Molecular phylogeny of the specialized schizothoracine fishes （Teleostei： Cyprinidae）,with their implications for the uplift of the Qinghai-Tibetan Plateau

Molecular phylogeny of three genera containing nine species and subspecies of the specialized schizothoracine fishes are investigated based on the complete nucleotide sequence of mitochondrial cytochrome b gene. Meantime relationships between the main cladogenetic events of the specialized schizothoracine fishes and the stepwise uplift of the Qinghai-Tibetan Plateau are also conducted using the molecular clock, which is calibrated by geological isolated events between the upper reaches of the Yellow River and the Qinghai Lake. Results indicated that the specialized schizothoracine fishes are not a monophyly. Five species and subspecies of Ptychobarbus form a monophyly. But three species of Gymnodiptychus do not form a monophyly. Gd.integrigymnatus is a sister taxon of the highly specialized schizothoracine fishes while Gdo pachycheilus has a close relation with Gd. dybowskii, and both of them are as a sister group of Diptychus maculatus. The specialized schizothoracines fishes might have originated during the Miocene(about 10 MaBP), and then the divergence of three genera happened during late Miocene (about 8 MaBP). Their main specialization occurred during the late Pliocene and Pleistocene (3.54-0.42 MaBP). The main cladogenetic events of the specialized schizothoracine fishes are mostly correlated with the geological tectonic events and intensive climate shift happened at 8, 3.6, 2.5 and 1.7 MaBP of the late Cenozoic.Molecular clock data do not support the hypothesis that the Qinghai-Tibetan Plateau uplifted to near present or even higher elevations during the Oligocene or Miocene, and neither in agreement with the view that the plateau uplifting reached only to an altitude of 2000 m during the late Pliocene(about 2.6 MaBP).     

Phylogenetic relationships and divergence times of the family Araucariaceae based on the DNA sequences of eight genes

Araucariaceae is one of the most primitive families of the living conifers, and its phylogenetic relationships and divergence times are critically important issues. The DNA sequences of 8 genes, i.e., nuclear ribosomal 18S and 26S rRNA, chloroplast 16S rRNA, rbcL, matK and rps4, and mitochondrial coxl and atpl, obtained from this study and GenBank were used for constructing the molecular phylogenetic trees of Araucariaceae, indicating that the phyiogenetic relationships among the three genera of this family should be （（Wollemia, Agathis）, Araucaria）. On the basis of the fossil calibrations of Wollemia and the two tribes Araucaria and Eutacta of the genus Araucaria, the divergence time of Araucariaceae was estimated to be （308± 53） million years ago, that is, the origin of the family was in the Late Carboniferous rather than Triassic as a traditional view. With the same gene combination, the diver- gence times of the genera Araucaria and Agathis were （246 ± 47） and （61 ±15） Ma, respectively. Statis- tical analyses on the phylogenetic trees generated by using different genes and comparisons of the divergence times estimated by using those genes suggested that the chloroplast matK and rps4 genes are most suitable for investigating the phylogenetic relationships and divergence times of the family Araucariaceae.     

Phylogenetic relationships of host insects of Cordyceps sinensis inferred from mitochondrial Cytochrome b sequences

This study used the sequence of the mitochondrial Cytochrome b(Cytb)to estimate phylogenetic relationships among host Hepialidae insects of Cordyceps sinensis.Genome DNA of host insect was extracted from the dead larva head part of 18 cordyceps populations and 2 species of Hepialus,and the Cytb fragment of host insect was amplified with PCR technique.The nucleotide sequence alignments and their homologous sequences of 24 species host Hepialidae insects of Cordyceps sinensis were obtained from GenBank and were used to construct phylogenetic trees based on neighbor-joining method.The results showed that genus Bipectilus diverged earlier than genus Hepialus and Hepialiscus.Hepialus host insects of Cordyceps sinensis have multitudinous species with different morphological characteristics and geographical distributions.The interspecific genetic differentiations are obvious in Hepialus.Thus,the genus Hepialus might be considered as polyphyletic origin.Cytb sequences have abundant variations among the host insects of Cordyceps sinensis on spe- cific and generic level.The divergence rate of Cytb sequences among the species in Hepialus ranged from 0.23% to 9.24%,except that Hepialus pratensis and Hepialus jinshaensis have the same sequence.Cytb sequence can be used for species identification of host insects of Cordyceps sinensis,but further confirmation in more host insect species is needed.To obtain the Cytb sequence of host insect by ampli- fying DNA extracted from the head part of dead larva in cordyceps turns out to be an effective and accurate approach,which will be useful for studies on phylogeny and genetic structure of host insects of cordyceps populations,especially for analyzing relationships between C. sinensis and its host insects.     

Investigations into the perplexing interrelationship of the Genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships within the genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodonti- dae) remain unresolved. Because of the use of Takifugu as model organisms, the resolution of these relationships is crucial for the interpretation of evolutionary trends in biology. Pufferfishes of this ge- nus are comprised of a comparatively small number of species and are mainly distributed along the coastal region of the western part of the Sea of Japan and the coastline of China. Mitochondrial gene sequences were employed to test the phylogenetic hypotheses within the genus. Seventeen species of the genus were examined. Molecular phylogenetic trees were constructed using the maximum parsi- mony, neighbor-joining, maximum likelihood and Bayesian methods. Our hypothesis of internal relationships within the genus differs from previous hypotheses. Our results indicate that (1) the genus Takifugu is a monophyletic assemblage; (2) the genus is divided into 6 subgroups based on the mo- lecular data; and (3) there is low genetic diversity among the species within this genus. In addition, speciation within Takifugu appears to be driven by hybridization and isolation by distribution. Our re- sults also suggested that the taxonomy in the genus should be clarified based on both molecular and morphological data.     

Asian origin for Polystichum （Dryopteridaceae） based on rbcL sequences

Chloroplast rbcL sequences of 60 species of Polystichum sensu lato (s.l.), including 23 new sequences from southwest China, were used to assess the phylogenetic relationships within the genus. On the basis of estimated evolution rate of rbcL gene and the genetic distance data that passed relative-rate tests, we further estimated the divergence times between some clades of the genus. The phylogenetic relationships were inferred using the neighbor-joining and maximum-parsimony methods, both methods producing trees with completely congruent topology. These trees reveal that all species of Polystichum s.l. in this study (including Cyrtomium and Cyrtomidictyum) form a monophyletic group. The basal split in Polystichum s.l. separates a clade with all Asian members from a clade containing other species from all over the world. The phylogenetic and divergence time estimation results lead us to suggest that Polystichum s.l. originated in Asia in the late Late Cretacous (≈76 Ma) and migrated into other places in the world in early Eocene(≈46 Ma).     

Investisations into the perplexing interrelationship of the Genus Takifugu Abe, 1949 （Tetraodontiformes,Tetraodontidae）

The phylogenetic relationships within the genus Takifugu Abe, 1949 （Tetraodontiformes, Tetraodontidae） remain unresolved. Because of the use of Takifugu as model organisms, the resolution of these relationships is crucial for the interpretation of evolutionary trends in biology. Pufferfishes of this genus are comprised of a comparatively small number of species and are mainly distributed along the coastal region of the western part of the Sea of Japan and the coastline of China. Mitochondrial gene sequences were employed to test the phylogenetic hypotheses within the genus. Seventeen species of the genus were examined. Molecular phylogenetic trees were constructed using the maximum parsi- mony, neighbor-joining, maximum likelihood and Bayesian methods. Our hypothesis of internal relationships within the genus differs from previous hypotheses. Our results indicate that （1） the genus Takifugu is a monophyletic assemblage; （2） the genus is divided into 6 subgroups based on the moecular data; and （3） there is low genetic diversity among the species within this genus. In addition, speciation within Takifugu appears to be driven by hybridization and isolation by distribution. Our results also suggested that the taxonomy in the genus should be clarified based on both molecular and morphological data.     

Estimating divergence times among subfamilies in Nymphalidae

The mitochondrial cytochrome oxidase subunit Ⅰ （COⅠ） gene and the nuclear elongation factor 1α （EF-1α） gene were sequenced from 13 species of Nymphalidae. Phylogenetic trees of Nymphalidae, which is the largest family in butterflies, were constructed based on the sequences determined from 13 species sequenced in our laboratory and an additional 43 species obtained from GenBank using the maximum likelihood （ML） and Bayesian methods. Relative-rate tests between lineages in these phylogenetic trees were performed. On the basis of the results of the relative-rate tests and fossil information of Satyrinae, Nymphalinae and Biblidinae, the average divergence times among the subfamilies are estimated as 44.2-87.1 million years ago （Ma）. These results will be helpful for better understanding of the origin and evolution of this family, as well as the divergence time of butterflies and other complex taxa.     

Analysis of RAPD and mitochondrial 16S rRNA gene sequences from Trichiurus lepturus and Eupleurogrammus muticus in the Yellow Sea

Random amplified polymorphic DNA (RAPD) technique is applied to 12 individuals from each species of the hairtail fishes Trichiurus lepturus and Eupleurogrammus muticus in the Yellow Sea. The percentage of polymorphic sites, degree of genetic polymorphism and genetic distance are compared and the phylogenetic tree is constructed by Neighbor-joining method. The partial mitochondrial 16S rRNA gene is amplified by polymerase chain reaction (PCR) and the PCR products are directly sequenced after being purified. These sequences, together with the homologous sequences of another Trichiuridae species Lepidopus caudatus obtained from GenBank, are used to analyze nucleotide difference and to construct a UPGMA phylogenetic tree by means of biological informatics. Analysis shows: (1) the RAPD technique is a highly sensitive method for investigating genetic diversity in T. lepturus, and E. muticus. T. lepturus exhibits a lower polymorphism and genetic diversity than E. muticus; (2) according to the analysis of the partial mitochondrial 16S rRNA gene sequences, a very low intraspecific variation and considerably high divergence among species were found, which reveals a dual nature of conservatism and variability in mitochondrial 16S rRNA gene; (3) five primers generate the species-specific RAPD sites and these sites can be served as the molecular markers for species identification and (4) it can be proved at DNA variation level that T. lepturus and E. muticus are of two species respectively pertaining to different genera, which supports the Nelson taxonomic conclusion.     

Molecular phylogenetics of Gymnocypris （Teleostei：Cyprinidae） in Lake Qinghai and adjacent drainages

149 complete mitochondrial DNA (mtDNA) cytochrome b (Cyt b) genes (1140 bp) of Gymnocypris przewaiskii, Gymnocypris eckloni and Gymnocypris scolistomus from the Lake Qinghai, Yellow River and Qaidam Basin were sequenced and analyzed. Consistent dendrogram indicated that the samples collected from the same species do not constitute a separate monophyletic group and all the samples were grouped into three highly divergent lineages (A, B and C). Among them, Lineage A contained all samples of G.przewaiskii from the Lake Qinghai and partial samples of the G. eckloni from the Yellow River. Lineage B contained the remaining samples of G. eckloni from the Yellow River.Lineage C was composed of a monophyletic group by G. eckloni from the Qaidam Basin. Analysis of molecular variance (AMOVA) indicated that most of genetic variations were detected within these three mtDNA lineages (93.12%), suggesting that there are three different lineages of Gymnocypris in this region. Our Cyt b sequence data showed that G.przewaiskii was not a polytypic species, and G. scolistomus was neither an independent species nor a subspecies of G.eckloni. The divergent mtDNA lineages of G. eckloni from theYellow River suggested that gene flow between the different populations was restricted to a certain extent by several gorges on the upper reach of the Yellow River. Lineage B of G. eckloni might be the genetic effect from the ancestor which was incorporated with the endemic schizothoracinefishes when the headward erosion of the Yellow Riverreached to its current headwaters of late. The G. eckloni from Basin Qaldam was a monophyletic group (lineage C) and Fst values within G. eckloni from the Yellow River were higher than 0.98, suggesting that the gene flow has been interrupted for a long time and the G. eckloni from Basin Qaidam might have been evolved into different species by ecology segregation. The correlation between the rakers number of Gymnocypris and population genetic variation was not significant.All Gymnocypris populations exhibited a low nucleotide diversity (n=0.00096-0.00485). Therefore the Gymnocypris populations from Basin Qaidam could have experienced severe bottleneck effect in history. Our result suggested Gymnocypris populations of Basin Qaidam should give a high priority in conservation programs.     

Phylogeny of higher taxa of hexapoda according to 12sRNA sequences

The phylogenetic relationship of Hexapoda has been debated for a long time, which will be resolved mainly depending on the settlement of monophyly, affinities and interrelationships among Protura, Collembola and Diplura. Mitochondrial 12sRNA gene about 355 bp fragments from one proturan species, two collembolan species, two dipluran species and one oribatid species were sequenced. The Kimura 2-parameter distances were calculated and a series of molecular phylogenetic trees were reconstructed by using the N-J method, from which the following points were drawn: (ⅰ) Protura and Collembola compose a monophyletic group representing absent-cerci; (ⅱ) Diplura is not a monophyletic group, in which Campodeoid with filiform cerci belongs to a clade and Japygoid with pincer cerci and Ectognatha compose another clade, that is, Insecta s. str. stemmed from Japygoid. So it would be suggested that the phylogenetic relationship of Hexapoda is [Parainsecta (Collembola + Protura) +Campodeoid +Insecta (Japygoid + Ectognatha)].     

淡水鱼类线粒体DNA D-loop基因的引物设计和应用

 线粒体DNA测序已广泛应用于鉴定和区分种类以及解决系统进化关系问题。本文选取已测定的主要淡水鱼类的线粒体DNA D-loop基因序列进行同源性比较,寻找保守序列,利用简并性原则设计一对通用的简并引物。利用设计的引物对广东省珠江流域主要的淡水鱼类线粒体DNA D-loop控制区基因进行扩增,均能获得单一的目的DNA片断,特异性扩增产物大小为1 kb左右。经测序及与GenBank同源序列的比较,证实为包含线粒体控制区全序列的扩增产物。本研究所设计的引物和应用的方法可以快速地同时对多种鱼类进行大规模的遗传背景分析,鉴定某些难于鉴别的近缘物种,为我国鱼类的种类鉴定、地理种群鉴别及种质资源的评估提供重要的工具。     

山西省雏蝗属部分种类的遗传关系分析

采用RAPD和SSRP-PCR技术分析雏蝗属(Chorth ippus F ieber)白纹雏蝗(C.a lbonem us Cheng et T u)、夏氏雏蝗(C.hsia i Cheng et T u)、小翅雏蝗(C.f a llax(Zub.))和北方雏蝗(C.hamm arstroem i(M ir.))的遗传关系,9条引物对98头个体产生131条清晰、稳定的谱带.Shannon信息指数表明,小翅雏蝗遗传多样性水平较高(0.2542),白纹雏蝗遗传多样性水平较低(0.1911).N e i's遗传距离显示,种群间遗传距离小于种间遗传距离.用N J法对N e i's遗传距离作聚类分析,构建分子系统树,结果显示:白纹雏蝗与夏氏雏蝗聚为一支,小翅雏蝗与北方雏蝗聚为一支,两支再相聚.聚类图所显示的种间亲缘关系的远近程度与形态分类学结果相一致,说明RAPD和SSRP-PCR技术在雏蝗属种间分子系统学研究中具有很好的适用性.     

藏山羊微卫星DNA多态性研究

选用10个SSR标记,经PCR扩增、9%非变性聚丙烯酰胺电泳和银染法显色,对高原型藏山羊、山谷型藏山羊进行遗传多态性研究,并以白玉黑山羊、建昌黑山羊、美姑山羊和新疆山羊作对照.结果表明,10个SSR标记在高原型藏山羊、山谷型藏山羊、白玉黑山羊、建昌黑山羊、美姑山羊和新疆山羊群体的平均H、PIC、Ne分别为0.673/0.631/4.3、0.680/0.649/4.7、0.777/0.660/4.3、0.797/0.716/5.1、0.793/0.561/3.2和0.680/0.629/4.6.高原型藏山羊与山谷型藏山羊(D=0.063)聚为1类;建昌黑山羊和美姑山羊(D=0.026)聚为1类后,再与白玉黑山羊聚为1类;最后两类与新疆山羊聚为1大类,与各群体的来源和生态地理分布一致.     

青藏高原城镇化时空格局变化及自然驱动因素分析

青藏高原的特殊自然条件导致了其城镇化发展水平较低,了解青藏高原城镇化发展的时空格局及其成因对推动当地未来的新型城镇化发展具有重要的指导意义。本研究利用1990～2020年历次人口普查数据,对青藏高原范围内县域单元的城镇化水平时空变化进行了分析,并利用地理探测器分析了导致城镇化格局形成的自然地理因素。结果表明：青藏高原地区城镇化率波动较大,区域差异明显,且差异有减小的趋势;城镇化水平较高的地区集中在行政中心与高原东部边缘地区,高原中部部分地区存在城镇化率下降的情况;地形因素是影响青藏高原地区城镇化发展的主要自然因素。     

基于Cyt b基因序列变异探讨鲱形目系统发育关系

鲱形目内科间分类关系一直以来存在较多争议,为探讨鲱形目系统发育关系,对黄鲫、凤鲚的线粒体Cyt b基因全序列进行扩增、克隆及测序,得到1 141 bp序列。结合Genbank中已发布的鲱形目鱼类Cyt b基因全长序列利用生物信息学软件进行比较分析后,共检测到553个变异位点,总变异为48.5%,其中含504个简约信息位点,未见序列插入或缺失。碱基的替换多在密码子第三位,序列中转换多于颠换,Ts/Tv为2.448,基因突变未达到饱和。基于K2P模型计算的遗传距离表明,宝刀鱼科同鲱科及鳀科的科间遗传距离(0.042~0.072)小于鲱科内属间遗传距离(0.065~0.106)。在以南美肺鱼为外群构建的NJ分子系统树中,齿头鲱科最先分化,是鲱形目中原始类群;鳓属、多齿鳓属独立聚为一支,且具较高的自举检验值,支持将其归入锯腹鳓科;宝刀鱼科并入到鲱科一支中去,与鲱科亲缘关系更近。     

Phylogenetic relationships of host insects of Cordyceps sinensis inferred from mitochondrial Cytochrome b sequences

This study used the sequence of the mitochondrial Cytochrome b (Cytb) to estimate phylogenetic relationships among host Hepialidae insects of Cordyceps sinensis. Genome DNA of host insect was extracted from the dead larva head part of 18 cordyceps populations and 2 species of Hepialus, and the Cytb fragment of host insect was amplified with PCR technique. The nucleotide sequence alignments and their homologous sequences of 24 species host Hepialidae insects of Cordyceps sinensis were obtained from GenBank and were used to construct phylogenetic trees based on neighbor-joining method. The results showed that genus Bipectilus diverged earlier than genus Hepialus and Hepialiscus. Hepialus host insects of Cordyceps sinensis have multitudinous species with different morphological characteristics and geographical distributions. The interspecific genetic differentiations are obvious in Hepialus. Thus, the genus Hepialus might be considered as polyphyletic origin. Cytb sequences have abundant variations among the host insects of Cordyceps sinensis on specific and generic level. The divergence rate of Cytb sequences among the species in Hepialus ranged from 0.23% to 9.24%, except that Hepialus pratensis and Hepialus jinshaensis have the same sequence. Cytb sequence can be used for species identification of host insects of Cordyceps sinensis, but further confirmation in more host insect species is needed. To obtain the Cytb sequence of host insect by amplifying DNA extracted from the head part of dead larva in cordyceps turns out to be an effective and accurate approach, which will be useful for studies on phylogeny and genetic structure of host insects of cordyceps populations, especially for analyzing relationships between C. sinensis and its host insects.     

Bayesian mixed models and divergence time estimation of Chinese cavefishes (Cyprinidae: Sinocyclocheilus)

The genus Sinocyclocheilus is distributed in Yun-Gui Plateau and its surrounding region only, within more than 10 cave species showing different degrees of degeneration of eyes and pigmentation with wonderful adaptations. To present, published morphological and molecular phylogenetic hypotheses of Sinocyclocheilus from prior works are very different and the relationships within the genus are still far from clear. We obtained the sequences of cytochrome b (cyt b) and NADH dehydrogenase subunit 4 (ND4) of 34 species within Sinocyclocheilus, which represent the most dense taxon sampling to date. We performed Bayesian mixed models analyses with this data set. Under this phylogenetic framework, we estimated the divergence times of recovered clades using different methods under relaxed mo-lecular clock. Our phyloegentic results supported the monophyly of Sinocyclocheilus and showed that this genus could be subdivided into 6 major clades. In addition, an earlier finding demonstrating the polyphyletic of cave species and the most basal position of S. jii was corroborated. Relaxed diver-gence-time estimation suggested that Sinocyclocheilus originated at the late Miocene, about 11 million years ago (Ma), which is older than what have been assumed.