International Conference Mathematical and Computational Biology 2011 International Journal of Modern Physics: Conference Series Vol. 9 (2012) 199 208 World Scientific Publishing Company DOI: 10.1142/S2010194512005260 A MORPHOMETRIC ANALYSIS OF HYLARANA SIGNATA GROUP (PREVIOUSLY KNOWN AS RANA SIGNATA AND RANA PICTURATA) OF MALAYSIA RAMLAH ZAINUDIN Department of Zoology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak, Malaysia zramlah@frst.unimas.my SITI NURLYDIA SAZALI Department of Zoology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak, Malaysia ssnurlydia@frst.unimas.my A study on morphometrical variations of Malaysian Hylarana signata group was conducted to reveal the morphological relationships within the species group. Twenty-seven morphological characters from 18 individuals of H. signata and H. picturata were measured and recorded. The numerical data were analysed using Discriminant Function Analysis in SPSS program version 16.0 and UPGMA Cluster Analysis in Minitab program version 14.0. The results show the complexity clustering between the examined species that might be due to ancient polymorphism of the lineages or cryptic species within the group. Hence, further study should include more representatives in order to fully elucidate the morphological relationships of H. signata group. Keywords: Hylarana signata; morphometric; cryptic species; cluster analysis. Introduction Hylarana signata and H. picturata are two frog species in the Hylarana signata group that is still not well-understood where the body measurement is generally less than 50mm for males and less than 70mm for females, respectively. H. signata was initially described as Polypedatus signatus (type locality Matang Sarawak) 1 before recognised as Rana signata 2,3. Ref. 4 however re-identified the species as Rana (Pulchrana) signata based on morphological characters and later to Pulchrana signata based on both molecular and morphological data 5 in which recently, classified as Hylarana signata 6. Distributions of H. signata include Peninsular Thailand and Malaysia, Sumatra (Indonesia) and also Malaysian Borneo 7,8. 199
200 R. Zainudin and S. N. Sazali H. picturata, on the other hand, was previously treated as a synonym of H. signata by Ref. 3 in which, Ref. 9 had separated this species from the synonym of H. signata without further discussion. Subsequently, this species was then assigned as Pulchrana picturata 5 and recently as H. picturata 10 by implications. Overall, H. picturata was stated to be distributed throughout Borneo and Peninsular Malaysia 7,8. The molecular findings on the phylogenetic relationships of Malaysian H. signata complex using ribosomal mitochondrial DNA gene 11 recently had revealed the complexity within Malaysian H. signata group and a possibility of new species that may emerge within this group. Using both morphological and protein electrophoresis variations, Ref. 7 showed monophyly of H. signata complex on the whole (Bornean and Philippines), but paraphyly of its Philippines members that leads to the emergence of five new species from one species of Philippines H. signata complex. Based on the complexity and unresolved relationship within Malaysian H. signata complex, this preliminary study is conducted to elucidate the morphometrical variations of these two species using 27 morphological characters from eighteen specimens, collected from different localities in Malaysia. Materials and Methods All the 27 morphological characters from 18 individuals of H. signata and H. picturata were measured following description made by Ref. 8 using a digital caliper (Mitutoyo TM ; calibrated to 0.01 mm) and a steel ruler, which later recorded appropriately in measurement data form. These data were analysed for Hierachical Cluster Analysis using Euclidean Distance of Unweighted Pair-Group Method Average (UPGMA) method in Minitab program version 14.0, whereas the Discriminant Function Analysis (DFA) and Canonical Variate Analysis (CVA) were run in program software Statistical Package for Social Science (SPSS) version 16.0. A probability of p < 0.05 was considered significant in all analysis. Results and Discussion Summary on the morphological data recorded and analysed in the present study are shown in Table 1. Hierachical cluster analyses generated from 27 morphological characters shows a clear clustering between the examined species of H. signata group where the species of H. picturata was separated alone, whereas H. signata had resulted into two different clusters (Fig. 1). The localities effect seems not to be appeared among specimens of H. picturata collected from various sites of Bhei, Baro, Mulu and Kana which being the representatives for upper Sarawak, Bako National Park as the isolated small island, and also Matang Wildlife Center representing Southern Sarawak.
A Morphometric Analysis of Hylarana signata Group of Malaysia 201 Table 1. Summary of the morphological characters measured in H. signata group. Species H. signata H. picturata Locality Character 1 Upper Sarawak (n = 4) SVL 51.20 ± 5.02 (46.23 57.18) HL 19.19 ± 2.44 (16.19 21.21) SNL 8.25 ± 1.62 (6.81 10.43) IOD 5.00 ± 0.66 (4.38 5.92) IND 3.41 ± 0.53 (2.65 3.88) ED 6.58 ± 0.95 (5.62 7.75) TAD 4.81 ± 1.21 (3.16 6.04) HW 16.19 ± 2.48 (12.92 18.72) UAL 10.00 ± 1.83 (8.18 12.50) FAL 13.01 ± 1.10 (11.99 14.54) FL 24.59 ± 1.76 (22.29 26.33) TBL 25.65 ± 1.39 (24.17 27.47) TSL 16.01 ± 2.04 (14.18 18.91) PL 23.91 ± 2.52 (21.52 26.49) ML 14.01 ± 1.78 (12.36 16.02) TOE4L 20.45 ± 2.03 (18.33 22.69) TOE1L 5.80 ± 1.94 (4.16 7.97) FIN1L 9.29 ± 0.38 (8.84 9.70) FIN3L 12.67 ± 1.37 (10.76 13.70) 2 Bako (n = 1) 55.0 19.67 8.32 5.25 3.19 8.89 4.37 16.26 12.95 13.74 20.98 26.17 12.98 23.0 14.37 22.47 5.25 9.48 12.34 3 Matang (n = 6) 41.00 ± 0.89 (40.00 42.00) 13.52 ± 0.84 (12.60 14.51) 6.33 ± 0.85 (5.13 7.69) 3.28 ± 0.64 (2.33 4.03) 2.32 ± 0.36 (1.86 2.90) 5.98 ± 0.58 (5.05 6.83) 3.37 ± 0.40 (2.95 3.97) 11.46 ± 0.77 (10.15 12.45) 8.95 ± 1.22 (7.64 10.36) 9.40 ± 0.64 (8.13 9.90) 18.18 ± 1.59 (16.58 20.19) 21.10 ± 0.93 (20.03 22.13) 11.46 ± 1.08 (9.68 12.75) 18.51 ± 1.94 (15.56 20.60) 10.79 ± 0.86 (9.55 11.95) 14.58 ± 2.12 (11.55 17.82) 5.66 ± 1.64 (3.00-7.28) 7.45 ± 1.04 (6.27 8.86) 8.91 ± 1.77 (5.87 11.37) 1 Upper Sarawak (n = 4) 42.06 ± 0.48 (41.43 42.56) 15.68 ± 1.26 (14.48 17.44) 6.64 ± 0.86 (5.83 7.75) 4.38 ± 0.32 (3.98 4.67) 2.66 ± 0.25 (2.48 3.02) 5.64 ± 1.11 (4.14 6.82) 3.38 ± 0.55 (2.55 3.70) 12.53 ± 1.21 (10.83 13.62) 8.85 ± 1.00 (7.47 9.84) 9.75 ± 1.37 (8.15 11.30) 21.40 ± 2.85 (17.90 24.21) 23.26 ± 1.77 (20.94 24.95) 12.69 ± 1.05 (11.50 13.94) 21.51 ± 2.29 (19.44 24.78) 11.87 ± 1.10 (10.53 13.14) 19.60 ± 2.03 (18.21 22.60) 7.06 ± 0.96 (6.08 8.36) 8.47 ± 1.42 (7.10 10.35) 11.89 ± 1.38 (10.50 13.73) 2 Matang (n = 2) 47.32 ± 9.45 (40.64 54.00) 18.28 ± 1.97 (16.89 19.67) 8.27 ± 0.07 (8.22 8.32) 5.24 ± 0.02 (5.22 5.25) 2.94 ± 0.35 (2.69 3.19) 7.36 ± 2.16 (5.83 8.89) 4.27 ± 0.15 (4.16 4.37) 14.81 ± 2.06 (13.35 16.26) 11.12 ± 2.60 (9.28 12.95) 12.43 ± 1.85 (11.12 13.74) 22.12 ± 1.61 (20.98 23.26) 24.44 ± 2.45 (22.70 26.17) 13.20 ± 0.31 (12.98 13.42) 21.91 ± 1.55 (20.81 23.00) 13.23 ± 1.62 (12.08 14.37) 19.81 ± 3.77 (17.14 22.47) 5.38 ± 0.18 (5.25 5.51) 8.28 ± 1.70 (7.08 9.48) 10.47 ± 2.65 (8.59 12.34) 3 Pahang (n = 1) 35.85 10.69 4.89 2.79 1.11 5.52 3.51 11.35 6.41 7.58 20.15 19.42 11.98 19.37 11.96 16.92 4.14 6.31 6.34
202 R. Zainudin and S. N. Sazali Table 1 (Continued). Species H. signata H. picturata Locality Character 1 Upper Sarawak (n = 4) NPL 2.80 ± 0.13 (2.66 2.98) NPW 1.64 ± 0.38 (1.32 2.19) TOE4DW 1.08 ± 0.16 (0.93 1.30) FIN1DW 1.28 ± 0.37 (0.76 1.63) FIN3DW 1.13 ± 0.27 (0.73 1.31) DLSL 45.44 ± 5.42 (38.14 50.36) DLSW 1.46 ± 0.39 (1.12 1.94) MTTL 7.76 ± 1.57 (6.34 9.24) 2 Bako (n = 1) 2.12 2.54 1.60 1.97 1.89 48.32 0.87 7.62 3 Matang (n = 6) 1.80 ± 0.48 (1.14 2.38) 1.33 ± 0.38 (0.95 1.95) 0.78 ± 0.21 (0.58 1.18) 0.68 ± 0.35 (0.15 1.08) 0.83 ± 0.13 (0.71 1.06) 27.80 ± 3.56 (21.62 31.10) 1.14 ± 0.26 (0.86 1.51) 5.81 ± 0.59 (4.84 6.34) 1 Upper Sarawak (n = 4) 1.87 ± 0.35 (1.41 2.17) 1.33 ± 0.20 (1.13 1.61) 0.98 ± 0.17 (0.73 1.12) 0.97 ± 0.15 (0.85 1.18) 0.88 ± 0.15 (0.75 1.18) 5.75 ± 5.85 (2.10 14.38) 1.59 ± 0.60 (1.11 2.42) 6.78 ± 0.86 (5.55 7.57) 2 Matang (n = 2) 2.13 ± 0.01 (2.12 2.13) 2.33 ± 0.30 (2.11 2.54) 1.34 ± 0.37 (1.07 1.60) 1.24 ± 0.02 (1.22 1.25) 0.97 ± 0.07 (0.92 1.02) 2.26 ± 0.18 (2.13 2.38) 1.08 ± 0.12 (0.99 1.16) 5.99 ± 1.62 (4.84 7.13) 3 Pahang (n = 1) However, the clustering was obviously different for H. signata where all specimens from Matang Wildlife Center were grouped into one sub clade, emerged from the same lineage of H. picturata. Another clade of H. signata was clustered independently which consisted of population from upper Sarawak and the only representative from Bako National Park. This finding suggests that the Malaysian H. signata group may comprised of more than two species instead of H. signata and H. picturata only, leading to cryptic species within the group complex 11,12. The morphometric analysis was congruence to the phylogenetic reconstruction using partial 16S rrna mitochondrial DNA gene 11 that revealed phylogeny complexity within Malaysia Hylarana signata group that might be due to ancient polymorphism of the lineages. Currently, the two species are diagnosed by being striped (signata) or spotted (picturata), also their tadpoles may be indistinguishable in the field 12. 2.51 2.43 0.53 0.11 0.53 5.74 1.15 4.89
A Morphometric Analysis of Hylarana signata Group of Malaysia 203 Fig. 1. UPGMA cluster analysis of Malaysian H. signata group. The analyses for discriminant function analysis were performed independently for each species. For H. signata alone, two significant functions were successfully extracted, in which the Functions 1 and 2 explained 99.2% and 0.8% of the variance, respectively (Table 2). This showed that the Function 1 with higher character loadings has higher variability of characters in the analysis. Tests of Function 1 through 2 showed significant value (Wilks lambda = 0.000) with p = 0.000 and also found significance in Function 2, (Wilks lambda = 0.061) with p = 0.002, respectively (Table 3). The highest character loading observed in both Function 1 and Function 2 were the TSL, with canonical
204 R. Zainudin and S. N. Sazali function coefficient values of -21.837 and 3.563, respectively (Table 4). This character was significantly informative for discriminating population of H. signata from upper Sarawak, Bako National Park and Matang Wildlife Reserve (Fig. 2). Table 2. Eigenvalues for DFA. Function Eigenvalue % of variance Cumulative % Canonical correlation 1 1909.406* 99.2 99.2 1.000 2 15.459* 0.8 100.0 0.969 *First 3 canonical discriminant functions were used in the analysis. Table 3. Wilks lambda for DFA. Test of Function(s) Wilks Lambda Chi-square df Sig. 1 through 2 0.000 62.136 10 0.000 2 0.061 16.805 4 0.002 Table 4. Standardised canonical discriminant function coefficients. Character Function 1 2 IOD 2.275 1.413 ED -6.867-1.132 FAL 17.705-1.650 TBL 12.688-0.482 TSL -21.837 3.563 *Diagnostic character in each function.
A Morphometric Analysis of Hylarana signata Group of Malaysia 205 Fig. 2. CVA plot of Functions 1 and 2 for H. signata. 1 = upper Sarawak, 2 = Bako National Park, 3 = Matang Wildlife Center. Meanwhile for H. picturata, two discriminant functions were recognised, in which the Function 1 and 2 explained the variance of 97.4% and 2.6% each (Table 5). The Wilks lambda statistic resulted significantly (Table 6) for the tests of Function 1 through 2 of 0.000 with p = 0.000, while Function 2 showed Wilk s lambda significant value of 0.099 and a probability of p = 0.031, respectively. In Table 7, the highest character loadings observed in Function 1 was the TBL with a canonical function coefficient value of - 5.346, while in Function 2 was the NPW, showing a canonical function coefficient value of -0.627 respectively. These two diagnostic characters were found to be useful for differentiating the population of H. picturata from three different localities (Fig. 3) of upper Sarawak, Matang Wildlife Center and also Pahang (Peninsular Malaysia).
206 R. Zainudin and S. N. Sazali Table 5. Eigenvalues for DFA. Function Eigenvalue % of variance Cumulative % Canonical correlation 1 338.686* 97.4 97.4 0.999 2 9.114* 2.6 100.0 0.949 *First 3 canonical discriminant functions were used in the analysis. Table 6. Wilks lambda for DFA. Test of Function(s) Wilks Lambda Chi-square df Sig. 1 through 2 0.000 24.426 6 0.000 2 0.099 6.942 2 0.031 Table 7. Standardised canonical discriminant function coefficients. Character Function 1 2 IOD 4.838 0.538 TBL -5.346 0.325 NPW 4.600-0.627 *Diagnostic character in each function.
A Morphometric Analysis of Hylarana signata Group of Malaysia 207 Conclusion Fig. 3. CVA plot of Functions 1 and 2 for H. picturata. 1= upper Sarawak, 2 = Matang Wildlife Center, 3 = Pahang. Morphometrical variations using 27 morphological characters successfully revealed phylogeny complexity within Malaysia Hylarana signata group that might be due to ancient polymorphism of the lineages or cryptic species that may have occurred within the group. Further studies using more representatives from other localities should be conducted in order to fully review the morphological relationships between the understudied species in this group complex, as well as to re-validate the findings with recent molecular studies. Acknowledgments This research was funded by UNIMAS Research Grant 01(124)513(05)12. We would like to thank the Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Universiti Kebangsaan Malaysia, Sarawak Forest Department, Sarawak Forestry Corporation, Borneo Highland Resort, Bario Management and Gunung Mulu National Park.
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