Characterization of the Complete Mitochondrial Genome of the Central Highland Grey-Shanked Douc Langur (Pygathrix cinerea), a Critically Endangered Species Endemic to Vietnam (Mammalia: Primates)
by
, , and
Mai Thi Phuong Nguyen
1,†,
Tram Thi Thuy Nguyen
2,3,†,
Tung Thanh Ha
1,
Chi Nguyen Quynh Ho
2,4,
Cuong Phan Minh Le
4,
Huy Nghia Quang Hoang
4,
Quynh Thi Nhu Nguyen
5,
Tao Thien Nguyen
6,
Dung Tri Luu
3,
Khoa Dang Dang
7,
Quan Ke Thai
8 and
Long Thanh Le
2,4,* 1
Tay Nguyen Institute for Scientific Research, Academy of Science and Technology, Dalat City 670000, Vietnam
2
Biotechnology Department, Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi 100000, Vietnam
3
Department of General and Biomedical Science, Ho Chi Minh City University of Physical Education and Sports, Ho Chi Minh 700000, Vietnam
4
Animal Biotechnology Department, Institute of Tropical Biology, Vietnam Academy of Science and Technology, Ho Chi Minh 700000, Vietnam
5
Department of Bioactive Compounds, Institute of Tropical Biology, Vietnam Academy of Science and Technology, Ho Chi Minh 700000, Vietnam
6
Institute of Genome Research, Vietnam Academy of Science and Technology, Hanoi 100000, Vietnam
7
Faculty of Biotechnology, Ho Chi Minh Open University, Ho Chi Minh 700000, Vietnam
8
Faculty of Natural Sciences Education, Saigon University, Ho Chi Minh 700000, Vietnam
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Curr. Issues Mol. Biol. 2024, 46(9), 9928-9947; https://doi.org/10.3390/cimb46090592
Submission received: 16 July 2024
/
Revised: 22 August 2024
/
Accepted: 2 September 2024
/
Published: 6 September 2024
(This article belongs to the Special Issue Mitochondrial Genome 2024)
Abstract
:The grey-shanked douc langur (Pygathrix cinerea) is a recently described, critically endangered primate, endemic to Vietnam. In this study, we describe the Central Highland species’ complete mitochondrial genome (mitogenome—mtDNA). It is a circular molecule with a length of 16,541 base pairs (bp). The genome consists of 37 genes, consistent with those found in most other vertebrates, including 13 protein coding genes, 22 transfer RNAs, and two ribosomal RNAs. A comparison with the mitogenomes of more than 50 primates showed that the mitogenome of Vietnamese Central Highland Pygathrix cinerea has a conservative gene order. We identified 43 nucleotide differences when comparing this genome with a previously published mitogenome of Pygathrix cinerea. It is evident that there are distinct differences between the Pygathrix cinerea we are currently studying and other Pygathrix cinerea specimens. These differences are unlikely to be solely the result of sequencing errors, as the mitogenomes were generated using high-quality methods. The genetic divergence observed between the two Pygathrix cinerea mitogenomes implies the potential existence of at least two distinct lineages or forms of this primate species within its native range in Vietnam.
1. Introduction
The grey-shanked douc langur (Pygathrix cinerea) is a critically endangered primate species endemic to Vietnam. It was initially described as a subspecies of the Indochinese douc langur (Pygathrix nemaeus) in 1997 but subsequent morphological and molecular studies have confirmed it as a distinct species within the genus Pygathrix [1,2,3,4]. The grey-shanked douc langur is one of the rarest primates in the world, with a highly fragmented population estimated at fewer than 1000 individuals [5,6,7,8]. At present, its distribution is primarily within the Vietnamese provinces of Quang Nam, Quang Ngai, Kon Tum, and Gia Lai, inhabiting a limited range of tropical forests [8]. Morphological variability within the species has been observed, with some populations exhibiting distinct phenotypic characteristics whereby coat coloration can range from light grey to dark charcoal, with some individuals exhibiting reddish-brown hues on the limbs and head [2,3]. The grey-shanked douc langur faces significant threats to its long-term survival. Its population is projected to decline substantially between 2050 and 2070, primarily due to habitat loss and fragmentation driven by deforestation, infrastructure development, and the expansion of agriculture [9]. Conservation efforts are critical to prevent the extinction of this endangered primate. Understanding the genetic diversity and evolutionary history of the grey-shanked douc langur is essential for effective conservation planning and management [10,11,12,13,14]. Analyzing the mitochondrial genome can determine the origins of and genetic relationships between populations of a species. Populations with low genetic diversity will need to be prioritized for conservation to maintain the vitality of the species. This helps better understand the genetic structure and evolutionary history of the species, allowing for more effective conservation strategies [3,15,16,17,18,19,20,21,22]. We sequenced the complete mitochondrial genome of a grey-shanked douc langur from Vietnam and compared it to available mtDNA sequences from other douc langur species. This study presents the first complete mitochondrial genome sequence of Pygathrix cinerea, a critically endangered primate endemic to Vietnam. While partial mitogenome sequences of this species were included in Liedigk et al. [4], a detailed analysis of its mitochondrial characteristics was lacking. We compared the complete mitogenome of Pygathrix cinerea to publicly available data from other primates, focusing on the precise location and orientation of genes within the mitochondrion. Our analysis revealed that the gene order and orientation in the Pygathrix cinerea mitogenome are generally conserved among douc langur species (Pygathrix). However, we identified significant differences in the positions and lengths of a few genes, highlighting potential variations in mitochondrial evolution within this group. Phylogenetic analysis was conducted to assess the evolutionary relationships among these species. The results of this study provide valuable information for understanding the characteristics, the evolutionary history, and conservation status of the Pygathrix cinerea, a critically endangered species.
2. Materials and Methods
2.1. DNA Extraction and Sequencing
DNA Extraction: Vietnamese Central Highland Pygathrix cinerea tissue (Supplementary Figure S1) was collected in the Biological Museum, Tay Nguyen Institute for Scientific Research, Vietnam Academy of Science and Technology. Its mitogenome was extracted using the QIAamp DNA Mini kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. Evaluation of DNA sample quality: The DNA concentration was determined using the fluorometric method (Qubit). The OD260/OD280 ratio was determined by absorbance measurement. The DNA size was determined by agarose gel electrophoresis. Samples meeting the quality criteria of concentration ≥2 ng/µL, amount ≥90 ng, and OD260/OD280 ≥ 1.70 were considered suitable for further experimental steps. Samples with DNA size <1000 bp were flagged.
Sequencing: Whole genome sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions. The library concentration was determined by the fluorometric method and the average library size was determined using a Bioanalyzer (Agilent, Santa Clara, CA, USA) according to Illumina’s library evaluation guidelines. Samples were considered suitable for sequencing when the concentration was ≥0.50 ng/µL (for genome size <1 Gb) or ≥2 ng/µL (for genome size >1 Gb). The libraries were then sequenced using the 150 PE Next Generation Sequencing method on a MiniSeq, MiSeq, or NovaSeq instrument (Illumina, San Diego, CA, USA) [23].
2.2. De Novo Assembly
De novo assembly is a method of constructing genome sequences from short or long reads without the need for them to be based on a reference genome sequence. We used the GetOrganelle (v1.7.7.0) pipeline with optimal parameters for the de novo assembly of the sample genome sequence. The assembly results are presented in Supplementary Table S4, showing a contig length of 16,656 bp consistent with the size of the mitochondrial genome, which was approximately 16 kb.
2.3. Bioinformatics Analysis
The raw sequencing data were purified using the fastp tool v0.23.1 [21]. Nucleotides with poor solution quality or that were unreliable or unknown (type N nucleotides) were eliminated based on the Phred-score value recorded for each nucleotide [22]. After being “purified”, the reads were assembled de novo using the GetOrganelle tool (v1.7.7.0) [24]. The raw sequencing data, after undergoing optimization, de novo assembly, and genome annotation, were stored in the FASTQ file format (Supplementary Data S2), containing read and sequence information and the corresponding quality score. The reading quality results are presented in Supplementary Table S3. The de novo assembly quality was assessed using the Quast v5.2.0 tool [22] and local alignment of reads onto assembled contigs. This allowed the detection of regions with unusually low depth coverage values compared to neighboring regions, which were noted in the assembly results. The assembly was annotated using the MITOS annotation system (revision 6b33f95) [25] with a specialized mitochondria database.
The mitogenome was annotated using Mitos WebServer version 2.1.9 [25] and Mito Fish version 4.03 [26]. Mito Annotator version 4.03 [26] was applied to create a genetic map of the entire Pygathrix cinerea mitogenome. Amino acid and nucleotide compositions were evaluated and compared for all 55 primate mitogenomes using MEGA 11: AT-skew = (A − T)/(A + T) and GC-skew = (G − T)/(G + T). The t-RNA sequences were aligned with the homologues of similar species. By comparing it to other Pygathrix mitogenomes, the A + T rich, PCGs, and r-RNAs were determined. The Relative Synonymous Codon Usage (RSCU) values of Pygathrix cinerea’s whole mitogenome were computed using MEGA 11. The secondary structures of transfer RNA (t-RNA) predictions were determined using tRNAscan-SE software v2.0 [27] and the Mitos WebServer [25].
2.4. Phylogenetic Tree
To determine the molecular location in the evolutionary tree of Pygathrix cinerea and its association with other primates, 13 PCGs of these 55 species’ sequence alignments were subjected to analysis with the Maximum Likelihood method using IQ-tree version 2.2.2.6 [28]. The appropriate substitution model for the data set was selected using ModelFinder version 2.2.0 [29]. Branch support was obtained using ultra-fast bootstrapping [30]. The GenBank accession numbers of all sequences used are shown in the Supplementary Materials.
3. Results and Discussion
3.1. Complete Mitochondrial Genome Analysis
The complete mitochondrial genome of Pygathrix cinerea comprises 16,541 base pairs (bp) and harbors the typical 37 genes found in primate mitogenomes (Figure 1 and Table 1). These genes include a control region, two ribosomal RNA (rRNA) genes (12S rRNA and 16S rRNA), 22 transfer RNA (tRNA) genes, and 13 protein-coding genes (PCGs). The majority of genes are located on the H strand, with the exception of ND6 and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu) residing on the L strand. The Pygathrix cinerea mitogenome exhibits a gene organization similar to that of other douc langur species. However, it is noteworthy that the complete mtDNA sequence of the Vietnamese Central Highland Pygathrix cinerea analyzed in this study is slightly larger (16,541 bp) than previously published sequences, including Pygathrix cinerea (JQ821842, 16,535 bp) and Pygathrix nigripes (MH064177, 16,536 bp) [4].
3.2. Nucleotide Composition Pattern
The Vietnamese Central Highland Pygathrix cinerea mitochondrial genome exhibits a high A + T content, accounting for 61.4%, which is similar to Pygathrix nigripes (61.1%) but differs slightly from other Pygathrix cinerea specimens (61.1%) and Pygathrix nemaeus (61.3%) (Table 2). This A + T bias is particularly pronounced in the trnF and trnL1 genes, reaching 71%. Consistent with other primates, Pygathrix cinerea mtDNA displays a strong A/T preference in codon usage (Figure 2). Comparative analysis of amino acid frequencies across the three Pygathrix species reveals a highly similar distribution, as evidenced by comparable A + T content, AT-skew, and GC-skew (Figure 3). These metrics, commonly used to assess nucleotide composition patterns in mitochondrial genomes, highlight the conserved nature of amino acid frequencies within the Pygathrix genus. The most frequent amino acids in Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea specimens include Ala (3.9–4%), Asp (1.8%), Cys (1.7%), Asn (6.1–7.1%), Gln (3.5–4.1%), Ile (7.9–9.1%), Phe (4.4–5.4%), and Thr (7.3–9.1%).
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).
3.6. Intergenic Region and Overlapping
3.6.1. Gene Overlaps
Comparative analysis of the mitochondrial genomes reveals several instances of gene overlaps:
- atp8 and atp6: a 22 bp overlap was consistently observed between the atp8 and atp6 genes in Vietnamese Central Highland Pygathrix cinerea, other Pygathrix cinerea specimens, and Pygathrix nigripes.
- trnV and rrnL: a 2 bp overlap was found in Vietnamese Central Highland Pygathrix cinerea, other Pygathrix cinerea individuals, and Pygathrix nigripes.
- trnI and trnQ: a 3 bp overlap was present in Vietnamese Central Highland Pygathrix cinerea, other Pygathrix cinerea individuals, and Pygathrix nigripes.
- nad4L and nad4: a 4 bp overlap was observed in Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea specimens.
- cob-0 and cob-1: an 8 bp overlap was uniquely found in Pygathrix nigripes.
These overlaps, particularly the 22 bp overlap between atp8 and atp6 and the 2 bp overlap between trnV and rrnL, are common among Pygathrix species and are located on the H strand.
3.6.2. Intergenic Spacer Regions
The Pygathrix cinerea mitochondrial genome contains approximately twenty non-coding intergenic spacer regions ranging in size from 1 bp to 68 bp (Table 1). While these regions were previously considered non-functional, recent studies suggest that some may play regulatory roles in gene expression.
The longest intergenic spacer in Pygathrix cinerea is 68 bp long, located between trnK and cox2. This spacer is significantly shorter than the 172 bp spacer found in other Pygathrix cinerea specimens and the 167 bp spacer found in Pygathrix nigripes.
Intergenic spacer lengths vary across Pygathrix species, with Pygathrix cinerea having shorter spacers than Pygathrix nemaeus (194 bp with 20 regions) and Macaca leonnina (182 bp with 22 regions) but longer spacers than Pygathrix nigripes (167 bp with 20 regions). The intergenic spacer regions between Pygathrix cinerea and Pygathrix nigripes are generally similar, except for the region between nad5 and trnL1, which is 15 bp in Pygathrix cinerea and 37 bp in Pygathrix nigripes.
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.
3.8. Evolutionary Analysis
The Tamura–Nei model and the Maximum Likelihood approach were used to deduce the evolutionary history [34]. The branches show the proportion of trees where the related taxa are grouped together. The resulting tree had the highest log likelihood (−433,849.22). By applying the Neighbor-Join and BioNJ algorithms on a matrix of pairwise distances computed using the Tamura–Nei model, the initial tree(s) for the heuristic search were automatically created. The topology with the superior log likelihood value was then selected. This analysis included 55 mitogenomes. The codon positions included were 1st, 2nd, 3rd and Noncoding. Evolutionary analyses were conducted in MEGA11 [33].
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.
4. Discussion
The mitochondrial genome of Vietnamese Central Highland Pygathrix cinerea reveals a fascinating blend of conserved features characteristic of primates and unique variations that offer insights into its evolution and conservation. Our analysis provides valuable data for understanding the genetic diversity within this threatened species and its potential implications for conservation efforts.
4.1. Control Region: A Window into Evolutionary History and Population Dynamics
The control region of Vietnamese Central Highland Pygathrix cinerea exhibits a significantly larger size (1094 bp) compared to other Pygathrix cinerea specimens (1092 bp); however, the two base pair difference is in a region of repetitive sequences and is therefore biologically insignificant, as this pattern is observed in other primate species that do not experience population bottlenecks or rapid evolutionary divergence [41]. Therefore, the minor size difference observed here does not warrant conclusions about distinct evolutionary lineages or population-level diversity.
This size variation, coupled with the presence of unique mutations, suggested a potentially distinct evolutionary lineage for this population, possibly reflecting a period of isolation or adaptation to specific environmental pressures [42]. These mutations might be associated with the effect of selection and genetic drift and supports the notion of a distinct evolutionary lineage [43]. However, the limited number of samples precludes any robust conclusions about the evolutionary history and population structure of this endangered primate.
The tandem repeats and GC-skew observed in the control region are consistent with other primates, highlighting the conserved nature of these regulatory elements. The specific arrangement and composition of these elements might influence replication initiation or transcription regulation [44].
4.2. Ribosomal RNA Genes: Essential for Protein Synthesis and Phylogenetic Resolution
The remarkable conservation of rRNA gene size and A + T content across Pygathrix species underscores their fundamental role in mitochondrial protein synthesis [45]. This consistency highlights the value of these genes as reliable markers for phylogenetic analysis, particularly for resolving relationships within closely related taxa [46,47]. While the rRNA genes are generally highly conserved, there are four nucleotide insertion positions found in rrnS and rrnL that may lead to regulate gene expression at the translational level and this rRNA modification pattern may occur in response to environmental shifts, developmental stages, and disease states [48] or indicate a unique evolutionary trajectory of Vietnamese Central Highland Pygathrix cinerea.
4.3. Transfer RNA Genes: Adapting to Specific Needs and Environmental Pressures
The presence of all 22 tRNA genes with consistent anticodons in Pygathrix cinerea is essential for the accurate translation of mitochondrial proteins. The conserved nature of these genes underscores their fundamental role in maintaining mitochondrial function. The variations in tRNA gene size and GC content, observed in Vietnamese Central Highland Pygathrix cinerea, could reflect differences in gene expression and regulation [49].
The identification of mismatched base pairs in several tRNA genes, particularly in the AA, TΨC, and AC stems, is intriguing. These mismatches could potentially influence tRNA structure and function.
4.4. Gene Overlaps and Intergenic Spacers: Unraveling Regulatory Mechanisms and Evolutionary Adaptations
The consistent gene overlaps observed between a pair of genes (atp8 and atp6; trnV and rrnL) in Pygathrix species suggest a potential functional significance beyond mere chance occurrences. These overlaps might play a regulatory role in gene expression, potentially influencing the transcription or translation of these genes [50].
The intergenic spacer regions, while previously considered non-functional, are now recognized as potential regulatory elements [51]. The variations in spacer lengths across Pygathrix species, with longer spacers in Pygathrix nemaeus and Macaca leonnina compared to Pygathrix cinerea, suggest a potential role in regulating gene expression [52].
The unique genetic features identified in Vietnamese Central Highland Pygathrix cinerea, particularly in the control region and tRNA genes, highlight the importance of conserving its genetic diversity. This diversity is crucial for adaptation and resilience in the face of environmental changes and threats. Understanding the genetic differences between Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea specimens can inform conservation efforts by identifying distinct populations that may require specific management strategies.
Further research is needed to investigate the functional significance of the observed variations in the control region, tRNA genes, and intergenic spacer regions. Comparative studies with a broader range of primate species could provide a more comprehensive understanding of the evolutionary dynamics and functional implications of these variations. Additionally, examining the expression patterns of these genes in response to environmental stressors could shed light on their potential role in adaptation and resilience.
5. Conclusions
This study presents the first comprehensive characterization of the complete mitochondrial genome of the Central Highland grey-shanked douc langur, Pygathrix cinerea, a critically endangered primate endemic to Vietnam. The mitogenome sequence obtained provides valuable molecular data on this poorly studied species. The Pygathrix cinerea mitogenome is shown to retain most of the conserved genes of ancestral features. The mtDNA of this Vietnamese Central Highland Pygathrix cinerea consists of 37 genes that are typically similar to most vertebrates, arranged in a specific pattern. It is a circular molecule with a length of 16,541 base pairs (bp). These genes include 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. This Vietnamese Central Highland Pygathrix cinerea is most closely related to Pygathrix nemaeus and the second closest relative is Pygathrix nigripes. Of particular note is that within the population of Pygathrix cinerea, there are significant differences in the gene structure with 43 distinct variations in the DNA that alter 23 amino acids in proteins, highlighting the differences between this Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea.
To better understand the genetic diversity, population dynamics, and conservation status of Pygathrix cinerea, future studies will need to incorporate a much larger sample size representing the major subpopulations across the species’ distribution. Comprehensive population genomic analyses incorporating nuclear as well as mitochondrial markers will be crucial to elucidate the evolutionary history and guide evidence-based conservation efforts for this threatened primate.
The mitogenome sequence reported here serves as an initial molecular resource, but additional research is needed to fully characterize the genetic diversity and evolutionary patterns within the grey-shanked douc langur. Expanding the genomic sampling and integrating it with ecological and demographic data will be necessary to inform effective conservation strategies for protecting this endangered species.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cimb46090592/s1. Figure S1: Vietnamese Central Highland Pygathrix cinerea; Data S2: Complete mtDNA genome of Vietnamese Central Highland Pygathrix cinerea; Table S3: Read quality (illumina) of analyzed samples; Table S4: De novo assembly result; Table S5: Mutation between three Pygathrixes; Table S6: Protein mutation between two Pygathrixes.
Author Contributions
Conceptualization, T.T.T.N., C.N.Q.H. and L.T.L.; methodology, T.T.T.N., C.N.Q.H., C.P.M.L. and L.T.L.; data collection and validation, T.T.T.N., T.T.H., C.P.M.L., H.N.Q.H., D.T.L., T.T.N. and K.D.D.; formal analysis, C.N.Q.H., Q.T.N.N., Q.K.T. and L.T.L.; data curation, T.T.T.N., T.T.H., C.N.Q.H., C.P.M.L., H.N.Q.H., Q.T.N.N., T.T.N., D.T.L., K.D.D. and L.T.L.; writing—original draft preparation, T.T.T.N. and L.T.L.; writing—review and editing, T.T.T.N., Q.K.T., M.T.P.N. and L.T.L.; project administration, M.T.P.N. and L.T.L.; funding acquisition, M.T.P.N. All authors have read and agreed to the published version of the manuscript.
Funding
This work was supported by Vietnam Academy of Science and Technology, grant ÐLTE00.08/22-23.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data are contained within the article and Supplementary Materials.
Acknowledgments
We would like to express their sincere gratitude to the Biological Museum, Tay Nguyen Institute for Scientific Research, Vietnam Academy of Science and Technology for their provision of resources, generous support, and assistance in facilitating this research.
Conflicts of Interest
The authors declare no conflicts of interest.
References
- von Nadler, T. A New Subspecies of Douc Langur, Pygathrix nemaeus ninereus ssp. nov. Zool. Gart. 1997, 67, 165–176. [Google Scholar]
- Finstermeier, K.; Zinner, D.; Brameier, M.; Meyer, M.; Kreuz, E.; Hofreiter, M.; Roos, C. A Mitogenomic Phylogeny of Living Primates. PLoS ONE 2013, 8, e69504. [Google Scholar] [CrossRef] [PubMed]
- Pozzi, L.; Hodgson, J.A.; Burrell, A.S.; Sterner, K.N.; Raaum, R.L.; Disotell, T.R. Primate Phylogenetic Relationships and Divergence Dates Inferred from Complete Mitochondrial Genomes. Mol. Phylogenetics Evol. 2014, 75, 165–183. [Google Scholar] [CrossRef]
- Liedigk, R.; Yang, M.; Jablonski, N.G.; Momberg, F.; Geissmann, T.; Lwin, N.; Hla, T.H.; Liu, Z.; Wong, B.; Ming, L.; et al. Evolutionary History of the Odd-Nosed Monkeys and the Phylogenetic Position of the Newly Described Myanmar Snub-Nosed Monkey Rhinopithecus strykeri. PLoS ONE 2012, 7, e37418. [Google Scholar] [CrossRef]
- Van Tuan, B.; Tam, N.A.; Vy, T.H.; Long, H.T.; Thao, N.T.T.; Phung, T.K.; Huy, H.Q.; Huan, P.M.; Nadler, T. Discovery of Isolated Populations of the ‘Critically Endangered’Grey-Shanked Douc Langur (Pygathrix cinerea) in Quang Nam Province, Vietnam. Vietnam. J. Primatol. 2019, 3, 19–25. [Google Scholar]
- Hoàng, T.M. Development of Primatology and Primate Conservation in Vietnam: Challenges and Prospects. Am. Anthropol. 2016, 118, 130–137. [Google Scholar] [CrossRef]
- Schwitzer, C.; Nash, S.D. Primates in Peril: The World’s 25 Most Endangered Primates 2012–2014; IUCN/SSC Primate Specialist Group (PSG): Gland, Switzerland; International Primatological Society (IPS): Gland, Switzerland; Conservation International (CI): Arlington County, VA, USA; Bristol Zoological Society (BZSP): Bristol, UK, 2014; ISBN 978-1-934151-69-3. [Google Scholar]
- Nadler, T. Status of Vietnamese Primates-Complements and Revisions. In Conservation of Primates in Indochina; Frankfurt Zoological Society and Conservation International: Frankfurt, Germany, 2010; pp. 3–16. [Google Scholar]
- Vu, T.T.; Tran, D.V.; Tran, H.T.P.; Nguyen, M.D.; Do, T.A.; Ta, N.T.; Cao, H.T.; Pham, N.T.; Phan, D.V. An Assessment of the Impact of Climate Change on the Distribution of the Grey-Shanked Douc Pygathrix cinerea Using an Ecological Niche Model. Primates 2020, 61, 267–275. [Google Scholar] [CrossRef] [PubMed]
- Nazeri, M.; Jusoff, K.; Madani, N.; Mahmud, A.R.; Bahman, A.R.; Kumar, L. Predictive Modeling and Mapping of Malayan Sun Bear (Helarctos malayanus) Distribution Using Maximum Entropy. PLoS ONE 2012, 7, e48104. [Google Scholar] [CrossRef]
- HH, C. The Earliest Fossil Primates and the Evolution of Prosimians: Introduction. In The Primate Fossil Record; Cambridge University Press: Cambrdige, UK, 2002. [Google Scholar]
- Silveira, L.C.L.; Saito, C.A.; Lee, B.B.; Kremers, J.; Da Silva Filho, M.; Kilavik, B.E.; Yamada, E.S.; Perry, V.H. Morphology and Physiology of Primate M- and P-Cells. In Progress in Brain Research; Elsevier: Amsterdam, The Netherlands, 2004; Volume 144, pp. 21–46. ISBN 978-0-444-50978-9. [Google Scholar]
- Shoshani, J.; Groves, C.P.; Simons, E.L.; Gunnell, G.F. Primate Phylogeny: Morphological vs Molecular Results. Mol. Phylogenetics Evol. 1996, 5, 102–154. [Google Scholar] [CrossRef]
- Tuttle, R.H. Primate Functional Morphology and Evolution; De Gruyter Mouton: Berlin, Germany, 1975; ISBN 978-90-279-7689-5. [Google Scholar]
- Curole, J.P.; Kocher, T.D. Mitogenomics: Digging Deeper with Complete Mitochondrial Genomes. Trends Ecol. Evol. 1999, 14, 394–398. [Google Scholar] [CrossRef]
- Lin, C.-P.; Danforth, B.N. How Do Insect Nuclear and Mitochondrial Gene Substitution Patterns Differ? Insights from Bayesian Analyses of Combined Datasets. Mol. Phylogenetics Evol. 2004, 30, 686–702. [Google Scholar] [CrossRef] [PubMed]
- Gissi, C.; Iannelli, F.; Pesole, G. Evolution of the Mitochondrial Genome of Metazoa as Exemplified by Comparison of Congeneric Species. Heredity 2008, 101, 301–320. [Google Scholar] [CrossRef] [PubMed]
- Cameron, S.L. Insect Mitochondrial Genomics: Implications for Evolution and Phylogeny. Annu. Rev. Entomol. 2014, 59, 95–117. [Google Scholar] [CrossRef] [PubMed]
- Zhao, L.; Zhang, X.; Tao, X.; Wang, W.; Li, M. Preliminary Analysis of the Mitochondrial Genome Evolutionary Pattern in Primates. Zool. Res. 2012, 33, E47–E56. [Google Scholar]
- Zardoya, R. Recent Advances in Understanding Mitochondrial Genome Diversity. F1000 Res. 2020, 9, 270. [Google Scholar] [CrossRef]
- Chen, S.; Zhou, Y.; Chen, Y.; Gu, J. Fastp: An Ultra-Fast All-in-One FASTQ Preprocessor. Bioinformatics 2018, 34, i884–i890. [Google Scholar] [CrossRef]
- Gurevich, A.; Saveliev, V.; Vyahhi, N.; Tesler, G. QUAST: Quality Assessment Tool for Genome Assemblies. Bioinformatics 2013, 29, 1072–1075. [Google Scholar] [CrossRef]
- Illumina, Inc. Quality Scores for Next-Generation Sequencing. In Technical Note: Sequencing; Illumina: San Diego, CA, USA, 2011; p. 31. [Google Scholar]
- Jin, J.-J.; Yu, W.-B.; Yang, J.-B.; Song, Y.; dePamphilis, C.W.; Yi, T.-S.; Li, D.-Z. GetOrganelle: A Fast and Versatile Toolkit for Accurate de Novo Assembly of Organelle Genomes. Genome Biol. 2020, 21, 241. [Google Scholar] [CrossRef]
- Bernt, M.; Donath, A.; Jühling, F.; Externbrink, F.; Florentz, C.; Fritzsch, G.; Pütz, J.; Middendorf, M.; Stadler, P.F. MITOS: Improved de Novo Metazoan Mitochondrial Genome Annotation. Mol. Phylogenetics Evol. 2013, 69, 313–319. [Google Scholar] [CrossRef]
- Iwasaki, W.; Fukunaga, T.; Isagozawa, R.; Yamada, K.; Maeda, Y.; Satoh, T.P.; Sado, T.; Mabuchi, K.; Takeshima, H.; Miya, M.; et al. MitoFish and MitoAnnotator: A Mitochondrial Genome Database of Fish with an Accurate and Automatic Annotation Pipeline. Mol. Biol. Evol. 2013, 30, 2531–2540. [Google Scholar] [CrossRef]
- Lowe, T.M.; Eddy, S.R. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Res. 1997, 25, 955–964. [Google Scholar] [CrossRef] [PubMed]
- Minh, B.Q.; Schmidt, H.A.; Chernomor, O.; Schrempf, D.; Woodhams, M.D.; Von Haeseler, A.; Lanfear, R. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 2020, 37, 1530–1534. [Google Scholar] [CrossRef]
- Kalyaanamoorthy, S.; Minh, B.Q.; Wong, T.K.F.; Von Haeseler, A.; Jermiin, L.S. ModelFinder: Fast Model Selection for Accurate Phylogenetic Estimates. Nat. Methods 2017, 14, 587–589. [Google Scholar] [CrossRef] [PubMed]
- Hoang, D.T.; Chernomor, O.; Von Haeseler, A.; Minh, B.Q.; Vinh, L.S. UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol. Biol. Evol. 2018, 35, 518–522. [Google Scholar] [CrossRef]
- Shadel, G.S.; Clayton, D.A. Mitochondrial Dna Maintenance In Vertebrates. Annu. Rev. Biochem. 1997, 66, 409–435. [Google Scholar] [CrossRef] [PubMed]
- Wang, Z.-L.; Li, C.; Fang, W.-Y.; Yu, X.-P. The Complete Mitochondrial Genome of Two Tetragnatha Spiders (Araneae: Tetragnathidae): Severe Truncation of tRNAs and Novel Gene Rearrangements in Araneae. Int. J. Biol. Sci. 2016, 12, 109–119. [Google Scholar] [CrossRef]
- Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
- Tamura, K.; Nei, M. Estimation of the Number of Nucleotide Substitutions in the Control Region of Mitochondrial DNA in Humans and Chimpanzees. Mol. Biol. Evol. 1993, 10, 512–526. [Google Scholar] [CrossRef]
- Mason, V.C.; Li, G.; Minx, P.; Schmitz, J.; Churakov, G.; Doronina, L.; Melin, A.D.; Dominy, N.J.; Lim, N.T.-L.; Springer, M.S.; et al. Genomic Analysis Reveals Hidden Biodiversity within Colugos, the Sister Group to Primates. Sci. Adv. 2016, 2, e1600633. [Google Scholar] [CrossRef]
- Avise, J.C. Phylogeography: The History and Formation of Species; Harvard University Press: Cambridge, MA, USA, 2000; ISBN 978-0-674-26870-8. [Google Scholar]
- Brown, W.M.; Prager, E.M.; Wang, A.; Wilson, A.C. Mitochondrial DNA Sequences of Primates: Tempo and Mode of Evolution. J. Mol. Evol. 1982, 18, 225–239. [Google Scholar] [CrossRef]
- Melnick, D.J.; Hoelzer, G.A. What Is mtDNA Good for in the Study of Primate Evolution? Evol. Anthropol. 1993, 2, 2–10. [Google Scholar] [CrossRef]
- Vilstrup, J.T.; Ho, S.Y.; Foote, A.D.; Morin, P.A.; Kreb, D.; Krützen, M.; Parra, G.J.; Robertson, K.M.; De Stephanis, R.; Verborgh, P.; et al. Mitogenomic Phylogenetic Analyses of the Delphinidae with an Emphasis on the Globicephalinae. BMC Evol. Biol. 2011, 11, 65. [Google Scholar] [CrossRef]
- Avise, J.C. Molecular Markers, Natural History and Evolution; Springer: Boston, MA, USA, 1994; ISBN 978-0-412-03781-8. [Google Scholar]
- Prado-Martinez, J.; Sudmant, P.H.; Kidd, J.M.; Li, H.; Kelley, J.L.; Lorente-Galdos, B.; Veeramah, K.R.; Woerner, A.E.; O’Connor, T.D.; Santpere, G. Great Ape Genetic Diversity and Population History. Nature 2013, 499, 471–475. [Google Scholar] [CrossRef]
- Moum, T.; Árnason, E. Genetic Diversity and Population History of Two Related Seabird Species Based on Mitochondrial DNA Control Region Sequences. Mol. Ecol. 2001, 10, 2463–2478. [Google Scholar] [CrossRef]
- Sigurðardóttir, S.; Helgason, A.; Gulcher, J.R.; Stefansson, K.; Donnelly, P. The Mutation Rate in the Human mtDNA Control Region. Am. J. Hum. Genet. 2000, 66, 1599–1609. [Google Scholar] [CrossRef]
- Cuppari, A.; Fernández-Millán, P.; Battistini, F.; Tarrés-Solé, A.; Lyonnais, S.; Iruela, G.; Ruiz-López, E.; Enciso, Y.; Rubio-Cosials, A.; Prohens, R.; et al. DNA Specificities Modulate the Binding of Human Transcription Factor A to Mitochondrial DNA Control Region. Nucleic Acids Res. 2019, 47, 6519–6537. [Google Scholar] [CrossRef] [PubMed]
- Brar, G.A.; Weissman, J.S. Ribosome Profiling Reveals the What, When, Where and How of Protein Synthesis. Nat. Rev. Mol. Cell Biol. 2015, 16, 651–664. [Google Scholar] [CrossRef] [PubMed]
- Yang, B.; Wang, Y.; Qian, P.-Y. Sensitivity and Correlation of Hypervariable Regions in 16S rRNA Genes in Phylogenetic Analysis. BMC Bioinform. 2016, 17, 135. [Google Scholar] [CrossRef] [PubMed]
- Kim, M.; Morrison, M.; Yu, Z. Evaluation of Different Partial 16S rRNA Gene Sequence Regions for Phylogenetic Analysis of Microbiomes. J. Microbiol. Methods 2011, 84, 81–87. [Google Scholar] [CrossRef]
- Sloan, K.E.; Warda, A.S.; Sharma, S.; Entian, K.-D.; Lafontaine, D.L.J.; Bohnsack, M.T. Tuning the Ribosome: The Influence of rRNA Modification on Eukaryotic Ribosome Biogenesis and Function. RNA Biol. 2017, 14, 1138–1152. [Google Scholar] [CrossRef]
- Seligmann, H.; Krishnan, N.M.; Rao, B.J. Possible Multiple Origins of Replication in Primate Mitochondria: Alternative Role of tRNA Sequences. J. Theor. Biol. 2006, 241, 321–332. [Google Scholar] [CrossRef] [PubMed]
- Sabath, N.; Graur, D. Detection of Functional Overlapping Genes: Simulation and Case Studies. J. Mol. Evol. 2010, 71, 308–316. [Google Scholar] [CrossRef] [PubMed]
- Yadav, M.L.; Mohapatra, B. Intergenic. In Encyclopedia of Animal Cognition and Behavior; Vonk, J., Shackelford, T., Eds.; Springer International Publishing: Cham, Switzerland, 2018; pp. 1–5. ISBN 978-3-319-47829-6. [Google Scholar]
- Sardana, R.; O’Dell, M.; Flavell, R. Correlation between the Size of the Intergenic Regulatory Region, the Status of Cytosine Methylation of rRNA Genes and Nucleolar Expression in Wheat. Mol. Gen. Genet. 1993, 236, 155–162. [Google Scholar] [CrossRef] [PubMed]
Figure 1.
Genome structure of the complete mitochondrial of Pygathrix cinerea. The circular mitochondrial genome is depicted, with genes represented by colored blocks. The control region is shown in grey, the 16S (large rRNA) and 12S (small rRNA) genes are shown in red, the 22 transfer RNA (tRNA) genes are labeled in dark blue, and the 13 protein-coding genes (PCGs) are shown in green and yellow. Genes located on the H strand are shown on the outer circle, while genes located on the L strand are shown on the inner circle.
Figure 1.
Genome structure of the complete mitochondrial of Pygathrix cinerea. The circular mitochondrial genome is depicted, with genes represented by colored blocks. The control region is shown in grey, the 16S (large rRNA) and 12S (small rRNA) genes are shown in red, the 22 transfer RNA (tRNA) genes are labeled in dark blue, and the 13 protein-coding genes (PCGs) are shown in green and yellow. Genes located on the H strand are shown on the outer circle, while genes located on the L strand are shown on the inner circle.
Figure 2.
Pygathrix cinerea mitochondrial protein-coding genes’ Relative Synonymous Codon Usage (RSCU).
Figure 2.
Pygathrix cinerea mitochondrial protein-coding genes’ Relative Synonymous Codon Usage (RSCU).
Figure 3.
Percentage frequency of amino acid composition in the whole mitogenome of Pygathrix species: Pygathrix cinerea PP623106, Pygathrix cinerea JQ821842, and Pygathrix nigripes MH064177.
Figure 3.
Percentage frequency of amino acid composition in the whole mitogenome of Pygathrix species: Pygathrix cinerea PP623106, Pygathrix cinerea JQ821842, and Pygathrix nigripes MH064177.
Figure 4.
Graphical representation of AT- and GC-skew in all 13 protein-coding genes of Pygathrix cinerea PP623106 mitogenome.
Figure 4.
Graphical representation of AT- and GC-skew in all 13 protein-coding genes of Pygathrix cinerea PP623106 mitogenome.
Figure 5.
Secondary structure of 22 tRNA genes of Vietnamese Highland Pygathrix cinerea. Red color is GC connection, blue color is AU connection.
Figure 5.
Secondary structure of 22 tRNA genes of Vietnamese Highland Pygathrix cinerea. Red color is GC connection, blue color is AU connection.
Figure 6.
Maximum likelihood phylogeny of 55 whole primate mitogenomes. The PP633106 Pygathrix cinerea in blue color is the Vietnamese Central Highland Pygathrix cinerea.
Figure 6.
Maximum likelihood phylogeny of 55 whole primate mitogenomes. The PP633106 Pygathrix cinerea in blue color is the Vietnamese Central Highland Pygathrix cinerea.
Table 1.
Location and length of 37 genes in the complete mitochondrial genome of Pygathrix cinerea.
| Name | Start | Stop | Strand | Length | Intergenic Nucleotides |
|---|---|---|---|---|---|
| trnF(ttc) | 137 | 207 | + | 71 | 0 |
| rrnS | 208 | 1156 | + | 949 | 0 |
| trnV(gta) | 1157 | 1222 | + | 66 | −2 |
| rrnL | 1221 | 2785 | + | 1565 | 0 |
| trnL2(tta) | 2786 | 2860 | + | 75 | 2 |
| nad1 | 2863 | 3813 | + | 951 | 4 |
| trnI(atc) | 3818 | 3887 | + | 70 | −3 |
| trnQ(caa) | 3885 | 3956 | - | 72 | 0 |
| trnM(atg) | 3957 | 4024 | + | 68 | 0 |
| nad2 | 4025 | 5062 | + | 1038 | 4 |
| trnW(tga) | 5067 | 5133 | + | 67 | 7 |
| trnA(gca) | 5141 | 5209 | - | 69 | 1 |
| trnN(aac) | 5211 | 5283 | - | 73 | 32 |
| trnC(tgc) | 5316 | 5381 | - | 66 | 0 |
| trnY(tac) | 5382 | 5446 | - | 65 | 1 |
| cox1 | 5448 | 6989 | + | 1542 | 0 |
| trnS2(tca) | 6990 | 7058 | - | 69 | 3 |
| trnD(gac) | 7062 | 7130 | + | 69 | 1 |
| cox2 | 7132 | 7791 | + | 660 | 68 |
| trnK(aaa) | 7860 | 7927 | + | 68 | 1 |
| atp8 | 7929 | 8111 | + | 183 | −22 |
| atp6 | 8090 | 8764 | + | 675 | 5 |
| cox3 | 8770 | 9552 | + | 783 | 1 |
| trnG(gga) | 9554 | 9620 | + | 67 | 0 |
| nad3 | 9621 | 9965 | + | 345 | 1 |
| trnR(cga) | 9967 | 10,031 | + | 65 | 0 |
| nad4l | 10,032 | 10,325 | + | 294 | −4 |
| nad4 | 10,322 | 11,689 | + | 1368 | 10 |
| trnH(cac) | 11,700 | 11,768 | + | 69 | 0 |
| trnS1(agc) | 11,769 | 11,827 | + | 59 | 0 |
| trnL1(cta) | 11,828 | 11,898 | + | 71 | 3 |
| nad5 | 11,902 | 13,701 | + | 1800 | 15 |
| nad6 | 13,717 | 14,235 | - | 519 | 0 |
| trnE(gaa) | 14,236 | 14,304 | - | 69 | 4 |
| Cob | 14,309 | 15,442 | + | 1134 | 7 |
| trnT(aca) | 15,450 | 15,514 | + | 65 | 2 |
| trnP(cca) | 15,517 | 15,583 | - | 67 |
Table 2.
Nucleotide composition indices in various regions of twenty-three representative primate mitogenomes.
Table 2.
Nucleotide composition indices in various regions of twenty-three representative primate mitogenomes.
| Accession Number | Whole | Protein-Coding Genes (PCGs) | Large Ribosomal RNA (rrnL) | Small Ribosomal RNA (rrnS) | |||||
|---|---|---|---|---|---|---|---|---|---|
| Length | AT (%) | Length (bp) | AT (%) | Length (bp) | AT (%) | Length (bp) | AT (%) | ||
| Vietnamese Central Highland Pygathrix cinerea | PP623106 | 16,541 | 61.1 | 11,292 | 61 | 1565 | 60 | 949 | 59.4 |
| Pygathrix cinerea | JQ821842 | 16,535 | 61.1 | 11,292 | 61 | 1562 | 59.9 | 948 | 59.4 |
| Pygathrix nemaeus | JF293096 | 15,467 | 61.3 | 11,292 | 61 | 1564 | 59.7 | 948 | 59.4 |
| Pygathrix nigripes | MH064177 | 16,534 | 61.4 | 11,304 | 62 | 1563 | 59.7 | 949 | 58.8 |
| Macaca leonina | KP330231 | 17,050 | 57.2 | 11,310 | 57 | 1562 | 57.8 | 947 | 54.6 |
| Presbytis femoralis | KU899140 | 16,548 | 61.9 | 11,277 | 62 | 1563 | 61 | 935 | 58.3 |
| Nasalis larvatus | DQ355298 | 16,570 | 60.9 | 11,298 | 61 | 1568 | 59.9 | 949 | 57.5 |
| Rhinopithecus roxellana | KM504390 | 16,552 | 61.5 | 11,292 | 62 | 722 | 59.6 | 949 | 58.2 |
| Rhinopithecus brelichi | JQ821836 | 16,553 | 61.6 | 11,295 | 62 | 1571 | 60 | 949 | 58.3 |
| Rhinopithecus bieti | JQ821839 | 16,550 | 61.5 | 11,298 | 62 | 1570 | 59.7 | 949 | 58 |
| Rhinopithecus avunculus | JF293093 | 16,552 | 61.6 | 11,289 | 62 | 1572 | 60.2 | 949 | 58.5 |
| Macaca arctoides | KM360179 | 16,559 | 56.7 | 11,313 | 56 | 1566 | 57.3 | 951 | 55.8 |
| Macaca mulatta | JQ821843 | 16,564 | 56.8 | 11,310 | 56 | 1560 | 57.4 | 947 | 55.6 |
| Papio hamadryas | NC001992 | 16,521 | 56.3 | 11,292 | 56 | 1572 | 57.7 | 947 | 55.1 |
| Callithrix jacchus | KM588314 | 16,499 | 59.7 | 11,280 | 59 | 1555 | 60.5 | 953 | 56.6 |
| Homo sapiens neanderthalensis | NC011137 | 16,565 | 55.6 | 11,283 | 55 | 1560 | 57.2 | 954 | 54.4 |
| Homo sapiens neanderthalens | OM062614 | 16,565 | 55.7 | 11,283 | 55 | 1560 | 57.3 | 954 | 54.4 |
| Cebus albifrons | NC002763 | 16,554 | 60.9 | 11,268 | 61 | 1556 | 60.2 | 958 | 57.6 |
| Moschiola indica | NC037993 | 16,444 | 61.4 | 11,310 | 61 | 1576 | 61.6 | 958 | 56.4 |
Table 3.
GC-skew and AT-skew in PCGs.
| Accession Number | T(U) | C | A | G | Total | AT-Skew | GC-Skew | |
|---|---|---|---|---|---|---|---|---|
| Vietnamese Central Highland Pygathrix cinerea | PP623106 | 30.9 | 26.1 | 30.5 | 12.5 | 868.6 | −0.00605 | −0.35278 |
| Pygathrix cinerea | JQ821842 | 30.9 | 26.1 | 30.5 | 12.5 | 868.6 | −0.0062 | −0.35277 |
| Pygathrix nemaeus | JF293096 | 30.9 | 26.1 | 30.5 | 12.5 | 868.6 | −0.00678 | −0.35155 |
| Pygathrix nigripes | MH064177 | 31.2 | 25.9 | 30.6 | 12.3 | 807.4 | −0.01002 | −0.35463 |
| Macaca leonina | KP330231 | 26.5 | 30.9 | 30.2 | 12.3 | 870 | 0.065732 | −0.42945 |
| Presbytis femoralis | KU899140 | 31.5 | 25.4 | 30.9 | 12.2 | 867.5 | −0.00896 | −0.34998 |
| Nasalis larvatus | DQ355298 | 30.5 | 26.7 | 30.6 | 12.2 | 869.1 | 0.001593 | −0.37082 |
| Rhinopithecus roxellana | KM504390 | 31.4 | 25.6 | 30.8 | 12.2 | 868.6 | −0.01054 | −0.35253 |
| Rhinopithecus brelichi | JQ821836 | 31.6 | 25.5 | 30.6 | 12.4 | 868.8 | −0.01638 | −0.34612 |
| Rhinopithecus bieti | JQ821839 | 31.5 | 25.6 | 30.7 | 12.2 | 869.1 | −0.01181 | −0.35612 |
| Rhinopithecus avunculus | JF293093 | 31.5 | 25.5 | 30.8 | 12.3 | 868.4 | −0.01138 | −0.34898 |
| Macaca arctoides | KM360179 | 26.4 | 31 | 30 | 12.5 | 870.2 | 0.063577 | −0.4244 |
| Macaca mulatta | JQ821843 | 26.6 | 31 | 29.8 | 12.6 | 870 | 0.056444 | −0.42052 |
| Papio hamadryas | NC001992 | 26.2 | 31.2 | 29.7 | 12.9 | 868.6 | 0.061768 | −0.41663 |
| Callithrix jacchus | KM588314 | 28.3 | 27.8 | 31.1 | 12.9 | 867.7 | 0.046296 | −0.36736 |
| Homo sapiens neanderthalensis | OM062614 | 26.2 | 31.9 | 28.8 | 13.1 | 867.8 | 0.046422 | −0.41631 |
| Cebus albifrons | NC002763 | 29.9 | 26.7 | 31.3 | 12.1 | 866.8 | 0.02306 | −0.37663 |
Table 4.
Different positions in control region of Vietnamese Central Highland Pygathrix cinerea, other Pygathrix cinerea specimens, and Pygathrix nigripes. The letters A, T, G, and C represent the four nucleotide bases in mtDNA: A (Adenine) T (Thymine) G (Guanine) C (Cytosine). The missing nucleotide base are represent in “.”.
Table 4.
Different positions in control region of Vietnamese Central Highland Pygathrix cinerea, other Pygathrix cinerea specimens, and Pygathrix nigripes. The letters A, T, G, and C represent the four nucleotide bases in mtDNA: A (Adenine) T (Thymine) G (Guanine) C (Cytosine). The missing nucleotide base are represent in “.”.
| Position | 1 | 1 | 1 | 1 | 1 | 1 | 2 | 2 | 2 | 2 | 2 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
| 4 | 5 | 6 | 6 | 8 | 8 | 5 | 5 | 8 | 9 | 9 | 1 | 0 | 1 | 2 | 2 | 3 | 3 | 3 | 3 | 3 | 4 | 4 | 5 | 7 | |
| 7 | 8 | 1 | 9 | 0 | 4 | 0 | 8 | 9 | 0 | 4 | 7 | 5 | 5 | 6 | 7 | 0 | 2 | 3 | 6 | 7 | 1 | 5 | 8 | 9 | |
| Pygathrix nigripes MH064177 | G | C | A | C | T | C | G | T | C | A | A | C | A | C | C | T | C | G | A | C | T | A | G | C | C |
| Pygathrix cinerea JQ821842 | A | T | G | T | C | T | A | C | T | G | G | A | G | T | T | C | A | A | G | T | A | G | A | T | T |
| Pygathrix cinerea PP623106 | A | T | G | T | C | T | A | C | T | G | G | A | G | T | T | C | A | A | G | T | A | G | A | T | T |
| Position | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 5 | 5 | 5 |
| 6 | 6 | 6 | 6 | 6 | 7 | 7 | 7 | 8 | 8 | 8 | 8 | 9 | 1 | 4 | 5 | 6 | 6 | 6 | 6 | 8 | 9 | 0 | 0 | 2 | |
| 2 | 3 | 5 | 6 | 8 | 3 | 5 | 6 | 1 | 2 | 4 | 9 | 0 | 6 | 8 | 1 | 3 | 4 | 7 | 9 | 3 | 0 | 6 | 9 | 8 | |
| Pygathrix nigripes MH064177 | A | G | T | A | C | T | T | A | T | C | G | C | C | T | T | T | T | A | A | T | T | C | A | T | G |
| Pygathrix cinerea JQ821842 | T | A | A | C | T | C | C | . | C | A | A | T | T | C | C | G | C | G | G | C | C | T | T | C | A |
| Vietnamese Central Highland Pygathrix cinerea PP623106 | C | A | A | T | . | C | C | G | C | A | A | T | T | C | C | G | C | G | G | C | C | T | T | C | . |
| Position | 1 | 1 | 1 | 1 | 1 | ||||||||||||||||||||
| 6 | 6 | 6 | 6 | 6 | 6 | 8 | 8 | 8 | 8 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 0 | 0 | 0 | 0 | 0 | ||
| 1 | 1 | 1 | 1 | 5 | 5 | 1 | 5 | 7 | 8 | 3 | 3 | 4 | 4 | 4 | 5 | 6 | 7 | 8 | 0 | 1 | 1 | 2 | 6 | ||
| 1 | 3 | 5 | 9 | 3 | 7 | 4 | 0 | 9 | 3 | 4 | 6 | 2 | 4 | 7 | 8 | 0 | 0 | 0 | 3 | 1 | 4 | 7 | 5 | ||
| Pygathrix nigripes MH064177 | T | T | T | A | G | T | A | T | C | C | T | C | C | T | A | C | A | T | A | T | C | C | T | A | |
| Pygathrix cinerea JQ821842 | C | . | C | G | A | C | G | C | T | . | C | T | T | A | G | T | G | C | G | C | T | . | A | G | |
| Vietnamese Central Highland Pygathrix cinerea PP623106 | C | C | C | G | A | C | G | C | T | T | C | T | T | A | G | T | G | C | . | C | T | T | A | G |
Table 5.
Pygathrix cinerea. The mismatched t-RNAs base pairs from Pygathrix cinerea. AA—amino acid acceptor, T-arm, AC—anticodon.
Table 5.
Pygathrix cinerea. The mismatched t-RNAs base pairs from Pygathrix cinerea. AA—amino acid acceptor, T-arm, AC—anticodon.
| tRNA | Mismatched Base Pairs | Stem | Frequency |
|---|---|---|---|
| Phenylalanine GAA | A-C | AC | 1 |
| Valine TAC | A-C | AC | 1 |
| Methionine CAT | A-G | AA | 1 |
| U-U | T-arm | 2 | |
| C-U | T-arm | 1 | |
| A-U | T-arm | 1 | |
| Glycine TCC | A-C | AC | 1 |
| Serine GCT | A-A | AA | 1 |
| A-A | AC | 1 | |
| Threonine TGT | U-C | AA | 1 |
Table 6.
Mutation of DNA in whole genome between Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea specimens. Dot “.” indicates nucleotide deletion; “-” indicates nucleotide insertion.
Table 6.
Mutation of DNA in whole genome between Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea specimens. Dot “.” indicates nucleotide deletion; “-” indicates nucleotide insertion.
| Location | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | ||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 1 | 2 | 2 | 2 | 3 | 3 | 3 | 3 | 4 | 4 | 4 | 4 | 5 | 5 | 5 | 5 | 6 | 7 | 7 | 8 | 0 | 0 | 2 | 3 | 3 | 3 | 4 | 4 | 4 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 6 | 6 | 6 | 6 | ||
| 2 | 2 | 7 | 5 | 7 | 7 | 0 | 1 | 2 | 9 | 4 | 6 | 8 | 9 | 0 | 4 | 5 | 6 | 6 | 0 | 5 | 9 | 3 | 5 | 8 | 1 | 9 | 9 | 7 | 7 | 9 | 0 | 3 | 5 | 8 | 8 | 8 | 8 | 9 | 0 | 3 | 4 | 4 | |
| 4 | 0 | 1 | 6 | 7 | 7 | 8 | 3 | 9 | 0 | 0 | 8 | 8 | 9 | 6 | 1 | 0 | 7 | 6 | 8 | 0 | 1 | 6 | 1 | 3 | 2 | 4 | 5 | 4 | 5 | 9 | 2 | 8 | 1 | 1 | 2 | 2 | 3 | 8 | 6 | 3 | 3 | 6 | |
| 2 | 6 | 1 | 6 | 2 | 3 | 0 | 4 | 6 | 6 | 7 | 9 | 7 | 7 | 4 | 4 | 7 | 8 | 3 | 3 | 9 | 7 | 0 | 9 | 7 | 5 | 2 | 7 | 9 | 9 | 8 | 7 | 2 | 1 | 6 | 0 | 2 | 0 | 2 | 7 | 7 | 4 | 8 | |
| Pygathrix cinerea NC 018063.1 | - | A | - | G | - | - | C | C | . | . | T | . | C | T | T | T | A | . | . | A | . | T | G | C | G | . | . | . | G | C | C | T | C | T | T | C | T | . | A | . | . | G | . |
| Vietnamese Central Highland Pygathrix cinerea PP623106 | T | . | A | . | C | A | . | . | G | T | . | C | . | . | . | . | . | C | G | A | T | . | . | . | . | G | T | T | . | . | . | . | . | T | C | T | . | G | . | C | T | . | T |
Table 7.
Mutation of protein in whole genome between Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea specimens. Dot “.” indicates nucleotide deletion; “-” indicates nucleotide insertion.
Table 7.
Mutation of protein in whole genome between Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea specimens. Dot “.” indicates nucleotide deletion; “-” indicates nucleotide insertion.
| Location | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 3 | 4 | 4 | 4 | 4 | 4 | 5 | 5 | 5 | 5 | 5 | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 5 | 8 | 9 | 9 | 0 | 0 | 0 | 6 | 8 | 8 | 8 | 5 | 3 | 6 | 6 | 9 | 9 | 0 | 3 | 3 | 4 | 4 | ||
| 8 | 7 | 5 | 2 | 2 | 2 | 4 | 9 | 6 | 0 | 3 | 9 | 0 | 7 | 4 | 5 | 1 | 2 | 0 | 2 | 5 | 4 | 9 | |
| 1 | 1 | 6 | 4 | 5 | 7 | 5 | 9 | 6 | 5 | 6 | 3 | 7 | 5 | 8 | 3 | 7 | 0 | 0 | 8 | 6 | 6 | 0 | |
| Pygathrix cinerea NC 018063.1 | - | - | D | - | - | T | S | . | I | I | Y | . | P | . | . | . | D | S | H | N | . | . | . |
| Vietnamese Central Highland Pygathrix cinerea PP623106 | L | I | . | H | N | . | . | R | . | . | . | S | . | W | Y | S | . | . | . | . | T | I | S |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
Nguyen, M.T.P.; Nguyen, T.T.T.; Ha, T.T.; Ho, C.N.Q.; Le, C.P.M.; Hoang, H.N.Q.; Nguyen, Q.T.N.; Nguyen, T.T.; Luu, D.T.; Dang, K.D.; et al. Characterization of the Complete Mitochondrial Genome of the Central Highland Grey-Shanked Douc Langur (Pygathrix cinerea), a Critically Endangered Species Endemic to Vietnam (Mammalia: Primates). Curr. Issues Mol. Biol. 2024, 46, 9928-9947. https://doi.org/10.3390/cimb46090592
AMA Style
Nguyen MTP, Nguyen TTT, Ha TT, Ho CNQ, Le CPM, Hoang HNQ, Nguyen QTN, Nguyen TT, Luu DT, Dang KD, et al. Characterization of the Complete Mitochondrial Genome of the Central Highland Grey-Shanked Douc Langur (Pygathrix cinerea), a Critically Endangered Species Endemic to Vietnam (Mammalia: Primates). Current Issues in Molecular Biology. 2024; 46(9):9928-9947. https://doi.org/10.3390/cimb46090592
Chicago/Turabian StyleNguyen, Mai Thi Phuong, Tram Thi Thuy Nguyen, Tung Thanh Ha, Chi Nguyen Quynh Ho, Cuong Phan Minh Le, Huy Nghia Quang Hoang, Quynh Thi Nhu Nguyen, Tao Thien Nguyen, Dung Tri Luu, Khoa Dang Dang, and et al. 2024. "Characterization of the Complete Mitochondrial Genome of the Central Highland Grey-Shanked Douc Langur (Pygathrix cinerea), a Critically Endangered Species Endemic to Vietnam (Mammalia: Primates)" Current Issues in Molecular Biology 46, no. 9: 9928-9947. https://doi.org/10.3390/cimb46090592
