Molecular Phylogenetics and Evolution 54 (2010) 627–633

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/ locate /ympev

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, Indiab Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, Indiac Department of Biological Sciences, University at Albany, State University of New York, Albany, NY 12222, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 June 2008Revised 9 October 2009Accepted 29 October 2009Available online 6 November 2009

Keywords:SemnopithecusLineage sortingCytochrome bProtamine P1Molecular systematicsSpeciation

1055-7903/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ympev.2009.10.034

* Corresponding author. Address: Centre for Ecologof Science, Bangalore 560012, India.

E-mail address: karanth@ces.iisc.ernet.in (K.P. Kar

Recent molecular studies on langurs of the Indian subcontinent suggest that the widely-distributed andmorphologically variable Hanuman langurs (Semnopithecus entellus) are polyphyletic with respect toNilgiri and purple-faced langurs. To further investigate this scenario, we have analyzed additionalsequences of mitochondrial cytochrome b as well as nuclear protamine P1 genes from these species.The results confirm Hanuman langur polyphyly in the mitochondrial tree and the nuclear markers sug-gest that the Hanuman langurs share protamine P1 alleles with Nilgiri and purple-faced langurs. We rec-ommend provisional splitting of the so-called Hanuman langurs into three species such that thetaxonomy is consistent with their evolutionary relationships.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

The Hanuman langur (Semnopithecus entellus, Subfamily Colobi-nae) is one of the most widely-distributed and morphologically var-iable species of colobine monkey (Newton, 1988). It is distributedthroughout most parts of India and Sri Lanka, and is also found inparts of Pakistan, Nepal, and Bangladesh. According to Roonwaland Mohnot (1977), it is predominantly a deciduous woodland spe-cies. Roonwal (1984) recognizes two major forms of Hanuman lan-gur based on tail morphology: the Northern type (here called as NT-Hanuman), which has a tail that loops forward, and the Southerntype (here called as ST-Hanuman), which has a tail that loops back-ward (Fig. 1). The NT-Hanuman is found north of the Tapti and God-avari rivers, over the plains of northern India and to an altitude of3000 m in the Himalayas. The ST-Hanuman is found south of theTapti and Godavari rivers in southern India and in Sri Lanka (Roon-wal, 1984). There is much disagreement in the literature regardingthe species and subspecies status of various populations of the so-called Hanuman langurs. Most authors have considered the Hanu-man langurs to be a single species, S. entellus, but have divided thisspecies into as many as 14 (Pocock, 1939), 15 (Napier and Napier,1967) and 16 (Roonwal and Mohnot, 1977) subspecies. Otherauthors have classified the Hanuman langurs into two (Brandon-

ll rights reserved.

ical Sciences, Indian Institute

anth).

316

Jones, 2004), four (Hill, 1939) and seven (Groves, 2001) distinct spe-cies. Hanuman langurs have also been extensively used as modelsystem in a various biomedical (Nandi et al., 2003; Dube et al.,2004; Lohiya et al., 2005), ecological (Kurup, 1984; Kamilar et al.,2006), behavioral (Newton, 1988; Sterck, 1999) and evolutionaryresearch (Karanth et al., 2008; Osterholz et al., 2008). Often thesehave been comparative studies wherein multiple populations ofHanuman langurs were studied from across the range. The decisionon the nature of these comparative studies, i.e., inter vs. intra spe-cific comparison, would depend on the species status of the variousHanuman langur populations. Thus for an appropriate interpreta-tion of results from these studies, it is imperative that the taxo-nomic status of Hanuman langurs is resolved.

Within the overall range of the Hanuman langurs two otherspecies of langurs are found, having more restricted distributions.These are Nilgiri langurs (S. johnii) found in the wet evergreen for-ests of south-west India and purple-faced langurs (S. vetulus) dis-tributed in the wet zone of Sri Lanka (Fig. 1). Thus langurmonkeys of the Indian subcontinent provide an interesting systemto study speciation because there are several closely-related spe-cies found in different habitats and distributed in different geo-graphical regions of this landmass.

Recent phylogenetic studies on the langurs of Asia suggested thatthe Hanuman langurs are polyphyletic with respect to Nilgiri andpurple-faced langurs in their mitochondrial DNA (Karanth et al.,2008; Osterholz et al., 2008). These results are indicative of an inter-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 ofBangladesh, Pakistan, and Nepal. The borderline between the Northern type and Southern type of the Hanuman langurs runs along the Tapti–Godavari rivers, approximatelywhere 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 SriLanka. The rest of the Indian subcontinent is predominantly dry, open country. Sampling locations are indicated by squares and triangles for wild and captive populationsrespectively. Langur illustrations modified from Roonwal (1984).

628 K.P. Karanth et al. / Molecular Phylogenetics and Evolution 54 (2010) 627–633

widely-distributed ‘‘proto” Hanuman langur might have divergedinto purple-faced langurs in Sri Lanka and Nilgiri langurs in South In-dia in the recent past. These diverged species might still share mito-chondrial DNA (mtDNA) haplotypes or nuclear alleles with a subsetof Hanuman langur populations resulting in their polyphyly. Thisspeciation model has been reported for the fascicularis group of themacaques (genus Macaca) (Melnick and Hoelzer, 1993) as well asfor Asian colobines (genus Trachypithecus) (Karanth et al., 2008).

The studies by Karanth et al. (2008) and Osterholz et al. (2008)were focused on resolving the phylogeny of Asian colobines anddid not delve into the Hanuman langur polyphyly issue. Addition-ally, sampling of Hanuman, Nilgiri and purple-faced langurs inthese studies were limited and derived mostly from zoos. To fur-ther investigate the Hanuman langur polyphyly issue, additionalsamples of this species were obtained from throughout its range.We have also used additional sequences of purple-faced and Nilgirilangurs downloaded from GenBank. Here we address two majorquestions. First, how are the various populations of Hanuman lan-

31

gurs related to each other and to the other langurs of the Indiansubcontinent? Second, based on genetic data do the Hanuman lan-gurs deserve being split into multiple species? To address thesequestions, two rapidly-evolving markers—the nuclear protamine1 (Prm1) gene and the mitochondrial cytochrome b (Cyt-b) gene—were used.

2. Methods

Three kinds of samples—namely hair, blood, and muscle tissue—were collected from wild and captive animals. Samples of thewidely-distributed Hanuman langurs were collected from fivelocations in the wild spanning the entire range of this species (Ta-ble 1 and Fig. 1). Blood samples were stored in digestion buffer(0.01 M Tris–HCl (pH 8), 0.01 M EDTA, 0.1 M NaCl, 10 ll 1 MDTT) in a 1:1 ratio, and tissue samples were stored in 95% ethanol.The methodology used for collecting these samples and for DNAextractions were the same as reported in Karanth et al. (2008).

7

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

Location Cyt-b (1140 bp) Acc# Prm1 (380 bp) Acc# Prm1 allele

Hanuman langurSouthern type

S1 Hyderabad AF293951 p1c AF294852 fc A2–A2S2 Hyderabad (1) AF293952 fc AF294852 fc A2–A2S3 Hyderabad AF293953 fc AF294851 fc A2–A2bS4 Colombo zoo AF293954 p1c AF294853 fc A1–A2S5 Anuradhapura (1) AF293955 fc AF294852 fc A1–A2S6 Anuradhapura AF293956 fc AF294853 fc A1–A2

Northern typeN1 Calcutta AF293958 fc AF294851 f A2b–A2bN2 Ramnagar AF293959 f AF294851 fc A2b–A2bN3 Jaipur AF293957 f AF294851 f A2b–A2bN4 (1)d AF012470 f AF294851 f A2b–A2bN5 (1)d AF295576 f PCR A2b–A2b

Purple-faced langurPF1 (1) AF295577 f AF294855 f A2–A2PF2 (1) AF294624 p1 AF119236 f A2–A2PF3 (2) AF020420 p2 NAPF4 (3) AY519454 p2 NA

Nilgiri langurNL1 (1) Anamalai AF294619 f AF294853 f A1–A1NL2 (1) AF294620 p1 AF294853 f A1–A1NL3 (3) AY519453 p2 AF294854 f A1b–A1bNL4 (2) AF020419 p2 NA

Genus Trachypithecus (SE Asian Colobines)Francois’ leaf monkey AF295578 fDusky leaf monkey AF295579 fSilvered leaf monkey AF295580 fPhayre’s leaf monkey AF294622 f

Outgroup (African Colobines)Guereza Colobus U38264 fRed 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 todetect 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, theidentification of the Prm1 allele was based on agarose gel visualization of PCR products rather than sequencing. Acc# = Accession number, NA = samples not available.

K.P. Karanth et al. / Molecular Phylogenetics and Evolution 54 (2010) 627–633 629

2.1. PCR amplification of the Prm1 and Cyt-b gene

Protamines are arginine-rich proteins that replace histones andbind sperm DNA during spermatogenesis in vertebrates (Rooneyand Zhang, 1999). The Prm1 gene has been used to resolve the phy-logeny of primate (Retief and Dixon, 1993) and other mammaliangroups (Retief et al., 1995; Krajewski et al., 1997; Blacket et al.,1999). In the case of Cyt-b gene, our studies have shown that thisgene is highly variable among the langurs (Karanth et al., 2008).Therefore, we used these two markers to study the nuclear andmitochondrial genetic diversity of the langurs of the Indian sub-continent. The primers used for amplification, PCR conditions andsequencing strategy for Prm1 and Cyt-b genes were same as thatin (Karanth et al., 2008).

All sequencing was performed using the Taq DyeDeoxy™ Ter-minator Cycle Sequencing Kit (Applied Biosystems), following themanufacturer’s instructions. Reactions were analyzed using eitheran ABI Model 373A or an ABI Model 377 Automated DNA Sequen-cer. Samples collected in India and Sri Lanka were sequenced in Dr.Lalji Singh’s laboratory. Sequences were edited using the programSeqEd (Applied Biosystems) and aligned manually in PAUP* (Swof-ford, 2001).

2.2. Analysis of the Cyt-b gene sequences

A total of 11 Hanuman langur and four each of Nilgiri and pur-ple-faced langur Cyt-b sequences were used to ascertain the evolu-

318

tionary relationships between these species. The programMODELTEST (Posada and Crandall, 1998) was used to choose sub-stitution models that best fit the dataset through hierarchical like-lihood ratio tests (Swofford et al., 1996), and to estimate thetransition–transversion rate ratio, gamma shape parameters, andbase frequencies under the best supported model. These values,along with the selected model, were used to derive the maximumlikelihood (ML) tree through a heuristic search with 10 replicatesof the random addition and TBR branch swapping options inPAUP*, wherein a molecular clock was not enforced. The above val-ues were used again to derive a neighbor-joining tree through10,000 bootstrap replications in PAUP*. In this dataset, transitionswere empirically estimated to be around 11 times more frequentthan transversions. To account for this difference in base substitu-tion, an 11:1 weighting scheme was used in all parsimony analysis.First, a branch-and-bound parsimony search was undertaken with‘simple addition of sequences’ option. To ascertain support for var-ious nodes, 1000 bootstrap replications were performed, using 10replicates of the random addition option. For all these analyses,the incomplete regions of partial sequences were coded as missingdata. Published sequences of Southeast Asian leaf monkeys (GenusTrachypithecus) that are sister groups to Semnopithecus were alsoincluded for comparison and the African colobines were used toroot these trees.

The program MacClade (Maddison and Maddison, 2000) wasused to infer amino acid sequences and to construct a constrainttree, wherein representatives of each species were forced to be

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‘‘species monophyly” tree as a null model for the data through alikelihood heuristic search. To examine support for the hypothesisof species monophyly, this constrained ML tree was compared withthe unconstrained ML tree from above using the one-tailed Shimo-daira–Hasegawa log-likelihood test (Shimodaira and Hasegawa,1999) as implemented in PAUP*, using the re-sampling estimatedlog-likelihood (RELL) technique approximation with 10,000 boot-strap replications.

2.3. Analysis of the Prm1 gene sequences

The sequence based phylogenetic analysis of Prm1 gene did notresolve the evolutionary relationships between the langurs of theIndian subcontinent due to a lack of phylogenetically informativecharacters (Karanth et al., 2008). Nevertheless, this gene does exhi-bit high levels of allelic variation among the langurs of the Indiansubcontinent. Therefore, we analyzed allelic data to determine ifthere were any fixed allelic differences between species or be-tween populations of Hanuman langurs. Two different approacheswere used to identify Prm1 alleles. First, the coding sequences fromvarious samples were aligned and their amino acid sequences wereinferred in the program MacClade (Maddison and Maddison, 2000).Alleles were classified on the basis of these amino acid sequences.For individuals that were heterozygous at this locus, the PCR prod-ucts were cloned and multiple clones were sequenced to deter-mine the sequences of the alleles. Heterozygous individuals weredetected through the presence of multiple peaks at a site in thechromatograms. In the case of some Hanuman langurs, the 30

non-coding region of this gene was found to have a tandem dupli-cation of 40 bp (here called the 40 bp repeat) in some individuals.The presence or absence of this 40 bp repeat in a sample can be de-tected by electrophoresing the PCR product in a 2% agarose gel.Therefore, for many Hanuman langur samples this particular allelewas identified on the basis of agarose gel electrophoresis. Four ofthese alleles with the 40 bp repeat were also sequenced to confirmthe presence of the repeat and to determine if there were any vari-ations in the sequence.

3. Results

3.1. The Cyt-b gene results

The program MODELTEST chose the HKY 85 (Hasegawa et al.,1985) + C substitution model with the following parameters:Base = (0.3231, 0.3018, 0.1111), TRatio = 11.0048, andShape = 0.2472. The maximum likelihood search yielded a singletree (In L = �5599.5731) that is shown in Fig. 2A. In this tree, thelangurs of the Indian subcontinent fall into three well-supportedclades: clade 1, consisting of NT-Hanuman individuals; clade 2,consisting of ST-Hanuman individuals from South India togetherwith the Nilgiri langur; and clade 3, consisting of ST-Hanumanindividuals from Sri Lanka together with the purple-faced langur.Thus, the mtDNA of the so-called Hanuman langur is not mono-phyletic and encompasses mtDNA variation of two other species(purple-faced langur and Nilgiri langur). In addition, within theclades 2 and 3, the mtDNAs of the so-called Hanuman langursare not monophyletic. For example, within clade 3, the ST-Hanu-man from Sri Lanka and the purple-faced langur are polyphyletic.The likelihood score of the best tree (In L = �5599.5731) was sig-nificantly higher than the species monophyly tree(ln L = �5830.8072), wherein the three species were constrainedto be reciprocally monophyletic (P = 0.000, Shimodaira–Hasegawatest). The parsimony branch-and-bound search yielded threeequally parsimonious trees (tree length = 2068). A strict consensus

31

of these three trees (not shown) was very similar to the likelihoodtree.

The overall tree topology was the same for various tree-buildingmethods and there was no significant difference between them(P > 0.05, Shimodaira–Hasegawa tests). The three tree-buildingmethods retrieved the three clades mentioned above and the rela-tionships among these clades were also identical. The only differ-ence between methods was with respect to the relationshipsbetween species in clades 2 and 3. These changes do not alterthe conclusions of the paper.

The aligned sequences of the Cyt-b gene region had no stop co-dons or indels. Base frequencies at the three codon positions werein concordance with expectations for vertebrate Cyt-b gene (Johnsand Avise, 1998; Prusak and Grzybowshi, 2004). Additionally, thirdcodon positions had a very low frequency of G’s, compared to othermtDNA genes. Thus, these sequences were considered as truemitochondrial Cyt-b sequences rather than nuclear-mitochondrialsequences (Zhang and Hewitt, 1996; Bensasson et al., 2001). Fur-ther, it must be noted that the work done by Osterholz et al.(2008) also support Hanuman langur polyphyly. In their studyCyt-b sequences of Hanuman, Nilgiri and purple-faced langurswere derived from other sources wherein different primers setswere used. Therefore it is very unlikely that multiple labs indepen-dently amplified nuclear copies of Cyt-b from three different spe-cies using different primers.

3.2. The Prm1 gene results

A total of four Prm1 alleles (A1, A1b, A2, and A2b) were foundamong the three species of langurs. The amino acid sequences ofthese alleles are given in Fig. 2B. All of the five NT-Hanuman sam-ples analyzed contained only allele A2b (Table 1), which has a40 bp repeat and therefore could be resolved on a 2% agarose gel.Four of these samples that contained A2b alleles were sequencedto confirm the presence of the repeat and to detect any new allele(due to sequence difference). All of the four Prm1 A2b sequenceswere identical (data not shown). ST-Hanuman, on the contrary,showed a total of three alleles (A1, A2, and A2b) in six samples ana-lyzed. Some of the individuals were heterozygous at this locus(Table 1).

The purple-faced langurs were found to have one allele (A2) inboth the samples analyzed. In the case of Nilgiri langur, a total ofthree samples were analyzed and two alleles were detected (A1and A1b). One of these alleles, A1 is also seen in ST-Hanuman lan-gurs. Thus, as in the case of mtDNA diversity, the Nilgiri langur andpurple-faced langur share at least one of their Prm1 alleles with ST-Hanuman langurs (see Fig. 2B).

4. Discussion

Results from this and earlier (Karanth et al., 2008; Osterholzet al. 2008) works indicate that the mtDNA of the so-called Hanu-man langurs is not monophyletic. Instead, the mtDNA of the ST-Hanuman from Sri Lanka branches with that of purple-faced langurof Sri Lanka, and the mtDNA of the ST-Hanuman from South Indiagroups with Nilgiri langur to the exclusion of NT-Hanuman(Fig. 2A). Furthermore, Prm1 allelic variation found in Nilgiri lan-gurs, purple-faced langurs, and NT-Hanuman langurs is a subsetof the variation seen in the ST-Hanuman langurs. These resultssuggest that the current day ST-Hanuman langur has maintainedhigh levels of mitochondrial and nuclear variation some of whichit shares with Nilgiri and purple-faced langurs.

A similar scenario has been reported for another Asian colobineof the genus Trachypithecus wherein the widely-distributedPhayre’s leaf monkey (T. phayrei) is polyphyletic with respect to

9

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.

K.P. Karanth et al. / Molecular Phylogenetics and Evolution 54 (2010) 627–633 631

Francois’ leaf monkey (T. francoisi) and dusky leaf monkey (T.obscurus) in the mtDNA tree but the nuclear Prm1 gene supporttheir monophyly (Karanth et al., 2008). This pattern is also ob-served among fascicularis group of the macaques (genus Macaca)

320

wherein the widely-distributed rhesus macaque (M. mulatta) ispolyphyletic with respect to Japanese (M. fuscata) and Taiwanesemacaque (M. cyclopis) (Melnick and Hoelzer, 1993). Nuclear allo-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-tively homogeneous species (Melnick and Hoelzer, 1992). Thus inboth the cases discussed above the nuclear markers suggest thatthese widely-distributed taxa constitute a single homogeneousspecies but their mtDNA have retained high levels of polymor-phism that is shared with sister taxa. In contrast, the so-calledHanuman langurs show slightly different pattern in that the nucle-ar marker does not support a single homogeneous species. Here,NT-Hanuman is distinct from ST-Hanuman at both nuclear andmitochondrial loci, and preliminary data suggest that ST-Hanumanmight have to be split into multiple species (see below).

4.1. Species status of NT- and ST-Hanuman langurs

In the mtDNA tree the NT-Hanuman is monophyletic (clade 1,Fig. 2A) and the average uncorrected pair-wise sequence differ-ences between clade 1 and 2 (after excluding the partial se-quences) is comparable to that of among-species comparisonswithin the genus Trachypithecus (around 7.9%). The mtDNA resultis complemented by the nuclear DNA (nDNA) result, wherein allfive NT-Hanuman samples collected from a wide area in northernIndia carry the A2b allele. This allele appeared to be rare in SouthIndian and Sri Lankan clade; only one sample of ST-Hanuman wasfound to have this allele (Table 1). Thus the NT-Hanuman appearedto be fixed for the A2b allele at the Prm1 locus. Additionally, theNT-Hanuman is morphologically distinct from ST-Hanuman andoccupies a distinct geographical region north of the Tapti–Godavaririver system (Fig. 1). These results suggest that NT-Hanumanmight warrant species status, but more studies need to be under-taken to rigorously test this hypothesis.

In the case of the ST-Hanuman, due to limited sample size, wemight have missed other divergent mtDNA haplotypes and Prm1alleles. Therefore, more work needs to be done to determine thespecies status of various ST-Hanuman populations. Neverthelessit is interesting to note that within clades 2 and 3, the Hanumanlangurs are polyphyletic with respect to Nilgiri and purple-facedlangurs. This tree topology is retrieved by all the tree-buildingmethods with minor rearrangements of the taxa.

Taxonomists have long considered Nilgiri and purple-faced lan-gurs as distinct species (see Karanth et al., 2008, and referencestherein). These two species are very distinct from Hanuman lan-gurs in their morphology and ecology and have until recently beenassigned to genus Trachypithecus along with Southeast Asian lan-gurs (Groves, 2001). Thus to reconcile our results with taxonomyit might be useful to tentatively split Hanuman langurs into threespecies namely; the NT-Hanuman, the ST-Hanuman from South In-dia, and the ST-Hanuman from Sri Lanka as has been suggested byOsterholz et al. (2008). The divergence between ST-Hanuman andtheir sister taxa in South India and Sri Lanka might have occurredrecently as suggested by the polyphyletic gene-tree. This is be-cause shortly after speciation the probability is high that two sistertaxa exhibit a polyphyletic gene-tree status (Avise, 1994) due toincomplete lineage sorting. Alternately, introgression of HanumanmtDNA into Nilgiri and purple-faced langurs or vice versa couldalso produce such a topology. There is one record of hybridizationbetween Hanuman and Nilgiri langurs reported from AnamalaiHills of South-west India (Hohmann, 1991). In this regard, it mustbe noted that the distance between the collection site of the ST-Hanuman samples from South India (Hyderabad) and the currentNilgiri langur distribution is around 1000 kms (Fig. 1). Given thatlangurs live in matrilineal society (Newton and Dunbar, 1994)and thus exhibit female natal philopatry it is unlikely that recentmtDNA introgression could have occurred over such a long dis-tance. Nevertheless, with the limited data presented here we can-not rule out ancient hybridization.

32

Taken together these analyses and observations suggest thatthe so-called Hanuman langurs might have to be split into multiplespecies as has been done in some classification schemes (Hill,1939; Groves, 2001; Brandon-Jones, 2004). But the results reportedhere are based on limited sampling of Hanuman langurs and doesnot include all the putative species/subspecies. Therefore, we haverefrained from designating species names to the three Hanumanlangur species discussed above. In the light of these findings, it isapparent that the results of comparative studies done on Hanumanlangurs from across the range need to be reinterpreted.

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(particularly a Y-chromosome marker and microsatellites). Theseanalyses will help us better understand the factors that are respon-sible for the maintenance of intraspecific mtDNA and nDNA diver-sity in this species complex and also to determine the classificationscheme that best fits the molecular data.

Acknowledgments

The authors thank R. Pethiagoda, M. Singh, S. Siva, S.N. Nigam, C.Roos, K. Launhardt, P. Dolhinow, R.S. Harave, A. Chaudary, S.M.Mohnot, and N.K. Lohiya for helping us with sample collection,and A.P. Jason de Koning for proofreading the manuscript. Field-work for this study was supported through SUNY benevolent,SUNY GSO, and Primate Conservation Inc. grants to K.P.K.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2009.10.034.

References

Avise, J.C., 1994. Molecular Markers, Natural History and Evolution. Chapman andHall, New York.

Bensasson, D., Zhang, D.-X., Hartl, D.L., Hewitt, G.M., 2001. Mitochondrialpseudogenes: evolutions misplaced witnesses. Trends Ecol. Evol. 16, 314–321.

Blacket, M.J., Krajewski, C., Labrinidis, A., Cambron, B., Cooper, S., Westerman, M.,1999. Systematic relationships within the dasyurid marsupial tribeSminthopsini—a multigene approach. Mol. Phylogenet. Evol. 2, 140–155.

Brandon-Jones, D., 2004. A taxonomic revision of the langurs and leaf monkeys(Primates: Colobinae) of South Asia. Zoos’ Print J. 19, 1552–1594.

Dube, A., Murthy, P.K., Puri, S.K., Misra-Bhattacharya, S., 2004. Presbytis entellus: aprimate model for parasitic disease research. Trends Parasitol. 20, 8.

Groves, C.P., 2001. Primate Taxonomy. Smithsonian Institute Press, Washington.Hasegawa, M., Kishino, H., Yano, T., 1985. Dating of the human-ape splitting by a

molecular clock of mitochondrial DNA. J. Mol. Evol. 9, 366–369.Hill, W.C., 1939. An annotated systematic list of the leaf-monkeys. Ceylon J. Sci.

Colombo B 21, 277–305.Hohmann, G., 1991. New evidence for hybridization in Presbytis johnii and Presbytis

entellus. J. Bombay Nat. Hist. Soc. 88, 315–319.Johns, G.C., Avise, J.C., 1998. A comparative summary of genetic distance in the

vertebrates from the mitochondrial cytochrome b gene. Mol. Biol. Evol. 15,1481–1490.

Kamilar, J.M., Franz, T.M., Koenig, A., Borries, C., 2006. How do ecological,demographic and anthropogenic factors affect Hanuman langur(Semnopithecus entellus) social organization? Am. J. Primatol. 68, 32.

Karanth, P.K., Singh, L., Collura, R., Stewart, C.-B., 2008. Molecular phylogeny andbiogeography of the langurs and leaf monkeys. Mol. Phylogenet. Evol. 46, 683–694.

Krajewski, C., Blacket, M., Buckley, L., Westerman, M., 1997. A multigene assessmentof phylogenetic relationships within the dasyurid marsupial subfamilySminthopsinae. Mol. Phylogenet. Evol. 8, 236–248.

Kurup, G.U., 1984. Census survey and population ecology of Hanuman langur,Presbytis entellus (Dufresne 1797) in south India. Proc. Indian Natl. Sci. Acad. 50,245–256.

Lohiya, N.K., Manivannan, B., Mishra, P., Sriram, S., Bhande, S., Panneerdoss, S., 2005.Preclinical evaluation for noninvasive reversal following long-term vasocclusion with styrene maleic anhydride in langur monkeys. Contraception71, 214–226.

Maddison, W.P., Maddison, D.R., 2000. MacClade 4: Analysis of Phylogeny andCharacter Evolution, Version 4. Sinauer Associates, Sunderland, MA.

1

K.P. Karanth et al. / Molecular Phylogenetics and Evolution 54 (2010) 627–633 633

Melnick, D.J., Hoelzer, G.A., 1992. Differences in male and female macaque dispersallead to contrasting distributions of nuclear and mitochondrial DNA variation.Int. J. Primatol. 13, 379–393.

Melnick, D.J., Hoelzer, G.A., 1993. What is mtDNA good for in the study of primateevolution? Evol. Anthropol. 2, 2–10.

Nandi, J.S., Tikute, S.A., Chhangani, A.K., Potdar, V.A., Tiwari-Mishra, M., Ashtekar,R.A., Kumari, J., Walimbe, A., Mohnot, S.M., 2003. Natural infection by simianretrovirus-6 (SRV-6) in Hanuman langurs (Semnopithecus entellus) from twodifferent geographical regions of India. Virology 311, 192–201.

Napier, J.R., Napier, P.H., 1967. A Handbook of Living Primates. Academic Press,London.

Newton, P.N., 1988. The variable social organization of Hanuman langur (Presbytisentellus), infanticide, and the monopolization of females. Int. J. Primatol. 9, 59–77.

Newton, P.N., Dunbar, R.I.M., 1994. Colobine monkey society. In: Davies, A.G., Oates,J.F. (Eds.), Colobine Monkeys: Their Ecology, Behaviour and Evolution.Cambridge University Press, Cambridge, pp. 311–346.

Osterholz, M., Walter, L., Roos, C., 2008. Phylogenetic position of the langur generaSemnopithecus and Trachypithecus among Asian colobines, and genus affiliationsof their species groups. BMC Evol. Biol. 8, 58.

Pocock, R.I., 1939. Primates and Carnivora (in part). Taylor and Francis, London.Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution.

Bioinformatics 14, 817–818.Prusak, B., Grzybowshi, T., 2004. Non-random base composition in codons of

mitochondrial cytochrome b gene in vertebrates. Acta Biochim. Pol. 51, 897–905.

Retief, J.D., Dixon, G.H., 1993. Evolution of pro-protamine P2 genes in primates. Eur.J. Biochem. 214, 609–615.

322

Retief, J.D., Krajewski, C., Westerman, M., Winkfein, R.J., Dixon, G.H., 1995.Molecular phylogeny and evolution of marsupial protamine P1 gene. Proc. R.Soc. Lond. B 259, 7–14.

Rooney, A.P., Zhang, J., 1999. Rapid evolution of a primate sperm protein: relaxationof functional constraint or positive Darwinian selection? Mol. Biol. Evol. 16,706–710.

Roonwal, M.L., 1984. Tail form and carriage in Asian and other primates, and theirbehavioral and evolutionary significance. In: Roonwal, M.L., Mohnot, S.M.,Rathore, N.S. (Eds.), Current Primate Research. Jodhpur University Press,Jodhpur, India, pp. 93–151.

Roonwal, M.L., Mohnot, S.M., 1977. Primates of South Asia: Ecology, Sociobiology,and Behavior. Harvard University Press, Cambridge, MA.

Shimodaira, H., Hasegawa, M., 1999. Multiple comparison of log-likelihoods withapplications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116.

Sterck, E.H.M., 1999. Variation in langur social organization in relation to thesocioecological model, human habitat alteration, and phylogenetic constraints.Primates 40, 199–213.

Swofford, D., 2001. PAUP*—Phylogenetic Analysis Using Parsimony and OtherMethods, Version 4. Sinauer Associates, Sunderland, MA.

Swofford, D.L., Olsen, G.J., Waddell, P.J., Hillis, D.M., 1996. Phylogenetic inference.In: Hillis, D.M., Moritz, C., Mable, B.K. (Eds.), Molecular Systematics. SinauerAssociates, Sunderland, MA, pp. 407–514.

Zhang, D.-X., Hewitt, G., 1996. Nuclear integrations: challenges for mitochondrialDNA markers. Trends Ecol. Evol. 11, 247–251.

Zhang, Y.-P., Ryder, O.A., 1998. Mitochondrial cytochrome b gene sequences of OldWorld monkeys: with special reference on evolution of Asian colobines.Primates 39, 39–49.