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Unequally Abundant Chromosomes and Unusual Collections of Transferred Sequences Characterize Mitochondrial Genomes of Gastrodia (Orchidaceae), One of the Largest Mycoheterotrophic Plant Genera.

The mystery of genomic alternations in heterotrophic plants is among the most intriguing in evolutionary biology. Compared to plastid genomes (plastomes) with parallel size reduction and gene loss, mitochondrial genome (mitogenome) variation in heterotrophic plants remains underexplored in many aspects. To further unravel the evolutionary outcomes of heterotrophy, we present a comparative mitogenomic study with 13 de novo assemblies of Gastrodia (Orchidaceae), one of the largest fully mycoheterotrophic plant genera, and its relatives. Analyzed Gastrodia mitogenomes range from 0.56 to 2.1 Mb, each consisting of numerous, unequally abundant chromosomes or contigs. Size variation might have evolved through chromosome rearrangements followed by stochastic loss of "dispensable" chromosomes, with deletion-biased mutations. The discovery of a hyper-abundant (∼15 times intragenomic average) chromosome in two assemblies represents the hitherto most extreme copy number variation in any mitogenomes, with similar architectures discovered in two metazoan lineages. Transferred sequence contents highlight asymmetric evolutionary consequences of heterotrophy: despite drastically reduced intracellular plastome transfers convergent across heterotrophic plants, their rarity of horizontally acquired sequences sharply contrasts parasitic plants, where massive transfers from their hosts prevail. Rates of sequence evolution are markedly elevated but not explained by copy number variation, extending prior findings of accelerated molecular evolution from parasitic to heterotrophic plants. Putative evolutionary scenarios for these mitogenomic convergence and divergence fit well with the common (e.g. plastome contraction) and specific (e.g. host identity) aspects of the two heterotrophic types. These idiosyncratic mycoheterotrophs expand known architectural variability of plant mitogenomes and provide mechanistic insights into their content and size variation.

Genome, Mitochondrial

The large mitochondrial genome of Syndiclis anlungensis (Lauraceae): Genome structure, comparative analysis, and phylogenetic relationships among Syndiclis species.

The complete mitochondrial genome (mitogenome) of Syndiclis anlungensis, a critically endangered tropical tree, was determined in this study. The mitogenome spans 2,368,454&#xa0;bp across four contigs and harbors 41 protein-coding genes, 22 tRNA genes, and three rRNA genes. Potential mutation regions, including 1317 repeat sequences and 698 simple sequence repeats (SSRs), were accurately located in the S. anlungensis mitogenome. Sixty-five transferred fragments of the repeats were found between its mitochondrial and chloroplast genomes. When compared to three other Laurales mitogenomes, extensive gene order shuffling is evident, leaving only five conserved gene clusters intact. Codon usage analysis reveals a pronounced A/T bias in both mitochondrial and chloroplast genes, and three mitochondrial genes (atp9, rps19, and sdh3) stand out for their high divergence across eleven Syndiclis taxa. Selection analyses indicate strong purifying pressure on rpl2, rpl16, and sdh3 (Ka/Ks&#xa0;<&#xa0;1), with no positive selection detected. Using 41 mitochondrial protein-coding gene sequences from sixteen and three individuals of Syndiclis and Beilschmiedia species, respectively, our phylogenetic tree recovers Syndiclis as monophyletic, with two well-supported clades: one includes S. anlungensis, S. chinensis, S. lotungensis, S. marlipoensis, and a putative new Syndiclis species from Yunnan; the other contains S. furfuracea, S. hongkongensis, S. kwangsiensis, and three putative new Syndiclis species from Guangdong and Vietnam.

Genome, Mitochondrial

Mitochondrial DNA diversity in Ecuadorian populations: Recurrence of variant 16136 within haplogroup B2.

The identification of lineage-defining variants, frequently found in the coding region of mitochondrial DNA (mtDNA), is essential for refining haplogroup classification. Most mtDNA studies in South American populations have focused on the control region (CR), which has provided important insights into population structure and maternal lineage origins, although information needed for more robust phylogenetic resolution has been neglected. This study investigates the maternal genetic structure of Ecuadorian populations by combining CR and whole mitogenome analyses. Sequences from the mtDNA CR were obtained from 461 individuals (253 Mestizos and 208 Native Americans), while complete mitogenomes were sequenced for 127 individuals to improve phylogenetic resolution by identifying lineage-defining variants present in coding region. Most mtDNA haplogroups in the two population groups analyzed were of Native American origin (A2, B2, B4, C1, D1, D4), with significant differences in the distribution of specific lineages between them. Among Mestizos, African haplogroups (all within the L branches) and Eurasian haplogroups (H, K, R, U) were detected at low frequencies, whereas no African lineages were observed among Native Americans. The results obtained highlighted a heterogeneity within Ecuadorian populations that must be considered when developing mtDNA haplotype databases for forensic purposes. Whole mitogenome sequences enabled the identification of variants that refined haplogroup classifications, provided a more accurate reconstruction of the maternal genetic diversity, and improve the discrimination between Native American and Asian maternal lineages within haplogroup B4b.

Humans

The complete and annotated mitochondrial genome of Hemileia vastatrix Race I, causal agent of coffee leaf rust.

Hemileia vastatrix is the fungal pathogen responsible for coffee leaf rust (CLR), the most economically important disease of Coffea arabica worldwide. Recently, the nuclear genome of this fungus was completely deciphered. However, the mitochondrial genome of H. vastatrix has remained undercharacterized. Here, we present the complete, circularized mitochondrial genome of H. vastatrix Race I (isolate HvRI), assembled using a hybrid approach combining PacBio HiFi long reads and BGIseq short reads. The genome is 173,525&#xa0;bp in length with a GC content of 33.1% and encodes 41 functional genes, including 15 protein-coding genes, 2 rRNAs, and 24 tRNAs. The assembly reveals significant structural complexity, driven by intron expansion in the cox1 and cob genes. Notably, the atp8 gene contains a group II intron, rare for this locus, whose internal open reading frame displays evidence of pseudogenization via internal stop codons.. We also characterized a putative replication initiation zone (~1.2&#xa0;kb) defined by a poly-G homopolymer and conserved regulatory motifs. The mitogenome of the HvRI isolate does not contain cob mutations that lead to amino acid substitutions G143A and F129L associated with the quinone outside inhibitor (QoI) fungicide resistance. This high-quality mitogenome is an important resource for comparative mitogenomics, population diversity studies, and the molecular surveillance of QoI fungicide resistance.

Genome, Mitochondrial

Complexity of schistosome vector bulinine snails in Kenya: Insights from nuclear genome size variation, complete mitochondrial genome sequence, and morphometric analysis.

Investigations of nuclear genome size, complete mitochondrial genome (mitogenome) sequence, and morphometrics were conducted on specimens of Bulinus snails (Gastropoda: Planorbidae) collected from 14 locations across the east coast, central Kenya, and western Kenya around the Lake Victoria region (November 2013 and January 2024). Flow cytometry measurements of DNA content (C-value) revealed unexpected variation in nuclear genome size, with diploid Bulinus africanus and B. forskalii species groups showing C-values ranging from 0.76 to 1.98 pg, while tetraploid B. truncatus had a C-value of 1.82 pg. Additionally, C-values for six B. globosus specimens from different localities ranged from 1.43 to 1.98 pg. These findings suggest that bulinine snails, particularly the B. africanus species group, have undergone genome expansion, whole genome duplication (polyploidization), or both, which have not been previously recognized. Next-generation sequencing was performed to determine and annotate 14 complete mitogenome sequences. Despite the well-conserved arrangement of protein-coding genes, two versions of mtDNA genome structure, distinguished by the tRNA-D (Asp) location, were found, designated as DCF (Asp-Cys-Phe) type (in the B. forskalii group and the B. truncatus/tropicus complex) and CF (Cys-Phe) type (in the B. africanus group). Phylogenetic analyses based on complete mtDNA sequences of bulinines from Kenya, along with cytochrome c oxidase subunit I (COX1) sequences from various localities across Africa, contributed to resolving species identities and provided further support for the presence of multiple or cryptic species in the taxon B. globosus. A landmark-based morphometric analysis was ineffective in distinguishing these species. This study reveals unexpected nuclear genome size variation, provides new mitogenome sequences, and highlights the limitations of morphological analysis. It offers valuable insights into the cytogenetics, polyploidy, genomics, taxonomy, and evolution of bulinines, which serve as intermediate hosts for schistosomes responsible for human urogenital schistosomiasis and intestinal schistosomiasis in domestic and wild mammals.

Animals

Assembling genomes of non-model plants: A case study with evolutionary insights from Ranunculus (Ranunculaceae).

Whereas genome sequencing and assembly technologies are improving, cost can still be prohibitive for plant species with large, complex genomes. As a consequence, genomics work on some taxa in evolutionarily pivotal positions in the vascular plant tree of life has been hampered. The species-rich genus Ranunculus (Ranunculaceae) is an important angiosperm group for the study of polyploidy, apomixis, and reticulate evolution. However, neither mitochondrial nor high-quality nuclear genome sequences are available. This limits phylogenomic, functional, and taxonomic analyses thus far. Here, we tested Illumina short-read, Oxford Nanopore Technology (ONT) and PacBio (HiFi) long-read, and hybrid-read assembly strategies. We sequenced the diploid progenitor species R. cassubicifolius (R.&#x2009;auricomus species complex) and selected the best assemblies in terms of completeness, contiguity, and quality scores. We first assembled the plastome (156&#x2009;kbp, 85 genes) and mitogenome (1.18&#x2009;Mbp, 40 genes) sequences using Illumina and Illumina-PacBio-hybrid strategies, respectively. We also present an updated plastome and the first mitogenome phylogeny of Ranunculaceae, including studies of gene loss (e.g., infA, ycf15, or rps) with evolutionary implications. For the nuclear genome sequence, we favored a PacBio-based assembly polished three times with filtered short reads and subsequently scaffolded into eight pseudochromosomes by chromatin conformation data (Hi-C). We obtained a haploid genome sequence of 2.69&#x2009;Gbp, with 94.1% complete BUSCO genes found and 35&#x2009;482 annotated genes, and inferred ancient gene duplications compared to existing Ranunculales genomes. The genomic information presented here will enable advanced evolutionary-functional analyses for the species complex, but also for the genus and beyond Ranunculaceae.

Ranunculus

Genetic (Genomic) and Morphological Evidence Suggest That the Korean Endemic Fat Minnow (Rhynchocypris kumgangensis) and the Deogyu Population Represent Distinct Species.

Geographically disconnected populations of freshwater fishes often show ecological and genetic divergence in response to opposing selection differentials in different habitat environments, sometimes leading to the formation of ecotypes and even speciation. Nevertheless, evidence for the speciation through allopatric processes in freshwater fish systems remains scarce. Kumkang fat minnow (Rhynchocypris kumgangensis), a Korean endemic coldwater fish, has recently been suggested to diverge into a separate species (Rhynchocypris deogyuensis). However, the level of their genetic and ecological divergence remains largely unknown. We analyzed population genetic structure of R. kumgangensis together with R. deogyuensis using mitochondrial DNA (mtDNA) and eight newly developed microsatellite markers. Genetic divergence at mitogenome level was further assessed between the presumed two species. Moreover, we examined morphology between two groups by analyzing morphometric traits and also conducted geometric morphometrics on body shape. We found distinct population structure between R. kumgangensis and R. deogyuensis at both mtDNA and microsatellites. One remnant population of R. deogyuensis harbored only a single haplotype and showed a very high level of inbreeding. Comparative analysis of the mitogenomes showed approximately 2.9% divergence between R. kumgangensis and R. deogyuensis, supporting the species-level divergence. The analyses of both morphometric traits and geometric morphometrics indicated significant morphological divergence between the two species. The observed low values of length-weight relationship and condition factor for R. deogyuensis are likely to be attributed to effects of the elevated level of inbreeding and depleted genetic diversity. Overall, our combined genetic/genomic and morphological analyses suggest the considerable divergence between R. kumgangensis and R. deogyuensis, supporting the hypothesis that they are distinct species. The mechanisms underpinning how they speciate still need to be studied further in detail.

Deogyu fat minnow

The Complete Mitochondrial Genome of a Newly Recorded Chinese Species of Diglyphus sabulosus (Hymenoptera: Eulophidae) and Insights into Its Phylogenetic Position.

Diglyphus Walker, 1844 is an economically important genus which many species acting as biocontrol agents against agromyzid leafminer pests, but there is a lack of mitogenomic data on the evolutionary relationships within this genus, hindering a comprehensive understanding of its evolutionary history. We used traditional morphological methods to identify species, and present the first complete mitochondrial genome sequence and characterization of features of Diglyphus sabulosus and further infer its phylogenetic position based on the amino acid sequences of 13 protein-coding genes (PCGs). The complete mitochondrial genome of D. sabulosus is 15,690&#xa0;bp in length, including 13 PCGs, 22 transfer RNA genes, 2 ribosomal RNA genes and a control region. The AT content of the whole genome sequence was 81.0%, indicating a significant AT bias. All protein-coding genes have the typical ATN as the start codon and TAA as the stop codon. Phylogenetic analysis inferred from the amino acid sequences of 13 PCGs revealed that all species within the family Eulophidae constituted a monophyletic clade, supporting the monophyly of this family. D. sabulosus and D. poppoea form a well-supported sister group, representing the species with the closest phylogenetic relationship within the analyzed taxa. In this study, the mitogenome structure was analyzed and the taxonomic status of D. sabulosus was clarified, thus providing a theoretical basis for understanding the phylogenetic relationships of Diglyphus.

Animals

Complete mitochondrial genomes of eight cyclophyllidean tapeworms: genome pattern and phylogenetic analysis.

Cyclophyllidean tapeworms are widespread parasites of significant medical and veterinary importance. However, mitochondrial (mt) genomic resources for cyclophyllideans from China, particularly those recovered from wildlife hosts, remain comparatively limited. In this study, we sequenced and characterized the complete mt genomes of eight cyclophyllidean isolates collected from diverse wild and domestic hosts in China, including two Hymenolepis sp. isolates and two Raillietina sp. isolates from China, and four additional isolates of previously sequenced Taenia species. The circular mt genomes ranged from 13,387 to 14,021&#xa0;bp in length, encoding 36 typical genes with variable non-coding regions. Comparative analysis revealed highly conserved gene composition and mostly conserved mt architecture, with localized rearrangement patterns detected among the cyclophyllidean lineages examined. In particular, all sampled Taeniidae exhibited a consistent trnL1-trnS2 arrangement, whereas the examined non-Taeniidae families showed the trnS2-trnL1 arrangement, confirming and extending, across additional wildlife-associated isolates, a previously proposed family-associated gene-order marker within Cyclophyllidea. Phylogenetic analyses based on concatenated amino acid sequences of the 12 protein-coding genes placed the eight isolates within their expected families, in topologies broadly consistent with previous mitogenomic studies. These data provide additional Chinese mitogenomic references, especially for underrepresented wildlife-associated isolates, and support family-associated gene-order patterns in Cyclophyllidea.

Animals

First complete mitochondrial genome of Uzelothrips scabrosus (Thysanoptera: Uzelothripidae) provides insights into gene rearrangements and phylogenetic position within Terebrantia.

The family Uzelothripidae is represented by a single genus Uzelothrips and can be distinguished from others by the presence of whip-like antennae, a circular ventral sensorium on antennal segment III, a well-developed tentorium, and a membranous ovipositor. Here, we generated the first complete mitochondrial genome of Uzelothrips scabrosus (15,674&#xa0;bp) using next-generation sequencing to explore the gene rearrangements and phylogenetic relationships. It consists of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and two putative control regions. The genome exhibits strong AT bias (71.35%) with negative AT and GC skew. Codon usage analyses indicate a strong bias towards A/U-ending codons and influenced by both natural selection and mutation pressure. All PCGs were under purifying selection, with cox1 being the most conserved and nad4L the most variable. The gene order of the family Uzelothripidae is highly rearranged compared to the ancestral insect gene order. Comparative analysis revealed that gene block B was the most widely conserved, whereas the remaining gene blocks exhibited family or lineage-specific conservation patterns, reflecting extensive mitochondrial gene rearrangements during the evolution of the Thysanoptera. Moreover, 228 synapomorphic and 68 autapomorphic gene boundaries were identified across thysanopteran mitogenomes. Phylogenies indicated that the family Uzelothripidae is in a sister relationship with Stenurothripidae, and the Uzelothripidae&#xa0;+&#xa0;Stenurothripidae clade is sister to Thripidae. This study provides the first mitogenomic insights into Uzelothripidae and highlights the need for broader taxon sampling and nuclear genomic data to resolve deep evolutionary relationships within Thysanoptera.

Comparative analysis

Chromosome-level de novo assembly of the nuclear and mitochondrial genomes of Arcopilus aureus, a filamentous fungus with multifaceted ecological and economic roles.

The filamentous fungus Arcopilus aureus (Sordariale: Chaetomiaceae) is notable for its multi-domain significance across agriculture, medicine, and industry. In this study, we generated a chromosome-level nuclear genome and a complete circular mitogenome for A. aureus by integrating data from next-generation sequencing, PacBio HiFi, and Hi-C technologies. The final nuclear genome assembly spans 33.77&#x2009;Mb (GC content: 57.67%), and was organized into seven chromosomal-sized scaffolds (only one gap) with an N50 size of 5.09&#x2009;Mb and BUSCO completeness of 95.91%. A total of 10,282 protein-coding genes, 228 non-coding RNAs, and ~1.77&#x2009;Mb of repetitive elements were predicted in the nuclear genome. By contrast, the mitogenome of A. aureus is 33,820&#x2009;bp in length, with a GC content of 25.96%. It harbors 15 typical mitochondrial protein-coding genes, one unidentified ORF, two rRNAs (small subunit rns and large subunit rnl), and 28 tRNAs. This high-quality genome assembly provides a valuable resource for understanding the ecology, genetics, and evolution of A. aureus, which facilitates elucidating its mechanisms of biocontrol, infection, and metabolite synthesis.

Genome, Mitochondrial

Mitochondrial genomic characteristics and phylogenetic analysis of Cunninghamella elegans (Mucorales: Cunninghamellaceae).

Cunninghamella, a filamentous fungal genus with important biomedical and biochemical value, lacks any fully annotated mitochondrial genome to date. Herein, we presented the first complete mitogenome of Cunninghamella elegans, a circular 41,552 bp molecule (GC 27.86%) encoding 14 conserved protein-coding genes, 2 rRNA genes, 24 tRNA genes, and 6 non-conserved ORFs. Structural comparison with related species (Absidia glauca and Gongronella sp. w5) revealed dynamic evolution in intron and repeat elements. Phylogenetics places C. elegans within Cunninghamellaceae, with Gongronella as its closest relative. This reference mitogenome will underpin future evolutionary and taxonomic investigations of this industrially and medically significant lineage.

Cunninghamella elegans

Natural Selection Drives Codon Usage Bias in the Mitochondrial Genome of Ligula intestinalis (Linnaeus, 1758) Gmelin, 1790 (Cestoda: Diphyllobothriidea): Insights from Comparative Genomics and Optimal Codon Identification.

Codon usage bias (CUB) is a useful indicator of evolutionary forces shaping mitochondrial genomes. Codon usage bias in mitochondrial genomes of Diphyllobothriidae and especially in Ligula intestinalis was characterized. The roles of natural selection and mutation pressure in framing this bias were evaluated on the basis of 12 protein-coding genes in Diphyllobothriidae. The complete mitogenome (13,725 bp) of L. intestinalis comprises 12 protein-coding genes (PCGs), 22 tRNAs, and two rRNAs, all positioned on the heavy strand, and contains an overall AT content of 66.15%. The mean CAI (0.176), CBI (-0.105), and ENC (45.33) and an evident preference for U-ending codons observed in all examined genes indicate weak CUB. Neutrality, ENC, and PR2 plots consistently demonstrate that natural selection is the predominant force driving CUB and contributes approximately 56% in L. intestinalis and 83% in other Diphyllobothriidea species, with mutation pressure playing a secondary role. Phylogenetic reconstruction supported the monophyly of Diphyllobothriidea, confirmed the paraphyly of Diphyllobothrium as traditionally defined, and placed Ligula and Digramma as sister taxa. These findings clarify the evolutionary constraints governing codon usage in cestode mitogenomes and provide practical resources for codon optimization in heterologous gene expression and genetic studies of this economically important parasite.

Diphyllobothriidea

Mitochondrial genomes and phylogenomic analysis of Plectostylus broderipii and Bostryx erythrostoma (Gastropoda, Stylommatophora, Orthalicoidea) from the Atacama Desert, Chile.

We report the first mitochondrial genomes of two Orthalicoidea land snails from the Atacama Desert of northern Chile, Bostryx erythrostoma and Plectostylus broderipii. Using Illumina short-read sequencing, we assembled and annotated mitochondrial genomes of 15,525 bp and 14,978 bp, respectively, with strong A+T bias (69.75% in B. erythrostoma; 72.08% in P. broderipii). Both genomes retain the standard metazoan mitochondrial complement of 37 genes, including 13 protein-coding genes (PCGs), 22 tRNAs, and two rRNAs, with differences in total length mainly due to variation in non-coding regions rather than coding-gene content. Comparative analysis showed that the two species share the same order of the 13 PCGs, whereas differences are concentrated in local tRNA/rRNA rearrangements and intergenic architecture. Both mitogenomes also exhibit negative AT skew and positive GC skew under a standardized orientation. Phylogenetic inference based on a concatenated 13-PCG nucleotide supermatrix resolved both taxa within Orthalicoidea with strong support: B. erythrostoma grouped with a congeneric Bostryx reference, and this clade clustered with Rabdotus mooreanus and Naesiotus nux, whereas P. broderipii was inferred as sister to this orthalicoid cluster.

Bothriembryontidae

Insights Into the Structural Features, Codon Usage Patterns, and Phylogenetic Analysis in Neoniphon argenteus (Teleostei: Holocentriformes) Based on Complete Mitochondrial Genome.

Neoniphon argenteus, a widely distributed nocturnal coral reef fish in the family Holocentridae, plays an important role in maintaining coral reef ecosystem health, yet its phylogenetic position remains poorly resolved. To bridge this gap, we sequenced and analyzed the complete mitochondrial genome of a specimen from the South China Sea to characterize its structural features, codon usage patterns, and phylogenetic relationships. The 16,569&#x2009;bp mitogenome (GenBank: PP190474.1) encodes 13 protein-coding genes (PCGs), 22 tRNAs, two rRNAs, and two non-coding regions, exhibiting a distinct A&#x2009;+&#x2009;T bias. All tRNAs fold into typical cloverleaf secondary structures except tRNA-Ser (AGN), which lacks the dihydrouridine (DHU) arm. The control region contains palindromic motifs (TACAT/ATGTA) capable of forming hairpin structures and five conserved sequence blocks, whereas the OL region harbors a conserved 5'-GCCGG-3' motif. RSCU analysis revealed 31 frequently used codons (RSCU >&#x2009;1) with a pronounced preference for A/C-ending codons. The &#x394;RSCU method identified 10 candidate optimal codons (GCA, CAA, GAA, GGA, AUU, CUA, CCA, CGA, ACA, and GUC). Selection pressure analysis using EasyCodeML and site-specific models indicated that all PCGs are predominantly under purifying selection, with no significant evidence of pervasive positive selection. ND6 exhibited elevated pairwise Ka/Ks ratios (mean&#x2009;=&#x2009;1.209&#x2009;&#xb1;&#x2009;0.047), consistent with reduced selective constraint rather than adaptive evolution. Phylogenetic analysis of 19 Holocentriformes species using maximum likelihood and Bayesian inference with partitioned models based on 13 PCGs and two rRNA genes (12S and 16S) assigned all taxa to two well-supported subfamilies (Holocentrinae and Myripristinae). Within Holocentrinae, Neoniphon species form a monophyletic clade nested within a paraphyletic Sargocentron, suggesting that the genus Sargocentron as currently defined is not monophyletic. This study provides useful baseline molecular data for further exploration of the evolutionary history of N. argenteus and other members of Holocentriformes.

Holocentridae

One-time duplication and ongoing loss of mitochondrial tRNA genes in Cryptocercus cockroaches.

Mitochondrial genome is a popular marker in phylogenetics and species diversity estimations. Mitogenome is relatively compact and conserved, while gene rearrangements were found in some species across various organisms. Models to explain the origin and evolution of gene rearrangement have been proposed but seldom demonstrated; empirical evidence from closely related species is particularly scarce. Here, through an intensive case study of the cockroach genus Cryptocercus Scudder, 1862, we elucidate the evolution of mitochondrial gene order. This study utilized 51 new samples and re-assembled raw reads of 26 published samples. A diversity of rearrangement patterns is recovered, especially in the tRNA gene cluster between ND3 and ND5, which is effectively explained by the duplication&#xa0;-&#xa0;random loss model. Specifically, the entire tRNA gene cluster was duplicated; this duplication is potentially facilitated by chance binding between the 3' end of ND5 gene and the ND3-trnA region during DNA replication. Furthermore, we reveal that one of the gene copies degenerated stochastically across lineages, directly contributing to the observed diversity in gene arrangement. Gene rearrangement patterns are apomorphies for certain clades, providing additional evidence for the inferred phylogeny and serving as potential indicators of species. This study underscores the importance of intensive sampling and rigorous data curation for deciphering the evolutionary mechanisms.

Duplication&#x2013;random loss model

The complete mitochondrial genome of Ramulus bifarius (Phasmida: Phasmatidae; Clitumninae).

We successfully acquired the complete mitochondrial genome information of Ramulus bifarius (S. C. Chen and Y. H. He, p. 476, Phasmatodea of China, 2008), which can be used for subsequent related molecular studies. The mitogenome of R. bifarius exhibits a circular duplex structure, with a total length of 16,915 bp and a high adenine and thymine bias of 76.7%. It contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a single control region.

China