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The timing of early cellular evolution, from the divergence of Archaea and Bacteria to the origin of eukaryotes, is poorly constrained. The ATP synthase complex is thought to have originated prior to the Last Universal Common Ancestor (LUCA) and analyses of ATP synthase genes, together with ribosomes, have played a key role in inferring and rooting the tree of life. We reconstruct the evolutionary history of ATP synthases using an expanded taxon sampling set and develop a phylogenetic cross-bracing approach, constraining equivalent speciation nodes to be contemporaneous, based on the phylogenetic imprint of endosymbioses and ancient gene duplications. This approach results in a highly resolved, dated species tree and establishes an absolute timeline for ATP synthase evolution. Our analyses show that the divergence of ATP synthase into F- and A/V-type lineages was a very early event in cellular evolution dating back to more than 4 Ga, potentially predating the diversification of Archaea and Bacteria. Our cross-braced, dated tree of life also provides insight into more recent evolutionary transitions including eukaryogenesis, showing that the eukaryotic nuclear and mitochondrial lineages diverged from their closest archaeal (2.67-2.19 Ga) and bacterial (2.58-2.12 Ga) relatives at approximately the same time, with a slightly longer nuclear stem-lineage. The timing of cellular evolution is poorly constrained. Here, the authors used improved molecular dating approaches to study the evolution of the ATP synthase in light of a dated tree of life thereby providing an absolute timescale for cellular evolution including eukaryotic origins.

The nature of the last universal common ancestor (LUCA), its age and its impact on the Earth system have been the subject of vigorous debate across diverse disciplines, often based on disparate data and methods. Age estimates for LUCA are usually based on the fossil record, varying with every reinterpretation. The nature of LUCA’s metabolism has proven equally contentious, with some attributing all core metabolisms to LUCA, whereas others reconstruct a simpler life form dependent on geochemistry. Here we infer that LUCA lived ~4.2 Ga (4.09–4.33 Ga) through divergence time analysis of pre-LUCA gene duplicates, calibrated using microbial fossils and isotope records under a new cross-bracing implementation. Phylogenetic reconciliation suggests that LUCA had a genome of at least 2.5 Mb (2.49–2.99 Mb), encoding around 2,600 proteins, comparable to modern prokaryotes. Our results suggest LUCA was a prokaryote-grade anaerobic acetogen that possessed an early immune system. Although LUCA is sometimes perceived as living in isolation, we infer LUCA to have been part of an established ecological system. The metabolism of LUCA would have provided a niche for other microbial community members and hydrogen recycling by atmospheric photochemistry could have supported a modestly productive early ecosystem. Integration of phylogenetics, comparative genomics and palaeobiological approaches suggests that the last universal common ancestor lived about 4.2 billion years ago and was a complex prokaryote-grade anaerobic acetogen that was part of an ecosystem.

Background Benincasa hispida Cogn. var. chieh-qua How, commonly referred to as the small wax gourd, is a widely cultivated cucurbit vegetable renowned for its considerable nutritional and medicinal benefits. However, the lack of reported mitochondrial genome (mitogenome) information for B. hispida severely restricts our understanding of the organelle evolution within the Cucurbitaceae family. This study bridges this knowledge gap by undertaking the sequencing, assembly, and comprehensive analysis of the complete mitogenome of B. hispida . Results Using an integrated sequencing approach that combined Illumina and Nanopore technologies, we performed de novo assembly of the mitogenome of B. hispida , followed by comparative analyses with its close relatives. The assembled mitogenome was characterized by a linear structure composed of 18 contigs, with a total length of 431,446 bp and a GC content of 44.9%. Annotation revealed 72 mitochondrial genes, including 40 protein-coding genes (PCGs), 27 tRNA genes, 4 rRNA genes, and 1 pseudogene ( rps14 ). Codon usage analysis showed a preference for codons that ended with A/T, and 488 RNA-editing sites, predominantly C-to-U conversions, were predicted, potentially enhancing the protein hydrophobicity and stability. A notable abundance of repetitive elements was observed, which comprised 106 simple sequence repeats, 20 tandem repeats, and 643 dispersed repeats, collectively accounting for 11.6% of the genome. Furthermore, we identified a significant presence of chloroplast-derived transfer fragments, with 56 homologous fragments that encompassed a length of 50,189 bp (11.6% of the mitogenome). A comparative evolutionary analysis revealed that most PCGs were under purifying selection, where cox1 and cox3 were highly conserved, while nad4 and rpl2 displayed signals indicative of positive selection. A nucleotide diversity analysis across 12 Cucurbitaceae species confirmed general sequence conservation, with rpl5 exhibiting the highest variability. Phylogenetic reconstruction based on 32 conserved mitochondrial PCGs from representative angiosperm species robustly placed B. hispida within the Cucurbitales clade, which revealed its closest relationship with Citrullus lanatus . Collinearity analysis further supported this close affinity, showing extensive homologous blocks between their mitogenomes, alongside structural rearrangements unique to B. hispida . Conclusions This study presents the first complete mitogenome resource for B. hispida , elucidating its structural complexity, evolutionary dynamics, and phylogenetic position. Prolific repeats and frequent intracellular gene transfers underscore their crucial roles in shaping the architecture and diversification of cucurbit mitogenomes. This genomic foundation paves the way for future research on molecular breeding, species identification, and evolutionary studies within the genus Benincasa and the broader Cucurbitaceae family.

Background The holothurians, commonly known as sea cucumbers, are marine organisms that possess significant dietary, nutritional, and medicinal value. However, the National Center for Biotechnology Information (NCBI) currently possesses only approximately 70 complete mitochondrial genome datasets of Holothurioidea, which poses limitations on conducting comprehensive research on their genetic resources and evolutionary patterns. In this study, a novel species of sea cucumber belonging to the genus Benthodytes , was discovered in the western Pacific Ocean. The genomic DNA of the novel sea cucumber was extracted, sequenced, assembled and subjected to thorough analysis. Results The mtDNA of Benthodytes sp. Gxx-2023 (GenBank No. OR992091) exhibits a circular structure spanning 17,386 bp, comprising of 13 protein-coding genes (PCGs), 24 non-coding RNAs (2 rRNA genes and 22 tRNA genes), along with two putative control regions measuring 882 bp and 1153 bp, respectively. It exhibits a high AT% content and negative AT-skew, which distinguishing it from the majority of sea cucumbers in terms of environmental adaptability evolution. The mitochondrial gene homology between Gxx-2023 and other sea cucumbers is significantly low, with less than 91% similarity to Benthodytes marianensis , which exhibits the highest level of homology. Additionally, its homology with other sea cucumbers is below 80%. The mitogenome of this species exhibits a unique pattern in terms of start and stop codons, featuring only two types of start codons (ATG and ATT) and three types of stop codons including the incomplete T. Notably, the abundance of AT in the Second position of the codons surpasses that of the First and Third position. The gene arrangement of PCGs exhibits a relatively conserved pattern, while there exists substantial variability in tRNA. Evolutionary analysis revealed that it formed a distinct cluster with B. marianensis and exhibited relatively distant phylogenetic relationships with other sea cucumbers. Conclusions These findings contribute to the taxonomic diversity of sea cucumbers in the Elasipodida order, thereby holding significant implications for the conservation of biological genetic resources, evolutionary advancements, and the exploration of novel sea cucumber resources.

Background Cercis chinensis is a leguminous species with notable ornamental value and considerable economic potential, and is primarily distributed in southern China. Among species in the genus Cercis , only the complete mitochondrial genome of Cercis canadensis has been publicly available. This lack of mitochondrial genomic data constrains our understanding of the evolutionary history, genetic diversity, and environmental adaptability of Cercis chinensis and its closely related species. In this study, we successfully completed the de novo assembly and annotation of Cercis chinensis mitochondrial genome. Results The mitochondrial genome of Cercis chinensis consists of two putative circular chromosomes, totaling 393,546 bp in length with a GC content of 45.29%. A total of 61 genes were annotated, including 39 protein-coding genes (PCGs), 20 transfer RNA (tRNA) genes, and 2 ribosomal RNA (rRNA) genes. Functional analysis predicted 546 C-to-U RNA editing sites, the majority of which result in nonsynonymous amino acid substitutions. A total of 155 repeat elements were identified, comprising simple sequence repeats (SSRs), tandem repeats, and dispersed repeats, highlighting the structural complexity of the mitochondrial genome. Furthermore, 18 mitochondrial plastid DNA transfer events (MTPTs) were detected, encompassing six complete genes, including 5 tRNA genes and 1 rRNA gene, originating from the chloroplast genome. The presence of intact tRNA and rRNA fragments among MTPTs suggests that such transfers may not only add to structural diversity but could also provide functional redundancy or influence RNA processing within the mitochondrial genome. Ka/Ks analysis indicated that most core PCGs are subject to purifying selection, reflecting high functional conservation across species. Notably, atp4 and nad4 exhibited signs of positive selection, suggesting their potential involvement in the adaptive evolution of Cercis chinensis . Phylogenetic analysis based on shared mitochondrial and chloroplast protein-coding genes placed Cercis chinensis in close relation to Bauhinia purpurea , thereby supporting its current taxonomic classification within the Fabaceae. Conclusion This study provides the first characterization of the mitochondrial genome of Cercis chinensis , detailing its structural organization, functional elements, and evolutionary dynamics. The assembled genome and associated analyses provide valuable insights into the mitochondrial architecture of Cercis chinensis and enrich the genomic resources for the Fabaceae family, enhancing our understanding of mitochondrial genome evolution in legumes and serving as a foundational reference for future research in species identification, molecular breeding, and phylogenetic studies of C. chinensis and related taxa. Highlights • The complete mitochondrial genome of Cercis chinensis was de novo assembled using third-generation sequencing technology. • The genome of Cercis chinensis consists of two circular chromosomes, in contrast to the single-chromosome structure reported in Cercis canadensis , but two species exhibit slight differences in genome size and GC content. • Comparative analysis revealed that some genes ( rpl2 , rps19 ) are lost in both Cercis chinensis and Cercis canadensis , while the cox2 is retained in C. chinensis , suggesting a lineage-specific gene retention mechanism within the genus Cercis . • Phylogenetic trees constructed based on 22 mitochondrial protein-coding genes and 68 chloroplast protein-coding genes confirmed the monophyly of Fabaceae, with both trees exhibiting highly congruent topologies.

Background Polygonatum cyrtonema Hua , a valuable medicinal and edible plant in China, has been increasingly cultivated to produce nutritional and health products to meet market demand. However, the absence of the mitochondrial genome hinders the research of evolutionary analysis, artificial cultivation, and medicinal resource development. This study aimed to systematically characterize the mitochondrial genome of P. cyrtonema and perform a preliminary bioinformatic analysis. Result In this study, the mitochondrial genome of P. cyrtonema was assembled and annotated by combining Illumina reads with long-read Oxford Nanopore Technologies, which formed a complex non-circular structure covering 664,991 bp in length, with 33 protein-coding genes, 20 tRNAs, and 3 rRNAs. Overall, a total of 194 simple repeats, 24 tandem repeats, and 294 dispersed repeats were identified, and 599 RNA editing sites were predicted, all of which were C-to-T types. In particular, 29 homologous fragments between the chloroplast and mitochondrial genome were detected and accounted for 2.4% of the mitochondrial genome. In addition, codon usage analysis, nucleotide diversity analysis, and Ka/Ks analysis suggested a slower rate of evolution and a relatively conserved structure of the mitochondrial genome. Phylogenetic relationships based on 33 species demonstrated that P. cyrtonema was most closely related to Polygonatum sibiricum . Conclusion Our results provide comprehensive information on the mitochondrial genome of P. cyrtonema , and demonstrate the availability of mitochondrial genome-based taxonomic classification for Polygonatum Mill.. Moreover, it offers a valuable foundation for future research in the cultivation and pharmacological development within Polygonatum species.

Background Porcine epidemic diarrhea (PED) is an infectious disease of the digestive tract caused by the porcine epidemic diarrhea virus (PEDV), characterized by vomiting, severe diarrhea, and high mortality rates in piglets. In recent years, the distribution of this disease in China has remarkably increased, and its pathogenicity has also increased. PEDV has been identified as the main cause of viral diarrhea in piglets. This study aimed to understand the genetic evolution and diversity of PEDV to provide a theoretical basis for the development of new vaccines and the prevention and treatment of PED. Methods A PEDV strain was isolated from the small intestine of a diarrheal piglet using Vero cells. The virus was identified using reverse transcription-polymerase chain reaction (RT-PCR), indirect immunofluorescence assay (IFA), and transmission electron microscopy. The whole genome sequence was sequenced, phylogenetic analysis was conducted using MEGA (version 7.0), and recombination analysis was performed using RDP4 and SimPlot. The S protein amino acid sequence was aligned using Cluster X (version 2.0), and the S protein was modeled using SWISS-MODEL to compare differences in structure and antigenicity. Finally, the piglets were inoculated with PEDV to evaluate its pathogenicity in newborn piglets. Result PEDV strain CH/HLJ/18 was isolated. CH/HLJ/18 shared 89.4–99.2% homology with 52 reference strains of PEDV belonging to the GII-a subgroup. It was a recombinant strain of PEDV BJ-2011-1 and PEDV CH_hubei_2016 with a breakpoint located in ORF1b. Unique amino acid deletions and mutations were observed in the CH/HLJ/18 S protein. The piglets then developed severe watery diarrhea and died within 7 d of inoculation with CH/HLJ/18, suggesting that CH/HLJ/18 was highly pathogenic to newborn piglets. Conclusion A highly pathogenic recombinant PEDV GII-a strain, CH/HLJ/18, was identified in China, with unique deletion and mutation of amino acids in the S protein that may lead to changes in protein structure and antigenicity. These results will be crucial for understanding the prevalence and variation of PEDV and for preventing and controlling PED.

Bordetella pertussis not expressing pertactin has increased in countries using acellular pertussis vaccines (ACV). The deficiency is mostly caused by pertactin gene disruption by IS481. To assess the effect of the transition from whole-cell vaccine to ACV on the emergence of B. pertussis not expressing pertactin in Spain, we studied 342 isolates collected during 1986-2018. We identified 93 pertactin-deficient isolates. All were detected after introduction of ACV and represented 38% of isolates collected during the ACV period; 58.1% belonged to a genetic cluster of isolates carrying the unusual prn::del(-292, 1340) mutation. Pertactin inactivation by IS481 insertion was identified in 23.7% of pertactin-deficient isolates, arising independently multiple times and in different phylogenetic branches. Our findings support the emergence and dissemination of a cluster of B. pertussis with an infrequent mechanism of pertactin disruption in Spain, probably resulting from introduction of ACV.

Background Forsythia suspensa (Thunb.) Vahl is a valuable ornamental and medicinal plant. Although the nuclear and chloroplast genomes of F. suspensa have been published, its complete mitochondrial genome sequence has yet to be reported. In this study, the genomic DNA of F. suspensa yellowish leaf material was extracted, sequenced by using a mixture of Illumina Novaseq6000 short reads and Oxford Nanopore PromethION long reads, and the sequencing data were assembled and annotated. Result The F. suspensa mitochondrial genome was obtained in the length of 535,692 bp with a circular structure, and the GC content was 44.90%. The genome contains 60 genes, including 36 protein-coding genes, 21 tRNA genes, and three rRNA genes. We further analyzed RNA editing of the protein-coding genes, relative synonymous codon usage, and sequence repeats based on the genomic data. There were 25 homologous sequences between F. suspensa mitochondria and chloroplast genome, which involved the transfer of 8 mitochondrial genes, and 9473 homologous sequences between mitochondrial and nuclear genomes. Analysis of the nucleic acid substitution rate, nucleic acid diversity, and collinearity of protein-coding genes of the F. suspensa mitochondrial genome revealed that the majority of genes may have undergone purifying selection, exhibiting a slower rate of evolution and a relatively conserved structure. Analysis of the phylogenetic relationships among different species revealed that F. suspensa was most closely related to Olea europaea subsp. Europaea. Conclusion In this study, we sequenced, assembled, and annotated a high-quality F. suspensa mitochondrial genome. The results of this study will enrich the mitochondrial genome data of Forsythia , lay a foundation for the phylogenetic development of Forsythia , and promote the evolutionary analysis of Oleaceae species.

To explore the dyeing technique and karyotype analysis of winter turnip rape (Brassica rape L.), the root tip of winter turnip rape Longyou 7 was used as the experimental material. Chromosome preparation technology was optimized, and karyotype analysis was carried out by changing the conditions of material collection time, pretreatment, fixation, and dissociation. The results showed that the optimal conditions for the preparation of dyeing winter turnip rape were as follows: the sampling time was 8:00–10:00, the ice–water mixture was pretreated at 4 °C for 20 h, the Carnot’s fixative solution I and 4 °C were fixed for 12 h, and the 1 mol/L HCl solution was bathed in a water bath at 60 °C for 10~15 min. Karyotype analysis showed that the number of chromosomes in winter turnip rape cells was 2n = 20, and the karyotype analysis formula was 2n = 2x = 20 = 16m + 4sm. The karyotype asymmetry coefficient was 58.85%, and the karyotype type belonged to type 2A, which may belong to the primitive type in terms of evolution. The results of this study provide a theoretical basis for further in-depth study of the phylogenetic evolution and genetic trend of Brassica rapa.