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, 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 sug- gest 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 spe- cies. 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 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.10.034 628 K.P. Karanth et al. / Molecular Phylogenetics and Evolution 54 (2010) 627–633 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.