Genetic diversity and the biogeographical process of Acheilognathus macropterus revealed by sequence variations of mitochondrial cytochrome b gene

In this study, thirty-six individuals of Acheilognathus macropterus were collected from the Heilongjiang River,the Yangtze River,and the Nandujiang River.Partial mitochondrial cytochrome b gene region (636 base pair) was sequenced to these samples and 22 haplotypes were found.With A.chankaensis and A.tokinensis as outgroups,their relationships were analyzed.The p-distances were calculated with Mega software and a molecular phyiogenetic tree was constructed using the neighbor-joining (NJ) method.The proportions of main morphological characters were compared as well.P-distances showed that the genetic differences in A.macropterus samples were far smaller than those between these samples and the outgroups.The molecular phylogenetic tree shows that samples with barbels and those without barbels were intermingled.There was no distinctive difference in proportions of morphological characteristics among them.These results suggested that samples with barbels and those without barbels (formally identified as A.taenianalis) are the same species;A.taenianalis is synonymous with A.macropterus.The thirtysix individuals were grouped into five clades and the positions of the samples in the clades were correspondingly grouped within their geographical distributions.Among the five clades,clades 1 and 5 included samples from the Heilongjiang River and Nandujiang River respectively.The samples from the Yangtze River scattered into clades 2,3,and 4.There were distinctive genetic differences (> 5%)among them.Interestingly,the distributions of the 21 samples in these three clades were not correlated to their geographical distributions.It is postulated that these genetic differences were due to the bitterlings' mating choice mechanism,the prozygotic isolation.The genetic differences between the fish from Nandujiang River and those from the mainland indicated that they were separated early.However,the small genetic differences among the samples and the positions of the fish from the Heilonjiang River in the molecular phylogenetic tree indicate that fish in Heilongjiang River might have dispersed from the Yangtze River to that area much later.     

大鳍鱊基于细胞色素b基因序列的遗传变异及生物地理过程

对采集于长江水系、黑龙江水系及海南岛的36尾大鳍(Acheilognathus macropterus)的细胞色素b基因序列进行了研究分析。发现有须个体和无须个体在分子系统树中处于混合分布状态,各个体相互间遗传变异远小于与外类群(A.chankaensis、A.tokinensis)间差异,统计了其主要形态度量数据,也显示各主要性状数据无显著差异,结果均支持有须个体和无须个体(曾经被鉴定为A.taenianalis)为同一物种,A.taenianalis为A.macropterus的同物异名。序列分析显示,36个个体明显聚合成5个大的分支,其中clade1分支全部由黑龙江水系类群组成,clade5分支由海南岛的1个个体组成,长江水系的所有个体分散在clade2、clade3、clade4这三个分支中,显示出一定的地理相关性。在clade2、3、4中,clade2包括了长江上游的5个个体和长江中游的2个个体,同时还包括了黑龙江的2个个体;clade3包括了中游的2个个体;clade4包括了中游的12个个体,三个分支间又存在明显的遗传变异率(均>5%),从21个个体在三个分支中的分布情况来看,三者间这种较大的遗传变异率不能由地理隔离解释,故推测,造成同一水系的A.macropterus间出现较明显遗传分化的原因可能与其特殊的性选择生殖方式有关,该生殖方式限制了不同生殖集群间同种鱼类的基因交流。基于系统分支图,计算了各分支间的遗传距离,由结果推测大鳍的生物地理过程为:海南岛水系类群同大陆类群较早产生隔离,黑龙江水系类群由长江水系类群在晚近时期向北扩散形成。       

Molecular phylogenetic relationships of China Seas groupers based on cytochrome b gene fragment sequences

The classification and evolutionary relationships are important issues in the study of the groupers. Cytochrome b gene fragment of twenty-eight grouper species within six genera of subfamily Epinephelinae was amplified using PCR techniques and the sequences were analyzed to derive the phylogenetic relationships of the groupers from the China Seas. Genetic information indexes, including Kimura-2 parameter genetic distance and T _S/T _V ratios, were generated by using a variety of biology softwares. With Niphon spinosus, Pagrus major and Pagrus auriga as the designated outgroups, phylogenetic trees, which invoke additional homologous sequences of other Epinephelus fishes from GenBank, were constructed based on the neighbor-joining (NJ), maximum-parsimony (MP), maximum-likelihood (ML) and minimum-evolution (ME) methods. Several conclusions were drawn from the DNA sequences analysis: (1) genus Plectropomus, which was early diverged, is the most primitive group in the subfamily Epinephelinae; (2) genus Variola is more closely related to genus Cephalopolis than the other four genera; (3) genus Cephalopolis is a monophyletic group and more primitive than genus Epinephelus; (4) Promicrops lanceolatus and Cromileptes altivelis should be included in genus Epinephelus; (5) there exist two sister groups in genus Epinephelus. These authors contributed equally to this work.     

Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt ^– parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COXI have been isolated. The possibility that the inactivation of the other mitochondrial genes is lethal for the cells is discussed.     

Multiphasic oxidation-reduction of cytochrome b in the succinate-cytochrome c reductase

The triphasic course previously reported for the reduction of cytochrome b in the succinate-cytochrome c reductase by either succinate or duroquinol has been shown to be dependent on the redox state of the enzyme preparation. Prior reduction with increasing concentrations of ascorbate leads to partial reduction of cytochrome c₁, and a gradual decrease in the magnitude of the oxidation phase of cytochrome b. At an ascorbate concentration sufficient to reduce cytochrome c₁ almost completely, the reduction of cytochrome b by either succinate or duroquinol becomes monophasic. Owing to the presence of a trace amount of cytochrome oxidase in the reductase preparation employed, the addition of cytochrome c makes electron flow from substrate to oxygen possible. Under such circumstances, the addition of a limited amount of either succinate or duroquinol leads to a multiphasic reduction and oxidation of cytochrome b. After the initial three phases as described previously, cytochrome b becomes oxidized before cytochrome c₁ when the limited amount of added substrate is being used up. However, at the end of the reaction when cytochrome c_a is being rapidly oxidized, cytochrome b becomes again reduced. The above observations support a cyclic scheme of electron flow in which the reduction of cytochrome b proceeds by two different routes and its oxidation controlled by the redox state of a component of the respiratory chain.     

基于Cyt b基因的扁圆吻鲴遗传多样性及其在鲴亚科系统发育地位探讨

扁圆吻鲴（Distoechodon compressus）是一种局域分布于我国福建、江西、台湾的小型经济鱼类,目前尚无其群体遗传学相关研究,此外扁圆吻鲴在鲴亚科（Xenocyprinae）鱼类中的分类地位也有争议。本研究基于线粒体Cyt b基因,对采自福建连城附近水域的三个扁圆吻鲴群体的遗传多样性、群体结构和历史动态进行分析。结果显示扁圆吻鲴的三个群体共检测到15个单倍型,整体单倍型多样性较高（0.554）而核苷酸多样性较低（0.001 42）,三个群体中池塘养殖群体的单倍型多样性和核苷酸多样性均最高,分别为0.600和0.002 24;各群体间未检测到显著的遗传分化,且遗传距离均极低（小于0.002）;群体历史动态分析结果也未检测到明显的群体扩张或瓶颈效应等。此外,分别基于邻接法（neighbor-joining, NJ）、最大似然法（maximum likelihood, ML）和贝叶斯法（Bayesian inference, BI）对鲴亚科已报道的基因序列进行系统发育分析,结果显示似鲴属（Xenocyprioides）位于系统发育树基部;鲴属（Xenocypris）和圆吻鲴属（Distoechodon）并未各自形成单系,而是不同属的各物种互为并系;在圆吻鲴属内部,扁圆吻鲴和圆吻鲴无法通过Cyt b基因进行区分,因此未来仍需更多的分子标记以支持鲴亚科的系统发育关系。本研究的开展有助于了解扁圆吻鲴的遗传多样性现状和群体结构,为资源保护管理提供了基础资料,同时也为鲴亚科的系统发育和进化历史提供了重要参考。     

栗背短脚鹎的种群分化与遗传多样性

栗背短脚鹎(Hemixos castanonotus)是我国南方山区常见的杂食性鸟类。为探明其遗传多样性及分化现状,采用线粒体Cyt b基因和7个核基因非编码区片段作为分子标记,对分布于广东、广西、海南、贵州和江西五省(自治区)的栗背短脚鹎11个地理种群进行了遗传分化及遗传多样性研究。基于所获得的Cyt b基因866 bp和7个核基因内含子序列6 808 bp进行分析。结果显示,在Cyt b基因中,共检测到37个单倍型,共享单倍型占单倍型总数的35.6%,推测这些共享单倍型可能属于祖先单倍型。分子方差分析结果显示,遗传变异主要来源于种群内部(79.77%)。Tajima’s D和Fu’s Fs中性检验分析结果均支持栗背短脚鹎种群可能曾经历过种群扩张现象。基于7个核基因内含子联合序列的贝叶斯天际线(BSP)分析,推断其种群在大约5.3 ~ 3.7百万年前(Mya)和约0.7 ~ 0.3百万年前(Mya)发生过扩张。基于Cyt b基因的贝叶斯系统发育分析,11个地理种群共分为两支,一支为海南猴猕岭地理种群,属指名亚种(H. c. castanonotus),其他10个地理种群聚为另一支,属H. c. canipennis亚种,并且后者尚未形成显著的地理结构,单倍型网络图分析也获得相似的结果。本研究所用分子数据基本支持两个亚种的分化,对于存在争议的广西南部分布的指名亚种,其分子数据与形态学亚种归属不一致,有待更深入研究。     

Ovipositor ultrastructure of the striped bitterling Acheilognathus yamatsutae (Teleostei: Acheilognathinae) during spawning season

The ovipositor of striped bitterling Acheilognathus yamatsutae was subjected to ultrastructure and histochemical analysis during spawning season using light and electron microscopy. Although the ovipositor of A. yamatsutae is a long cylindrical tube with smooth external surface, it was possible to confirm the presence of well-developed fingerprint structure using scanning electron microscopy. Internal aspect analysis of ovipositor revealed formation of 5–8 longitudinal folds. Cross section analysis revealed that the ovipositor is composed of an outer epithelial layer, a mid connective tissue layer, and an inner epithelial layer. The outer epithelial layer contains 7–9 cell layers composed mainly of epithelial and mucous cells. Result of AB–PAS (pH 2.5) and AF–AB reaction showed that mucous cells contained mainly acidic carboxylated mucosubstances. The connective tissue layer was loose and made mainly of collagen fibers and some muscle fibers, along with blood vessels and a small number of chromatophores. The inner epithelial layer, which is a single layer, is composed of columnar epithelia. Observation under transmission electron microscope enabled distinction of the outer epithelial layer into superficial, intermediate and basal layers. Although the types of cells in the superficial tissue layer were diverse, they all shared the development of glycocalyx covered microridges. The majority of epithelial cells in the intermediate layer were cuboidal shaped, while those in the basal layer were columnar. Two types (A and B) of secretory cells were observed in the outer epithelial layer. The connective tissue layer had two types of chromatophores including xantophore and melanophore, in addition to a well-developed nerve fiber bundles. Columnar epithelial cells, mitochondria-rich cells and rodlet cells were observed in the inner epithelial layer. Microvilli were well developed on the free surface of columnar epithelial cells.     

Phylogenetic Study of Complete Cytochrome b Genes in Musk Deer (Genus Moschus) Using Museum Samples

As an endangered animal group, musk deer (genus Moschus) are not only a great concern of wildlife conservation, but also of special interest to evolutionary studies due to long-standing arguments on the taxonomic and phylogenetic associations in this group. Using museum samples, we sequenced complete mitochondrial cytochrome b genes (1140 bp) of all suggested species of musk deer in order to reconstruct their phylogenetic history through molecular information. Our results showed that the cytochrome b gene tree is rather robust and concurred for all the algorithms employed (parsimony, maximum likelihood, and distance methods). Further, the relative rate test indicated a constant sequence substitution rate among all the species, permitting the dating of divergence events by molecular clock. According to the molecular topology, M. moschiferus branched off the earliest from a common ancestor of musk deer (about 700,000 years ago); then followed the bifurcation forming the M. berezovskii lineage and the lineage clustering M. fuscus, M. chrysogaster, and M. leucogaster (around 370,000 years before present). Interestingly, the most recent speciation event in musk deer happened rather recently (140,000 years ago), which might have resulted from the diversified habitats and geographic barriers in southwest China caused by gigantic movements of the Qinghai-Tibetan Plateau in history. Combining the data of current distributions, fossil records, and molecular data of this study, we suggest that the historical dispersion of musk deer might be from north to south in China. Additionally, in our further analyses involving other pecora species, musk deer was strongly supported as a monophyletic group and a valid family in Artiodactyla, closely related to Cervidae.     

西南地区野生刺梨的遗传多样性和遗传结构研究

为了探究西南地区野生刺梨(Rosa roxburghii)的遗传多样性和起源演化,该研究基于2段单拷贝核基因(GAPDH和ncpGS)和3段叶绿体基因(atpF-trnH、trnL-trnF和trnG-trnS)的拼接序列,对刺梨27个野生居群共320个个体进行PCR扩增和测序,并用相关软件对测序结果进行分析。结果表明:(1)在单拷贝核基因和叶绿体基因水平上刺梨均表现出较低的遗传多样性(scnDNA: Hd=0.469 2, π=0.000 49; cpDNA: Hd=0.653 4, π=0.000 65),并且不同居群间存在较大差异。(2)分子方差分析(AMOVA)结果均显示,遗传变异主要发生在居群内,表明居群内的变异是野生刺梨遗传变异的主要来源,居群间存在明显的遗传分化( cpDNA:F_ST=0.336 47,G_ST=0.273,N_ST= 0.308; scnDNA:F_ST=0.094 87,N_ST=0.076,G_ST=0.056),刺梨的分布不具有明显的谱系地理结构(P>0.05)。(3)中性检验 Tajima''s D值均为不显著负值,符合中性进化模型。Fu''s Fs值均为显著负值,结合失配分析曲线,推测刺梨种群在小范围内经历过扩张,但总体上维持稳定状态。(4)根据单倍型多样性得出,毕节地区的居群遗传多样性水平较高并且拥有丰富的单倍型,推测可能为冰期避难所,因此应对其实施就地保护。对于拥有特殊性状和特有单倍型的居群也应采取优先保护措施。该文为野生刺梨资源保护和遗传育种提供了一定的参考价值。     

Molecular Systematics of Selene (Perciformes: Carangidae) Based on Cytochrome b Sequences

The marine fishes of the genus Selene are morphologically unique, although little is known about how these species are related to other members of the family Carangidae (Perciformes). In addition, questions remain about the potential validity of two putative species and how species groups with unique body forms within Selene are related. We used DNA sequences of the mitochondrial cytochrome b gene to reconstruct the phylogeny of the seven species of Selene along with five additional species of carangids. Maximum-likelihood and maximum-parsimony analyses were used to examine the sequence data and both phylogenetic methods were compared. Maximum-likelihood produced a monophyletic Selene, whereas parsimony analyses did not. Both maximum-likelihood and maximum-parsimony produced similar support for species groups within Selene. Maximum-likelihood produced two monophyletic subgroups within the genus Selene, the "long-finned" and "short-finned" Selene. Maximum-parsimony produced the same monophyletic "long-finned" group but a paraphyletic "short-finned" group. Both analyses confirm that S. brownii and S. setapinnis are distinct species, expunging the question of conspecificity. The phylogenetic placement of the most basal taxon within Selene, S. orstedii, was problematic and differed among analyses. More data are needed to resolve with confidence its correct phylogenetic placement and, thus, the monophyly of the genus Selene.     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

A neonatal polyvisceral failure linked to a de novo homoplasmic mutation in the mitochondrially encoded cytochrome b gene

Mutations within the mitochondrially encoded cytochrome b (MTCYB) gene are heteroplasmic and lead to severe exercise intolerance. We describe an unusual clinical presentation secondary to a novel homoplasmic mutation within MTCYB. The m.15635T > C transition (S297P) was carried by a newborn who presented with a polyvisceral failure. This mutation was responsible for a complex III deficiency. It was homoplasmic in all tissues tested and was undetectable in patient’s mother. Functional analyses, including studies on patient’s cybrid cell lines, demonstrate the pathogenicity of this variant. Our data show that mutations within MTCYB can be responsible for severe phenotype at birth.     

Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12

Summary The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b ₅₆₁ and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b ₅₆₁ is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b ₅₆₁ with those of other bacterial b-type cytochromes was observed.     

Phylogeographic relationships among worldwide populations of the cosmopolitan marine species, the striped gray mullet (Mugil cephalus), investigated by partial cytochrome b gene sequences

Several factors such as geographical barriers, demographic history, biological and ecological traits may contribute to delineate the evolutionary history of a species. The gray mullet, Mugil cephalus, represents an interesting case of a marine species with a coastal ecology and a cosmopolitan distribution. In this study, partial cytochrome b sequences were resolved for 177 M. cephalus specimens sampled from 14 different geographic sites, in order to investigate the genetic divergence and the phylogeographic relationship among populations at a global scale. Demographic parameters were also assessed. Analysis of partial cyt b sequences showed high levels of differentiation among populations from distant geographic areas as most populations harbored private alleles and showed reciprocal monophyly. Both phylogenetic trees and haplotype network indicate the East Australian haplotypes as the closest to the ancestor sequences, which leads to the hypothesis of an Indo-West Pacific center of origin for M. cephalus. The analyses of the molecular variance showed that the genetic variation in M. cephalus is mainly harbored among populations rather than within populations. The high variability indices (h, π) calculated on M. cephalus individuals pooled together and the very low variability indices detected at some geographic sampling sites suggest that gray mullet populations have undergone a long-term reproductive isolation characterized by events of population abundance reductions which may have favored genetic differentiation among populations.     

Analysis of the Genetic Diversity and the Phylogenetic Evolution of Chinese Sheep Based on Cyt b Gene Sequences

The complete sequences of Cyt b gene from 20 individuals belonging to eight Chinese indigenous sheep breeds and one foreign breed were studied. The results showed that the hapolotype diversity of Chinese sheep breeds was 97.1%. The mean nucleotide composition of all the sequences was 27.1% T, 28.5% C, 31.4% A, and 13.0% G. The nucleotide diversity was 0.602%. A total of 43 mutation sites were detected, including 40 transitions and 3 transversions. Fu's test of selective neutrality showed that the sheep populations had no population demographic expansion (0.10 > P > 0.05). The different clustering methods, namely neighbor-joining, minimum evolution, and unweighted pair group method with arithmetic means, all showed a similar result, which indicated that Chinese local sheep had three maternal resources.     

Genetic diversity and structure of Cordyceps sinensis populations from extensive geographical regions in China as revealed by inter-simple sequence repeat markers

Cordyceps sinensis is one of the most valuable medicinal caterpillar fungi native to China. However, its productivity is extremely limited and the species is becoming endangered. The genetic diversity of eighteen C. sinensis populations across its major distributing regions in China was evaluated by inter-simple sequence repeat (ISSR) markers. A total of 141 markers were produced in 180 individuals from the 18 populations, of which 99.3% were polymorphic. The low average of Shannon (0.104) and Nei index (0.07) of the 18 populations indicates that there are little genetic variations within populations. For all 18 populations, estimates of total gene diversity (H_T), gene diversity within populations (H_S), coefficient of genetic differentiation (G_ST), and gene flow (Nm) were 0.170, 0.071, 0.583, and 0.357, respectively. This pattern suggests that the genetic diversity of C. sinensis is low and most of the ISSR variations are found among populations with little gene exchange. The 18 populations are divided into five groups based on the genetic distance and the grouping pattern matches with the geographic distribution along the latitudinal gradient. The five groups show obvious difference in the G_ST and Nm values. Therefore, the genetic diversification of C. sinensis populations may be determined by geographic isolation and the combined effects of life history characters and the interaction with host insect species. The information illustrated by this study is useful for selecting in situ conservation sites of C. sinensis.