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.

Interannual fluctuations in floral longevity, pollinator visitation and pollination limitation of an alpine plant (Gentiana straminea Maxim., Gentianaceae) at two altitudes in the Qinghai-Tibetan Plateau

Prolonged floral longevity and bumblebees as dominate pollinators in alpine ecosystem have been suggested to overcome pollination limitation of alpine plants arising from the decrease of pollinator activity with increasing altitude. However, this conclusion has never been examined in the Qinghai-Tibetan Plateau (QTP), the highest and largest plateau in the world. In this study, we intended to test year-to-year correlations between floral longevity, visiting frequency and pollen limitation of this species between two populations (at 3200 m and 4000 m) of Gentiana straminea in this plateau. Pollinator exclusion elongated both male and female phases greatly at both sites, and durations of both male and female phases in natural condition varied greatly over three years. The visiting frequency of bumblebees varied greatly at the higher altitude, but seemed to be stable at the lower altitude. Seed production was pollination limited in both populations in most studied years. The floral durations, pollinator frequency and pollination limitation showed no significant and consistent variations with the increase of altitude. The previous hypothesis that the prolonged floral longevity of alpine plants can compensate for low levels of pollinator visitation therefore could not be confirmed, and our results further suggested that in the QTP platform, the altitude shows no consistent effect on the reproductive performance of this species, despite that the fluctuation of visit frequency intensified at the higher altitude.

Floral closure in response to temperature and pollination in Gentiana straminea Maxim. (Gentianaceae), an alpine perennial in the Qinghai-Tibetan Plateau

Floral closure may be induced by pollination and various other factors, but is rarely studied comprehensively. Different kinds of floral closure should have various effects on reproductive fitness of plants. Two contrasting types of floral closure were observed in the flowers of Gentiana straminea Maxim. in the eastern Qinghai-Tibetan Plateau. The first type occurred prior to pollination during both gender phases, in response mainly to decreasing air temperatures. Flowers closed when decreasing temperatures approached 20 degrees C and subsequently began to reopen the following day during mid-morning when air temperatures warmed to approximately 13-15 degrees C. This kind of floral closure can protect pollen grains on either stamens or stigmas, increasing fitness of both male and female. Following pollination, permanent floral closure occurred, although there was a delay between the dates of pollination and permanent closure, during which flowers continued to show temporary closure in response to low temperature episodes. The time required for permanent, pollination-induced closure varied according to the age of the gender phase, including a prolonged time before closure if pollination occurred early in the female phase. The retaining of permanent closed flowers increased both approaching (to inflorescences) and visiting (to unpollinated flowers) frequencies of individual plants when with fewer open flowers and the persisting corolla is further beneficial for seed sets of these pollinated flowers. Thus, two separate types of floral closure, one in response to environmental cues and the other in response to the age of each gender stage, appeared to have a strong influence on reproductive fitness in this species. These results revealed a different adaptive strategy of alpine plants in the sexual reproduction assurance in addition to the well-known elevated floral longevity, dominant role of more effective pollinators and increased reproduction allocation in the arid habitats.

Reproductive ecology of the Qinghai-Tibet Plateau endemic Gentiana straminea (Gentianaceae), a hermaphrodite perennial characterized by herkogamy and dichogamy

The combined occurrence of both herkogamy and dichogamy in a hermaphrodite species has been considered to strongly favour outcrossing. In this study, we investigated in detail the reproductive ecology of Gentiana straminea Maxim. (Gentianaceae), a hermaphrodite perennial endemic to the Qinghai-Tibet Plateau. In a series of observations and experiments over four consecutive years, we examined whether the combination of dichogamy and herkogamy in individual flowers completely prevents geitonogamous pollen transfer in this species. The mode of floral development clearly indicates that autonomous self-pollination is completely avoided through herkogamy and dichogamy in individual flowers. 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. Many flowers opened simultaneously on individual plants, the ratio of male to female phase flowers was close to 2:1 in each inflorescence, at the full anthesis phase, and they were randomly distributed amongst the upper, middle and lower parts of each stem's inflorescence. On average, Bombus sushikini Skorikov, the most frequent visitor and only legitimate pollinator of G. straminea, visited nearly two flowers per inflorescence, and four flowers per plant. Among the pollinators' foraging bouts, the proportions of geitonogamous visits to inflorescences or flowers within an individual plant were 29% and 37%, respectively. Therefore, despite the strict dichogamous and herkogamous characteristics of the individual flowers, geitonogamous selfing might still prevail in G. straininea because of the size of its floral displays and the continuous visiting behavior of B. sushkini. (C) 2005 Elsevier SAS. All rights reserved.

Leaf orientation, incident sunlight, and photosynthesis in the alpine species Suassurea superba and Gentiana straminea on the Qinghai-Tibet Plateau

The extremely high level of solar radiation on the Qinghai-Tibet Plateau may induce photoinhibition and thus limit leaf carbon gain. To assess the effect of high light, we examined gas exchange and chlorophyll fluorescence for two species differing in light interception: the prostrate Saussurea superba and the erect-leaved Gentiana straminea. In controlled conditions with favorable water and temperature, neither species showed apparent photoinhibition in gas exchange measurements. In natural environment, however, their photosynthetic rate decreased remarkably at high light. Photosynthesis depression was aggravated under high leaf temperature or soil water stress. Relative stomatal limitation was much higher in S. superba than in G. straminea and it remarkably increased in the later species at midday when soil was dry. F-v/F-m as an indicator for photoinhibition was generally higher in S. superba than in the other species. F-v/F-m decreased significantly under high light at midday in both species, even when soil moisture was high. F-0 linearly elevated with the increment of leaf temperature in G. straminea, but remained almost constant in S. superba. Electron transport rate (ETR) increased with photosynthetically active photon flux density (PPFD) in S. superba, but declined when PPFD was high than about 1000 mumol m(-2) s(-1) in G. straminea. Compared to favorable environment, the estimated daily leaf carbon gain at PPFD above 800 mumol m(-2) s(-1) was reduced by 32% in S. superba and by 17% in G. straminea when soil was moist, and by 43% and 53%, respectively, when soil was dry. Our results suggest that the high radiation induces photoinhibition and significantly limits photosynthetic carbon gain, and the limitation may further increase at higher temperature and in dry soil.

Photosynthesis of Saussurea superba and Gentiana straminea is not reduced after long-term enhancement of UV-B radiation

Experiments were conducted in an alpine Kobresia humilis meadow near Haibei Alpine Meadow Ecosystem Research Station (37degrees29'-37degrees45'N, 101degrees12'-101degrees33'E; altitude 3200 m). Effects of enhanced ultraviolet-B (UV-B) radiation on photosynthesis of the alpine plants of Saussurea superba and Gentiana straminea were investigated. Both species were exposed to a UV-B-BE density at 15.80 kJ m(-2) per day, simulating nearly 14% ozone (O-3) reduction during the plant growing season. Neither photosynthetic CO2 uptake rate nor photosynthetic O-2 evolution rate were decreased after a long period of enhanced UV-B radiation treatment. On the contrary, there was a tendency to increase of both parameters in both species. The photosynthetic pigments were also increased, when expressed on a leaf area basis. UV-B absorbing compounds, detected by the absorbance values at 300 mm, had a tendency to increase in both species after enhanced UV-B radiation. After long-term exposure of plants to enhanced UV-B radiation, leaf morphology was also affected. Leaf thickness in both S. superba and G. straminea were increased significantly (P < 0.001). This supports our hypothesis that the increase of leaf thickness in both species after long-term exposure of enhanced UV-B radiation could compensate for the photodestruction of photosynthetic pigments when light passes through the leaf. Therefore, photosynthesis is not reduced in either species when expressed on leaf area basis. (C) 2003 Elsevier B.V. All rights reserved.

Taxonomy, phylogeny and reproductive ecology of Gentiana lutea L.

This research focuses on taxonomy, phylogeny and reproductive ecology of Gentiana lutea. L.. Taxonomic analysis is a critical step in botanical studies, as it is necessary to recognize taxonomical unit. Herbarium specimens were observed to assess the reliability of several subspecies-diagnostic characters. The analysis of G. lutea genetic variability and the comparison with that of the other species of sect. Gentiana were performed to elucidate phylogenetic relationships among G. lutea subspecies and to propose a phylogenetic hypothesis for the evolution and the colonization dynamics of the section. Appropriate scientific information is critical for the assessment of species conservation status and for effective management plans. I carried out field work on five natural populations and performed laboratory analyses on specific critical aspects, with special regard to G. lutea breeding system and type and efficiency of plant-pollinator system. Bracts length is a reliable character to identify subsp. vardjanii, however it is not exclusive, hence to clearly identify subsp. vardjanii, other traits have to be considered. The phylogenetic hypotheses obtained from nuclear and chloroplast data are not congruent. Nuclear markers show a monophyly of sect. Gentiana, a strongly species identity of G. lutea and clear genetic identity of subsp. vardjanii. The little information emerging from plastid markers indicate a weak signal of hybridization and incomplete sorting of ancestral lineages. G. lutea shows a striking variation in intra-floral dichogamy probably evolved to reduce pollen-stigma interference. Although the species is partially self-compatible, pollen vectors are necessary for a successful reproduction, and moreover it shows a strong inbreeding depression. G. lutea is a generalist species: within its spectrum of visitors is possible to recognize "nectar thieves" and pollinators with sedentary or dynamic behaviour. Pollen limitation is frequent and it could be mainly explained by poor pollen quality.

Morphological and germination response variability in seeds of wild yellow gentian (Gentiana lutea L.) accessions from northwest Spain

Gentiana lutea L. (yellow gentian, Gentianaceae) is an important medicinal plant under protection as endangered species in most European countries. The aim of this work was to evaluate variation in seed mass, seed water content, and seed germination among 56 wild accessions of G. lutea. The effect of gibberellic acid (GA3), putrescine, moist chilling, and level of ripeness of seeds on subsequent germination was also investigated. Seeds of G. lutea showed physiological dormancy (final germination percentages ranged from 0% to 11%, depending on the accession) and GA3 enhanced seed germination drastically in all the accessions. The highest germination (99%) of GA3-treated seeds was reached at 15 °C. Final germination percentage and germination rate (as expressed by mean germination time), as well as seed mass and seed water content, varied significantly among accessions. In general, 1 year moist chilling did not significantly enhance G. lutea seed germination. For most accessions, no significant differences were found between fully ripe seeds and less ripe seeds for seed water content, seed mass, and seed germination. Applications of GA3 were always most effective than those of putrescine for increasing seed germination.

龙胆科蔓龙胆属及其相关类群的系统学研究

本文回顾了龙胆科蔓龙胆属、双蝴蝶属、龙胆属狭蕊组和翼萼蔓属的研究历史，对这些类群的外部形态、花部解剖结构、染色体、花粉形态及胚胎学进行了研究，并结合已有的资料，探讨了这些类群的分类学问题。主要结论如下： 1．外部形态 蔓龙胆属、双蝴蝶属、龙胆属狭蕊组和龙胆属其他组之 间以及蔓龙胆属和翼萼蔓属之间在外部形态上均存在差异。 2．花部解剖结构 蔓龙胆属、双蝴蝶属、龙胆属狭蕊组和龙胆属其他组的花部解剖结构存在较多地相似之处，同时蔓龙胆属和双蝴蝶属的花被维管束和腺体比龙胆属狭蕊组和龙胆属其他组的特化。而翼萼蔓属的花部解剖结构与蔓龙胆属的差异较大，与扁蕾属的花部解剖结构较相似。 3．染色体 本文对蔓龙胆属、双蝴蝶属、龙胆属狭蕊组和翼萼蔓属的7种植物的染色体数目、基数和核型对称性作了研究，其中6种植物的染色体为首次报道，它们是：披针叶蔓龙胆C．delavayi、双蝴蝶T.chnense、峨眉双蝴蝶T．cordatum、锯齿龙胆 G．serra、毛脉龙胆G．souliei、和翼萼蔓P．volubilis。蔓龙胆属、双蝴蝶属和龙胆属狭蕊组的核型不对称性稍强于龙胆属其他组，但在染色体组成上十分相似。翼萼蔓属和蔓龙胆属的染色体差异较大，面与扁蕾属的染色体较相似。 4.花粉形态蔓龙胆属、双蝴蝶属、龙胆属狭蕊组和龙胆属其他组均具有球形或长球形、3孔沟、沟长而宽、外壁纹饰为条纹一无穿孔和条纹一穿孔的花粉类型。而翼萼蔓属的花粉为球形、纹饰为网状纹馋与蔓龙胆属不同，与扁蕾属的花粉较相似。 5．胚胎学 本文对披针叶蔓龙胆C，delavayi、双蝴蝶T．chinense、峨眉双蝴蝶T．cordatum、条纹龙胆G. striata和翼萼蔓P．volubilis作了较详细的胚胎学研究，这些类群在花药壁的分化和形成、胚珠和胎座类型、卵器中的助细胞和反足细胞、种皮以及胚发育等特征上存在差异，并且在胚胎学特征上各自拥有原始和进化的性状。蔓龙胆属、双蝴蝶属和龙胆属之间的胚胎学性状差异较大，龙胆属狭蕊组与双蝴蝶属的胚胎学性状较相似，而与蔓龙胆属和龙胆属其他组的差吴较大。翼萼蔓属的胚胎学性状与扁蕾属的较相似，而与蔓龙胆属的差异较大。 6．综合分析外部形态、花部解剖结构、染色体、花粉形态及胚胎学性状，结果表明，蔓龙胆属、双蝴蝶属、龙胆属狭蕊组和龙胆属其他组是亲缘关系十分相近的类群，在某些特征上已存在明显地演化极向，但在大多数特征上演化极向不明。蔓龙胆属和双蝴蝶属分别作为独立属处理较为合适，而且两属的亲缘关系很近。狭蕊组仍应放在龙胆属，蔓龙胆属比双蝴蝶属与龙胆属的亲缘关系更近一些。翼萼蔓属也应作为一独立属处理，而且该属可能与扁蕾属的亲缘关系较近，而与蔓龙胆属亲缘关系较远。 7．本文还讨论了蔓龙胆属、双蝴蝶属、龙胆属狭蕊组和翼萼蔓属的地理分布。