Molecular phylogenetic relationships based on mitochondrial genomes of novel deep-sea corals(Octocorallia:Alcyonacea):Insights into slow evolution and adaptation to extreme deep-sea environments

A total of 10 specimens of Alcyonacea corals were collected at depths ranging from 905 m to 1633 m by the manned submersible Shenhai Yongshi during two cruises in the South China Sea(SCS).Based on mitochondrial genomic characteristics,morphological examination,and sclerite scanning electron microscopy,the samples were categorized into four suborders(Calcaxonia,Holaxonia,Scleraxonia,and Stolonifera),and identified as 9 possible new cold-water coral species.Assessments of GC-skew dissimilarity,phylogenetic distance,and average nucleotide identity(ANI)revealed a slow evolutionary rate for the octocoral mitochondrial sequences.The nonsynonymous(Ka)to synonymous(Ks)substitution ratio(Ka/Ks)suggested that the 14 protein-coding genes(PCGs)were under purifying selection,likely due to specific deep-sea environmental pressures.Correlation analysis of the median Ka/Ks values of five gene families and environmental factors indicated that the genes encoding cytochrome b(cyt b)and DNA mismatch repair protein(mutS)may be influenced by environmental factors in the context of deep-sea species formation.This study highlights the slow evolutionary pace and adaptive mechanisms of deep-sea corals.

KaKs_Calculator 2.0:A Toolkit Incorporating Gamma-Series Methods and Sliding Window Strategies

We present an integrated stand-alone software package named KaKs_Calculator 2.0 as an updated version. It incorporates 17 methods for the calculation of nonsynonymous and synonymous substitution rates; among them, we added our modified versions of several widely used methods as the gamma series including y-NG, y-LWL, ）,-MLWL, y-LPB, y-MLPB, y-YN and y-MYN, which have been demonstrated to perform better under certain conditions than their original forms and are not implemented in the previous version. The package is readily used for the identification of positively selected sites based on a sliding window across the sequences of interests in 5＇ to 3＇ direction of protein-coding sequences, and have improved the overall performance on sequence analysis for evolution studies. A toolbox, including C＋＋ and Java source code and executable files on both Windows and Linux platforms together with a user instruction, is downloadable from the website for academic purpose at https：//sourceforge.net/projects/kakscalculator2/.

半滑舌鳎Gadd45g3基因的克隆与表达分析

生长阻滞和DNA损伤诱生蛋白45 （growth arrest and DNA damage inducible protein 45,Gadd45）基因在胚胎发育和性别决定中起重要作用.本研究通过RACE方法在半滑舌鳎（Cynoglossus semilaevis）中克隆得到了1个Gadd45g同源基因,命名为Gadd45g3.该基因全长1 147 bp,编码155个氨基酸残基.氨基酸序列分析表明,半滑舌鳎Gadd45g3与其他鱼类Gadd45g-like的同源性为79％～88％,与硬骨鱼和高等脊椎动物的Gadd45g同源性为58％～67％.对多个物种的Gadd45g同源基因的CDS区进行Ka/Ks分析,发现Ka/Ks均小于0.3.FISH结果显示,半滑舌鳎Gadd45g3基因位于常染色体上.实时荧光定量PCR结果显示,Gadd45g3在雌雄成鱼的脑中都有很高的表达,说明其参与了神经发育;在成熟性腺中,Gadd45g3的表达具有精巢特异性,说明Gadd45g3是性别相关基因;而在不同发育时期的性腺中,Gadd45g3的表达量在孵化后20 d的雄性仔鱼中显著高于同龄雌鱼（P＜0.05）,但在孵化后95 d的雌雄仔鱼中均有高表达,说明在半滑舌鳎性别决定和性腺发育中起着重要作用.

分子进化研究中的几个统计量及其应用

为在分子水平上快速精确地研究生物的进化关系,提出了分子进化研究中的几个常用统计量及其计算方法.根据这些统计量探测正选择作用,并确定特定基因的进化速度或年龄.这些统计量包括核苷酸同义替换数ds(Ks)、非同义替换数dn(Ka)、Ka／Ks、DNA多态性和遗传距离等.通过植物抗性基因的一个实例揭示了这些统计量在阐明进化机制过程中的重要性.

Characterization and comparison of chloroplast genomes from two sympatric Hippophae species(Elaeagnaceae)

The genus Hippophae includes deciduous shrubs or small trees,which provide many ecological,economic,and social benefi ts.We assembled and annotated the chloroplast genomes of sympatric Hippophae gyantsensis(Rousi)Lian and Hippophae rhamnoides Linn subsp.yunnanensis Rousi and comparatively analyzed their sequences.The fulllength chloroplast genomes of H.gyantsensis and H.rhamnoides subsp.yunnanensis were 155,260 and 156,415 bp,respectively;both featured a quadripartite structure with two copies of a large inverted repeat(IR)separated by small(SSC)and large(LSC)single-copy regions.Each Hippophae chloroplast genome contained 131 genes,comprising 85 protein-coding,8 ribosomal RNA,and 38 transfer RNA genes.Of 1302 nucleotide substitutions found between these twogenomes,824(63.29%)occurred in the intergenic region or intron sequences,and 478(36.71%)were located in the coding sequences.The SSC region had the highest mutation rate,followed by the LSC region and IR regions.Among the protein-coding genes,three had a ratio of nonsynonymous to synonymous substitutions(Ka/Ks)>1 yet none were signifi-cant,and 66 had Ka/Ks<1,of which 46 were signifi cant.We found 20 and 16 optimal codons,most of which ended with A or U,for chloroplast protein-coding genes of H.gyantsensis and H.rhamnoides subsp.yunnanensis,respectively.Phylogenetic analysis of fi ve available whole chloroplast genome sequences in the family Elaeagnaceae—using one Ziziphus jujube sequence as the outgroup—revealed that all fi ve plant species formed a monophyletic clade with two subclades:one subclade consisted of three Hippophae species,while the other was formed by two Elaeagnus species,supported by 100%bootstrap values.Together,these results suggest the chloroplast genomes among Hippophae species are conserved,both in structure and gene composition,due to general purifying selection;like many other plants,a signifi cant AT preference was discerned for most proteincoding genes in the Hippophae chloroplast genome.This study provides a valuable reference tool for future research on the general characteristics and evolution of chloroplast genomes in the genus Hippophae.

不同海拔喜马拉雅旱獭线粒体基因组比较分析

以分布在不同海拔(2279、3273、4452 m)的喜马拉雅旱獭(Marmota himalayana)为供试材料,通过二代测序技术对线粒体基因组序列进行比较分析,挖掘喜马拉雅旱獭低氧适应相关基因。结果表明:不同海拔喜马拉雅旱獭线粒体序列长度之间并无差异,但蛋白质编码基因突变位点个数随海拔增加而增多,ND5基因的突变位点个数和非同义突变位点个数都为最多,其Ka/Ks>1,表明ND5基因存在正选择效应,可作为适应高海拔低氧环境的候选基因。与玉树(海拔4452 m)相比,乐都(海拔2279 m)和黄南(海拔3273 m)的喜马拉雅旱獭亲缘关系相对较近。喜马拉雅旱獭13个蛋白质编码基因的核苷酸序列共编码氨基酸3812个,去掉起始密码子和终止密码子后共编码氨基酸3802个。在20种氨基酸中,Leu的使用率最高,达16.18%,Gys的使用率最低,仅有0.71%。RSCU值>1的密码子有32种,RSCU值<1的密码子有32种,其中AGG的RSCU为0。

Transcriptomic comparison to identify rapidly evolving genes in Braya humilis

The Brassicaceae species Braya humilis shows broad adaptation to different climatic zones and latitudes. However, the molecular adaptation mechanism of B. humilis is poorly understood. In China, B. humilis is mainly distributed on the QinghaiTibetan Plateau(QTP) and in the adjacent arid region. Previous transcriptome analysis of B. humilis has revealed that 39 salt and osmotic stress response genes are subjected to purifying selection during its speciation. To further explore the adaptation mechanism of B. humilis to an arid environment, OrthoMCL program was employed in this study and 6,268 pairs of orthologous gene pairs with high confidence were obtained between B. humilis and Arabidopsis thaliana. A comparative evolutionary analysis based on nonsynonymous to synonymous substitution ratio(Ka/Ks) was then conducted. There were 64 pairs exhibiting a Ka/Ks ratio more than 0.5 and among which, three instrumental candidate genes, T20487,T22576, and T23757, were identified with strong selection signatures(Ka/Ks >1). The corresponding A. thaliana orthologs are double-stranded RNA-binding domain protein, MADS-box family protein, and NADH-dehydrogenase subunit6, which is encoded by mitochondria genome. This report not only demonstrates the adaptation contribution of fast evolving nuclear genes, but also highlights the potential adaptive value of mitochondria gene to the speciation and adaptation of B. humilis toward the extreme environment in an arid region.

How Do Variable Substitution Rates Influence Ka and Ks Calculations?

The ratio of nonsynonymous substitution rate （Ka） to synonymous substitution rate （Ks） is widely used as an indicator of selective pressure at sequence level among different species, and diverse mutation models have been incorporated into several computing methods. We have previously developed a new γ-MYN method by capturing a key dynamic evolution trait of DNA nucleotide sequences, in consideration of varying mutation rates across sites. We now report a further improvement of NG, LWL, MLWL, LPB, MLPB, and YN methods based on an introduction of gamma distribution to illustrate the variation of raw mutation rate over sites. The novelty comes in two ways： （1） we incorporate an optimal gamma distribution shape parameter a into γ-NG, γ-LWL, γ-MLWL, γ-LPB, γ-MLPB, and γ-YN methods; （2） we investigate how variable substitution rates affect the methods that adopt different models as well as the interplay among four evolutional features with respect to Ka/Ks computations. Our results suggest that variable substitution rates over sites under negative selection exhibit an opposite effect on estimates compared with those under positive selection. We believe that the sensitivity of our new methods has been improved than that of their original methods under diverse conditions and it is advantageous to introduce novel parameters for Ka/Ks computation.

哺乳动物中不同表达丰度外显子的进化特征

通过比较基因组学方法,利用哺乳动物基因组数据对不同表达丰度外显子的进化特征进行了研究.以非编码区和假基因为对照,对外显子进行序列相似度、phastCons和Ka/Ks分析,结果显示,高表达外显子十分保守而低表达和低表达的外显子也在物种间表现出保守的进化特征.低表达丰度的外显子构成了物种转录组中大多数的转录本种类,结果显示它们的进化都受到功能限制,暗示它们可能在生物进化过程中为增加生物复杂度做出贡献.

基于基因进化树和地理数据库追踪禽流感病毒变异

目的禽流感疫情的爆发和传播受到多种自然因素的影响。今欲尝试将地理信息系统与基因进化树分析相结合，以建立一种基于基因序列变异追踪中国禽流感病毒地理传播的技术。方法禽流感病毒基因来源于美国国立医学图书馆（National Library for Medicine，NLM）数据库，所获得的基因组数据利用E—Utilities软件包转化为结构体后，可用Matlab软件阅读。结构体主要字段包括PB2、PBl、PA、HA、NP、HA、M1和NSl8个片段，分别代表流感病毒的8个不同的基因片段。基于结构体字段，利用计算生物学的方法比较不同传播能力禽流感病毒的同义突变／非同义突变基因（Ka／Ks）比例，确定不同选择压力之下A型禽流感病毒的基因突变模式。进而选择Ka／Ks比例最大的基因片段，采用Jukes—Cantor算法估计氨基酸序列变异的进化距离，然后对不同爆发点的H5N1型禽流感进行进化树聚类。将聚类信息输入Google Earth，并利用不同图层地理信息对影响爆发点分布的因素做单因素分析。结果比较分析A型禽流感所有的8个基因序列可以看出，NSl、HA和NA蛋白的Ka／Ks比值较大。三者中，HA基因的Ka／Ks比值最大，可以代表病毒的传播能力。利用分级聚类的思路对HA基因转录的氨基酸相似程度进行比较，发现自2003年以来亚洲地区爆发的H5N1型禽流感之间的关系可以表示为一个由30个节点构成的进化树，其中14个节点为分支节点，16个节点为叶子结点。把分支树的前三个节点作为分类标准，可以把所有16个病毒株分为四类。这四类病毒在地理空间的分布呈现一定规律。计算发现禽流感爆发相关地理因素排序分别为：内陆水体〉主要铁路交通线〉家禽密度。结论对中国HSNl病毒株基因序列变异的地理分布分析显示，禽流感病毒爆发与候鸟迁徙、家禽运输密切相关。

Evaluation of Six Methods for Estimating Synonymous and Non-synonymous Substitution Rates

Methods for estimating synonymous and nonsynonymous substitution rates among protein-coding sequences adopt different mutation (substitution) models with subtle yet significant differences, which lead to different estimates of evolutionary information. Little attention has been devoted to the comparison of methods for obtaining reliable estimates since the amount of sequence variations within targeted datasets is always unpredictable. To our knowledge, there is little information available in literature about evaluation of these different methods. In this study, we compared six widely used methods and provided with evaluation results using simulated sequences. The results indicate that incorporating sequence features (such as transition/transversion bias and nucleotide/codon frequency bias) into methods could yield better performance. We recommend that conclusions related to or derived from Ka and Ks analyses should not be readily drawn only according to results from one method.

Genome-wide identification, characterization and evolution of cuticular protein genes in the malaria vector Anopheles sinensis （Diptera： Culicidae）

Thirteen cuticular protein （CP） families have been recognized in arthropods. In this study, 250 Anopheles sinensis CP genes were identified and named based on genome and transcriptome sequences. They were classified into 10 families based on mo- tifs and phylogenetic analyses. In 11 other insect species, nine had CP numbers 〉 150 while Apis mellifera and Tribolium castaneum had CP numbers less than 52. The CPs of eight species occupied 〉 1.4% of the total genomic gene number, whereas in three species the CPs occupied 〈 1%. The phylogenies for each CP family in An. sinensis were constructed and discussed. The 250 CPs each had 1-8 exons with 144 CPs （57.6%） having two exons. The intron length ranged from 66--3888 bp with 174 introns （54.0%） being 66--100 bp long. Except for two CPs on two contigs, 248 CPs were mapped onto 28 scaffolds with 136 genes （54.4%） restricted to five scaffolds. A total of 107 CPs were clustered and located at 27 loci. The CPR family had the conserved motif GSYS- LVEPDGTVRTV. The RR- 1 subfamily had an additional 21 amino acid （aa） motifs with the YVADENGF sequence that is common in insects. The RR-2 subfamily had an additional 50 aa motifs with two additional regions RDGDWKG and G-x（3）-VV. A comparison with 115 orthologous counterparts of An. gambiae CPs suggested purifying selection for all of these genes. This study provides basic information useful for further studies on biological functions of An. sinensis CPs as well as for comparative genomics of insect CPs.

Molecular characterization and phylogenetic analysis of three odorant binding protein gene transcripts in Dendrolimus species （Lepidoptera： Lasiocampidae）

Pine caterpillar moths, Dendrolimus spp. （Lepidoptera： Lasiocampidae）, are serious economic pest of pines. Previously, phylogenetic analyses of Dendrolimus using different methods yielded inconsistent results. The chemosensory systems of insects may play fundamental roles in promoting speciation. Odorant-binding proteins （OBPs） participate in the first step of odor detection. Studying the evolution of OBPs in closely related species may help us to identify their role in speciation. We identified three OBPs - one pheromone-binding protein and two general odorant-binding proteins - from male antennae of four Dendrolimus species, D. superans （Butler）, D. punctatus （Walker）, D. kikuchii Matsumura, and D. houi Lajonquiere, the olfactory recognition systems of which had not been previously investigated. We analyzed their molecular characteristics and compared their sequences to those of OBPs in D. tabulaeformis Tsai et Liu. Ka/Ks ratio analyses among the five Dendrolimus species indicate that PBP1 genes experienced more evolutionary pressure than the GOBPs. Phylogenetic relationships of PBP1 and GOBP1 both indicated that D. houi was the basal species, then branched D. kikuchii, while D. tabulaeformis, D. punctatus, and D. superans evolved more recently. These relationships are consistent with the changes in sex pheromone components of these five species. Dendrolimus tabulaeformis and D. punctatus are closely related sister species. However, the distances among GOBP2 sequences in the five Dendrolimus were very short, and the relationships of D. houi and D. la＇kuchii could not be resolved. Integrating our results with those of previous studies, we hypothesized that D. kikuchii, D. punctatus and D. superans evolved from the basal ancestor because of sex pheromone mutations and environmental pressure.

Evolutionary characteristics of exons expressed at different abundance levels in mammals

With comparative genomics approaches, we evaluated the evolutionary characteristics of conservation of exons which are expressed abundantly, moderately or lowly in mammals. Using non-coding regions and pseudogenes as controls, sequence identity, phastCons and Ka/Ks analyses were carried out and our results showed that as the exons of high abundance are highly conserved, the minor and low exons also showed conservative characteristics in evolution. Our findings suggested that the exons with less abundance which constitute a large proportion of distinct species in transcriptome of organisms are under functional constraint and might play certain roles in enriching biological complexity in the evolution of organisms.

基于行星会合指数对太阳轨道运动速度和加速度指数的建立——对太阳自转运动周期性振荡成因机制的探讨

Tlatov(2007)研究表明太阳自转具有准22年振荡周期,并认为是太阳系自引力造成的.根据刘复刚和王建(2013)创建的行星会合指数KP(令KP=K是为了和其他指数的表现形式相统一)和获得的修正系数发现:太阳轨道运动具有平均准22.1826年运动周期,认为太阳轨道角动量和太阳自转角动量的周期性叠加致使太阳自转角速度具有准22.20年周期性变化.基于行星会合指数KP同时标定了太阳质心S相对于一个无法观测到的太阳系质心C位置关系的变化规律,进而通过太阳轨道运动指数KS、速率指数Kv、加速度指数Kα对太阳轨道运动特征进行了系统描述,这使得从行星系统之视角分析、揭示太阳运动和太阳活动成为可能,并对太阳自转运动的准11年和22年周期振荡受控于太阳轨道运动的调控进行了分析,这从探索途径和思想方法上是一种突破.依K_P表达式给出的太阳轨道运动轨迹与Jose(1965)、杨志根等(1988)、Scafetta(2014)、Mc Cracken等(2014)给出的图像不但形态特征相同而且图像的相位也完全一致.行星会合指数KP更重要的作用是标定了行星系统质心距离太阳位置关系的变化.本文从行星系统质心出发,根据行星系统质心P与太阳质心S绕太阳系质心C同步运动这一基本原理来进一步揭示太阳轨道运动规律.由于研究问题的出发点和视角不同,按这一途径可根据8大行星公开的天文数据分析太阳轨道运动规律.这种方法不但把纷繁复杂的行星系统统一起来,呈现出行星系统固有的整体运动规律,而且在整个太阳系中建立了与太阳处于相同子系统地位的行星系统质心.在此基础上,通过行星系统质心P与太阳S间位置关系变化,系统分析了太阳S和太阳系质心C的统一与分野规律.其重要意义是通过行星系质心运动规律发现它与太阳轨道运动特征间的本质联系,这使得从行星系统质心的运动特征对太阳运动与太阳活动关系的探寻成为可能.