Unique and dominant nucleotide bases variation representatives of each group

d. Unique and dominant nucleotide bases variation representatives of each group

Grouping of Himantura uarnak species complex has been known to have four major groups, namely Group I Himantura leoparda var.1, II Himantura undulata, III Himantura uarnak and IV H. leoparda var.2. To determine the unique nucleotide bases in this complex species, the representation from each individual was displayed by a standard determination based on the highest number of individuals represented. Individual representatives from each groups were IR001 Group I, IR007 Group II, IR086 Group III and IR003 Group IV. Nucleotide base changes that occurred in the representation of each group was dominated by changes in the pyrimidine ↔pyrimidine bases at 30 sites bases, alteration of purine ↔purine bases at 9 sites and one base sites from purine change →pyrimidine, which was the 39 site, where total change in the nucleotide base was in 40 sites Table 4.4. Table 4.4 Variation of unique nucleotides in each representative of Indonesia ingroup by cyt b genetic marker Group 12 21 36 39 54 63 70 75 81 87 90 93 96 102 GroupI, IR001 C A T A T T C C C C T C C C GroupII, IR007 . . . C . C . . . T C . . T GroupIII, IR086 . G C T . . . . . . C T T . GroupIV, IR003 T . . . C . T T T . C . . . Group 105 112 117 118 123 129 135 138 153 156 159 162 171 GroupI, IR001 G A C A C C T A T C T T G GroupII, IR007 . G . G T . . G . T . C . GroupIII, IR086 . . . . T . . . C . . . A GroupIV, IR003 A . T G T T C . . . C . . Group 180 186 192 196 201 202 207 213 222 228 231 234 237 GroupI, IR001 C A C T G C G T T T T C C GroupII, IR007 T . T C A . A C C C . T . GroupIII, IR86 T . T C A . A C . . C T T GroupIV, IR003 T G . . A T A C C C . . T Nucleotide changes that occurred in Group I and Group II consisted of 20 base sites, i.e. the 39, 63, 87, 90, 102, 112, 118, 123, 138, 156, 162, 180, 192, 196, 201, 207, 213, 222, 228 and 234 sites. Transition base substitution of pyrimidine ↔pyrimidine C →T and T→C was in amount of 14, while the purine↔purine transition A→G and G →A were 5 times and the substitution of a purine→pyrimidine transversions was 1 time i.e. sites 39 A →C. Nucleotide changes in the Group I and Group III were in 18 sites, i.e. the 21, 36, 39, 90, 93, 96, 123, 153, 171, 180, 192, 196, 201, 207, 213, 231, 234 and 237. The base change consisted of 13 substitutions transitions pyrimidine ↔pyrimidine, while the transitions purine ↔purine were 4 times and 1 time transversion substitutions purine →pyrimidine. Nucleotide changes in the Group I and Group IV were 22 times, which were 12, 54, 70, 75, 81, 90, 105, 117, 118, 123, 129, 135, 159, 180, 186, 201, 202, 207, 213, 222, 228 and 237 base sites. The base change had 17 substitutions transitions pyrimidine ↔pyrimidine and 5 transitions purine↔purine. Nucleotide changes in the Group II and Group III occurred in site 21, 36, 39, 63, 87, 93, 96, 102, 112, 118, 138, 153, 156, 162, 171, 222, 228, 231 and 237. The base change occurred at 19 sites consisting of 14 base substitutions transition from pyrimidine ↔pyrimidine and 5 transitions purine ↔purine occurs at 5 bases site. Consistency of the grouping results proves that the Indonesia ingroup of Himantura uarnak species complex was divided into 4 main groups, which each of them refers to a particular species. Forming of four main groups by COI and cyt b marker yielded two clades namely A and B. Clade A in the COI and cyt b marker consisting of Group I, Group II and Group III, while Clade B comprises Group IV. Four main groups on the formation of both markers were slightly the same. Based on the grouping results, it was assumed that gene flow being occurred from Group III to Group I and Group IV. This can be explained by observing changes in individuals grouping of IR086, IR087, IR102 IR088 against individuals IR110 and IR111. Based on mtDNA marker of COI gene, individual IR086 and IR087 were in Group I, while by the cyt b marker they were in Group III. By using the COI marker, individual IR088 was in Group IV, while by the cyt b it was in Group III. Individuals IR102, IR110 and IR111 by COI marker were in Group III, while by the cyt b marker they scattered in Group I IR102 and IR110 and Group IV IR111. This explanation confirmed that there were contribution of nucleotide bases of Group III to Group I and Group IV. This nucleotide bases contribution causes additional combinations of genetic variations in Group I and Group IV Gravel 2012; Morjan Rieseberg 2004; Zhaxybayeva Gogarten 2004. Based on the observations in Figure 4.4 and Figure 4.5 it was clear that Group IV is a distinct species, i.e. Himantura leoparda, while Group I was still a question mark, and there is assumption that the Group I were a separated species although for the time being still classified in H.a leopard var.1. Observation on the formation of Group II in clade A with single individual, i.e. IR007, where both by genetic markers COI and cyt b inferred as H. undulata, assumed possibly as a beginning individual of H. uarnak complex species which formed as a result of gene flow that occurs in group of H. uarnak Group III Gravel 2012. Other allegations were Group I was a cryptic species of H. leoparda based on the nucleotide sequences that have been compared between Group I and IV. It was known that in both groups occur only transitions base substitutions, i.e. alteration in nucleotide bases derived from purine ↔purine A→G and G→A and pyrimidine ↔pyrimidine C→T and T→C. So alteration of the nucleotide bases does not change the amino acid composition from each individual in each Group I and IV. Classification results by cyt b markers have proved the truth of grouping by using genetic marker COI and intron pholymorphic. In this case, classification of group species based on morphological observations was inconsistency in some type of spotting pattern but the type of reticulation was a basic reference for molecular identification. However the validation of group classifications were still needed, so the three previously results were compared to spotting pattern on the morphological character. Conclusions 1. Four main lineages clade were observed as for COI mithocondria genetic marker. 2. Both Clade I and IV comprised H. leoparda reference specimens from Manjaji 2004

CHAPTER V DISTINGUISHSING SPECIES AMONG MEMBERS OF