3.3. Protein-Coding Genes
The Pygathrix cinerea mitogenome harbors the 13 canonical protein-coding genes (PCGs), collectively spanning 11,292 bp and representing 68.27% of the mitochondrial genome. Twelve of these PCGs reside on the H-strand (majority strand), while nad6 is located on the L-strand (minority strand). The overall A + T content of the PCGs is 61.4%, ranging from 58.7% (cox3) to 68.9% (atp8). This A + T bias is consistent with the overall nucleotide composition of the Pygathrix cinerea mitogenome.
To assess nucleotide composition bias across the PCGs, we calculated AT- and GC-skews. The majority of
Pygathrix cinerea PCGs exhibit negative AT-skewness (
Table 3), indicating a higher frequency of thymines than adenines. This pattern is also observed in other primates, where the majority of PCGs display negative GC-skewness, ranging from −0.35 to −0.42, suggesting a C-biased nucleotide composition.
nad6, however, deviates from this trend, exhibiting a positive GC-skew (0.644) and a negative AT-skew (−0.378). This pattern is similar to that observed in other
Pygathrix cinerea (−0.387 and 0.648) and
Pygathrix nigripes (−0.341 and 0.596) samples (
Figure 4).
All 13 PCGs in Pygathrix cinerea initiate with ATN codons (ATG or ATT), consistent with other Pygathrix species, However, cob-1 in Pygathrix nigripes exhibits a distinct start codon, TTG, suggesting potential variation in translation initiation mechanisms within the Pygathrix genus.
3.4. A + T-Rich Region
The control region (D-loop), located between the
trnP gene and
trnF gene, spans 1094 base pairs (positions 15,584–16,541 and continuing at 1–136). This size is typical for vertebrate mitochondrial genomes [
31].
The size of this region in Vietnamese Central Highland
Pygathrix cinerea is larger than in other
Pygathrix cinerea specimens (1092 bp; 15,444–16,535 nt) and in
Pygathrix nigripes (1092 bp; 15,443–16,534 nt),
Macacac mulatta (1085 bp; 15,480–16,564),
Papio hamadryas (1076 bp; 15,446–16,521 nt), and
Callithrix jacchus (1079 bp; 15,421–16,499 nt), but shorter than in
Homo sapiens (1121 bp; 16,022–16,567nt, 1–575 nt). This size variation is primarily attributed to differences in copy numbers and tandem repeats within the region [
32].
The control region exhibits a high degree of variation compared to other areas of the mitochondrial genome, reflecting the presence of multiple tandem repeats (TRs) and variations in their copy numbers [
32]. The total GC-skew in the control region was −0.349, AT-skew was 58%, and A + T content was −0.013. No discernible repeat deletions were observed in the control region of Vietnamese Central Highland
Pygathrix cinerea.
A comparison with other primate species revealed over 80 mutations between
Pygathrix cinerea and
Pygathrix nigripes within the control region. Most mutations were similar between different
Pygathrix cinerea samples, except for nine positions (highlighted in orange in
Table 4). These nine nucleotide mutations were either deletions or insertions, and they were unique to Vietnamese Central Highland
Pygathrix cinerea and other
Pygathrix cinerea specimens.
The short sequence (TATAA) was identified within the control region, occurring four times in Pygathrix cinerea and twice in other species. The sequence (AATAAT) also occurred twice in other Pygathrix cinerea specimens but was not present in Pygathrix nigripes. Additionally, the control region contains a high AT content (AATTATATAATCTATTA), which may represent a distinctive feature of Vietnamese Central Highland Pygathrix cinerea.
3.5. tRNA and rRNA Genes
As predicted by Mitos WebServer, all 22 transfer RNA (tRNA) genes could be folded into a secondary clover-leaf structure (
Figure 5). Eight tRNA genes were located on the L strand, while the remaining genes were located on the H strand (
Table 1). All 22 tRNA genes identified in the
Pygathrix cinerea mitochondrial genome have anticodons that are consistent with those found in other primates. The tRNA genes range in size from 59 bp (for
trnS1) to 75 bp (for
trnL2). The average base composition of the tRNA genes is A: 32.7%, T: 30.4%, G: 19.3%, and C: 17.5%.
trnT has the highest GC content (50.8%), while
trnD and
trnH have the lowest (23.2%). In addition to the typical clover-leaf structure, seven tRNA genes in
Pygathrix cinerea contain a total of eleven mismatched base pairs. These mismatches are found in the amino acid acceptor (AA) stem, the pseudouridine (TΨC) stem, and the anticodon (AC) stem (
Table 5).
The mitochondrial genome of
Pygathrix cinerea also contains two ribosomal RNA (rRNA) genes: the large ribosomal subunit (
16S rRNA) and the small ribosomal subunit (
12S rRNA). The
16S rRNA gene is located between the
trnV and
trnL2 genes, while the
12S rRNA gene is located between the
trnF and
trnV genes. This arrangement is typical for most vertebrates, where the
trnV gene separates the large and small ribosomal subunits [
33]. The
12S rRNA gene is 949 bp long, and the
16S rRNA gene is 1565 bp long. These sizes are similar to those found in other
Pygathrix cinerea specimens (948 bp and 1562 bp, respectively). The A + T content of the
16S rRNA gene is 60%, and the A + T content of the
12S rRNA gene is 59.4%. These values are consistent with those observed in other primates, including other
Pygathrix cinerea specimens (59.9% for
16S and 59.4% for
12S) and
Pygathrix nigripes (60% for
16S and 58.8% for
12S) (
Table 2).
3.7. Mutations
While many of the positions differed between Vietnamese Central Highland
Pygathrix cinerea and other
Pygathrix species (
Supplementary Table S5), only a few of them resulted in changes to the encoding of amino acids (
Supplementary Table S6), which demonstrates the distinctive features of Vietnamese Central Highland
Pygathrix cinerea compared to other species. In particular, 43 mutation positions in the DNA (
Table 6) that encoded for 23 amino acids differed between Vietnamese Central Highland
Pygathrix cinerea and other
Pygathrix cinerea specimens
(Table 7). Among them, four nucleotide insertion positions were found in
rrnS and
rrnL; others included 30 nucleotide substitution or deletion positions in PCG and nine nucleotide substitution or deletion positions in the control region. The insertion T at position 242 encodes for
rrnS (208–1156); A at position 1711; C at position 2772; A at position 2773, leading to the insertion of one more amino acid leucine at the 81st position in
rrnS; the amino acid isoleucine at position 571 in
rrnL; the amino acid histidine at the 924th position in
rrnL; and the amino acid asparagine at position 925 in
rrnL, none of which were found in the other
Pygathrix cinerea specimens. In addition, in the
rrnL genome, the substitution mutation at the 2566th position also differed, from glycine in the other
Pygathrix cinerea specimens to asparagine in Vietnamese Central Highland
Pygathrix cinerea. Most PCGs exhibited substitutions or deletion mutations that led to changes in the amino acid which it codes. The deletion from C in other
Pygathrix cinerea specimens in Vietnamese Central Highland
Pygathrix cinerea at positions 3080 and 3134 resulted in changing threonine to isoleucine at the 1027th and 1045th positions. In contrast, at the 3296th position, nucleotide G, which changes lysine to arginine at position 1099th in
nad1, occurs in Vietnamese Central Highland
Pygathrix cinerea but not in other species.
In nad2, trbY, and nad4, there was one deletion in each PCG of the Vietnamese Central Highland Pygathrix cinerea mitogenome, which changed the protein that they code. More specifically, in nad2, trbY, and nad4, the deletion at positions 4997, 5414, and 10,519 derived isoleucine to threonine at positions 1666 and 1805, and cysteine to serine at position 3507 in Vietnamese Central Highland Pygathrix cinerea, respectively.
There were two deletion mutations in cox1 at the 5507th and 5678th loci. In comparison with other Pygathrix cinerea specimens, at position 5507 in the Vietnamese Central Highland Pygathrix cinerea mitogenome, A was not seen, while C did not occur at the 5678th position in the other Pygathrix cinerea specimens, meaning that cysteine was present at position 1836 instead of tyrosine and serine was present at the 1893rd position instead of cysteine, respectively.
G was present at position 13,125 in Vietnamese Central Highland Pygathrix cinerea instead of the deletion found in other Pygathrix cinerea specimens, meaning that there was tryptophan instead of no amino acid at position 4375 in the nad5 genome. At protein positions 413 and 414, which correspond to the 1206th base pair, there was still an insertion mutation.
In nad6 at the 13,942nd and 13,957th loci, nucleotide T was found in Vietnamese Central Highland Pygathrix cinerea; however, it was not found in other samples. This led to the amino acids being present at different positions (4648th and 4653rd), replacing histidine with tyrosine and proline with serine.
More specially, three positions did not occur in the cob genome of Vietnamese Central Highland Pygathrix cinerea in comparison with other Pygathrix cinerea specimens. At position 14,749, G was deleted, and at positions 14,659 and 14,998, C was found. Therefore, there were three differences in the amino acids compared with other Pygathrix cinerea specimens in terms of cob protein: aspartic acid instead of cysteine at the 4917th position, phenylalanine instead of serine at the 4920th position, and tyrosine instead of histidine at the 5000th position.
3.9. Phylogeny of 55 Primate Mitogenomes
The phylogeny of primates is shown in
Figure 6. The tree topology of the primates was consistent according to the Maximum Likelihood method and analysis with bootstrap support (>70). The closest living relatives of primates are
Hominidae. The tree was rooted with the outgroup
Cynocephalus variegatus based on Mason et al. [
35]. The entire Primates tree has its roots stemming from the species of Dermoptera, specifically
Cynocephalus variegatus, and the resulting structure is consistent with the structure obtained from previous studies [
36,
37,
38,
39,
40]. The phylogenetic tree constructed using the complete mitochondrial genomes of over 50 primate species clearly shows that the
Pygathrix species (
Pygathrix cinerea,
Pygathrix nemaeus, and
Pygathrix nigripes) form a well-supported monophyletic clade. In previous studies, the relative position of
Pygathrix and
Nasalis was closer than
Rhinopithecus, and the present study revealed the same thing [
2,
4]. A common origin of the group is now widely accepted, although the phylogenetic relationships among its genera and species are largely unknown [
4]. Our analysis strongly supports this relationship among primates and confirms that the douc langurs (genus
Pygathrix) are a distinct evolutionary lineage within the odd-nosed monkey group. The
Pygathrix clade is most closely related to the genera
Nasalis (proboscis monkey) and
Simias (
Mentawai langur). Together, these three genera form a larger monophyletic group of odd-nosed monkeys, which is consistent with previous morphological and molecular studies.
The mitochondrial genome sequence of Pygathrix cinerea generated in this study (GenBank accession PP623106) is significantly different from a previously published Pygathrix cinerea mitogenome (GenBank accession JQ821842) with 43 nucleotide differences, suggesting the presence of distinct genetic lineages within this species. This divergence between the mitogenomes indicates the previously published sequence (JQ821842) may have come from a different geographic population or subspecies of Pygathrix cinerea, which could account for the observed genetic differences or the possibility of cryptic subspecies or evolutionarily significant units within the grey-shanked douc langur population. The mitogenome sequence generated in this study is firmly placed within the Pygathrix clade, confirming the species assignment of the sample as Pygathrix cinerea. However, the distinct genetic differences from the previously published Pygathrix cinerea mitogenome suggest the need for more population investigation into the evolutionary history and population structure of this critically endangered primate species.