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.

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.

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.

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.

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 Five-dot Sergeant Parathyma sulpitia(Nymphalidae:Limenitidinae) and its phylogenetic implications

The complete mitochondrial genome of the Parathyma sulpitia （Lepidoptera, Nymphalidae, Limenitidinae） was determined. The entire mitochondrial DNA （mtDNA） molecule was 15 268 bp in size. Its gene content and organization were the same as those of other lepidopteran species, except for the presence of the 121 bp long intergenic spacer between trnSI（AGN）and trnE. The 13 protein-coding genes （PCGs） started with the typical ATN codon, with the exception of the coxl gene that used CGA as its initial codon. In addition, all protein-coding genes terminated at the common stop codon TAA, except the nad4 gene which used a single T as its terminating codon. All 22 tRNA genes possessed the typical clover leaf secondary structure except for trnSI（AGN）, which had a simple loop with the absence of the DHU stem. Excluding the A＋T-rich region, the mtDNA genome of P. sulpitia harbored 11 intergenic spacers, the longest of which was 121 bp long with the highest A＋T content （100%）, located between trnSI（AGN） and trnE. As in other lepidopteran species, there was an 18-bp poly-T stretch at the 3＇-end of the A＋T-rich region, and there were a few short microsatellite-like repeat regions without conspicuous macro-repeats in the A＋T-rich region. The phylogenetic analyses of the published complete mt genomes from nine Nymphalidae species were conducted using the concatenated sequences of 13 PCGs with maximum likelihood and Bayesian inference methods. The results indicated that Limenitidinae was a sister to the Heliconiinae among the main Nymphalidae lineages in this study, strongly supporting the results of previous molecular data, while contradicting speculations based on morphological characters.

Complete mitochondrial genome of the Thai Red Junglefowl (Gallus gallus) and phylogenetic analysis

DEAR EDITOR,In this study, we sequenced the complete mitochondrial genome （mitogenome） of the Thai Red Junglefowl （RJF; Gallus gallus） using the next-generation sequencing （NGS） platform of the Ion Torrent PGM. Samples were taken from Mae Wang District, Chiang Mai Province, northern Thailand Our data showed the complete mitogenome to be 16 785 bp in length, composed by 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one control region. The genome nucleotide composition was 30.3% A, 23.7% T, 32.5% C, and 13.5% G, resulting in a high percentage of A＋T （50.4%）. Phylogenetic analysis revealed that the mitogenome belonged to haplogroup X, whereas those of all domestic chickens belong to haplogroups A to G. This newly released mitogenome sequence will advance further evolutionary and population genetics study of the RJF and domestic chicken The availability of the G. gallus mitogenome will also contribute to further conservation genetics research of a unique species, listed as ＇data deficient＇ in Thailand.

A Monophyletic Status of Axis Genus in Subfamily Cervinae Supported by the Complete Mitochondrial Genome of Chinese Hog Deer(Axis porcinus)

Hog deer(Axis porcinus)is a small mammal and listed in the International Union for Conservation of Nature.However,phylogenetic position of hog deer within Axis genus has remained controversial.In the present study,we first assembled complete mitochondrial genome of Chinese hog deer reared in Chengdu Zoo,Sichuan,by the second-generation sequencing technology.This newly assembled mitochondrial genome of hog deer is 16376 bp in length and consists of 13 protein-encoding genes,23 transfer RNA genes and 2 ribosomal RNA genes.Phylogenetic analyses based on complete mitochondrial genome and cytochrome b gene sequences revealed that hog deer is closely clustered together and placed with sister taxon of spotted deer(A.Axis),which therefore supported monophyletic statue of Axis genus.Furthermore,considerable genetic differentiation,up to 139 mutations of complete mitochondrial genome was revealed between geographical populations of hog deer in France and Southeast Asia.However,only six variable sites(nucleotide diversity of 0.00007)and four haplotypes(haplotype diversity of 0.533)were totally detected among ten newly sequenced Chinese hog deer.The results provide a better understanding on the phylogeny of hog deer.

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.

Complete Mitochondrial Genome of the Red Fox (Vuples vuples) and Phylogenetic Analysis with Other Canid Species

The whole mitochondrial genome sequence of red fox （Vuples vuples） was determined. It had a total length of 16 723 bp. As in most mammal mitochondrial genome, it contained 13 protein coding genes, two ribosome RNA genes, 22 transfer RNA genes and one control region. The base composition was 31.3% A, 26.1% C, 14.8% G and 27.8% T, respectively. The codon usage of red fox, arctic fox, gray wolf, domestic dog and coyote followed the same pattern except for an unusual ATT start codon, which initiates the NADH dehydrogenase subunit 3 gene in the red fox. A long tandem repeat rich in AC was found between conserved sequence block 1 and 2 in the control region. In order to confirm the phylogenetic relationships of red fox to other canids, phylogenetic trees were reconstructed by neighbor-joining and maximum parsimony methods using 12 concatenated heavy-strand protein-coding genes. The result indicated that arctic fox was the sister group of red fox and they both belong to the red fox-like clade in family Canidae, while gray wolf, domestic dog and coyote belong to wolf-like clade. The result was in accordance with existing phylogenetic results.

The Complete Mitochondrial Genome of Salt-water Crocodile (Crocodylus porosus) and Phylogeny of Crocodilians

The nucleotide sequence of the complete mitochondrial DNA （mtDNA） molecule of the salt-water crocodile （Crocodylus porosus） was determined in this article. The molecule is 16,917 base pairs Cop） in length, and codes for 22 tRNAs, 13 protein-coding genes, 2 rRNAs, as well as a control region （D-loop）, as is characteristic for mitochondrial genomes of other metazoans. The gene order conforms to that of other crocodilians sequenced, but the arrangement of some tRNA genes differs from other vertebrates. It shows that the gene order of crocodilians is remarkably conserved. In this study, the relationships among crocodilians were examined in the phylogenetic analysis based on the control conserved regions of 17 crocodilians. The results suggest that the gharial （Gavialis gangeticus） joins the false gharial （Tomistoma schlegelii） on a common branch, and then constitutes a sister group to traditional Crocodylidae. Thus, the result supports that G gangeticus belongs to Crocodylidae. The analyses also suggest that the African slender-shouted crocodile （Crocodylus cataphractus） can be treated as an isolated genus, and constitutes a sister group to Crocodylus.

The complete mitochondrial genome of the Keeled box turtle Pyxidea mouhotii and phylogenetic analysis of major turtle groups

The complete mitochondrial genome （16,837 bp） from the Keeled box turtle （Pyxidea mouhotii） was determined. The genome content, gene order, and base composition conformed to the consensus vertebrate type mtDNA. However, a remarkable feature was found in this molecule： a large number of （ATTATATC） n direct tandem repeats followed by （TA） n microsatellite at the 3＇ end of the control region （D-loop）, which might be useful as molecular markers for studying population genetics and helpful for species identification and conservation. Besides, to review phylogenetic relationships among major turtle lineages, maximum-likelihood （ML） and Bayesian （BI） analyses were conducted based on concatenated sequences of 13 protein-coding genes from 16 taxa. The resultant ML and BI analyses showed homological topologies, which only differed on the exact placement of Platysternon. Nevertheless, the results strongly supported that 1） Pyxidea mouhotii and Cuora aurocapitata formed a monophyletic clade, whereas Cyclemys atripons was not closer to the Pyxidea-Cuora than to Chinemys reevesii, suggesting that Cyclemys and the Cuora group （containing Pyxidea） may have originated from two ancestors; 2） the Geoemydidae with Testudinidae was a sister group rather than with the Emydidae.

Comparative analysis of mitochondrial genome of a deepsea crab Chaceon granulates reveals positive selection and novel genetic features

Deep-sea organisms survive in an extremely harsh environment.There must be some genetic adaptation mechanisms for them.We systematically characterized and compared the complete mitochondrial genome(mitogenome)of a deep-sea crab(Chaceon granulates)with those of shallow crabs.The mitogenome of the crab was 16126 bp in length,and encoded 37 genes as most of a metazoan mitogenome,including 13 protein-coding genes(PCGs),22 transfer RNA(tRNA)genes,and 2 ribosomal RNA(rRNA)genes.The gene arrangement and orientation was conserved in the crabs.However,a unique mitogenome element regulator,the origin of light-strand replication(O L),was firstly predicted in the present crab mitogenome.In addition,further positive selection analysis showed that two residues(33 S in ND3 and 502 I in ND5)in C.granulates mitogenome were positively selected,indicated the selective evolution of the deep-sea crab.Therefore,the mitogenome of deep-sea C.granulates showed a unique OL element and positive selection.These special features would influence the mitochondrial energy metabolism,and be involved in the adaptation of deepsea environment,such as oxygen deficits and low temperatures.

The first mitochondrial genome for the butterfly family Riodinidae(Abisara fylloides) and its systematic implications

The Riodinidae is one of the lepidopteran butterfly families. This study describes the complete mitochondrial genome of the butterfly species Abisara fylloides, the first mitochondrial genome of the Riodinidae family. The results show that the entire mitochondrial genome of A. fylloides is 15301 bp in length, and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and a 423 bp A＋T-rich region. The gene content, orientation and order are identical to the majority of other lepidopteran insects. Phylogenetic reconstruction was conducted using the concatenated 13 protein-coding gene （PCG） sequences of 19 available butterfly species covering all the five butterfly families （Papilionidae, Nymphalidae, Peridae, Lycaenidae and Riodinidae）. Both maximum likelihood and Bayesian inference analyses highly supported the monophyly of Lycaenidae＋Riodinidae, which was standing as the sister of Nymphalidae. In addition, we propose that the riodinids be categorized into the family Lycaenidae as a subfamilial taxon.

Characterization of the Complete Mitochondrial Genome of the Hybrid Epinephelus moara♀ × Epinephelus lanceolatus♂,and Phylogenetic Analysis in Subfamily Epinephelinae

This study presents the complete mitochondrial genome of the hybrid Epinephelus moara♀× Epinephelus lanceolatus♂. The genome is 16886 bp in length, and contains 13 protein-coding genes, 2 r RNA genes, 22 t RNA genes, a light-strand replication origin and a control region. Additionally, phylogenetic analysis based on the nucleotide sequences of 13 conserved protein-coding genes using the maximum likelihood method indicated that the mitochondrial genome is maternally inherited. This study presents genomic data for studying phylogenetic relationships and breeding of hybrid Epinephelinae.

Nuclear pseudogenes of mitochondrial DNA as a variable part of the human genome

Novel pseudogenes homologous to the mitochondrial(mt) 16S rRNA gene were detected via different approaches. Eight pseudogenes were sequenced. Copynumber polymorphism of the mtDNA pseudogenes wasobserved among randomly chosen individuals, and evenamong siblings. A mtDNA pseudogene in the Ychromosome was observed in a YAC clone carrying onlyrepetitive sequence tag site (STS). PCR screening of human yeast artificial chromosome (YAC) libraries showedthat there were at least 5.7×105 hp of the mtDNA pseudogenes in each haploid nuclear genome. Possible involvement of the mtDNA pseudogenes in the variable part ofthe human nuclear genome is discussed.

The mitochondrial genome of Chaeturichthys stigmatias provides novel insight into the interspecific difference with Amblychaeturichthys hexanema

Chaeturichthys stigmatias and Amblychaeturichthys hexanema belong to the family Gobiidae,which are offshore warm fish species and widely distribute in the western Pacific Ocean.In this study,the mitochondrial cytochrome c oxidase subunit I(COI)sequences and 12 S ribosomal RNA(12 S rRNA)sequences were used to analyze the interspecific differences between the two species.The phylogenetic analysis showed that the interspecific distance was significantly higher than the intraspecific genetic distance.The Neighbor-Joining tree showed two separate clusters,without sharing haplotype.The mitochondrial genome sequence of C.stigmatias was also reported.This genome was 17134 bp in size,with a high A+T content of 55.9%.The phylogenetic analysis based on the tandem 13 coding protein genes nucleotide sequences indicated that C.stigmatias showed a close relationship with A.hexanema.This study can provide the basic genetic data for two species and will help for constructing the phylogeny of the Gobiiade.

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.

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.

Sequencing and phylogenetic analysis of the Pyrgilauda ruficollis （Aves, Passeridae） complete mitochondrial genome

In this study, both long PCR and conserved primers walking sequencing methods were used to determine the complete sequence of the of Pyrgilauda ruficollis mitochondrial genome （KC836121）. The results showed that the complete mitochondrial genome ofP. ruficollis is 16909 bp in length with 55.0% A＋T content, harboring the typical 37 genes. The mitogenome had the same gene order with that ofPodoces hendersoni. All protein coding genes started with ATG codon, except ND3 with GTG For the stop codon usage, most genes terminate with codons TAA or TAG; but ND5 terminated with AGA, while ND1 and COl genes with AGG; and both the genes COIII and ND4 have an incomplete termination codon （T）. The secondary structures of 22 tRNA genes were also predicted, showing that all tRNAs can form typical clover-leaf secondary structures, except for the tRNAs^r （AGN） which loses the DHU arm, while tRNAPhe harbor an extra nucleotide inserted in the TvC arm. The predicted secondary structures of 12S rRNA and 16S rRNA exhibit 47 helices in 4 domains and 60 helices in 6 domains respectively.-The control region ofP. ruficollis with the length of 1305 bp was located between tRNA6lu and tRNAphe, and typical domains of which could be found as other bird groups. Using the data from 13 mitochondrial protein-coding genes, results of a final phylogenetic analysis strongly supports the traditional view that P ruficollis is closely related with Passeridae and Fringillidae.