麻花艽和管花秦艽(龙胆科)之间自然杂交类型的分子验证

野外考察发现麻花艽(Gentiana stramineaMaxim.)和管花秦艽(G.siphonantha Maxim.ex Kusn.)同域分布时存在大量形态位于二者之间的个体。经形态变异研究后发现它们可能是这两个物种之间的杂交后代。对两个亲本种以及假设杂交群体共55个个体的核糖体ITS序列和叶绿体trnS-G序列的分析结果表明:中间形态个体是麻花艽和管花秦艽的自然杂交后代。此外分析了两个亲本种以及杂交群内个体间trnS-G和ITS序列的变异状况以及分子标记结果与形态鉴定不一致的可能原因;指出可能是杂交诱导的叶绿体基因组重组以及早期物种分化中的谱系筛选不彻底等原因造成了亲本种群体内序列变异的多样化。     

棉毛橐吾和穗序橐吾的自然杂交研究

在棉毛橐吾（Ligulariavellerea）和穗序橐吾（L．subspicata）同域分布的居群中存在形态特征介于两者之间的个体。经分析,这些个体有3种不同的形态类型,可能是这两个物种之间的杂交后代。为检验这一假设,对所采集的假设亲本和中间个体材料进行了核糖体内转录间隔区ITS4—5和叶绿体atpB．rbcL测序。假设杂交个体的ITS直接测序结果具明显的叠加性,之后的克隆测序也分离出两种与假设亲本种相同的序列类型．因此。这些假设杂交个体得到了证实。据母系遗传的叶绿体atpB—rbcL测序结果,穗序橐吾同是它们的质体贡献者,即虽然这些杂交个体具不同的形态特征,但都是以棉毛橐吾和穗序橐吾为亲本且以穗序橐吾为质体贡献者的杂交后代。     

管花秦艽的传粉生态学研究——兼与同域分布近缘种的比较

在青藏高原东北部连续两年观察了晚秋开花植物管花秦艽Gentiana siphonantha的传粉生态学特征,并在此基础上进一步比较分析了与该物种同域分布且亲缘关系较近、但开花较早的麻花艽G straminea之间的传粉生态学特征.管花秦艽的花发育过程表现出雌雄异熟和雌雄异位的特点,不存在花内的自花传粉,套袋隔离的花不结实也支持这一结论;株内自交的高结实率表明该物种是自交亲和的.盛花期每植株平均有15朵开放的花,雄性和雌性阶段的花比例为1.2:1;自然条件下产生种子必须依赖传粉媒介;苏氏熊蜂是最有效的传粉昆虫,且访花过程中埘雄性和雌性阶段花不具明显的偏向性;株内连续访花的频率高达87.8%,从而导致同株异花传粉自交的广泛存在.与同域分布的麻花艽相比,管花秦艽的单花花期、雄性和雌性期持续时间缩短.但盛花期开花数量明显增加.令人感兴趣的是尽管两个近缘种的花形态特征存在显著差异,但都是由同一种熊蜂传粉.这一特点与过去认为花颜色和花管长度是物种分化过程中与不同传粉昆虫协同进化导致牛殖隔离的假说不相符合.管花秦艽单花的访花频率和同株异花连续访花的比例都明显高于麻花艽.两个物种不同花序设计导致访花昆虫行为的改变可能是造成这一差异的主要原因.两个物种具有不同的开花时间,但仍然存在一定的花期重叠,表现出不完全的传粉生殖隔离状态.     

结合分子手段和形态分析验证大白杜鹃与马缨杜鹃的自然杂交

中国西南地区是世界杜鹃属(Rhododendron)植物的分布和分化中心,开展杜鹃属自然杂交研究可为分类修订和新品种选育提供依据。本文以贵州百里杜鹃自然保护区(百里保护区)和云南石宝山分布的大白杜鹃(Rhododendron decorum)、马缨杜鹃(R.delavayi)及两种间疑似自然杂交后代和人工杂交后代个体为研究材料,通过形态特征比较、核基因间隔序列ITS以及叶绿体基因片段trnL-trn F、rbcL和trnH-psb A测序特征分析,发现疑似自然杂交后代与人工杂交后代形态特征介于大白杜鹃与马缨杜鹃之间;4个DNA片段序列均能区分大白杜鹃和马缨杜鹃,同一物种的不同居群在叶绿体基因片段rbcL、trnH-psb A序列存在差异位点;所有人工杂交后代个体和1个石宝山疑似杂交个体均以大白杜鹃为母本,所有百里保护区疑似杂交个体和6个石宝山疑似杂交个体以马缨杜鹃为母本。本研究证实了大白杜鹃与马缨杜鹃在百里保护区和云南石宝山存在自然杂交现象,杂交为双向且自然杂交后代绝大多数以马缨杜鹃为母本。     

粗茎秦艽与西藏秦艽(龙胆科)种间的形态学、染色体与分子界定

粗茎秦艽Gentiana crassicaulis Duthie ex Burk和西藏秦艽G. tibetica King ex Hook. f.的主要区别是前者花冠小、檐部蓝色, 后者花大、檐部紫褐色; 但二者在西藏东南部地区的形态界限不清楚, 具有较多的中间形态过渡类型。本文以该地区居群作为主要研究对象, 并以云南和西藏其他地区的典型形态类群为对照, 比较研究了两个物种间的形态学、染色体和分子标记界定。共研究了10个居群69个个体的trnS-G和ITS序列、6个居群26株植株的染色体倍性和54份标本的形态变异。结果表明, 花冠大小与染色体倍性密切相关。云南等地分布的粗茎秦艽均为二倍体(2n=26), 花冠较小; 西藏西部的西藏秦艽均为四倍体(2n=52), 花冠较大。而西藏东南部的形态过渡类型也为四倍体, 花冠较大, 且其中的部分植株花颜色变浅, 类似西藏秦艽。但母系与双亲遗传背景的分子标记都表明西藏东南部居群与典型形态特征上的粗茎秦艽聚为一支, 应属于粗茎秦艽; ITS序列上也未发现杂合位点。这些分子证据都不支持近期杂交渗入造成形态过渡性的假设。综合分析表明多倍化造成了粗茎秦艽种内形态特征的变异, 其四倍体个体与四倍体西藏秦艽在形态特征上的趋同, 最终导致了二者在西藏东南部地区形态界限的模糊。但是, 分子证据可以很好地区分这种多倍化造成的形态趋同。我们的研究结果还表明, 综合利用分子、染色体和广义形态证据来解决近缘种之间种范围的界定具有更大的优势。此外, 还讨论了粗茎秦艽不同染色体倍性的分布以及该高山物种可能的起源与扩散途径。     

基于ITS序列和trnL-F序列探讨小叶锦鸡儿、中间锦鸡儿和柠条锦鸡儿的种间关系

中间锦鸡儿Caragana davazamcii Sancz.的分类处理一直存在争议, 它与小叶锦鸡儿C. microphylla Lam.和柠条锦鸡儿C. korshinskii Kom.的关系尚不清楚。该种被处理为一个独立的种或后两个种的变种。本文利用ITS序列和叶绿体trnL-F序列, 综合形态和地理分布, 探讨了中间锦鸡儿的起源及与另外两个种的种间关系。结果显示, 中间锦鸡儿的trnL-F序列与小叶锦鸡儿完全一致, 而与柠条锦鸡儿有明显差异。中间锦鸡儿的ITS序列高度纯合, 不支持该种可能是杂交起源的假设。相反, 另外两个种的ITS序列均出现多个位点杂合, 克隆后均得到2种不同的序列。中间锦鸡儿的ITS序列与小叶锦鸡儿2种序列中的1种完全一致。该结果表明, 中间锦鸡儿可能作为亲本之一参与了小叶锦鸡儿的杂交起源, 或者基因流是造成这两个种形态相似的主要原因。中间锦鸡儿与柠条锦鸡儿形态上的相似可能是趋同进化的表现。     

本地种云南柳与外来种旱柳（杨柳科）的同倍体自然杂交?

对分布于云南的旱柳( Salix matsudana)和云南柳( Salix cavaleriei)之间的一个自然杂交种进行了研究。野外观察表明疑似杂交种异蕊柳( Salix × heteromera)形态上介于旱柳和云南柳之间,并得到了基于叶形态特征的主成份分析的印证。核基因ITS序列数据表明这三个种存在ITS序列的种内和个体内的多态性,且疑似杂交种的ITS序列的基因型总是疑似亲本的嵌合体。因此可以判定异蕊柳是旱柳和云南柳的自然杂交后代。流式细胞分析表明这三个种均为四倍体,因而,本杂交事件为同倍体杂交。基于四个叶绿体序列片段的数据表明本自然杂交事件是不对称的,云南柳是异蕊柳的母本。常见外来种旱柳与稀有本地种云南柳的杂交可能导致稀有种云南柳的濒危甚至灭绝。研究表明柳属植物的引种应非常谨慎。     

本地种云南柳与外来种旱柳(杨柳科)的同倍体自然杂交(英文)

对分布于云南的旱柳(Salix matsudana)和云南柳(Salix cavaleriei)之间的一个自然杂交种进行了研究。野外观察表明疑似杂交种异蕊柳(Salix×heteromera)形态上介于旱柳和云南柳之间,并得到了基于叶形态特征的主成份分析的印证。核基因ITS序列数据表明这三个种存在ITS序列的种内和个体内的多态性,且疑似杂交种的ITS序列的基因型总是疑似亲本的嵌合体。因此可以判定异蕊柳是旱柳和云南柳的自然杂交后代。流式细胞分析表明这三个种均为四倍体,因而,本杂交事件为同倍体杂交。基于四个叶绿体序列片段的数据表明本自然杂交事件是不对称的,云南柳是异蕊柳的母本。常见外来种旱柳与稀有本地种云南柳的杂交可能导致稀有种云南柳的濒危甚至灭绝。研究表明柳属植物的引种应非常谨慎。     

窄叶鲜卑花(Sibiraea angustata)nrDNA ITS和cpDNA trnL-F序列分子进化特点的分析

应用PCR产物直接测序法分析了窄叶鲜卑花居群间nrDNA(核糖体DNA)ITS序列和cpDNA(叶绿体DNA)trnL-F的碱基差异,并与cpDNAtrnS-G序列和rpl20-rps12序列进行比较,从而初步研究两套植物基因组的变异速率。采用改良的CTAB法从硅胶干燥的窄叶鲜卑花叶片中提取总DNA,并对nrDNA ITS和cpDNAtrnL-F区域进行扩增、纯化、测序。nrDNA ITS序列共有601 bp,有变异位点3处,变异位点百分率为0.05%,(G+C)含量为41.4%。cpDNAtrnL-F序列共有927 bp,有变异位点1处,变异位点百分率0.01%,(G+C)含量为32.6%,两种序列的核苷酸多样性非常低。比较发现,窄叶鲜卑花nrDNA ITS区域较cpDNAtrnS-G序列和rpl20-rps12序列保守,变异速率较慢,比cpDNAtrnL-F序列变异速率稍快。通过对ITS序列单倍型(haplotype)进行分析发现,窄叶鲜卑花现有分布范围经历了居群近期范围扩张,与叶绿体基因组(trnS-G和rpl20-rps12序列)得出的结论一致。因此,窄叶鲜卑花nrDNA ITS序列适合该种的谱系地理学研究。     

野牡丹属两个海南特有种与同属广布种自然杂交的分子证据

自然杂交既可以在物种形成、遗传交换和适应性进化方面发挥重要作用,也可以造成稀有种的下降甚至灭绝以及超级入侵种的产生。涉及到稀有种的自然杂交研究可以为稀有种的保护提供重要的信息。紫毛野牡丹(Melastoma penicillatum)和枝毛野牡丹(M.dendrisetosum)是野牡丹属的两个海南特有种,其中枝毛野牡丹已处于濒危状态。我们的野外调查和形态观察发现,海南吊罗山存在紫毛野牡丹和野牡丹(M.candidum)以及枝毛野牡丹和毛菍(M.sanguineum)的嫌疑杂种个体。本研究测定并分析了这些嫌疑杂种及其嫌疑亲本的4个核基因和5个叶绿体基因间隔区序列,发现嫌疑杂种个体在嫌疑亲本物种的固定替换位点上均为嫌疑亲本物种碱基的杂合状态,单倍型网络分析表明所有嫌疑杂种个体在每个核基因上均具有来自两个嫌疑亲本的等位基因,证实了紫毛野牡丹和野牡丹,以及枝毛野牡丹和毛菍之间确实存在自然杂交。另外,相对于其他野牡丹属物种,枝毛野牡丹具有极低的遗传多样性。因为紫毛野牡丹和野牡丹、枝毛野牡丹和毛菍之间均存在很强的生态隔离,所以因公路修建而造成的生境干扰可能造成了这两个杂交事件的发生。因此避免生境干扰是保护这两个海南特有种最关键的手段。除此之外,在实验室条件下进行种子萌发或枝条扦插繁殖,然后将幼苗种植到合适生境中是扩大种群的另一个可行方法。     

Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morpho-logy always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermedi-ate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence compar-ison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) inter-genic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we exam-ined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species.     

Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morphology always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermediate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence comparison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) intergenic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we examined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species. __________ Translated from Acta Botanica Yunnanica, 2007, 29 (1): 91–97 [译自:云南植物研究]     

Pollination ecology of Gentiana siphonantha (Gentianaceae) and a further comparison with its sympatric congener species

In this study, we studied pollination ecology of Gentiana siphonantha (Gentianaceae), a late-autumn flowering alpine perennial in the northeastern Qinghai-Tibetan Plateau for two years for the first time. We also aimed to compare the pollination differentiation between this species and sympatrically distributed G. straminea that has a close phylogenetic origin but is flowering early. Flowers of G. siphonantha are characteristic of di-chogamy and herkogamy and this floral development prevents occurrence of autonomous self-pollination. This implication was confirmed by the breeding experiments, since no seed was produced when flowers were isolated. However, this gentian proved to be highly self-compatible when geitonogamous selfing was artificially induced. Each individual plant of this species has an average of 14.6 flowers ranging from 4 to 31, at both staminate and pistillate stages with a ratio of 1.2:1 in full anthesis. Both floral development and breeding experiments suggested that pollen vectors were indispensable for successful seed sets of this species. A great variety of insects were observed to visit this species, but the most common and only legitimate pollinator is Bumbus sushikini. A statistic observation suggested that this pollinator showed no preference to either staminate or pistillate flowers and visited them at random. We further calculated the frequency of their visits between and within individual plants. Among the pollinators’ bouts, the proportions of geitonogamous visits within an individual plant occupy about 87.8%. Such a case implied that geitonogamous selfing prevails in this species in spite of floral dichogamy and her-kogamy that were suggested to promote outcrossing. Compared with sympatric G. straminea, the total floral longevity and the male and female duration of this species are shorter. However, the number of flowers of each individual plant and branch increased when in full anthesis. It is interesting that both closely related species shared the same pollinator despite their distinct difference in flower morphology. This finding is inconsistent with the previous hypothesis that both flower color and corolla tube depth have coevolved with different pollinators during speciation and formation of reproductive isolation. Both visit frequencies of the individual flower and geitonoga-mous visits within the individual plant are higher in G. siphonantha than in G. straminea. This difference may result from their different inflorescence designs that actively act upon behaviors of pollinator. Although these two species differed in flowering phenology, their flowering stages overlapped for a few days, suggesting incomplete pollination isolation between them.     

Natural Hybrids between Ligularia vellerea and L.subspicata (Asteraceae: Senecioneae)

Three types of intermediate morphological individuals were found in the sympatric distribution areas of Ligularia vellerea and Lsubspicata. These intermediate individuals were hypothesized to be the hybrids of these two species based on the detailed comparison of their morphological characters. To test this hypothesis, we compared the DNA sequence of Lvellerea, Lsubspicata and the intermediate individuals by using the internal transcribed spacer region of nuclear ribosomal and atpB rbcL intergenic spacer region of the chloroplast. Direct sequencing of ITS clearly had additives and then cloned sequencing cloned out two kinds of sequences that were identical to those putative parental species, thus these putative hybrids were approved. With different morphological characters among three types, their plastid donors were all Lsubspicata according to the maternal sequences of atpB rbcL.     

甘草属种间杂交种叶绿体DNA父系遗传的发现及分析

通过对甘草属乌拉尔甘草(Glycyrrhiza uralensis)、光果甘草(G.glabra)、胀果甘草(G.inflata)及其人工杂交种组合G.uralensis♀×G.glabra♂、G.glabra♀×G.uralensis♂、G.uralensis♀×G.inflata♂、G.inflata♀×G.uralensis♂共68份材料的核基因ITS序列、叶绿体rbc L、mat K、trn H-psb A基因的序列分析,探讨了甘草属叶绿体DNA遗传方式。结果表明:(1)亲本种和人工杂交种ITS序列长度均为614 bp,其中34份人工杂交种ITS序列存在4处变异位点,且人工杂交种均检测出来自父本、母本ITS序列相同位点碱基的叠加,检测率为100%。(2)亲本种与人工杂交种的叶绿体基因rbc L、mat K、trn H-psb A序列长度相同,共有4处变异位点,人工杂交种在变异位点处的碱基与其相对应的父本碱基一致率高达97.1%。以上结果说明,该研究获得34份人工杂交种为100%杂交成功的F_1子代,核基因ITS序列可用于甘草属杂交种的遗传鉴定;甘草属叶绿体rbcL、mat K、trn H-psb A基因具有父系遗传特性,推测甘草属质体的遗传方式主要表现为父系遗传,这种质体遗传方式的发现为甘草属杂交种和遗传多样性研究提供了新的认识,也为杂交种的亲本鉴定提供分子依据。     

DNA marker based identification of spontaneous hybrids between Pinus mugo and P. sylvestris at the Lithuanian sea‐side

The objective was to identify spontaneous hybrids between P. mugo and P. sylvestris using organelle DNA markers in sympatric zones at the sea‐side spit of Kursiu Nerija in western Lithuania. A field inventory was carried out over the entire Lithuanian part of the spit and 203 individuals morphologically intermediate between P. sylvestris and P. mugo were tested for their male parent with chloroplast DNA PCR‐RFLP markers and for their female parent with mitochondrial DNA PCR markers. Unfortunately, the mitochondrial DNA Nad7 marker failed to identify the female parent species in our study. However, the chloroplast DNA PCR‐RFLP marker revealed that out of 203 tested individuals only 23 had a paternity different than that indicated by morphology. Of these, 13 individuals were morphologically identified as P. sylvestris but possessed cpDNA of P. mugo (putative hybrids with P. sylvestris (female) ×P. mugo (male parent), and 10 individuals morphologically identified as P. mugo possessed cpDNA of P. sylvestris and may be hybrids with P. mugo (female) ×P. sylvestris (male parent). The remainder of the 177 individuals identified in the field inventory were were considered as pure species. In conclusion, our study indicates ongoing spontaneous hybridization between P. mugo and P. syvestris in Kursiu Nerija. Human impact via seed transfer on altered hybridization rates as well as evolutionary consequences are discussed.