using MEGA5 Tamura et al. 2011. Sequences of nucleotide bases from each cyt b fragments that have been edited are stored in GenBank http:www.ncbi.nlm.nih.gov,
with access code JX274304-JX274416.
Data Analysis A total of 131 sequences cyt b ingroup consisted of 113 sequences as results of this
study IR001-IR113, 14 homologous cyt b sequences from Manjaji 2004 that H. uarnak 1CSIROH5476.03, H. uarnak 2CSIROH5477.01, H. uarnak 3CSIROH5484.01, H. uarnak
6_Manjaji, H. uarnak8_Manjaji, H. undulata2 CSIROH5483.01, H. undulata3 CSIROH5481.01, H. sp. A1_Manjaji, H. sp. A2_UMS_MMSK c4, H. sp. A4_Manjaji,
H. sp. A5_Manjaji, H. sp.A6 CSIROH5284.05 paratype_H. leoparda, H. sp.A7 CSIROH5478.01 paratype_H. leoparda, H. sp.A9_Manjaji. Four B CYT homologous
sequences from Zanzibar ZANZ6_cytb_KangNing, ZANZ6_cyt_b_Kang_Ning 2, ZANZ6_long, ZANZ6_original_KNS, and 1 H. signifer3_Manjaji outgroup sequences
Manjaji 2004.
A total of 131 sequences are aligned using ClustalW on MEGA alignment editor version 5 Tamura et al. 2011. Analysis of nucleotide diversity based on some basic
parameters, which are conserved nucleotides vs. variable nucleotides, singleton nucleotides, parsimony-informative nucleotides that analyzed using MEGA 5 program,
whereas haplotype diversity using the same program with the manual observation. The best substitution model according to Bayesian Information Criterion BIC is Kimura 2
parameter Kimura 1980, where the evolutionary rate variation among sites was modeled with a discrete gamma distribution + G Yang 1994. This model is used to generate the
phylogeny from Neighbor-Joining from cyt b sequences and test bootstap Felsenstein 1985 with 1000 replications.
Results and Discussion
a. Selection of primer pair from genetic markers cyt b
This study used two pairs primer with targeted genetic marker was cyt b gene. Two pairs primer are 1 forward and reverse CBF2 CBR1 and 2 forward and reverse GLU1L
CB2H. Based on the results test, it is known that the pair primer of CBF2 and CBR1 does not give good results for DNA amplification to proceed the sequencing phase. Most of the
amplified DNA using primers CBF2 and CBR1 has a double band as shown in Figure 4.3.
Figure 4.2 DNA amplification produced by using alternative primer of cyt b CBF2 and CBR1
Amplification success rate can be determined by a single band of amplified DNA on electrophoresis results. The amplified results by using the primer were not good,
therefore there is no further sequencing process.
707 bp
Figure 4.3 DNA fragment so long as ± 420bp of DNA amplification produced by using
cyt b marker by primer GLU1L and CB2H Amplification produced by using another primer pair namely GLU1L and CB2H
entirely result 420 bp amplified DNA, which are ready to be purified and prepared for next sequencing process. A total of 113 individuals Himantura uarnak species complex
originating from Indonesia produced DNA fragment as long as ± 420 bp Figure 4.4.
b. Nucleotide and haplotype variation
After alignment with other individuals from Manjaji 2004, along 420 bp only 239 bp can be compared. The 239 bp of DNA fragment from 113 individuals Indonesia
ingroup produced 196 nucleotide with the same bases conserved = invariable sites and 43 nucleotide bases of unique bases variable site from analyzed individuals. This is also
done after adding some other individuals based on its ingroup and outgroup Table 4.2. Table 4.2 Nucleotide variations based on observations of the individual number of
ingroup and outgroup by cyt b marker Length of sequence 239 bp
conserved variable
si sv
R Pi
s n=113
ingroup Indonesia 196
43 15
34
9 n=131
ingroup Indonesia, Manjaji 2004, Zanzibar 188
51 40
5 8
46 5
n=132 ingroup + outgroup
175 64
57 6
9,5
46 18
Based on the number of samples and sample origin, nucleotide variations occur variably, which are 17.9 n = 113, 21.3 n = 131 and 26.7 n = 132. The amount
of unique nucleotide from a total of 239 nucleotides in the 113 individual nucleotide bases can be grouped based on the formation of different species groups distinct. Grouping
sample group from Indonesian origin after traced with sequence results of Manjaji 2004 and individuals from Zanzibar were generating a phylogenetic tree with 4 different groups
Figure 4.4. Nucleotides from Indonesia ingroup n = 113 had a substitution on the purine and pyrimidine bases. Substitution of the nucleotide bases were translated into
transition = si nucleotide substitutions between two purines or two pyrimidines and transverse = sv nucleotide substitutions between a purine and a pyrimidine.
A total of 113 samples originating from Indonesia, where 29 individuals were in Group I, one individual in Group II, 23 individuals in Group III and 60 individuals in
±420 bp
Group IV. Group I was divided into 8 subgroups haplotypes, haplotype 1 was comprised from 1 individual from Manjaji 2004, namely H. sp.A6 CSIROH5284.05 paratype H.
leoparda, haplotype 2 consisted of 1 individual IR113, haplotype 3 consisted of 24 individuals from Indonesia represented by IR001 which cluster together with 4
individuals from Manjaji 2004 as haplotypes 4 and haplotype 5 consist of 1 individual IR112 that clustered with haplotype 6 consist of 3 individuals represented by IR024 and
4 individuals from Zanzibar as haplotype 7 and H. sp.A9_Manjaji as haplotype 8. Alteration of nucleotide bases of Group I in Indonesia ingroup only occur at three sites
namely bases 105, 117 and 231. Bases change occurred in purine
→purine bases G→A in 1 base site 105 and 2 alteration of pyrimidine
↔pyrimidine bases C↔T namely site 117 and 231 Appendix 10.
Group II was divided into 3 haplotypes Appendix 11, haplotype 1 was represented by 1 individual Indonesia ingroup which is IR007 that clustered with 1
individual from Manjaji 2004, i.e. H. undulata 2 CSIROH5483.01 as haplotype 2 and haplotype 3 was H. undulata 3 CSIROH5481.01 Manjaji 2004. Alteration of nucleotide
bases of Group II was known in 5 sites base 120, 123, 156, 213 and 237 on the pyrimidine
↔pyrimidine bases C→T and T→C. Group III was divided into 6 haplotypes Appendix 12 were represented by
individual Manjaji 2004 consist of H. uarnak 6 and H. uarnak 8 as haplotype 1 and haplotype 2. The next sub-group consist of four haplotypes was IR106 as haplotypes 3 and
IR091 as a sub group from haplotypes 4. IR096 as haplotype 5 and IR086 sub group consists of 17 individuals where 14 individuals originating from Indonesia ingroup and 3
individuals came from Manjaji 2004 as haplotype 6. Group III undergone changes in purine
→purine nucleotide bases A→G at 1 site base 105, whereas alteration in the pyrimidine
↔pyrimidine bases C→T and T→C happened on 3 sites, namely 129, 159 and 222.
Group IV was divided into 6 haplotypes which 5 haplotypes from Indonesia, namely haplotype 1 consist of 1 individual represented by IR111 which clustered
separately from 5 other haplotypes. Haplotype 2 consist of 1 haplotype, i.e H.sp.A7_CSIRO_H5478.01 haplotype from Manjaji 2004 that clustered by itself but still
combined with haplotypes 2, 3, 4, and 5. Haplotype 3 consisted of 54 individuals were represented by IR003, haplotype 4 consist of 3 individuals represented by IR013,
haplotypes 5 and 6 only consist by 1 individual, i.e IR050 and IR063 respectively. Nucleotide changes in Group IV occurs in purine
↔purine bases A→G and G→A at 2 base site site 171 and 217 while the pyrimidine
↔pyrimidine bases C→T and T→C occurs at 6 sites i.e. 38, 93, 135, 213 and 221
Appendix 13. Formation of haplotype and nucleotide bases changes that occurred in Indonesia ingroup n = 113 and Manjaji 2004
produces grouping groups as shown in Figure 4.4.
Figure 4.4 The phylogeny tree of Himantura uarnak species complex based on cyt b marker along the 239 bp
Group I
Group II
Group III
Group IV
c. Manjaji 2004 and Zanzibar sample position in Indonesia ingroup n = 113
