Molecular Phylogenetics and Evolution 39 (2006) 887–892 www.elsevier.com/locate/ympev Short communication Molecular phylogenetic aYnities of the simakobu monkey (Simias concolor) Danielle J. Whittaker a,¤, Nelson Ting b, Don J. Melnick c,d a Department of Anthropology, Queens College, Flushing, NY 11367, USA Department of Anthropology, City University of New York Graduate Center, New York, NY 10016, USA c Department of Ecology, Evolution, and Environmental Biology, Columbia University, New York, NY 10027, USA d Center for Environmental Research and Conservation (CERC), Columbia University, New York, NY 10027, USA b Received 25 July 2005; revised 21 December 2005; accepted 23 December 2005 Available online 3 March 2006 1. Introduction The simakobu (Simias concolor), or pig-tailed snubnosed langur, is one of four endemic primate species on the Mentawai Islands oV the west coast of Sumatra, Indonesia, and is currently considered a monotypic genus (BrandonJones et al., 2004; Miller, 1903). This species, like many other colobines, is a specialized folivore found in tropical rainforests. The simakobu monkey is a member of the “odd-nosed” group of Asian colobines, an informal grouping that also includes the proboscis monkey of Borneo (Nasalis larvatus), the snub-nosed monkeys of China and Vietnam (Rhinopithecus spp.), and the douc langurs of Vietnam, Laos, and Cambodia (Pygathrix spp.). There is no agreement, however, on the relationships among these genera, nor whether they even form a natural taxonomic or phylogenetic group (Delson, 1975; Groves, 1970, 2001; Jablonski, 1998; though see Sterner et al., in press). Geographically, the simakobu is found closest to the proboscis monkey, and some morphological data have indicated that these species are sister taxa. Both Simias and Nasalis exhibit narrow, long-faced skulls; long narrow nasal bones; and similar hair patterns (Groves, 1970). These two species appear to share more traits than do Rhinopithecus and Pygathrix, and some researchers have suggested that Simias should be subsumed into the genus Nasalis (Delson, 1975; Groves, 1970), perhaps as a subgenus (Delson, 1975); Groves (2001; Brandon-Jones, et al., 2004) appears to have retreated from the close linkage of these genera, though is not necessarily opposed to it (Groves, pers. comm.). * Corresponding author. Fax: +1 718 997 2885. E-mail address: djwhittaker@mindspring.com (D.J. Whittaker). 1055-7903/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.12.013 Alternative hypotheses suggest that there is no evidence that Simias and Nasalis are phylogenetically linked (Napier, 1985), and that the former may be a basal colobine unrelated to other members of the “odd-nosed” group (Jablonski, 1998). Other Mentawai primates have also been suggested as basal members of their respective lineages. While BrandonJones (1998) considers Simias a member of the genus Nasalis, he has suggested that the other three Mentawai primate species display primitive characteristics and are basal to Southeast Asian primate lineages. According to Brandon-Jones (1998), the primates of Sumatra went extinct during Pleistocene glaciations, and the Mentawai Islands may have provided a reservoir from which primates recolonized Sumatra during interglacials (Brandon-Jones, 1998). However, this scenario is not supported by more recent biogeographic analyses of Presbytis and genetic analyses of the Mentawai gibbon (Hylobates klossii) (Meijaard and Groves, 2004; Takacs et al., 2005; Whittaker, 2005). To date, few behavioral or ecological studies have been conducted focusing on the simakobu monkey. The proboscis monkey was previously believed to be dependent on mangrove and coastal forests, while the simakobu is typically found in inland evergreen tropical rainforest. This habitat diVerence has led some researchers to conclude that these species are quite distinct ecologically and thus should be separated at the genus level (Napier, 1985; Oates et al., 1994). However, more recent data have shown that proboscis monkeys are more widely distributed than previously thought, occurring throughout Borneo (Meijaard and Nijman, 2000). The odd-nosed genera Simias, Nasalis, and Pygathrix seem to follow a similar adaptive strategy, including the exploitation of high-quality foods during part of the year with the ability to subsist on low-quality diets at 888 D.J. Whittaker et al. / Molecular Phylogenetics and Evolution 39 (2006) 887–892 other times (Bennett and Davies, 1994). These three genera also appear to have some Xexibility in their social systems: in the simakobu monkey, social groups contain one male and either one or several females, plus their oVspring (Fuentes and Tenaza, 1995; Tenaza and Fuentes, 1995; Watanabe, 1981), while proboscis monkeys are typically found in one-male multi-female groups, but with overlapping home ranges (Bennett and Sebastian, 1988). Neither the simakobu nor the proboscis monkey have been able to out-compete sympatric colobine species, in apparent contrast to members of the genus Pygathrix (Bennett and Davies, 1994). These ecological data suggest many similarities between Simias and Nasalis, though no Wrm conclusions can be drawn without further Weld studies of Simias. We present here the results of the Wrst molecular study to test hypotheses regarding the phylogenetic position and taxonomic status of the simakobu monkey. 2. Methods Hairs were collected from a wild-caught infant simakobu pet in the village of Sikakap on North Pagai Island (Fig. 1) and stored dry in a tube. The monkey was likely caught in the Pagai Islands, but its origin could have been from another Mentawai Island. Total genomic DNA was extracted from four hairs, using the Qiagen DNA Micro Kit and manufacturer-supplied protocols for hairs without roots. We ampliWed and sequenced 424 bp of mitochondrial DNA using universal cytochrome b primers (L14724, H15149) (Kocher et al., 1989). This region includes part of the tRNAthr (24 bp) and cytochrome b genes (400 bp). Polymerase chain reaction (PCR) was performed in 50 l reactions containing 4 l of template DNA, 1 M each of primers L14724 and H15149, 0.25 mM dNTPs, 2.0 mM MgCl2, and 1.25 U AmpliTaq DNA polymerase (Applied Biosystems). The PCR was run in a GeneAmp PCR System 9700 (Applied Biosystems) thermocycler with the following conditions: initial 2 min at 94 °C; followed by 45 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 2 min, and extension at 72 °C for 3 min; with a Wnal extension at 72 °C for 7 min. A negative control containing no DNA was included in addition to the six replications of the Simias sample. The PCR products were electrophoresed on an agarose gel, stained with ethidium bromide, and digitally photographed under UV light. The PCR products were puriWed using the QIAquick PCR PuriWcation Kit (Qiagen) according to the manufacturer’s instructions. Cycle sequencing was performed in 10 l volume reactions, containing 0.8 l of the puriWed PCR product and 0.5 l sequencing primer in a BigDye Terminator 3.1 Cycle Sequence reaction mixture (Applied Biosystems). Cycle sequence products were puriWed using the ethanol/EDTA/sodium acetate protocol speciWed by Applied Biosystems. Sequencing was performed on an ABI 3730XL capillary sequencer. A consensus sequence from both strands and from six replications was generated using Sequencher 3.1. The Simias sequence has been submitted to GenBank (Accession No. DQ143883). Comparative sequence data from other cercopithecid genera were obtained from GenBank (Table 1), including Papio and Macaca as outgroups. The sequences downloaded from GenBank were compared Table 1 List of samples used in study. Taxonomy follows Brandon-Jones et al. (2004) Fig. 1. Map showing location of the Mentawai Islands. Location of the village of Sikakap on North Pagai Island is indicated with an asterisk (*). Taxon GenBank Acquisition Number Simias concolor Nasalis larvatus (2) Rhinopithecus roxellana Rhinopithecus avunculus Rhinopithecus bieti Pygathrix nemaeus Semnopithecus entellus Semnopithecus johnii Semnopithecus vetulus Trachypithecus francoisi Trachypithecus obscurus Colobus guereza Colobus angolensis Colobus polykomos Procolobus badius Papio hamadryas Macaca mulatta DQ143883 AF020418, U62663 AF020416 AF020415 AF020413 AF295582 AF020417 AF020419 AF020420 AF295578 AY863425 AY863427 AF295583 AF020411 AF295575 Y18001 NC005943 D.J. Whittaker et al. / Molecular Phylogenetics and Evolution 39 (2006) 887–892 to those from unpublished mitochondrial genomes ampliWed in overlapping halves (Sterner et al., in press) to ensure that the analysis would not be aVected by nuclear pseudogenes. The sequences were aligned using the program CLUSTALX (Jeanmougin et al., 1998). To evaluate the relationship of Simias to the other colobine species, we used a Bayesian Markov Chain Monte Carlo simulation to estimate the most likely phylogenetic trees with Mr. Bayes 3.1 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Modeltest 3.6 (Posada and Crandall, 1998) was Wrst run to estimate the nucleotide substitution rate and the frequency of transitions versus transversions, and these parameters were entered into Mr. Bayes. Pairwise distances (p) were generated in PAUP 4.0 (SwoVord, 2002). To test whether colobine lineages are evolving in a manner consistent with a molecular clock, a relative rate test was performed using the program RRTree (Robinson-Rechavi and Huchon, 2000). 3. Results A total of 424 bp of the cytochrome b and adjacent tRNAthr genes were analyzed. Using the hierarchical likelihood ratio test, Modeltest 3.6 selected the Tamura-Nei (1993) model with rate variation among sites. This model assumes unequal base frequencies (observed from the data) and higher likelihood of transitions than transversions (Tamura and Nei, 1993). The parameters specify a substitution rate of 1.0 for transversions, 18.26 for A–G transitions, and 17.98 for C–T transitions. The amongsite rate variation follows a gamma distribution with a shape parameter of 0.28. The Bayesian analysis was run with four chains for 500,000 generations, sampling every 889 100th generation, with a burn-in percentage of 25% or 1250 generations. The phylogenetic tree produced by the Bayesian analysis (Fig. 2) strongly supports the sister taxon relationship of Simias and Nasalis with a credibility value of 100. This tree supports reciprocally monophyletic African and Asian colobine groups, as well as the monophyly of each genus for which multiple species were included. Within the Asian clade, Semnopithecus clusters with the odd-nosed colobines and away from Trachypithecus (Fig. 2A), a conclusion congruent with colobine phylogenies produced from whole mitochondrial genomes (Sterner et al., in press). An analysis of the mitochondrial ND3–ND4 region (Wang et al., 1997) further supports the separation of Trachypithecus from the other Asian colobines. However, in our analysis, this node has a low credibility value of 83, and collapsing this node as well as the Rhinopithecus/Pygathrix/Semnopithecus clade (with a credibility value of 88) results in an unresolved Asian colobine polytomy (Fig. 2B). Molecular pairwise distances (Table 2) also suggest a close relationship between the simakobu and the proboscis monkey. With the exception of Simias and Nasalis, all of the species that have a distance of about 10% or less are widely considered congeners. Pairwise distances between genera range from 10 to 25%. The molecular pairwise distance between Simias and Nasalis (about 6%) is comparable to that between congeneric species (610%), rather than between genera (10– 25%). The relative rate test found no signiWcant diVerence between the rates of change in Asian and African colobine lineages in relation to the cercopithecine outgroup taxa (p > 0.83), suggesting that the rate of change in these lineages is consistent with the expectations of a molecular clock. Fig. 2. Cladograms produced by Bayesian analysis. Numbers above the branches represent clade credibility scores. (A) The node supporting a Rhinopithecus/Pygathrix/Semnopithecus clade exclusive to Nasalis/Simias has low support, as does the node supporting a sister taxon relationship of Trachypithecus to the other Asian colobines. (B) Nodes with clade credibility values of less than 90 have been manually collapsed, resulting in an Asian colobine polytomy. Note that a close relationship between Nasalis and Simias is still supported. 890 D.J. Whittaker et al. / Molecular Phylogenetics and Evolution 39 (2006) 887–892 Table 2 Molecular pairwise distance matrix derived from the cytochrome b data Taxon S. concolor N. larvatus1 N. larvatus2 R. roxellana R. avunculus R. bieti P. nemaeus S. entellus S. johnii S. vetulus T. francoisi T. obscurus C. guereza C. angolensis C. polykomos P. badius P. hamadryas M. mulatta S. concolor N. larvatus1 N. larvatus2 R. roxellana R. avunculus R. bieti P. nemaeus S. entellus S. johnii S. vetulus T. francoisi T. obscurus C. guereza C. angolensis C. polykomos P. badius P. hamadryas M. mulatta — 0.059 0.062 0.125 0.144 0.132 0.136 0.156 0.161 0.147 0.136 0.140 0.172 0.173 0.182 0.179 0.174 0.200 0.063 — 0.005 0.135 0.139 0.149 0.170 0.168 0.154 0.154 0.133 0.133 0.198 0.195 0.200 0.193 0.174 0.193 0.067 0.005 — 0.139 0.151 0.151 0.172 0.166 0.158 0.156 0.139 0.138 0.199 0.201 0.200 0.194 0.180 0.198 0.148 0.159 0.166 — 0.057 0.047 0.116 0.158 0.142 0.125 0.123 0.140 0.193 0.181 0.213 0.174 0.171 0.212 0.176 0.165 0.182 0.060 — 0.064 0.141 0.173 0.161 0.145 0.140 0.152 0.208 0.208 0.220 0.181 0.169 0.217 0.157 0.178 0.182 0.050 0.068 — 0.124 0.154 0.137 0.135 0.140 0.149 0.193 0.171 0.213 0.178 0.174 0.217 0.161 0.211 0.213 0.134 0.167 0.143 — 0.195 0.175 0.170 0.161 0.155 0.167 0.172 0.172 0.199 0.185 0.193 0.186 0.202 0.199 0.192 0.212 0.183 0.246 — 0.080 0.090 0.174 0.164 0.188 0.210 0.194 0.168 0.200 0.189 0.195 0.184 0.192 0.168 0.194 0.161 0.214 0.088 — 0.095 0.162 0.152 0.179 0.193 0.189 0.173 0.178 0.190 0.170 0.179 0.183 0.143 0.169 0.156 0.205 0.099 0.104 — 0.160 0.164 0.193 0.190 0.206 0.178 0.183 0.199 0.160 0.153 0.161 0.142 0.165 0.167 0.192 0.214 0.196 0.191 — 0.086 0.145 0.158 0.152 0.175 0.178 0.180 0.167 0.155 0.163 0.168 0.185 0.182 0.186 0.198 0.180 0.201 0.095 — 0.180 0.165 0.185 0.192 0.173 0.173 0.215 0.256 0.257 0.251 0.275 0.250 0.202 0.236 0.218 0.242 0.170 0.225 — 0.062 0.010 0.155 0.166 0.189 0.219 0.252 0.263 0.233 0.281 0.214 0.212 0.275 0.243 0.237 0.192 0.204 0.067 — 0.068 0.162 0.160 0.188 0.230 0.261 0.259 0.279 0.292 0.280 0.207 0.245 0.231 0.258 0.179 0.233 0.010 0.073 — 0.165 0.176 0.190 0.225 0.247 0.249 0.216 0.225 0.222 0.253 0.201 0.209 0.216 0.215 0.249 0.187 0.198 0.199 — 0.185 0.220 0.213 0.210 0.219 0.209 0.202 0.211 0.225 0.251 0.217 0.222 0.219 0.209 0.198 0.191 0.212 0.227 — 0.145 0.252 0.236 0.245 0.272 0.279 0.278 0.234 0.231 0.229 0.246 0.216 0.205 0.231 0.232 0.232 0.284 0.170 — Figures below the diagonal are uncorrected (p) distances; Wgures above the diagonal are Tamura–Nei distances. Note that individuals diVering less than 10% (shaded cells) are generally considered congenerics, with the exception of Simias and Nasalis. D.J. Whittaker et al. / Molecular Phylogenetics and Evolution 39 (2006) 887–892 891 4. Summary and discussion Acknowledgments An analysis of part of the mitochondrial cytochrome b gene strongly supports a sister taxon relationship between the simakobu and proboscis monkeys, and places this lineage within a monophyletic Asian colobine clade. This study is not inconsistent with the classiWcation of the simakobu within the genus Nasalis, as suggested by earlier morphological analyses (Delson, 1975, 2000; Groves, 1970). Few studies have been conducted on the behavior and ecology of the simakobu, but some data indicate that Simias and Nasalis are following similar adaptive strategies (Bennett and Davies, 1994). The hypothesis that Simias is a basal colobine (Jablonski, 1998) is not supported, nor is the corresponding suggestion for Nasalis (Groves, 1989). Recent genetic analyses of the Mentawai primates H. klossii and Macaca pagensis have consistently shown these species to be derived members of their respective clades with their closest relatives on nearby Sumatra (Roos et al., 2003; Takacs et al., 2005; Whittaker, 2005), and not primitive or ancestral as has sometimes been suggested. The phylogenetic tree presented here shows well-supported monophyletic Asian and African clades, but provides little resolution otherwise. Nasalis and Simias form a clade, but relationships among most of the Asian colobine genera appear as an unresolved polytomy. Further research is necessary to understand the phylogenetic and biogeographic history of the Asian colobines. Currently, two subspecies are deWned for S. concolor based on diVerences in pelage coloration: S. concolor siberu on the northernmost island of Siberut and S. c. concolor on the three southern islands. The same pattern is described in P. potenziani and M. pagensis, though not for H. klossii (Whittaker, 2005). Preliminary genetic analysis of M. pagensis suggests a paraphyletic origin (Roos et al., 2003). Future studies need to examine intraspeciWc patterns of divergence in Mentawai primates. The simakobu monkey is among the least understood, but most endangered, of all the Asian primates. The IUCN Red List currently lists this species as “Endangered,” under criteria A1cd+2c (IUCN, 2004). However, a recent reassessment of the available evidence suggests that the status of S. concolor should be upgraded to “Critically Endangered” based on criteria A2cd (Whittaker, 2005). The simakobu is also included in Conservation International’s list of the 25 Most Endangered Primates (Mittermeier et al., 2002). Taxonomic uniqueness is sometimes used as a criterion for setting conservation priorities (Olson and Dinerstein, 1998). Should the simakobu monkey be reclassiWed as a member of the genus Nasalis, its conservation priority (or that of the Mentawai Islands as a whole) may be aVected based on such methods. We believe that taxonomy should reXect phylogeny, and we do not mean to imply that this unique primate, or any of the Mentawai primates, requires any less attention or conservation action. This research was supported by an NSF Doctoral Dissertation Improvement Grant (BCS-0335949) and Primate Conservation, Inc. (D.J.W.). We thank the Republic of Indonesia, the Indonesian Institute of Sciences (LIPI), Amsir Bakar of Andalas University and Noviar Andayani of University of Indonesia for support and permission to conduct research in Indonesia. 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