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。     

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 straminea Maxim. ) 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。     

粗茎秦艽种子粗提物与破眠方法研究

为了探究影响粗茎秦艽（Gentiana crassicaulis Duthie ex Burk.）种子休眠的因素,破除休眠,寻找其种子快速萌发的方法,以干燥的粗茎秦艽种子为材料,测定种子吸水率及粗提物的活性,使用不同浓度的赤霉素（GA₃）、高锰酸钾（KMnO₄）、聚乙二醇（PEG6000）和过氧化氢（H₂O₂）溶液进行浸种处理,比较不同处理条件对粗茎秦艽种子萌发的影响。结果显示,粗茎秦艽种皮对种子吸水没有明显阻碍作用;不同浓度的种子粗提物对白菜、小麦的萌发和生长均表现出抑制作用;不同浓度的粗提物对粗茎秦艽种子自身的萌发也表现出一定的抑制作用,当粗提物浓度达到0.1 g/mL时,抑制作用最显著（P < 0.05）;高锰酸钾处理可提高粗茎秦艽种子的萌发率,浓度为1.5%时效果较显著（P < 0.05）,而过氧化氢处理对粗茎秦艽种子的萌发效果不如前者,此外,用500 mg/L的赤霉素浸种和300 mg/L的聚乙二醇预处理也可显著打破粗茎秦艽种子休眠（P < 0.01）。研究结果表明粗茎秦艽种子的内源抑制物是影响其休眠的因素之一;种皮的机械阻碍也在一定程度上影响了种子萌发;粗茎秦艽种子具有综合性休眠特性。高锰酸钾预处理、赤霉素浸种和聚乙二醇引发均可打破种子休眠、缩短种子出芽时间,提高种子的发芽能力。     

达乌里秦艽的传粉生物学研究

对达乌里秦艽3个自然居群和1个人工栽培居群进行了两年的传粉生态学研究.研究发现:(1)达乌里秦艽套袋花均不结实,说明雌雄异熟和雌雄异位这两种花部机制成功阻止了主动自交,且不存在无融合生殖现象.(2)隔离传粉昆虫导致花寿命明显延长,授粉会导致花冠提前闭合,说明达乌里秦艽的花寿命具有可塑性,可通过延长花寿命确保成功繁殖.(3)在一个自然居群中证明存在花粉限制,且该居群的花寿命显著长于另外两个没有花粉限制的自然居群.(4)在整个雌性持续期柱头都具有一定的可授性,且在第2天可授性最高,随后逐渐降低;开花后期柱头可授性降低,使植物维持花开放资源投入的产出将有可能降低.研究表明,达乌里秦艽在严重缺乏传粉者的情况下,延长花寿命仅能部分弥补传粉者的不足,该物种在部分居群中仍有可能存在花粉限制.     

粗茎秦艽及其栽培

粗茎秦艽为龙胆科龙胆属植物,又叫粗茎龙胆(Gentiana crassicaulis),别名秦艽、小秦艽、川秦艽、秦纠、左秦艽、大艽、左扭、罗卜艽、牛尾艽等。出《神农本草经》,列为中品,历来粗茎秦艽与秦艽一样入药,现仍收载药典,以根入药。性味辛、苦、平。有祛风湿、清湿热、止脾痛之功。常用于风湿脾痛、筋脉拘挛、骨节烦痛、日脯潮热、小儿疳积发热等症。据报道,根含生物碱:秦艽碱乙、秦艽碱丙、糖类、挥发油和龙胆苦碱等。我省所用的秦艽,一直是靠从省外进货,主要是从西北,每年进货五千公斤左右(现价每公斤7     

粗茎秦艽不同部位龙胆苦甙含量的分析

采用高压液相法分析了栽培粗茎秦艽不同部位的有效药用成分龙胆苦甙(gentiopicrin)的含量,并与野生麻花秦艽、达乌里秦艽龙胆苦甙的含量相比较。结果表明粗茎秦艽根、茎、叶均具有较高的开发利用价值,为合理开发利用粗茎秦艽提供了科学依据。     

粗茎秦艽根茎品质与栽培土壤化学因子的相关性分析

对云南丽江鲁店乡18个样点粗茎秦艽（Gentiana crassicaulis Duth.ex Burk.）的根茎品质指标以及栽培土壤基本养分和矿质元素含量进行了测定;在此基础上,分析了根茎品质指标与土壤化学指标的相关性,并采用逐步回归分析方法筛选出影响粗茎秦艽根茎品质的主要土壤化学因子。测定结果表明：各样点间粗茎秦艽根茎的总灰分、酸不溶性灰分、水分、醇溶性浸出物、马钱苷酸和龙胆苦苷含量的差异较大,平均值分别为2.84%、0.28%、6.21%、28.57%、1.53%和4.70%,均符合相关的药材标准。土壤p H值为p H 4.67~p H 6.83,平均值为p H 5.51;土壤中有机质、速效N、速效P和速效K含量差异明显,平均值分别为7.38%、128.09μg·g-1、86.85μg·g-1和232.33μg·g-1;土壤中交换性Ca、交换性Mg、有效Zn、有效Mn、有效Fe和有效Cu含量也有较大差异,平均值分别为1 391.16、91.87、2.81、56.18、51.07和0.92μg·g-1。相关性分析结果表明：土壤速效P含量与粗茎秦艽根茎中醇溶性浸出物和龙胆苦苷含量分别呈极显著和显著正相关;土壤有效Fe含量与根茎中总灰分、酸不溶性灰分、醇溶性浸出物和龙胆苦苷含量呈极显著或显著正相关;土壤有效Cu含量与根茎中总灰分含量呈极显著正相关。逐步回归分析结果表明：影响粗茎秦艽根茎中总灰分含量的土壤化学因子是有效Zn、有效Fe和有效Cu含量,影响根茎中酸不溶性灰分和龙胆苦苷含量的土壤化学因子是有效Fe含量,影响根茎中醇溶性浸出物含量的土壤化学因子是速效P含量;而根茎中水分和马钱苷酸含量与各土壤化学因子均无明显的回归关系。综合分析结果显示：云南丽江粗茎秦艽种植区域的土壤均呈弱酸性,基本养分充足、矿质元素含量丰富,适宜于粗茎秦艽的生长;土壤有效Fe含量对粗茎秦艽根茎的品质指标影响最大,在实际生产中应适当喷施含Fe的微肥。     

甘肃濒危植物秦艽的龙胆苦苷含量和遗传多样性的研究北大核心CSCD

通过对不同产地秦艽的含量和遗传多样性进行研究,为甘肃秦艽的就地保护提供理论依据。龙胆苦苷含量测定采用高效液相色谱法,遗传多样性和遗传结构进行研究采用等位酶分析技术。不同产地秦艽的有效成分龙胆苦苷的含量存在一定的差异,对秦艽的6个居群152株通过12个酶位点的分析表明,秦艽具有较低的遗传多样性。每位点平均等位基因数（A）为0．7694,多态位点百分率（P）为16．16,秦艽居群间的遗传分化系数（白t）为0．1247,说明其87．63％的遗传变异存在于居群问。通过对不同地区秦艽药材的有效成分含量和等位酶的分析,为保护野生品种、确保引种栽培秦艽药材的质量提供了科学依据。     

6种秦艽镇痛和抗炎活性的比较

目的:比较六种秦艽甲醇提取物抗炎镇痛活性。方法:采用二甲苯致小鼠耳廓肿胀、棉球诱发小鼠肉芽肿试验、热板法和冰醋酸致小鼠扭体试验。结果:粗茎秦艽对由于腹腔注射冰醋酸引起急性腹膜炎而产生持久的疼痛刺激的镇痛效果最好,达乌里秦艽对热刺激引起的疼痛的缓解效果最好,大叶秦艽对二甲苯引起的炎症反应的抑制效果最佳,而麻花秦艽对棉球诱导的慢性炎症反应的抑制率最高。分析结果显示,黄管秦艽和管花秦艽在镇痛、抗炎活性上与上述4种药典收录的秦艽没有显著性差异。结论:黄管秦艽和管花秦艽在镇痛抗炎方面可作为秦艽的替代品。     

4种秦艽属植物不同器官中4种环烯醚萜苷成分含量的比较分析

利用HPLC法测定了秦艽(Gentiana macrophylla Pall.)、粗茎秦艽(G.crassicaulis Duthie ex Burk.)、麻花秦艽(G.straminea Maxim.)和小秦艽(G.dahurica Fisch.)根、茎、叶和花中4种环烯醚萜苷成分(包括马钱苷酸、獐牙菜苦苷、龙胆苦苷和獐牙菜苷)的含量,并对来源于不同产地秦艽和小秦艽不同器官4种环烯醚萜苷成分的含量进行了比较。结果显示:4种秦艽属植物不同器官4种环烯醚萜苷成分含量差异明显,来源于3个产地(陕西太白、甘肃西河乡和马狭)的秦艽和2个产地(青海互助和陕西麟游)的小秦艽不同器官中4种环烯醚萜苷含量也均具有明显差异。4种秦艽属植物根、茎、叶和花中龙胆苦苷和马钱苷酸总量分别为质量分数5.996%～10.869%、0.310%～4.065%、0.235%～4.138%和0.545%～5.591%;根中獐牙菜苦苷和獐牙菜苷的质量分数分别为0.516%～0.953%和0.042%～0.210%,茎中分别为0.173%～0.383%和0.031%～1.700%,叶中分别为0.068%～0.684%和0.020%～3.208%,花中分别为0.460%～0.832%和0.138%～3.827%。粗茎秦艽各器官龙胆苦苷和马钱苷酸总量均最高,小秦艽各器官龙胆苦苷和马钱苷酸总量均最低,总体上,秦艽和粗茎秦艽中龙胆苦苷和马钱苷酸总量高于小秦艽和麻花秦艽;小秦艽茎、叶和花中獐牙菜苷含量均最高,而秦艽茎和叶及粗茎秦艽花中獐牙菜苷含量均最低。4种秦艽属植物根部龙胆苦苷和马钱苷酸含量均明显高于茎、叶和花,獐牙菜苦苷在根和花中的积累较多,獐牙菜苷在花中的含量均相对最高。产自青海互助的小秦艽茎、叶和花中獐牙菜苷含量均最高,质量分数分别为2.884%、5.215%和7.321%。研究结果表明:4种环烯醚萜苷成分含量不但与植物种类和器官有关,而且与产地和采样时间也有关。     

Divergent and reticulate species relationships in Leucaena (Fabaceae) inferred from multiple data sources: insights into polyploid origins and nrDNA polymorphism

Previous analyses of species relationships and polyploid origins in the mimosoid legume genus Leucaena have used chloroplast DNA (cpDNA) restriction site data and morphology. Here we present an analysis of a new DNA sequence data set for the nuclear ribosomal DNA (nrDNA) 5.8S subunit and flanking ITS 1 and ITS 2 spacers, a simultaneous analysis of the morphology, ITS and cpDNA data sets for the diploid species, and a detailed comparison of the cpDNA and ITS gene trees, which include multiple accessions of all five tetraploid species. Significant new insights into species relationships and polyploid origins, including that of the economically important tropical forage tree L. leucocephala, are discussed. Heterogeneous ITS copy types, including 26 putative pseudogene sequences, were found within individuals of four of the five tetraploid and one diploid species. Potential pseudogenes were identified using two pairwise comparison approaches as well as a tree-based method that compares observed and expected proportions of total ITS variation contributed by the 5.8S subunit optimized onto branches of one of the ITS gene trees. Inclusion of putative pseudogene sequences in the analysis provided evidence that some pseudogenes in allopolyploid L. leucocephala are not the result of post-allopolyploidization gene silencing, but were inherited from its putative diploid maternal progenitor L. pulverulenta.     

基于cpDNA和ITS序列对庭藤复合群的居群遗传学研究

采用2个叶绿体基因组片段(ndh J-trn F和trn D-trn T)和核基因组ITS序列对分布于中国东部和相邻地区的庭藤复合群(Indigofera decora complex)的24个居群进行分析,探讨该复合群的遗传多样性、单倍型分布、遗传结构和居群历史动态。cp DNA和ITS序列分析表明,庭藤复合群的单倍型多样性较高,而总体遗传多样性偏低(cp DNA片段:Hd=0.778,π=0.00123;ITS:Hd=0.909,π=0.01290);皖浙地区居群的遗传多样性相对较高,推测其可能是该复合群遗传多样性的分布中心。遗传变异主要存在于复合群居群间,种内居群间遗传分化较大(cp DNA片段:FST=0.848;ITS:FST=0.787);基因流较小(cp DNA片段:Nm=0.228;ITS:Nm=0.241),主要是地理隔离(遗传漂变)或生境片段化促进了物种形成;复合群的不同物种间存在由基因渐渗引起的单倍型共享现象,可能是种间不完全的生殖隔离所致。花木蓝的居群单独聚为一枝,且与其他种无共享单倍型,因此应保留花木蓝种的分类地位,而其他种的分类地位有待进一步研究。     

Cryptic repeated genomic recombination during speciation in Gossypium gossypioides

The Mexican cotton Gossypium gossypioides is a perplexing entity, with conflicting morphological, cytogenetic, and molecular evidence of its phylogenetic affinity to other American cottons. We reevaluated the evolutionary history of this enigmatic species using 16.4 kb of DNA sequence. Phylogenetic analyses show that chloroplast DNA (7.3 kb), nuclear ribosomal internal transcribed spacers (ITS; 0.69 kb), and unique nuclear genes (8.4 kb) yield conflicting resolutions for G. gossypioides. Eight low-copy nuclear genes provide a nearly unanimous resolution of G. gossypioides as the basalmost American diploid cotton, whereas cpDNA sequences resolve G. gossypioides deeply nested within the American diploid clade sister to Peruvian G. raimondii, and ITS places G. gossypioides in an African (rather than an American) clade. These data, in conjunction with previous evidence from the repetitive fraction of the genome, implicate a complex history for G. gossypioides possibly involving temporally separated introgression events from genetically divergent cottons that are presently restricted to different hemispheres. Based on repetitive nuclear DNA, it appears that G. gossypioides experienced nuclear introgression from an African species shortly after divergence from the remainder of the American assemblage. More recently, hybridization with a Mexican species may have resulted in cpDNA introgression, and possibly a second round of cryptic nuclear introgression. Gossypium gossypioides provides a striking example of the previously unsuspected chimeric nature of some plant genomes and the resulting phylogenetic complexity produced by multiple historical reticulation events.     

Chloroplast DNA phylogeny of the woody Sonchus alliance (Asteraceae: Sonchinae) in the Macaronesian Islands

Determining accurate phylogenetic relationships among the members of the woody Sonchus alliance presents challenges because of an insufficient level of molecular variation and the convergent evolution of similar morphological traits in island settings. To obtain a better resolved phylogeny and to test the potential role of hybridization and introgression, we sequenced all members of the alliance with multiple populations for the ITS of nrDNA and over 4000 base pairs of coding and noncoding regions of cpDNA. The cpDNA phylogeny is not well resolved in the core members of the alliance (i.e., subg. Dendrosonchus and genus Taeckholmia), but like the ITS tree, it has identified basal lineages of monotypic genera. The cpDNA data set was not significantly different from that of ITS, and subsequent combined analysis provided a better resolved and supported phylogeny within the alliance. The combined ML tree identified the same basal lineages, suggested nonmonophyly of Dendrosonchus and Taeckholmia, and did not support either Boulos' or Aldridge's infrasubgeneric classification system. Assessment of the role of hybridization and introgression was limited due to poor resolution in the cpDNA phylogeny. The combined analysis supports a Gran Canaria origin for the alliance and two subsequent long distance dispersal events to Madeira and Cape Verde islands.     

Genetic differentiation and delimitation of Pugionium dolabratum and Pugionium cornutum (Brassicaceae)

Species delimitation has been a major research focus in evolutionary biology. However, the genetic delimitation of recently diverged species varies depending on the markers examined. In this study, we aimed to examine genetic differentiation and delimitations between only two species of Pugionium Gaertn (Brassicaceae)—Pugionium cornutum (L.) Gaertn and Pugionium dolabratum Maxim—that occur in the desert habitats of central Asia and have parapatric distributions. We genotyped 169 individuals from 25 populations, using two chloroplast (cp) DNA fragments (trnV-trnM and trnS-trnfM), seven simple repeated sequence (SSR) loci and the nuclear ribosomal internal transcribed spacer (ITS). Four cp haplotypes were identified, three of which commonly occur in the two species, suggesting incomplete species-specific lineage sorting. Between-species cpDNA differentiation (F _CT) was low, even lower than among populations of the same species. However, we found higher than average SSR F _CT values, and both Bayesian clustering of SSR variables and maximum-likelihood genetic analyses divided all sampled individuals into two groups, agreeing well with morphological separation, although gene flow between species was obvious according to the SSR loci data. However, two types of ITS sequences were highly consistent with the morphological delimitation of the two species in all sampled individuals. These results together suggest that these two species shared numerous ancestral cpDNA polymorphisms and point to the importance of nuclear DNA (ITS or genetic accumulation at multiple loci) in delimiting recently diverged species.     

More introgression with less gene flow: chloroplast vs. mitochondrial DNA in the Picea asperata complex in China, and comparison with other Conifers

Recent work has suggested that rates of introgression should be inversely related to levels of gene flow because introgressed populations cannot be 'rescued' by intraspecific gene flow if it is too low. Mitochondrial and chloroplast DNA (mtDNA and cpDNA) experience very different levels of gene flow in conifers due to their contrasted maternal and paternal modes of transmission, hence the prediction that mtDNA should introgress more readily than cpDNA in this group. Here, we use sequence data from both mtDNA and cpDNA to test this hypothesis in a group of closely related spruces species, the Picea asperata complex from China. Nine mitochondrial and nine chloroplast haplotypes were recovered from 459 individuals in 46 natural populations belonging to five species of the Picea asperata complex. Low variation was found in the two mtDNA introns along with a high level of differentiation among populations ( G _ST = 0.90). In contrast, we detected higher variation and lower differentiation among populations at cpDNA markers ( G _ST = 0.56), a trend shared by most conifer species studied so far. We found that cpDNA variation, although far from being fully diagnostic, is more species-specific than mtDNA variation: four groups of populations were identified using cpDNA markers, all of them related to species or groups of species, whereas for mtDNA, geographical variation prevails over species differentiation. The literature suggests that mtDNA haplotypes are often shared among related conifer species, whereas cpDNA haplotypes are more species-specific. Hence, increased intraspecific gene flow appears to decrease differentiation within species but not among species.     

Morphological, cytogenetic, and molecular evidence for introgressive hybridization in birch.

Extensive morphological variation of tetraploid birch (Betula pubescens) in Iceland is believed to be due to gene flow from diploid dwarf birch (B. nana) by means of introgressive hybridization. A combined morphological and cytogenetic approach was used to investigate this phenomenon in two geographically separated populations of natural birch woodland in Iceland. The results not only confirmed introgressive hybridization in birch, but also revealed bidirectional gene flow between the two species via triploid interspecific hybrids. The populations showed continuous morphological variation connecting the species, but karyotypically they consisted of only three types of plants: diploids, triploids, and tetraploids. No aneuploids were found. Some of the tetraploid plants had B. pubescens morphology as expected, but most of them had intermediate characters. Most of the diploid plants were B. nana, but some were intermediates and a few had B. pubescens morphology. The triploid plants were either intermediates or they resembled one of the two species. Similar introgressive variation was observed among the diploid and triploid progeny of open-pollinated B. nana in a garden. Birch samples including field plants and artificial hybrids were further examined using a molecular method based on genomic Southern hybridization. The experiments verified introgression at the DNA level.     

Ancient and current gene flow between two distantly related Mediterranean oak species, Quercus suber and Q. ilex

Background and Aims

Quercus suber and Q. ilex are distantly related and their distributions partially overlap. They hybridize occasionally, but the complete replacement of Q. suber chloroplast DNA (cpDNA) by that of Q. ilex was identified in two specific geographical areas. The objective of this study was to determine whether the contrasting situation reflected current or recent geographical interspecies gene flow variation or was the result of ancient introgression.

Methods

cpDNA PCR-RFLPs (restriction fragment length polymorphisms) and variation at ten nuclear microsatellite loci were analysed in populations of each species, in 16 morphologically intermediate individuals and the progeny of several of them. Interspecies nuclear introgression was based on individual admixture rates using a Bayesian approach with no a priori species assignment, and on a maximum-likelihood (ML) method, using allele frequencies in the allopatric populations of each species as controls. Gene flow was compared specifically between populations located within and outside the specific areas.

Key Results

High interspecies nuclear genetic differentiation was observed, with twice the number of alleles in Q. ilex than in Q. suber. According to Bayesian assignment, approx. 1 % of individuals had a high probability of being F₁ hybrids, and bidirectional nuclear introgression affected approx. 4 % of individuals in each species. Hybrid and introgressed individuals were identified predominantly in mixed stands and may have a recent origin. Higher proportions including allospecific genes recovered from past hybridization were obtained using the ML method. Similar rates of hybridization and of nuclear introgression, partially independent of cpDNA interspecies transfer suggestive of gene filtering, were obtained in the populations located within and outside the areas of complete cpDNA replacement.

Conclusions

The results did not provide evidence for geographical variation in interspecies gene flow. In contrast, historical introgression is supported by palynological records and constitutes the more reliable origin of cpDNA replacement in specific regions.Key words: cpDNA PCR-RFLPs, nuclear microsatellite (nSSR) variation, hybridization, interspecies genetic introgression, Quercus suber, Quercus ilex     

Out of the Qinghai-Tibet Plateau: evidence for the origin and dispersal of Eurasian temperate plants from a phylogeographic study of Hippophaë rhamnoides (Elaeagnaceae)

Numerous temperate plants now distributed across Eurasia are hypothesized to have originated and migrated from the Qinghai-Tibet Plateau (QTP) and adjacent regions. However, this hypothesis has never been tested through a phylogeographic analysis of a widely distributed species. Here, we use Hippopha? rhamnoides as a model to test this hypothesis. We collected 635 individuals from 63 populations of the nine subspecies of H. rhamnoides. We sequenced two maternally inherited chloroplast (cp) DNA fragments and also the bi-paternally inherited nuclear ribosomal ITS. We recovered five major clades in phylogenetic trees constructed from cpDNA and internal transcribed spacer (ITS) sequence variation. Most sampled individuals of six subspecies that are distributed in northern China, central Asia and Asia Minor/Europe, respectively, comprised monophyletic clades (or subclades) nested within those found in the QTP. Two subspecies in the QTP were paraphyletic, while the placement of another subspecies from the Mongolian Plateau differed between the ITS and cpDNA phylogenetic trees. Our phylogeographic analyses supported an 'out-of-QTP' hypothesis for H. rhamnoides followed by allopatric divergence, hybridization and introgression. These findings highlight the complexity of intraspecific evolutions and the importance of the QTP as a center of origin for many temperate plants.