松科nrDNA 的进化研究Evolution of nuclear ribosomal DNA in Pinaceae

核核糖体DNA（nrDNA）已被作为一个重要的标记，用于推断很多分类等级上的系统发育关系。相对于在被子植物中的快速致同进化，nrDNA在裸子植物中的致同进化速率低，且ITS和5S-NTS区有着较大的长度变异，这种现象在松科植物中尤为明显。在本研究中，我们克隆并测定了银杉属的5S rDNA以及冷杉属、银杉属、雪松属、油杉属、长苞铁杉属、金钱松属与铁杉属的ITS序列。基于获得的新数据，再结合前人报导的其它属的数据，我们探讨了如下四个问题： （1）松科 nrDNA ITS1 亚重复单位的组成、分布及进化;（2）ITS1区的长度变异与亚重复单位数目的关系以及它们的系统学意义;（3）松科ITS1的二级结构特征;（4）银杉5S rDNA编码区及非转录间隔区的结构特征。主要研究结果如下： 1. ITS区的序列分析ITS区的克隆及序列分析发现：（1） 松科ITS1的长度变异范围为 944-3271 bp, 这是目前已报导的真核生物中属间ITS变异最大的类群之一;（2） 所有松科植物的ITS区域都包含亚重复单位，亚重复单位的数目从2到9，并且这些亚重复单位可分为两种类型，即不含保守核心序列（5’-GGCCACCCTAGTC ） 的长亚重复单位（LSR）和含上述保守核心序列的短亚重复单位（SSR）;（3） ITS1区的巨大长度变异主要归因于亚重复单位的数量变异; （4） ITS1区的GC含量与 它的序列长度和亚重复单位的数目有一定关系，并能够提供一些系统发育信息，特别是支持云杉属、松属和银杉属三者具有很近的亲缘关系。 2. ITS1亚重复单位的系统发育分析为了研究亚重复单位的进化关系，我们用最大似然法和最大简约法构建了松科ITS1亚重复单位的系统发育树。结果表明：（1）在ML和MP树中可发现有共同的五个分支; （2） 银杉比松科其它属拥有更多的SSR，且该属的所有9个SSR在系统树中构成一个单系支，表明它们是在银杉属内发生重复的;（3）一些SSR在属间和种间具有同源性，可为nrDNA ITS 的进化历史以及松科的系统发育 研究提供重要信息;（4）亚重复单位的多次重复以及伴随的重组可能是导致LSR 和SSR在松科不同属中分布式样不同的原因。 3. 松科ITS1的二级结构 用 Mfold 3.2 软件对松科所有11个属的ITS1区进行了二级结构预测，共获得了563个最低自由能折叠。结合以前关于松科二级结构的报导，我们分析的结果表明：（1） 松科ITS1的二级结构主要由几个延展的发夹结构组成;（2） 构象的复杂性与亚重复的数目呈正相关;（3）配对的亚重复单位通常在保守核心区（5’-GGCCACCCTAGTC ） 处有部分重叠，并且构成一个长茎，而其它的亚重复单位通常会自身折叠，且保守核心区的部分出现在发夹结构的环中。 4. 银杉5S rDNA 序列分析 我们对来自银杉不同群体的3个个体的5S rDNA进行了克隆，共获得 45 条序列，分析结果表明：（1） 绝大多数银杉5S rDNA编码区长度为120 bp, 以GGG 开头，以CTC结尾，编码区出现的碱基替代主要为转换;（2） 银杉与其它裸子植物相比，5S rDNA基因编码区具很高的相似性（90-99%）; （3）间隔区含有一个poly-C和一个poly-T结构、两个TC丰富区以及五个GC丰富区。根据长度和序列特征，银杉的5S rDNA间隔区可分为三种类型：Type A 长751-764 bp，Type B 长770-807 bp （含一个32 bp的插入），Type C 长581-594 bp; （5）长间隔区（Type A，Type B ）中含有两个148-175 bp的串联亚重复单位，该亚重复单位与短间隔区（Type C ）中的一段143 bp的序列具有较高的相似性（56.0-66.8%）。 5. 银杉5S rRNA的二级结构 Mfold 3.2 预测结果表明：（1）银杉5S rRNA二级结构包括5个双螺旋区（干区）（Ⅰ-Ⅴ）、2个发夹结构环区（C和D）、3个中间环区（B1、B2 和 E）和1个铰链区（A）, 铰链区为三个双螺旋的结合处;（2） 二级结构中的环区通常比双螺旋区更加保守;（3）在5个双螺旋中，I 和 IV 区有较高的碱基替代率。

DEVELOPMENT OF MICROSATELLITE LOCI FOR PINUS KORAIENSIS (PINACEAE)

Premise of the study: Microsatellite markers were developed for Pinus koraiensis to characterize its genetic diversity and understand its population structure. Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining (FIASCO) Repeats protocol, 20 primer sets were developed in Chinese populations of P. koraiensis. Three of the markers showed polymorphism with two alleles per locus when assessed in a sample of two populations of P. koraiensis from the Changbai Mountain in the Jilin Province of China. Five and three loci were successfully amplified in P. taiwanensis and P. massoniana, respectively. The amplification size of these loci matches those in P. koraiensis. Conclusions: These markers may be useful for further investigation of population genetics of P. koraiensis.

Phylogeography of Pinus tabulaeformis Carr. (Pinaceae), a dominant species of coniferous forest in northern China

How coniferous trees in northern China changed their distribution ranges in response to Quaternary climatic oscillations remains largely unknown. Here we report a study of the phylogeography of Pinus tabulaeformis, an endemic and dominant species of coniferous forest in northern China. We examined sequence variation of maternally inherited, seed-dispersed mitochondrial DNA (mtDNA) (nad5 intron 1 and nad4/3-4) and paternally inherited, pollen- and seed-dispersed chloroplast DNA (cpDNA) (rpl16 and trnS-trnG) within and among 30 natural populations across the entire range of the species. Six mitotypes and five chlorotypes were recovered among 291 trees surveyed. Population divergence was high for mtDNA variation (G(ST) = 0.738, N-ST = 0.771) indicating low levels of seed-based gene flow and significant phylogeographical structure (N-ST > G(ST), P < 0.05). The spatial distribution of mitotypes suggests that five distinct population groups exist in the species: one in the west comprising seven populations, a second with a north-central distribution comprising 15 populations, a third with a southern and easterly distribution comprising five populations, a fourth comprising one central and one western population, and a fifth comprising a single population located in the north-central part of the species' range. Each group apart from the fourth group is characterized by a distinct mitotype, with other mitotypes, if present, occurring at low frequency. It is suggested, therefore, that most members of each group apart from Group 4 are derived from ancestors that occupied different isolated refugia in a previous period of range fragmentation of the species, possibly at the time of the Last Glacial Maximum. Possible locations for these refugia are suggested. A comparison of mitotype diversity between northern and southern subgroups within the north-central group of populations (Group 2) showed much greater uniformity in the northern part of the range both within and between populations. This could indicate a northward migration of the species from a southern refugium in this region during the postglacial period, although alternative explanations cannot be ruled out. Two chlorotypes were distributed across the geographical range of the species, resulting in lower levels of among-population chlorotype variation. The geographical pattern of variation for all five chlorotypes provided some indication of the species surviving past glaciations in more than one refugium, although differentiation was much less marked, presumably due to the greater dispersal of cpDNA via pollen.

Mitochondrial and chloroplast phylogeography of Picea crassifolia Kom. (Pinaceae) in the Qinghai-Tibetan Plateau and adjacent highlands

The disjunct distribution of forests in the Qinghai-Tibetan Plateau (QTP) and adjacent Helan Shan and Daqing Shan highlands provides an excellent model to examine vegetation shifts, glacial refugia and gene flow of key species in this complex landscape region in response to past climatic oscillations and human disturbance. In this study, we examined maternally inherited mitochondrial DNA (nad1 intron b/c and nad5 intron 1) and paternally inherited chloroplast DNA (trnC-trnD) sequence variation within a dominant forest species, Picea crassifolia Kom. We recovered nine mitotypes and two chlorotypes in a survey of 442 individuals from 32 populations sampled throughout the species' range. Significant mitochondrial DNA population subdivision was detected (G(ST) = 0.512; N-ST = 0.679), suggesting low levels of recurrent gene flow through seeds among populations and significant phylogeographical structure (N-ST > GST, P < 0.05). Plateau haplotypes differed in sequence from those in the adjacent highlands, suggesting a long period of allopatric fragmentation between the species in the two regions and the presence of independent refugia in each region during Quaternary glaciations. On the QTP platform, all but one of the disjunct populations surveyed were fixed for the same mitotype, while most populations at the plateau edge contained more than one haplotype with the mitotype that was fixed in plateau platform populations always present at high frequency. This distribution pattern suggests that present-day disjunct populations on the QTP platform experienced a common recolonization history. The same phylogeographical pattern, however, was not detected for paternally inherited chloroplast DNA haplotypes. Two chlorotypes were distributed throughout the range of the species with little geographical population differentiation (G(ST) = N-ST = 0.093). This provides evidence for highly efficient pollen-mediated gene flow among isolated forest patches, both within and between the QTP and adjacent highland populations. A lack of isolation to pollen-mediated gene flow between forests on the QTP and adjacent highlands is surprising given that the Tengger Desert has been a geographical barrier between these two regions for approximately the last 1.8 million years.

辽宁省抚须石新世木化石群的初步研究

我国古新世植物化石发现较少，因而目前对古新世的植被面貌;生态环境等植物学;植物地理学和地质学方面的认识尚感不足。抚顺古新过木化石群的研究提供了这方面的新资料无疑在植物学;质植物地理学和地质学等方面都有一定意义。 本研究表明，抚顺古新世木化石群皆由松柏目Pinales裸子植物组成，共有三科七属七种和三个未定种。其中杉科Taxodiaceae三属三种和一个未定种，松科Pinaceae和柏科Cupressaceae各二属二种和一个未定种。共有四个新种。本化石群的地质时代被确定为古新世。通过对此化石植物群的研究，作者认为右新世时抚顺地区存在着以杉和柏科植物为主体的北亚热带一北温暖带边缘地带暖性针叶林。根据孢粉资料(木化石未见)同时还有繁盛的被子植物存在。当时当地的生态环境类似我国现今长江流域南部偏北地带，如川鄂;江浙一带的山地，系属亚热带山区气候。 此外，根据新的化石资料，本文还对扁柏型木属Chamaecyparioxylon做了补充修正，报导了木化石中真菌菌丝的发现。它被初步鉴定为藻状菌纲毛霉目 Mucorales真菌。

裸子植物的分类和系统发育研究—松科的分类、地理和种系发生研究，中国红豆杉科、三尖杉科、南洋杉科和银杏科的分类学研究

球果类植物是现存裸子植物中最为繁盛的一群，也是目前组成北温带森林植被的重要成分，我国是球果类植物科属的重要分布、分化或保存中心。对这些科属的分类和系统发育的研究，无疑对北温带的森林植被的研究，以及林业生产均具有重要的理论和实用价值．本研究即是围绕着球果类中的Pinaceae，Taxaceae及Cephalotaxaceae等科展开的研究，内容主要包括以下几方面： 一、在《中国植物志》第七卷的基础上，完成了《FLORA OF CHINA》中裸子植物Ginkgoaceae，Arancariaceae Pinaceae，Taxaceae，和Cephalotaxaceae各科的修订和编写． 建立2个新种、2个新变种、12个新组合、64个新异名、2个新分布，2个新启用名，分类群及其名称的变动和增补，以及增补异名57个。 二、根据植物的地理分布与系统发育统一的原理，在资料分析和实验的基础上，运用表征分析和分支分析研究方法，同时结合化石和地史资料，针对松科的分类、地理分布、以及起源分化和扩散等问题进行了一系列系统的研究，主要结论如下： 1．提出松科分类的新系统，将松科划分为三个亚科：松亚科Subfam．Pinoideae:仅一属Pinus Linn．： 落叶松亚科Subfam．Laricoideae Melchior＆Werderm, emend．N．Li et L.K．Fu：包括四属Larix Mill.，Cathaya Chun&Ku:tng, Picea A Dretr. andPseudotsuga Carr．;冷杉亚科Subfam．Abietoideae Pilger, emend．Frankis:包括五属Abies Mill．，Cedrus Trew, Pseudolarix Gord．，Keteleeria Carr.,and Tsuga Carr． 2．构思和绘出了以地质年代为纵座标的松科系统发育树。 3．进行松科植物分区，将松科划分为6区、4亚区。并分别附有分区图、各区所分布种数的统计表、以及各属目前的地理分布和化石分布图。 4．松科在地质时期曾是一个很庞大的类群，现代松科只是其祖先中少部分喜温性分支的后裔，其兴旺与温带成份的出现关系密切，可能是一类衍生类群． 5．松科的起源时间虽然可追溯到侏罗纪甚至三迭纪，但现代松科各属的出现时间大约是在早白垩纪至第三纪之间。 6．松科各属可能并非起源于同一时间和地点。松属可能是现存的、最早分化出来的类群，于侏罗纪至早白垩纪间起源于欧美古陆;其它属则到晚白垩纪至第三纪早期（有1-2属至中期）才陆续从其祖先复合体中分化出来。 7．现代松科的早期扩散可能存在着以下三条主要的迁陡路线，即：欧美路线、欧亚路线和古白令路线．