a. Nucleotide Variations
Along the 620 bp fragment resulted from 111 individuals of Indonesia ingroup produce 515 in the same bases conserved = invariable site and 105 nucleotides are
unique identifier sequence bases variable site from individuals analyzed. The same things also done after the additions of individual based on another ingroup and outgroup
Table 3.3.
Table 3.3 Nucleotide variations based on observations of Himantura uarnak ingroup and outgroup by COI genetic marker
Length of sequence 620bp conserved
variable si
sv R
Pi s
n=111 ingroup Indonesia
515 105
28 4
7 86
19 n=116
ingroup Indonesia, Taiwan, Zanzibar 508
112 29
4 7,25
101 11
n=117 ingroup + outgroup
444 176
30 5
6
102 74
Note: Pi=parsimony-informative sites, s=singleton sites, si= transitionsal pairs, sv= transversional pairs dan R= the ratio of sisv
Based on the distinguished group, the unique identifier are different from the rest that amounted of 16.9 n = 111, 18.06 n = 116 and 28.39 n = 117. The
nucleotide diversity will showed how many genes are inherited jointly by the parents, which is in the molecular basis known as variable site. How big is the uniqueness from a
total of 620 nucleotides in the 111 individual nucleotide bases can be grouped based on the formation of different species groups distinct. Grouping species group on ingroup are
individuals that came from Indonesia formation result of phylogenetic tree topology and outgroup produces 4 different major groups.
The nucleotide bases pair from Indonesia ingroup n = 111 run into substitution on the purine and pyrimidine bases. Substitution of nucleotide bases were translated into
transitional nucleotide substitutions between two purine purine ↔purine or two
pyrimidines pyrimidine ↔pyrimidine and transversions between the purine nucleotide
bases and the pyrimidine purine →pyrimidine Table 3.3. Total ingroup individuals
originating from Indonesia were as many as 111, which divided into two major groups namely A consisting of Group I, II and III, while B consist only of 1 group Group IV.
Indonesia ingroup with n = 111 individuals consisting of 29 individuals include in Group I, one individual in Group II, 23 individuals in Group III, and 58 other individuals in
Group IV.
b. Haplotype variation from Indonesia ingroup n = 111 and other ingroup Zanzibar, Taiwan and the data of GeneBank
Group I was divided into 6 haplotypes, with haplotype 1 represented by IR086 consisting of 2 individuals. Haplotypes 2 by IR112 which also consist of 2 individuals,
haplotype 3 only consist of one individual is represented by IR024. Haplotypes 4 represented by IR037 consisting of 2 individuals. Haplotypes 5 comprise only 1 individual
which is represented by IR052. Haplotype 6 was haplotype with the highest number of 21 individuals represented by IR085. Haplotypes of ingroup outside Indonesia was individual
which come from Zanzibar by 1 individual ZANZ_6. Alteration in the nucleotide bases of Group I was dominated by the substitution transition be changed from
pyrimidine
↔pyrimidine bases C↔T occurs in as many as 6 base sites. The base change
occurred at the 241, 404, 497, 518, 560 and 614 base sites, only two base changes occur in Indonesia, namely the 404 and 614 base ingroup. Other sites base change between
Indonesia ingroup by comparison was ZANZ_6. DNA modification through base substitution transition purine
↔purine A↔G occurred in 5 sites at the 171, 275, 389, 482 and 596, which happens to all ingroup of Indonesia. Transversion substitutions
purine →pyrimidine base A→C occurred in the 296 sites among ingroup of Indonesia by
purine A and ZANZ_6 became pyrimidine C. Group II only consisted of one ingroup of haplotypes originating from Indonesia,
IR007, while another weas individual which come from GeneBank with access code DQ108167. A change in the nucleotide bases in Group II was dominated by the transition
substitution from pyrimidine ↔pyrimidine bases C↔T occurs in four sites, i.e. the 149,
251, 266 and 560. Substitution of transition from purine →purine bases A→G occurs at
the 608 base sites. The base change was result of comparisons between Indonesia ingroup IR007 with the GeneBank DQ108167 data.
Group III was divided into 5 haplotype consisting of haplotype 1 IR089 represented by 10 individuals. One individual 637_H. uarnak coming from Taiwan as
haplotype 2 clustered with haplotype 3 consisting of 5 individuals represented by IR101. Haplotype 4 was represented by individual IR094 of 6 individuals clustered with
haplotypes 5 were represented by the IR107 and IR109 consist of 2 individuals. Substitution of transition si in Group III occurred on 38 sites for pyrimidine
↔pyrimidine C
↔T, purine↔purine bases A↔G occurs at 16 base sites, while substitution transverse sv in the purine
↔pyrimidine bases A↔T, C→G, C→A and T→G were in 5 of base sites. Changes at 38 sites pyrimidine
↔pyrimidine bases were on the 20, 50, 59, 63, 89, 113, 128, 140, 185, 188, 197, 202, 229, 238, 251, 252, 260, 299, 311, 326, 329, 356,
383, 386, 410, 422, 428, 491, 494, 500, 513, 527, 539, 552, 563, 587, 605 and 614. Alterations at 16 base sites were purine
↔purine consisting of the 29, 80, 92, 116, 134, 137, 171, 220, 232, 254, 263, 275, 371, 485, 512, and 530 base sites. Changes in
purine ↔pyrimidine bases found at the 5 base sites i.e. the 32, 170, 194, 241, 257, 284 and
497. Almost all changes in nucleotide bases were the result of comparisons between Indonesia ingroup in Group III with individual H. uranak from Taiwan, except on-site of
the 371 and 605 comparison occur only in Indonesia ingroup.
Group IV was divided into 17 haplotypes; with haplotype 1 represented by IR062 separated itself from other groups of individuals. Haplotypes 2 represented by IR069
consisting of 16 individuals, subsequent haplotype 3 and 4 were represented by the IR007 and IR022. Nine individuals were in the haplotype 5 represented by IR066, then haplotype
6 was represented by IR053 consist of 4 individuals. Haplotypes 7 represented by IR060 consisting of 1 individual. Haplotypes 8 represented by IR048 and IR036, subsequent
haplotype 9, represented by the IR032 which consist of four individuals clumped together. Haplotypes 10, 11, 12, 13, 14 and 15 respectively represent by IR017, IR078, IR071,
IR018, IR010 and IR070. Haplotypes 16 represented by 12 individuals and haplotype 17 composed from 1 individual, the IR088. Other haplotypes derived from GeneBank with
the access code JF493652 and JF492651.
Nucleotide substitution changes through the transition si occurs in pyrimidine
↔pyrimidine bases C↔T i.e. a total of 39 base sites at the 50, 63, 66, 98, 101, 128, 140, 143, 155, 173, 185, 188, 191, 202, 217, 229, 235, 238, 251, 252, 299, 311, 318,
326, 329, 338, 377, 399, 407, 419, 488, 491, 500, 527, 539, 552, 563, 587 and 614 sites. Change of base substitutions through purine
↔purine base consist of 16 sites at the 44, 80, 131, 137, 195, 223, 232, 254, 275, 281, 380, 401, 455, 485, 530 and 545 base sites. While
the base change by substitution transversions purine ↔pyrimidine sv occurred 8 times
with 1 site having 3 times base change C →G→A i.e. on the 287 site. Changes in
purine ↔pyrimidine bases in 7 other base sites consist of change T→A 2 times in the 10
and 194 sites, change T
→G occurs in the 152 sites, change C
→A consist of 2 the site ie the 241
and 497 and the last change is the change G
↔C 2 sites that consist of the 371 and
534 base sites. Formation of haplotype and nucleotide bases changes that occurred in each
group which is clustered generates a phylogenetic tree as shown in Figure 3.3.
Figure 3.3 Phylogeny tree of Himantura uarnak species complex COI of 620 bp Group IV
Group I
Group II
Group III
c. Dominant haplotype variation from each representative Indonesia ingroup
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