Contributions to Zoology, 73 (4) (2004)Thomas Geissmann; Colin P. Groves; Christian Roos: The Tenasserim Lutung, Trachypithecus barbei (Blyth, 1847) (Primates: Cercopithecidae): Description of a live specimen, and a reassessment of phylogenetic affinities, taxonomic history, and distribution

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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).

 

Table 1: Origin of samples for the genetic study Abbreviations: ZMB: Zoologisches Museum der Humboldt Universität Berlin; EPRC: Endangered Primate Rescue Center, Cuc Phuong National Park; DPZ: Deutsches Primatenzentrum.

Species

Origin

Institution/Sponsor a

Accession-Nr

Trachypithecus barbei

-

Bangkok Zoo, Thailand

AY519462

T. obscurus

-

Wuppertal Zoo, Germany

AY519459

T. phayrei phayrei

SW-Burma

ZMB, Germany

AY519460

T. p. crepusculus

Vietnam

EPRC, Vietnam

AY519461

T. francoisi francoisi

-

Bristol Zoo, Great Britain

AY519458

T. cristatus

-

Singapore Zoo, Singapore

AY519456

T. germaini

-

Bangkok Zoo, Thailand

AY519457

T. auratus auratus

-

Bristol Zoo, Great Britain

AY519455

T. pileatus

-

GenBank

AF294626

T. geei

-

GenBank

AF294618

T. johnii

-

Erfurt Zoo, Germany

AY519453

T. vetulus

-

Bristol Zoo, Great Britain

AY519454

Semnopithecus entellus priam

-

Krefeld Zoo, Germany

AY519452

S. entellus hector

Nepal

DPZ, Germany

AY519451

Presbytis comata comata

-

Howletts Zoo, Great Britain

AY519449

P. melalophos mitrata

-

Howletts Zoo, Great Britain

AY519450

Colobus guereza

-

Munich Zoo, Germany

AY519463

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).