Evolutionary conservation and lineage-specific virulence repurposing of the core housekeeping gene ushA in Enterobacteriaceae.

Comparing Phylogeny and the Predicted Pathogenicity of Protein Variations Reveals Equal Purifying Selection across the Global Human mtDNA Diversity

Molecular characterization of Indian Sugarcane streak mosaic virus isolates reveals recombination and negative selection in the P1 gene

Simple SummaryMicrohyla fissipes, a species within the family Microhylidae, is distributed exclusively in Asia. This frog holds significant scientific and ecological importance, but research on this species remains limited to date. In this study, we report the characteristics of the mitochondrial genome of M. fissipes and, using these data, investigate the phylogenetic relationships within the family Microhylidae. The results indicate that the mitogenomic size of M. fissipes is 16,723 bp, arranged in a circular configuration, containing 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, and 1 non-coding region. The nucleotide composition is 28.9% adenine (A), 31.01% thymine (T), 25.51% cytosine (C), and 14.58% guanine (G). Phylogenetic analysis indicated that Microhylidae can be divided into four monophyletic genera (Microhyla, Glyphoglossus, Kaloula, Anilany), with M. fissipes being most closely related to Microhyla heymonsi. Analysis indicates that the selection pressure ratios for most genes are less than 1, suggesting that these genes have undergone strong purifying selection. However, in the COX2 and COX3 genes, the Ka/Ks ratios between some species exceed 1, indicating that the evolutionary process aims to eliminate harmful mutations. This study enriches the basic biological data for M. fissipes and provides a fundamental reference for the conservation of M. fissipes and Microhyla.The microhylid frog Microhyla fissipes is a protected terrestrial wildlife species in China, recognized for its ecological, economic, and scientific value. However, its mitochondrial genome remains poorly characterized. To address this gap, we sequenced and annotated the complete mitogenome of M. fissipes to elucidate its structural organization and phylogenetic placement within Microhylidae. The assembled mitogenome is 16,723 bp in length and contains 37 genes, including 13 protein-coding genes, 2 rRNAs, and 22 tRNAs, along with one control region and the origin of heavy-strand replication. We also identified eight overlapping regions and eleven intergenic spacers. The overall base composition showed an A + T bias (59.91%) with negative AT-skew (−0.04) and GC-skew (−0.27). All tRNAs displayed typical cloverleaf secondary structures, except for trnS1, which lacked the D-arm. Phylogenetic reconstruction using both maximum likelihood and Bayesian inference strongly supported the monophyly of Microhylidae and revealed a sister-group relationship between Microhyla and Kaloula. Within Microhyla, M. fissipes was most closely related to M. heymonsi, with which it formed a well-supported clade that also included Microhyla okinavensis, Microhyla mixtura, and Microhyla beilunensis. Selection pressure analysis on protein-coding genes indicated widespread purifying selection (Ka/Ks < 1) across most genes, except for ATP8, COX2, and COX3, which may be under relaxed selective constraints. These findings offer valuable genomic resources for the conservation of M. fissipes and provide new insights into the phylogeny and evolution of microhylid frogs.

Camellia luteoflora Y.K. Li ex Hung T. Chang & F.A. Zeng belongs to the Camellia L. genus (Theaceae Mirb.). As an endemic, rare, and critically endangered species in China, it holds significant ornamental and economic value, garnering global attention due to its ecological rarity. Despite its conservation importance, genomic investigations on this species remain limited, particularly in organelle genomics, hindering progress in phylogenetic classification and population identification. In this study, we employed high-throughput sequencing to assemble the first complete mitochondrial genome of C. luteoflora and reannotated its chloroplast genome. Through integrated bioinformatics analyses, we systematically characterized the mitochondrial genome’s structural organization, gene content, interorganellar DNA transfer, sequence variation, and evolutionary relationships.Key findings revealed a circular mitochondrial genome spanning 587,847 bp with a GC content of 44.63%. The genome harbors70 unique functional genes, including 40 protein-coding genes (PCGs), 27 tRNA genes, and 3 rRNA genes. Notably, 9 PCGs contained 22 intronic regions. Codon usage analysis demonstrated a pronounced A/U bias in synonymous codon selection. Structural features included 506 dispersed repeats and 240 simple sequence repeats. Comparative genomics identified 19 chloroplast-derived transfer events, contributing 29,534 bp (3.77% of total mitochondrial DNA). RNA editing prediction revealed 539 C-to-T conversion events across PCGs. Phylogenetic reconstruction using mitochondrial PCGs positioned C. luteoflora in closest evolutionary proximity to Camellia sinensis var. sinensis. Selection pressure analysis (Ka/Ks ratios < 1 for 11 PCGs) and nucleotide diversity assessment (Pi values: 0–0.00711) indicated strong purifying selection and low sequence divergence.This study provides the first comprehensive mitochondrial genomic resource for C. luteoflora, offering critical insights for germplasm conservation, comparative organelle genomics, phylogenetic resolution, and evolutionary adaptation studies in Camellia species.

The study of chloroplast genome evolutionary dynamics provides critical insights into plant adaptive evolution and phylogenetic relationships. This research conducted a systematic comparative analysis of chloroplast genomes across 35 species within the Rutaceae family. All genomes displayed the typical quadripartite structure, with sizes ranging from 155 to 161 kb, GC contents between 38.17% and 38.83%, and gene counts varying from 122 to 144. Structural conservation was high across species, with variations mainly localized at the boundaries of inverted repeat (IR) regions. AT-rich mononucleotide simple sequence repeats (SSRs) were dominant and primarily distributed in non-coding regions. Collinearity analysis revealed high sequence conservation alongside lineage-specific rearrangements. Relative synonymous codon usage (RSCU) analysis revealed significant heterogeneity among species, with values ranging from 0.386 to 1.797. ENC-GC3s, GC3-GC12, and PR2 analyses indicated a marked deviation from neutral evolution. Selection pressure analysis indicated strong purifying selection (Ka/Ks < 0.2) acting on photosynthetic system genes, while certain genes (e.g., matK, rpl20) exhibited signals of positive selection, highlighting adaptive evolutionary features in specific genomic regions. Phylogenetic reconstruction placed Murraya paniculata within a clade containing other Murraya species, closely related to Citrus and Clausena, reflecting morphological and biogeographic patterns. This study provides a molecular framework for taxonomic revision in Rutaceae and enhances understanding of chloroplast genome evolution in the family.

Genetic variation in mitochondrial genes could underlie metabolic adaptations because mitochondrially encoded proteins are directly involved in a pathway supplying energy to metabolism. Macquarie perch from river basins exposed to different climates differ in size and growth rate, suggesting potential presence of adaptive metabolic differences. We used complete mitochondrial genome sequences to build a phylogeny, estimate lineage divergence times and identify signatures of purifying and positive selection acting on mitochondrial genes for 25 Macquarie perch from three basins: Murray-Darling Basin (MDB), Hawkesbury-Nepean Basin (HNB) and Shoalhaven Basin (SB). Phylogenetic analysis resolved basin-level clades, supporting incipient speciation previously inferred from differentiation in allozymes, microsatellites and mitochondrial control region. The estimated time of lineage divergence suggested an early- to mid-Pleistocene split between SB and the common ancestor of HNB+MDB, followed by mid-to-late Pleistocene splitting between HNB and MDB. These divergence estimates are more recent than previous ones. Our analyses suggested that evolutionary drivers differed between inland MDB and coastal HNB. In the cooler and more climatically variable MDB, mitogenomes evolved under strong purifying selection, whereas in the warmer and more climatically stable HNB, purifying selection was relaxed. Evidence for relaxed selection in the HNB includes elevated transfer RNA and 16S ribosomal RNA polymorphism, presence of potentially mildly deleterious mutations and a codon (ATP6113) displaying signatures of positive selection (ratio of nonsynonymous to synonymous substitution rates (dN/dS) >1, radical change of an amino-acid property and phylogenetic conservation across the Percichthyidae). In addition, the difference could be because of stronger genetic drift in the smaller and historically more subdivided HNB with low per-population effective population sizes.

Pandalus platyceros Brandt in von Middendorf, 1851, the spot prawn, is a commercially important pandalid shrimp that sustains a small fishery in the northeastern Pacific Ocean. We report, for the first time, the complete mitochondrial genome of P. platyceros, while also testing whether linked-reads sequencing (10X Genomics) data can be used to assemble complete and accurate mitochondrial genomes. The pipeline GetOrganelle assembled and circularized the complete mitochondrial chromosome of P. platyceros with an average coverage of 28.2x from a dataset of 5 M pairs of linked reads. The AT-rich mitochondrial genome of P. platyceros is 16,628 bp in length and comprised of 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 24 transfer RNA genes. One copy of all tRNA genes was present, except for tRNA-G, which had three copies. A single 1,077 bp-long intergenic space was assumed to be the D-loop/Control region. Selective pressure analysis indicated the PCGs were under purifying selection, although levels differed among genes. The highest KA:KS ratios were found in nad4 and nad4l, suggesting weaker purifying selection and environmental constraints on these genes. The KA:KS ratios for cob and cox1 were a magnitude lower than the ratios in other PCGs, suggesting strong purifying selection acting upon these genes. A maximum likelihood phylogenetic analysis based on all PCGs that included a total of 91 species of shrimps supported the monophyly of the infraorder Caridea and family Pandalidae. Furthermore, the monophyly of other caridean families, including Alvinocaridae, Atyidae, Thoridae, Lysmatidae, and Palaemonidae was also supported by the same analysis. Our results thus suggest that mitochondrial PCGs have enough phylogenetic information to resolve relationships at high taxonomic levels (families) in Caridea. This study contributes new genomic resources for this commercially important species and demonstrates that linked-reads sequencing can be used to assemble accurate mitochondrial genomes.

As the null hypothesis of genome evolution, population genetic theory suggests that selection strength controls genome size. Through the process of genetic drift, this theory predicts that compact genomes are maintained by strong purifying selection while complex genomes are enabled by weak purifying selection. It offers a unifying framework that explains why prokaryotic genomes are much smaller than their eukaryotic counterparts. However, recent findings suggest that bigger prokaryotic genomes appear to experience stronger purifying selection, indicating that purifying selection may not dominate prokaryotic genome evolution. Since archaeal genomes were underrepresented in those studies, generalization of the conclusions to both archaeal and bacterial genomes may not be warranted. In this study, we revisited this matter by focusing on archaeal and bacterial genomes separately. We found that bigger bacterial genomes indeed experienced stronger purifying selection, but the opposite was observed in archaeal genomes. This new finding would predict an enrichment of noncoding sequences in large archaeal genomes, which was confirmed by an analysis of coding density. In contrast, coding density remained stable regardless of bacterial genome size. In conclusion, this study suggests that purifying selection may play a more important role in archaeal genome evolution than previously hypothesized, indicating that there could be a major difference between the evolutionary regimes of Archaea and Bacteria. IMPORTANCE The evolution of genome complexity is a fundamental question in biology. A hallmark of eukaryotic genome complexity is that larger genomes tend to have more noncoding sequences, which are believed to be minimal in archaeal and bacterial genomes. However, we found that archaeal genomes also possessed this eukaryotic feature while bacterial genomes did not. This could be predicted from our analysis on genetic drift, which showed a relaxation of purifying selection in larger archaeal genomes, also a eukaryotic feature. In contrast, the opposite was evident in bacterial genomes.

Identification of cancer driver genes using somatic mutation patterns indicative of positive selection has become a major goal in cancer genomics. However, cancer cells additionally depend on a large number of genes involved in basic cellular processes. While such genes should in theory be subject to strong purifying (negative) selection against damaging somatic mutations, these patterns have been elusive and purifying selection remains inadequately explored in cancer. Here, we hypothesized that purifying selection should be evident in hemizygous genomic regions, where damaging mutations cannot be compensated for by healthy alleles. Using a 7,781-sample pan-cancer dataset, we first confirmed this in POLR2A, an essential gene where hemizygous deletions are known to confer elevated sensitivity to pharmacological suppression. We next used this principle to identify several genes and pathways that show patterns indicative of purifying selection to avoid deleterious mutations. These include the POLR2A interacting protein INTS10 as well as genes involved in mRNA splicing, nonsense-mediated mRNA decay and other RNA processing pathways. Many of these genes belong to large protein complexes, and strong overlaps were observed with recent functional screens for gene essentiality in human cells. Our analysis supports that purifying selection acts to preserve the remaining function of many hemizygously deleted essential genes in tumors, indicating vulnerabilities that might be exploited by future therapeutic strategies.

On the Expansion of “Dangerous” Gene Repertoires by Whole-Genome Duplications in Early Vertebrates

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.

Bacteria, Archaea, and Eukarya all share a common set of metabolic reactions. This implies that the function and topology of central metabolism has been evolving under purifying selection over deep time. Central metabolism may similarly evolve under purifying selection during long-term evolution experiments, although it is unclear how long such experiments would have to run (decades, centuries, millennia) before signs of purifying selection on metabolism appear. I hypothesized that central and superessential metabolic enzymes would show evidence of purifying selection in the long-term evolution experiment with Escherichia coli (LTEE). I also hypothesized that enzymes that specialize on single substrates would show stronger evidence of purifying selection in the LTEE than generalist enzymes that catalyze multiple reactions. I tested these hypotheses by analyzing metagenomic time series covering 62,750 generations of the LTEE. I find mixed support for these hypotheses, because the observed patterns of purifying selection are idiosyncratic and population-specific. To explain this finding, I propose the Jenga hypothesis, named after a children's game in which blocks are removed from a tower until it falls. The Jenga hypothesis postulates that loss-of-function mutations degrade costly, redundant, and non-essential metabolic functions. Replicate populations can therefore follow idiosyncratic trajectories of lost redundancies, despite purifying selection on overall function. I tested the Jenga hypothesis by simulating the evolution of 1,000 minimal genomes under strong purifying selection. As predicted, the minimal genomes converge to different metabolic networks. Strikingly, the core genes common to all 1,000 minimal genomes show consistent signatures of purifying selection in the LTEE.

Yellow leaf of sugarcane is caused by Sugarcane yellow leaf virus (SCYLV) and has been a threat to global sugar industry. Understanding the diversity and genetic structure of plant virus populations may help to develop strategies to control virus-induced diseases. In this study, demography, genetic differentiation and selection pressure were assessed based on 57 nucleotide sequences of the P0 (567 nt) and 54 nucleotide sequences of the P1 (1413 nt) genes of SCYLV isolates. Phylogenetic analysis revealed three phylogeographical groups: African (Clade I), Chinese/American (Clade II), and Asian (Clade III). Genetic distance values between groups (0.034–0.250) were higher than within-group values (0.011–0.033). The African group was in a state of expansion, with lower nucleotide sequence diversity. Except for those isolates within the Asian group, SCYLV showed infrequent gene flow within or between phylogenetic groups. Analysis of selection pressure suggested that these two genes have been subjected to negative (purifying) selection. However, some sites, especially in the P0 coding region of the Chinese/American group, have undergone positive selection. Overall, aside from recombination and founder effects, gene flow and purifying selection are two important factors driving SCYLV evolution.

When isolated but reproductively compatible populations expand geographically and meet, simulations predict asymmetric introgression of neutral loci from a local to invading taxon. Genetic introgression may affect phylogenetic reconstruction by obscuring topology and divergence estimates. We combined phylogenetic analysis of sequences from one mtDNA and 12 nuDNA loci with analysis of gene flow among 5 species of Pacific Locustella warblers to test for presence of genetic introgression and its effects on tree topology and divergence estimates. Our data showed that nuDNA introgression was substantial and asymmetrical among all members of superspecies groups whereas mtDNA showed no introgression except a single species pair where the invader's mtDNA was swept by mtDNA of the local species. This introgressive sweep of mtDNA had the opposite direction of the nuDNA introgression and resulted in the paraphyly of the local species' mtDNA haplotypes with respect to those of the invader. Тhe multilocus nuDNA species tree resolved all inter- and intraspecific relationships despite substantial introgression. However, the node ages on the species tree may be underestimated as suggested by the differences in node age estimates based on non-introgressing mtDNA and introgressing nuDNA. In turn, the introgressive sweep and strong purifying selection appear to elongate internal branches in the mtDNA gene tree.

The activity of transposable elements (TEs) contributes significantly to pathogen genome evolution. TEs often destabilize genome integrity but may also confer adaptive variation in pathogenicity or resistance traits. De-repression of epigenetically silenced TEs often initiates bursts of transposition activity that may be counteracted by purifying selection and genome defenses. However, how these forces interact to determine the expansion routes of TEs within a pathogen species remains largely unknown. Here, we analyzed a set of 19 telomere-to-telomere genomes of the fungal wheat pathogen Zymoseptoria tritici. Phylogenetic reconstruction and ancestral state estimates of individual TE families revealed that TEs have undergone distinct activation and repression periods resulting in highly uneven copy numbers between genomes of the same species. Most TEs are clustered in gene poor niches, indicating strong purifying selection against insertions near coding sequences, or as a consequence of insertion site preferences. TE families with high copy numbers have low sequence divergence and strong signatures of defense mechanisms (i.e., RIP). In contrast, small non-autonomous TEs (i.e., MITEs) are less impacted by defense mechanisms and are often located in close proximity to genes. Individual TE families have experienced multiple distinct burst events that generated many nearly identical copies. We found that a Copia element burst was initiated from recent copies inserted substantially closer to genes compared to older copies. Overall, TE bursts tended to initiate from copies in GC-rich niches that escaped inactivation by genomic defenses. Our work shows how specific genomic environments features provide triggers for TE proliferation in pathogen genomes.