Phylogenetic Position of the Genus Trachypithecus as Inferred from Y-chromosome and Autosomal DNA Sequences

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1 Malaysian Journal of Biochemistry and Molecular Biology (), 8-8 Phylogenetic Position of the Genus Trachypithecus as Inferred from Y-chromosome and Autosomal DNA Sequences Md-Zain BM, Morales JC, Hassan MN, Jasmi A and Melnick DJ School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 6, Bangi, Selangor, Malaysia; Center For Environmental Research and Conservation, Columbia University In The City Of New York, USA; Department of Wildlife and National Parks (PERHILITAN), Cheras, 6, Kuala Lumpur, Malaysia. Abstract We present a molecular study examining the phylogenetic position of the genus Trachypithecus in relation to other Asian colobine genera. We used two unlinked nuclear gene regions to estimate the phylogenetic positions among the species classified as Trachypithecus. We sequenced base pairs of the Y-chromosome TSPY and SRY genes and 69 base pairs of the autosomal IRBP intron- locus from two species of Trachypithecus (T. cristatus and T. obscurus). Other members of Asian colobines were used as the outgroups. Our interpretation based on character and distance analyses suggests that Trachypithecus forms its own monophyletic clade distinct from other Asian colobines. Relative genetic distances and bootstrap support values from two unlinked nuclear regions further confirm the monophyly of Trachypithecus. Keywords: Tradipithecas, lead monkeys, nuclear DNA sequences. Y-chromosome, autosomal DNA. Introduction Many primatologists do not agree on a common delimitation of species within the Presbytis group [,]. Formerly, based on morphological characters, Trachypithecus and Semnopithecus were grouped into Presbytis [,,]. Some Chinese primatologists agree with this arrangement [6,7]. Hill [8] separated these groups from Presbytis at the genus level and Hooijer [9] and Eudey [] subsequently agreed with this assignment. However, Brandon-Jones [], Strasser and Delson [] and Delson [] recognize Trachypithecus as the subgenus of the Semnopithecus. The separation of Trachypithecus from Presbytis has also been adopted by several other researchers [,]. However, the variability in the use of the Trachypithecus- Presbytis clades and their presumed relationship to one another has produced taxonomic and phylogenetic confusion. For this reason, this taxon should be reanalyzed using other systematic approaches such as those provided by molecular analysis. In this study, we examined whether Trachypithecus is a monophyletic group distinct from Presbytis. This was done by using molecular techniques to determine whether gene sequences found in species of Trachypithecus are phylogenetically distinct from gene sequences found in species of the genus Presbytis. In this study, we used two loci on the Y-chromosome (testis-specific protein (TSPY) and sex-determining region (SRY) and one locus on an autosomal chromosome (interstitial retinal-binding region (IRBP-intron ) to resolve intrageneric phylogenetic relationships. These two unlinked regions of the nuclear genome were selected based on their capacity to resolve phylogenetic relationships in cercopithecines and differing modes of inheritance [6,7]. Using independent gene sequences in different genomes, each with their own unique inheritance pattern offers the greatest opportunity to capture all of the phylogenetic information present in a study group. Material and Methods Samples All samples used in this study are listed in Table. We used two species to represent the genus Trachypithecus: T. cristatus and T. obscurus. T. cristatus has a narrow distribution on the Malay Peninsula and Central Thailand, but is more geographically widespread in Borneo, Sumatra and Indochina. The range of T. obscurus is more restricted, extending from the Isthmus of Kra to the Malay Peninsula. We also used five species of the genus Presbytis: P. hosei, P. rubicunda, P. melalophos, P. thomasi and P. comata. We also used Nasalis larvatus, Pygathrix nemaeus, Colobus guereza, Macaca nemestrina and M. fascicularis as outgroups in oder to properly root the relationships between the two formerly congeneric groups. Author for correspondence: Dr. Badrul Munir Md-Zain, School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 6, Bangi, Selangor, Malaysia. Tel: Fax: abgbadd@pkrisc.cc.ukm.my

2 9 Table : Details of genetic samples. Taxon Code Origin P. melalophos siamensis BM Besut, Terengganu, Malaysia P. melalophos robinsoni BM Selama, Perak, Malaysia P. melalophos femoralis BM6 Kluang, Johor, Malaysia P. melalophos mitrata DJ6 Simpai, Sumatra, Indonesia P. melalophos natunae DJ69 Natuna Islands, Indonesia P. rubicunda Tawau Tawau, Sabah, Malaysia P. thomasi DJ66 North Sumatra, Indonesia P. comata DJ 7 West Java, Indonesia P. hosei BM67 Tawau, Sabah, Malaysia T. cristatus BMB Kuala Selangor, Malaysia T. cristatus BMA Kota Kuala Muda, Kedah, Malaysia T. cristatus BMB Kuala Selangor, Malaysia T. obscurus BM8 Sik, Kedah, Malaysia T. obscurus BM Melaka, Malaysia N. larvatus BM9 Kuching, Sarawak, Malaysia N. larvatus BM9 Simunjan, Sarawak, Malaysia N. larvatus DJ97 Borneo Py. nemaeus DJ9 Quang Nam, Vietnam C. guereza Cg Kenya, Africa M. fascicularis DM687 Indonesia M. fascicularis UKM Selangor, Malaysia M. nemestrina BM96 Kuching, Sarawak, Malaysia Table : Oligonucleotide primer used in this study and their PCR conditions Gene Region TSPY SRY IRBP intron- Forward/Reverse Primer Sequences TSPY-A ('-AGC CAG GAA GGC CTT TTC TCG-') TSPYR ('-CTG TGC ATA AGA CCA TGC TGA G-') SRYF SW ('- CTT GAG AAT GAA TAC ATT GTC AGG G -') SRYR SWB ('- AGG TCT TTG TAG CCA ATG TTA CCC G-') IRBP-FA (' GTG AAC TCT GGA CAC ACG CCC AGG TTG TAG GT -') IRBP-RA (' GCC GTC ACA CTG CTG GTC AGA ATG A-') List of Internal Primers TSPY Sequence (' to ') 7F CTT CAT GGT GCC ARG CAG 7F CGG CAG TTC TCT GCA T 9F TCA TCT CTG CCA GAT CAT E9F GCA GGA TCC ART AGG ATT G E7F GTC CGT CTT ATC CAT GYC GA EF CGG AAT GTG CCT GGT TG ER CTA CCR TGC ATG TGT TAC A ER CAA CCA GGC ACA TTC CG E69R TCG RCA TGG ATA AGA CGG AC E9R CAA TCC TAY TGG ATC CTG C 9R ATG ATC TGG CAG AGA TGA 7R GAT CAT GTA GCT CAG CAT GTC T 8R ATG CAG AGA ACT GCC G 7R CAA AGG GTC ACA TSG ACG C SRY Sequence (' to ') F AGT GAA GCG ACC CAT GAA YG R GTA TCC CAG MTG CTT GCT GAT C IRBP Sequence (' to ') Intron- F CAG TGC TCA CCA TAC TTG AGC T EF AGC ATT CTG GCT CAC GTT GCA TTC ER AGC TCA AGT ATG GTG AGC ACT G R GAA TGC AAC GTG AGC CAG AAT GCT F GAC TCA ATT CTA CAG ATA GTG AGG TC R ATG CCT GTG TAT TAC AGA AGC ATG Thermocycling parameters were cycles of denaturing at 9 C ( min), annealing at 8 C ( min), and extension for min at 7 C.

3 DNA sequencing Total DNA were extracted from tissue and blood with the Qiagen Tissue Kit (QIAGEN) using modifications of the blood and tissue procedures. Successful amplifications were obtained using the conditions listed in Table. The oligonucleotide primer pairs used to amplify the Y- chromosome and autosomal gene sequences [8] are listed in Table. Each PCR reaction contained. unit of Taq DNA polymerase (Perkin Elmer), pm/µl of each primer, µl of dntps, 8 µl of Buffer A, and. µl of DMSO. Table lists the conditions that were used to get successful amplifications in the two unlinked DNA regions. PCR amplifications for sequencing were done in ul volumes and loaded into.% agarose gel for electrophoresis. PCR products were cleaned using the Qiagen PCR Purification Kit (QIAGEN) and made ready to proceed with cycle sequencing. Cycle sequencing was carried out with internal primers [9] in a Perkin Elmer Model 8 thermal cycler with Big-Dye sequencing kit (Perkin Elmer) following the supplied protocol modified by having all reaction volumes equal 9 ul. Sequencing products were cleaned of excess dyes with CentriSep Spin Columns (Princeton separations), and then electrophoresed on a.% polyacrylamide gel (9: Acryl/ Bis gel stock, AMRESCO) and scored by an ABI 77 PRISM automated DNA sequencer (Perkin Elmer). Sequence Alignment and Data Analyses Alignment of nuclear DNA (ndna) sequences as made by codon position, using the AutoAssembler software (Applied Biosystem Inc., Perkin-Elmer []) against the homologous regions of human nuclear DNA []. Sequence data from this study were analyzed using a number of different algorithms employed for automated phylogenetic reconstruction using PAUP version. []. Using Presbytis, Nasalis, Pygathrix, Colobus, and Macaca as outgroup reference taxa, maximum parsimony (MP) and neighbor joining (NJ) analyses on the Y-chromosome, and autosomal sequences, were conducted separately. Trees we obtained by heuristic or branch and bound searches. The data were also subjected to bootstrap analysis with replications in order to gain some estimate of the strength of support for each clade []. Neighbor joining analyses was conducted with PAUP. based on the distance measure of Tajima and Nei []. The branch lengths for the most parsimonious MP tree were obtained using the describe tree option of PAUP. Homoplasy was quantified using the consistency index (CI) and the homoplasy index (HI). Results and Discussion Sequence variation The sequences of the TSPY and SRY genes were combined because of their close linkage [] and because previous studies have demonstrated that their phylogenetic signals do not conflict with one another [6]. The combined DNA sequences of the TSPY and SRY genes contain bp, of which 6 bp were included in our analyses. For the autosomal IRBP intron-, we were able to sequence 69 bp for some individuals but only 6 bp were consistently obtained and included in our phylogenetic analyses. Thus, the following analyses are based on approximately.7 Kb of DNA sequence. Table is a compilation of data on TI/TV ratios. Our results indicate that TSPY/SRY possesses the low ratio of TI/TV (.:) and the high is in the IRBP gene (.78:). These results indicate that autosomal region evolved more rapidly in six genera compared to the Y-chromosome region. Table : Summary of variations along the sequences across taxaa. CHROMOSOME-Y IRBP Total characters examined 6 6 Constant characters Parsimony-uninformative characters Parsimony-informative characters 8 6 % informative no. characters.9.8 Ratio of TI/TV calculated from pairwise base differences (PAUP)..78 Tree length 9 a All gaps were excluded from analyses. The numbers of unambiguous transitions and transversions were generated from pairwise base differences using PAUP. Phylogenetic resolution For the TSPY and SRY genes, MP yielded only one most parsimonious tree (length=, CI=.9, HI=.868; Figure ). NJ analysis (tree not shown) also yielded a single tree whose topology is congruent with that of the MP tree. In all of these analyses, Trachypithecus represented by T. cristatus and T. obscurus form a single monophyletic clade with % bootstrap support.

4 P.m.robinsoni.BM P.m.femoralis.BM6 (97) (86) P.m.natunae.DJ69 P.m.siamensis.BM 66 () P.m.mitrata.DJ6 P.rubicunda.Tawau 6 (97) P.thomasi.DJ66 (99) P.comata.DJ7 9 T.cristatus.BMA () 7 () T.obscurus.BM8 (78) 6 N.larvatusDJ97 6 Py.nemaeus.DJ9 M.fascicularis.DM687 M.nemestrina.BM96 Figure : Bootstrap tree of the combined TSPY/SRY data set using the heuristic search from the maximum parsimony analysis of PAUP. The bootstrap support values are shown below the branches of the tree. Presbytis also form a single monophyletic clade with % bootstrap support. Trachypithecus, Nasalis and Presbytis form a deeper, single clade with 99% bootstrap support, to the exclusion of Pygathrix. MP analysis produced a single bootstrap tree (length=9, CI=.98, HI=.97) for the IRBP intron- gene sequence (Figure ). NJ analysis (tree not shown) also yielded a single tree with virtually the same topology as the MP tree. As in the case of the Y-chromosome gene trees, these autosomal gene trees indicate a single clade for the Presbytis species with % bootstrap support. A second distinct clade, composed of members of the genus Trachypithecus and the genus Nasalis, was defined and supported at the 6% level. Bootstrap values of % support the membership of clades containing T. cristatus and T. obscurus and Nasalis respectively. We have generated tree topologies from two unlinked regions using character state and distance methods of analysis. All tree topologies agree that Trachypithecus species form a single monophyletic clade distinct from the genus Presbytis. This distinction is strongly supported by % bootstrap values for the TSPY/SRY and IRBP

5 P.m.siamensis.BM (6) P.m.robinsoni.BM P.m.femoralis.BM6 (6) P.m.natunae.DJ69 7 () (98) (9) (6) P.m.mitrata.DJ6 P.rubicunda.Tawau P.comata.DJ7 P.thomasi.DJ66 9 P.hosei.BM67 (6) () (96) T.cristatus.BMB T.cristatus.BMB T.onscurus.BM8 (6) (6) () T.obscurus.BM N.larvatus.BM9 N.larvatus.BM9 Py.nemaeus.DJ9 C.guereza.Cg 6 (98) 9 M.fascicularis.UKM M.nemestrina.BM96 Figure : Bootstrap tree of IRBP data set using the heuristic search from the maximum parsimony analysis of PAUP. The bootstrap support values are shown below the branches of the tree. intron- data sets. Therefore, we believe these results strongly support the taxonomic arrangement of Oates et al. [] and Brandon-Jones et al [6] in which Trachypithecus species are separated from Presbytis species with each being placed in their own distinct genus. Support for a distinct monophyletic relationship among Trachypithecus can also be derived from the Tajima and Nei distance matrix. Md-Zain [9] lists the sequence divergence values calculated using Tajima and Nei s algorithm [] for the TSPY/SRY and IRBP intron-. For TSPY/SRY, the average intra-generic genetic distance ranges from.% in the Presbytis genus to.8% in the Trachypithecus genus. The average genetic distance between Presbytis and Trachypithecus is observed to be.9%. The average intra-generic genetic distance for IRBP intron- ranges from.7% in the Trachypithecus genus to.% in the Presbytis genus. The average difference between Trachypithecus and Presbytis is about an order of magnitude greater at.% (Table ). These results show that intergeneric differences between Trachypithecus and Presbytis are much greater than the interspecific differences in either genus: 7 times

6 Table : Average percentage of genetic distance among and between Trachypithecus Presbytis, Nasalis, and Pygathrix using the Tajima and Nei distance. TSPY/SRY Trachypithecus Presbytis Nasalis Pygathrix Trachypithecus.89 Presbytis.8. Nasalis Pygathrix IRBP Trachypithecus Presbytis Nasalis Pygathrix Trachypithecus.7 Presbytis..9 Nasalis.8. - Pygathrix for Y-chromosome and 8 times for IRBP intron-. Data from these two unlinked regions clearly shows a concurrent pattern of genetic distances, that indicate considerably greater genetic divergence between Trachypithecus and Presbytis than within either clade. Therefore, we strongly believe that these phenetic differences further validate the phylogenetic separation of Trachypithecus and Presbytis into two separate genera. Frequently Trachypithecus has been grouped with Presbytis [,] as one large heterogeneous genus. Some primatologists agreed with this arrangement [6,7]. However, Hill [8] and Hooijer [9] subdivided the genus Presbytis by elevating the subgenera Semnopithecus and Trachypithecus to generic level and retaining Presbytis for a distinct subset of species. Ecological, behavioral, and morphological data clearly support the separation of Trachypithecus from Presbytis [,6]. Our molecular data have further corroborated this taxonomic distinction. Tree topologies from different phylogenetic analyses clearly indicate that Trachypithecus and Presbytis form their own distinct monophyletic clades. Bootstrap values strongly support the topologies obtained, which in turn support the phylogenetic hypothesis of two separate genera. Genetic distance patterns are also congruent with these results. Acknowledgements The authors thank the Faculty of Science and Technology (Universiti Kebangsaan Malaysia), Center for Environmental Research and Conservation (Columbia University, New York) and NYCEP. We are deeply indebted to several institutions for providing us with necessary facilities and assistance for tissue sample collection including UKM, PERHILITAN, Zoo Taiping, Zoo Melaka, Singapore Zoo, Sarawak Forestry Department, Pusat Kepelbagaian Biologi Sarawak, Sabah Park, and the government of Malaysia, Indonesia and Vietnam. This research was made possible under grants awarded by the United States National Science Foundation (Grant SBR ), John D. and Catherine T. MacArthur Foundation, UKM J//98 and IRPA 89EA. References. Groves CP. A theory of human and primates evolution. Oxford: Clarendon Press Brandon-Jones D. A revision of the Asian pied leaf monkeys (mammalia: Cercopithecidae: Superspecies Semnopithecus auratus), with a description of a new subspecies. Raffles. Bull. Zool. 99; : -. Pocock RI. The langurs, or leaf monkeys, of British India. Journal of the Bombay Natural History Society 98; : 7-.. Napier PH. Catalogue of primates in the British Museum (Natural History) and elsewhere in the British Isles. III. Family Cercopithecidae, subfamily Colobinae. London: British Museum (Natural History) 98.. Wolfheim JH. Primates of the world. Seattle: University of Washington Press Peng YZ, Ye ZZ, Pan RL. The classification and phylogeny of snub-nosed monkeys based on gross morphological characters. Zool. Res. 988; 9: Li ZY. Preliminary investigation of the habitats of Presbytis francoisi and Presbytis leucocephalus with notes on the activity pattern of Presbytis leucocephalus. Folia Primatol. 99; 6: Hill WCO. A monograph on the purple-faced leaf monkeys (Pithecus vetulus). Ceylon J. Sci. 9; 9: Hooijer DA. Quaternary langurs and macaques from the Malay Archipelago. Zool. Verhandelingen 96; : -6.. Eudey AA. Action plan for Asian primates conservation: Switzerland: Internl. Union Conserv. Nat., Gland Brandon-Jones D. Colobus and leaf monkeys. In : MacDonald D. eds. The Encyclopaedia of Mammals. Vol.. London: Allen and Unwin 98.. Strasser E, Delson E. Cladistic analysis of cercopithecid relationships. J. Human Evolution 987; 6: Delson E. Evolutionary history of the colobine monkeys in paleoenvironmental perspective. In : Davies AG and

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