Chromosome-level genome and population genomics of the intermediate horseshoe bat(Rhinolophus affinis)reveal the molecular basis of virus tolerance in Rhinolophus and echolocation call frequency variation

Horseshoe bats(genus Rhinolophus,family Rhinolophidae)represent an important group within chiropteran phylogeny due to their distinctive traits,including constant high-frequency echolocation,rapid karyotype evolution,and unique immune system.Advances in evolutionary biology,supported by high-quality reference genomes and comprehensive whole-genome data,have significantly enhanced our understanding of species origins,speciation mechanisms,adaptive evolutionary processes,and phenotypic diversity.However,genomic research and understanding of the evolutionary patterns of Rhinolophus are severely constrained by limited data,with only a single published genome of R.ferrumequinum currently available.In this study,we constructed a high-quality chromosome-level reference genome for the intermediate horseshoe bat(R.affinis).Comparative genomic analyses revealed potential genetic characteristics associated with virus tolerance in Rhinolophidae.Notably,we observed expansions in several immune-related gene families and identified various genes functionally associated with the SARS-CoV-2 signaling pathway,DNA repair,and apoptosis,which displayed signs of rapid evolution.In addition,we observed an expansion of the major histocompatibility complex class II(MHC-II)region and a higher copy number of the HLA-DQB2 gene in horseshoe bats compared to other chiropteran species.Based on whole-genome resequencing and population genomic analyses,we identified multiple candidate loci(e.g.,GLI3)associated with variations in echolocation call frequency across R.affinis subspecies.This research not only expands our understanding of the genetic characteristics of the Rhinolophus genus but also establishes a valuable foundation for future research.

Analysis of environmental selection pressure of superoxide dismutase in deep-sea sea cucumber

Manganese superoxide dismutase(MnSOD)is an antioxidant that exists in mitochondria and can effectively remove superoxide anions in mitochondria.In a dark,high-pressure,and low-temperature deep-sea environment,MnSOD is essential for the survival of sea cucumbers.Six MnSODs were identified from the transcriptomes of deep and shallow-sea sea cucumbers.To explore their environmental adaptation mechanism,we conducted environmental selection pressure analysis through the branching site model of PAML software.We obtained night positive selection sites,and two of them were significant(97F→H,134K→V):97F→H located in a highly conservative characteristic sequence,and its polarity c hange might have a great impact on the function of MnSOD;134K→V had a change in piezophilic a bility,which might help MnSOD adapt to the environment of high hydrostatic pressure in the deepsea.To further study the effect of these two positive selection sites on MnSOD,we predicted the point mutations of F97H and K134V on shallow-sea sea cucumber by using MAESTROweb and PyMOL.Results show that 97F→H,134K→V might improve MnSOD’s efficiency of scavenging superoxide a nion and its ability to resist high hydrostatic pressure by moderately reducing its stability.The above results indicated that MnSODs of deep-sea sea cucumber adapted to deep-sea environments through their amino acid changes in polarity,piezophilic behavior,and local stability.This study revealed the correlation between MnSOD and extreme environment,and will help improve our understanding of the organism’s adaptation mechanisms in deep sea.

Genetic mechanism of body size variation in groupers:Insights from phylotranscriptomics

Animal body size variation is of particular interest in evolutionary biology,but the genetic basis remains largely unknown.Previous studies have shown the presence of two parallel evolutionary genetic clusters within the fish genus Epinephelus with evident divergence in body size,providing an excellent opportunity to investigate the genetic basis of body size variation in vertebrates.Herein,we performed phylotranscriptomic analysis and reconstructed the phylogeny of 13 epinephelids originating from the South China Sea.Two genetic clades with an estimated divergence time of approximately 15.4 million years ago were correlated with large and small body size,respectively.A total of 180 rapidly evolving genes and two positively selected genes were identified between the two groups.Functional enrichment analyses of these candidate genes revealed distinct enrichment categories between the two groups.These pathways and genes may play important roles in body size variation in groupers through complex regulatory networks.Based on our results,we speculate that the ancestors of the two divergent groups of groupers may have adapted to different environments through habitat selection,leading to genetic variations in metabolic patterns,organ development,and lifespan,resulting in body size divergence between the two locally adapted populations.These findings provide important insights into the genetic mechanisms underlying body size variation in groupers and species differentiation.

Comparative transcriptomes reveal the disjunction adaptive strategy of Thuja species in East Asia and North America

The genus Thuja is ideal for investigating the genetic basis of the East Asia-North America disjunction.The biogeographical background of the genus is debatable and an adaptive strategy is lacking.Through the analysis and mining of comparative transcriptomes,species differentiation and positively selected genes(PSGs)were identified to provide information for understanding the environmental adaptation strategies of the genus Thuja.De novo assembly yielded 44,397-74,252 unigenes of the five Thuja species with contig N50length ranging from 1,559 to 1,724 bp.Annotations revealed a similar distribution of functional categories among them.Based on the phylogenetic trees constructed using the transcriptome data,T.sutchuenensis was divided first,followed by T.plicata and T.occidentalis.The final differentiation of T.koraiensis and T.standishii formed a clade.Enrichment analysis indicated that the PSGs of the North American Thuja species were involved in plant hormone signal transduction and carbon fixation of photosynthetic organisms pathways.The PSGs of East Asian Thuja were related to phenolic,alkaloid,and terpenoid synthesis,important stress-resistant genes and could increase plant resistance to external environmental stresses.This study discovered numerous aroma synthetic-related PSGs including terpene synthase(TPS)genes and lipid phosphate phosphatase 2(LPP2),associated with the synthetic aroma of T.sutchuenensis.Physiological indicators,such as the contents of soluble sugars,total chlorophyll,total phenolics,and total flavonoids were determined,which are consistent with the PSGs enrichment pathways associated with adaptive strategies in the five Thuja species.The results of this study provide an important basis for future studies on conservation genetics.

Adaptive Evolution of Large Subunits of RubisCO in Magnoliophyta Crops

[Objective] The aim was to study adaptive evolution of the large subunits of RubisCO in Magnoliophyta crops. [Method] Taking Magnoliophyta crops such as corn and rice as research materials, the analysis on molecular adaptive evolution was carded out by using codon replacement and maximum likelihood methods. [ Result] The RubisCO suffered positive selection effect and six amino acid sites were identified. [ Conclusion] The six amino acid sites are of important guiding significance for studying catalytic activity of RubisCO large subunits and crop improvement.

Evidence for Positive Darwinian Selection of Vip Gene in Bacillus thuringiensis

Vegetative insecticidal proteins （VIPs）, produced during the vegetative stage of their growth in Bacillus thuringiensis, are a group of insecticidal proteins and represent the second generation of insecticidal trans-genes that will complement the novel δendotoxins in future. Fewer structural and functional relationships of Vip proteins are known in comparison with those of δ-endotoxins. In this study, both the maximum-likelihood methods and the maximum parsimony based sliding window analysis were used to evaluate the molecular evolution of Vip proteins. As a result, strong evidence was found that Vip proteins are subject to the high rates of positive selection, and 16 sites are identified to be under positive selection using the Bayes Empirical Bayesian method. Interestingly, all these positively selected sites are located from site-705 to site-809 in the C-terminus of the Vip proteins. Most of these sites are exposed and clustered in the loop regions when mapped onto its computational predicted secondary tertiary and a part of the tertiary structure. It has been postulated that the high divergence in the C-terminal of Vip proteins may not result from the lack of functional constraints, but rather from the rapid mutation to adapt their targeted insects, driven by positive selection. The potential positive selection pressures may be an attempt to adapt for the ＂arm race＂ between Vip proteins and the targeted insects, or to enlarge their target＇s host range. Sites identified to be under positive selection may be related to the insect host range, which may shed a light on the investigation of the Vip proteins＇ structural and functional relationships.

GCH1 plays a role in the high-altitude adaptation of Tibetans

Tibetans are welt adapted to high-altitude hypoxia. Previous genome-wide scans have reported many candidate genes for this adaptation, but only a few have been studied. Here we report on a hypoxia gene （GCH1, GTP-cyclohydrolase I）, involved in maintaining nitric oxide synthetase （NOS） function and normal blood pressure, that harbors many potentially adaptive variants in Tibetans. We resequenced an 80.8 kb fragment covering the entire gene region of GCH1 in 50 unrelated Tibetans Combined with previously published data, we demonstrated many GCHI variants showing deep divergence between highlander Tibetans and lowlander Han Chinese. Neutrality tests confirmed a signal of positive Darwinian selection on GCH1 in Tibetans. Moreover, association analysis indicated that the Tibetan version of GCH1 was significantly associated with multiple physiological traits in Tibetans, including blood nitric oxide concentration, blood oxygen saturation and hemoglobin concentration. Taken together, we propose that GCH1 plays a role in the genetic adaptation of Tibetans to high altitude hypoxia.

Current status of genetic transformation technology developed in cucumber(Cucumis sativus L.)

Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cucumber, and its frequency has been up to 23%. For example, significantly enhancement of the transformation efficiency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efficient positive selection system in lots of experiments. This review is to summarize some key factors influencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efficiency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.

Positive Selection Analysis of VP1 Genes of Worldwide Human Enterovirus 71 Viruses

Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.

c-Fos enhances the survival of thymocytes during positive selection by upregulating Bcl-2

T cells are derived from progenitor thymocytes, of which only a minority receive the appropriate TCR signal, undergo positive selection and mature. Owing to the very short lifespan of thymocytes, the prerequisite for posi- tive selection is survival. TCR signal-induced Bcl-2 expression is believed to play a dominant role in the survival of positively selecting thymocytes, but how Bcl-2 is directly regulated is unknown. Here we report that the immediate early gene （IEG） c-Fos can stimulate the expression of Bcl-2, depending on a specific AP-l-binding site in the Bcl-2 promoter. In c-Fos transgenic （Fos-Tg） mice, c-Fos binds to this site and promotes the expression of Bcl-2. As a result, Fos-Tg thymocytes exhibited enhanced survival, and more mature single-positive （SP） thymocytes were generated, even on a unique TCR background. The TCR repertoire remained normal in Fos-Tg mice. Our results identified c-Fos as the mediator of the stimulatory effect of TCR signaling on Bcl-2 expression. Therefore, c-Fos, as an IEG, because of its early response ability, can quickly rescue the survival of short-lived thymocytes during positive selection. Our results provide novel insight into the mechanism regulating the survival of positively selecting thymocytes.

Genome-wide evolution of MAPKs family and their expression in response to bacterial infection in seahorse Hippocampus erectus

Seahorses have evolved many unique biological traits,including a male brood pouch,the absence of caudal and pelvic fins,and the lack of spleen and gut-associated lymphatic tissue.The mitogenactivated protein kinases(MAPKs)are known to be involved in various important biological processes including growth,differentiation,immunity,and stress responses.Therefore,we hypothesized that the adaptive evolution and expression of the MAPK gene family in seahorse may differ from those of other teleost species.We identified positive selection sites in the erk2,erk5,jnk1,and p38αMAPK genes of the lined seahorse Hippocampus erectus and tiger-tailed seahorse Hippocampus comes.A novel expression profile of MAPK cascade genes was found in seahorse larvae during the first day after birth based on the RNA-seq data of H.erectus,which refl ected vital signs of immune response to its parental immune system.The expression patterns of the four positively selected MAPK genes were analyzed following the bacterial challenge of Vibrio fortis,revealing their upregulation pattern in brood pouch and other immune tissues.This study enriched our knowledge of the evolution of the H.erectus MAPK subfamilies,and could help better understanding the functional role of MAPKs in teleosts.

Genes for the high life： New genetic variants point to positive selection for high altitude hypoxia in Tibetans

People living on the high plateaus of the world have long fascinated biological anthropologists and geneticists because they live in ＂thin air＂ and epitomize an extreme of human biological adaptation.

Evolutionary implications of Avian Infectious Bronchitis Virus(AIBV)analysis

For developing efficient vaccines, it is essential to identify which amino acid changes are most important to the survival of the virus. We investigate the amino acid substitution features in the Avian Infectious Bronchitis Virus （AIBV） antigenic domain of a vaccine serotype （DE072） and a virulent viral strain （GA98） to better understand adaptive evolution of AIBV. In addition, the SARS Coronavirus （SARS-CoV） was also analyzed in the same way. It is interesting to find that extreme comparability exists between AIBV and SARS in amino acid substitution pattern. It suggests that amino acid changes that result in overall shift of residue charge and polarity should be paid special attention to during the development of vaccines.

Positively selected genes of the Chinese tree shrew （Tupaia belangeri chinensis） locomotion system

While the recent release of the Chinese tree shrew （Tupaia belangeri chinensis） genome has made the tree shrew an increasingly viable experimental animal model for biomedical research, further study of the genome may facilitate new insights into the applicability of this model. For example, though the tree shrew has a rapid rate of speed and strong jumping ability, there are limited studies on its locomotion ability. In this study we used the available Chinese tree shrew genome information and compared the evolutionary pattern of 407 locomotion system related orthologs among five mammals （human, rhesus monkey, mouse, rat and dog） and the Chinese tree shrew. Our analyses identified 29 genes with significantly high co （Ka/Ks ratio） values and 48 amino acid sites in 14 genes showed significant evidence of positive selection in the Chinese tree shrew. Some of these positively selected genes, e.g. HOXA6 （homeobox A6） and AVP （arginine vasopressin）, play important roles in muscle contraction or skeletal morphogenesis. These results provide important clues in understanding the genetic bases of locomotor adaptation in the Chinese tree shrew.

Comparative analysis of the complete chloroplast genomes of six white oaks with high ecological amplitude in China

Quercus L.has significant societal,ecological and economic benefits in the Northern Hemisphere.However,species identification among oaks is notoriously difficult.China harbours highly diverse oaks,of which the diversity of white oaks is the most extensive;however,to date,the evolution of chloroplast(cp)genomes in white oaks in China has not been comprehensively studied.Thus,we sequenced the complete cp genomes(161,254 bp,161,229 bp and 161,254 bp in size)of three white oak species(Quercus serrata Thunb.var.brevipetiolata A.DC.Nakai,Quercus wutaishansea Mary and Quercus mongolica Fischer ex Ledebour,respectively).Six white oak species(Quercus aliena Blume,Quercus dentata Thunb.,Quercus aliena Blume var.acutiserrata Maximowicz ex Wenzig,Q.serrata var.brevipetiolata,Q.wutaishanica and Q.mongolica)and five other Fagaceae species(Quercus rubra L.,Quercus variabilis Bl.,Quercus aquifolioid.es Rehd.et Wils.,Fagus engleriana Seem.and Castanea henryi Skan Rehd.et Wils.)were retrieved for comparative analyses.We detected11 highly divergent regions(psbA,matK/rps16,rps16,trnSGCU/trnG-GCC,trnR-UCU/atpA,trnT-GGU/psbD,ndhJ,ndhJ/ndhK,accD,ndhF and ycfl)through comparative analyses and these regions might be used as molecular markers.Theωratio of the rps12,rpoC2 and ycf1 genes was greater than 1 in several comparison groups between white oaks and the petA gene was subjected to significant positive selection between the comparison of six white oaks and Q.variabilis.Phylogenetic analyses revealed that six white oaks were grouped with Q.rubra,forming a single clade.

New insights into the evolutionary history of Megacodon:Evidence from a newly discovered species

Megacodon is an ideal genus to study speciation and ecological adaptation in the Sino-Himalayan region.The genus contains two species distributed at different elevations and in two separate areas.However,studies of this genus have long been impeded by a lack of fieldwork on one of its species,Megacodon venosus.In this study,we collected specimens of two Megacodon species and found an extraordinary new species of Megacodon in Lushui county of north-west Yunnan province,which we have since named Megacodon lushuiensis.We propose new species based on both morphological and molecular evidence.The finding of this new species emphasized the importance of ecological divergence in the divergence of Megacodon stylophorus and its parapatric low-elevation Megacodon species.To identify genetic determinants that underlie adaptations to different elevations,we characterized transcriptomes of the new species M.lushuiensis,which is distributed at low elevations,and M.stylophorus,which is distributed at high elevations.Comparative transcriptome analysis identified 8926 orthogroups containing single-copy genes,and 370 orthogroups containing significantly positively selected genes.The set of positively selected genes was enriched into 25 Gene Ontology terms,including "response to water deprivation","response to osmotic stress",and "cellular response to external stimulus".Our results provide new insights into how ecological adaptation and speciation occurred in Megacodon and highlight the role of heterogeneous habitats in the speciation of plants in the Sino-Himalayan region.

Positive selection analysis reveals the deep-sea adaptation of a hadal sea cucumber ( Paelopatides sp.) to the Mariana Trench

The Mariana Trench,the deepest trench on the earth,is ideal for deep-sea adaptation research due to its unique characters,such as the highest hydrostatic pressure on the Earth,constant ice-cold temperature,and eternal darkness.In this study,tissues of a the hadal holothurian(Paelopatides sp.)were fi xed with RNA later in situ at~6501-m depth in the Mariana Trench,which,to our knowledge,is the deepest in-situ fi xed animal sample.A high-quality transcript was obtained by de-novo transcriptome assembly.A maximum likelihood tree was constructed based on the single copy orthologs across nine species with their available omics data.To investigate deep-sea adaptation,113 positively selected genes(PSGs)were identifi ed in Paelopatides sp.Some PSGs such as microphthalmia-associated transcription factor(MITF)may contribute to the distinct phenotype of Paelopatides sp.,including its translucent white body and degenerated ossicles.At least eight PSGs(transcription factor 7-like 2[TCF7L2],ETS-related transcription factor Elf-2-like[ELF2],PERQ amino acid-rich with GYF domain-containing protein[GIGYF],cytochrome c oxidase subunit 7a,[COX7A],type I thyroxine 5′-deiodinase[DIO1],translation factor GUF1[GUF1],SWI/SNF related-matrix-associated actin-dependent regulator of chromatin subfamily C and subfamily E,member 1[SMARCC]and[SMARCE1])might be related to cold adaptation.In addition,at least nine PSGs(cell cycle checkpoint control protein[RAD9A],replication factor A3[RPA3],DNA-directed RNA polymerases I/II/III subunit RPABC1[POLR2E],putative TAR DNA-binding protein 43 isoform X2[TARDBP],ribonucleoside-diphosphate reductase subunit M1[RRM1],putative serine/threonine-protein kinase[SMG1],transcriptional regulator[ATRX],alkylated DNA repair protein alkB homolog 6[ALKBH6],and PLAC8 motif-containing protein[PLAC8])may facilitate the repair of DNA damage induced by the high hydrostatic pressure,coldness,and high concentration of cadmium in the upper Mariana Trench.

Transcriptome assembly of Modiolus modiolus and comparative analysis with Bathymodiolus platifrons

The genetic basis for bivalves＇ adaptation and evolution is not well understood. Even few studies have focused on the mechanism of molluscan molecular evolution between the coastal intertidal zone and deep-sea environment.In our studies, we first conducted the transcritpome assembly of Modiolus modiolus mussels living in coastal intertidal zones. Also, we conducted transcriptome comparison analyses between M. modiolus and Bathymodiolus platifrons living in hydrothermal vents and cold methane/sulfide-hydrocarbon seeps. De novo assemblies of the clean reads yielded a total of 182 476 and 156 261 transcripts with N50 values of 1 769 and 1 545 in M. modiolus and B. platifrons. A total of 27 868 and 23 588 unigenes were identified, which also displayed the similar GO representation patterns. Among the 10 245 pairs of putative orthologs, we identified 26 protein-coding genes under strong positive selection（Ka/Ks〉1） and 12 genes showing moderate positive selection（0.5

Identification of genes under positive selection reveals evolutionary adaptation of Ulva mutabilis

Ulvophytes are attractive model systems for understanding the evolution of growth,development,and environmental stress responses.They are untapped resources for food,fuel,and high-value compounds.The rapid and abundant growth of Ulva species makes them key contributors to coastal biogeochemical cycles,which can cause significant environmental problems in the form of green tides and biofouling.Until now,the Ulva mutabilis genome is the only Ulva genome to have been sequenced.To obtain further insights into the evolutionary forces driving divergence in Ulva species,we analyzed 3905 single copy ortholog family from U.mutabilis,Chlamydomonas reinhardtii and Volvox carteri to identify genes under positive selection(GUPS)in U.mutabilis.We detected 63 orthologs in U.mutabilis that were considered to be under positive selection.Functional analyses revealed that several adaptive modifications in photosynthesis,amino acid and protein synthesis,signal transduction and stress-related processes might explain why this alga has evolved the ability to grow very rapidly and cope with the variable coastal ecosystem environments.

Transcriptomic comparison to identify rapidly evolving genes in Braya humilis

The Brassicaceae species Braya humilis shows broad adaptation to different climatic zones and latitudes. However, the molecular adaptation mechanism of B. humilis is poorly understood. In China, B. humilis is mainly distributed on the QinghaiTibetan Plateau(QTP) and in the adjacent arid region. Previous transcriptome analysis of B. humilis has revealed that 39 salt and osmotic stress response genes are subjected to purifying selection during its speciation. To further explore the adaptation mechanism of B. humilis to an arid environment, OrthoMCL program was employed in this study and 6,268 pairs of orthologous gene pairs with high confidence were obtained between B. humilis and Arabidopsis thaliana. A comparative evolutionary analysis based on nonsynonymous to synonymous substitution ratio(Ka/Ks) was then conducted. There were 64 pairs exhibiting a Ka/Ks ratio more than 0.5 and among which, three instrumental candidate genes, T20487,T22576, and T23757, were identified with strong selection signatures(Ka/Ks >1). The corresponding A. thaliana orthologs are double-stranded RNA-binding domain protein, MADS-box family protein, and NADH-dehydrogenase subunit6, which is encoded by mitochondria genome. This report not only demonstrates the adaptation contribution of fast evolving nuclear genes, but also highlights the potential adaptive value of mitochondria gene to the speciation and adaptation of B. humilis toward the extreme environment in an arid region.