Mitochondrial genomes of Tapes dorsatus and Cardita variegata:insights into Heteroconchia phylogeny

Heteroconchia,a widespread and abundant aquatic invertebrate,is an important clade of bivalve mollusks.The relationship between the three branches of Heteroconchia,Palaeoheterodonta,Archiheterodonta,and Euheterodonta has become a main controversy in molecular studies of the relationships between bivalves.In the present study,we assembled the complete mitochondrial genomes of Tapes dorsatus(Veneridae)and Cardita variegata(Carditidae)using high-throughput sequencing.C.variegata is the first mitochondrial genome belonging to the family Carditidae to be reported.We used 12 protein coding genes(excluding atp8)from the complete mitochondrial genomes of 146 species to recover the internal relationships of Heteroconchia.Our results support the traditional view of early branching of Palaeoheterodonta and the recovery of the monophyly of Palaeoheterodonta,Anomalodesmata,Imparidentia.Rearrangement analysis show that gene arrangement within Venerida was highly variable.Time-calibrated phylogenetic studies based on a relaxed molecular clock model suggested that Veneridae originated approximately 337.62 million years ago(Ma)and split into two major clades,whereas Carditidae originated approximately 510.09 Ma.Our results provide evidence of the internal relationships of Heteroconchia.

Molecular phylogenetic relationships based on mitochondrial genomes of novel deep-sea corals(Octocorallia:Alcyonacea):Insights into slow evolution and adaptation to extreme deep-sea environments

A total of 10 specimens of Alcyonacea corals were collected at depths ranging from 905 m to 1633 m by the manned submersible Shenhai Yongshi during two cruises in the South China Sea(SCS).Based on mitochondrial genomic characteristics,morphological examination,and sclerite scanning electron microscopy,the samples were categorized into four suborders(Calcaxonia,Holaxonia,Scleraxonia,and Stolonifera),and identified as 9 possible new cold-water coral species.Assessments of GC-skew dissimilarity,phylogenetic distance,and average nucleotide identity(ANI)revealed a slow evolutionary rate for the octocoral mitochondrial sequences.The nonsynonymous(Ka)to synonymous(Ks)substitution ratio(Ka/Ks)suggested that the 14 protein-coding genes(PCGs)were under purifying selection,likely due to specific deep-sea environmental pressures.Correlation analysis of the median Ka/Ks values of five gene families and environmental factors indicated that the genes encoding cytochrome b(cyt b)and DNA mismatch repair protein(mutS)may be influenced by environmental factors in the context of deep-sea species formation.This study highlights the slow evolutionary pace and adaptive mechanisms of deep-sea corals.

Gene characterization and phylogenetic analysis of four mitochondrial genomes in Caenogastropoda

Caenogastropoda is a highly diverse group,containing~60%of all existing gastropods.Species in this subclass predominantly inhabit marine environments and have a high ecological and economic value.Owing to the increase in relevant phylogenetic studies,our understanding of between species relatedness in Caenogastropoda has improved.However,the biodiversity,taxonomic status,and phylogenetic relationships of this group remain unclear.In the present study,we performed next-generation sequencing of four complete mitochondrial genomes from three families(Buccinidae,Columbellidae,and Cypraeidae)and the four mitogenomes were classical circular structures,with a length of 16177 bp in Volutharpa ampullacea,16244 bp in Mitrella albuginosa,16926bp in Mauritia arabica asiatica and 15422 bp in Erronea errones.Base composition analysis indicated that whole sequences were biased toward A and T.Then compared them with 171 complete mitochondrial genomes of Caenogastropoda.The phylogenetic relationship of Caenogastropoda derived from Maximum Likelihood(ML)and Bayesian Inference(BI)trees constructed based on CDS sequences was consistent with the results of traditional morphological analysis,with all three families showing close relationships.This study supported Caenogastropoda at the molecular level as a separate clade of Mollusca.According to our divergence time estimations,Caenogastropoda was formed during the middle Triassic period(~247.2–237 Ma).Our novel mitochondrial genomes provide evidence for the speciation of Caenogastropoda in addition to elucidating the mitochondrial genomic evolution of this subclass.

Evolutionary timescale of chalcidoid wasps inferred from over one hundred mitochondrial genomes

Chalcidoidea is one of the most biologically diverse groups among Hymenoptera.Members are characterized by extraordinary parasitic lifestyles and extensive host ranges,among which several species attack plants or serve as pollinators.However,higher-level chalcidoid relationships remain controversial.Here,we performed mitochondrial phylogenomic analyses for major clades(18out of 25 families)of Chalcidoidea based on 139 mitochondrial genomes.The compositional heterogeneity and conflicting backbone relationships in Chalcidoidea were assessed using various datasets and tree inferences.Our phylogenetic results supported the monophyly of 16families and polyphyly of Aphelinidae and Pteromalidae.Our preferred topology recovered the relationship(Mymaridae+(Signiphoridae+Leucospidae)+(Chalcididae+((Perilampidae+Eucharitidae)+remaining Chalcidoidea))).The monophyly of Agaonidae and Sycophaginae was rejected,while the gall-associated((Megastigmidae+Ormyridae)+(Ormocerinae+Eurytomidae))relationship was supported in most results.A six-gene inversion may be a synapomorphy for most families,whereas other derived gene orders may introduce confusion in phylogenetic signals at deeper nodes.Dating estimates suggested that Chalcidoidea arose near the Jurassic/Cretaceous boundary and that two dynamic shifts in diversification occurred during the evolution of Chalcidoidea.We hypothesized that the potential codiversification between chalcidoids and their hosts may be crucial for accelerating the diversification of Chalcidoidea.Ancestral state reconstruction analyses supported the hypothesis that gallinducers were mainly derived from parasitoids of gallinducers,while other gall-inducers were derived from phytophagous groups.Taken together,these findings advance our understanding of mitochondrial genome evolution in the major interfamilial phylogeny of Chalcidoidea.

Assembly and phylogenomic analysis of cotton mitochondrial genomes provide insights into the history of cotton evolution

Cotton is a major crop that provides the most important renewable textile fibers in the world.Studies of the taxonomy and evolution of cotton species have received wide attentions,not only due to cotton’s economic value but also due to the fact that Gossypium is an ideal model system to study the origin,evolution,and cultivation of polyploid species.Previous studies suggested the involvement of mitochondrial genome editing sites and copy number as well as mitochondrial functions in cotton fiber elongation.Whereas,with only a few mitogenomes assembled in the cotton genus Gossypium,our knowledge about their roles in cotton evolution and speciation is still scarce.To close this gap,here we assembled 20 mitogenomes from 15 cotton species spanning all the cotton clades(A–G,K,and AD genomes)and 5 cotton relatives using short and long sequencing reads.Systematic analyses uncovered a high level of mitochondrial gene sequence conservation,abundant sequence repeats and many insertions of foreign sequences,as well as extensive structural variations in cotton mitogenomes.The sequence repeats and foreign sequences caused significant mitogenome size inflation in Gossypium and its close relative Kokia in general,while there is no significant difference between the lint and fuzz cotton mitogenomes in terms of gene content,RNA editing,and gene expression level.Interestingly,we further revealed the specific presence and expression of two novel mitochondrial open reading frames(ORFs)in lint-fiber cotton species.Finally,these structural features and novel ORFs help us gain valuable insights into the history of cotton evolution and polyploidization and the origin of species producing long lint fibers from a mitogenomic perspective.

Assembly and marker analysis of mitochondrial genomes provide insights into origin,evolution and spread of Brassica juncea(L.)Czern.et Coss.

The release of mitochondrial genome sequences provides the basis for characterizing interspecific and intraspecific variation in Brassica mitochondrial genomes.However,few B.juncea(mustard)mitochondrial genomes have been published.We assembled the mitochondrial genomes of three B.juncea subspecies and compared them with previously published genomes.The genomes were phylogenetically classified into A,B,C,and Bna clades.Two variant sites,a transversion(C→A)at nt 79,573 and a 31-bp copy-number variation between nts 65,564 and 65,596,were identified.Based on these variant sites,mitotype-specific sequence markers were developed to characterize the variation among worldwide 558 B.juncea accessions.Three mitochondrial genome types(mitotypes MT1–MT3)were identified.In terms of geographical distribution,MT1 and MT2 accessions were distributed mainly to the north and MT3 to the south of 34°N.Root mustards carried only MT1,leaf and stem mustards carried mainly MT3,and seed mustards carried all three mitotypes,implying that the mitotypes underwent selection during B.juncea domestication.A new form of oil mustard evolved by hybridization between two gene pools in southwest China.

Novel evolutionary insights into nemacheilid cavefi sh: evidence from comparative analysis of mitochondrial genomes

Cavefi sh can be important models for understanding the relationships among evolution,adaptation,and development in extreme environments.However,cavefi sh remain poorly studied,particularly at the genome level.Here,we sequenced the complete mitogenome of three cavefi sh in the family Nemacheilidae(Paranemachilus pingguoensis,Oreonectes polystigmus,and Heminoemacheilus longibarbatus),which were collected from karst caves in South China.The mitogenomes each contained 37 genes(13 protein coding,22 tRNA,and two rRNA genes)and a single control region,with the same genetic arrangement and distribution as those found in vertebrates.The non-synonymous/synonymous mutation ratios(Ka/Ks)of the mitogenomes indicated that the protein-coding genes(PCGs)of the three cavefi sh evolved under purifying selection.The mitogenomes of the three cavefi sh exhibit nucleotide composition biases for PCGs,tRNAs,rRNAs,and the whole genome,indicating that the mitochondrial DNA might have been subjected to evolutionary selection in response to extreme cave environments.Divergence time and evolutionary history analyses suggested that the speciation and diversifi cation of the cavefi sh coincided with the Miocene uplift of the southern Qinghai-Tibet Plateau,which greatly changed cave habitats.Overall,our study sheds light on the mitogenomes,phylogeny,and evolutionary history of nemacheilid cavefi sh.

Relaxation of Selective Constraint on the Ultra-Large Mitochondrial Genomes of Arcidae (Mollusca: Bivalvia)

The mitochondrial genomes(mitogenomes)are purportedly under selection for smaller size to improve their replica-tion and translation efficiency.However,the mitogenomes of Arcidae species are larger than those of other bivalves,and are among the largest metazoan mitogenomes reported to date.In order to explore the differences of base composition and selective constraints between the large and small mitogenomes,we compared the mitogenomes of 9 large arcid mitogenomes and 77 small bivalves mitogenomes.Base composition analyses indicated that Arcidae mitogenomes have significantly greater GC skews in both their whole genomes and coding sequences.This result suggests that the replication of the large mitogenomes in Arcidae may be slower than those in other bivalves,exposing the parental strand to deamination for a longer time.Selection pressure analyses showed that the mitochondrial protein-coding genes of Arcidae species have significantly higher Ka/Ks ratios than other bivalves,suggesting that they have accumulated more nonsynonymous nucleotide substitutions.Seven protein-coding genes(atp6,cox1-3,nad1,nad4 and nad5)show significant difference for Ka/Ks ratios between the Arcidae and non-Arcidae groups.However,these divergences are not observed in the nuclear gene within histone H3.From these observations,we concluded that the large mitoge-nomes of Arcidae species experienced more relaxed selective constraints.As some Arcidae species are more tolerant to hypoxia that can lead to low metabolic rate,the relaxed selective constraints of mitogenomes may be energy-related.This study provides new insights into the evolution of Arcidae mitogenomes.

Phylogenomic resolution of Imparidentia(Mollusca:Bivalvia)diversifcation through mitochondrial genomes

Despite signifcant advances in the phylogenomics of bivalves over the past decade,the higher-level phylogeny of Imparidentia(a superorder of Heterodonta)remains elusive.Here,a total of fve new mitochondrial sequences(Chama asperella,Chama limbula,Chama dunkeri,Barnea manilensis and Ctena divergens)was added to provide resolution in nodes that required additional study.Although the monophyly of Lucinida remains less clear,the results revealed the overall backbone of the Imparidentia tree and the monophyly of Imparidentia.Likewise,most relationships among the fve major Imparidentia lineages—Lucinida,Cardiida,Adapedonta,Myida and Venerida—were addressed with a well-supported topology.Basal relationships of Imparidentia recovered Lucinidae as the sister group to all remaining imparidentian taxa.Thyasiridae is a sister group to other imparidentian bivalves(except Lucinidae species)which is split into Cardiida,Adapedonta and the divergent clade of Neoheterodontei.Neoheterodontei was comprised of Venerida and Myida,the former of which now also contains Chamidae as the sister group to all the remaining venerid taxa.Moreover,molecular divergence times were inferred by calibrating nine nodes in the Imparidentia tree of life by extinct taxa.The origin of these major clades ranged from Ordovician to Permian with the diversifcation through the Palaeozoic to Mesozoic.Overall,the results obtained in this study demonstrate a better-resolved Imparidentia phylogeny based on mitochondrial genomes.

Structural and Expressional Variation Analyses of Mitochondrial Genomes Reveal Candidate Transcripts for the S^V Cytoplasmic Male Sterility in Wheat(Triticum aestivum L.)

Common wheat （Triticum aestivum L.） is one of the most important crops, and intra-specific wheat hybrids have obvious heterosis in yield and protein quality. Therefore, utilization of hybrid wheat varieties offers an effective way to increase yield and nutrition. Cytoplasmic male sterility （CMS） systems are a useful genetic tool for hybrid crop breeding, and are ideal models for studying the genetic interaction and cooperative function of mitochondrial and nuclear genomes in plants （Schnable and Wise, 1998; Hanson and Bentolila, 2004）.

Three New Ranidae Mitogenomes and the Evolution of Mitochondrial Gene Rearrangements among Ranidae Species

Various types of gene rearrangements have been discovered in the mitogenoes of the frog family Ranidae. In this study, we determined the complete mitogenome sequence of three Rana frogs. By combining the available mitogenomic data sets from GenBank, we evaluated the phylogenetic relationships of Ranidae at the mitogenome level and analyzed mitogenome rearrangement cases within Ranidae. The three frogs shared an identical mitogenome organization that was extremely similar to the typical Neobatrachian-type arrangement. Except for the genus Babina, the monophyly of each genus was well supported. The genus Amnirana occupied the most basal position among the Ranidae. The [Lithobates ＋ Rana] was the closest sister group of Odorrana. The diversity of mitochondrial gene arrangements in ranid species was unexpectedly high, with 47 mitogenomes from 40 ranids being classified into 10 different gene rearrangement types. Some taxa owned their unique gene rearrangement characteristics, which had significant implication for their phylogeny analysis. All rearrangement events discovered in the Ranidae mitogenomes can be explained by the duplication and random loss model.

Conserved gene arrangement in the mitochondrial genomes of barklouse families Stenopsocidae and Psocidae

Substantial variation in gene organization and arrangement has been reported for sequenced mitochondrial(mt) genomes from the suborders of the insect order Psocoptera. In this study we sequenced the complete mt genome of Stenopsocus immaculatus, the first representative of the family Stenopsocidae from the suborder Psocomorpha. Relative to the ancestral pattern, rearrangements of a protein-coding gene(nad3) and five t RNA genes(trnQ, trnC, trnN, trnS1, trnE) were found. This pattern was similar to that of two barklice from the family Psocidae, with the exception of the translocation of trnS1,trnE and trnI. Based on comparisons of pairwise breakpoint distances of gene rearrangements, gene number and chromosome number, it was concluded that mt genomes of Stenopsocidae and Psocidae share a relatively conserved pattern of gene rearrangements; mt genomes within the Psocomorpha have been generally stable over long evolutionary history; and mt gene rearrangement has been substantially faster in the booklice(suborder Troctomorpha) than in the barklice(suborders Trogiomorpha and Psocomorpha). It is speculated that the change of life history and persistence of unusual reproductive systems with maternal inheritance contributed to the contrasting rates in mt genome evolution between the barklice and booklice.

Assembly and analysis of the Populus deltoides mitochondrial genome:the first report of a multicircular mitochondrial conformation for the genus Populus

Genomics research of Populus deltoides,an important timber species that is widely planted worldwide,is an important part of poplar breeding.Currently,the nuclear and chloroplast genome of P.deltoides have been sequenced,but its mitochondrial genome(mitogenome)has not been reported.To further explore the evolution and phylogeny of P.deltoides,the mitogenome of P.deltoides I-69 was assembled using reads from Nanopore and Illumina sequencing platforms and found to consist of 802,637 bp and three circular chromosomes(336,205,280,841,and 185,591 bp)containing 58 genes(34 protein-coding genes,21 tRNA genes,and 3 rRNA genes).RNA analysis in combination with several species showed signifi cantly fewer RNA editingsites in the mitogenomes of poplar and other angiosperms than in gymnosperms.Sequence transfer analysis showed extensive mitogenome rearrangements in Populus species,and with evolution from lower to higher plants,tRNA transfer from chloroplasts to mitochondria became increasingly frequent.In a phylogenetic analysis,the evolutionary status of P.deltoides was determined,and the section Populus was supported.Our results based on the fi rst report of a multicircular conformation of the Populus mitogenome provide a basis for further study of the evolution and genetics of P.deltoides and other Populus species and for breeding programs.

Characterization and Phylogenetic Implications of the Complete Mitogenomes of Two Species in the Genus Zhangixalus(Anura:Rhacophoridae)

Mitochondrial genomes(mitogenomes)in frogs are essential for reconstructing the phylogenetic relationships and shedding light on the molecular evolution in these animals.However,there are only seven complete Rhacophoridae genomes that have been reported to date.In this paper,two complete mitogenomes of the Chinese whipping frog(Zhangixalus chenfui)and Emei tree frog(Z.omeimontis)were described,each of which was 20520 and 19782 bp in size,and had A+T contents of 64.26%and 63.83%.The two mitogenomes each included two non-coding control region(D-loop,CR),two ribosomal RNA genes(rRNAs),13 protein-coding genes(PCGs),and 22 transfer RNA genes(tRNAs),and it was found that the mitogenome of Z.chenfui also includes three tandem tRNAMet gene sequences.A typical clover-leaf structure was found for all tRNAs except for tRNASer1(AGN),which showed a reduced DHU arm.The putative D-loop region contains multiple types of tandem repeats regions.Both of these two mitogenomes showed similar pattern of gene rearrangement(tRNA-Ser-ND6-tRNA-Glu-CytbCR1-ND5-CR2-tRNA-Thr-tRNA-Leu-tRNA-Pro).Additionally,three consecutive tRNAMet genes were found for the first time in Z.chenfui,a species in the Rhacophoridae family.For all phylogenetic analyses,which were based on 13 protein-coding genes from 91 Ranoidea mitogenomes,the same phylogenetic trees were observed using either maximum likelihood or Bayesian approaches.These results suggest that the phylogenetic hypotheses for the Ranoidea(including Rhacophoridae,Mantellidae,Ranidae and Dicroglossidae)derived from these mitogenomic data could provide substantiation for the relationships of(Dicroglossidae(Ranidae,(Mantellidae,Rhacophoridae))),and support the presence of a monophyletic group in four families.Moreover,Z.omeimontis and Z.dennysi were found to cluster on the same branch,indicating that they were more closely related as a group.This group may in turn form sister groups with Z.arboreus and Z.schlegelii.However,Z.chenfui is located on the base of other species in the genera.Increased mitogenome sampling should be conducted to provide a more satisfactory resolution to the phylogeny of the Rhacophorus,Rhacophoridae,and Mantellidae.

Evolutionary relationships of mitogenomes in a recently radiated Old World avian family

Environmentally heterogeneous mountains provide opportunities for rapid diversification and speciation.The family Prunellidae(accentors)is a group of birds comprising primarily mountain specialists that have recently radiated across the Palearctic region.This rapid diversification poses challenges to resolving their phylogeny.Herein we sequenced the complete mitogenomes and estimated the phylogeny using all 12(including 28 individuals)currently recognized species of Prunellidae.We reconstructed the mitochondrial genome phylogeny using 13 protein-coding genes of 12 species and 2 Eurasian Tree Sparrows(Passer montanus).Phylogenetic relationships were estimated using a suite of analyses:maximum likelihood,maximum parsimony and the coalescent-based SVDquartets.Divergence times were estimated by implementing a Bayesian relaxed clock model in BEAST2.Based on the BEAST time-calibrated tree,we implemented an ancestral area reconstruction using RASP v.4.3.Our phylogenies based on the maximum likelihood,maximum parsimony and SVDquartets approaches support a clade of large-sized accentors(subgenus Laiscopus)to be sister to all other accentors with small size(subgenus Prunella).In addition,the trees also support the sister relationship of P.immaculata and P.rubeculoides+P.atrogularis with 100%bootstrap support,but the relationships among the remaining eight species in the Prunella clade are poorly resolved.These species cluster in different positions in the three phylogenetic trees and the nodes are often poorly supported.The five nodes separating the seven species diverged simultaneously within less than half million years(i.e.,between 2.71 and 3.15 million years ago),suggesting that the recent radiation is likely responsible for rampant incomplete lineage sorting and gene tree conflicts.Ancestral area reconstruction indicates a central Palearctic region origin for Prunellidae.Our study highlights that whole mitochondrial genome phylogeny can resolve major lineages within Prunellidae but is not sufficient to fully resolve the relationship among the species in the Prunella clade that almost simultaneously diversify during a short time period.Our results emphasize the challenge to reconstruct reliable phylogenetic relationship in a group of recently radiated species.

Characterization of Rheum palmatum mitochondrial genome and comparative analysis among Caryophyllales species

Objective:This work aimed to report the first complete mitochondrial genome(mitogenome)of Rheum palmatum,summarize the features of Caryophyllales mitogenomes,and to reveal the potential of utilizing the mitogenomes of R.palmatum and other Caryophyllales species for inferring phylogenetic relationships and species identification.Methods:Both Illumina short reads and PacBio HiFi reads were utilized to obtain a complete mitogenome of R.palmatum.A variety of bioinformatics tools were employed to characterize the R.palmatum mitogenome,compare the reported mitogenomes in Caryophyllales and conduct phylogenetic analysis.Results:The mitogenome of R.palmatum was assembled into a single master circle of 302,993 bp,encoding 35 known protein-coding genes,18 transfer RNA genes,and three ribosome RNA genes.A total of 249 long repeats and 49 simple sequence repeats were identified in this mitogenome.The sizes of mitogenomes in Caryophyllales varied from 253 kb to 11.3 Mb.Among them,23 mitogenomes were circular molecules,one was linear,and one consisted of relaxed circles,linear molecules,and supercoiled DNA.Out of the total mitogenomes,11 were single-chromosome structure,whereas the remaining 14 were multi-chromosomal organizations.The phylogenetic analysis is consistent with both the Engler system(1964)and the Angiosperm Phylogeny Group III system.Conclusions:We obtained the first mitogenome of R.palmatum,which consists of a master circle.Mitogenomes in Caryophyllales have variable genome sizes and structures even within the same species.Circular molecules are still the dominant pattern in Caryophyllales.Single-chromosome mitogenomes account for nearly a half of all the mitogenomes in Caryophyllales,in contrast to previous studies.It is feasible to utilize mitochondrial genomes for inferring phylogenetic relationships and conducting species identification.

High Altitude Adaptation and Phylogenetic Analysis of Tibetan Horse Based on the Mitochondrial Genome

To investigate genetic mechanisms of high altitude adaptations of animals living in the Tibetan Plateau, three mitochondrial genomes （mt-genome） of Tibetan horses living in Naqu （4,500 m） of Tibetan, Zhongdian （3,300 m） and Deqin （3,100 m） of Yunnan province were sequenced. The structures and lengths of these three mt-genomes are similar to the Cheju horse, which is related to Tibetan horses, but little shorter than the Swedish horse. The pair-wise identity of these three horses on nucleotide level is more than 99.3%. When the gene encoding the mitochondrial protein of Tibetan horses was analyzed, we found that NADH6 has higher non-synonymous mutation rate in all of three Tibetan horses. This implies that NADH6 may play a role in Tibetan horses＇ high altitude adaptation. NADH6 is one of the subunits of the complex I in the respiratory chain. Furthermore, 7 D-loop sequences of Tibetan horse from different areas were sequenced, and the phylogeny tree was constructed to study the origin and evolutionary history of Tibetan horses. The result showed that the genetic diverse was high among Tibetan horses. All of Tibetan horses from Naqu were clustered into one clade, and Tibetan horses from Zhongdian and Deqin were clustered into others clades. The first molecular evidence of Tibetan horses indicated in this study is that Tibetan horse population might have multiple origins.

Complete mitochondrial genome of the laced fritillary Argyreus hyperbius(Lepidoptera:Nymphalidae)

We investigated the complete mitochondrial genome（mitogenome） of Argyreus hyperbius.The 151 56 bp long genome harbored the gene content（13 protein coding genes,22 tRNA genes,2 rRNA genes and an A＋T-rich region） and the gene arrangement was identical to all known lepidopteran mitogenomes.Mitogenome sequence nucleotide organization and codon usage analyses showed that the genome had a strong A＋T bias,accounting for A＋T content of 80.8%,with a small negative AT skew（？0.019）.Eleven intergenic spacers totaling 96 bp,and 14 overlapping regions totaling 34 bp were scattered throughout the whole genome.As has been observed in other lepidopteran species,12 of the 13 protein-coding genes（PCGs） were initiated by ATN codons,while the COI gene was tentatively designated by the CGA codon.A total of 11 PCGs harbored the complete termination codon TAA,while the COI and COII genes ended at a single T residue.All of the 22 tRNA genes showed typical clover structures except that the tRNASer（AGN） lacks the dihydrouridine（DHU） stem which is replaced by a simple loop.The intergenic spacer sequence between the tRNASer（AGN） and ND1 also contained the ATACTAA motif,which is conserved in all other lepidopterans as well.Additionally,the 349 bp A＋T-rich region was not comprised of large tandem repetitive sequences,but harbored a few structures common to other lepidopteran insects,such as the motif ATAGA followed by a 20 bp poly-T stretch,a microsatellite-like（AT）9 element preceded by the ATTTA motif,and a 5 bp poly-A site present immediately upstream of tRNAMet.The mitochondrial genomic sequence features found in this study not only contribute to genetic diversity information of the group,but also are useful in future studies of the endangered nymphalid butterfly in population genetic dynamics,species conservation,phylogeography and evolution.

Complete Sequence and Gene Organization of the Mitochondrial Genome of Tokay (Gekko gecko)

Long-PCR amplification, clone and primer-walking sequencing methods were employed in determine the complete sequence of mitochondrial genome of tokay (Gekko gecko). The genome is 16 435 bp in size, contains 13 protein-coding, 2 ribosomal and 22 transfer RNA genes. The mt genome of Gekko is similar to most of the vertebrates in gene components, order, orientation, tRNA structures, low percentage of guanine and high percentage of thymine, and skews of base GC and AT. Base A was preferred at third codon positions for protein genes is similar to amphibians and fishes rather than amnion vertebrates. The standard stop codes (TAA) present only in three protein genes, less than those of most vertebrates. Transfer RNA genes range in length from 63 to 76 nt, their planar structure present characteristic clover leaf, except for tRNA-Cys and tRNA-Ser (AGY) because of lacking the D arm.

Morphological and molecular analyses of a Philine kinglipini outbreak in summer of 2022 in Jiaozhou Bay,China

A sudden and unprecedented outbreak of molluscan Philine kinglipini occurred in summer 2022 in Jiaozhou Bay,Shandong,China,causing substantial damage to local mariculture industry of the Manila clam Ruditapes philippinarum.Although P.kinglipini has been found in many coastal regions of China,the molecular research of P.kinglipini has not been extensively studied,making it difficult to accurately identify and track P.kinglipini samples in field using molecular methods.Samples were collected during the outbreak and their morphological features and molecular sequences were analyzed.Results show that the causative species of the outbreak was P.kinglipini.The mitochondrial genome(mt DNA)of P.kinglipini was constructed for the first time,based on which phylogenetic analysis of the mt DNAs of P.kinglipini and related species in the order Cephalaspidea was carried out.As revealed by metabarcoding analysis of 18S rDNA V4,the seasonal change of P.kinglipini and closely related Philine species was striking with peaks between April and August.Therefore,metabarcoding analysis is applicable tool for monitoring the bloom development of P.kinglipini and related species.This study generated for the first time essential molecular marker sequences and mtDNA of P.kinglipini,which provided a reference for future characterization and monitoring of its outbreaks and for phylogenetic analysis of Philine species.