Materials and methods
To test the phylogenetic relationship of the study animal to other langurs, CR sequenced a fragment of the mitochondrial cytochrome b gene. DNA was extracted from hair samples (T. barbei, T. phayrei crepusculus, T. auratus auratus, T. cristatus, T. germaini, T. francoisi francoisi, T. vetulus, T. johnii, P. comata comata, P. melalophos mitrata and S. entellus hector), peripheral blood lymphocytes (T. obscurus, S. entellus priam and C. guereza) and museum skin (T. phayrei phayrei) by standard me-thods as outlined in Walsh et al. (1991)and Sambrook et al. (1989) and the QIAamp DNA Mini Kit, respectively. A 620 bp long fragment of the gene was PCR-amplified (Saiki et al., 1988) using the oligonucleotide primers 5’-CTCCTCATT GA AACATGAAATAT-3’ and 5’-CTTTGTTGTTTG GATTTGTG-3’. The resulting PCR products were separated on 1% agarose gels and visualized by ethidium staining. The fragments were excised from the gel and the DNA extracted using the Qiagen Gel Extraction Kit. Direct sequencing reactions were performed with the same primers as indicated above with the Big Dye Terminator Cycle Sequencing Kit (Perkin-Elmer) following the manufacturer’s recommendations. All sequence reactions were run on an automated ABI377 sequencer (Perkin-Elmer). Sequences were deposited at GenBank and are available under the accession numbers AY519449 – AY519463 (see also Table 1).
To get a more complete overview on Trachypithecus evolution, the data set was expanded with homologous sequences from T. pileatus and T. geei, both deposited at GenBank. Sequence differences and distances in the 573 bp long alignment were estimated by two measures of sequence divergence. First, the observed proportion of base differences between taxa was calculated by PAUP 4.0b10 (Swofford, 1999). Second, a maximum-likelihood (ML) estimate was obtained with the PUZZLE software, version 5.0 (Strimmer and Von Haeseler, 1996) with base frequencies (28.9% A, 30.2% C, 11.7% G, 29.1% T) and a transition:transversion ratio (9.11) estimated from the data set.
Phylogenetic tree reconstructions were carried out using three algorithms: maximum-parsimony (MP) (Fitch, 1971) and neighbor-joining (NJ) (Saitou and Nei, 1987) as implemented in PAUP and maximum-likelihood, included in PUZZLE. Support of internal branch lengths was either determined by bootstrap analyses (MP and NJ) performed with 1000 replications or indicated by the ML quartet puzzling support values (1000 puzzling steps).