Besar Ketol, Andy Kho, Abdullah, M. T. and Yuzine B. Esa Animal Resource Science and Management Program Faculty of Resource Science and Technology Universiti Malaysia Sarawak Email : kbesar@frst.unimas.my Abstracts: A study on genetic relationship among Rhinolophus species was conducted in Borneo. A total number of thirty-two individuals from ten species of Rhinolophus including one species (R. yunenensis) from Thailand were analysed using DNA sequencing technique. A total of nine samples including four samples from Genbank of 409 bp length of sequence were examined. Of the 409 nucleotide positions examined, 149 positions (36.4%) were variable sites and 104 positions (25%) were phylogenetically informative sites. Cyt b mitochondrial DNA was targeted to amplify 450 base pair sequences and phylogenetic relationships were constructed using distance neighbor joining and maximum parsimony analyses. The results showed that genus Rhinolophus is monopheletic, which formed two clusters. Phylogenetis analyses indicated that relationship within Rhinolophus is close ranging from 6.8% to 17.8%. Further studies should be conducted using full sequence of Cyt b (1140bp) and another five species of Rhinolophus. Keywords : Rhinolophus; mtDNA; Cytochrome b; Maximum Parsimony; Neighbor Joining. Methodology: A total of 24 tissue samples of Rhinolophus species were collected from Unimas Museum from different localities in northern and western Borneo, four samples were collected from Bau, Sarawak and four samples from Niah National Park. Mist-nets and harp-trap apparatus were used for this purpose. Tissue samples were preserved in 70-80 % ethanol. Total DNA was extracted from muscle tissue using CTAB (Grewe et al. 1993). The quality and approximate yield of DNA were determined by electrophoresis in a 1% agarose gel containing ethidium bromide run at 90V for 30 mins. A set of primers were used to partially amplify the cyt b gene; 5’-TGACT TGAAR AACCA YCGTT G- 3’, known as GluDG-L (Palumbi et al. 1991) and 5’-CCCTC AGAAT GATAT TTGTC CTCA- 3’, known as CB2-H. The PCR products were further purified using Fermentas DNA purification kits. The purified PCR products were directly sent for sequencing by a company using the ABI PRISM BigDye terminator cycle sequencing kit (ACGT) only for the forward strand. Phylogenetic relationships were inferred using two methods: distance analysis using the neighbour-joining method (NJ) and unweighted maximum parsimony (MP) analysis (using close-neighbor-interchange, CNI option) implemented in MEGA (version 2.1; Kumar et al. 2001). The NJ clustering was performed using the Kimura two-parameter evolution model (Kimura, 1980). The phylogenetic trees inferred from the cyt b sequences were rooted with sequences of Pteropus vampyrus, Rousettus amplexicaudatus and Eonyteris spelaea from gene bank were used as out-groups. Phylogenetic confidence was estimated by bootstrapping with 1000 replicate data sets. Results: From 20 samples sent for sequencing only five were successful. A total of 409bp length of sequence cyt b gene were examined with the nucleotide composition of genus Rhinolophus was A (26.0%), C (29.9%), G (16.6%) and T (27.5%). Of the 409 nucleotide positions examined, 149 positions (36.4%) were variable sites and 104 positions (25%) were phylogenetically informative site. Two phylogenetic trees neighbor-joining and maximum parsimony tree were successfully constructed (Figures 2 and 3). All the trees showed that Rhinolophus is monophyletic and this is well supported by bootstraps value of 100%. A Phylogenetic Relationship In The Genus Rhinolophus (Family: Rhinolophidae) From Borneo Inferred Using Partial MtDNA Cytochrome b Gene Figure 2: Maximum parsimony tree of genus Rhinolophus inferred from cyt b mtDNA with Kimura-2 parameter distance (Kimura, 1980) and 1000 replication was used (PAUP).Numbers at nodes indicate the bootstrap values in percentage. Figure 3: Neighbor-joining tree of genus Rhinolophus inferred from cyt b mtDNA with Kimura-2 parameter distance (Kimura, 1980) and 1000 replication was used (MEGA). Numbers at nodes indicate the bootstrap values in percentage. Table 1: Percent sequence divergence for all pairwise comparison of cyt b nucleotide sequence (409 bp). Sequence divergence was calculated using Kimura-2 parameter (Kimura, 1980). Acknowledgements: I would like to thank my supervisor, Ass. Prof. Dr Mohd Tajuddin Abdullah, and all lecturers from Animal Science Resource Management Program, laboratory assistants, especially Isa Sait, Jailani Morteda and Raymond Atet, postgraduate students, Andy Khoo, Jeffrine Rovie Ryan, Jaya Raj Kumaran, Imelda Vivian Paul and coursemates Ahmad Mashur, Wahap Marni, Ratnawati Hazali for their support, and valuable advise. Financial support by Nichimen Forestry Scholarship [(NFSF(O)720831-13- 5543] is gratefully appreciated. Research permits NPW.907.4-47, NPW.907.4-48, NPW.907.4-49 and NPW. 907.4-51 to study bats in Sarawak were granted by the Director of Sarawak Forestry Department is gratefully acknowledged. This work is supported by IRPA research grant 09-02-09-1022-EA001 awarded to MTA, Mr Yuzine Esa and Dr. Awang Ahmad Sallehin Awang Husaini. Discussion: In general, both MP and NJ Phylogenetic tree suggest that the genus Rhinolophus is monophyletic with a strong bootstrap value support of 100% and distance divergence ranged from 6.8% to 17.8% (Table 1). The structure of the phylogenetic tree has highly supported that the relationship within Rhinolophus is monophyletic. This result is consistent with the findings by Wang et al. (2001). In MP, Rhinolophus is divide into two clades with a bootstrap value 78%, which consist of R. acuminatus, R. affinis and R. creaghi for clade 1. Mean while, for clade 2 the bootstrap value is 79%, which consist of R. pusillus, R. borneensis and R. luctus. MP phylogenetic tree produced better result than NJ with above 70 % of bootstrap value. While in NJ Rhinolophus is also divide into two clades with support boostrap value 100% but, clade 1 is separated in to two sub-clades with a low bootstrap value ranging from 46% to 79% consisting of five species from Rhinolophus and in clade 2 consisting only R. luctus. The morphological characteristics of the species may be reflected in phylogenetic tree. Both R. affinis and R. acuminatus are placed in clade 1 with a low sequence divergence 6.8%. Morphologically both species haves similar coloration, almost overlapping length of forearm and weight. However, there is a difference in connecting process where R. affinis has rounded while R. acuminatus is pointed (Payne et al., 1995). The forearm length and body weight of R. pusillus in Figure 1: Rhinolophus borneensis (Photo by Ee Ling) Conclusion: This is the first study on the molecular phylogeny of five species in the genus Rhinolophus in Borneo. However, failed to obtained five more Rhinolophus species because of sequencing failure. Based on the result of this study there is a need for further studies on complete phylogeny of the genus Rhinolophus by using complete cyt b sequence (1140bp). It is also necessary to use others mtDNA genes to compare their relationship due to different rate of evolution in the genes. According to Wang et al. (2003) it is possible that the phylogenetic tree obtained from one gene does not accurately reflect evolutionary relationship among taxa. Further study should also include the Nycteridae and Megadernatidae to prove either that both families has closed relative based on complexity of facial ornamentation to family Rhinolophidae (Findley, 1993). clade 2 were smaller compared to R. borneensis in clade 1. Their forearm length are between 37 to 44mm and weight between 7.5 to 12g while R. affinis, R. acuminatus and R. creaghi have bigger forearm length between 48.9 to 51.3mm and weight between 10 to 14g. The major different between R. pusillus and R. borneensis is in their noseleaf size where R. borneensis have a larger noseleaf size compared to R. pusillus (Payne et al., 1995). Forearm length and body weight of R. pusillus and R. borneensis in clade 2 were smaller compared to those in clade 1. Their forearm length are between 37 to 44mm and weight between 7.5 to 12g while R. affinis, R. acuminatus and R. creaghi have forearm length between 48.9 to 51.3mm and weight between 10 to 14g. The major different between R. pusillus and R. borneensis is in their noseleaf size where R. borneensis have a larger noseleaf size compared to R. pusillus (Payne et al., 1995). R. luctus is the largest species in the genus Rhinolophus. Morphologically R. luctus is bigger in size with body weight ranging from 29 to 37g and longer forearm length ranging from 63 to 67mm compared to other species of Rhinolophus (Payne et al., 1995). R. luctus also has a clear long and woolly fur. This might be the major characteristic that has caused R. luctus was placed at the base of NJ tree within genus Rhinolophus and with sequence divergence ranging from 11.7% to 17.8% within the group. In general, cyt b gene was successfully used to infer the phylogenetics differences between Rhinolophus and elucidate the relationship between species of Rhinolophus in Borneo. Objectives/aims: To identify the genetic relationship among the species within the genus of Rhinolophus in Borneo by using partial cyt b sequence. Introduction: Bats are difficult to be identified from distance unless this animal can be captured for a close up examination. Teeth of the bats should be examined to confirm their identity. The special characters in identifying the species from genus Rhinolophus includes, coloration, measurement of forearm, presence of lappet, size of ear and the most important is the shape of sella (Payne et al., 1995; Yasuma and Andau, 1999). The shape of sella is useful as key character between Rhinolophus species. However, confusion between immature and adult specimen having similar morphology also contributes toward wrong identification. According to Payne et al. (1995) immature are generally much duller in colour, grey or grey brown and if held against a bright light, the wing joints of immature will appear banded where the cartilage has not yet turned to bone. Meanwhile lack of knowledge and experience in this field also has put up the difficulties in the process of identification of species. In Borneo there are 10 species of genus Rhinolophus namely R. philippinensis, R. creaghi, R. acuminatus, R. arcuatus, R. borneensis, R. pusillus, R. affinis, R. luctus, R. sedulus and R. trifoliatus (Payne et al., 1995; Yasuma and Andau, 1999). According to Findley (1993), Rhinolophidae is closely related to Megadermatidae and Nycteridae, which can be identify by the complexity of facial ornamentation. The controversy about relationship between Aselliscus and other genera in Hipposideridae and Rhinolophidae had attracted the attention of some taxonomists. Through molecular technique based on mtDNA cytochrome b (cyt b) sequence gene, Wang et al. (2001) suggested that each Hipposideridae and Rhinolophidae is monophyletic group and Aselliscus should remain as a genus within Hipposideridae with the mean percentage sequence differences (16.43%) while transition:transversion ratios (2.032) between Aselliscus and Hipposideridae. However, through the DNA technique the identity of the species Rhinolophus can be resolved. Based on the divergence in the sequence data, a new phylogeny can be reconstructed and proposed.