Analysis of the Genetic Relationships among Thai Gibbon Species Using AFLP Markers

MATERIALS AND METHODS

Sample collection: Blood samples from 2 black handed gibbons (H. agilis from Southern Thailand), 5 white handed gibbons (H. lar Northeastern Thailand) and 3 pileatus gibbons (H. pileatus from Northeastern Thailand) were collected (Fig. 1). The samples were divided into two groups, the first for cytogenetic analysis and the second for molecular analysis.

Fig. 1:	Map of Thailand indicating sample collection of gibbons (•), where N is Northern Thailand, NE is Northeastern Thailand, C is Central Thailand, W is Western Thailand, E is Eastern Thailand and S is Southern Thailand

Cytogenetic analysis: Blood samples were subjected to cytogenetic studies by lymphocyte culture of whole blood. The cultured cells were examined by the colchicines-hypotonic fixation-air drying technique followed by a conventional technique (Tanomtong et al., 2005). Chromosomal checks were performed with 20 cells of each individual by light microscopy.

Molecular analysis
DNA isolation: Genomic DNA was extracted from blood samples following Sambrook and Russel (2001). The quality and quantity of extracted DNA was assessed by 0.8% agarose gel electrophoresis and spectrophotometry.

AFLP reaction: The procedures of AFLP method (Vos et al., 1995) were performed according to the protocol of the Kit (AFLP^R Analysis System I, Invitrogen, USA). After adaptor ligation and preselective amplification, selective amplification was conducted with seven primer combinations: E-ACC/M-CAG, E-AAC/M-CAT, E-AAC/M-CAG, E-AAG/M-CAT, E-AAG/M-CTT, E-AGG/M-CTT and E-AGC/M-CAT. The PCR products amplified with different primer combinations were loaded onto 6.0% denaturing polyacrylamide gels and electrophoresed for 3 h and detected by Silver Quest^TM Silver Staining Kit (Invitrogen, USA).

Data analysis: Each AFLP band was considered as an independent character and the bands were scored visually as having each band either absent (0) and present (1) across all samples with the same primer pairs. Qualitative differences in band intensity were not considered. With the band data, a pair-wise genetic similarity matrix was generated among Hylobates lar, H. agilis and H. pileatus individuals using Ochiai similarity coefficients which were then converted to a genetic distance matrix. Based on the genetic distance matrix, cluster analyses were performed and the corresponding dendrograms were constructed for the three gibbon species using the Single Linkage Cluster method. Cophenetic correlations were computed from the clustering matrix in order to get the best fit dendrogram. All of these analyses were performed using the Fingerprinting II program (BioRad, USA).

RESULTS

Results from lymphocyte culture of whole blood and conventional staining of the three gibbons species, namely Hylobates lar, H. agilis and H. pileatus indicate that all species have an identical number of 44 diploid chromosomes (2n) consisting of 42 autosomes and 2 sex chromosomes. A representative chromosome of the three gibbon species is from a male of H. pileatus as shown in Fig. 2.

AFLP fragments were established for five individuals of H. lar, two individuals of H. agilis and three individuals of H. pileatus. Seven primer combinations generated a total of 1669 scorable bands, averaging at 238 bands per primer. Of these bands, 32.59% (544 bands) were polymorphic. Figure 3 presents AFLP fingerprinting of three gibbons with seven primer combinations.

Fig. 2:	Diploid chromosome number of male H. pileatus, 2n = 44 consisting of 42 autosomes and 2 sex chromosomes

Fig. 3:	Specific banding pattern of three gibbon species with the seven successful primer combinations

Fig. 4:	Dendrogram depicting the seven AFLP primer combinations produced by single linkage cluster analysis that are used to clarify the genetic relationships among the three gibbon species

Table 1:	Average similarity index values based on AFLP analysis of Hylobatid species and P. pygmaeus

A dendrogram was constructed based on the AFLP scoring data and using single linkage cluster analysis. The three hylobatid species are clustered together, whereas Pongo pygmaeus is separated as an outgroup with a bootstrap value of 83%. Within the hylobatid cluster, H. lar is more closely related to H. agilis than H. pileatus (Fig. 4). Average values of inter-specific similarity index (S) among all hylobatid studied species are 67.15% (between H. lar and H. pileatus) to 71.03% (between H. lar and H. agilis) (Table 1).

DISCUSSION

The identity of diploid chromosome number (2n = 44) of three gibbon species agrees with Ankel-Simons (2000) and Geissmann (2002), reflecting the close genetic relationships among species of gibbons. Moreover, Ankel-Simons (2000) suggested that chromosome polymorphism and inter-specific chromosomal variability are common among prosimian primates. Primate cytogenetics can still not reconstruct reliably the relatedness of gibbon species. The AFLP molecular marker was thus used to elucidate their relatedness. The advantage of AFLP fingerprints is that they are highly reproducible in the samples studied.

The sample studied contained a small number of some species such as H. agilis because blood samples are difficult to collect from primates in the forest. However, at least two samples could be used for preparing DNA fingerprints. Within the Hylobates species (Fig. 4), H. lar is very closely related to H. agilis more than H. pileatus with high bootstrap value (78%) and high average S-value (71.03%). This result is supported by other work using DNA sequences (Garza and Woodruff, 1992; Hayashi et al., 1995; Takacs et al., 2005), morphology (Kimble and Martin, 1993) and by vocal data (Geissmann, 2002).

Based on the geography of the Khao Yai National Park, Thailand, two members of the lar group, H. lar and H. pileatus, meet and occasionally hybridize. In the Khao Yai overlap zone, pure H. lar can be found 5 km into the pileatus side of the zone and pure H. pileatus go 9-12 km into the lar side. The change of the morphological index from 90% lar to 90% pileatus occurs over about 9 km (Kimbel and Matin, 1993). However, the genetic relationships between H. lar and H. pileatus is low with a 67.15% S value, while the S value between H. agilis and H. pileatus is 69.97%. Therefore, we suggest that although H. lar and H. pileatus have overlapping habitation zones and habitat has been lost, they still show genetic stability within a species. In order to resolve the genetic relationships within the lar group properly, it is necessary to study a larger sample and cover distributed areas, as well as to consider comparative behavioral studies. Thus, these results suggest that genetic analysis based on AFLP fingerprinting has a good capacity for study of species diversity, especially in gibbons. As AFLP analysis does not require prior genetic information of the taxa studied and AFLP fingerprints are reproducible, the technique should be of value in genetic analysis of a wide variety of primates (Chaveerach et al., 2006; Tanee et al., 2006). Moreover, the seven successful primer combinations can be used to sufficiently determine genetic differences among gibbon species.

ACKNOWLEDGMENT

This work was supported in part by Khon Kaen University’s Graduate Research Fund, Academic Year 2004.