation within Borneo, indicated by the few individuals that fell outside of the main subpopulation clusters. There was only one case in which an orangutan was not located in the expected geographical cluster. Hair samples were collected from the nest of a wild individual, TNK 38, in Kutai National Park. However, on phylogenetic analysis, this individual was consistently located in the cluster from Northwest Kalimantan/Sarawak (fig. 2 ). Occasionally, ex-captive orangutans were released into Kutai National Park unofficially prior to the establishment of new regulations in 1995 governing orangutan reintroduction practice. Thus, the probable explanation is that this animal represents an ex-captive orangutan originating from the Northwest Kalimantan/Sarawak population that was subsequently released into Kutai National Park.
In conclusion, this study provides evidence for (1) marked divergence and speciation of Sumatran and Bornean orangutans and (2) at least four distinct subpopulations of Bornean orangutans. Based on mtDNA analysis, these populations are estimated to be equally genetically distinct from each other. It can be debated whether there is sufficient divergence for the four Bornean populations to be classified as subspecies. However, they are clearly genetically isolated populations that are in the process of divergence. In this regard, these populations should be treated as "subpopulations," and their genetic diversity should be maintained. Furthermore, given the most recent catastrophic destruction of orangutan habitat by fires and logging, more detailed studies will be required to further define other distinct populations as well as to generate predictive data about the future survival of smaller orangutan populations isolated in increasingly fragmented habitats. These findings provide evidence to strengthen arguments for further conservation and management efforts to secure greater areas of orangutan habitat. The orangutan subpopulations should be protected in each geographic region to ensure their genetic diversity and survival.
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Acknowledgements |
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Footnotes |
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1 Keywords: orangutan
Pongo pygmaeus,
mitochondrial DNA
control region
genetic diversity
biogeography
2 Address for correspondence and reprints: Jonathan L. Heeney, Department of Virology, Biomedical Primate Research Center, P.O. Box 3306, 2280 GH Rijswijk, the Netherlands. heeney{at}bprc.nl
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