Molecular phylogenetic systematics of twelve species of Acipenseriformes based on mtDNA ND4L -ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DMA (mtDNA) ND4L and partial A7D4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser; (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form of A. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.     

Molecular phylogenetic systematics of twelve species of Acipenseriformes based on mtDNAND4L—ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DNA (mtDNA)ND4L and partialND4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species ofHuso can be ascribed toAcipenser, (ii)A. dabryanus is the mostly closely related toA. sinensis, and most likely the landlocked form ofA. sinensis; (iii) genusAcipenser in trans-Pacific region might have a common origin; (iv) mtDNAND4L andND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.     

长蠹科几种检疫性害虫的ND4基因序列及系统进化

长蠹科昆虫严重危害林木和仓贮物品。应用非损伤性DNA测序技术测定了来自不同国家的长蠹科害虫的线粒体DNA ND4 基因的部分序列。在获得的204 bp的序列中,5种昆虫的序列变异丰富,多数变异发生在密码子的第3位点上。用PAUP3.1.1数据分析软件构建了5个种的合意简约树。并将实验结果与形态学特征比较分析,探讨5个种及所在属的系统进化。结果表明：双钩异翅长蠹所在的异翅长蠹属分化最早,其次是竹大长蠹所在的大长蠹属、双棘长蠹和黑双棘长蠹所在的双棘长蠹属及红艳长蠹所在的钻木长蠹属。双棘长蠹和黑双棘长蠹隶属同一个属,遗传关系最近,分化最晚,与形态学研究结果相吻合。     

几种鲟鱼基因组大小、倍体的特性及鲟形目细胞进化的探讨

采用Ｆｅｕｌｇｅｎ－显微分光光度计方法,以鸡红细胞为标准ＤＮＡ（３．２２ｐｇ／２Ｃ）测定了长江白鲟、达氏鲟、中华鲟、史氏鲟和北美匙吻鲟的体细胞基因组大小（ＤＮＡ含量）。结果表明,上述五种鲟鱼ＤＮＡ含量分别为４．１１、８．２６、９．０７、６．０７和３．９６ｐｇ。长江白鲟和北美匙吻鲟均属于四倍体鱼类,分布在长江水系的中华鲟和达氏鲟两种鲟属鱼类为八倍体类型。史氏鲟初步判断为八倍体,据分析可能存在四倍体的     

Genetic characterization of the mitochondrial DNA from Lepeophtheirus salmonis (Crustacea; Copepoda). A new gene organization revealed

The mitochondrial DNA (mtDNA) from the salmon louse, Lepeophtheirus salmonis, is 15445 bp. It includes the genes coding for cytochrome B (Cyt B), ATPase subunit 6 and 8 (A6 and A8), NADH dehydrogenase subunits 1-6 and 4L (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6), cytochrome c oxidase subunits I-III (COI, COII and COIII), two rRNA genes (12S rRNA and 16S rRNA) and 22 tRNAs. Two copies of tRNA-Lys are present in the mtDNA of L. salmonis, while tRNA-Cys was not identified. Both DNA strands contain coding regions in the salmon louse, in contrast to the other copepod characterized Tigriopus japonicus, but only a few genes overlap. In vertebrates, ND4 and ND4L are transcribed as one bicistronic mRNA, and are therefore localized together. The same organization is also found in crustaceans, with the exceptions of T. japonicus, Neocalanus cristatus and L. salmonis that deviate from this pattern. Another exception of the L. salmonis mtDNA is that A6 and A8 do not overlap, but are separated by several genes. The protein-coding genes have a bias towards AT-rich codons. The mitochondrial gene order in L. salmonis differs significantly from the copepods T. japonicus, Eucalanus bungii, N. cristatus and the other 13 crustaceans previously characterized. Furthermore, the mitochondrial rRNA genes are encoded on opposite strands in L. salmonis. This has not been found in any other arthropods, but has been reported in two starfish species. In a phylogenetic analysis, using an alignment of mitochondrial protein sequences, L. salmonis groups together with T. japonicus, being distant relatives to the other crustaceans.     

中华鲟(Acipenser sinensis)及相关种类的mtDNA控制区串联重复序列及其进化意义

采用 PCR技术和 DNA测序技术 ,发现了我国一级珍稀保护动物中华鲟 ( Acipensersinensis)线粒体 DNA( mt DNA)的控制区 ( D- loop)存在数目不等的串联重复序列 ,该重复序列造成了中华鲟广泛的异质性现象 .从分子水平进行了不同类型重复序列变化规律的研究 ,同时还初探了重复序列在我国其它几种鲟鱼类的存在情况 ,发现在白鲟 ( Psephurus gladius)、达氏鲟 ( A.dabryanus)和史氏鲟 ( A.schrenckii)均存在类似的重复序列结构 .序列比较分析表明 ,不同鲟鱼类重复序列在鲟鱼类进化过程中扮演着一定的角色 ,很有可能碱基差异大小与它们的亲缘关系的远近呈正相关 .     

Mitochondrial DNA divergence and phylogenetic relationships in mullets (Pisces: Mugilidae) of the Sea of Japan and the Sea of Azov revealed by PCR-RFLP-analysis

Three Mugilid species: Mugil cephalus (Linnaeus, 1758) and Liza haematocheila (Temminck et Schlegel, 1845; syn. Mugil soiuy, M. haematocheilus, L. soiuy, Chelon haematocheilus) from the Sea of Japan, as well as M. cephalus and Liza aurata (Risso, 1810) from the Sea of Azov were investigated on the basis of PCR-RFLP analysis of mitochondrial DNA (mtDNA) fragments, which included 12S/16S rRNA, and ND3/ND4L/ND4 genes. Among 61 individuals of three Mugilid species thirteen different haplotypes were detected. Eight and thirteen restriction endonucleases were found to be species-specific in 12S/16SrRNA and ND3/ND4L/ND4 respectively. This method may be useful for species identification. M. cephalus showed the largest genetic divergence while L. haematocheila and L. aurata were closely related and clustered together. The level of mtDNA differentiation between the two M. cephalus samples from the Sea of Japan and the Sea of Azov, i.e., nucleotide substitutions of approximately 3%, appeared to be relatively high.     

Structure and expression of the overlapping ND4L and ND5 genes of Neurospora crassa mitochondria

Summary Genes homologous to the mammalian mitochondrial NADH dehydrogenase subunit genes ND4L and ND5 were identified in the mitochondrial genome of the filamentous fungus Neurospora crassa, and the structure and expression of these genes was examined. The ND4L gene (interrupted by one intervening sequence) potentially encodes an 89 residue long hydrophobic protein that shares about 26% homology (or 41% homology if conservative amino acid substitutions are allowed) with the analogous human mitochondrial protein. The ND5 gene (which contains two introns) encodes a 715 residue polypeptide that shares 23% homology with the human analogue; a 300 amino acid long region is highly conserved (50% homology) in the two ND5 proteins. The stop codon of the ND4L gene overlaps the initiation codon of the downstream ND5 gene, and the two genes are contranscribed and probably cotranslated. A presumed mature dicistronic (ND4L plus ND5) RNA was detected. The postulated mRNA (about 3.2 kb) contains 5 and 3 non-coding regions of about 86 and 730 nucleotides, respectively; this species is generated from very large precursor RNAs by a complex processing pathway. The ND4L and ND5 introns are all stable after their excision from the precursor species.Abbreviations bp base pairs - rRNA ribosomal RNA - ND NADH dehydrogenase - URF unidentified reading frame - kDal kilodaltons; a.a., amino acid     

Phylogenetic relationships of the North American sturgeons (order Acipenseriformes) based on mitochondrial DNA sequences

The evolutionary relationships of the extant species within the order Acipenseriformes are not well understood. Nucleotide sequences of four mitochondrial genes (12S rRNA, COII, tRNA(Phe), and tRNA(Asp) genes) in North American sturgeon and paddlefish were examined to reconstruct a phylogeny. Analysis of the combined gene sequences suggests a basal placement of the paddlefish with regard to the sturgeons. Nucleotide sequences of all four genes for the three Scaphirhynchus species were identical. The position of Scaphirhynchus based on our data was uncertain. Within the genus Acipenser, the two Acipenser oxyrinchus subspecies were very similar in sequence and found to be basal to the remaining Acipenser species examined. Based on our data, Acipenser transmontanus and Acipenser medirostris were sister taxa, as were Acipenser fulvescens and Acipenser brevirostrum. Comparison of our results with hypotheses of sturgeon relationships proposed by previous authors is presented. The sequence data presented here are phylogenetically useful and provide a solid foundation of genetic information for the North American Acipenseriformes that can be expanded to include Eurasian species to provide a global picture of sturgeon evolution.     

以ND4L和ND4基因为标记探讨黑腹果蝇种组的系统发育关系

多年来的形态学、染色体组学以及DNA序列几个方面的研究均没有很好地阐明黑腹果蝇种组内的系统发育关系。本实验测定了33个样品的ND4和31个样品的ND4L基因序列,以D．obscuroides为外群,用最大简约法和Bayesian法分别构建进化树。结果表明两种方法构建的拓扑结构一致,而且大部分支系的支持率较高。整个黑腹果蝇种组分成三大谱系：1）montium种亚组;2）ananssae种亚组;3）Oriental种亚组（melanogaster、ficsphila、eugracilis、elegans、suzukii、takahashii）。montium是最早分化的种亚组。在第三谱系中,melanogaster分化得最早;然后依次是ficsphila,eugracilis,elegans;suzukii与takahashii为姐妹种亚组,最后分化。     

几种鳄分子系统发生的探讨

百年来关于扬子鳄的分类学位置存在着很多争议,本测得扬子鳄、暹罗鳄和湾鳄的mtDNA ND4和Cytb基因,并从GenBank中获得密西西比鳄和海龟的DN4基因和Cytb基因相应片段。用Clustal X1.8进行对位排列,以海龟为外群构建分子进化系统树。结果显示,在鳄类动物中,扬子鳄与密西西比鳄的亲缘关系最近,两ND4基因序列碱基差异的20．68％,而Cytb基因序列碱基差异为14．43％,但扬子鳄与密西西相比与鳄的分类问题仍将有待进一步探讨。     

Phylogeny of the Acipenseriformes: cytogenetic and molecular approaches

The review of the data on karyology and DNA content in Acipenseriformes shows that both extant families, the Polyodontidae and Acipenseridae, originated from a tetraploid ancestor which probably had a karyotype consisting of 120 macro- and microchromosomes and DNA content of about 3.2–3.8 pg per nucleus. The tetraploidization of the presumed 60-chromosome ancestor seems to have occurred at an early time of evolution of the group. The divergence of the Acipenseridae into Scaphirhyninae and Acipenserinae occurred without polyploidization. Within the genus Acipenser, polyploidization was one of the main genetic mechanisms of speciation by which 8n and 16n-ploid species were formed. Individual gene trees constructed for sequenced partial fragments of the 18S rRNA (230 base pairs, bp), 12S rRNA (185 bp), 16S rRNA (316 bp), and cytochrome b (270 bp) genes of two Eurasian (A. baerii and A. ruthenus) and two American (A. transmontanus and A. medirostris) species of Acipenser, Huso dauricus, Pseudoscaphirhynchus kaufmanni, Scaphirhynchus albus, and Polyodon spathula showed a low level of resolution; the analysis of a combined set of data for the four genes, however, gave better resolution. Our phylogeny based on molecular analysis had two major departures from existing morphological hypotheses: Huso dauricus is a sister-species to Acipenser instead of being basal to all acipenseriforms, and Scaphirhynchus and Pseudoscaphirhynchus do not form a monophyletic group. The phylogenetic tree constructed for the cytochrome b gene fragments (with inclusion of 7 additional species of Acipenser) supported the conclusion that octoploid species appeared at least three times within Acipenser.     

Free radicals-mediated damage in transmitochondrial cells harboring the T14487C mutation in the ND6 gene of mtDNA

We have studied the production of reactive oxygen species (ROS) in transmitochondrial cells, harboring homoplasmic levels of the T14487C mtDNA mutation in the ND6 gene of mitochondrial DNA (mtDNA). Previous work has shown that this mutation causes complex I deficiency. Here, we show that this mutation causes an overproduction of ROS leading to an increase in the oxidation of lipids and mtDNA without modification of antioxidant enzyme activities. We suggest that mutations in mtDNA affecting complex I activity may result in oxidative cellular damage, and reinforce the possible role of ROS-mediated mechanisms participating in some mtDNA-related disorders.     

Comparative phylogeography of two open forest frogs from eastern Australia

We investigated the phylogeography of two closely related Australian frog species from open forest habitats, Limnodynastes tasmaniensis and L. peronii , using mitochondrial ND4 sequence data. Comparison of our results with previous work on Litoria fallax allowed us to test the generality of phylogeographic patterns among non-rainforest anurans along the east coast of Australia. In general, there was no strong evidence for congruence between overall patterns of genetic structure in the three species. However, phylogenetic breaks congruent with the position of the Burdekin Gap were detected at some level in all species. As previously noted for closed forest taxa, this area of dry habitat appears to have been an important influence on the evolution of several open forest taxa. There were broad geographic similarities in the phylogenetic structuring of southern populations of L. peronii and L. tasmaniensis. Contrarily, although the McPherson Range has previously been noted to coincide geographically with a major mtDNA phylogenetic break in Litoria fallax this pattern is not apparent in L. peronii or L. tasmaniensis. It appears that major phylogeographic splits within L. peronii and L. tasmaniensis may predate the Quaternary. We conclude that phylogeographies of open forest frogs are complex and more difficult to predict than for rainforest taxa, mainly due to an absence of palæomodels for historical distributions of non-rainforest habitats.     

Unusual mitochondrial genome structure of the freshwater goby Odontobutis platycephala: rearrangement of tRNAs and an additional non-coding region

Mitochondrial DNA was isolated from the Korean freshwater gobioid fish Odontobutis platycephala by long-polymerase chain reaction with conserved primers and this mtDNA was sequenced by primer walking using flanking sequences as sequencing primers. The resultant O. platycephala mtDNA sequence was found to be 17 588 bp in size with a mostly conserved structural organization when compared with that of other teleost fish. Rearrangements of tRNAs (tRNA-Ser, tRNA-Leu, tRNA-His) and an additional non-coding region (533 bp) were present between the ND4 and ND5 genes. In the present paper, the basic characteristics of the O. platycephala mitochondrial genome is reported, including its structural organization, base composition of rRNAs, tRNAs and protein-encoding genes, characteristics of mitochondrial tRNAs and the peculiar rearrangement features of some parts of the mtDNA. Phylogenetic analysis performed using the cytochrome b gene sequences of 16 Korean freshwater fishes (15 gobioids) with the Bayesian algorithm showed that O. platycephala forms a clade (1·00 of posterior probability) with other species of Odontobutis . This suggests that the observed rearrangement between the ND4 and ND5 genes in the O. platycephala mitogenome reflects independent events.     

A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis

Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species‐specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real‐time high‐resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species‐specific genetic mutations that result in PCR products with unique melt profiles. A real‐time HRM PCR species‐diagnostic assay (RT‐HRM‐PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing.     

Morphological and mitochondrial DNA divergence validates blackmouth, Galeus melastomus, and Atlantic sawtail catsharks, Galeus atlanticus, as separate species

A total of 60 morphometric traits and nucleotide sequences of the entire mtDNA NADH dehydrogenase subunit 2 (ND2) gene [1047 base pair (bp)] in 23 individuals of blackmouth, Galeus melastomus , and 13 individuals of sawtail catsharks, Galeus atlanticus , caught in Southern Portugal, were examined to test the validity of these two taxa. These sharks closely resemble each other, have overlapping geographical ranges and are difficult to identify by morphological characters. Non-metric multidimensional scaling of morphometric variables indicates a clear separation between the two species, with 10 characters each contributing 2·12–2·45% of the total variability between species. Maximum likelihood, parsimony and neighbour-joining trees revealed two major mtDNA haplotype clades, corresponding to the two species, with an average corrected sequence divergence between them of 3·39 ± 0·56%. Within species divergences between haplotypes averaged 0·27 ± 0·18% in G. melastomus and 0·12 ± 0·08% in G. atlanticus . A total of 35 diagnostic nucleotide site differences and four restriction fragment length polymorphism recognition sites in the ND2 gene can be used to distinguish the two species.     

Comparative Analysis of Mitochondrial Genomes in <Emphasis Type="Italic">Bombina</Emphasis> (Anura; Bombinatoridae)

The complete mitochondrial genomes of two basal anurans, Bombina bombina and B. variegata (Anura; Bombinatoridae), were sequenced. The gene order of their mitochondrial DNA (mtDNA) is identical to that of canonical vertebrate mtDNA. In contrast, we show that there are structural differences in regulatory regions and protein coding genes between the mtDNA of these two closely related species. Corrected sequence divergence between the mtDNA of B. bombina and B. variegata amounts to 8.7% (2.3% divergence in amino acids). Comparisons with two East Asian congeners show that the control region contains two repeat regions, LV1 and LV2, present in all species except for B. bombina, in which LV2 has been secondarily lost. The rRNAs and tRNAs are characterized by low nucleotide divergence. The protein coding genes are considerably more disparate, although functional constraint is high but variable among genes, as evidenced by dN/dS ratios. A mtDNA phylogeny established the distribution of autapomorphic nonsynonomous substitutions in the mitogenomes of B. bombina and B. variegata. Nine of 98 nonsynonomous substitutions led to radical amino acid replacements that may alter mitochondrial protein function. Most radical substitutions were found in ND2, ND4, or ND5, encoding mitochondrial subunits of complex I of the electron transport system. The extensive divergence between the mitogenomes of B. bombina and B. variegata is discussed in terms of its possible role in impeding gene flow in natural hybrid zones between these two species.     

基于线粒体基因组全序列的鲟形目鱼类(Pisces: Acipenseriformes)的分子系统发育重建

为厘清鲟形目鱼类的系统发育, 研究新测定了中华鲟(Acipenser sinensis)、长江鲟(A. dabryanus)、短吻鲟(A. brevirostrum)、纳氏鲟(A. naccarii)、鳇(Huso dauricus)和匙吻鲟(Polyodon spathula)共6种鲟类的线粒体全基因组序列。联合已测的17种鲟类的线粒体基因组数据, 利用最大似然法和贝叶斯法重建了鲟形目鱼类的分子系统发育关系, 并采用似然值检验对不同的树拓扑结构进行了评价。结果表明, 6种新测鲟类的线粒体基因组大小为16521—16766 bp, 编码13个蛋白质编码基因、22个转运RNA基因和2个核糖体基因, 与大多数已测的鲟类的线粒体基因组结构高度相似。基于23种鲟形目鱼类线粒体基因组数据, 系统发育分析的结果表明: (1)鲟形目的两个科, 匙吻鲟科(Polyodontidae)和鲟科(Acipenseridae)均为单系; (2)鲟科的内部亲缘关系复杂, 鲟属和鳇属的物种均不构成单系群。鲟科鱼类按分子系统发育重建结果可以分为3个类群: 尖吻鲟类(A. sturio - A. oxyrinchus clade)、大西洋鲟类(Atlantic clade)和太平洋鲟类(Pacific clade)。树拓扑结构的检验结果表明, 鲟科的系统发育关系为(尖吻鲟类(太平洋鲟类, 大西洋鲟类))。铲鲟属(Scaphirhynchus)是大西洋鲟类的基部类群。研究也说明线粒体基因组数据在鲟形目鱼类系统与进化研究方面具有重要应用价值。