麂属动物的DNA进化

本文分析了麂属动物及其近缘种的线粒体DNA（Mitochondrial DNA, mtDNA)和核塘体DNA（Ribosomal DNA, rDNA）限制性片段长度多态性（RFLP），建立了麂属动物mtDNA和rDNA的限制性内切酶间谱。据此计算出各个物种的种内及种间的遗传距离，构建了麂属动物的种内及种间的分子聚类图。结果表明，在现生麂类中，黑麂和贡山麂之间的关缘关系最近，其次是费氏麂；印度麂是一个特化的特种，它和黑麂支系（包括费氏麂、贡山麂和黑麂）可能是从小麂的祖先类群中独立分化出来的。在近缘物种中，毛冠鹿与麂属动物的亲缘关系较近。结合前人有关的工作，计算19种鹿科动物之间的遗传距离并绘制它们的分子聚类图。

中国貉(Nyctereutes procyonoides)线粒体DNA多态性及其与亚种分化的关系

我们用8种限制性核酸内切酶(Hpa I、Bgl I、EcoR I、Sca I、BamH I、EcoRV、Stu I、和Apa I)分析来自东北、华北、华东、西南、华南地区的中国貉(Nyctereutes procyonoides)5个地理群体共11只个体的mtDNA多态性，计算各群体间的遗传距离。分别用UPG法和NJ法构建中国貉的系统发生关系。结果表明，云南、广西、安徽群体与华北、东北群体分别构成中国貉的南、北两个支系；根据遗传距离的计算，华北貉可以构成一个独立的亚种；指名亚种中的广西、安徽两群体间的遗传差异较大(p=o.oo97)，而广西貉与西南亚种（云南貉）则非常接近(p=o.0047)。这提示，指名亚种内已发生明显的遗传分化。华东（福建）、华南群体（两广）的分类地位及其与其它各群体的遗传关系有待进一步探讨。在所研究的5个群体中，MtDNA差异与群体间的地理距离表现出某种相关性。

川西青杨组(Section Tacamahaca Spach)不同种的亲缘关系分析

青杨组（Section Tacamahaca Spach）杨树是我国重要的乡土经济树种，目前对其分子遗传变异和系统进化的研究还很少，尤其是在青杨组杨树遗传资源极为丰富的川西地区，杨树的分子进化及亲缘关系的研究极为缺乏，非常不利于该树种遗传资源的开发和利用。本研究从川西地区收集了青杨（Populus cathayana）、青海杨（P. prezewalskii）、滇杨（P. yunnanensis）、康定杨（P. kangdingensis）、西南杨（P. schneideri）、小叶杨（P.simonii）和三脉青杨（P. trinervis）这7 个青杨组树种的10 个群体，利用多种分子标记手段对其种间的亲缘关系进行比较，并结合形态和地史资料进行了全面的研究和评价，得到了如下的主要研究结果： 1. SSR 和ISSR 位点变异丰富。通过10 对引物对50 个杨树个体的DNA 样品进行了SSR 分析，所有位点展现了丰富的群体间和种间的多态性，多态位点率达到了100%，每位点的等位基因数变化范围为5 ~ 17，平均为11.9 个；通过11 条ISSR 随机引物对供试的混合DNA 样品进行分析，共检测到130 个标记，其中多态性标记为119 个，多态百分率为91.5%。研究认为，SSR 单个标记能展现高水平信息，而ISSR 单个引物能探测更多数量多态性。通过两个标记的遗传距离、聚类图和PCA 分析，表明：同一种内不同群体间的同源性最高；康定杨和西南杨有较近的亲缘关系；小叶杨和三脉青杨聚合在一起，显示了其相互较近的亲缘关系；滇杨与其它杨树种可能存在着较远的亲缘关系。 2. 采用4 对选择性引物对7 个青杨组杨树种10 个群体进行AFLP 分析，总共扩增出284 个位点，其中200 个位点显示出了多态性，多态位点百分比为70.4%，平均多态带为50 条。TE-AFLP 的分析总共扩增出192 个位点，其中139 个位点显示出了多态性，多态位点百分比为72.4%，平均多态带为34.7 条。比较的结果表明AFLP、TE-AFLP 的遗传信息含量比较接近，略小于ISSR，大约仅为SSR 的1/3；但这两个基于AFLP 的标记系统的信息探察能力也远大于ISSR 和SSR 标记系统。这两个分子标记的聚类结果，显示小叶杨、三脉青杨和滇杨三个种聚为一组，其中小叶杨与三脉青杨的亲缘关系更近；其它几个杨树种聚为一类，西南杨与青杨表现出较近的亲缘关系。 3. 所有7 对cpSSR 引物中，仅有4 个叶绿体位点在种间具有多态性，而在种内群体中并不具有多态性，共检测出13 个条带，组合成了4 种不同的单倍型；对于cpDNA的5 对引物，共检测出了73 条酶切片段，其中52 条是多态带，组合成了9 种不同的单倍型；而5 对mtDNA 通用引物未能检测出多态性的条带，表现出线粒体的保守性。叶绿体的聚类分析认为，小叶杨、三脉青杨和滇杨有较近的母性起源，且依次聚合；其余四种杨树聚为一类，并且康定杨与西南杨表现出最近的亲缘关系，并依次与青杨和青海杨聚合。 4. 根据本文的分子数据，结合形态和生境分布资料分析认为：青杨组杨树种内群体间的遗传变异程度是小于种间的遗传差异，显示了与传统分类一致的结果；三脉青杨和小叶杨有很近的亲缘关系，可能拥有相同的祖先类群；滇杨与小叶杨和三脉青杨之间具有一定的亲缘关系，特别是在其母性祖先的起源上有着一定的同源性；西南杨与青杨和康定杨均保持着较近的亲缘关系，且有可能是这两个种原始祖先杂交后所形成的。 Although western Sichuan is regarded as a natural distribution and variation center forthe Section Tacahamaca of the Populus species in China, little is currently known about themajority of poplar species occurring in this region. In the present study, molecular data wereutilized to determine the genetic relationships among Populus species in Section Tacamahacain western Sichuan including P. cathayana, P. prezewalskii, P. yunnanensis, P. kangdingensis,P. schneideri, P. simonii and P.trinervis. The results are as fellows: 1. The genetic variation at SSR and ISSR loci was abundant. All the 10 SSR loci werepolymorphic, and the number of alleles per locus varied from 5 to 17 with a mean valueequaling 11.9. Based on the 11 ISSR primers, 130 clear and reproducible DNA fragmentswere generated, of which 119 (91.5%) were polymorphic. Our results reveal that single SSRlocus can present more genetic information, while more polymorphic bands can be detectedby single ISSR primer. Moreover, the genetic distance, cluster and PCA analysisdemonstrated that: a close relationship among accessions of the same species and suggestedmonophyly in P. przewalskii and P. cathayana; P. schneideri is genetically highly similar to P.kangdingensis; P. trinervis and P. simonii have a close genetic affinity; P. yunnanensis isdistinct from the other species. 2. Genetic relationships of poplar species in Section Tacamahaca from western Sichuanwere evaluated by means of AFLP and TE-AFLP. For four AFLP primer combinations, atotal of 284 bands were obtained of which 200 (70.4%) were polymorphic with the average of50 polymorphic bands. For four TE-AFLP primer combinations, a total of 192 band wereobtained of which 139 (72.4%) were polymorphic with the average of 34.7 polymorphicbands. Our results indicate that the genetic information of AFLP is similar to that ofTE-AFLP, and little less than that of ISSR, but only about 1/3 of that of SSR. However, theability of information detection of the two AFLP-based markers is much higher than that ofISSR and SSR. In addition, the cluster analysis of AFLP, TE-AFLP and combined data revealthat: P. yunnanensis, P. trinervis and P. simonii clustered together, and P. trinervis and P.simonii showed more closed affinity; the other four poplar species clustered together, P.cathayana and P. schneideri showed more closed origin especially. 3. The cpSSR analysis for seven Populus species belonging to the Section Tacamahaca.Four out of the seven analyzed chloroplast loci were polymorphic, whereas none of the lociwere polymorphic across the accessions within a species. 13 bands and 4 different kinds ofhaplotypes were reduced. Based on 5 pairs of cpDNA primers, 73 fragments (52 polymorphic)and 9 kinds of haplotypes were produced. However, none of the polymorphic was detected bythe 5 mtDNA primer pairs, revealing conservation of mtDNA region. The cluster analysis ofcpDNA revealed that: similar maternal phylogeny among P. yunnanensis, P. trinervis and P.simonii; the other four species clustered together, P. schneideri and P. kangdingensis showedmore closed maternal lineage especially. 4. Our molecular data, morphological characters and nature habitat revealed that: sameto the traditional taxonomy assignment, genetic variation within a same Populus species islower than that among Populus species in Section Tacamahaca; P. yunnanensis may share itschloroplast ancestor with P. trinervis and P. simonii; moreover, sister genetic relationship of P.trinervis and P. simonii indicated their similar origin; P. schneideri clustered with P.kangdingensis and P. cathayana, respectively, and may have derived from an ancienthybridization event involving the ancestors of the two species.

香港地区湍蛙遗传多样性研究

依据线粒体上ND2和CO1两个变异较大的基因序列分析了香港地区香港湍蛙7种群、华南湍蛙1种群，以及大陆其他地区华南湍蛙7种群，戴云湍蛙1种群，武夷湍蛙1种群的系统发育关系，进而探讨香港湍蛙的遗传多样性、香港湍蛙特有性、如何确定香港湍蛙最佳保护单元以及这四种湍蛙的物种分类地位。 1． 香港湍蛙保护遗传学研究 香港湍蛙核苷酸传多样性较低，从其遗传多样性信息、单倍型网络分析、中性检验值以及岐点分布结果一致显示香港湍蛙很可能经历了瓶颈后的扩张，种群正在由一个较小的有效种群大小迅速增长, 有足够的时间通过变异用于积累单倍型的多态性, 而对于提高核苷酸多样化而言, 时间尚短（Nei M et al，1975，Avise J C，2000；李明等，2003）。 分子变异分析结果显示香港湍蛙种群间存在较多的基因交流，且系统发育树上各种群间交叉在一起，没有形成与地理单元相关的分支，而从其单倍型网络看，他们源于共同的祖先，是一个单系群，与地理单元间没有形成显著的遗传分化。因此应作为一个进化显著单元（ESU）。结合其与其他湍蛙发育关系及遗传距离以及野外采集信息认为香港湍蛙只在香港地区有分布，属于香港特有种。该物种内遗传多样性较低，又属于世界自然保护联盟红皮书中的近危种，同时也是《野生动物保护条例》中的受保护野生动物，且由于香港城市建设等使得其栖息环境受到威胁，因此在香港特别行政区应该受到重点保护。 从单倍型分布和核苷酸多样性可以看出大榄涌种群和城门种群具有较高的单倍型多样性和核苷酸多样性，应该作为保护的重点区域。 2. 华南湍蛙东、南沿海种群间系统关系 华南湍蛙分布广，各种群存在着丰富的遗传多样性信息且中部种群广西龙胜和湖南张家界种群核苷酸多样性明显高于其他边缘种群华南湍蛙。种群间几乎没有基因交流，且各种群间无共享单倍型，可见已形成了显著的遗传分化。各种群间遗传距离都较远，其中广东南昆山种群以及福建三港种群与其他种群距离最远，因此可以推测其他种群（广东深圳、香港大屿山、广西龙胜和防城以及湖南张家界种群）可能为独立进化的种群。但是否是一新种或一隐存种，还需要结合形态学进行更深入的研究。 本研究中无论从系统关系看还是从遗传距离看，大屿山种群与深圳种群最近，支持陈坚峰等将其定为华南湍蛙，即华南湍蛙新增一个分布点：香港大屿山。 系统树上广西防城种群（支B）与龙胜和湖南种群（支A）形成姐妹群。香港大屿山种群与深圳种群先形成姐妹群（支C），但却没有与其距离很近的广东南岭及南昆山种群（支D）形成姐妹群，可能粤北和粤中的环境及气候较复杂因此与粤南其他种群形成了明显的隔离。同时可以看出华南湍蛙种群遗传分化与地理距离没有显著的相关性。 3. 四种湍蛙间的系统关系 根据线粒体CO1基因建立四种湍蛙间的系统关系及其遗传距离，很清楚地看到，香港湍蛙与戴云湍蛙关系很近，而华南湍蛙则与武夷湍蛙较近。然而，戴云湍蛙同一个种群内部共有两个单倍型DY1和DY2，且两个单倍型间遗传距离大于DY1与香港湍蛙间遗传距离，更远远大于香港湍蛙种群内部的距离，即戴云湍蛙内部两个单倍型间遗传距离达到了种级水平，同样在系统发育树上这两个单倍型与香港湍蛙形成并系。但是，戴云湍蛙种内在形态上差异不显著。因此，其是否属于萌芽物种分化形成（budding speciation）或已经完全分化为两个不同的种值得进一步研究？ 与戴云湍蛙香港湍蛙关系类似，从系统树上看华南湍蛙不形成单系，而是分成两个大支，与武夷湍蛙形成并系，且福建和南昆山的华南湍蛙与武夷湍蛙遗传距离远大于武夷湍蛙种内福建种群与浙江种群的遗传距离，达到了种级分化水平。由此，可以推断武夷湍蛙是有效种。系统树上广东深圳、香港大屿山、广西防城和龙胜以及湖南张家界种群与华南湍蛙福建及南昆山各种群间遗传距离已超出了种内各种群间的遗传距离，但是至于这一支是否应为另外一个种，有必要扩大采样，并结合核基因及形态信息进行进一步研究。 MtDNA of ND2 and CO1 gene were used to investigate genetic diversity of Amolops in Hongkong .We collected seven populations of A. hongkongensis,，one population of A.ricketti from Hong Kong and other seven populations of A.ricketti from East and South of Chinese mainland. As well as one population of A. daiyunensis and one population of A.wuyiensis Phylogenetic relationship were analyzed of four species. Discussed whether A.hongkongensis is an endemic species and how can we make the conservation and management decisions. 1. Conservation Genetics of A. hongkongensis A. hongkongensis has a low nucleotide diversity, the results of genetic diversity, haplotype network, neutrality test and the mismatch distributions indicate that A. hongkongensis experienced a recent expansion after a bottle neck. They had enough time to accumulated haplotype diversity, but it’s too short to have a high nucleotide diversity（Nei M et al，1975，Avise J C，2000；Li et al，2003）. The result of AMOVA reveals that it has much gene exchange among the populations of A. hongkongensis. The clades of the phylogenetic tree were mixed together, no significant genetic differentiation among 8 populations and they share the same ancestor from the network analysis, these indicate that they are monophyly and should be protected as one ESU. Combined with the information of relationships of interspecies, genetic distance and distribution investigate, We conclude that A. hongkongensis is an endemic species of Hong Kong. Considering on the status of low genetic diversity in A.hongkongensis, and this species was listed in the IUCN red list as near threatened, as well as listed in the . Furthermore, it’s habitat loss and degradation more rapidly as the human activity got higher and higher. So it’s urgent to protect them in Hong Kong. Our results suggest that Tai Lam Wu and TAI MO Shan -Shing Mun populations have the higher priority to be protected because their higher genetic diversity. 2．Phylogenetic relationships among populations of Amolops ricketti from the Southern and eastern China A. ricketti has the considerable genetic diversity of mitochondrial haplotypes within and among populations, and Mitochondrial DNA diversity was higher in populations at the central area of the present distribution range of the frog，i. e. the Longsheng population and Zhangjiajie population, than at the edges of their distribution range. They have no share haplotype among populations, and have a significant genetic differentiation. Genetic distance is high among the populations, especially the distance of Nankun and Sangang group with others, which suggested that they evolved independently. May be there is a cryptic species or a new species, a further study is needed. The results of gene tree and the genetic distance clearly demonstrate that the population from LanTau island is A. ricketti, so we agree with Chen et al(2005) . That means A.ricketti have a new distribution site: LanTau island, HongKong. Phylogenetic relationships were analyzed through NJ and Mrbayes methods and got a consistent topological structure, the structure indicated that the ingroup were comprised four groups. Populations Longsheng and Zhangjiajie were first clustered as clade A; Populations Fangcheng was clustered together (clade B) as a sister group to clade A；Populations Shenzhen and Lantau island were sister groups and clustered as clade C；Then the clade D included populations Nankunshan and Nanling in Guangdong province and Sangang in Fujian province. 3. Phylogenetic Relationships among these four specises Phylogenetic relationships based on 1503bp CO1 gene and the genetic distance show that A. hongkongensis close to A. daiyunensis whereas A.ricketti near to A.wuyiensis. Nevertheless, there are two haplotypes in A.daiyunensis and the genetic distance between them higher than the distance between DY1 with A. hongkongensis. A. hongkongensis is nested in the paraphyletic ancestral species A. daiyunensis. Without significant difference in the morphological characters, So, we considered both A.daiyunensis and A.hongkongensis are valid species, may be this represents a case of ‘budding speciation’ like Batrachuperus pinchonii(Fu and Zeng,2008) in the population of A. daiyunensis. Just like two species above A. wuyiensis and A. ricketti are not monophyly, instead, A.wuyiensis is nested in the paraphyletic ancestral species A.ricketti. We need do more research to make sure whether they are new species.

黄河上游青海沙蜥种组的系统地理学研究

沙蜥属（Phrynocephalus）的卵胎生类群主要分布在我国青藏高原，包括南疆沙蜥（P. forsythii）、西藏沙蜥（P. theobaldi）、红尾沙蜥（P. erythrurus）、贵德沙蜥（P. putjatia）和青海沙蜥（P. vlangalii）。其卵胎生生殖方式适应了高寒生境，与青藏高原隆升有关。纵观前人的研究，上述几种卵胎生沙蜥的分类、系统发育关系以及生物地理都还存在疑问。本文研究了分布在若尔盖湿地的青海沙蜥红原亚种（P. v hongyuanensis）以及分布在黄河上游其它地区青海沙蜥种组的地理分布格局，并探讨了其形成机制。 青海沙蜥在黄河上游主要分布于若尔盖湿地以及青海湖周边地区。若尔盖湿地青海沙蜥红原亚种的生境由于沼泽的形成被切割成不连续的斑块，通过遗传分析可以推测这种特殊生境对它们遗传结构的影响。其次，贵德沙蜥、青海沙蜥的青海湖周边各居群以及若尔盖湿地居群之间的系统地理格局还未见报道。因此本文以居群为单位，将它们作为一个复合体，通过系统地理研究，可以了解其种群遗传结构，据此分析相关的地质历史事件对其分布的影响。主要结果如下： 1. 若尔盖湿地青海沙蜥红原亚种的种群遗传结构： 共研究了三个地理单元（红原（HY）、辖曼（XM）、玛曲（MQ））的7个采集点的72个个体。所有ND4-tRNALeu序列比对得到785 bp的片断，定义了9种单倍型。结果显示总的核苷酸多样性较低，单倍型多样性较高。分子变异分析（AMOVA）显示3个单元间差异显著（P<0.01），遗传变异主要存在于地理单元间，占62.61%。除MQ单元，XM各居群与HY居群混杂在一起，单倍型网络图没有显示出单倍型和地理位置的对应关系。XM单元单倍型的不配对分布（Mismatch distribution）为明显左移的单峰，且Fu’s Fs test为负值，表明XM单元可能经历了近期种群扩张，有足够的时间积累单倍型的多态性，还不足以大幅提高核苷酸多样性，这是其单倍型多样性较高和核苷酸多样性较低的原因。MQ单元遗传多样性低而与其他单元显著分化，推测这与3万年前黄河在若尔盖玛曲之间贯通有关。近期沼泽的形成对XMb居群的隔离时间短，使得其遗传多样性低但还不足以形成大的遗传差异。无论黄河的贯通还是沼泽的形成其隔离形成的时间都不长，其作用改变了单倍型出现的频率，也出现了一些特有单倍型，但共享单倍型还广泛存在，还不足以使得不同居群之间形成较大的遗传距离。 2. 黄河上游青海沙蜥种组的分布格局与地史过程的关系： 黄河上游青海沙蜥种组包括贵德沙蜥、青海沙蜥指名亚种的青海湖周边各居群、青海沙蜥红原亚种若尔盖湿地居群、以及青海湖以西的部分居群（序列由Genbank下载获得），总计22个居群189个样品。所有ND4-tRNALeu序列比对得到703个位点，定义了39种单倍型。以南疆沙蜥为外群构建的贝叶斯树以及MP法构建的无根树，都分为A、B两大组。其中A包括若尔盖湿地居群以及玛多居群（A1）、青海湖以西的居群和兴海居群（A2）、西藏沙蜥；B包括青海湖以南的居群和天祝居群（B1）、青海湖以东北的居群（B2）。单倍型网络图分别对应了系统发育树上的各支。按照系统发育结果分组进行分子变异分析，得到组间变异占88.63％，各组间差异显著（P=0.000）。种群遗传结构分析得到，A1和B2可能经历了近期的种群扩张，前者扩张时间约为0.105－0.189 Ma B.P.（million years before present），后者为0.057－0.102 Ma B.P.，可能与末次间冰期的气候变暖有关。A2和B1对应的两个地理单元都具有较强的种群遗传结构，较为稳定。 青海沙蜥种组A、B两大支之间遗传距离大，分化明显，分化大约发生在4.29－2.38 Ma B.P.，推测青藏运动的A幕运动后复杂的地形变化可能是它们产生分化的原因。B1和B2分化大约发生在1.73－0.96 Ma B.P.，这与湟水流域构造运动发生的时间相符。在早、中更新世时期，B1支内部各居群可能有交流，中更新世末共和盆地出现的抬升以及河流溯源改道等事件可能是引起这支内部多个单倍型丢失的原因。A1、A2支的分化可能与倒数第三次冰期降临之后气候变冷、阿尼玛卿山的大冰帽有关。 The viviparous group of genus Phrynocephalus is mainly distributed in the Qinghai –Tibetan Plateau, including P. forsythii、P. theobaldi、P. erythrurus、P. putjatia and P. vlangalii. These species are adapted well to the cold clime there, and the origin of this group was the result of a vicariance event associated with the uplifting of the Qinghai -Tibetan Plateau. Although many works have been done, there are still several questions about classification、phylogenetic relationships and the biogeography of this group. The phylogeographic pattern of the P. vlangalii complex on the upper reaches of the Yellow River and the P. v. hongyuanensis in Zoige Wetland were studied in this thesis. On the upper reaches of the Yellow River, P. vlangalii complex are distributed in Zoige Wetland and the southeast and northeast region of Kuku-noor Lake. Because of the forming of the wetland in Zoige, the habitats for sand lizards are divided into many discontinuous ones, and it is necessary to analyze genetic structure in these unique habitats. The phylogeographic patter among P. putjatia、populations of P. vlangalii in the southeast region of Kuku-noor Lake and populations of P. vlangalii in Zoige Wetland hasn’t been studied yet, and the complicated geological events of the Plateau may play an important role in the populations’ diversity and species forming there. So these populations were gathered as a complex, and phylogeographic analysis were used to clarify these doubts. According to the two topics above, this thesis has two parts of results as follows: 1. Three geographic units of P. vlangalii hongyuanensis in Zoige Wetland were defined, and they were Xiaman （XM）、Hongyuan （HY） and Maqu （MQ）. 785bp fragments of the mtDNA ND4-tRNAleu were determined from 72 samples and nine haplotypes were identified. As a whole, the nucleotide diversity was low，but the haplotype diversity was high. Analysis of molecular variance （AMOVA） showed that the three units were distinctly different（P<0.01），and 62.61% of the total genetic diversity was attributable to variation among units. There were 3 haplotypes shared among XM and HY，and no geographic clustering was observed except MQ from the TCS network. The results from the mismatch distribution analysis and Fu’s Fs test implied that there might be a recent population expansion in the XM unit, and this may be the reason why XM had a high haplotype diversity but a low nucleotide diversity. We estimate that the MQ and XMb have lower diversities because of some very recent geographic events, such as the formation of the Yellow river’s upriver and the Zoige Wetland. Although they are distinctly different, not enough time has passed for them to have diverged a great genetic distance. 2. 189 samples in 22 populations of P. vlangalii complex were collected, including P. putjatia、populations of P. vlangalii in the southeast and northeast region of Kuku-noor Lake、 populations of P. vlangalii in Zoige Wetland and the data from Genbank. 703bp ND4-tRNALeu sequences identified 39 haplotypes. P. forsythii was selected as outgroup, and both the Bayesian tree and the MP unrooted tree were divided into two groups（A、B）. A included populations in Zoige Wetland and Xinghai（A1）、populations in the west of Kuku-noor Lake（A2）、P. theobaldi, and B included populations in the southeast of Kuku-noor Lake and Tianzhu（B1）、populations in the northeast of Kuku-noor Lake（B2）. The haplotype network agreed with these groups. AMOVA showed that these five groups were distinctly different（P<0.01）, and 88.63% of the total genetic diversity was attributable to variation among groups. There might be recent population expansion in A1 and A2, which corresponded to the dry climate of the last interglacial period. The expansion times were 0.189-0.105 Ma B.P. and 0.102-0.057 Ma B.P., respectively. A2 and B1 had strong genetic structure. The large genetic distance between A and B showed that they had been separated from each other for a long time（about 4.29-2.38 Ma B.P.）, and it corresponded to the A phase of Qingzang Movement. The diversity between B1 and B2 at 1.73-0.96 Ma B.P. may be caused by the geological event in Huangshui valley. In early Pleistocene, populations in B1 may have gene flow because of geographic linkage, and later the uplift of the Plateau and the change of river route there made a few haplotypes lost. A1 and A2 were divided into two parts by A’nyemaqen Mountains at 0.66-0.37 Ma B.P., which maybe corresponded to glaciations at about 0.7 Ma B.P.

重离子束辐照诱变啤酒酵母线粒体DNA研究

摘要 II 5. 在不同注量离子束辐照后筛选出来的呼吸缺陷型酵母菌株中扩增获得位 于第 12 染色体上的 SOF1 基因，而在同样的扩增体系中没有得到野生型 菌株的该基因。 6. 选取离子束辐照后筛选出来的呼吸缺陷型酵母菌株再次进行辐照，发现 其在低剂量范围（＜0.93Gy）辐照下非常敏感，而在高剂量范围（＞ 0.93Gy）又表现出一定程度的辐射抗性。 结论： 1. 离子束辐照酵母细胞，直接或间接作用于酵母线粒体DNA，导致线粒体 DNA损伤，形成呼吸缺陷的酵母菌株。 2. I 类内含子和 II 类内含子对于离子束辐照的敏感性不同： II 类内含子比较 稳定，II 类内含子可能利用自身编码的反转录酶通过目的DNA引导的反 转录机制对受到辐照损伤的II 类内含子进行修复。 3. 离子束辐照后 SOF1 基因可能发生了突变，影响酵母细胞的生长。 4. 呼吸缺陷型酵母菌株因其线粒体 DNA发生变化及线粒体功能的改变， 使 呼吸缺陷型酵母菌株在不同剂量区的离子束辐照下表现不同辐射敏感 性。目的： 研究啤酒酵母的线粒体 DNA 在重离子辐照作用下的突变效应及其突变机 理。 材料与方法： 利用兰州重离子研究装置(HIRFL)加速的氖、碳离子辐照酵母细胞，用 TTC 显色培养基筛选呼吸缺陷型酵母菌株，并用 mtDNA 限制性酶切手段分析其突变 规律。采用 PCR扩增并对目的产物测序的方法对辐照后线粒体DNA上的 I 类内 含子和 II类内含子进行研究。 结果： 1. TTC 显色实验表明：离子束辐照导致酵母线粒体上的电子传递链发生改 变，产生的还原氢减少，造成呼吸缺陷。 2. 利用限制性酶切实验对线粒体 DNA进行研究，结果表明：离子束辐照诱 变筛选出来的呼吸缺陷型酵母菌株其线粒体DNA变化明显： 主要表现为 酶切条带缺失严重。即使在同一注量下筛选出来的呼吸缺陷型酵母菌株， 其酶切图谱也不相同。 3. 通过 PCR 手段对辐照后酵母线粒体 DNA 碱基序列进一步进行分析，发 现经不同注量离子束辐照后筛选出来的呼吸缺陷型酵母菌株，其I 类内含 子（ai4 and ai5）经设计不同引物进行扩增，没有获得目的条带，说明此 序列发生了突变，可能对离子束辐照比较敏感。 4. 经不同注量离子束辐照后筛选出来的呼吸缺陷型酵母菌株，其 II 类内含 子（ai2）的碱基序列与野生型相比没有变化，表现出在离子束辐照作用 下比较稳定的特性。

苯并[a]芘对赤子爱胜蚓线粒体编码基因表达影响研究

根据ISO11268-1:1993方法,完成了土壤中苯并[a]芘对赤子爱胜蚓(Eisenia fetida)的人工污染模拟实验.并采用Clontech PCR-SelectTMcDNASubtration Kit构建了污染组和对照组蚯蚓之间的抑制消减文库.经测序和比对分析,在苯并[a]芘诱导上调文库中发现5个线粒体DNA(mtDNA)编码的基因,且在上调文库中具有较高的频率.它们分别为:细胞色素c氧化酶亚基Ⅰ、Ⅱ、Ⅲ,NADH氢脱氢酶亚基1,ATP合成酶亚基.研究表明,土壤中苯并[a]芘污染改变了蚯蚓线粒体基因组的表达水平,线粒体基因组表达异常是多环芳烃污染胁迫的重要分子生物标记物.

PHYLOGEOGRAPHIC PATTERNS INDICATE TRANSATLANTIC MIGRATION FROM EUROPE TO NORTH AMERICA IN THE RED SEAWEED CHONDRUS CRISPUS (GIGARTINALES, RHODOPHYTA)1

Inferring how the Pleistocene climate oscillations have repopulated the extant population structure of Chondrus crispus Stackh. in the North Atlantic Ocean is important both for our understanding of the glacial episode promoting diversification and for the conservation and development of marine organisms. C. crispus is an ecologically and commercially important red seaweed with broad distributions in the North Atlantic. Here, we employed both partial mtDNA Cox1 and nrDNA internal transcribed spacer region 2 (ITS2) sequences to explore the genetic structure of 17 C. crispus populations from this area. Twenty-eight and 30 haplotypes were inferred from these two markers, respectively. Analysis of molecular variance (AMOVA) and of the population statistic Theta(ST) not only revealed significant genetic structure within C. crispus populations but also detected significant levels of genetic subdivision among and within populations in the North Atlantic. On the basis of high haplotype diversity and the presence of endemic haplotypes, we postulate that C. crispus had survived in Pleistocene glacial refugia in the northeast Atlantic, such as the English Channel and the northwestern Iberian Peninsula. We also hypothesize that C. crispus from the English Channel refugium repopulated most of northeastern Europe and recolonized northeastern North America in the Late Pleistocene. The observed phylogeographic pattern of C. crispus populations is in agreement with a scenario in which severe Quaternary glaciations influenced the genetic structure of North Atlantic marine organisms with contiguous population expansion and locally restricted gene flow coupled with a transatlantic dispersal in the Late Pleistocene.

Population genetic structure of crimson snapper Lutjanus erythropterus in East Asia, revealed by analysis of the mitochondrial control region

The population genetic structure of the crimson snapper Lutjanus erythropterus in East Asia was examined with a 427-bp hypervariable portion of the mtDNA control region. A total of 262 samples were collected and 75 haplotypes were obtained. Neutrality tests (Tajima's and Fu's) suggested that Lutjanus erythropterus in East Asia had experienced a bottleneck followed by population expansion since the late Pleistocene. Despite the low phylogeographic structures in mtDNA haplotypes, a hierarchical examination of populations in 11 localities from four geographical regions using analysis of molecular variance (AMOVA) indicated significant genetic differentiation among regions (Phi(CT) = 0.08564, p < 0.01). Limited gene flow between the eastern region (including a locality in the western Pacific Ocean and two localities in the East Sea) and three geographic regions of the South China Sea largely contributed to the genetic subdivision. However, comparisons among three geographic regions of the South China Sea showed little to no genetic difference. Populations of Lutjanus erythropterus in East Asia are inferred to be divided into two major groups: an eastern group, including populations of the western Pacific Ocean and the East Sea, and a South China Sea group, consisting of populations from northern Malaysia to South China. The results suggest that fishery management should reflect the genetic differentiation and diversity in East Asia. (c) 2006 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.

The complete mitochondrial genome of the large yellow croaker, Larimichthys crocea (Perciformes, Sciaenidae): Unusual features of its control region and the phylogenetic position of the Sciaenidae

To understand the systematic status of Larimichthys crocea in the Percoidei, we determined the complete mitochondrial (mt) genome sequence using 454 sequencing-by-synthesis technology. The complete mt genome is 16,466 bp in length including the typical structure of 22 tRNAs, 2 rRNAs, 13 protein-coding genes and the noncoding control region (CR). Further sequencing for the complete CR was performed using the primers Cyt b-F and 12S-R on six L crocea individuals and two L polyactis individuals. Interestingly, all seven CR sequences from L crocea were identical while the three sequences from L polyactis were distinct (including one from GenBank). Although the conserved blocks such as TAS and CSB-1, -2, and -3 are readily identifiable in the control regions of the two species, the typical central conserved blocks CSB-D, -E, and -F could not be detected, while they are found in Cynoscion acoupa of Sciaenidae and other Percoidei species. Phylogenetic analysis shows that L crocea is a relatively recently emerged species in Sciaenidae and this family is closely related to family Pomacanthidae within the Percoidei. L crocea, as the first species of Sciaenidae with complete mitochondrial genome available, will provide important information on the molecular evolution of the group. Moreover, the genus-specific pair of primers designed in this study for amplifying the complete mt control region will be very useful in studies on the population genetics and conservation biology of Larimichthys. (c) 2008 Elsevier B.V. All rights reserved.

Using the ribosomal internal transcribed spacer (ITS) as a complement marker for species identification of red macroalgae

Barcodes based on mitochondrial cytochrome oxidase (mtDNA CO1) sequences are being used for broad taxonomic groups of animals with demonstrated success in species identification and cryptic species discovery, but it has become clear that complementation by a nuclear marker system is necessary, in particular for the barcoding of plants. Here, we propose the nuclear internal transcribed spacer (ITS) as a potentially usable and complementary marker for species identification of red macroalgae, as well as present a primary workflow for species barcoding. Data show that for most red macroalgal genera (except members of the family Delesseriaceae), the size of ITS region ranges from 600 to 1200 bp, and contains enough variation to generate unique identifiers at either the species or genus levels. Consistent with previous studies, we found that the ITS sequence can resolve closely related species with the same fidelity as mtDNA CO1. Significantly, we confirmed that length polymorphism in the ITS region (including 5.8S rRNA gene) can be utilized as a character to discriminate red macroalgal species. As a complementary marker, the verifiable nuclear ITS region can speed routine identification and the detection of species, advance ecological and taxonomic inquiry, and permit rapid and accurate analysis of red macroalgae.

Broader pattern of tandem repeats in the mitochondrial control region of Perciformes

Perciformes, the largest order of vertebrates with 20 suborders, is the most diverse fish order that dominates vertebrate ocean life. The complete mitochondrial control region (CR) of Trichiurus japonicus (Trichiuridae, Scombroidei) and Pampus sp. (Stromateidae, Stromateoidei) were amplified and sequenced. Together with data from GenBank, the tandem repeats in the mitochondrial CR from 48 species, which covered nine suborders of Perciformes, are reported in this study. The tandem repeats tend to be long in the suborder Percoidei and Stromateoidei. The identical repeats in 21 species of Cichlidae suggest a common origin and have existed before species divergence. Larimichthys crocea shows tandem repeats instead of the typical structure of the central conserved sequence blocks, which was first reported in Perciformes and vertebrates. This might have resulted from interruption of the polymerase activity during the H-strand synthesis. The four broader patterns presented here for the tandem repeats, including those in both the 5' and 3' ends, only in the either 5' or 3' end, and in the central conserved domain of the control region, will be useful for understanding the evolution of species.