Mitochondrial Protein-coding Genes Research Articles

The complete mitochondrial genome of the leopard shark Triakis semifasciata (Triakidae)

ABSTRACT The leopard shark Triakis semifasciata (family Triakidae) is threatened by habitat loss and targeted by recreational and commercial fishermen in the Eastern Pacific Ocean, from Oregon, USA, to the Gulf of California, Mexico. Despite environmental issues, there are few genetic and genomic resources available for this species. This study assembled the complete mitochondrial genome of T. semifasciata from a skin metagenomic sample and described it in detail. The phylogenetic position of T. semifasciata amongst closely related species was also examined using mitochondrial protein-coding genes (PCGs). The mitochondrial genome of T. semifasciata is 16,613 bp in length and consists of 13 PCGs, two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes. A 30 bp long region was identified as the origin of replication for the light strand (OL) between the trnN and trnC genes, and a 974 bp long putative control region (CR) contains the origin of replication for the heavy strand (OH). The gene order in T. semifasciata is identical to that of cofamilial species. An analysis of Ka/Ks ratios for all PCGs yielded values < 1, indicating that all PCGs experience strong purifying selection. All tRNAs exhibit a canonical ‘cloverleaf’ secondary structure except for tRNA-Ser1 which lacks the stem of the dihydrouridine (DHU) arm and in place possesses a simple loop. A phylomitogenomic maximum likelihood (ML) analysis did not support the monophyly of the family Trakidae and placed T. semifasciata in a sister position to Hemitriakis japanica. However, the aforementioned sister position was poorly supported by ML bootstrap values. This study represents a new genomic resource for this commercially and recreationally important species and confirms that mitochondrial genomes can be assembled from skin metagenomic samples as shown before in an unrelated shark species.

TRAP1 modulates mitochondrial biogenesis via PGC-1α/TFAM signalling pathway in colorectal cancer cells

Metabolic rewiring promotes cancer cell adaptation to a hostile microenvironment, representing a hallmark of cancer. This process involves mitochondrial function and is mechanistically linked to the balance between mitochondrial biogenesis (MB) and mitophagy. The molecular chaperone TRAP1 is overexpressed in 60–70% of human colorectal cancers (CRC) and its over-expression correlates with poor clinical outcome, being associated with many cancer cell functions (i.e. adaptation to stress, protection from apoptosis and drug resistance, protein synthesis quality control, metabolic rewiring from glycolysis to mitochondrial respiration and vice versa). Here, the potential new role of TRAP1 in regulating mitochondrial dynamics was investigated in CRC cell lines and human CRCs. Our results revealed an inverse correlation between TRAP1 and mitochondrial-encoded respiratory chain proteins both at transcriptional and translational levels. Furthermore, TRAP1 silencing is associated with increased mitochondrial mass and mitochondrial DNA copy number (mtDNA-CN) as well as enhanced MB through PGC-1α/TFAM signalling pathway, promoting the formation of new functioning mitochondria and, likely, underlying the metabolic shift towards oxidative phosphorylation. These results suggest an involvement of TRAP1 in regulating MB process in human CRC cells.Key messagesTRAP1 inversely correlates with protein-coding mitochondrial gene expression in CRC cells and tumours.TRAP1 silencing correlates with increased mitochondrial mass and mtDNA copy number in CRC cells.TRAP1 silencing favours mitochondrial biogenesis in CRC cells.

Influence of life-history traits on mitochondrial DNA substitution rates exceeds that of metabolic rates in teleost fishes

Abstract Understanding the molecular relevance of metabolic rate (MR) is crucial for unveiling the mechanisms driving the evolution of animals. In this study, we investigated the association between mitochondrial DNA characteristics and both resting and maximal MRs in conjunction with life-history traits among 139 species of teleost fish. We gathered fish MR data from various sources and procured sequences of 13 mitochondrial protein-encoding genes. We calculated the absolute substitution rate for entire nucleotide sequences and 4-fold degenerate sites of each gene, along with encoding amino acid sequences. Using the phylogenetic comparative method, we then explored the associations between MR and mitochondrial DNA absolute substitution rate. Additionally, we screened MR-associated single nucleotide variants in mitochondrial DNA. The findings indicate no positive correlation between MRs and any substitution rate values of both combined sequences and individual mitochondrial protein-coding genes, refuting the MR hypothesis. Instead, both maximum body size and longevity correlated negatively with molecular substitution rates, suggesting their influences on both mutation and fixation within mitochondrial genes in fish. Results also revealed significant correlations between base variation at ATP6_169 and both resting MR and maximum MR, identifying the unique ATP6_169G in Scombridae fish, which results in an extremely low isoelectric point (pI) value of the ATP6 protein. Considering its functional significance, the ATP6_169G in Scombridae fish might link to their lifestyle characterized by fast locomotion and high metabolic demands alongside a slower molecular evolutionary rate.

Evolutionary Insights from the Mitochondrial Genome of Oikopleura dioica: Sequencing Challenges, RNA Editing, Gene Transfers to the Nucleus, and tRNA Loss.

Sequencing the mitochondrial genome of the tunicate Oikopleura dioica is a challenging task due to the presence of long poly-A/T homopolymer stretches, which impair sequencing and assembly. Here, we report on the sequencing and annotation of the majority of the mitochondrial genome of O. dioica by means of combining several DNA and amplicon reads obtained by Illumina and MinIon Oxford Nanopore Technologies with public RNA sequences. We document extensive RNA editing, since all homopolymer stretches present in the mitochondrial DNA correspond to 6U-regions in the mitochondrial RNA. Out of the 13 canonical protein-coding genes, we were able to detect eight, plus an unassigned open reading frame that lacked sequence similarity to canonical mitochondrial protein-coding genes. We show that the nad3 gene has been transferred to the nucleus and acquired a mitochondria-targeting signal. In addition to two very short rRNAs, we could only identify a single tRNA (tRNA-Met), suggesting multiple losses of tRNA genes, supported by a corresponding loss of mitochondrial aminoacyl-tRNA synthetases in the nuclear genome. Based on the eight canonical protein-coding genes identified, we reconstructed maximum likelihood and Bayesian phylogenetic trees and inferred an extreme evolutionary rate of this mitochondrial genome. The phylogenetic position of appendicularians among tunicates, however, could not be accurately determined.

Analysis of the complete mitochondrial genome of Panax quinquefolius reveals shifts from cis-splicing to trans-splicing of intron cox2i373

Panax quinquefolius is a perennial plant with medicinal values. In this study, we assembled the complete mitochondrial genome (mitogenome) of P. quinquefolius using PMAT assembler. The total length of P. quinquefolius mitogenome is 573,154 bp. We annotated a total of 34 protein-coding genes (PCGs), 35 tRNA genes, and 6 rRNA genes in this mitogenome. The analysis of repetitive elements shows that there are 153 SSRs, 24 tandem repeats and 242 pairs of dispersed repeats this mitogenome. Also, we found 24 homologous sequences with a total length of 64,070 bp among its mitogenome and plastome, accounting for 41.05 % of the plastome, and 11.18 % of the mitogenome, showing a remarkable frequent sequence dialogue between plastome and mitogenomes. Besides, a total of 583 C to U RNA editing sites on 34 PCGs of high confidence were predicted by using Deepred-mt. We also inferred the phylogenetic relationships of P. quinquefolius and other angiosperms based on mitochondrial PCGs. Finally, we observed a shift from cis- to trans-splicing in P. quinquefolius for two mitochondrial introns, namely cox2i373 and nad1i728, and a pair of 48 bp short repetitive sequences may be associated with the breaking and rearrangement of the cox2i373 intron. The fragmentation of the cox2i373 intron was further confirmed by our PCR amplification experiments. In summary, our report on the P. quinquefolius mitogenome provides a new perspective on the intron evolution of the mitogenome.

The de novo assembly and characterization of the complete mitochondrial genome of bottle gourd (Lagenaria siceraria) reveals the presence of homologous conformations produced by repeat-mediated recombination.

Bottle gourd is an annual herbaceous plant that not only has high nutritional value and many medicinal applications but is also used as a rootstock for the grafting of cucurbit crops such as watermelon, cucumber and melon. Organellar genomes provide valuable resources for genetic breeding. A hybrid strategy with Illumina and Oxford Nanopore Technology sequencing data was used to assemble bottle gourd mitochondrial and chloroplast genomes. The length of the bottle gourd mitochondrial genome was 357547 bp, and that of the chloroplast genome was 157121 bp. These genomes had 27 homologous fragments, accounting for 6.50% of the total length of the bottle gourd mitochondrial genome. In the mitochondrial genome, 101 simple sequence repeats (SSRs) and 10 tandem repeats were identified. Moreover, 1 pair of repeats was shown to mediate homologous recombination into 1 major conformation and 1 minor conformation. The existence of these conformations was verified via PCR amplification and Sanger sequencing. Evolutionary analysis revealed that the mitochondrial genome sequence of bottle gourd was highly conserved. Furthermore, collinearity analysis revealed many rearrangements between the homologous fragments of Cucurbita and its relatives. The Ka/Ks values for most genes were between 0.3~0.9, which means that most of the genes in the bottle gourd mitochondrial genome are under purifying selection. We also identified a total of 589 potential RNA editing sites on 38 mitochondrial protein-coding genes (PCGs) on the basis of long noncoding RNA (lncRNA)-seq data. The RNA editing sites of nad1-2, nad4L-2, atp6-718, atp9-223 and rps10-391 were successfully verified via PCR amplification and Sanger sequencing. In conclusion, we assembled and annotated bottle gourd mitochondrial and chloroplast genomes to provide a theoretical basis for similar organelle genomic studies.

Two complete mitochondrial genomes of Boulenophrys (Anura: Megophryidae: Megophryinae): characteristics and phylogenetic implications

The Chinese horned toads, Boulenophrys boettgeri (Boulenger, 1899) and Boulenophrys kuatunensis (Pope, 1929), are two captivating species within the family Megophryidae, which inhabit the mountainous streams in the Eastern of China. In this study, two new complete mitochondrial genomes of B. boettgeri and B. kuatunensis were sequenced, assembled, and annotated using next-generation sequencing. The length of mitochondrial genomes of B. boettgeri and B. kuatunensis was 16,597 and 17,921 bp, respectively, with both containing 13 protein coding genes, 22 tRNA genes, two rRNA genes, and one putative control region. Phylogenetic relationships based on protein-coding mitochondrial genes showed that the two Boulenophrys species formed a cluster with other Boulenophrys species. The two new sequences provide valuable insights into the mitochondrial genomes of these two species, offering important data for understanding the phylogenetic relationships of the genus Boulenophrys.

The Phylogenetic Relationships of Major Lizard Families Using Mitochondrial Genomes and Selection Pressure Analyses in Anguimorpha.

Anguimorpha, within the order Squamata, represents a group with distinct morphological and behavioral characteristics in different ecological niches among lizards. Within Anguimorpha, there is a group characterized by limb loss, occupying lower ecological niches, concentrated within the subfamily Anguinae. Lizards with limbs and those without exhibit distinct locomotor abilities when adapting to their habitats, which in turn necessitate varying degrees of energy expenditure. Mitochondria, known as the metabolic powerhouses of cells, play a crucial role in providing approximately 95% of an organism's energy. Functionally, mitogenomes (mitochondrial genomes) can serve as a valuable tool for investigating potential adaptive evolutionary selection behind limb loss in reptiles. Due to the variation of mitogenome structures among each species, as well as its simple genetic structure, maternal inheritance, and high evolutionary rate, the mitogenome is increasingly utilized to reconstruct phylogenetic relationships of squamate animals. In this study, we sequenced the mitogenomes of two species within Anguimorpha as well as the mitogenomes of two species in Gekkota and four species in Scincoidea. We compared these data with the mitogenome content and evolutionary history of related species. Within Anguimorpha, between the mitogenomes of limbless and limbed lizards, a branch-site model analysis supported the presence of 10 positively selected sites: Cytb protein (at sites 183 and 187), ND2 protein (at sites 90, 155, and 198), ND3 protein (at site 21), ND5 protein (at sites 12 and 267), and ND6 protein (at sites 72 and 119). These findings suggested that positive selection of mitogenome in limbless lizards may be associated with the energy requirements for their locomotion. Additionally, we acquired data from 205 mitogenomes from the NCBI database. Bayesian inference (BI) and Maximum Likelihood (ML) trees were constructed using the 13 mitochondrial protein-coding genes (PCGs) and two rRNAs (12S rRNA and 16S rRNA) from 213 mitogenomes. Our phylogenetic tree and the divergence time estimates for Squamata based on mitogenome data are consistent with results from previous studies. Gekkota was placed at the root of Squamata in both BI and ML trees. However, within the Toxicofera clade, due to long-branch attraction, Anguimorpha and (Pleurodonta + (Serpentes + Acrodonta)) were closely related groupings, which might indicate errors and also demonstrate that mitogenome-based phylogenetic trees may not effectively resolve long-branch attraction issues. Additionally, we reviewed the origin and diversification of Squamata throughout the Mesozoic era, suggesting that Squamata originated in the Late Triassic (206.05 Mya), with the diversification of various superfamilies occurring during the Cretaceous period. Future improvements in constructing squamate phylogenetic relationships using mitogenomes will rely on identifying snake and acrodont species with slower evolutionary rates, ensuring comprehensive taxonomic coverage of squamate diversity, and increasing the number of genes analyzed.

Comparative analysis of the mitochondrial genomes of four Dendrobium species (Orchidaceae) reveals heterogeneity in structure, synteny, intercellular gene transfer, and RNA editing.

The genus Dendrobium, part of the Orchidaceae family, encompasses species of significant medicinal, nutritional, and economic value. However, many Dendrobium species are threatened by environmental stresses, low seed germination rates, and overharvesting. Mitochondria generate the energy necessary for various plant life activities. Despite their importance, research on the mitochondrial genomes of Dendrobium species is currently limited. To address this gap, we performed a comprehensive genetic analysis of four Dendrobium species-D. flexicaule, D. nobile, D. officinale, and D. huoshanense-focusing on their mitochondrial and chloroplast genomes to elucidate their genetic architecture and support conservation efforts. We utilized advanced sequencing technologies, including Illumina for high-throughput sequencing and Nanopore for long-read sequencing capabilities. Our findings revealed the multichromosomal mitochondrial genome structures, with total lengths ranging from 596,506 bp to 772,523 bp. The mitochondrial genomes contained 265 functional genes, including 64-69 protein-coding genes, 23-28 tRNA genes, and 3 rRNA genes. We identified 647 simple sequence repeats (SSRs) and 352 tandem repeats, along with 440 instances of plastid-to-mitochondrial gene transfer. Additionally, we predicted 2,023 RNA editing sites within the mitochondrial protein-coding genes, predominantly characterized by cytosine-to-thymine transitions. Comparative analysis of mitochondrial DNA across the species highlighted 25 conserved genes, with evidence of positive selection in five genes: ccmFC, matR, mttB, rps2, and rps10. Phylogenetic assessments suggested a close sister relationship between D. nobile and D. huoshanense, and a similar proximity between D. officinale and D. flexicaule. This comprehensive genomic study provides a critical foundation for further exploration into the genetic mechanisms and biodiversity of Dendrobium species, contributing valuable insights for their conservation and sustainable utilization.

Comparative Analysis of the Mitochondrial Genomes of Three Species of Yangiella (Hemiptera: Aradidae) and the Phylogenetic Implications of Aradidae.

The mitochondrial genomes of three species of Yangiella were sequenced, annotated, and analyzed. The genome length of the three species of the genus is 15,070-15,202 bp, with a typical gene number, including a control region, 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and 13 protein-coding genes (PCGs). It was found that the mitochondrial genome of Yangiella had AT bias. Except for the lack of a DHU arm of the trnS1 gene, the other tRNAs had a typical cloverleaf structure, and the codon usage preferences of the three species exhibited high similarity. In addition, tRNA gene rearrangements were observed among the three subfamilies of Aradidae (Mezirinae, Calisiinae, Aradinae), and it was found that codon usage preferences appeared to be less affected by base mutation and more by natural selection. The Pi and Ka/Ks values indicated that cox1 was the most conserved gene in the mitochondrial genome of Aradidae, while atp8 and nad6 were rapidly evolved genes. Substitution saturation level analysis showed that the nucleic acid sequence of mitochondrial protein-coding genes in Aradidae did not reach saturation, suggesting the rationality of the phylogenetic analysis data. Bayesian and maximum likelihood methods were used to analyze the phylogeny of 16 species of Hemiptera insects, which supported the monophyly of Aneurinae, Carventinae, and Mezirinae, as well as the monophyly of Yangiella. Based on fossils and previous studies, the differentiation time was inferred, indicating that Yangiella diverged about 57 million years ago.

Mitochondrial genome of the fluke pond snail, Austropeplea cf. brazieri (Gastropoda: Lymnaeidae)

BackgroundLymnaeid snails of the genus Austropeplea are an important vector of the liver fluke (Fasciola hepatica), contributing to livestock production losses in Australia and New Zealand. However, the species status within Austropeplea is ambiguous due to heavy reliance on morphological analysis and a relative lack of genetic data. This study aimed to characterise the mitochondrial genome of A. cf. brazieri, an intermediate host of liver fluke in eastern Victoria.MethodsThe mitochondrial genome was assembled and annotated from a combination of second- and third-generation sequencing data. For comparative purposes, we performed phylogenetic analyses of the concatenated nucleotide sequences of the mitochondrial protein-coding genes, cytochrome c oxidase subunit 1 and 16S genes.ResultsThe assembled mt genome was 13,757 base pairs and comprised 37 genes, including 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The mt genome length, gene order and nucleotide compositions were similar to related species of lymnaeids. Phylogenetic analyses of the mt nucleotide sequences placed A. cf. brazieri within the same clade as Orientogalba ollula with strong statistical supports. Phylogenies of the cox1 and 16S mt sequences were constructed due to the wide availability of these sequences representing the lymnaeid taxa. As expected in both these phylogenies, A. cf. brazieri clustered with other Austropeplea sequences, but the nodal supports were low.ConclusionsThe representative mt genome of A. cf. brazieri should provide a useful resource for future molecular, epidemiology and parasitological studies of this socio-economically important lymnaeid species.Graphical abstract

Molecular phylogeny reveals Varroa mites are not a separate family but a subfamily of Laelapidae

Varroa mites, notorious for parasitizing honeybees, are generally classified as Varroidae. Their extremely modified morphologies and behaviors have led to debates regarding their phylogenetic position and classification as an independent family. In this study, two different datasets were employed to reconstruct the phylogenies of Varroa mites and related Laelapidae species: (1) 9257 bp from the whole 13 mitochondrial protein-coding genes of 24 taxa, (2) 3158 bp from 113 taxa using Sanger sequencing of four nuclear loci. Both mitochondrial and nuclear analyses consistently place Varroa mites within the Laelapidae. Here we propose to place Varroa mites in the subfamily Varroinae stat. nov., which represents a highly morphologically adapted group within the Laelapidae. Ancestral state reconstructions reveal that bee-associated lifestyles evolved independently at least three times within Laelapidae, with most phoretic traits originating from free-living ancestors. Our revised classification and evolutionary analyses will provide new insight into understanding the Varroa mites.

Comparative mitogenomic analysis provides evolutionary insights into Formica (Hymenoptera: Formicidae).

Formica is a large genus in the family Formicidae with high diversity in its distribution, morphology, and physiology. To better understand evolutionary characteristics of Formica, the complete mitochondrial genomes (mitogenomes) of two Formica species were determined and a comparative mitogenomic analysis for this genus was performed. The two newly sequenced Formica mitogenomes each included 37 typical mitochondrial genes and a large non-coding region (putative control region), as observed in other Formica mitogenomes. Base composition, gene order, codon usage, and tRNA secondary structure were well conserved among Formica species, whereas diversity in sequence size and structural characteristics was observed in control regions. We also observed several conserved motifs in the intergenic spacer regions. These conserved genomic features may be related to mitochondrial function and their highly conserved physiological constraints, while the diversity of the control regions may be associated with adaptive evolution among heterogenous habitats. A negative AT-skew value on the majority chain was presented in each of Formica mitogenomes, indicating a reversal of strand asymmetry in base composition. Strong codon usage bias was observed in Formica mitogenomes, which was predominantly determined by nucleotide composition. All 13 mitochondrial protein-coding genes of Formica species exhibited molecular signatures of purifying selection, as indicated by the ratio of non-synonymous substitutions to synonymous substitutions being less than 1 for each protein-coding gene. Phylogenetic analyses based on mitogenomic data obtained fairly consistent phylogenetic relationships, except for two Formica species that had unstable phylogenetic positions, indicating mitogenomic data are useful for constructing phylogenies of ants. Beyond characterizing two additional Formica mitogenomes, this study also provided some key evolutionary insights into Formica.

Assessment of fish diversity in the East China Sea hairtail national aquatic germplasm resources conservation zone using DNA barcoding

Based on the fishery resource survey data from 2019 to 2022, the species composition and taxonomic diversity of fish in the East China Sea hairtail national aquatic germplasm resources conservation zone (ECSHZ) were studied through the analysis of 185 COI sequences. The results showed that the 185 fish samples collected belonged to 105 species, 21 orders, 46 families, and 89 genera in terms of their taxonomic status. There are discrepancies between morphological identification and genetic identification results for four fish species. The intraspecific genetic distance ranged from 0 % to 1.0 %, with an average value of 0.3 %. Meanwhile, the interspecies within genus distance ranged from 5.3 % to 22.7 %, with an average of 14.5 %. The outcomes of the division using Automatic Barcoding Gap Discovery (ABGD) and Bayesian phylogenetics and phylogeography (BPP) were concordant, identifying a total of 105 species groups. ABGD and BPP have shown that there was no overlap between intra-species variation and inter-species variation, supporting the existence of a barcoding gap. The results demonstrate the superiority of DNA barcoding in species identification. A phylogenetic tree was constructed using the maximum likelihood method based on the COI gene, which revealed certain limitations of COI in representing phylogenetic relationships at the genus and higher taxonomic levels. Another phylogenetic tree was constructed using 13 mitochondrial protein-coding genes with more genetic information, allowing for a better study of evolutionary relationships of ECSHZ fish species. The tree supported that Carangidae, Polynemidae, Paralichthyidae, Pleuronectidae, Sphyraenidae, and Cynoglossidae were clustered in an independent clade, with Priacanthidae and Lobotidae separating from Perciformes. This study offers DNA barcodes for ECSHZ fish species, providing crucial data support for conserving fish diversity.

RNA degradation in human mitochondria: the journey is not finished.

Mitochondria are vital organelles present in almost all eukaryotic cells. Although most of the mitochondrial proteins are nuclear-encoded, mitochondria contain their own genome, whose proper expression is necessary for mitochondrial function. Transcription of the human mitochondrial genome results in the synthesis of long polycistronic transcripts that are subsequently processed by endonucleases to release individual RNA molecules, including precursors of sense protein-encoding mRNA (mt-mRNA) and a vast amount of antisense noncoding RNAs. Because of mitochondrial DNA (mtDNA) organization, the regulation of individual gene expression at the transcriptional level is limited. Although transcription of most protein-coding mitochondrial genes occurs with the same frequency, steady-state levels of mature transcripts are different. Therefore, post-transcriptional processes are important for regulating mt-mRNA levels. The mitochondrial degradosome is a complex composed of the RNA helicase SUV3 (also known as SUPV3L1) and polynucleotide phosphorylase (PNPase, PNPT1). It is the best-characterized RNA-degrading machinery in human mitochondria, which is primarily responsible for the decay of mitochondrial antisense RNA. The mechanism of mitochondrial sense RNA decay is less understood. This review aims to provide a general picture of mitochondrial genome expression, with a particular focus on mitochondrial RNA (mtRNA) degradation.

De Novo Assembly and Comparative Analysis of Mitochondrial Genomes of Two Pueraria montana Varieties.

Pueraria montana is a species with important medicinal value and a complex genetic background. In this study, we sequenced and assembled the mitochondrial (mt) genomes of two varieties of P. montana. The mt genome lengths of P. montana var. thomsonii and P. montana var. montana were 457,390 bp and 456,731 bp, respectively. Both P. montana mitogenomes showed a multi-branched structure consisting of two circular molecules, with 56 genes annotated, comprising 33 protein-coding genes, 18 tRNA genes (trnC-GCA and trnM-CAU are multi-copy genes), and 3 rRNA genes. Then, 207 pairs of long repeats and 96 simple sequence repeats (SSRs) were detected in the mt genomes of P. montana, and 484 potential RNA-editing sites were found across the 33 mitochondrial protein-coding genes of each variety. Additionally, a syntenic sequence analysis showed a high collinearity between the two mt genomes. This work is the first to analyze the mt genomes of P. montana. It can provide information that can be used to analyze the structure of mt genomes of higher plants and provide a foundation for future comparative genomic studies and evolutionary biology research in related species.

Genomic analysis and phylogenetic characterization of Himalayan snow trout, Schizothorax esocinus based on mitochondrial protein-coding genes.

Mitochondrial DNA (mtDNA) has become a significant tool for exploring genetic diversity and delineating evolutionary links across diverse taxa. Within the group of cold-water fish species that are native to the Indian Himalayan region, Schizothorax esocinus holds particular importance due to its ecological significance and is potentially vulnerable to environmental changes. This research aims to clarify the phylogenetic relationships within the Schizothorax genus by utilizing mitochondrial protein-coding genes. Standard protocols were followed for the isolation of DNA from S. esocinus. For the amplification of mtDNA, overlapping primers were used, and then subsequent sequencing was performed. The genetic features were investigated by the application of bioinformatic approaches. These approaches covered the evaluation of nucleotide composition, codon usage, selective pressure using nonsynonymous substitution /synonymous substitution (Ka/Ks) ratios, and phylogenetic analysis. The study specifically examined the 13 protein-coding genes of Schizothorax species which belongs to the Schizothoracinae subfamily. Nucleotide composition analysis showed a bias towards A + T content, consistent with other cyprinid fish species, suggesting evolutionary conservation. Relative Synonymous Codon Usage highlighted leucine as the most frequent (5.18%) and cysteine as the least frequent (0.78%) codon. The positive AT-skew and the predominantly negative GC-skew indicated the abundance of A and C. Comparative analysis revealed significant conservation of amino acids in multiple genes. The majority of amino acids were hydrophobic rather than polar. The purifying selection was revealed by the genetic distance and Ka/Ks ratios. Phylogenetic study revealed a significant genetic divergence between S. esocinus and other Schizothorax species with interspecific K2P distances ranging from 0.00 to 8.87%, with an average of 5.76%. The present study provides significant contributions to the understanding of mitochondrial genome diversity and genetic evolution mechanisms in Schizothoracinae, hence offering vital insights for the development of conservation initiatives aimed at protecting freshwater fish species.

BIOINFORMATICS AND MOLECULAR ANALYSES IDENTIFIED THE CONTROL REGION AS THE MOST POWERFUL MITOGENOMIC MARKER FOR DISTINGUISHING THE MAIN MATERNAL HAPLOGROUPS IN GOATS

Identification of genetic markers to distinguish animals within and between species demands extensive genomic and bioinformatics investigation. Previous studies have not carefully taken into consideration the effect of mitogenomic components on the genetic differentiation of the maternal lineages in goats. As a precaution, the complete goat mitogenome was downloaded from the NCBI database and used in the current study to assess the effects of the choice of mitogenomic fragments on phylogenetic studies and to identify any potential polymorphic region by which the main maternal haplogroups of goats can be classified. Phylogenetic results confirmed that all 13 individual mitochondrial protein-coding genes and 2 ribosomal genes are not applicable to differentiate the maternal lineages. Instead, a single novel polymorphic region with a length of 756 bp within the control region was successfully amplified by newly designed primers. Both phylogenetic analysis and principal components analysis of the sequenced mitogenomic region of the mtDNA control region efficiently differentiated the main maternal haplogroups in goats. Higher numbers of polymorphic sites were found in the control region and the mitogenomic marker region. Highly significant correlations were discovered between the polymorphic sites and the length of each individual mitogenomic component. Our results demonstrate useful guidance and cautionary notes for researchers who are interested in the investigation of genetic diversity in animal species using mtDNA sequences. The bioinformatics and molecular methods used herein can be powerful in selecting a minimum amount of data using PCR amplification when the entire sequences of the mitogenome are unavailable.

Different Evolutionary Trends of Galloanseres: Mitogenomics Analysis.

The two existing clades of Galloanseres, orders Galliformes (landfowl) and Anseriformes (waterfowl), exhibit dramatically different evolutionary trends. Mitochondria serve as primary sites for energy production in organisms, and numerous studies have revealed their role in biological evolution and ecological adaptation. We assembled the complete mitogenome sequences of two species of the genus Aythya within Anseriformes: Aythya baeri and Aythya marila. A phylogenetic tree was constructed for 142 species within Galloanseres, and their divergence times were inferred. The divergence between Galliformes and Anseriformes occurred ~79.62 million years ago (Mya), followed by rapid evolution and diversification after the Middle Miocene (~13.82 Mya). The analysis of selective pressure indicated that the mitochondrial protein-coding genes (PCGs) of Galloanseres species have predominantly undergone purifying selection. The free-ratio model revealed that the evolutionary rates of COX1 and COX3 were lower than those of the other PCGs, whereas ND2 and ND6 had faster evolutionary rates. The CmC model also indicated that most PCGs in Anseriformes exhibited stronger selective constraints. Our study suggests that the distinct evolutionary trends and energy requirements of Galliformes and Anseriformes drive different evolutionary patterns in the mitogenome.

Mitochondrial Genomic Evidence of Selective Constraints in Small-Bodied Terrestrial Cetartiodactyla.

Body size may drive the molecular evolution of mitochondrial genes in response to changes in energy requirements across species of different sizes. In this study, we perform selection pressure analysis and phylogenetic independent contrasts (PIC) to investigate the association between molecular evolution of mitochondrial genome protein-coding genes (mtDNA PCGs) and body size in terrestrial Cetartiodactyla. Employing selection pressure analysis, we observe that the average non-synonymous/synonymous substitution rate ratio (ω) of mtDNA PCGs is significantly reduced in small-bodied species relative to their medium and large counterparts. PIC analysis further confirms that ω values are positively correlated with body size (R2 = 0.162, p = 0.0016). Our results suggest that mtDNA PCGs of small-bodied species experience much stronger purifying selection as they need to maintain a heightened metabolic rate. On the other hand, larger-bodied species may face less stringent selective pressures on their mtDNA PCGs, potentially due to reduced relative energy expenditure per unit mass. Furthermore, we identify several genes that undergo positive selection, possibly linked to species adaptation to specific environments. Therefore, despite purifying selection being the predominant force in the evolution of mtDNA PCGs, positive selection can also occur during the process of adaptive evolution.