3.1. Mitogenome structure and organization
In the present study, we determined the mitochondrial genome sequences of B. kachuga (16,155 bp), and B. dhongoka (15,620 bp). The newly sequenced mitogenomes have been deposited in the GenBank database under accession numbers MZ242095 (for B. kachuga), and MZ242096 (for B. dhongoka). Similar to most vertebrates, the Batagur mitogenome comprised 37 genes: 13 PCGs (COXI-III, ND1–6, ND4L, CYTB, ATP6, and ATP8), 22 putative tRNA genes, two rRNA (12S and 16S) genes, and a non-coding region (Fig. 1). Among these, 28 genes (2 rRNA, 14 tRNA, and 12 PCGs) were located on the Heavy strand (H-strand), while the remaining nine genes (ND6 and 8 tRNAs) were found on the Light strand (L-strand) (Table 1). The gene order and arrangement were similar to those of other reported Testudines species (Kundu et al., 2019; 2020).
Table 1. Annotation of the mitochondrial genomes of Batagur kachuga and Batagur dhongoka.
Gene
|
Strand
|
Anticodon
|
Batagur kachuga
|
Batagur dhongoka
|
|
|
|
Position
|
Intergenic
|
Start/Stop
|
Position
|
Intergenic
|
Start/Stop
|
tRNA-Phe
|
H
|
GAA
|
1-70
|
0
|
–
|
1-70
|
0
|
–
|
12S rRNA
|
H
|
–
|
71-1035
|
0
|
–
|
71-1035
|
0
|
–
|
tRNA-Val
|
H
|
TAC
|
1036-1105
|
0
|
–
|
1036-1104
|
0
|
–
|
16S rRNA
|
H
|
–
|
1105-2699
|
–1
|
–
|
1104-2710
|
–1
|
–
|
tRNA-Leu
|
H
|
TAA
|
2701-2776
|
+1
|
–
|
2712-2787
|
+1
|
–
|
ND1
|
H
|
–
|
2777-3744
|
0
|
ATG/ TA–
|
2788-3755
|
0
|
ATG/ TA–
|
tRNA-Ile
|
H
|
GAT
|
3745-3814
|
0
|
–
|
3756-3826
|
0
|
–
|
tRNA-Gln
|
L
|
TTG
|
3814-3884
|
–1
|
–
|
3826-3896
|
–1
|
–
|
tRNA-Met
|
H
|
CAT
|
3884-3952
|
–1
|
–
|
3896-3964
|
–1
|
–
|
ND2
|
H
|
–
|
3953-4991
|
0
|
ATG/ T–
|
3965-5003
|
0
|
ATG/ T–
|
tRNA-Trp
|
H
|
TCA
|
4992-5064
|
0
|
–
|
5004-5076
|
0
|
–
|
tRNA-Ala
|
L
|
TGC
|
5066-5134
|
+1
|
–
|
5078-5146
|
+1
|
–
|
tRNA-Asn
|
L
|
GTT
|
5136-5208
|
+1
|
–
|
5148-5220
|
+1
|
–
|
tRNA-Cys
|
L
|
GCA
|
5235-5300
|
+26
|
–
|
5244-5309
|
+23
|
–
|
tRNA-Tyr
|
L
|
GTA
|
5301-5371
|
0
|
–
|
5310-5380
|
0
|
–
|
COI
|
H
|
–
|
5373-6923
|
+1
|
GTG/ AGG
|
5382-6932
|
+1
|
GTG/ AGG
|
tRNA-Ser
|
L
|
TGA
|
6912-6982
|
–12
|
–
|
6921-6991
|
–12
|
–
|
tRNA-Asp
|
H
|
GTC
|
6983-7052
|
+0
|
–
|
6992-7061
|
+0
|
–
|
COII
|
H
|
–
|
7053-7739
|
0
|
ATG/ TAA
|
7062-7748
|
0
|
ATG/ TAA
|
tRNA-Lys
|
H
|
TTT
|
7741-7813
|
+1
|
–
|
7750-7822
|
+1
|
–
|
ATP8
|
H
|
–
|
7815-7982
|
+1
|
ATG/ TAA
|
7824-7991
|
+1
|
ATG/ TAA
|
ATP6
|
H
|
–
|
7973-8656
|
–10
|
ATG/ TAA
|
7982-8665
|
–10
|
ATG/ TAA
|
COIII
|
H
|
–
|
8656-9439
|
–1
|
ATG/ T–
|
8665-9448
|
–1
|
ATG/ T–
|
tRNA-Gly
|
H
|
TCC
|
9440-9507
|
0
|
–
|
9449-9516
|
0
|
–
|
ND3
|
H
|
–
|
9509-9858
|
+1
|
ATG/ T–
|
9518-9867
|
+1
|
ATG/ T–
|
tRNA-Arg
|
H
|
TCG
|
9859-9928
|
0
|
–
|
9868-9937
|
0
|
–
|
ND4L
|
H
|
–
|
9929-10225
|
0
|
ATG/ TAA
|
9938-10234
|
0
|
ATG/ TAA
|
ND4
|
H
|
–
|
10219-11595
|
–7
|
ATG/ TAG
|
10228-11604
|
–7
|
ATG/ TAG
|
tRNA-His
|
H
|
GTG
|
11604-11673
|
+8
|
–
|
11617-11685
|
+12
|
–
|
tRNA-Ser
|
H
|
GCT
|
11674-11740
|
0
|
–
|
11686-11752
|
0
|
–
|
tRNA-Leu
|
H
|
TAG
|
11740-11812
|
–1
|
–
|
11752-11824
|
–1
|
–
|
ND5
|
H
|
–
|
11813-13621
|
0
|
ATG/ TAA
|
11825-13633
|
0
|
ATG/ TAA
|
ND6
|
L
|
–
|
13617-14141
|
–5
|
ATG/ AGA
|
13629-14153
|
–5
|
ATG/ AGG
|
tRNA-Glu
|
L
|
TTC
|
14142-14209
|
0
|
–
|
14154-14221
|
0
|
–
|
CYTB
|
H
|
–
|
14215-15354
|
+5
|
ATG/ TAG
|
14227-15366
|
+5
|
ATG/ TAA
|
tRNA-Thr
|
H
|
TGT
|
15359-15431
|
+4
|
–
|
15371-15443
|
+4
|
–
|
tRNA-Pro
|
L
|
TGG
|
15432-15502
|
0
|
–
|
15444-15514
|
0
|
–
|
Partial CR
|
H
|
–
|
15503-16155
|
0
|
–
|
15515-15620
|
0
|
–
|
The sequenced mitogenomes displayed significant variation in size (Table 2). Notably, the length of B. dhongoka was the smallest among the available mitogenomes of Testudines due to partial amplification of CR. In addition, the differences in mitogenome length may be attributed to the size variation of the non-coding region among Testudines (Zheng et al., 2013).
Table 2
Nucleotide composition in regions of the mitogenomes for Geoemydidae species.
Species | Mitogenome (bp) | A + T % | AT-skew | GC-skew | PCGs (bp) | A + T % | AT-skew | GC-skew | rRNAs (bp) | A + T % | AT-skew | GC-skew |
B. kachuga | 16,153 | 58.27 | 0.162 | −0.371 | 11,377 | 57.82 | 0.092 | −0.389 | 2,561 | 58.26 | 0.290 | −0.175 |
B. dhongoka | 15,619 | 57.76 | 0.165 | −0.372 | 11,378 | 57.44 | 0.089 | −0.381 | 2,573 | 58.26 | 0.303 | −0.181 |
B. trivittata | 16,463 | 58.12 | 0.156 | −0.366 | 11,379 | 57.44 | 0.092 | −0.386 | 2,568 | 57.78 | 0.289 | −0.173 |
P. tentoria | 16,657 | 59.44 | 0.120 | −0.331 | 11,295 | 58.52 | 0.052 | −0.348 | 2,562 | 58.86 | 0.265 | −0.155 |
P. sylhetensis | 16,568 | 59.27 | 0.124 | −0.334 | 11,268 | 58.77 | 0.056 | −0.345 | 2,561 | 59.00 | 0.273 | −0.173 |
M. sinensis | 16,461 | 60.02 | 0.126 | −0.340 | 11,395 | 59.54 | 0.064 | −0.354 | 2,570 | 58.94 | 0.272 | −0.177 |
M. caspica | 16,741 | 61.21 | 0.112 | −0.334 | 11,382 | 60.11 | 0.054 | −0.348 | 2,568 | 59.54 | 0.272 | −0.170 |
M. mutica | 16,775 | 60.79 | 0.122 | −0.335 | 11,392 | 59.37 | 0.058 | −0.345 | 2,568 | 59.46 | 0.266 | −0.164 |
M. rivulata | 16,766 | 61.22 | 0.121 | −0.332 | 11,382 | 60.09 | 0.054 | −0.346 | 2,567 | 59.36 | 0.272 | −0.169 |
M. reevesii | 16,783 | 61.04 | 0.119 | −0.329 | 11,377 | 59.84 | 0.063 | −0.350 | 2,573 | 59.89 | 0.265 | −0.176 |
M. japonica | 16,443 | 60.48 | 0.125 | −0.341 | 11,385 | 60.06 | 0.061 | −0.356 | 2,570 | 59.72 | 0.270 | −0.171 |
M. leprosa | 17,067 | 61.90 | 0.111 | −0.347 | 11,382 | 60.66 | 0.052 | −0.344 | 2,567 | 60.03 | 0.268 | −0.165 |
M. annamensis | 16,844 | 60.56 | 0.113 | −0.338 | 11,391 | 59.39 | 0.055 | −0.351 | 2,715 | 59.33 | 0.266 | −0.181 |
M. megalocephala | 16,783 | 61.25 | 0.111 | −0.338 | 11,385 | 60.11 | 0.052 | −0.346 | 2,574 | 59.82 | 0.267 | −0.176 |
M. nigricans | 16,779 | 60.93 | 0.118 | −0.336 | 11,382 | 59.65 | 0.056 | −0.346 | 2,570 | 59.80 | 0.277 | −0.186 |
C. dentata | 16,484 | 61.50 | 0.114 | −0.320 | 11,376 | 60.98 | 0.043 | −0.328 | 2,565 | 60.50 | 0.274 | −0.160 |
C. atripons | 16,500 | 61.62 | 0.117 | −0.321 | 11,387 | 61.05 | 0.044 | −0.329 | 2,561 | 60.71 | 0.275 | −0.165 |
C. pulchristriata | 16,527 | 61.57 | 0.116 | −0.324 | 11,380 | 60.80 | 0.044 | −0.333 | 2,564 | 60.68 | 0.276 | −0.164 |
C. tcheponensis | 16,593 | 60.97 | 0.121 | −0.323 | 11,377 | 60.13 | 0.042 | −0.327 | 2,576 | 59.66 | 0.284 | −0.170 |
C. oldhamii | 16,656 | 61.18 | 0.122 | −0.324 | 11,370 | 60.13 | 0.042 | −0.329 | 2,569 | 59.90 | 0.283 | −0.163 |
C. fusca | 16,491 | 61.29 | 0.118 | −0.326 | 11,382 | 60.80 | 0.045 | −0.338 | 2,563 | 60.32 | 0.274 | −0.162 |
C.galbinifrons | 17,244 | 61.70 | 0.106 | −0.348 | 11,399 | 60.03 | 0.049 | −0.342 | 2,571 | 60.13 | 0.271 | −0.184 |
C. flavomarginata | 16,721 | 61.67 | 0.102 | −0.331 | 11,377 | 60.51 | 0.038 | −0.339 | 2,562 | 60.34 | 0.262 | −0.159 |
C. aurocapitata | 16,890 | 60.98 | 0.100 | −0.331 | 11,373 | 59.30 | 0.051 | −0.343 | 2,577 | 59.48 | 0.274 | −0.160 |
C. pani | 16,922 | 61.10 | 0.102 | −0.331 | 11,393 | 59.39 | 0.047 | −0.345 | 2,568 | 59.50 | 0.276 | −0.161 |
C. trifasciata | 16,675 | 60.68 | 0.115 | −0.332 | 11,382 | 59.60 | 0.049 | −0.341 | 2,568 | 59.34 | 0.282 | −0.174 |
C. picturata | 16,623 | 60.85 | 0.116 | −0.335 | 11,395 | 60.03 | 0.049 | −0.340 | 2,553 | 59.61 | 0.282 | −0.179 |
C. amboinensis | 16,708 | 60.57 | 0.116 | −0.337 | 11,397 | 59.49 | 0.057 | −0.354 | 2,572 | 59.05 | 0.275 | −0.171 |
C. bourreti | 16,661 | 60.77 | 0.117 | −0.334 | 11,394 | 59.86 | 0.050 | −0.340 | 2,571 | 59.43 | 0.282 | −0.181 |
H. annandalii | 16,604 | 61.85 | 0.136 | −0.356 | 11,380 | 60.96 | 0.071 | −0.378 | 2,563 | 61.60 | 0.276 | −0.162 |
H. grandis | 16,581 | 62.38 | 0.112 | −0.334 | 11,379 | 61.96 | 0.051 | −0.348 | 2,566 | 61.10 | 0.267 | −0.160 |
H. depressa | 16,773 | 62.52 | 0.119 | −0.330 | 11,382 | 61.50 | 0.048 | −0.336 | 2,565 | 61.55 | 0.257 | −0.156 |
S. bealei | 16,564 | 61.01 | 0.120 | −0.330 | 11,373 | 60.39 | 0.053 | −0.347 | 2,574 | 59.98 | 0.270 | −0.165 |
S. quadriocellata | 16,555 | 61.11 | 0.121 | −0.333 | 11,366 | 60.32 | 0.052 | −0.346 | 2,859 | 59.39 | 0.261 | −0.164 |
N. platynota | 16,981 | 62.49 | 0.100 | −0.347 | 11,398 | 61.60 | 0.043 | −0.348 | 2,573 | 61.01 | 0.263 | −0.164 |
The base composition for the A + T bias of the B. kachuga and B. dhongoka mitogenome is 58.27% and 57.76%, respectively (Table 2). It was identical to the nucleotide composition biases reported in other Testudines, ranging from 58.12% (B. trivittata) to 62.52% (Heosemys depressa). A significant bias towards A/T was also observed in the mitogenomes and the PCGs and rRNA of Batagur and other and Testudines. In Batagur, AT skewness was positive, which indicated that nucleotide adenine was more prevalent than thymine, whereas negative GC skewness showed that the cytosine was more common than the guanine base in complete mitogenome. Similar results have also been reported in several turtles, such as Pangshura spps. (Kundu et al., 2019; 2020), Cyclemys dentata (Huang et al., 2015), Pyxidea mouhotii (Zhang et al., 2008b), Cuora trifasciata (Li et al., 2015), and B. trivittata (Feng et al., 2017).
The nine pairs of the overlapping region in the mitogenomes were observed among tRNAVal/16SrRNA, tRNAIle/tRNAGln, tRNAGln/tRNAMet, COI/tRNASer, ATP8/ATP6, ATP6/COIII, ND4L/ND4, tRNASer/tRNALeu and ND5/ND6. The overlapping regions were ranged from − 1 to – 12 bp. The longest overlap was observed between COI and tRNASer (12 bp), whereas it was smallest (1 bp) between tRNAVal/16SrRNA, tRNAIle/tRNAGln, tRNAGln/tRNAMet, ATP6/COIII, and tRNASer/tRNALeu. The non-coding CR region was located between tRNAPro and tRNAPhe. In addition, the longest intergenic spacers (26 bp in B. kachuga and 23 bp in B. dhongoka) were observed between the tRNAAsn and tRNACys mitochondrial regions (Table 1). The mitogenome-based genetic differentiation showed 7.6% variation between B. dhongoka and B. kachuga, 7.7% between B. kachuga and B. trivittata, whereas it was slightly low between B. dhongoka and B. trivittata at 6.3% (Fig. 2).