Taxonomic problems in the Drosophila melanica species group (Diptera : Drosophilidae) from southern China, with special reference to karyotypes and reproductive isolation

Karyotypes and reproductive isolation were studied in two allopatric populations of Drosophila tsigana, one from Guizhou Province in southern China and the other from Hokkaido in northern Japan, and in one population of a closely related species, D. longi

四川黄龙杓兰属植物的传粉生物学

兰科植物传粉生物学的研究以往多集中于单个物种上，很少对两个以上的物种同时进行研究。但后一类研究对于理解一个地区或一个代表类群的传粉适应是大有裨益和非常必要的，毕竟单一种植物与其传粉者在一个居群或是一年中的相互关系所提供的有效信息是非常有限的。杓兰属Cypripedium L.是兰科植物中比较原始的类群，全世界约有50种;中国是杓兰属植物的分布中心，有30多种。但是，有关该属植物的传粉生物学研究集中在欧、美的种类，中国绝大部分物种尚未进行这方面的研究。本文通过对分布于四川省黄龙寺自然保护区的8种杓兰属植物的传粉生物学研究，探讨了该属植物的传粉机制、适应进化及生殖隔离等问题。 1． 杓兰属植物的繁育系统 虽然所研究的8种杓兰人工自交授粉均可以成功结实，但在自然条件下都必须依赖于昆虫才能结实成功，表明杓兰属植物的繁殖系统以异交授粉为主。 2． 杓兰属植物的传粉系统以及传粉系统的进化趋势 杓兰属植物一向被认为是典型的蜂类传粉物种，本文所包括的西藏杓兰C. tibeticum King ex Rolfe、离萼杓兰C. plectrochilum Franch.、绿花杓兰C. henryi Rolfe与褐花杓兰C. smithii Schltr.的传粉生物学研究也证明了这一点。但研究发现最进化的“无苞组”的3种杓兰，即无苞杓兰C. bardolphianum W. W. Smith et Farrer、小花杓兰C. micranthum Franch.与四川杓兰C. sichuanense Perner都是由蝇类传粉的，而黄花杓兰C. flavum P. F. Hunt et Summerh.则可由蜂类和蝇类共同传粉。结合杓兰的种间系统关系，本文认为杓兰属中存在从蜂类传粉系统向蝇类传粉系统的进化趋势。 3．杓兰属植物传粉系统的特化机制 传粉观察表明8种杓兰均有多种多样的访花昆虫，但只有1种或1类具有相同功能的昆虫能成为其传粉者。这说明杓兰属植物是具备特化传粉系统的种类。以离萼杓兰为例进行的花色、花香及花结构的分析表明，杓兰拥有特化的传粉者几乎完全是由于受到花结构的限制，特别是雄蕊到唇瓣底高度（AL）、柱头到唇瓣底高度（SL）、唇瓣入口直径（DL）与唇瓣出口宽度（EL）的大小。这些因素决定了昆虫是否能进入唇瓣，是否能碰触到柱头和花粉，是否能从出口挤出来。因此，杓兰的唇瓣的主要功能不仅是象原来所认为的作为“陷阱”来诱捕昆虫，而且同样作为一种促进产生“特化传粉”的机制而存在。 4． 杓兰属植物吸引昆虫的机制 杓兰属植物具有复杂的吸引昆虫的机制。离萼杓兰、黄花杓兰主要以泛化的食源性欺骗机制来吸引昆虫，绿花杓兰能通过其唇瓣和退化雄蕊的光滑特性诱使其传粉昆虫被动进入唇瓣中，西藏杓兰可以通过“筑巢式欺骗”来吸引昆虫，无苞杓兰则可通过模拟成熟果实来吸引其特化的传粉者—果蝇Drosophila spp.。 5． 杓兰属植物的花部特征与传粉系统的适应 在整个杓兰属内，不同种类植物的花色与花香和传粉者种类间没有统一的规律。但是，杓兰属植物的唇瓣大小与其传粉者大小之间存在比较明显的适应关系。体积最大的西藏杓兰、褐花杓兰与黄花杓兰由体型最大的熊蜂Bombus spp.传粉，体积中等的离萼杓兰、绿花杓兰、四川杓兰由中等大小的蜂或蝇传粉，而体积最小的无苞杓兰与小花杓兰由体型很小的果蝇传粉。 在杓兰属中，大部分种类的花粉只是粘性的团状，只有一部分能在一次访问中被昆虫带出，如黄花杓兰、西藏杓兰、离萼杓兰及绿花杓兰的花粉团。与此不同，在2个“无苞组”的杓兰，即无苞杓兰、小花杓兰中，它们的花粉凝聚成块状，而且它们的传粉昆虫(果蝇)的一次访问可带出一侧雄蕊的全部花粉。它们的花粉成块可能是对果蝇这类小昆虫传粉的一种适应。 6． 杓兰属植物的生殖隔离机制 本文的研究表明，杓兰属植物之间人工杂交授粉可以成功结实，它们主要是通过受精前隔离机制保持物种界限的。它们的受精前隔离机制多种多样。具有相同传粉者—果蝇的无苞杓兰与小花杓兰通过地理隔离机制保持物种界限; 同域的西藏杓兰与黄花杓兰通过利用不同大小的熊蜂作为传粉者来保持生殖隔离;同域的离萼杓兰与绿花杓兰可能通过花香成分的不同特化吸引同一属中不同种的传粉昆虫;而同域的西藏杓兰与褐花杓兰之间并不具备完善的生殖隔离机制。

广布小蝶兰及两种根茎兰属植物的传粉生物学研究

植物与传粉者的关系并非一门独立的学科，它包含很多进化生物学的内容。传粉生物学研究为我们理解植物进化的一些关键问题，如生殖隔离、物种形成、适应进化等提供重要证据。兰科植物由于其精巧的花部结构及其独特的传粉机制，备受传粉生物学家的关注。从达尔文时代以来，有大量的文献记载了欧洲兰族植物的传粉生物学的各个方面，其中，共涉及4种欺骗性传粉方式，它们几乎代表了兰科植物中近1/3没有报偿兰花所有的欺骗类型。然而，到目前为止，尽管在中国有丰富的兰族植物资源，中国兰科植物的传粉生物学研究仍然很缺乏。 在本研究中，作者以黄龙自然保护区内兰族的2种根茎兰属植物和1种小蝶兰属植物为材料，研究它们的传粉生物学，并探讨他们之间的生殖隔离机制。 1． 根茎兰属Galearis 及小蝶兰属Ponerorchis植物的交配系统 所研究的2种根茎兰属植物和1种小蝶兰属植物人工自交授粉均可以成功结实。在自然条件下，广布小蝶兰和二叶根茎兰都必须依赖于昆虫才能结实成功，而黄龙根茎兰则是以自花授粉为主。 2． 广布小蝶兰Ponerorchis chusua的传粉系统 在黄龙自然保护区进行了连续2 年的野外观测和实验，结果表明，广布小蝶兰是一种自交亲和，但需要昆虫传粉的欺骗性植物。广布小蝶兰的主要传粉者为雌性Bombus rufofasciatus，雌性B. consobrinus wittenburg，B. cucorun的蜂王和雄性B. pyrosona。熊蜂对广布小蝶兰具有典型的欺骗性兰花传粉的特征，即访问十分迅速，并且一般一个植株只访问一朵花，避免了同株异花授粉。 3． 二叶根茎兰Galearis diantha的传粉系统 野外观察表明，二叶根茎兰只有一种有效的传粉者，其访问频率不高，访 问时间短，并且会一次访问一个植株的两朵花，不可避免的产生同株异花授粉。自然结实率为23.0－26.8％，人工授粉实验证明传粉者限制是结实率较低的主要原因。 4. 黄龙根茎兰Galearis huanglongensis的传粉系统 研究结果表明，黄龙根茎兰为自动自花授粉的植物，黄龙根茎兰的花粉约在花开放4天后，花粉块从花粉囊中掉出，由于重力作用，花粉块柄弯曲使花粉接触到柱头，完成传粉。 5. 根茎兰属Galearis及小蝶兰属Ponerorchis植物的生殖隔离机制 本文的研究表明，根茎兰属内的杂交及与小蝶兰属间植物之间人工杂交授粉均可以成功结实，它们物种之间的生殖隔离主要是通过受精前隔离机制。具有相同传粉者（B. rufofasciatus）的广布小蝶兰与二叶根茎兰通过机械隔离机制保持物种界限。

高山松（Pinus densata）同倍性杂交物种形成的生态遗传学研究

种间杂交在自然界中普遍存在。杂交可以为群体带来多方面的遗传改变，具有重要的进化生物学意义。越来越多的证据表明，同倍性杂交物种形成是一种重要物种形成途径，在动、植物物种进化中扮演着重要的角色。同倍性杂交物种形成意味着一个稳定的、可育的、与亲本种间形成生殖隔离的杂种群体建立起来，但不改变染色体的数目。这种生殖隔离被认为是通过快速的染色体重组、生态分化、或空间分离形成的。对于现存杂交种的对比分析和对同倍性杂交物种形成的理论模拟都表明，适应性分化和生态转变在同倍性杂交物种形成中起着至关重要的作用。来自于生态成种的研究也表明生态选择自身就可以导致生殖隔离的形成和新物种的形成。尽管如此，我们对同倍性杂交物种形成过程的研究仍然较多地关注于染色体重组及相应的遗传效应；对于自然选择，特别是生态选择在同倍性杂交成种过程中的作用模式缺乏全面地了解。 本研究以同倍性杂交种－高山松为研究对象，利用地理信息学技术、基于天然群体取样的性状对比分析和人工杂交手段，全面地定量描述了高山松及其亲本种间的生态分化、生态转变下的天然群体生殖适应性、以及生态选择下的形态变异和种间杂交可配性。以此探讨了高山松的杂交成种过程，生态转变和自然选择的重要性以及它们与杂交种适应性、形态变异和生殖隔离之间的关系。利用地理信息学手段，对高山松及其亲本种间的生态分化对比分析表明：高山松与其亲本种间形成了明显的生态转变；在自然选择的作用下，高山松已经具备了对于青藏高原东南缘横断山区高山地带独特的生态适应性；相对于亲本种，它的适应性特征体现为混合型，在某些性状上它高出亲本种的适应性，在另一些性状上，它体现出居中、较低或者类似于亲本种适应性；高山松适生区由于地形地貌的复杂性，往往不连续，呈间断状。基于四个反映结实效率的性状、小孢子母细胞减数分裂异常现象及花粉萌发率在种间的对比分析表明，这些反映生殖状态的指标，在三个种及群体间并没有显著差异；相对于亲本种的天然群体，居于青藏高原的高山松群体，表现出了正常的授粉效率和生殖适应性。高山松天然群体正常的生殖效率表明，它经历了生态转变过程中的选择作用，已经具备了在新生境下，繁殖发展下去的生殖适应性。 在大规模群体取样的基础上，通过对反映植物个体发育和营养状态的球果和针叶形态性状的对比分析，我们发现，高山松及其亲本种间的形态变异存在以下特征：第一，绝大多数性状，在种间和种内群体间都存在着显著的差异，并且大部分性状的差异主要体现在种间；第二，高山松在大部分形态性状上介于两个亲本种之间，少数性状表现为超亲分离、类似于某个亲本或无种间差别；第三，在多数性状上，和亲本种相比，高山松群体都拥有较大的变异幅度。第四，在临近亲本种的群体中，高山松的形态特征更类似于所临近的亲本种。上述形态变异特征及其与气象、土壤、经纬度、海拔等因子间普遍的相关性，意味着生态选择在高山松成种过程中起了重要作用。总的来说，形态性状变异的总体模式揭示出：高山松在选择作用下的遗传和表型的精炼过程中已经形成了自身适应于高原环境的、稳定而有效的形态发育模式和资源利用方式，甚至是恰当的防御机制。 本研究通过云南松×油松的单交实验和油松×高山松部分双列杂交实验，检验了高山松及其亲本种间的杂交可配性。结果表明，就平均水平而言，高山松及其亲本种间的种间杂交结实可配性低于种内杂交，但个别种间杂交组合的结实可配性可能高于种间杂交；高山松及其亲本种间正在或已经形成了部分的合子后生殖障碍；现存的高山松群体及其亲本种间的生态地理隔离可能是很有效的生殖隔离形式，一方面做为合子前生殖隔离，它能避免种间相互传粉、阻碍种间杂交的发生，另一方面做为合子后生殖隔离，它能清除掉可能存在的劣势回交产物。 综合已有的证据，我们提出了高山松同倍性杂交物种形成模型：高山松的同倍性杂交成种是与青藏高原隆升过程以及隆升过程中所产生的全新生态空间紧密相关的；青藏高原隆升导致了生态环境的剧变，创造了全新的生态位，在选择的作用下，拥有综合了双亲遗传基础的杂种基因型开始占领这个全新的生态位；在分化的生态选择的持续作用下，杂种基因型在新的生境下稳定下来，形成了独立于亲本种的资源利用方式、形态特征和繁育系统，具备了完善的生殖和生态适应性；此时，一个与亲本种间以生态隔离存在的同倍性杂交种－高山松形成了；随着分化的生态选择的持续进行,高山松与亲本种间的内在生殖隔离不断加强。

The youngest split in sympatric schizothoracine fish (Cyprinidae) is shaped by ecological adaptations in a Tibetan Plateau glacier lake

Although new empirical evidence shows that sympatric speciation has occurred in some species, there are few indisputable model organisms for this process of speciation. The two subspecies (Gymnocypris eckloni eckloni and G. e. scoliostomus) of the schizothoracine Gymnocypris fish species complex from a small glacier lake in the Tibetan Plateau, Lake Sunmcuo, fit several of the key characteristics of the sympatric speciation model. We used combined mitochondrial control region sequences and the cytochrome b gene (1894 bp) to address the phylogenetics and population genetics of 232 specimens of G. e. eckloni and G. e. scoliostomus, as well as all of its closely related sister species. We found that: (i) a total of four old lineages were uncovered in the widespread G. e. eckloni, of which only one was shown to be shared with all G. e. scoliostomus individuals and (ii) the new subspecies (G. e. scoliostomus) evolved in Lake Sunmcuo from the ancestral G. e. eckloni population within approximately 0.057 Ma. These two taxa of the species complex are morphologically distinct, and reproductive isolation is further suggested. Ecological disruptive selection based on morphological traits (e.g. mouth cleft characters) and food utilization may be a mechanism of incipient speciation of two sympatric populations within Lake Sunmcuo. This study provides the first genetic evidence for sympatric speciation in the schizothoracine fish.

Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca)

Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of Crassostrea: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)-the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)-the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of Crassostrea. This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.

LABORATORY HYBRIDIZATION BETWEEN CRASSOSTREA ARIAKENSIS AND C. SIKAMEA

TO understand possible reproductive interaction between Crassostrea ariakensis (Fujita, 1913) and C. sikamea (Amemiya, 1928), which coexist ill estuaries of China and Japan, we conducted 2 X 2 factorial crosses between the two species. Asymmetry in fertilization success was observed where C. sikamea eggs can be fertilized be C. ariakensis the receprocal cross resulted in no fertilization. Fertilization Success ill C.sikamea female X C. ariakemvis male (SA) crosses was lower than that in the two intraspecific crosses and produced larvae that had similar growth the rate as their maternal species during the first nine days because of maternal effects. After that, genome incompatibility casted negative effects on the growth and survival of the hybrid larvae. Most hybrid larvae died during metamorphosis. but a small number of spat survived. Genetic analysis revealed that the survived SA spat contained DNA from both species and were the hybried. This study demonstrates that hybridization between C. ariakensis and C. sikamea is possible in one direction.

Molecular evidence for double maternal origins of the diploid hybrid Hippophae goniocarpa (Elaeagnaceae)

Homoploid hybrid plant species are rare, and the mechanisms of their speciation are largely unknown, especially for homoploid hybrid tree species. Two contrasting hypotheses have been proposed to explain the origin of Hippophae goniocarpa: (1) it is a diploid hybrid originating from H. rhamnoides ssp. sinensis x H. neurocarpa ssp. neurocarpa, and (2) it originated via marginal differentiation from H. rhamnoides ssp. sinensis. Regardless of which of these hypotheses is true (if either), previous studies have suggested that H. rhamnoides ssp. sinensis is the only maternal donor for this hybrid species. In this study, we aim to elucidate the maternal composition of H. goniocarpa and to test the two hypotheses. For this purpose, we sequenced the maternal chloroplast DNA trnL-F region of 75 individuals representing H. goniocarpa, H. rhamnoides ssp. sinensis, and H. neurocarpa ssp. neurocarpa in two co-occurring sites of the taxa. Seven haplotypes were identified from three taxonomic units, and their phylogenetic relationships were further constructed by means of maximum parsimony, maximum likelihood, and network analyses. These seven haplotypes clustered into two distinct, highly divergent lineages. Two haplotypes from one lineage were found in H. rhamnoides ssp. sinensis, and five (representing the other lineage) in H. neurocarpa ssp. neurocarpa. Hippophae goniocarpa shared four common haplotypes from both lineages, but the haplotypes detected from the two populations differed to some extent, and in each case were identical to local haplotypes of the putative parental species. Thus, both H. rhamnoides ssp. sinensis and H. neurocarpa ssp. neurocarpa appear to have together contributed to the maternal establishment of H. goniocarpa. These results clearly demonstrate that the marginal origin hypothesis should be rejected, and support the hybrid origin hypothesis. Hippophae goniocarpa exhibits a sympatric distribution with its two parent species, without occupying new niches or displaying complete ecological isolation. However, this species has effectively developed reproductive isolation from its sympatric parent species. Our preliminary results suggest that H. goniocarpa may provide a useful model system for studying diploid hybrid speciation in trees. (c) 2008 The Linnean Society of London.

Isolation and characterization of polymorphic microsatellites in a sex-reversal fish, rice field eel (Monopterus albus)

The rice field eel (Monopterus albus) is a fish of economic importance in China and some Asian countries. From a (GT)(n)-enriched genomic library, 30 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Thirteen loci exhibited polymorphism with two to 13 alleles (mean 7.9 alleles/locus) in a test population and observed heterozygosity ranged from 0.3125 to 0.9688 (mean 0.7140). These loci should provide sufficient level of genetic variation to study the fine-scale population structure and reproductive ecology of the species.