Comparative analyses of mitogenomes in the social bees with insights into evolution of long inverted repeats in the Meliponini

The insect mitogenome is typically a compact circular molecule with highly conserved gene contents.Nonetheless,mitogenome structural variations have been reported in specific taxa,and gene rearrangements,usually the tRNAs,occur in different lineages.Because synapomorphies of mitogenome organizations can provide information for phylogenetic inferences,comparative analyses of mitogenomes have been given increasing attention.However,most studies use a very few species to represent the whole genus,tribe,family,or even order,overlooking potential variations at lower taxonomic levels,which might lead to some incorrect inferences.To provide new insights into mitogenome organizations and their implications for phylogenetic inference,this study conducted comparative analyses for mitogenomes of three social bee tribes(Meliponini,Bombini,and Apini)based on the phylogenetic framework with denser taxonomic sampling at the species and population levels.Comparative analyses revealed that mitogenomes of Apini and Bombini are the typical type,while those of Meliponini show diverse variations in mitogenome sizes and organizations.Large inverted repeats(IRs)cause significant gene rearrangements of protein coding genes(PCGs)and rRNAs in Indo-Malay/Australian stingless bee species.Molecular evolution analyses showed that the lineage with IRs have lower dN/dS ratios for PCGs than lineages without IRs,indicating potential effects of IRs on the evolution of mitochondrial genes.The finding of IRs and different patterns of gene rearrangements suggested that Meliponini is a hotspot in mitogenome evolution.Unlike conserved PCGs and rRNAs whose rearrangements were found only in the mentioned lineages within Meliponini,tRNA rearrangements are common across all three tribes of social bees,and are significant even at the species level,indicating that comprehensive sampling is needed to fully understand the patterns of tRNA rearrangements,and their implications for phylogenetic inference.

The first complete mitogenome of Acharax sp.(Protobranchia,Solemyida,Solemyidae):comparisons with other Solemyidae bivalves and deep-sea adaptive characteristics

Solemyidae is an ancient group of protobranch bivalves,and most solemyids are symbiotic with chemoautotrophic and gill-hosted bacteria,enabling them to survive in unusual habitats such as deepsea chemosynthetic environments.However,evolution of the mitogenomes in this family and their phylogenetic relationships remain poorly understood.The complete mitogenome of Acharax sp.was determined and compared with other available mitogenomes of solemyids.The mitogenome of Acharax sp.is 18970 bp in length and consists of 13 protein-coding genes,2 ribosomal RNA genes,and 22 transfer RNA genes.The gene arrangement was identical to those of other sequenced solemyids.For the present five mitogenomes of Solemyidae species,all protein-coding genes were initiated with the typical ATD(ATA,ATG,and ATT)codon and terminated with the TAA/TAG codon.Furthermore,the incomplete termination codon was detected.The Ka/Ks ratio analyses indicated that 13 protein-coding genes of five Solemyidae mitogenomes suffered strong purifying selection.Compared to 45 existing shallow water equivalents,the 18 available mitogenomes from the deep-sea,including the Acharax sp.in this study,show significantly more nonpolar amino acids in the 13 protein-coding genes,which indicates the adaptation to the deep-sea environment.The phylogenetic tree based on 48 Bivalvia complete mitogenomes provided further information to support the scientific classification of protobranchs.The relationships among Solemyidae were assessed based on 2 mitochondrial(16S rRNA and COX1)and 3 nuclear(18S rRNA,28S rRNA,and histone H3)gene sequences from 17 in-group species.The two genera Acharax and Solemya formed a monophyletic clade each,and Acharax sp.clustered with previously reported Acharax bivalves with high support values.

Two complete mitogenomes of Ocypodoidea(Decapoda: Brachyura), Cleistostoma dilatatum(Camptandriidae) and Euplax sp.(Macrophthalmidae) and its phylogenetic implications

Complete mitochondrial genomes(mitogenomes) can provide useful information for phylogenetic relationships,gene rearrangement, and molecular evolution. In the present study, two newly sequenced mitogenomes of Ocypodoidea(Cleistostoma dilatatum and Euplax sp.) were reported for the first time, which are 15 444 bp and16 129 bp in length, respectively. Cleistostoma dilatatum is the first species in the family Camptandriidae whose complete mitogenome was sequenced. Each mitogenome contains an entire set of 37 genes and a putative control region, but their gene arrangements are largely different. Tandem duplication and random loss model is proposed to account for their gene arrangements. Comparative genomic analyses of 19 mitogenomes clustering in one branch reveal that 18 of them shared the same gene rearrangement, while that of C. dilatatum mitogenome was consistent with the ancestral gene arrangement of Brachyura. The dN/dS ratio analysis shows that all PCGs are evolving under purifying selection. Phylogenetic analyses show that all Macrophalmidae species cluster together as a group, and then form a sister clade with Camptandriidae. Moreover, the polyphyly of three superfamilies(Ocypodoidea, Eriphioidea, and Grapsoidea) is reconfirmed. These findings help to confirm the phylogenetic position of Camptandriidae, as well as provide new insights into the phylogeny of Brachyura.

The complete mitogenome of the Chinese bush cricket,Gampsocleis gratiosa(Orthoptera:Tettigonioidea)

The complete mitochondrial genome （mitogenome） of Gampsocleis gratiosa was determined. The 15,929 bp in the size of G. gratiosa mitogenome contains a typical gene content, base composition, and codon usage found in metazoan. All 13 protein coding genes （PCGs） of the G gratiosa mitogenome start with a typical ATN codon. The usual termination codons （TAA and TAG） were found from 10 PCGs. However, the atp6, had4, and had5 had incomplete termination codon （T）. The anticodons of all tRNAs are identical to those observed in Drosophila yakuba and Locusta migratoria, and can be folded in the form of a typical clover leaf structure except for trnS （AGN）. The secondary structure of trnS （AGN） was drawn according with the Steinberg-Cedergren tertiary structure. The A＋T content （67.4%） of the A＋T-rich region is relatively lower among the mitogenome regions, in contrast, it usually contains the highest A＋T content for most insects. Two isolated sequence repeat regions （202 bp） were found in the A＋T-rich region with mapping and secondary structure information.

Comparative mitogenome phylogeography of two anteater genera (Tamandua and Myrmecophaga;Myrmecophagidae, Xenarthra): Evidence of discrepant evolutionary traits

The species within Xenarthra(sloths,anteaters,and armadillos)are quintessential South American mammals.Of the three groups,Vermilingua(anteaters)contains the fewest extant and paleontological species.Here,we sampled and sequenced the entire mitochondrial genomes(mitogenomes)of two Tamandua species(Tamandua tetradactyla and T.mexicana)(n=74)from Central and South America,as well as Myrmecophaga tridactyla(n=41)from South America.Within Tamandua,we detected three different haplogroups.The oldest(THI)contained many specimens with the T.tetradactyla morphotype(but also several with the T.mexicana morphotype)and originated in southeastern South America(currently Uruguay)before moving towards northern South America,where the THII haplogroup originated.THII primarily contained specimens with the T.mexicana morphotype(but also several with the T.tetradactyla morphotype)and was distributed in Central America,Colombia,and Ecuador.THI and THII yielded a genetic distance of 4%.THII originated in either northern South America or“in situ”in Central America with haplogroup THIII,which consisted of~50%T.mexicana and 50%T.tetradactyla phenotypes.THIII was mostly located in the same areas as THII,i.e.,Central America,Ecuador,and Colombia,though mainly in the latter.The three haplogroups overlapped in Colombia and Ecuador.Thus,T.tetradactyla and T.mexicana were not reciprocally monophyletic.For this reason,we considered that a unique species of Tamandua likely exists,i.e.,T.tetradactyla.In contrast to Tamandua,M.tridactyla did not show different morphotypes throughout its geographical range in the Neotropics.However,two very divergent genetic haplogroups(MHI and MHII),with a genetic distance of~10%,were detected.The basal haplogroup,MHI,originated in northwestern South America,whereas the more geographically derived haplogroup,MHII,overlapped with MHI,but also expanded into central and southern South America.Thus,Tamandua migrated from south to north whereas Myrmecophaga migrated from north to south.Our results also showed that temporal mitochondrial diversification for Tamandua began during the Late Pliocene and Upper Pleistocene,but for Myrmecophaga began during the Late Miocene.Furthermore,both taxa showed elevated levels of mitochondrial genetic diversity.Tamandua showed more evidence of female population expansion than Myrmecophaga.Tamandua experienced population expansion~0.6–0.17 million years ago(Mya),whereas Myrmecophaga showed possible population expansion~0.3–0.2 Mya.However,both taxa experienced a conspicuous female decline in the last 10000–20000 years.Our results also showed little spatial genetic structure for both taxa.However,several analyses revealed higher spatial structure in Tamandua than in Myrmecophaga.Therefore,Tamandua and Myrmecophaga were not subjected to the same biogeographical,geological,or climatological events in shaping their genetic structures.

Mitogenome-based phylogeny of mosquitoes(Diptera:Culicidae)

Mosquitoes are of great medical significance as vectors of many deadly diseases.Mitogenomes have been widely used in phylogenetic studies,but mitogenome knowledge within the family Culicidae is limited,and Culicidae phylogeny is far from resolved.In this study,we surveyed the mitogenomes of 149 Culicidae species,including 7 newly sequenced species.Comparative analysis of 149 mosquito mitogenomes shows gene composition and order to be identical to that of an ancestral insect,and the AT bias,length variation,and codon usage are all consistent with that of other reported Dipteran mitogenomes.Phylogenetic analyses based on the DNA sequences of the 13 protein-coding genes from the 149 species robustly support the monophyly of the subfamily Anophelinae and the tribes Aedini,Culicini,Mansonini,Sabethini,and Toxorhynchitini.To resolve ambiguous relationships between clades within the subfamily Culicinae,we performed topological tests and show that Aedini is a sister to Culicini and that Uranotaenini is a sister to(Mansonini)+(Toxorhynchitini+Sabethini).In addition,we estimated divergence times using a Bayesian relaxation clock based on the sequence data and 3 fossil calibration points.The results show mosquitoes diverged during the Early Jurassic with massive Culicinae radiations during the Cretaceous,coincident with the emergence of angiosperms and the burst of mammals and birds.Overall,this study,which uses the largest number of Culicidae mitogenomes sequenced to date,comprehensively reveals the mitogenome characteristics and mitogenome-based phylogeny and divergence times of Culicidae,providing information for further studies on the mitogenome,phylogeny,evolution,and taxonomic revision of Culicidae.

The phylogeny of Orthoptera inferred from mtDNA and description of Elimaea cheni(Tettigoniidae:Phaneropterinae)mitogenome

The complete 15,831 bp nucleotide sequence of the mitochondrial genome from Elimaea cheni （Phaneropterinae） was determined. The putative initiation codon for cox1 was TTA. The phylogeny of Orthoptera based on different mtDNA datasets were analyzed with maximum likelihood （ML） and Bayesian inference （BI）. When all 37 genes （mtDNA） were analyzed simultaneously, the monophyly of Caelifera and Ensifera were recovered in the context of our taxon sampling. The phylogeny of Orthoptera was largely consistent with previous phylogenetic hypotheses. Rhaphidophoridae to be a sister group of Tettigoniidae, and the relationships among four subfamilies of Tettigoniidae were （Phaneropterinae ＋ （Conocephalinae ＋ （Bradyporinae ＋ Tettigoniinae）））. Pyrgomorphidae was the most basal group of Caelifera. The relationships among six acridid subfamilies were （Oedipodinae ＋ （Acridinae ＋ （Gomphocerinae ＋ （Oxyinae ＋ （Callip- taminae ＋ Cyrtacanthacridinae）））））. However, we did not recover a monophyletic Grylloidea. Myrmecophilidae clustered into one clade with Gryllotalpidae instead of with Gryllidae. ML and BI analyses of all protein coding genes （using all nucleotide sequence data or excluding the third codon position, and amino acid sequences） revealed a topology identical to that of the entire mtDNA genome dataset. However, 22 tRNAs genes excluding the DHU loop and TψC loop （TRNA）, and two rRNA genes （RRNA） perform poorly when analyzed as single dataset. Our results suggest that the best phylogenetic inferences were ML and BI methods based on total mtDNA. Excluding tRNA genes, rRNA genes and the third codon position of protein coding genes from dataset and converting nucleotide sequences to amino acid sequences do not positively affect phylogenetic reconstruction.

Comparative mitochondrial genome analysis of Cynoglossidae(Teleost:Pleuronectiformes)and phylogenetic implications

Generally,a teleostean group(e.g.,family or genus)owns one type or a set of similar mitochondrial gene arrangement.It is interesting,however,that four different types of gene arrangement have been found in the mitochondrial genome(mitogenome)of Cynoglossidae species.So far,the possible mechanisms of mitogenomic gene rearrangement and its potential implications have aroused widespread attention and caused lots of controversy.Here,a total of 21 Cynoglossidae mitogenomes and a newly sequenced mitogenome of Cynoglossus puncticpes(Pleuronectiformes:Cynoglossidae)were compared.The length ranges from 16417 bp to 18369 bp,which is mainly caused by the length heteroplasmy of control region(CR).Further analysis reveals that the difference of tandem repeats acts as a determining factor resulting in the length heterogeneity.Like most gene rearrangements of Cynoglossinae mitogenomes,tRNA-Gln gene encoded by the L-strand has translocated to the H-strand(Q inversion),accompanied by the translocation of CR in C.puncticpes mitogenome.The typical IQM order(tRNA-Ile-Gln-Met)changed to QIM order.Tandem duplication/random loss and mitochondrial recombination were accepted as the most possible models to account for the rearrangements in C.puncticpes mitogenome.Phylogenetic trees showed a strong correlation between the gap spacer in the rearranged QIM area and phylogeny,which provides a fresh idea for phylogenetic studies in future.

The complete mitochondrial genome of Parachiloglanis hodgarti and its phylogenetic position within Sisoridae

The complete mitogenome of Parachiloglanis hodgarti was sequenced and characterized,which is the first mitogenome of the genus Parachiloglanis within Sisoridae.The mitogenome is 16511-bp long and included 13 protein-coding genes,22 transfer RNAs,two ribosomal RNAs,and one control region.The genome composition was A+T biased(58.64%)and exhibited a positive AT-skew(0.095)and a negative GC-skew(-0.283).Compared with other Sisoridae fishes,the characteristics of nucleotide skews,codon usage,and amino acid usage of the P.hodgarti mitogenome are more similar to those of non-Glyptosternoid fishes.Furthermore,the phylogenetic trees show that Sisoridae fishes fall into four major clades and P.hodgarti(CladeⅠ)is basal to the Sisoridae family,forming a sister clade to the Glyptosternoids(CladeⅣ).The topological structures constructed in this study raised doubts over the traditional classification system.These results will help better understand the feature s of the P.hodgarti mitogenome and provide a reference for further phylogenetic research on Sisoridae species.

A new genus of Asiatic short-tailed shrew (Soricidae, Eulipotyphla) based on molecular and morphological comparisons

Blarinellini is a tribe of soricine shrews comprised of nine fossil genera and one extant genus. Blarinelline shrews were once widely distributed throughout Eurasia and North America, though only members of the Asiatic short-tailed shrew genus Blarinella currently persist （mostly in southwestern China and adjacent areas）. Only three forms of Blarinella have been recognized as either species or subspecies However, recent molecular studies indicated a strikingly deep divergence within the genus, implying the existence of a distinct genus-level lineage. We sequenced the complete mitochondrial genomes and one nuclear gene of three Asiatic short-tailed and two North American shrews and analyzed them morphometrically and morphologically. Our molecular analyses revealed that specimens ascribed to B. griselda formed two deeply diverged lineages, one a close relative to B. quadraticauda, whereas the other--comprised of topotype specimens from southern Gansu-iverged from other Blarinella in the middle Miocene （ca. 18.2 million years ago （Ma）, 95% confidence interval=13.4-23.6 Ma）. Although the skulls were similarly shaped in both lineages, we observed several diagnostic characteristics, including the shape of the upper p4. In consideration of the molecular and morphological evidence, we recognize B. griselda as the sole species of a new genus, namely, Pantherina gen. nov. Interestingly, some characteristics of Pantherina griselda are more similar to fossil genera, suggesting it represents an evolutionarily more primitive form than Blarinella.Recognition of this new genus sheds light on the systematics and evolutionary history of the tribe Blarinellini throughout Eurasia and North America.

Complete mitochondrial genome of the leaf muntjac(Muntiacus putaoensis) and phylogenetics of the genus Muntiacus

The leaf muntjac （Muntiacus putaoensis） is an endemic deer species found in the east trans- Himalayan region. In recent years, population numbers have decreased due to heavy hunting and habitat loss, and little genetic data exists for this species, thus our knowledge of distribution rangs and population sizes likewise remain limited. We obtained mtDNA genes and the complete mitochondrial genome sequence of M. putaoensis using PCR, followed by direct sequencing. The complete mitogenome sequence was determined as a circular 16 349 bp mitochondrial genome, containing 13 protein-coding genes, two rRNA genes 22 tRNA genes, and one control region, the gene composition and order of which were similar to most other vertebrates so far reported. Most mitochondrial genes, except for ND6 and eight tRNAs, were encoded on the heavy strand. The overall base composition of the heavy strand was 33.1% A, 29.3% T, 24.2% C, and 13.4% G, with a strong AT bias of 62.4%. There were seven regions of gene overlap totaling 95 bp and 11 intergenic spacer regions totaling 74 bp. Phylogenetic analyses （ML and BI） among the Muntiacus genus based on the sequenced of mitogenome and ND4L-ND4 supported M. putaoensis as a member of Muntiacus, most closely related to M. vuquangensis. However, when analyses based on cyt b included two more muntjacs, M. truongsonensis was most closely related to M. putaoensis rather than M. vuquangensis, and together with M. rooseveltorum, likely forming a M. rooseveltorum complex of the species. This study will help in the exploration of the evolutionary history and taxonomic status of the leaf muntjac, as well as its protection as a genetic resource.

Comparative mitochondrial genome analysis of Varunidae and its phylogenetic implications

Complete mitochondrial genomes(mitogenomes)can indicate phylogenetic relationships,as well as useful information for gene rearrangement mechanisms and molecular evolution.Currently,the phylogenetic location of the genus Varuna(Brachyura:Varunidae)has not been well resolved mainly because of limited representatives(only two extant species).Here,we determined a new mitogenome of this genus(Varuna litterata)and added the published mitogenomes to reconstruct the phylogeny of Varunidae.The 16368-bp mitogenome contains the entire set of 37 genes and a putative control region.The characteristics of this newly sequenced mitogenome were described and compared with the other 15 Varunidae mitogenomes.All 16 analyzed mitogenomes have identical gene order and similar molecular features.The sliding window and genetic distance analyses demonstrate highly variable nucleotide diversity,with comparatively low variability of COI and COII,and high variability of ND6.The nonsynonymous/synonymous substitution rates(dN/dS ratio)analysis shows that all 13 PCGs are under purifying selection and ATP8 gene evolves under the least selective pressure.Twelve tRNA genes,two rRNAs,one PCG,and the putative control region are found to be rearranged with respect to the pancrustacean ground pattern gene order.Tandem duplication/random loss model is adopted to explain the large-scale gene rearrangement events occurring in Varunidae mitogenomes.Phylogenetic analyses show that all Varunidae species are placed into one group,and form a sister clade with Macrophthalmidae.Nevertheless,the phylogenetic relationships within Varunidae are not completely consistent based on the two different datasets used in this study.These findings will contribute to a better understanding of gene rearrangement and molecular evolution in Varunidae mitogenomes,as well as provide insights into the phylogenetic studies of Brachyura.

Mitochondrial DNA diversity and origin of indigenous pigs in South China and their contribution to western modern pig breeds

Indigenous pigs in South China are valuable genetic resources with many specific and unique characters, which have played an important role in the establishment of some western modern pig breeds. However, the origin and genetic diversity of indigenous pigs in South China have not been fully understood. In the present study, we sequenced 534 novel mitochondrial DNA (mtDNA) D-loop and assembled 54 complete mitogenome sequences for all 17 indigenous pig breeds from Fujian, Guangdong, Guangxi and Hainan in South China. These data were analyzed together with previously published homologous sequences relevant to this study. We found that all 13 coding genes of the mitogenomes were under purifying selection, but ND1 had the most variable sites and CYTB contained the most non-synonymous SNPs. Phylogenetic analysis showed that all indigenous pigs in South China were clustered into the D haplogroup with D1a1, D1b, D1c and D1e sub-haplogroups found to be dominant. Haplotype and nucleotide diversities of D-loop sequences ranged from 0.427 to 0.899 and from 0.00342 to 0.00695, respectively, among which all pigs in Guangdong had the lowest diversity. The estimates of pairwise FST, gene flow (Nm) and genetic distance (Da) indicated that most of these indigenous pig breeds differentiated from each other significantly (P<0.05). Among the western modern breeds, Berkshire and Yorkshire had significant Asian matrilineal footprints from indigenous pigs in South China, especially the Spotted pigs distributed in Guangdong and Guangxi. The neutrality test (Fu’s FS) indicated that indigenous pigs from Fujian and Guangxi had gone through recent population expansion events (P<0.05). It is concluded that indigenous pigs in South China were most likely derived from the Mekong region and the middle and downstream regions of Yangtze River through Guangxi and Fujian. Our findings provide a complete and in-depth insight on the origin and distribution pattern of maternal genetic diversity of indigenous pigs in South China.

Complete Mitochondrial Genome of Myra affinis(Decapoda:Brachyura:Leucosiidae)and Its Phylogenetic Implications for Brachyura

Knowledge of complete mitochondrial genomes(mitogenomes)can help understand the molecular evolution and phylogenetic relationships of various species.To date,the phylogenetic status of Leucosiidae within Brachyura remains unresolved because of the limited number of mitogenomes available.In the present study,the complete mitogenome of Myra affinis was sequenced using next-generation sequencing to supplement the limited mitogenome information of Leucosiidae.The 15349 bp-long mitogenome includes 13 protein-coding genes(PCGs),22 transfer RNAs(tRNAs),2 ribosomal RNAs(rRNAs),and a putative control region.The overall base composition is 34.5%A,36.1%T,11.5%G,and 17.9%C,with a high AT bias(70.6%).All 13 PCGs start with the standard ATN codon and stop with the TAN codon or incomplete T.Except for tRNA-Ser,all other tRNAs have a typical cloverleaf-like secondary structure.Phylogenetic analyses based on the maximum likelihood and Bayesian inference methods are employed to generate identical phylogenetic topologies,thereby supporting the sister relationship between Leucosiidae and Matutidae for the first time.The monophyly of Eubrachyura is well established,and its sister relationship with Raninoida is strongly supported.The results of this work will not only help achieve a better understanding of the characteristics of the M.affinis mitogenome and the phylogenetic position of Leucosiidae,but also provide relevant information for further studies on the phylogeny of Brachyura.

Comparative mitochondrial genome analysis of Sesarmidae and its phylogenetic implications

Here,we sequenced the complete mitogenome of Parasesarma eumolpe(Brachyura:Grapsoidea:Sesarmidae)for the first time.The characteristics of this newly sequenced mitogenome were described and compared with other Sesarmidae species.The 15646-bp mitogenome contains 13 protein-coding genes(PCGs),two ribosomal RNA genes(r RNAs),22 transfer RNA genes(t RNAs),and an A-T rich region.All of the PCGs are initiated by the start codon ATN and terminated by the standard TAN codon or an incomplete T.The pairwise Ka/Ks ratio analysis shows that all 13 PCGs are under purifying selection,whereas the ATP8 gene is an outlier,with pairwise comparison values ranging from neutral selection(0.000)to positive selection(1.039).The gene arrangement of P.eumolpe compared with ancestral Decapoda shows the translocation of two t RNAs(t RNA-His and t RNA-Gln),which is identical to other Sesarmidae species.Phylogenetic analyses show that all Sesarmidae species are placed into one group,and the polyphyly of Eriphioidea,Ocypodoidea,and Grapsoidea is well supported.The relationship between gaps in the QIM region and the phylogeny of Sesarmidae is analyzed.It is obvious that both the G5(the gap between Q and I)and G6(the gap between I and M)decrease progressively with the evolution process.These results will help to better understand the genomic evolution within Sesarmidae and provide insights into the phylogeny of Brachyura.

The Mitochondrial Genome of Pseudocalotes microlepis (Squamata: Agamidae) and its Phylogenetic Position in Agamids

We describe the complete mitochondrial genome of a small-scaled forest agamid （Pseudocalotes microlepis）, which is 17 873 bp in size, containing 13 PCGs, 2 rRNAs, 22 tRNAs and non-coding regions. The mitogenome has a typical gene order among squamates. 13 PCGs include 2 start codons （ATG and ATA）, 3 stop codons （TAG, TAA and AGG）, and an incomplete stop codon （T-）. Codon usage analyses showed that CUA-Leul and CGG-Arg are the most frequently and rarely used codon, respectively. All 22 tRNAs were predicted to form canonical cloverleaf secondary structures, except for two tRNAs （tRNAcys and tRNASer （AGY）） lacking the dihydorouridine （DHU） arm. The large non- coding region （control region） is 2 687 bp long （28.3% T, 18.2% C, 42.3% A, and 11.2% G）, with four different types of repeating sequences. The phylogenetic tree resulting from BEAST analyses based on concatenated 2 rRNAs and 13 PCGs in sequence revealed that the newly sequenced P. microlepis, where the genus Acanthosaura were aggregated. Together with Calotes versicolor, they constitute the subfamily Draconinae.

Complete Mitochondrial Genome Analysis of Daphniopsis tibetana(Branchiopoda:Diplostraca):New Insights into the Taxonomy of the Genus and Its Phylogenetic Implications for Branchiopoda

Daphniopsis tibetana is widely distributed in Xinjiang,Qinghai,Tibet of China,as well as in Russia and India,which is the dominant zooplankton in many high-altitude(4000 m)salt lakes.D.tibetana can adapt to saline waters,whereas the other species of the superorder Cladocera can only inhabit in freshwater.However,the phylogenetic status of D.tibetana in Branchiopoda remains unclear primarily because of limited mitogenome.In this study,complete mitochondrial genome sequences of D.tibetana were sequenced and annotated for the first time to obtain a comprehensive understanding of its phylogenetic status.The complete mitogenome of D.tibetana is 16196 bp in length.It contains 37 genes,including two ribosomal RNAs(12S and 16S rRNAs)genes,22 transfer RNA(tRNA)genes,13 protein-coding genes,and one non-coding region.The overall base composition is 29.6%A,33.2%T,19.0%G,and 18.2%C with a high AT bias(62.8%).Except for trnS1(GCT),most tRNAs have a typical cloverleaf secondary structure.Phylogenetic analysis based on maximum likelihood and Bayesian inference generates identical phylogenetic topology and shows the phylogenetic status of D.tibetana,which reconfirm the distinction between the genera Daphniopsis and Daphnia.Meanwhile,the class Branchiopoda is clustered into three clades(Anostraca,Notostraca,and Diplostraca)with high support values.These results provide not only a comprehensive understanding of the characteristics of D.tibetana mitogenome and its phylogenetic position in Diplostraca,but also information for future research on the phylogeny of Branchiopoda.

Evolution of Phenotype and Mitochondrial Genome Reveals Limbless and Body-elongated Squamates may Change Their Energy Basis for Locomotion

Limb reduction in Squamata present the dramatic characteristic to focus and usually accompanied with particularly morphological modifications, impacting tremendous locomotion changing and might generate different energy requirement. Herein, we combined both morphological and mitochondrial genomic data to explore the evolution of phenotypic transformation and mitochondrial genome of limbless and body-elongated squamates. We collected phenotypic measurements of 503 individuals, representing limbed or limbless taxa across all major lineages in Squama ta to investiga te the morphological correla tions with limb-reduction. Furthermore, we provided the mitochondrial genome of the representative limbless and elongated species Dibamus bourreti(Angel, 1935) to detect selective constraints on limbless clades with published mitogenomes of other squa ma te reptiles. Our results evidenced tha t body elongation had certain negative relationship with limbreduction in Squamata lineage and Lacertilia lineage(R = –0.495, P < 2.2 e-16;R= –0.332, P = 1.1 e-13, respectively), while tail length showed slight correlation in both clades(R = 0.156, P = 4.3 e-04;R= 0.192, P = 2.1 e-05, respectively). Besides, detection demonstrated that ATP6 has experienced accelerated evolution among limbless lineages, suggesting selective pressure on mitogenomes may play an essential role in energy disparity for locomotion of limbed and limbless squamates.

Characterizing the complete mitochondrial genome of Psephenothrips eriobotryae Dang&Qiao(Thysanoptera:Phlaeothripidae)with massive gene arrangement in Phlaeothripidae

Each species within the order Thysanoptera has a distinct mitochondrial gene order displaying a high level of gene rearrangement,particularly massive in Phlaeothripidae.To understand the evolutionary patterns of rearrangement in this family,we sequenced the complete mitochondrial genome of Psephenothrips eriobotryae,a notable potential pest of loquat trees.Its mitogenome is a circular DNA molecule(15,413 bp in size)composed of 37 genes:13 PCGs,22 tRNAs,two rRNAs,and two putative control regions(CRs).By comparing with the ancestral mitogenome of arthropods,P.eriobotryae exhibits notable gene rearrangement variations.However,it shares four conserved gene blocks with three closely related species in Phlaeothripinae.In the phylogenetic tree,P.eriobotryae was clustered with these three leaffeeding Phlaeothripinae species.The presence of more similar conserved gene blocks probably indicates a strong correlation between gene rearrangements and the phylogeny within Phlaeothripidae.Furthermore,the mitogenome of P.eriobotryae shows some characteristics:(a)two putative CRs in which CR2 is the copy of partial CR1 with 99.62%sequence similarity,and CR1 consists of two 237 bp repeat unites;(b)high A+T content of 82.6%in overall base composition;(c)trn S1 lacking a complete dihydrouridine(DHU)arm;(d)the protein coding gene,atp8 started with TTG instead of the more common ATN,while nad3 terminated by TAG rather than TAA;(e)translocations and inversions observed in fifteen of the 37 genes.

The complete mitochondrial genome of Cervaphis quercus （Insecta： Hemiptera： Aphididae： Greenideinae）

The mitochondrial genome of Cervaphis quercus has been sequenced and annotated. The entire genome of 15 272 bp encodes two ribosomal RNA genes （rrnL and rrnS）, 22 transfer RNA （tRNA） genes, 13 protein-coding genes and a control region. The genome has the same gene order as that found in the inferred ancestral insect. Nucleotide composition is highly A＋T biased. All protein-coding genes use standard mitochondrial initiation codons. Secondary structure models of the two ribosomal RNA genes ofC. quercus are similar to those proposed for other insects. All tRNAs have the classic clover-leaf structure, except for the dihydrouridine （DHU） arm of trnS （AGN）, which forms a simpleloop. The presence of structural elements in the control region is also discussed, with an emphasis on the possible regulation of replication and/or transcription. Comparison withmitochondrial genomes of other aphid species shows their gene arrangements are conserved; however, the variety of repeat regions in species from a different aphid subfamily,Aphidinae, suggests that they resulted from independent evolutionary events.