The phylogeny of Ephemeroptera in Pterygota revealed by the mitochondrial genome of Siphluriscus chinensis (Hexapoda: Insecta)

The mayfly species Siphluriscus chinensis (Siphluriscidae) has valuable structures useful for phylogeny reconstruction, given its putative basal position within the Ephemeroptera. Here its nearly complete mitochondrial genome is sequenced. We built phylogenetic trees through multiple analytical strategies with some other insect mitogenomes. Structurally, the obtained mitochondrial genome of S. chinensis is 16,616 bp in length, ¹ containing 37 genes and an extra trnK-like (trnK2 (AAA)) gene. The 12 PCGs start with typical ATN codons, except the nad1 gene which starts with an unnormalized TTG. Like other known mayfly mitogenomes, the strand bias has negative AT-skew and negative GC-skew. Phylogenetically, our topologies suggest that Odonata is the basally diverged clade in Pterygota; Ephemeroptera is the sister group of the Neoptera; and S. chinensis is indeed the most basal mayfly branch.     

"古翅类"系统发育关系研究进展(昆虫纲,有翅类)

古翅类的系统发育问题是六足总纲中有争议的热点问题之一。对现存古翅类(孵蝣目 蜻蜒目)与新翅类之间的系统发育关系有3种主要观点：古翅类(＝蜉蝣目 蜻蜒目) 新翅类,蜉蝣目 (蜻蜒目 新翅类),蜻蜒目 (蜉蝣目 新翅类)。第1种观点得到化石、形态和部分基因证据支持,第2种观点得到较多形态特征支持,支持第3种观点的证据较少。这一问题的解决有赖于更多昆虫种类、化石以及分子证据的发现和研究。     

Mitochondrial Genome of the Stonefly Kamimuria wangi (Plecoptera: Perlidae) and Phylogenetic Position of Plecoptera Based on Mitogenomes

This study determined the mitochondrial genome sequence of the stonefly, Kamimuria wangi. In order to investigate the relatedness of stonefly to other members of Neoptera, a phylogenetic analysis was undertaken based on 13 protein-coding genes of mitochondrial genomes in 13 representative insects. The mitochondrial genome of the stonefly is a circular molecule consisting of 16,179 nucleotides and contains the 37 genes typically found in other insects. A 10-bp poly-T stretch was observed in the A+T-rich region of the K. wangi mitochondrial genome. Downstream of the poly-T stretch, two regions were located with potential ability to form stem-loop structures; these were designated stem-loop 1 (positions 15848–15651) and stem-loop 2 (15965–15998). The arrangement of genes and nucleotide composition of the K. wangi mitogenome are similar to those in Pteronarcys princeps, suggesting a conserved genome evolution within the Plecoptera. Phylogenetic analysis using maximum likelihood and Bayesian inference of 13 protein-coding genes supported a novel relationship between the Plecoptera and Ephemeroptera. The results contradict the existence of a monophyletic Plectoptera and Plecoptera as sister taxa to Embiidina, and thus requires further analyses with additional mitogenome sampling at the base of the Neoptera.     

基于线粒体基因组序列的鞘翅目肉食亚目水生类群系统发育分析

【目的】明确肉食亚目(Adephaga)水生类群线粒体基因组的基本特征,并基于线粒体基因组序列分析肉食亚目水生类群的系统发育关系。【方法】基于Illumina HiSeq X Ten测序技术测定了圆鞘隐盾豉甲Dineutus mellyi和齿缘龙虱Eretes sticticus的线粒体全基因组序列,对其进行了基因注释,并对其tRNA基因二级结构进行了预测分析。加上已公布的鞘翅目(Coleoptera)肉食亚目水生类群17个种的线粒体基因组序列,对该类群共19个种线粒体的蛋白质编码基因(protein-coding genes, PCGs)开展了比较基因组学分析,包括AT含量、密码子偏好性、选择压力等。基于13个PCGs的氨基酸序列和核苷酸序列,利用最大似然法(ML)和贝叶斯法(BI)分别构建鞘翅目肉食亚目水生类群的系统发育关系,并通过FcLM分析进一步评估伪龙虱科(Noteridae)和瀑甲科(Meruidae)的系统发育位置。【结果】圆鞘隐盾豉甲和齿缘龙虱的线粒体基因组全长分别为16 123 bp(GenBank登录号: MN781126)和16 196 bp(GenBank登录号: MN781132),都包含13个PCGs、22个tRNA基因、2个rRNA基因和1个D-loop区(控制区)。19个肉食亚目水生类群线粒体基因组PCGs的碱基组成都呈现A+T偏好性,在密码子使用上也都偏向于使用富含A+T的密码子;在进化过程中13个PCGs的进化模式相同,都受到纯化选择。基于线粒体基因组13个PCGs的氨基酸序列的肉食亚目水生类群的系统发育关系为(豉甲科Gyrinidae+(沼梭甲科Haliplidae+((壁甲科Aspidytidae+(两栖甲科Amphizoidae+龙虱科Dytiscidae))+(水甲科Hygrobiidae+(瀑甲科Meruidae+伪龙虱科Noteridae)))))。【结论】研究结果表明,豉甲科是肉食亚目水生类群的基部类群,接下来是沼梭甲科和龙虱总科;伪龙虱科和瀑甲科互为姐妹群,并一起作为龙虱总科内部的一个分支;两栖甲科与龙虱科具有更近的亲缘关系。     

Complete mitochondrial genome of Oxya chinensis （Orthoptera, Acridoidea）

The complete sequence of Oxya chinensis （0. chinensis） mitochondrial genome is reported here. It is 15,443 bp in length and contains 75.9% A＋T. The protein-coding genes have a similar A＋T content （75.2%）. The initiation codon of the cytochrome oxidase subunit I gene in the mitochondrial genome of O. chinensis appears to be ATC, instead of the tetranucleotides that have been reported in Locusta migratoria （L migratoria） mitochondrial genome. The sizes of the large and small ribosomal RNA genes are 1319 and 850 bp, respectively. The transfer RNA genes have been modeled and showed strong resemblance to the dipteran transfer RNAs, and all anticodons are identical to those of dipteran. The A＋T-rich region is 562 bp, shorter than that of other known Orthoptera insects. The six conserved domains were identified within the A＋T-rich region by comparing its sequence with those of other grasshoppers. The result of phylogenetic analysis based on the dataset containing 12 concatenated protein sequences confirms the close relation-ship of O. chinensis with L migratoria.     

The complete mitochondrial genome of the cockroach <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> (Blattaria: Polyphagidae) and the phylogenetic relationships within the Dictyoptera

We present the complete mitochondrial DNA sequence of Eupolyphaga sinensis. This closed circular molecule is 15553 bp long and consists of 37 genes that encode for 13 inner membrane proteins, 2 ribosomal RNAs and 22 transfer RNAs. The genome shares the gene order and orientation with previously known Blattaria mitochondrial genomes. All tRNAs could be folded into the typical cloverleaf secondary structure, but the tRNASer (AGN) appears to be missing the DHU arm. The A + T-rich region is 857 bp long and longer than other cockroaches. Based on the concatenated amino acid sequences of all protein coding genes of E. sinensis in conjunction with those 23 other arthropod sequences, we reconstruct the phylogenetic tree. Phylogenetic analyses shows that Blataria (including Isoptera) and the Mantodea are sister groups. Furthermore the relationship of the three basal clades of winged insects are different from the three previous hypotheses ((Ephemeroptera + Odonata) +Neoptera, Ephemeroptera + (Odonata + Neoptera), Odonata + (Ephemeroptera +Neoptera)). The Ephemeroptera (Parafronurus youi) clusters with the Plecoptera (Pteronarcys princes).     

Ultrastructure of the sperm of Dolania americana Edmunds and Traver (Ephemeroptera : Behningiidae)

The ultrastructure of the mature sperm of the mayfly, Dolania americana Edmunds and Traver (Ephemeroptera : Behningiidae), is described from scanning and transmission electron microscopy. The head is 0.7–1 μm wide and 4.6–6.9 μm long, rodlike, and topped by a short, rounded acrosome 0.4 μm long and 0.6 μm wide. The flagellum is 5–6 times the head length and is flattened, except for a thin, tubelike terminal portion. The axoneme pattern is 9-9-1 (9 outer singlet microtubules, 9 doublet microtubules, and a central dark element) and is new for Ephemeroptera. The inner dynein arms are conspicuous and outer arms are lacking, and radial spokes and a central sheath are prominent. A densely-staining and bi-lobed accessory body lies adjacent to the axoneme. A mitochondrial derivative with regularly arranged transverse-to-oblique cristae lies adjacent to the accessory body.     

眼镜王蛇线粒体基因组全序列分析

参照近缘物种线粒体基因序列共设计和合成了8对引物, 结合Ex Taq-PCR、TA克隆和步移测序技术, 文章首次获得眼镜王蛇线粒体基因组全序列(GenBank登录号: EU_921899)。该基因组全长17 267 bp, 共编码13个蛋白、2个rRNA、23个tRNA-- 其中tRNA-Ile基因发生了复制, 属于一种新的蛇类物种线粒体基因排列模式, 另外还含有2个非编码的富含AT的控制区。除了8个tRNA基因和1个蛋白基因由L链编码外, 其余均由H链编码, 其中H链编码基因的A和T含量接近, 而L链上A的含量则明显高于T。基于21种蛇合并的“12S＋16S”rRNA基因序列的系统发育分析表明, 眼镜王蛇属与眼镜蛇属亲缘关系较近, 两者与环蛇属共同构成一个单系群。作为国内外眼镜王蛇线粒体基因组全序列的首次报道, 上述结果对于蛇类物种分子系统发育和进化研究具有重要意义。     

The complete mitochondrial genome of Scutigerella causeyae (Myriapoda: Symphyla) and the phylogenetic position of Symphyla

The first complete mitochondrial genome of a representative of the Symphyla, Scutigerella causeyae (Arthropoda: Myriapoda), was sequenced using a PCR-based approach. Its gene order shows different positions for three tRNA genes compared to the ancestral arthropod pattern. Presence of a pseudogene with partial sequence similarity to rrnS favours the duplication-random loss model as an explanation for at least one of the translocations. None of the genome rearrangements hypothesized for S. causeyae are shared by any of the other four myriapod mitochondrial genomes sequenced so far (two from Chilopoda and two from Diplopoda). Different rearrangement events must have occurred independently in the lineages leading to S. causeyae, Lithobius forficatus, Scutigera coleoptrata and Diplopoda. Phylogenetic analyses could not unequivocally elucidate the position of Symphyla among myriapods. While the nucleotide dataset of eleven protein-coding genes gives weak support for an affinity to Chilopoda, this is not recovered with the corresponding amino acid dataset.     

Morphology of Male Reproductive Systems in Ephemeroptera: Intrinsic Musculature

Although the Ephemeroptera have been studied over a long period of time, there are still few studies on the morphology of male reproductive system. The spermatic ducts are considered conserved among different Ephemeroptera groups. However, previous studies distinguished different organizational patterns of the spermatic duct intrinsic musculature. This study describes the morphology of the spermatic ducts, especially their musculature, in six species of Ephemeroptera, representing five families. We have observed variations in the organizational pattern of the spermatic ducts, even between species from the same family. Moreover, all species studied had intrinsic musculature in the spermatic ducts although with different organizational patterns. Thus, we believe that this musculature is important to move the spermatozoa along the ducts of all Ephemeroptera and not only of those with aflagellated spermatozoa (Leptophlebiidae). The variations in musculature organization must be related to differences in reproductive physiology (i.e., copula duration) and not only with spermatozoa characteristics.     

Variation of the mitochondrial genome in the evolution of Drosophila

The evidence on mitochondrial genome variation and its role in evolution of the genus Drosophila are reviewed. The mitochondrial genome is represented by a circular double-stranded DNA molecule 16 to 19 kb in length. The genome contains no introns involved in recombination. The entire mitochondrial genome can be arbitrarily divided into three parts: (1) protein-coding genes; (2) genes encoding rRNA and tRNA; and (3) the noncoding regulatory region (A + T region). The selective importance of mutations within different mtDNA regions is therefore unequal. In Drosophila, the content of the A + T pairs in mtDNA is extremely low and a pattern of nucleotide substitution is characterized by a low transition/transversion ratio (and a low threshold of mutation saturation). The deletions and duplications are of common occurrence in the mitochondrial genome. However, this genome lacks such characteristic for the nuclear genome aberrations as the inversions and transpositions. The phenomena of introgression and heteroplasmy provide an opportunity to study the adaptive role of the mitochondrial genome and its role in speciation. Analysis of evidence concerning mtDNA variation in different species of the genus Drosophila made it possible to ascertain data on phylogenetic relationships among species obtained by studying nuclear genome variation. In some species, mtDNA variation may serve as a reliable marker for population differentiation within a species, although evidence on the population dynamics of the mtDNA variation is very scarce.     

Complete mitochodrial genome of the crab spider Ebrechtella tricuspidata (Araneae: Thomisidae): A novel tRNA rearrangement and phylogenetic implications for Araneae

Mitochondrial genomes are widely used for phylogenetic and phylogeographic analyses among arthropods, but there is a lack of sufficient mitochondrial genome sequence data for spiders. Herein, we sequenced and characterized the complete mitochondrial genome of a crab spider Ebrechtella tricuspidata (Araneae: Thomisidae). The circular mitochondrial genome is 14,352 bp long, including a standard set of 37 genes and an A + T-rich region. Nucleotide composition is highly biased toward A + T nucleotides (77.3%). A novel gene order rearrangement is detected by a tRNA (trnL1) translocation. Tandem repeats are not identified in the A + T-rich region. Most of the tRNAs are greatly reduced in size and cannot be folded into typical cloverleaf-shaped secondary structures. The phylogenetic analysis confirms that the mitochondrial genome sequences are useful in resolving higher-level relationship of Araneae. Overall, our data present in this study will elevate our knowledge on the architecture and evolution of spider mitochondrial genome.     

Complete Mitochondrial Genome of the Free-Living Earwig, Challia fletcheri (Dermaptera: Pygidicranidae) and Phylogeny of Polyneoptera

The insect order Dermaptera, belonging to Polyneoptera, includes ～2,000 extant species, but no dermapteran mitochondrial genome has been sequenced. We sequenced the complete mitochondrial genome of the free-living earwig, Challia fletcheri, compared its genomic features to other available mitochondrial sequences from polyneopterous insects. In addition, the Dermaptera, together with the other known polyneopteran mitochondrial genome sequences (protein coding, ribosomal RNA, and transfer RNA genes), were employed to understand the phylogeny of Polyneoptera, one of the least resolved insect phylogenies, with emphasis on the placement of Dermaptera. The complete mitochondrial genome of C. fletcheri presents the following several unusual features: the longest size in insects is 20,456 bp; it harbors the largest tandem repeat units (TRU) among insects; it displays T- and G-skewness on the major strand and A- and C-skewness on the minor strand, which is a reversal of the general pattern found in most insect mitochondrial genomes, and it possesses a unique gene arrangement characterized by a series of gene translocations and/or inversions. The reversal pattern of skewness is explained in terms of inversion of replication origin. All phylogenetic analyses consistently placed Dermaptera as the sister to Plecoptera, leaving them as the most basal lineage of Polyneoptera or sister to Ephemeroptera, and placed Odonata consistently as the most basal lineage of the Pterygota.     

枣食芽象甲线粒体基因组全序列测定与系统发育分析

【目的】从线粒体基因组水平上探讨枣食芽象甲Scythropus yasumatsui与近缘种的系统发育关系。【方法】利用Illumina MiSeq测序平台对枣食芽象甲线粒体基因组进行测序,对基因组序列进行拼装、注释和特征分析;利用贝叶斯法和最大似然法构建基于象甲科13个物种的线粒体基因组13个蛋白质编码基因核苷酸序列的系统发育树。【结果】结果表明,枣食芽象甲线粒体基因组全长为16 472 bp (GenBank登录号: MF807224),包含13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和2个非编码控制区,37个基因的排列顺序与祖先昆虫的线粒体基因排列顺序一致。13个蛋白质编码基因的起始密码子为ATN,其中除了cob和nad1基因的完全终止密码子为TAG外,其余11个基因的完全终止密码子为TA(A)。22个tRNA基因中除了trnS1缺少DHU臂,反密码子由GCT变为TCT外,其余均能形成典型的三叶草结构。基于13个蛋白质编码基因序列构建的系统发育树结果显示,象甲科8个亚科系统发育关系为：(((隐喙象亚科(Cryptorhynchinae)+(象虫亚科(Curculioninae)+魔喙象亚科(Molytinae)))+长小蠹亚科(Platypodinae))+(粗喙象亚科(Entiminae)+Cyclominae亚科))+隐颏象亚科(Dryophthorinae)+小蠹亚科(Scolytinae))。【结论】在13种象甲科昆虫物种中,同属于粗喙象亚科的枣食芽象甲与南美果树象甲Naupactus xanthographus在系统发育树中聚为同一分支,表明基于线粒体基因组全序列的分子系统发育结果与传统的形态分类结果是一致的。     

Variation of the Mitochondral Genome in the Evolution of Drosophila

The evidence on mitochondrial genome variation and its role in evolution of the genus Drosophila are reviewed. The mitochondrial genome is represented by a circular double-stranded DNA molecule 16 to 19 kb in length. Mitochondrial genes lack introns and recombination. The entire mitochondrial genome can be arbitrarily divided into three parts: (1) protein-coding genes; (2) genes encoding rRNA and tRNA; and (3) the noncoding regulatory region (A + T region). The selective importance of mutations within different mtDNA regions is therefore unequal. In Drosophila, the content of the A + T pairs in mtDNA is extremely high and a pattern of nucleotide substitution is characterized by a low transition/transversion ratio (and a low threshold of mutation saturation). The deletions and duplications are of common occurrence in the mitochondrial genome. However, this genome lacks such characteristic for the nuclear genome aberrations as inversions and transpositions. The phenomena of introgression and heteroplasmy provide an opportunity to study the adaptive role of the mitochondrial genome and its role in speciation. Analysis of evidence concerning mtDNA variation in different species of the genus Drosophilamade it possible to ascertain data on phylogenetic relationships among species obtained by studying nuclear genome variation. In some species, mtDNA variation may serve as a reliable marker for population differentiation within a species, although evidence on the population dynamics of the mtDNA variation is very scarce.     

The complete mitochondrial genome of the nudibranch Roboastra europaea (Mollusca: Gastropoda) supports the monophyly of opisthobranchs

The complete nucleotide sequence (14,472 bp) of the mitochondrial genome of the nudibranch Roboastra europaea (Gastropoda: Opisthobranchia) was determined. This highly compact mitochondrial genome is nearly identical in gene organization to that found in opisthobranchs and pulmonates (Euthyneura) but not to that in prosobranchs (a paraphyletic group including the most basal lineages of gastropods). The newly determined mitochondrial genome differs only in the relative position of the trnC gene when compared with the mitochondrial genome of Pupa strigosa, the only opisthobranch mitochondrial genome sequenced so far. Pupa and Roboastra represent the most basal and derived lineages of opisthobranchs, respectively, and their mitochondrial genomes are more similar in sequence when compared with those of pulmonates. All phylogenetic analyses (maximum parsimony, minimum evolution, maximum likelihood, and Bayesian) based on the deduced amino acid sequences of all mitochondrial protein-coding genes supported the monophyly of opisthobranchs. These results are in agreement with the classical view that recognizes Opisthobranchia as a natural group and contradict recent phylogenetic studies of the group based on shorter sequence data sets. The monophyly of opisthobranchs was further confirmed when a fragment of 2,500 nucleotides including the mitochondrial cox1, rrnL, nad6, and nad5 genes was analyzed in several species representing five different orders of opisthobranchs with all common methods of phylogenetic inference. Within opisthobranchs, the polyphyly of cephalaspideans and the monophyly of nudibranchs were recovered. The evolution of mitochondrial tRNA rearrangements was analyzed using the cox1+rrnL+nad6+nad5 gene phylogeny. The relative position of the trnP gene between the trnA and nad6 genes was found to be a synapomorphy of opisthobranchs that supports their monophyly.     

Complete nucleotide sequence and gene organization of the mitochondrial genome of Paa spinosa (Anura: Ranoidae)

The mt genome of Paa spinosa (Anura: Ranoidae) is a circular molecule of 18,012 bp in length, containing 38 genes (including an extra copy of tRNA-Met gene). This mt genome is characterized by three distinctive features: a cluster of rearranged tRNA genes (LTPF tRNA gene cluster), a tandem duplication of tRNA-Met gene (Met1 and Met2), and distinct repeat regions at both 5′ and 3′-sides in the control region. Comparing the locations and the sequences of all tRNA-Met genes among Ranoidae, and constructing NJ tree of the nucleotide of those tRNA-Met genes, we suggested a tandem duplication of tRNA-Met gene can be regarded as a synapomorphy of Dicroglossinae. To further investigate the phylogenetic relationships of anurans, phylogenetic analyses (BI, ML and MP) based on the nucleotide dataset and the corresponding amino acid dataset of 11 protein-coding genes (except ND5 and ATP8) arrived at the similar topology.     

The sperm of Hexagenia (Pseudeatonica) albivitta Walker (Ephemeroptera: Fossoriae: Ephemeridae)

This study describes the sperm morphology of the mayfly Hexagenia (Pseudeatonica) albivitta (Ephemeroptera). Its spermatozoon measures approximately 30 μm of which 9 μm corresponds to the head. The head is composed of an approximately round acrosomal vesicle and a cylindrical nucleus. The nucleus has two concavities, one in the anterior tip, where the acrosomal vesicle is inserted and a deeper one at its base, where the flagellum components are inserted. The flagellum is composed of an axoneme, a mitochondrion and a dense rod adjacent to the mitochondrion. A centriolar adjunct is also observed surrounding the axoneme in the initial portion of the flagellum and extends along the flagellum for at least 2 μm, surrounding the axoneme in a half‐moon shape. The axoneme is the longest component of the flagellum, and it follows the 9+9+0 pattern, with no central pair of microtubules. At the posterior region of the flagellum, the mitochondrion has a dumb‐bell shape in cross sections that, together with the rectangular mitochondrial‐associated rod, is responsible for the flattened shape of the flagellum. An internal membrane is observed surrounding both mitochondrion and its associated structure.     

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 phylogenetie 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+(Calliptaminae +Cyrtacanthaeridinae))))).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 T()C loop(TRNA),and two rRNA genes(RRNA)perform poorly when analyzed as single dataset.Our results suggest that the best phylogenetie 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.