Dominant haplotype variation from each representative Indonesia ingroup

c. Dominant haplotype variation from each representative Indonesia ingroup

The nucleotide diversity and uniqueness of each group species were presented in Appendix 5-8. Table 3.4 in below shown nucleotide variations of each representative group, Group I Himantura leoparda var.1, II Himantura undulata, III Himantura uarnak and IV Himantura leoparda. Table 3.4 Variation of unique nucleotides in each representative of Indonesia ingroup by COI genetic marker Group 20 29 32 44 50 59 63 66 80 89 92 Group I_IR001 C G T G T C C C G T A Group II_IR007 . A A A C T T . A . G Group III_IR089 T A A . C T T . A C G Group IV_IR003 . . . . C . T T A . . Group 98 101 113 116 128 131 134 137 140 143 149 Group I_IR001 C T C A T G A G C T T Group II_IR007 . . . . C A . A . C C Group III_IR089 . . T G C . G A T . . Group IV_IR003 T C . . C A . A T . . Group 152 155 170 171 173 185 188 194 197 200 203 Group I_IR001 T T C G T T C T T A T Group II_IR007 . C . . . C T A C G C Group III_IR089 . . G A . C T A C G C Group IV_IR003 G C . . C C T A . . C Group 215 224 230 233 239 242 251 252 254 257 260 Group I_IR001 A A C G C C C T G A T Group II_IR007 G . T A T A T C . C C Group III_IR089 . . T A T A T C A T C Group IV_IR003 . G T A T A T C A . . Group 263 266 275 281 284 299 311 318 326 329 338 Group I_IR001 G C G A T T C T C T C Group II_IR007 . T A . . C T . . C . Group III_IR089 A . A . G C T . T C . Group IV_IR003 . . A G . C T C T C T Group 356 359 371 377 378 380 383 386 389 407 410 Group I_IR001 C A G T C A C T A C T Group II_IR007 T G A . T . T . G T . Group III_IR089 T . . . . . T C . . C Group IV_IR003 . . C C . G . . . T . Group 419 422 428 455 458 485 488 491 494 497 500 Group I_IR001 T C C A G G T C C C T Group II_IR007 . T T . A A . T T A . Group III_IR089 . T T . . A . T T G C Group IV_IR003 C . . G . A C T . A C Group 512 513 515 527 530 539 548 552 557 560 563 Group I_IR001 G T A T G C A T A T T Group II_IR007 A C G C A T G C G C C Group III_IR089 A C . C A T . C . . C Group IV_IR003 . . . C A T . C . . C Group 587 602 614 Group I_IR001 C T C Group II_IR007 T C T Group III_IR089 T . T Group IV_IR003 T . T The representatives of each group species, was known to have 62 nucleotide changes in the ratio of Group I with Group II. The changes consist of base substitutions purine ↔purine A↔G, pyrimidine↔pyrimidine C↔T, purine↔pyrimidine C↔A and T →A. A total of 20 nucleotide bases undergo substitution A↔G nucleotide at the site of the 29, 44, 80, 92, 131, 137, 200, 215, 233, 275, 359, 371, 389, 458, 485 512, 515, 530, 548 and 551. Base change by substitution pyrimidine ↔pyrimidine C↔T, there were 37, while the changes in purine ↔pyrimidine bases C↔A and T→A, respectively were observed at 3 sites the 242, 257 and 417 sites and 2 sites the 32 and 194 sites. Comparison of Group I and Group III is known to have a total of 61 nucleotide changes. The changes consist of base substitutions purine ↔purine A↔G, pyrimidine ↔pyrimidine C↔T, purine↔pyrimidine C→A, A↔T, C→G and T→G. Changes in purine ↔purine base substitutions A↔G there were as many as 17 consist of the 29, 44, 80, 92, 116, 131, 134, 137, 171, 200, 233, 254, 263, 275, 485, 512 and 530 base site, while the base change pyrimidine ↔pyrimidine C↔T as many as 37, followed by changes in purine ↔pyrimidine bases C→A, A↔T, C→G and T→G, each consisting of 1, 3, 2 and 1 sites. The difference of Group I and Group IV were 51 base sites, with changes in purine ↔purine bases A↔G occurs at 12 sites consisting of base sites at the 80, 131, 137, 224, 233, 254, 275, 281 , 380, 455, 485 and 530, pyrimidine ↔pyrimidine C↔T there were as many as 34 bases sites and purine ↔pyrimidine base change C→A, A↔T, G→C and T →G there were as many as 5 base site , ie to base site at the 152, 194, 242, 371 and 497. Differences in the nucleotide sites in Group II and Group III there were 36 sites and 91 sites total bases from nucleotide bases compared. The differences were the result of changes in purine ↔purine bases A↔G as many as 16 sites of nucleotide base sites at the 44, 116, 131, 134, 171, 215, 254, 263, 359, 371, 389, 458, 497, 515, 548 and 557. Changes pyrimidine ↔pyrimidine bases C↔T were 17 nucleotide bases comprising the 20 site, 89, 113, 140, 143, 149, 155, 257, 266, 326, 378, 386, 407, 410, 500, 560 and 602, and the change from purine ↔pyrimidine bases C→G, C→T and T→G nucleotide bases as much as 3 sites, namely the base at the 170, 257 and 284. Nucleotide similarities between Group I and IV were known as 40 base sites, whereas Group II and III equations there were 55 base sites. Equation Group I, II and III were known to have as many as 14 bases sites, as well as the equation of Group I, III and IV consisted of 14 the same as base sites Appendix 9. Based on the observations that have been carried out indicated a mixture of Himantura leoparda Group IV=Cluster 4 and H. uarnak Group III=Cluster 3 produced a few hybrids. Another second generation hybrid was produced between Cluster 4 and Cluster 1 both “H. leoparda”. Group II= Cluster 2 was one group that has been identified as H. undulata. If we compare with the results of morphological approach, H. undulata was a group of Himantura uarnak species complex which are dominant in Indonesian waters. Based on the COI marker, H. undulata was separate group from H. leoparda Group I, IV and H. uarnak Group III. Group III was one of other group that identified as H. uarnak and the result was not a surprise because based on morphological observations, it’s indeed a separate species from H. undulata and H. leoparda. Group I and IV were the two groups of species that have been released as H. leoparda by Manjaji- Matsumoto Last 2008. Group I is the mitochondrial DNA clade that characterize as Cluster 1 which is a full species. Based on 91 nucleotide bases different were found in 4 representative groups comparised, known 14 sites base were the same on Group I, III and IV. If we calculated to the total bases site of 620 bp, bases equation of Group I, III and IV were 543 bases or 87.58. Phylogeny based on Maximum Likelihood ML using mitochondrial DNA, based on partial COI marker genes supports monophyly from Himantura uarnak species complex. In Indonesia ingroup were grouped into four major lineages based on mtDNA COI Group I-IV Figure 3.3. Group I clustered in Group II and Group III, while Group IV have been separated from the three groups. Nucleotide diversity composition for four representative comparison groups had an average score those rates of less than 50. This condition will affect the performance of phylogeny reconstruction methods. Intra nucleotide diversity in the group was relatively low, where Group I into Group IV were characterized by no many differences observed. These results indicate the possibility that the Himantura uarnak species complex has undergone admixture between sister species. Species in this group allegedly evolved from a single ancestral species that inhabit the same geographical area. The existence of cryptic species were found in H. leoparda Group I and IV showed that the species was sympatric with a wide geographic area. Both species were scattered in the Sunda Strait, north of Java Sea and Bali. Sympatric speciation occurs due to the overlapping relationship or even identical in the range of the same geographic area. This illustrates were the distribution of speciation that led to the formation of sister species, in which the variation in the mating habits simultaneously, within a geographic area and occur continuously giving rise to different species Cracraft 1989; Futuyma 2009. The differences were a result of limited cross-breeding, where the two groups that deviate intercourse Templeton 1989; Mayr 1992; Queiroz 2007. Although the morphological observations it seem were not right when it compared with the pattern of group species based on molecular. Inaccuracies of grouping patterns indicate that the genetic approach is much more promising and has a great opportunity to reveal the identity of a species Ward et al. 2005; 2008. That does not mean that the disclosure of species identity simply can be done by using molecular approaches. Morphology, which is one component of observation in the taxonomy, and until this day, it is still being used in revealing the identity of a species Last Compagno 1999; Manjaji 2004; Manjaji-Matsumoto Last 2008. Just necessary prudence in determining the character in the identification and morphological approach will be more successful when combined with molecular approaches. The results of molecular approached by using 5 size-polymorphic intron loci of nuclear DNA had shown that the complex species of H. uarnak consists of 4 clusters based on intron markers or 4 group based on mitochondrial DNA who refer to each particular species. Molecular approached by using genetic markers COI has proven that the results of previous research were accurate. Although according to morphological observations get different result. Verification on two previous results needs to be done by using genetic markers cyt b. Conclusions 1. Four main groups from mitochondrial DNA of COI genetic markers describe the grouping of certain species. 2. Hybridization was found in one group of Indonesia. 3. Cryptic species was found in Himantura leoparda i.e. Himantura leoparda var.1 Group I. 4. Found a sympatric speciation in Himantura uarnak species complex.

CHAPTER IV CYTOCHROME B cyt b MARKER IN