Primates, Lorisidae: Loris) in Sri Lanka Based on Mtdna CO1 Barcoding

Home , Lorisidae, Slender loris

Turkish Journal of Zoology Turk J Zool (2019) 43: 609-616 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1901-20

Phylogenetic relationship among slender loris species (Primates, Lorisidae: Loris) in Sri Lanka based on mtDNA CO1 barcoding

1 2, 3 Saman GAMAGE , Faiz MARIKAR *, Colin GROVES , 4 5 1 Craig TURNER , Kalinga PADMALAL , Sarath KOTAGAMA  1 Department of Zoology, Faculty of Science, University of Colombo, Colombo, Sri Lanka 2 Department of Staff Development Centre, General Sir John Kotelawela Defence University, Ratmalana, Sri Lanka 3 School of Archaeology and Anthropology, Australian National University, Canberra, Australia 4 Zoological Society of London, Regent’s Park, London, UK 5 Department of Zoology, The Open University of Sri Lanka, Nawala, Nugegoda, Sri Lanka

Received: 16.01.2019 Accepted/Published Online: 23.09.2019 Final Version: 01.11.2019

Abstract: There are 2 species of slender lorises found in Sri Lanka: the red slender loris,Loris tardigradus (Linnaeus, 1758), endemic to the wet zone and montane areas, and the grey slender loris, Loris lydekkerianus (Cabrera, 1908), which is widespread. In addition, currently there are 2 subspecies recognized for each of these 2 slender loris species of Sri Lanka. Yet their taxonomy, evolutionary history, and biogeography are still poorly understood, and there are some preliminary data suggesting the presence of other subspecies. In this study, we have analysed the partial mitochondrial cytochrome oxidase I gene sequence of 21 Loris specimens collected from 7 different regions in Sri Lanka. The phylogenetic tree reconstructed based on the neighbour joining method and Bayesian approach revealed 2 clades with high branch supports (>60%), consistent with the 2 slender loris species. Moreover, the samples within the Loris lydekkerianus clade were divided into 3 taxa and the samples within the Loris tardigradus clade were divided into 4 taxa, all consistent with the 7 sampling zones. The results of the study have provided the first DNA barcoding analysis on slender lorises in Sri Lanka to resolve phylogenetic relationships. Further studies on Loris are necessary for decoding their taxonomic issues in Sri Lanka.

Key words: Cytochrome oxidase I, taxonomy, Loris lydekkerianus, Loris tardigradus

1. Introduction lydekkerianus uva (Gamage,et al., 2017). Gamage et al. The slender lorises (Suborder: Strepsirrhini, Family: (2017) discussed a further subgroup of slender loris from Lorisidae, Genus: Loris É. (Geoffroy Saint-Hilaire, 1796) the Rakwana mountain range, which they nominated as are small nocturnal primates inhabiting India and Sri the Rakwana slender loris. Lanka (Groves, 1998, 2001; Nekaris and Jayawardene, In this study, we analysed partial cytochrome oxidase 2004; Gamage et al., 2017). Based on museum specimens, I (CO1) sequences of 21 slender loris individuals sampled Groves (1998) recognised 2 species of slender loris within from 7 different locations in Sri Lanka, as the CO1 gene Sri Lanka: Loris tardigradus (Linnaeus, 1758) and Loris is widely used in phylogenetic and phylogeographical lydekkerianus (Cabrera, 1908). A Sri Lankan islandwide studies in mammals, including primates (Moore, 1995). study conducted by Gamage et al. (2017) further The aim of our study was to answer 2 specific questions: confirmed that the classification of these 2 species needed 1) are the 2 species (L. tardigradus, and L. lydekkerianus) to be reviewed. The classification of slender lorises in Sri phylogenetically distinct in the Sri Lankan context? Lanka suggested by Gamage et al. (2017) is as follows: And 2) is there genetic support for the recent subspecies the southwestern red slender loris Loris tardigradus classification suggested by Gamage et al. (2017)? tardigradus (Linnaeus, 1758), the montane slender loris Lori stardigradus nycticeboides, the northwestern red 2. Materials and methods slender loris Loris tardigradus parvus (Gamage et al., 2017), 2.1. Study area, sampling, and ethical clearance the highland grey slender loris Loris lydekkerianus grandis Fifty-six live slender lorises were captured from 7 different (Hill,1953), northern grey slender loris Loris lydekkerianus forest regions to represent the 7 nominal groups suggested nordicus (Hill, 1953), and the Uva red slender loris Loris by Gamage et al. (2017). The loris species found in Sri * Correspondence: [email protected] 609

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Lanka are distributed throughout the country and mostly 2.3. Phylogenetic analysis located in 7 regions: Group 1-Northwestern; Group CO1 sequences were aligned according to DNA 2-Southwestern; Group 3-Rakwana; Group 4-Montane; sequences using MEGA 5 (Rambaut, 1996)1, and the Group 5-Highland; Group 6- Uva; Group 7-Northern. data were analysed using the neighbour joining method The blood samples were collected using a lancet to (NJ) criterion and maximum parsimony (MP) method puncture the ear, and the blood was soaked on Whatman (Felsenstein, 1981). Phylogenetic trees were built through filter papers. Each filter paper was air dried for 2 h, stored the NJ and MP approach using RAxML v. 8.1.22 (Ronquist in an envelope, and placed in a portable desiccator until et al., 2003) and Bayesian approach using Mr Bayes v. 3.2.5 transported to the molecular laboratory. (Jansen et al., 2003). A test for equal-base frequencies was All of the lorises were released where they were captured performed first, followed by a test to rate heterogeneity after blood samples were taken. The research was carried among sites or vice versa, in selecting the best-fit model out under Department of Wildlife Conservation Sri Lanka (Posada and Crandall, 2001). The sequence (CO1 region) of permit number WL/3/2/1/9, and guided and supervised Loris tardigradus, available on GenBank, was also included by the National Research Committee of the Department in this study, and registered GenBank sequences for slow of Wildlife Conservation. The American Society of loris (Nycticebus) species (N. pygmaeus, N. javanicus, N. Primatologists’ principles for the ethical treatment of pri menagensis, N. coucang, and N. bengalensis) were used as mates were strictly followed, as well as the International outgroups to root the tree. Table 1 presents the collection Primatological Society’s international guidelines for the sites, voucher numbers, and GenBank accession numbers acquisition, care, and breeding of nonhuman primates. for each sample. Sequences of Indian lorises were not 2.2. DNA extraction, amplification, and sequencing available in GenBank. The percent pairwise uncorrected The total genomic DNA was extracted using DNeasy® Blood distances between the Sri Lankan slender loris taxa were and Tissue Extraction Kit (QIAGEN Inc., Dusseldorf, calculated on the CO1 gene using MEGA 5. Germany) following the manufacturer’s protocol. The target 657-bp fragment of CO1 was amplified using 3. Results M13-tailed cocktail primers (C_VF1LFt1 – C_VR1LRt1- 3.1. Analysis–phylogenetic analysis Mammal cocktail; Ivanova et al., 2007) by polymerase Using the neighbour joining (NJ) method, we chose the chain reaction (PCR) using AccuPower® PCR PreMix tree having the highest likelihood value as our best tree; its (Bioneer Corp., Daejeon, Korea). The following thermal topology was congruent with the topology of the maximum cycle parameters were used for 50 µL amplification parsimony (MP) tree (Figure 1). The MP tree and the tree reactions: initial denaturation for 5 min at 94 °C, followed constructed by the NJ or ML method were very similar by 34 cycles of 1 min at 94 °C for denaturing, 1 min at 45 (node values of MP/NJ/ML are given in Figure 2). The °C for annealing, and 1 min at 72 °C for extension, with monophyly of the genus Loris was well-supported (100% a subsequent final extension at 72 °C for 7 min. The PCR of bootstrap support). The concatenated and partitioned products were tested by electrophoresis on a 2% agarose NJ and Bayesian trees showed similar topologies. In both gel. Only single, sharp, and clean bands were purified analyses, there are several short internodes and polytomies; using a QIAquick® PCR purification kit (QIAGEN Inc., however, the clades of significance to Sri Lankan loris were Dusseldorf, Germany) to be used in sequencing PCR well supported. reactions. After purification, the PCR products were 3.2. Analysis–phylogenetic relationship of Sri Lankan sequenced in both directions by using the automated slender lorises sequencer (ABI Prism 3730 XL DNA Analyzer) in BOWEI The genus was split into 2 clades, separating the wet zone (PR of China, Nanjing) with the primers M13 forward (with weak support of 63%) from the dry/intermediate 5’-TGTAAAACGACGGCCAGT-3’ and M13 reverse zone (96%); the northwestern red slender loris was well- 5’-CAGGAAACAGCTATGAC-3’ (Messing, 1983). The supported (Figure 2) as the sister group to the Rakwana resulting chromatograms were evaluated for miscalls and slender loris. Based on the CO1 region, the southwestern ambiguities and assembled into contigs using SeqManTM red slender loris (Loris tardigradus tardigradus) and the Pro (version 7.1.0, 2006; DNAstar® Inc., Madison, WI, montane slender loris (Loris tardigradus nycticeboides) are USA). Of the 44 successfully amplified samples, only 21 genetically very similar. The 3 dry/intermediate zone taxa, yielded readable sequences. The sample collection sites, highland grey (Loris lydekkerianus grandis), Uva (Loris voucher numbers, and GenBank accession numbers for lydekkerianus uva), and northern grey (Loris lydekkerianus each these 21 slender loris individuals of Sri Lanka are nordicus), formed a single clade, of which L. l. grandis and described in Table 1. L. l. uva are genetically very similar. Table 2 presents the 1 Rambaut A (1996). Se-Al: Sequence Alignment Editor. Available at URL: http://evolve.zoo. ox.ac.uk/ (accessed: 25 May 2014).

610 GAMAGE et al. / Turk J Zool Specimen No Specimen SLCPL01 2015.06 SLCP 2015.17 SLCP 2015.09 SLCP SLCPL02 SLCPL03 SLCPL04 2012.02 SLCP SLCPL05 2015.18 SLCP SLCPL06 SLCPL07 2015.08 SLCP SLCPL08 2015.16 SLCP SLCPL09 2015.12 SLCP SLCPL10 SLCPL11 2015.14 SLCP SLCPL12 E E E E E E E E E E E E E E E E E E E E E ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ 09.68 45.61 38.60 29.93 52.80 58.69 09.43 10.74 25.71 59.39 23.22 37.78 22.16 51.41 24.00 23.69 46.37 44.37 36.11 1217 40.31 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ N/80°07 N/80°18 N/80°08 N/80°08 N/80°11 N/80°08 N/80°24 N/80°31 N/80°25 N/80°25 N/80°45 N/80°45 N/80°35 N/80°36 N/80°45 N/80°45 N/81°43 N/81°26 N/80°30 N/80°30 N/80°21 ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ 00.20 42.48 54.46 20.42 28.76 19.20 00.84 41.01 21.31 35.19 03.62 36.05 38.70 40.38 30.35 06.90 36.31 58.82 52.22 07.21 45.43 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ Lat/Long 7°13 8°12 7°15 7°15 6°45 6°25 6°19 6°06 6°25 6°25 6°55 6°57 6°23 6°24 7°31 7°32 6°56 7°19 8°20 8°20 8°16 Collected site Kotakanda-Gampaha Thambuthegama-Anuradapura, Mirigama-Gampaha1 Mirigama-Gampaha2 Ingiriya-Kalutara Yagirala-Kalutara Kalubovitiyana-Matara Oliyaga-Matara 1 Kudawa-Sinharaja 2 Kudawa-Sinharaja Conical-NuwaraEliya, Eliya Single-tree-Nuwara Ensalwatta-Deniyaya Morningside-Rathnapura Knuckles Mahalakotuwa- Pitawala-Knuckles Lahugala-Ampara Nilgala-Monaragala 1 Mhintale-Anuradapura 2 Mhintale-Anuradapura Talawa-Anuradapura s s s s s s s s parvus parvus parvus tardigradu tardigradu tardigradu tardigradu tardigradu tardigradu tardigradu tardigradu s s s s s s s s s s s s Accession No Accession GenBank* Loris tardigradu nordicus Loris lydekkerianus Loris tardigradu Loris tardigradu Loris tardigradu Loris tardigradu Loris tardigradu Loris tardigradu Loris tardigradu Loris tardigradu nycticeboides Loris tardigradus nycticeboides Loris tardigradus Loris tardigradu Loris tardigradu Loris lydekkerianus grandis Loris lydekkerianus grandis uva Loris lydekkerianus uva Loris lydekkerianus nordicus Loris lydekkerianus nordicus Loris lydekkerianus nordicus Loris lydekkerianus Loris tardigradu Loris lydekkerianus Species KX761807 KX761808 KX761809 KX761810 KX761811 KX761812 KX761813 KX761814 KX761815 KX761816 KX761817 KX761818 KX761819 KX761820 KX761821 KX761822 KX761823 KX761824 KX761825 KX761826 KX761827 AB371094.1 KX761827.1 . Collection sites, voucher numbers, and GenBank accession numbers for each sample were used as given below. used given as were sample each for numbers GenBank accession and numbers, voucher . Collection sites, Number 1 21 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Reference Reference Table 1 *http://ncbi.nlm.nih.gov/nucleotides

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Figure 1. A neighbour-joining tree using 604 cytochrome C oxidase sub-unit I (CO1) sequences from 7 different slender loris (Loris) taxas, rooted using slow loris (Nycticebus) sequences deposited in the GenBank. percentage of pairwise uncorrected molecular distances Gamage et al. (2017). In particular, the northwestern red between the different slender loris groups given by Gamage slender loris (Loris tardigradus parvus) shows >2% of et al. (2017), which ranged from 0.2% to 5.6%. Table 2 divergence from all other wet zone relatives, and Rakwana shows a comparison of the classification given by Gamage slender loris shows >1.2% of divergence from all other wet et al. with the haplogroups based on CO1 sequences. zone relatives. Based on skull morphology, Groves and Thirty-five polymorphic sites were observed in the Meijaard (2005) placed the montane slender loris (taxon CO1 region (Table 3). The highest variability was noted in nycticeboides) in Loris lydekkerianus as a subspecies, the L. t. parvus (Group 1), Rakwana slender loris (Group yet Nekaris and Jayawardene (2004) transferred it to L. 3), and the L. l. nordicus (Group 7). Sequence alignment tardigradus, while Yapa and Ratnaveera (2013) suggested was closely clustered in the L. t. tardigradus (Group 2) and the taxon nycticeboides might be a distinct species. In a the L. t. nycticeboides (Group 4) from the wet zone clade; recent study, Gamage et al. (2017) found uniqueness of in the dry/intermediate zone clade, the L. l. grandis (Group facial/pelage features, external body morphology, and skull 5) and the L. l. uva (Group 6) showed similar clustering. morphology for the taxon nycticeboides. However, based on partial CO1 sequences, there is no significant distinction 4. Discussion between taxon L. t. tardigradus (southwestern red slender Analysis of the CO1 gene partial sequence shows evidence loris) and taxon nycticeboides (montane slender loris), and of 2 discrete clades to some extent (wet zone lorises and thus taxon nycticeboides has been placed very close to L. dry/intermediate zone lorises). This supports the 2-species t. tardigradus. Both taxa can be categorized into a single classification given by Groves et al. (2009), but the skull haplogroup (haplogroup 2 = southwestern slender loris). and pelage analyses employ the further divisions of Moreover, the northwestern red slender loris and Rakwana

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Figure 2. A neighbour-joining tree using 604 cytochrome c oxidase subunit I (CO1) sequences from 7 different slender loris (Loris) taxas found in Sri Lanka with their external appearance. slender loris is genetically different from haplogroup 2 other, and are thus categorized into 2 separate haplogroups (southwestern slender loris). Northwestern red slender (Table 3). The recent study by Gamage (Gamage et al., 2017) loris and Rakwana slender loris share mutations, but they found distinctiveness of facial/pelage features, external also have mutations that differentiate them from each body morphology, and skull morphology on the slender

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Table 2. Percentage (%) pairwise uncorrelated molecular distance between different slender loris taxa given by Gamage et al. (2017) using the 604 base pair fragment of the CO1 region, analysed using Mega 5: Group 1-L. t. parvus; Group 2-L. t. tardigradus; Group 3-Rakwana slender loris; Group 4-L. t. nycticeboides; Group 5-L. l. grandis; Group 6- L. l. uva; Group 7-L. l. nordicus.

Groups Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 2 2.4 Group 3 2.0 1.2 Group 4 2.4 0.3 1.2 Group 5 4.7 2.2 3.4 2.5 Group 6 4.7 2.2 3.4 2.5 0.2 Group 7 5.6 3.1 4.1 3.4 0.8 1.0 lorises found from the eastern dry and intermediate zone Education), and the Forest Department staff for their in Sri Lanka, which was named as a new subspecies of support and providing necessary permission. Thanks are Loris lydekkerianus uva. Hitherto, CO1 region data did not also due to the Secretary to the Ministry of Environment support this deviation, and the taxon uva is very close to and members of the Loris Steering Committee for their the taxon grandis; thus, both taxa can be categorized into advice on this work. Special thanks to Helga Schulze for a single haplogroup (haplogroup 3 = eastern grey slender providing valuable literature and comments. We also loris). Based on museum specimens, Groves (1998) found thank C. J. Hettiarachchi for his contribution to the GIS no difference between taxagrandis and nordicus, yet the work. We give special acknowledgment to the dedicated recent study by Gamage et al. (2017) clearly distinguished and professional Slender Loris Conservation Project field both taxa using facial/pelage features, external body team: C. A. Mahanayakage, G. D. C. Pushpa-Kumara, V. morphology, and skull morphology. The CO1 region data Sumanasekara, G. Rathnayake, D. R. Vidanapathirana, also supported this deviation. S. Ariyarathna, V. Rathnayake, D. Samarasingha, D. De Silva, T. Senevirathna, R. Peries, U. Wickramasingha, N. 5. Taxonomic conclusions Goonasekara, L. Rathnayake, S. Jayalath, P. Yahanpath, Analysis of the CO1 region shows evidence of 2 discrete and M. A. C. Maheshani. We also wish to thank Saroopa clades (wet zone and dry/intermediate zone clades); Samaradivakara and Ishara Herath (Genetech Molecular however, the wet zone clade is not monophyletic. Thus, Diagnostics) for their assistance in the molecular work. the current 2-species classification of slender lorises The program is funded through the EDGE of Existence (genus Loris) in Sri Lanka is acceptable to some extent. Programme of the Zoological Society of London and the In addition, these 2 clades can be further categorized into Synchronicity Foundation; we are extremely grateful 5 haplogroups. The CO1 region data strongly supported for their support. Furthermore, the first author (SNG) the proposed new subspecies from the wet zone clade, gratefully acknowledges the Conservation Leadership northwestern red slender loris (Loris tardigradus parvus); Programme (CLP) and the People’s Trust for Endangered CO1 region data further shows there is a possible new Species (PTES), UK, for providing equipment. The subspecies from the Rakwana mountain range (Rakwana authors also acknowledge the staff of the University of slender loris). However, our sample size is too small to give Colombo, the Open University of Sri Lanka, and the Field a name at this stage. CO1 data did not strongly support the Ornithology Group of Sri Lanka for their kind support separation of taxon nycticeboides from Loris tardigradus during the study. All work carried out falls under permits tardigradus, or Loris lydekkerianus grandis from Loris and ethics approvals issued to and held by Dr. U. K. G. lydekkerianus uva. K. Padmalal and the Open University of Sri Lanka by the Department of Wildlife Conservation. We followed the Acknowledgements American Society of Primatologists’ principles for the We thank the Director General of the Department of ethical treatment of primates and the IPS (International Wildlife Conservation (DWC), its Research Committee, Primatological Society) international guidelines for the the Deputy Director of Research, and DWC staff for acquisition, care, and breeding of nonhuman primates. their support and providing necessary permission for the We would like to extend our gratitude to anonymous collection of voucher specimens (permit no: WL/3/2/1/9). reviewers and the editor, who graciously reviewed this We extend our thanks to the Conservator General of manuscript and provided helpful suggestions for its Forests and the Conservator of Forests (Research and improvement.

614 GAMAGE et al. / Turk J Zool 567 G G G G G G G G G G G G G G A A A A A A A A 540 C C C C C C C C C C C C C C T T T T T T T T 501 C C C C C T T C C C C C C C C C C C C C C C 492 T T T T T T T T T T T T T T C C C C C C C C 426 C C C C C C C C C C C C C C T T T T T T T T 384 A A A A A A A A A A A A A A A A A A G G G G 369 A A A A A A A A A A A A A A G G G G G G G G 357 T T T T T T T T T T T T T T C C C C C C C C 345 T T T T T T T T T T T T T T C C C C C C C C 315 A A A A A A A T T A A A A A A A A A A A A A 257 G G G G G G G G G G G G G G A A A A A A A A 242 T T T T T T T T T T T T T T C C C C C C C C 236 G G G G G G G G G G G G G G T T T T T T T T 227 A A A A A A A A A A A A A A G G G G G G G G 185 C C C T T C C C C C C C C C T T T T T T T T 46 G G G G G G G G G G G G G G A A A A A A A A 45 T T T T T T T T T T T G G G T T T T T T T T 26 G G G G G G G G G G G A A A G G G G G G G G 23 T T T T T T T T T T T A A A T T T T T T T T 20 G G G G G G G G G G G A A A G G G G G G G G 19 A A A A A A A A A G G A A A A A A A A A A A 17 G G G G G G G G G G G A A A G G G G G G G G 15 G G G G G G G G G T T G G G G G G G A A A A 14 C C C C C C C C C C C C C C C C C C T T T T 13 C C C C C C C C C C C C C C C C C C T T T T 12 T T T T T T T T T T T G G G T T T T T T T T 11 A A A A A A A A A A A G G G A A A A A A A A 10 A A A A A A A A A A A A A A A A A A C C C C 9 T T T T T T T T T T T G G G T T T T T T T T 8 C C C C C C C C C C C G G G C C C C C C C C 7 A A A A A A A A A A A A A A C C T T T T T T 6 C C C C C C C C C T T T T T C C C C C C C C 5 C C C C C C C C C G G G G G C C C C C C C C 3 A A A A A A A A A T T T T T A A A A A A A A 2 T T T T T T T T T G G G G G T T T T T T T T Sequence variations of CO1 region among the different groups of slender lorises. slender of groups the different among CO1 region of Sequence variations Nucleotide positon Nucleotide no. sample and Group 012763 NC 2 (1)-Kudawa Group 2 (2)-Kudawa Group 2 (3)-Kalubovitiyana Group 2 (4)-Oliyagan Group 2 (5)-Ingiriya Group 2 (6)-Yagirala Group 4 (1)-Conical Hill Group 4 (2)- Kikiliamana Group 3 (1)-Morningside Group 3 (2)-Morningside Group 1 (1)-Kotakanda Group 1 (2)-Mirigama Group 1 (2)-Mirigama Group 6 (1)-Lahugala Group 6 (2)-Nilgala Group 5 (1)-Knuckles Group 5 (2)-Knuckles Group 7 (1)-Mihintale Group 7 (2)-Mihintale Group 7 (3)-Talawa Group 7 (4)-Thambuttegama Group Table 3. Table

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