Mitochondrial and Nuclear Markers Suggest Hanuman Langur (Primates: Colobinae) Polyphyly: Implications for Their Species Status

Molecular Phylogenetics and Evolution 54 (2010) 627–633

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Molecular Phylogenetics and Evolution

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Short Communication Mitochondrial and nuclear markers suggest Hanuman langur (Primates: Colobinae) polyphyly: Implications for their species status

K. Praveen Karanth a,c,*, Lalji Singh b, Caro-Beth Stewart c a Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560012, India b Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India c Department of Biological Sciences, University at Albany, State University of New York, Albany, NY 12222, USA article info abstract

Article history: Recent molecular studies on langurs of the Indian subcontinent suggest that the widely-distributed and Received 30 June 2008 morphologically variable Hanuman langurs (Semnopithecus entellus) are polyphyletic with respect to Revised 9 October 2009 Nilgiri and purple-faced langurs. To further investigate this scenario, we have analyzed additional Accepted 29 October 2009 sequences of mitochondrial cytochrome b as well as nuclear protamine P1 genes from these species. Available online 6 November 2009 The results conﬁrm Hanuman langur polyphyly in the mitochondrial tree and the nuclear markers suggest that the Hanuman langurs share protamine P1 alleles with Nilgiri and purple-faced langurs. We rec- Keywords: ommend provisional splitting of the so-called Hanuman langurs into three species such that the Semnopithecus taxonomy is consistent with their evolutionary relationships. Lineage sorting Cytochrome b Ó 2009 Elsevier Inc. All rights reserved. Protamine P1 Molecular systematics Speciation

1. Introduction Jones, 2004), four (Hill, 1939) and seven (Groves, 2001) distinct species. Hanuman langurs have also been extensively used as model The Hanuman langur (Semnopithecus entellus, Subfamily Colobi- system in a various biomedical (Nandi et al., 2003; Dube et al., nae) is one of the most widely-distributed and morphologically var- 2004; Lohiya et al., 2005), ecological (Kurup, 1984; Kamilar et al., iable species of colobine monkey (Newton, 1988). It is distributed 2006), behavioral (Newton, 1988; Sterck, 1999) and evolutionary throughout most parts of India and Sri Lanka, and is also found in research (Karanth et al., 2008; Osterholz et al., 2008). Often these parts of Pakistan, Nepal, and Bangladesh. According to Roonwal have been comparative studies wherein multiple populations of and Mohnot (1977), it is predominantly a deciduous woodland spe- Hanuman langurs were studied from across the range. The decision cies. Roonwal (1984) recognizes two major forms of Hanuman lan- on the nature of these comparative studies, i.e., inter vs. intra spe- gur based on tail morphology: the Northern type (here called as NT- ciﬁc comparison, would depend on the species status of the various Hanuman), which has a tail that loops forward, and the Southern Hanuman langur populations. Thus for an appropriate interpreta- type (here called as ST-Hanuman), which has a tail that loops back- tion of results from these studies, it is imperative that the taxo- ward (Fig. 1). The NT-Hanuman is found north of the Tapti and God- nomic status of Hanuman langurs is resolved. avari rivers, over the plains of northern India and to an altitude of Within the overall range of the Hanuman langurs two other 3000 m in the Himalayas. The ST-Hanuman is found south of the species of langurs are found, having more restricted distributions. Tapti and Godavari rivers in southern India and in Sri Lanka (Roon- These are Nilgiri langurs (S. johnii) found in the wet evergreen for- wal, 1984). There is much disagreement in the literature regarding ests of south-west India and purple-faced langurs (S. vetulus) dis- the species and subspecies status of various populations of the so- tributed in the wet zone of Sri Lanka (Fig. 1). Thus langur called Hanuman langurs. Most authors have considered the Hanu- monkeys of the Indian subcontinent provide an interesting system man langurs to be a single species, S. entellus, but have divided this to study speciation because there are several closely-related spe- species into as many as 14 (Pocock, 1939), 15 (Napier and Napier, cies found in different habitats and distributed in different geo- 1967) and 16 (Roonwal and Mohnot, 1977) subspecies. Other graphical regions of this landmass. authors have classiﬁed the Hanuman langurs into two (Brandon- Recent phylogenetic studies on the langurs of Asia suggested that the Hanuman langurs are polyphyletic with respect to Nilgiri and purple-faced langurs in their mitochondrial DNA (Karanth et al., * Corresponding author. Address: Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560012, India. 2008; Osterholz et al., 2008). These results are indicative of an inter- E-mail address: [email protected] (K.P. Karanth). esting speciation model wherein certain ancestral populations of a

Fig. 1. Distribution of the langurs of the Indian subcontinent and sampling locations. Hanuman langurs are found throughout most of India and Sri Lanka, as well as in parts of Bangladesh, Pakistan, and Nepal. The borderline between the Northern type and Southern type of the Hanuman langurs runs along the Tapti–Godavari rivers, approximately where the dashed line is drawn on this map. The Nilgiri langur is distributed along the wet zone of south-west India, and purple-faced langur is found in the wet zone of Sri Lanka. The rest of the Indian subcontinent is predominantly dry, open country. Sampling locations are indicated by squares and triangles for wild and captive populations respectively. Langur illustrations modiﬁed from Roonwal (1984). widely-distributed ‘‘proto” Hanuman langur might have diverged gurs related to each other and to the other langurs of the Indian into purple-faced langurs in Sri Lanka and Nilgiri langurs in South In- subcontinent? Second, based on genetic data do the Hanuman lan- dia in the recent past. These diverged species might still share mito- gurs deserve being split into multiple species? To address these chondrial DNA (mtDNA) haplotypes or nuclear alleles with a subset questions, two rapidly-evolving markers—the nuclear protamine of Hanuman langur populations resulting in their polyphyly. This 1(Prm1) gene and the mitochondrial cytochrome b (Cyt-b) gene— speciation model has been reported for the fascicularis group of the were used. macaques (genus Macaca)(Melnick and Hoelzer, 1993) as well as for Asian colobines (genus Trachypithecus)(Karanth et al., 2008). 2. Methods The studies by Karanth et al. (2008) and Osterholz et al. (2008) were focused on resolving the phylogeny of Asian colobines and Three kinds of samples—namely hair, blood, and muscle tissue— did not delve into the Hanuman langur polyphyly issue. Addition- were collected from wild and captive animals. Samples of the ally, sampling of Hanuman, Nilgiri and purple-faced langurs in widely-distributed Hanuman langurs were collected from ﬁve these studies were limited and derived mostly from zoos. To fur- locations in the wild spanning the entire range of this species (Ta- ther investigate the Hanuman langur polyphyly issue, additional ble 1 and Fig. 1). Blood samples were stored in digestion buffer samples of this species were obtained from throughout its range. (0.01 M Tris–HCl (pH 8), 0.01 M EDTA, 0.1 M NaCl, 10 ll1M We have also used additional sequences of purple-faced and Nilgiri DTT) in a 1:1 ratio, and tissue samples were stored in 95% ethanol. langurs downloaded from GenBank. Here we address two major The methodology used for collecting these samples and for DNA questions. First, how are the various populations of Hanuman lan- extractions were the same as reported in Karanth et al. (2008). K.P. Karanth et al. / Molecular Phylogenetics and Evolution 54 (2010) 627–633 629

Table 1 Sequences used to determine the evolutionary relationship between various populations of the Hanuman langur and their relationship with other langurs of the Indian subcontinent.

Location Cyt-b (1140 bp) Acc# Prm1 (380 bp) Acc# Prm1 allele Hanuman langur Southern type S1 Hyderabad AF293951 p1c AF294852 fc A2–A2 S2 Hyderabad (1) AF293952 fc AF294852 fc A2–A2 S3 Hyderabad AF293953 fc AF294851 fc A2–A2b S4 Colombo zoo AF293954 p1c AF294853 fc A1–A2 S5 Anuradhapura (1) AF293955 fc AF294852 fc A1–A2 S6 Anuradhapura AF293956 fc AF294853 fc A1–A2 Northern type N1 Calcutta AF293958 fc AF294851 f A2b–A2b N2 Ramnagar AF293959 f AF294851 fc A2b–A2b N3 Jaipur AF293957 f AF294851 f A2b–A2b N4 (1)d AF012470 f AF294851 f A2b–A2b N5 (1)d AF295576 f PCR A2b–A2b Purple-faced langur PF1 (1) AF295577 f AF294855 f A2–A2 PF2 (1) AF294624 p1 AF119236 f A2–A2 PF3 (2) AF020420 p2 NA PF4 (3) AY519454 p2 NA Nilgiri langur NL1 (1) Anamalai AF294619 f AF294853 f A1–A1 NL2 (1) AF294620 p1 AF294853 f A1–A1 NL3 (3) AY519453 p2 AF294854 f A1b–A1b NL4 (2) AF020419 p2 NA Genus Trachypithecus (SE Asian Colobines) Francois’ leaf monkey AF295578 f Dusky leaf monkey AF295579 f Silvered leaf monkey AF295580 f Phayre’s leaf monkey AF294622 f Outgroup (African Colobines) Guereza Colobus U38264 f Red Colobus AF294625 f

Sample location: 1 = Karanth et al. (2008),2=Zhang and Ryder (1998),3=Osterholz et al. (2008), d = langurs originally procured from Delhi zoo; Sequence length: p1 = partial sequence of 830 bp; p2 = partial sequence of 393–576 bp; f = full length sequence. The letter ‘‘c” indicates that for these samples the PCR products were cloned to detect either Prm1 alleles or Cyt-b nuclear pseudogenes, for the rest of the samples the genes were sequenced directly from PCR products; PCR = for these samples, the identiﬁcation of the Prm1 allele was based on agarose gel visualization of PCR products rather than sequencing. Acc# = Accession number, NA = samples not available.

2.1. PCR amplification of the Prm1 and Cyt-b gene tionary relationships between these species. The program MODELTEST (Posada and Crandall, 1998) was used to choose sub- Protamines are arginine-rich proteins that replace histones and stitution models that best fit the dataset through hierarchical like- bind sperm DNA during spermatogenesis in vertebrates (Rooney lihood ratio tests (Swofford et al., 1996), and to estimate the and Zhang, 1999). The Prm1 gene has been used to resolve the phy- transition–transversion rate ratio, gamma shape parameters, and logeny of primate (Retief and Dixon, 1993) and other mammalian base frequencies under the best supported model. These values, groups (Retief et al., 1995; Krajewski et al., 1997; Blacket et al., along with the selected model, were used to derive the maximum 1999). In the case of Cyt-b gene, our studies have shown that this likelihood (ML) tree through a heuristic search with 10 replicates gene is highly variable among the langurs (Karanth et al., 2008). of the random addition and TBR branch swapping options in Therefore, we used these two markers to study the nuclear and PAUP*, wherein a molecular clock was not enforced. The above val- mitochondrial genetic diversity of the langurs of the Indian sub- ues were used again to derive a neighbor-joining tree through continent. The primers used for amplification, PCR conditions and 10,000 bootstrap replications in PAUP*. In this dataset, transitions sequencing strategy for Prm1 and Cyt-b genes were same as that were empirically estimated to be around 11 times more frequent in (Karanth et al., 2008). than transversions. To account for this difference in base substitu- All sequencing was performed using the Taq DyeDeoxy™ Ter- tion, an 11:1 weighting scheme was used in all parsimony analysis. minator Cycle Sequencing Kit (Applied Biosystems), following the First, a branch-and-bound parsimony search was undertaken with manufacturer’s instructions. Reactions were analyzed using either ‘simple addition of sequences’ option. To ascertain support for var- an ABI Model 373A or an ABI Model 377 Automated DNA Sequen- ious nodes, 1000 bootstrap replications were performed, using 10 cer. Samples collected in India and Sri Lanka were sequenced in Dr. replicates of the random addition option. For all these analyses, Lalji Singh’s laboratory. Sequences were edited using the program the incomplete regions of partial sequences were coded as missing SeqEd (Applied Biosystems) and aligned manually in PAUP* (Swof- data. Published sequences of Southeast Asian leaf monkeys (Genus ford, 2001). Trachypithecus) that are sister groups to Semnopithecus were also included for comparison and the African colobines were used to root these trees. 2.2. Analysis of the Cyt-b gene sequences The program MacClade (Maddison and Maddison, 2000) was used to infer amino acid sequences and to construct a constraint A total of 11 Hanuman langur and four each of Nilgiri and pur- tree, wherein representatives of each species were forced to be ple-faced langur Cyt-b sequences were used to ascertain the evolu- 630 K.P. Karanth et al. / Molecular Phylogenetics and Evolution 54 (2010) 627–633 monophyletic. This constraint tree was used in PAUP* to derive a of these three trees (not shown) was very similar to the likelihood ‘‘species monophyly” tree as a null model for the data through a tree. likelihood heuristic search. To examine support for the hypothesis The overall tree topology was the same for various tree-building of species monophyly, this constrained ML tree was compared with methods and there was no significant difference between them the unconstrained ML tree from above using the one-tailed Shimo- (P > 0.05, Shimodaira–Hasegawa tests). The three tree-building daira–Hasegawa log-likelihood test (Shimodaira and Hasegawa, methods retrieved the three clades mentioned above and the rela- 1999) as implemented in PAUP*, using the re-sampling estimated tionships among these clades were also identical. The only differ- log-likelihood (RELL) technique approximation with 10,000 boot- ence between methods was with respect to the relationships strap replications. between species in clades 2 and 3. These changes do not alter the conclusions of the paper. The aligned sequences of the Cyt-b gene region had no stop co- 2.3. Analysis of the Prm1 gene sequences dons or indels. Base frequencies at the three codon positions were in concordance with expectations for vertebrate Cyt-b gene (Johns The sequence based phylogenetic analysis of Prm1 gene did not and Avise, 1998; Prusak and Grzybowshi, 2004). Additionally, third resolve the evolutionary relationships between the langurs of the codon positions had a very low frequency of G’s, compared to other Indian subcontinent due to a lack of phylogenetically informative mtDNA genes. Thus, these sequences were considered as true characters (Karanth et al., 2008). Nevertheless, this gene does exhi- mitochondrial Cyt-b sequences rather than nuclear-mitochondrial bit high levels of allelic variation among the langurs of the Indian sequences (Zhang and Hewitt, 1996; Bensasson et al., 2001). Fur- subcontinent. Therefore, we analyzed allelic data to determine if ther, it must be noted that the work done by Osterholz et al. there were any fixed allelic differences between species or be- (2008) also support Hanuman langur polyphyly. In their study tween populations of Hanuman langurs. Two different approaches Cyt-b sequences of Hanuman, Nilgiri and purple-faced langurs were used to identify Prm1 alleles. First, the coding sequences from were derived from other sources wherein different primers sets various samples were aligned and their amino acid sequences were were used. Therefore it is very unlikely that multiple labs indepen- inferred in the program MacClade (Maddison and Maddison, 2000). dently amplified nuclear copies of Cyt-b from three different spe- Alleles were classified on the basis of these amino acid sequences. cies using different primers. For individuals that were heterozygous at this locus, the PCR products were cloned and multiple clones were sequenced to determine the sequences of the alleles. Heterozygous individuals were 3.2. The Prm1 gene results detected through the presence of multiple peaks at a site in the chromatograms. In the case of some Hanuman langurs, the 30 A total of four Prm1 alleles (A1, A1b, A2, and A2b) were found non-coding region of this gene was found to have a tandem dupli- among the three species of langurs. The amino acid sequences of cation of 40 bp (here called the 40 bp repeat) in some individuals. these alleles are given in Fig. 2B. All of the five NT-Hanuman sam- The presence or absence of this 40 bp repeat in a sample can be de- ples analyzed contained only allele A2b (Table 1), which has a tected by electrophoresing the PCR product in a 2% agarose gel. 40 bp repeat and therefore could be resolved on a 2% agarose gel. Therefore, for many Hanuman langur samples this particular allele Four of these samples that contained A2b alleles were sequenced was identified on the basis of agarose gel electrophoresis. Four of to confirm the presence of the repeat and to detect any new allele these alleles with the 40 bp repeat were also sequenced to confirm (due to sequence difference). All of the four Prm1 A2b sequences the presence of the repeat and to determine if there were any vari- were identical (data not shown). ST-Hanuman, on the contrary, ations in the sequence. showed a total of three alleles (A1, A2, and A2b) in six samples analyzed. Some of the individuals were heterozygous at this locus (Table 1). 3. Results The purple-faced langurs were found to have one allele (A2) in both the samples analyzed. In the case of Nilgiri langur, a total of 3.1. The Cyt-b gene results three samples were analyzed and two alleles were detected (A1 and A1b). One of these alleles, A1 is also seen in ST-Hanuman lan- The program MODELTEST chose the HKY 85 (Hasegawa et al., gurs. Thus, as in the case of mtDNA diversity, the Nilgiri langur and 1985)+C substitution model with the following parameters: purple-faced langur share at least one of their Prm1 alleles with ST- Base = (0.3231, 0.3018, 0.1111), TRatio = 11.0048, and Hanuman langurs (see Fig. 2B). Shape = 0.2472. The maximum likelihood search yielded a single tree (In L = 5599.5731) that is shown in Fig. 2A. In this tree, the langurs of the Indian subcontinent fall into three well-supported 4. Discussion clades: clade 1, consisting of NT-Hanuman individuals; clade 2, consisting of ST-Hanuman individuals from South India together Results from this and earlier (Karanth et al., 2008; Osterholz with the Nilgiri langur; and clade 3, consisting of ST-Hanuman et al. 2008) works indicate that the mtDNA of the so-called Hanu- individuals from Sri Lanka together with the purple-faced langur. man langurs is not monophyletic. Instead, the mtDNA of the ST- Thus, the mtDNA of the so-called Hanuman langur is not mono- Hanuman from Sri Lanka branches with that of purple-faced langur phyletic and encompasses mtDNA variation of two other species of Sri Lanka, and the mtDNA of the ST-Hanuman from South India (purple-faced langur and Nilgiri langur). In addition, within the groups with Nilgiri langur to the exclusion of NT-Hanuman clades 2 and 3, the mtDNAs of the so-called Hanuman langurs (Fig. 2A). Furthermore, Prm1 allelic variation found in Nilgiri lan- are not monophyletic. For example, within clade 3, the ST-Hanu- gurs, purple-faced langurs, and NT-Hanuman langurs is a subset man from Sri Lanka and the purple-faced langur are polyphyletic. of the variation seen in the ST-Hanuman langurs. These results The likelihood score of the best tree (In L = 5599.5731) was sig- suggest that the current day ST-Hanuman langur has maintained nificantly higher than the species monophyly tree high levels of mitochondrial and nuclear variation some of which (ln L = 5830.8072), wherein the three species were constrained it shares with Nilgiri and purple-faced langurs. to be reciprocally monophyletic (P = 0.000, Shimodaira–Hasegawa A similar scenario has been reported for another Asian colobine test). The parsimony branch-and-bound search yielded three of the genus Trachypithecus wherein the widely-distributed equally parsimonious trees (tree length = 2068). A strict consensus Phayre’s leaf monkey (T. phayrei) is polyphyletic with respect to K.P. Karanth et al. / Molecular Phylogenetics and Evolution 54 (2010) 627–633 631

Fig. 2. (A) Maximum likelihood tree based on Cyt-b gene of the langurs of the Indian subcontinent. The numbers by the nodes indicate likeihood, parsimony and neighbor- joining bootstrap supports respectively and the stars indicate 100% support. The arrows indicate the three major clades that were retrieved by all tree-building methods. (B) Prm1 amino acid sequences from the langurs. The site (amino acid 25) shown in bold was used to identify alleles in the coding sequence of the gene. indicate 40 bp repeat; —, deletion. See Table 1 abbreviations.

Francois’ leaf monkey (T. francoisi) and dusky leaf monkey (T. wherein the widely-distributed rhesus macaque (M. mulatta)is obscurus) in the mtDNA tree but the nuclear Prm1 gene support polyphyletic with respect to Japanese (M. fuscata) and Taiwanese their monophyly (Karanth et al., 2008). This pattern is also ob- macaque (M. cyclopis)(Melnick and Hoelzer, 1993). Nuclear allo- served among fascicularis group of the macaques (genus Macaca) zyme studies on this system, however, indicate that eastern and 632 K.P. Karanth et al. / Molecular Phylogenetics and Evolution 54 (2010) 627–633 western populations of rhesus macaques are members of one rela- Taken together these analyses and observations suggest that tively homogeneous species (Melnick and Hoelzer, 1992). Thus in the so-called Hanuman langurs might have to be split into multiple both the cases discussed above the nuclear markers suggest that species as has been done in some classification schemes (Hill, these widely-distributed taxa constitute a single homogeneous 1939; Groves, 2001; Brandon-Jones, 2004). But the results reported species but their mtDNA have retained high levels of polymor- here are based on limited sampling of Hanuman langurs and does phism that is shared with sister taxa. In contrast, the so-called not include all the putative species/subspecies. Therefore, we have Hanuman langurs show slightly different pattern in that the nucle- refrained from designating species names to the three Hanuman ar marker does not support a single homogeneous species. Here, langur species discussed above. In the light of these findings, it is NT-Hanuman is distinct from ST-Hanuman at both nuclear and apparent that the results of comparative studies done on Hanuman mitochondrial loci, and preliminary data suggest that ST-Hanuman langurs from across the range need to be reinterpreted. might have to be split into multiple species (see below). To address the Hanuman langur species and lineage sorting is- sues, more samples of Hanuman langurs from South India, Bangla- desh, and Sri Lanka need to be analyzed with additional markers 4.1. Species status of NT- and ST-Hanuman langurs (particularly a Y-chromosome marker and microsatellites). These analyses will help us better understand the factors that are respon- In the mtDNA tree the NT-Hanuman is monophyletic (clade 1, sible for the maintenance of intraspecific mtDNA and nDNA diver- Fig. 2A) and the average uncorrected pair-wise sequence differ- sity in this species complex and also to determine the classification ences between clade 1 and 2 (after excluding the partial se- scheme that best fits the molecular data. quences) is comparable to that of among-species comparisons within the genus Trachypithecus (around 7.9%). The mtDNA result Acknowledgments is complemented by the nuclear DNA (nDNA) result, wherein all five NT-Hanuman samples collected from a wide area in northern The authors thank R. Pethiagoda, M. Singh, S. Siva, S.N. Nigam, C. India carry the A2b allele. This allele appeared to be rare in South Roos, K. Launhardt, P. Dolhinow, R.S. Harave, A. Chaudary, S.M. Indian and Sri Lankan clade; only one sample of ST-Hanuman was Mohnot, and N.K. Lohiya for helping us with sample collection, found to have this allele (Table 1). Thus the NT-Hanuman appeared and A.P. Jason de Koning for proofreading the manuscript. Field- to be fixed for the A2b allele at the Prm1 locus. Additionally, the work for this study was supported through SUNY benevolent, NT-Hanuman is morphologically distinct from ST-Hanuman and SUNY GSO, and Primate Conservation Inc. grants to K.P.K. occupies a distinct geographical region north of the Tapti–Godavari river system (Fig. 1). These results suggest that NT-Hanuman might warrant species status, but more studies need to be under- Appendix A. Supplementary data taken to rigorously test this hypothesis. In the case of the ST-Hanuman, due to limited sample size, we Supplementary data associated with this article can be found, in might have missed other divergent mtDNA haplotypes and Prm1 the online version, at doi:10.1016/j.ympev.2009.10.034. alleles. 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