Phylogenetic relationships and divergence dates of softshell turtles (Testudines: Trionychidae) inferred from complete mitochondrial genomes

The softshell turtles (Trionychidae) are one of the most widely distributed reptile groups in the world, and fossils have been found on all continents except Antarctica. The phylogenetic relationships among members of this group have been previously studied; however, disagreements regarding its taxonomy, its phylogeography and divergence times are still poorly understood as well. Here, we present a comprehensive mitogenomic study of softshell turtles. We sequenced the complete mitochondrial genomes of 10 softshell turtles, in addition to the GenBank sequence of Dogania subplana, Lissemys punctata, Trionyx triunguis, which cover all extant genera within Trionychidae except for Cyclanorbis and Cycloderma. These data were combined with other mitogenomes of turtles for phylogenetic analyses. Divergence time calibration and ancestral reconstruction were calculated using BEAST and RASP software, respectively. Our phylogenetic analyses indicate that Trionychidae is the sister taxon of Carettochelyidae, and support the monophyly of Trionychinae and Cyclanorbinae, which is consistent with morphological data and molecular analysis. Our phylogenetic analyses have established a sister taxon relationship between the Asian Rafetus and the Asian Palea + Pelodiscus + Dogania + Nilssonia + Amyda, whereas a previous study grouped the Asian Rafetus with the American Apalone. The results of divergence time estimates and area ancestral reconstruction show that extant Trionychidae originated in Asia at around 108 million years ago (MA), and radiations mainly occurred during two warm periods, namely Late Cretaceous–Early Eocene and Oligocene. By combining the estimated divergence time and the reconstructed ancestral area of softshell turtles, we determined that the dispersal of softshell turtles out of Asia may have taken three routes. Furthermore, the times of dispersal seem to be in agreement with the time of the India–Asia collision and opening of the Bering Strait, which provide evidence for the accuracy of our estimation of divergence time. Overall, the mitogenomes of this group were used to explore the origin and dispersal route of Trionychidae and have provided new insights on the evolution of this group.     

新疆裂腹鱼类的系统发生关系及物种分化时间

文章采用线粒体COI、ND4、16S rRNA基因联合数据分析了新疆分布的裂腹鱼类4属10个种或亚种的分子系统发育,并通过鲤亚科的分化时间、黄河上游和青海湖隔离的地质时间来校正分子钟,进而探讨了裂腹鱼类分化时间与塔里木盆地周边地区主要地质事件和气候变化的关系。结果发现,扁吻鱼属未能形成独立的分支,与塔里木裂腹鱼、厚唇裂腹鱼聚在一起。进一步基于COI基因序列,利用Kimura双参数模型计算遗传距离,发现扁吻鱼属和裂腹鱼属间的遗传距离未达到属间区别,扁吻鱼可能是裂腹鱼属内特化的物种;聚类分析结果不同于裂腹鱼属的形态学分类方法,不支持裂腹鱼属鱼类的亚属划分;原始等级裂腹鱼类(包括裂腹鱼属和扁吻鱼属)两个族群的分化时间(8.18 Ma)与新疆裸重唇鱼和斑重唇鱼的分化时间(7.67 Ma)均发生于晚中新世,这可能与8Ma左右青藏高原北缘和北天山抬升、塔里木盆地气候干旱化等重大地质事件和气候转型导致青藏高原北缘(昆仑山)和北天山水系间出现分离相关。而塔里木盆地裂腹鱼属鱼类现今的分布格局可能与第四纪喜马拉雅运动使盆地形成西高东低的地势、罗布泊成为塔里木盆地周边高山发源河流的汇水中心相关。     

Revisiting the phylogeny of Ranunculeae: Implications for divergence time estimation and historical biogeography

Previous studies based on different molecular datasets have generated conflicting topologies for Ranunculeae. Here, we revisit the phylogeny of Ranunculeae by analyzing the individual matK/trnK, psbJ-petA, and internal transcribed spacer (ITS) data, the combined matK/trnK, psbJ-petA, and ITS dataset, and the combinedrbcL, trnL-F, matK/trnK, psbJ-petA, and ITS dataset. Based on the tree-based comparisons, blast searches against NCBI of the sequences, and close examination of the alignment, we found that 10 psbJ-petA sequences previously used were questionable (erroneous or problematic) and responsible for previous conflicting topologies. After omitting these questionable sequences, we provide a new phylogeny for Ranunculeae, in which Beckwithia–Cyrtorhyncha, Kumlienia, andPeltocalathos were replaced. These new replacements are supported by corresponding morphological characters. Moreover, three previously proposed intercontinental disjunct distributions within Ranunculus were also refuted. In our framework, our divergence time and biogeographic analyses indicate that divergence time estimates and the ancestral areas reconstructed for 10 of the 15 nodes in the genus-level phylogeny were influenced by elimination of the questionable sequences. The most recent common ancestor of Ranunculeae was inferred to be present in Europe and North America during the late Eocene. Clades I and II began to diversify in Europe and North America, respectively, and subsequently migrated to other continents. This study shows that it is necessary to analyze individual chloroplast DNA region datasets separately to detect questionable sequences early in the study. The combined dataset including the questionable sequences resulted in an erroneous phylogenetic tree, and the use of this tree subsequently affected age estimates and biogeographic analyses.     

Complete mitochondrial genomes of three skippers in the tribe Aeromachini (Lepidoptera: Hesperiidae: Hesperiinae) and their phylogenetic implications

The mitochondrial genome is now widely used in the study of phylogenetics and molecular evolution due to its maternal inheritance, fast evolutionary rate, and highly conserved gene content. To explore the phylogenetic relationships of the tribe Aeromachini within the subfamily Hesperiinae at the mitochondrial genomic level, we sequenced and annotated the complete mitogenomes of 3 skippers: Ampittia virgata, Halpe nephele, and Onryza maga (new mitogenomes for 2 genera) with a total length of 15,333 bp, 15,291 bp, and 15,381 bp, respectively. The mitogenomes all contain 13 protein‐coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a noncoding A + T‐rich region and are consistent with other lepidopterans in gene order and type. In addition, we reconstructed the phylogenetic trees of Hesperiinae using maximum likelihood (ML) and Bayesian inference (BI) methods based on mitogenomic data. Results show that the tribe Aeromachini in this study robustly constitute a monophyletic group in the subfamily Hesperiinae, with the relationships Coeliadinae + (Euschemoninae + (Pyrginae + ((Eudaminae + Tagiadinae) + (Heteropterinae + ((Trapezitinae + Barcinae) + Hesperiinae))))). Moreover, our study supports the view that Apostictopterus fuliginosus and Barca bicolor should be placed out of the subfamily Hesperiinae.     

Evolutionary implications of analyses of complete mitochondrial genomes across order Zoantharia (Cnidaria: Hexacorallia)

Cnidarians are early-diverging metazoans, but evolutionary aspects of some taxa are still poorly understood, as in the order Zoantharia (Anthozoa: Hexacorallia). Zoantharians have been divided into two suborders based on the arrangement of the fifth septae as complete (Macrocnemina) or incomplete (Brachycnemina). Previous molecular phylogenetic analyses have indicated the need for re-evaluation as Macrocnemina has been found to be paraphyletic. Despite many phylogenetic studies, the recovery of complete mitochondrial genomes (mt-genomes) for systematic and evolutionary studies of zoantharians has been limited. The present study represents the first to sequence the complete mt-genomes of members of eight of nine zoantharian families. Although all examined mt-genomes had the same gene order arrangement, there were variations among mt-genomes' sizes, nucleotide substitution rates, and introns. Only two species did not have the cox1 intron, which harbors a gene coding a homing endonuclease of the LAGLIDADG type. Our mitogenomic analyses also showed relatively high nucleotide diversity in mt-DNA regions other than the standard regions traditionally considered for DNA barcoding of this group. Phylogenetic analyses using 13 mt-genome protein-coding genes recovered a fully resolved tree with clear separation between macrocnemic representatives. Ancestral state reconstruction analyses revealed three main transitions in arrangement of the marginal musculature through the evolutionary history of the order. An “early” transition from reticulate mesogleal to a cteniform endodermal arrangement was followed by transitions that occurred in the common ancestor of the Brachycnemina and family Hydrozoanthidae. Our results indicate the need for clarification of higher-level phylogeny and taxonomy of Zoantharia.     

Phylogenetic relationships of nine mullet species (Mugilidae) in the Mediterranean Sea

Phylogenetic relationships among four genera and nine species (Mugil cephalus, Mugil soiuy, Liza ramada, Liza aurata, Liza abu, Liza saliens, Liza carinata, Chelon labrosus, Oedalechilus labeo) of the Mediterranean mullets (Mugilidae) were investigated by means of allozyme electrophoresis using a seven-enzyme system (CK*, GAPDH*, G3PDH*, IDHP*, ME*, MDH*, PGM*) comprising eleven putative loci. The highest genetic divergence was 1.299, detected between M. cephalus and L. aurata and the lowest (0.280) was found between L. carinata and L. saliens. The amount of genetic divergence between the genera Chelon and Oedalechilus did not appear to be high (0.285). In a UPGMA tree, all nine species were grouped in two main branchings. In the first branch, C. labrosus and O. labeo clustered as closest taxa and were sister group to L. ramada. The other four Liza species produced two sub-branching in this group; L. carinata branched together with L. saliens, and L. aurata branched together with L. abu. In the second branch the two species of the genus Mugil, M. cephalus and M. soiuy, clustered together and were clearly isolated from the other three genera.     

The complete mitochondrial genome of Pardosa pusiola (Araneae,Lycosidae) and its phylogenetic implications

The mitochondrial genome (mitogenome) is useful for identification and phylogenetic analyses among arthropods, but there are no sufficient mitogenome data for wolf spiders. To enrich the mitogenome database of wolf spiders, the complete mitogenome of Pardosa pusiola was sequenced by high-throughput sequencing. It is 14,284 bp, comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a control region (CR). It represents a high bias toward A and T nucleotides with an A + T content of 76.49%. The mitogenome exhibited a negative AT skew (−0.13) and a positive GC skew (0.32). Most PCGs started with ATN codons and ended with TAA, TAG, or an incomplete T. In addition, most tRNAs had aberrant secondary structures with the absence of DHU arm or TΨC arm. Analysis performed with CREx software demonstrated that large-scale rearrangements of tRNAs were observed in the mitogenome of P. pusiola as compared with the putative ancestral mitogenome. The Bayesian inference (BI) and maximum likelihood (ML) phylogenetic trees based on the 13 PCGs of 25 spiders had the same topology, which could be presented as (Araneidae + (Agelenidae + (Dictynidae + Desidae)) + (Salticidae + (Thomisidae + (Oxyopidae + (Pisauridae + Lycosidae))))). This study offers a useful genetic resource for the taxonomy and phylogeny of spiders.     

基于线粒体基因组的华蝉族系统发育地位研究(半翅目: 蝉科)(英文)

【目的】本研究旨在明确无鼓膜发音器的华蝉族(Sinosenini)昆虫在蝉总科(Cicadoidea)的系统发育地位。【方法】依据在陕西宁陕采集的合哑蝉Karenia caelatata成虫标本,对华蝉族的合哑蝉K. caelatata线粒体基因组进行测序、注释和生物信息学分析;并与蝉总科其他类群的线粒体基因组进行了比较,然后利用最大似然法(ML)和贝叶斯法(BI)分别构建了蝉总科分子系统发育树。【结果】合哑蝉线粒体基因组长14 960 bp (GenBank登录号: MN922304),其基因组成、蛋白编码基因的核苷酸组成和密码子使用等,与蝉总科其他类群具相似特征。核苷酸多样性分析表明,atp8, nad6和nad2为易变基因,而cox1比较保守。非同义替换率和同义替换率比表明,蝉总科昆虫线粒体基因组进化处于高水平的纯化选择下。系统发育分析结果支持蝉次目(Cicadomorpha)的单系性,该次目3个总科的关系为：角蝉总科Membraciodea+(蝉总科Cicadoidea+沫蝉总科Cercopodidea)。无鼓膜发音器的哑蝉属与蝉亚科(Cicadinae)的蜩蝉族(Dundubiini)相关类群聚在一起,且与寒蝉属Meimuna关系最近;黑蝉族(Cicadatrini)的草蝉属Mogannia和音蝉属Vagitanus则与姬蝉亚科(Cicadettinae)相关类群聚在一起;日宁蝉属Yezoterpnosia并非一个单系群。【结论】华蝉族应从姬蝉亚科转移至蝉亚科并与蜩蝉族(Dundubiini)合并,而黑蝉族应从蝉亚科转移至姬蝉亚科。研究结果为进一步解析具有不同发声机制的蝉科昆虫系统演化提供了新信息。     

Taenia asiatica and Taenia saginata: genetic divergence estimated from their mitochondrial genomes

We conducted a differential identification of Taenia asiatica and Taenia saginata, through the mapping of mitochondrial genomes and the sequencing of the cox1 and cob genes. The entire mitochondrial genomes of T. asiatica and T. saginata were amplified by long-extension PCR and cloned; each was approximately 14 kb in size. Restriction maps of T. asiatica and T. saginata mitochondrial genomes were then constructed using 13 restriction enzymes. The resulting restriction patterns enable us to estimate their genetic divergence at 4.8%. The actual sequence divergence was computed 4.5% from the cox1 gene, and 4.1% from the cob gene. These results support the designation of T. asiatica as a separate species from T. saginata.     

A comparison of mitochondrial tRNAs in five vertebrates

Sequences of several vertebrate mitochondrial tRNAs were aligned and compared. The comparisons were made in pairs of the tRNAs for an identical amino acid. There are 22 genes for different tRNAs in each vertebrate mitochondrial DNA. The closest similarities were between rat and mouse, the next were between mammals, and the widest difference was between human or rat and Xenopus laevis. However, there were very wide variations between different amino acids in each set of comparisons. The time lapse for each percent of difference greatly increased with evolutionary separation. Most of the nucleotide substitutions appeared to be neutral in character.Based on a presentation made at a workshop- Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code-held at Berkeley, CA, July 17–20, 1994     

Shifting distributions and speciation: species divergence during rapid climate change

Questions about how shifting distributions contribute to species diversification remain virtually without answer, even though rapid climate change during the Pleistocene clearly impacted genetic variation within many species. One factor that has prevented this question from being adequately addressed is the lack of precision associated with estimates of species divergence made from a single genetic locus and without incorporating processes that are biologically important as populations diverge. Analysis of DNA sequences from multiple variable loci in a coalescent framework that (i) corrects for gene divergence pre-dating speciation, and (ii) derives divergence-time estimates without making a priori assumptions about the processes underlying patterns of incomplete lineage sorting between species (i.e. allows for the possibility of gene flow during speciation), is critical to overcoming the inherent logistical and analytical difficulties of inferring the timing and mode of speciation during the dynamic Pleistocene. Estimates of species divergence that ignore these processes, use single locus data, or do both can dramatically overestimate species divergence. For example, using a coalescent approach with data from six loci, the divergence between two species of montane Melanoplus grasshoppers is estimated at between 200,000 and 300,000 years before present, far more recently than divergence estimates made using single-locus data or without the incorporation of population-level processes. Melanoplus grasshoppers radiated in the sky islands of the Rocky Mountains, and the analysis of divergence between these species suggests that the isolation of populations in multiple glacial refugia was an important factor in promoting speciation. Furthermore, the low estimates of gene flow between the species indicate that reproductive isolation must have evolved rapidly for the incipient species boundaries to be maintained through the subsequent glacial periods and shifts in species distributions.     

Complete mitochondrial genomes of three neobatrachian anurans: a case study of divergence time estimation using different data and calibration settings

We sequenced the whole mitochondrial (mt) genomes of three neobatrachian species: Japanese tree frog Hyla japonica, Japanese common toad Bufo japonicus, and narrow-mouthed toad Microhyla okinavensis. The gene arrangements of these genomes diverged from that of basal anurans (suborder Archaeobatrachia), but are the same as that of the members of derived frogs (i.e., superfamily Hyloidae and Ranoidae in suborder Neobatrachia), suggesting the one-time occurrence of a gene rearrangement event in an ancestral lineage of derived anurans. Furthermore, several distinct repeat motifs including putative termination-associated sequences (TASs) and conserved sequence blocks (CSBs) were observed in the control regions (CRs) of B. japonicus and H. japonica, while no repeat motifs were found in that of M. okinavensis. Phylogenetic analyses using both nucleotide and amino acid data of mt genes support monophyly of neobatrachians. The estimated divergence time based on amino acid data with multiple reference points suggests that the three living amphibian orders may have originated in the Carboniferous period, and that the divergences of anurans had occurred between the Permian and Tertiary periods. We also checked the influence of the data types and the settings of reference times on divergence time estimation. The resultant divergence times estimated from several datasets and reference time settings suggest that the substitution saturation of nucleotide data may lead to overestimated (i.e., older) branching times, especially for early divergent taxa. We also found a highly accelerated substitution rate in neobatrachian mt genes, and fast substitution possibly resulted in overestimation. To correct this erroneous estimation, it is efficient to apply several reference points among neobatrachians.     

Phylogenetic analyses and improved resolution of the family Bovidae based on complete mitochondrial genomes

Efforts have been made to investigate the phylogeny of the family Bovidae; however, the relationships within this group still remain controversial. To further our understanding of the relationships, we sequenced the mitochondrial genome of the Himalayan goral, Naemorhedus goral, an IUCN Redlist near threatened conservation dependent species. Then we conducted molecular phylogenetic relationships of the Bovidae based on Bayesian and Maximum Likelihood methods. The results indicate that the basal divergence within the Bovidae is between the Bovinae and a strongly supported clade of the remaining Bovidae species. The two Neotragus species (the suni and pygmy antelope) clustered with the impala, Aepyceros melampus (Aepycerotinae), and together they formed the most basal of the non-Bovinae. All the genera of the Antilopinae clustered together except Neotragus, which suggested that the Antilopinae was a paraphyletic subfamily. The present study confirmed a close relationship between the genera Capricornis and Naemorhedus while supporting their designation as separate genera and suggested that the Capricornis-Naemorhedus-Ovibos clade (serows, gorals, and the muskox) should be placed in the Caprinae. Bison, Bos, and Tragelaphus (bison & cattle and kudus and nyalas) were paraphyletic. The very close relationship between Bison and Bos suggested that Bos and Bison should be integrated into a single Bos genus. Saiga and Pantholops (the Chiru or Tibetan Antelope), unique genera which have sometimes been lumped together, were placed in different groups: Saiga within the Antilopinae and Pantholops at the base of the Caprinae. Our results also supported a new taxonomy which places the three species of Hemitragus into three monospecific genera: the genus Hemitragus is restricted to the Himalayan tahr, and two new genera are created: Arabitragus for the Arabian tahr and Nilgiritragus for the Nilgiri tahr.     

两种毛蚜线粒体基因组比较分析

【目的】线粒体基因组分析已被应用于昆虫系统发育研究。本研究以蚜科Aphididae重要类群毛蚜亚科物种为代表,测定并比较分析了该类蚜虫的线粒体基因组特征,探讨了基于线粒体基因组信息的蚜虫系统发育关系重建。【方法】以毛蚜亚科三角枫多态毛蚜Periphyllus acerihabitans Zhang和针茅小毛蚜Chaetosiphella stipae Hille Ris Lambers,1947为研究对象,利用长短PCR相结合的方法测定线粒体基因组的序列,分析了基因组的基本特征;基于在线t RNAscan-SE Search Server搜索方法预测了t RNA的二级结构;基于12个物种(本研究获得的2个物种和10个Gen Bank上下载的物种数据)的蛋白编码基因(PCGs)序列,利用最大似然法和贝叶斯法重建了蚜科的系统发育关系。【结果】两种毛蚜均获得了约94%的线粒体基因组数据,P.acerihabitans获得了14 908 bp,控制区为1 205 bp;C.stipae获得了13 893 bp,控制区为609 bp。两种毛蚜同时获得33个基因,包含接近完整的13个蛋白编码基因(PCGs)(nad5不完整),18个tRNA,2个rRNA基因;ka/ks值表明,C.stipae的进化速率更快。从基因组组成、基因排列顺序、核苷酸组成分析、密码子使用情况、t RNA二级结构等特征来分析,两种蚜虫线粒体基因组基本特征相似。系统发育重建结果表明毛蚜亚科、蚜亚科的单系性得到了支持,毛蚜亚科位于蚜科的基部位置。【结论】两种毛蚜线粒体基因组的基本特征相似,符合蚜虫线粒体基因组的一般特征,两种线粒体基因组的长度差异主要来自控制区长度的不同;系统发育重建支持毛蚜亚科与蚜亚科的单系性,毛蚜亚科位于蚜科较为基部的位置。研究结果为蚜虫类系统发育重建提供了参考。     

Genetic markers in Andean Puya species (Bromeliaceae) with implications on plastome evolution and phylogeny

The Andean plant endemic Puya is a striking example of recent and rapid diversification from central Chile to the northern Andes, tracking mountain uplift. This study generated 12 complete plastomes representing nine Puya species and compared them to five published plastomes for their features, genomic evolution, and phylogeny. The total size of the Puya plastomes ranged from 159,542 to 159,839 bp with 37.3%–37.4% GC content. The Puya plastomes were highly conserved in organization and structure with a typical quadripartite genome structure. Each of the 17 consensus plastomes harbored 133 genes, including 87 protein‐coding genes, 38 tRNA (transfer RNA) genes, and eight rRNA (ribosomal RNA) genes; we found 69–78 tandem repeats, 45–60 SSRs (simple sequence repeats), and 8–22 repeat structures among 13 species. Four protein‐coding genes were identified under positive site‐specific selection in Puya. The complete plastomes and hypervariable regions collectively provided pronounced species discrimination in Puya and a practical tool for future phylogenetic studies. The reconstructed phylogeny and estimated divergence time for the lineage suggest that the diversification of Puya is related to Andean orogeny and Pleistocene climatic oscillations. This study provides plastome resources for species delimitation and novel phylogenetic and biogeographic studies.     

Concordant divergence in proteins and mitochondrial DNA between two vole species in the genusClethrionomys

The bank vole (Clethrionomys glareolus) and the northern red-backed vole (C. rutilus) are two closely related species where interspecific crosses result in fertile female but sterile male offspring. Mitochondrial DNA (mtDNA) fromC. rutilus has passed the species barrier and is found inC. glareolus from northern Fennoscandia. The present report shows that the genetic distance between the two species, calculated from enzyme data (Nei'sD), is 0.64. Isoelectric focusing of muscle proteins resolved around 55 bands, of which each species had 6 or 7 bands not present in the other species. Sequence divergence of mtDNA from the two species is 13.9%. A comparison between protein and mtDNA distances in other species pairs reveals a high correlation between the two measures, indicating that differences in mtDNA between taxa are not random when compared to divergence in protein-coding nuclear genes. The relationship between genetic divergence in proteins and that in mtDNA betweenClethrionomys glareolus andC. rutilus is similar to that found in other species pairs. It is also shown that despite large differences on the protein level it is still, in some cases, possible for species pairs to produce fertile hybrid females.This study was sponsored by the Swedish Natural Science Research Council, the Erik Philip-Sörensen Foundation, and the Nilsson-Ehle Foundation.     

Epinephelus moara: a valid species of the family Epinephelidae (Pisces: Perciformes)

This study documents the major external and internal morphological differences between Epinephelus bruneus and Epinephelus moara, and analyses the complete mitogenomes of both species. The partial cytochrome oxidase subunit I (coI) sequence divergence between E. bruneus and E. moara is significantly higher than specimens within the same species (P < 0·05). Analyses of gene flow (N_m = 0·02) and genetic differentiation (?_st = 0·92995, P > 0·05) reveal reproductive isolation between E. bruneus and E. moara. These results support the hypothesis that E. moara is a valid species. Further molecular comparisons between E. bruneus and E. moara obtained in this study and a specimen identified in GenBank as E. bruneus from South Korea reveal that the latter is identical to E. moara rather than to E. bruneus.