Use of DNA barcode sensu lato to identify traditional Tibetan medicinal plant Gentianopsis paludosa (Gentianaceae)

Gentianopsis paludosa (Hook. f.) Ma (Gentianaceae) is an important species in Tibetan folk medicine commonly used to clear away the "heat evils" and toxic materials. A survey of market samples revealed that nine adulterant species, Gentianopsis barbata, G. contorta, G. grandis, Halenia elliptica, Lomatogonium macranthum, L.rotatum, Swertia angustifolia, S. bifolia and S. erythrosticta, are often marketed as G. paludosa. Methods to distinguish G. paludosa from its adulterants are limited by current morphological and chemical methods. DNA sequence analysis of the nuclear ribosomal DNA internal transcribed spacer region (ITS) was used in the differentiation of G.paludosa from the adulterant species. The data showed that the internal transcribed spacer regions differ significantly between G. paludosa and all nine adulterant species, so that they could be easily distinguished at the DNA level.     

Reduced fecundity in small populations of the rare plant Gentianopsis ciliate (Gentianaceae)

Habitat destruction is the main cause for the biodiversity crisis. Surviving populations are often fragmented, i.e., small and isolated from each other. Reproduction of plants in small populations is often reduced, and this has been attributed to inbreeding depression, reduced attractiveness for pollinators, and reduced habitat quality in small populations. Here we present data on the effects of fragmentation on the rare, self-compatible perennial herb Gentianopsis ciliata (Gentianaceae), a species with very small and presumably well-dispersed seeds. We studied the relationship between population size, plant size, and the number of flowers produced in 63 populations from 1996-1998. In one of the years, leaf and flower size and the number of seeds produced per fruit was studied in a subset of 25 populations. Plant size, flower size, and the number of seeds per fruit and per plant increased with population size, whereas leaf length and the number of flowers per plant did not. The effects of population size on reproduction and on flower size remained significant if the effects were adjusted for differences in plant size, indicating that they could not be explained by differences in habitat quality. The strongly reduced reproduction in small populations may be due to pollination limitation, while the reduced flower size could indicate genetic effects.     

Genetic diversity and population structure of Swertia tetraptera (Gentianaceae), an endemic species of Qinghai-Tibetan Plateau

Swertia tetraptera Maxim is an annual alpine herb endemic to the Qinghai-Tibetan Plateau (QTP). Its populations are locally scattered as isolated patches throughout this region. Genetic variation within and among thirty-four populations of this species was assessed using ISSR fingerprinting with 10 primers. High levels of genetic diversity exist within species (P = 98.9%, I = 0.3475; He = 0.2227), while the within-population diversity is low (P = 32.7%, I = 0.177; He = 0.12). High levels of genetic differentiation were detected among populations based on various statistics, including Nei’s genetic diversity analysis (G_ST = 0.4608), Bayesian analysis (θ^B = 0.476) and AMOVA (F_ST = 0.57). That is, populations shared low levels of genetic identity (I = 0.2622–0.0966). This genetic structure was probably due to severe genetic drift, breeding system and limited gene flow. The observed genetic structure of the populations implies that different populations across the distribution range of the species should be sampled to maintain high genetic diversity when a conservation strategy is implemented.     

青藏高原高山流石滩特有植物绵参的谱系地理学研究

绵参(Eriophyton wallichii)是中国青藏高原地区高山流石滩特有植物.为探讨第四纪冰期气候波动对青藏高原高山流石滩植物居群遗传结构以及谱系历史的影响,检测了绵参20个居群187个个体的核糖体内转录间隔区(ITS)的序列变异,共发现19个单倍型,且结果表明大部分居群拥有独自的单倍型.AMOVA分析表明,居群间的变异占了总变异量的89.54％,居群遗传分化指数很高(GST=0.863,NST=0.957),但无明显的谱系结构.我们推测这种独特的谱系地理结构可能主要是由于高山流石滩独特的地形和环境及“孤岛效应”形成的,与先前研究过的青藏高原植物不同,绵参呈现出高度分化的遗传结构,常常呈现出“一个居群,一种单倍型”的模式,这可能是由于第四纪冰期及间冰期尽管绵参的分布范围有扩大和收缩,但始终由于特殊的高山生境和流石滩基质,导致了该物种长期以来居群间很少有基因交流并一直处于隔离状态,造成了现在这样的谱系地理结构.     

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°C and subsequently began to reopen the following day during mid-morning when air temperatures warmed to approximately 13–15 °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.     

青藏高原高寒草甸罂粟科植物种子萌发特性研究

对9种罂粟科植物种子在5个温度(5℃、10℃、15℃、20℃、25℃)下的萌发特性进行了研究.结果显示:(1)采自低海拔的小果博落回、秃疮花和白屈菜在15～25℃的萌发率高于5～10℃;采自高海拔的全缘叶绿绒蒿和多刺绿绒蒿达到最大萌发率的温度分别为5℃和10℃;而采自高海拔的红花绿绒蒿和五脉绿绒蒿只有通过5℃冷处理后才能萌发,表明种子的最佳萌发温度与采集地海拔相关.(2)萌发速率随温度升高增大,秃疮花和苣叶秃疮花在25℃萌发速率最大,其余种25℃的萌发速率略小于20℃的萌发速率.(3)萌发时滞表现出随温度升高而缩短的趋势,秃疮花、白屈菜和苣叶秃疮花在25℃萌发时滞最短,其余种25℃萌发时滞又长于20℃萌发时滞.(4)一年生植物多刺绿绒蒿在所有温度下的萌发率和萌发速率均最高,而萌发时滞最短,这可能是一年生植物适应特殊环境的一种对策.     

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.     

高寒草甸常见雀形目鸟类的卵壳特征

作为胚胎发育的屏障,卵壳特征如卵壳厚度和气孔密度具有重要的生态学意义。本文研究了高寒草甸繁殖的7种代表性雀形目鸟类角百灵（Eremophila alpestris）、小云雀（Alauda gulgula）、黄头鹡鸰（Motacilla citreola）、树麻雀（Passer montanus）、粉红胸鹨（Anthus roseatus）、黄嘴朱顶雀（Carduelis flavirostris）、赭红尾鸲（Phoenicurus ochruros）的卵与卵壳特征,以探索在高寒缺氧环境下,不同科鸟类的适应性调节。结果发现,7种鸟类中,除小云雀和角百灵之外,其他鸟类的卵与卵壳特征均有显著的种间差异（除卵壳厚度和气孔直径之外）;对7种鸟类卵壳厚度、气孔密度、卵体积、卵重之间的线性拟合显示,卵壳厚度、卵体积与气孔密度均无显著相关性（卵壳厚度：P = 0.11,卵体积：P = 0.09）,卵重、卵体积与卵壳厚度呈显著正相关（卵重：r2 = 0.46,P ＜ 0.001;卵体积：r2 = 0.44,P ＜ 0.001）;对7种鸟类卵的数据与繁殖期雌鸟平均体重的线性拟合结果显示：平均卵重、卵壳厚度的差异与雌鸟体重成正相关（卵重：r2 = 0.66,P = 0.03;卵壳厚度：r2 = 0.92,P ＜ 0.01）;对6种鸟类（缺乏粉红胸鹨窝卵数数据）卵的数据与孵卵相关变量的线性拟合结果显示：卵壳厚度及气孔率与窝卵数成负相关（卵壳厚度：r2 = 0.64,P = 0.056;气孔率：r2 = 0.87,P ＜ 0.01）,6种鸟类（缺乏树麻雀巢杯指数数据）气孔率与巢杯指数或巢型无显著相关性（巢杯指数：P = 0.49,巢型：P = 0.435）。卵表面积和总气孔数解释了大部分气孔率差异（87%）,卵重和气孔率与孵卵期无显著相关（P = 0.77）,气孔率显著低于预期气孔率（P＜0.001）。这些结果表明,种间的遗传性（如成鸟的形态、窝卵数等）决定了大部分卵与卵壳特性的差异,但是为适应高寒低氧的气候特征,不同种的鸟都具有相对厚的卵壳和低的气孔率特征,说明环境因素同样影响卵的进化。另外,单纯的卵特性（卵体积和气孔率）并不能决定孵卵期的长短,亲鸟的孵卵行为同样具有重要的影响,角百灵虽然离巢频率相对较高,但是孵卵期却相对较短,这可能与其较高的气孔率有关。     

Carbon Dioxide Exchange Between the Atmosphere and an Alpine Shrubland Meadow During the Growing Season on the Qinghai-Tibetan Plateau

Abstract: In the present study, we used the eddy covariance method to measure CO₂ exchange between the atmosphere and an alpine shrubland meadow ecosystem (37°36'N, 101°18'E; 3 250 m a.s.l.) on the Qinghai-Tibetan Plateau, China, during the growing season in 2003, from 20 April to 30 September. This meadow is dominated by formations of Potentilla fruticosa L. The soil is Mol-Cryic Cambisols. During the study period, the meadow was not grazed. The maximum rates of CO₂ uptake and release derived from the diurnal course of CO₂ flux were -9.38 and 5.02 μmol·m^-2·s^-1, respectively. The largest daily CO₂ uptake was 1.7 g C·m^-2·d^-1 on 14 July, which is less than half that of an alpine Kobresia meadow ecosystem at similar latitudes. Daily CO₂ uptake during the measurement period indicated that the alpine shrubland meadow ecosystem may behave as a sink of atmospheric CO₂ during the growing season. The daytime CO₂ uptake was correlated exponentially or linearly with the daily photo synthetic photon flux density each month. The daytime average water use efficiency of the ecosystem was 6.47 mg CO₂/g H₂O. The efficiency of the ecosystem increased with a decrease in vapor pressure deficit.
(Managing editor: Ya-Qin HAN)     

Carbon Dioxide Exchange Between the Atmosphere and an Alpine Shrubland Meadow During the Growing Season on the Qinghai-Tibetan Plateau

In the present study, we used the eddy covariance method to measure CO2 exchange between the atmosphere and an alpine shrubland meadow ecosystem (37°36′ N, 101o18′ E; 3 250 m a.s.l.) on the Qinghai-Tibetan Plateau, China, during the growing season in 2003, from 20 April to 30 September. This meadow is dominated by formations ofPotentillafruticosa L. The soil is Mol-Cryic Cambisols. During the study period, the meadow was not grazed. The maximum rates of CO2 uptake and release derived from the latitudes. Daily CO2 uptake during the measurement period indicated that the alpine shrubland meadow ecosystem may behave as a sink of atmospheric CO2 during the growing season. The daytime CO2 uptake was correlated exponentially or linearly with the daily photosynthetic photon flux density each month. The daytime average water use efficiency of the ecosystem was 6.47 mg CO2/g H2O. The efficiency of the ecosystem increased with a decrease in vapor pressure deficit.     

高山植物黄管秦艽cDNA文库构建与表达序列标签（EST）分析

黄管秦艽（Gentiana officinalis）是一种重要的藏药高山植物,本研究构建了该物种开花期的eDNA文库。经检测达到中等cDNA文库水平,文库滴度为1．2×10^7pfu／ml,重组率95．9％,插入片段平均长度大于500bp。对343个随机挑选的重组克隆进行部分测序,获得的ESTs经编辑后共有181条有效序列。经生物信息学方法分析181条表达序列标签（EST）代表144个单克隆序列,其中55个与已鉴定的基因同源,35个序列与未鉴定的EST匹配,54个未找到同源序列;后两者共有89个EST序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现,相关基因主要编码以下蛋白：与蛋白表达相关的占35％;光合作用相关的占笠％;新陈代谢相关的占18％;抗性相关的占11％;质膜运输和细胞分裂相关的分别占5％;染色体变化和细胞信号转导的分别占2％。根据有效EST序列设计引物,通过RT-PCR进一验证了所得EST的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

高山植物黄管秦艽cDNA 文库构建与表达序列标签(EST) 分析

黄管秦艽( Gentiana officinalis) 是一种重要的藏药高山植物, 本研究构建了该物种开花期的cDNA 文库。经检测达到中等cDNA 文库水平, 文库滴度为1 . 2×107 pfu􊄯ml , 重组率95.9% , 插入片段平均长度大于500 bp。对343 个随机挑选的重组克隆进行部分测序, 获得的ESTs 经编辑后共有181 条有效序列。经生物信息学方法分析181 条表达序列标签(EST) 代表144 个单克隆序列, 其中55 个与已鉴定的基因同源, 35 个序列与未鉴定的EST 匹配, 54 个未找到同源序列; 后两者共有89 个EST 序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现, 相关基因主要编码以下蛋白: 与蛋白表达相关的占35%; 光合作用相关的占22%; 新陈代谢相关的占18%; 抗性相关的占11%; 质膜运输和细胞分裂相关的分别占5% ; 染色体变化和细胞信号转导的分别占2%。根据有效EST 序列设计引物, 通过RT-PCR 进一验证了所得EST 的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

Effects of Nitrogen and Phosphorus Fertilization on Soil Carbon Fractions in Alpine Meadows on the Qinghai-Tibetan Plateau

In grassland ecosystems, N and P fertilization often increase plant productivity, but there is no concensus if fertilization affects soil C fractions. We tested effects of N, P and N+P fertilization at 5, 10, 15 g m⁻² yr⁻¹ (N₅, N₁₀, N₁₅, P₅, P₁₀, P₁₅, N₅P₅, N₁₀P₁₀, and N₁₅P₁₅) compared to unfertilized control on soil C, soil microbial biomass and functional diversity at the 0–20 cm and 20–40 cm depth in an alpine meadow after 5 years of continuous fertilization. Fertilization increased total aboveground biomass of community and grass but decreased legume and forb biomass compared to no fertilization. All fertilization treatments decreased the C:N ratios of legumes and roots compared to control, however fertilization at rates of 5 and 15 g m⁻² yr⁻¹ decreased the C:N ratios of the grasses. Compared to the control, soil microbial biomass C increased in N₅, N₁₀, P₅, and P₁₀ in 0–20 cm, and increased in N₁₀ and P₅ while decreased in other treatments in 20–40 cm. Most of the fertilization treatments decreased the respiratory quotient (qCO₂) in 0–20 cm but increased qCO₂ in 20–40 cm. Fertilization increased soil microbial functional diversity (except N₁₅) but decreased cumulative C mineralization (except in N₁₅ in 0–20 cm and N₅ in 20–40 cm). Soil organic C (SOC) decreased in P₅ and P₁₅ in 0–20 cm and for most of the fertilization treatments (except N₁₅P₁₅) in 20–40 cm. Overall, these results suggested that soils will not be a C sink (except N₁₅P₁₅). Nitrogen and phosphorus fertilization may lower the SOC pool by altering the plant biomass composition, especially the C:N ratios of different plant functional groups, and modifying C substrate utilization patterns of soil microbial communities. The N+P fertilization at 15 g m⁻² yr⁻¹ may be used in increasing plant aboveground biomass and soil C accumulation under these meadows.     

Effect of Degradation Intensity on Grassland Ecosystem Services in the Alpine Region of Qinghai-Tibetan Plateau,China

The deterioration of alpine grassland has great impact on ecosystem services in the alpine region of Qinghai-Tibetan Plateau. However, the effect of grassland degradation on ecosystem services and the consequence of grassland deterioration on economic loss still remains a mystery. So, in this study, we assessed four types of ecosystem services following the Millennium Ecosystem Assessment classification, along a degradation gradient. Five sites of alpine grassland at different levels of degradation were investigated in Guoluo Prefecture of Qinghai Province, China. The species composition, aboveground biomass, soil total organic carbon (TOC), and soil total nitrogen (TN) were tested to evaluate major ecological services of the alpine grassland. We estimated the value of primary production, carbon storage, nitrogen recycling, and plant diversity. The results show the ecosystem services of alpine grassland varied along the degradation gradient. The ecosystem services of degraded grassland (moderate, heavy and severe) were all significantly lower than non-degraded grassland. Interestingly, the lightly degraded grassland provided more economic benefit from carbon maintenance and nutrient sequestration compared to non-degraded. Due to the destruction of the alpine grassland, the economic loss associated with decrease of biomass in 2008 was $198/ha. Until 2008, the economic loss caused by carbon emissions and nitrogen loss on severely degraded grassland was up to $8 033/ha and $13 315/ha, respectively. Urgent actions are required to maintain or promote the ecosystem services of alpine grassland in the Qinghai-Tibetan Plateau.     

Carbon Balance in an Alpine Steppe in the Qinghai-Tibet Plateau

Carbon fluxes were measured using a static chamber technique in an alpine steppe in the Qinghai-Tibet Plateau from July 2000 to July 2001. It was shown that carbon emissions decreased in autumn and increased in spring of the next year, with higher values in growth seasons than in winters. An exponential correlation （Ecarbon = 0.22（exp（0.09T） ＋ In（0.31P ＋ 1））, R^2 = 0.77, P 〈 0.001） was shown between carbon emissions and environmental factors such as temperature （T） and precipitation （P）. Using the daily temperature （T） and total precipitation （R）, annual carbon emission from soil to the atmosphere was estimated to be 79.6 g C/m^2, 46% of which was emitted by microbial respiration. Considering an average net primary production of 92.5 g C/m^2 per year within the 2 year experiment, alpine steppes can take up 55.9 g CO2-C/m^2 per year. This indicates that alpine steppes are a distinct carbon sink, although this carbon reservoir was quite small.     

Carbon Balance in an Alpine Steppe in the Qinghai-Tibet Plateau

Carbon fluxes were measured using a static chamber technique in an alpine steppe in the Qinghai-Tibet Plateau from July 2000 to July 200t. It was shown that carbon emissions decreased in autumn and increased in spring of the next year, with higher values in growth seasons than in winters. An exponential correlation (Ecarbon = 0.22(exp(0.09T) + In(0.31P + 1)), R2 = 0.77, P < 0.001) was shown between carbon emissions and environmental factors such as temperature (T) and precipitation (P). Using the daily temperature (T) and total precipitation (R), annual carbon emission from soil to the atmosphere was estimated to be 79.6 g C/m2, 46% of which was emitted by microbial respiration. Considering an average net primary production of 92.5 g C/m2 per year within the 2 year experiment, alpine steppes can take up 55.9 g CO2-C/m2 per year. This indicates that alpine steppes are a distinct carbon sink, although this carbon reservoir was quite small.     

青藏高原高山植物麻花艽的传粉生态学研究

对青藏高原东部麻花艽 (Gentiana straminea)海北居群的传粉生态学进行了 3a连续的观察和实验。试验表明自然去雄、人工自交和杂交处理均结实 ,而人工去雄套袋和自然套袋不结实。麻花艽自交亲合 ,但必须依赖传粉媒介才能完成授粉过程 ,不存在无融合生殖。野外捕捉到 14种访花昆虫 ,它们分别属于 2个纲 ,7个目 ,8个科。观察和分析了各种昆虫的访花行为后 ,认为苏氏熊蜂 (Bombus sushikini)是麻花艽有效而稳定的传粉者。测量表明麻花艽花蜜通道的深度和苏氏熊蜂的舌长基本吻合。苏氏熊蜂的访花频率在 10 :0 0～ 12 :0 0 ,13:0 0～ 15 :0 0和 16 :0 0～ 18:0 0时间段没有差别 ,单花的访花频率为 0 .0 0 5次 / (花· min)。和其它高山植物相比 ,青藏高原高山植物麻花艽的访花频率较高。熊蜂传粉和高频率的访花维持了麻花艽在极端寒旱的青藏高原环境下的有性生殖。此外 ,高频率的访花对于维持该地区高山植物的生殖保障具有重要的现实意义 ,但是否具有普遍性 ,还有待研究更多的高山代表类群。     

青藏高原特有植物祁连獐牙菜（龙胆科）的花综合征与虫媒传粉

雌雄异熟和异型花在一些被子植物中的共同存在被认为是一种有效避免两性花植物自交并促进异交的机制。本文报道青藏高原特有植物祁连獐牙菜Swertia przewalskii Pissjauk.的花同时具有雌雄异熟和异型花这两种特征。在此基础上,我们详细观察了该植物访花昆虫的访花行为,主要探讨这种促进异花传粉的机制是否能完全避免株内异花传粉的自交。繁育系统试验证明祁连獐牙菜单花套袋不结实,说明雌雄异熟和异型花的花特征完全避免了单花的自花传粉,自然结实必须依赖昆虫传粉才能完成。然而,该植物却是高度自交亲和的,因为人     

青藏高原高寒草甸3种植物对模拟增温的生理生化响应

采用国际冻原计划(ITEX)模拟增温对植物影响的研究方法,将开项式生长室(OTC)按不同直径设置5个增温梯度,并按其直径从小到大的顺序依次标记为A、B、C、D、E 5个处理,研究了增温效应对青藏高原高寒草甸3种植物生理生化特性的影响。研究表明:（1） A~E 5个增温处理使OTC内部气温依次比对照升高了2.68 ℃、1.57 ℃、1.20 ℃、1.07 ℃ 和0.69 ℃,土壤温度依次比对照升高了1.74 ℃、1.06 ℃、0.80 ℃、0.60 ℃和0.30 ℃。（2）增温对3种植物的叶绿素含量、可溶性糖含量、可溶性蛋白含量、SOD活性、谷胱甘肽等生理生化特性产生了一系列的影响,3种植物对增温效应的响应也不尽相同。（3）增温对青藏高原3种植物生理生化特性的影响明显且复杂,适度增温0.69 ℃~1.07 ℃（D、E处理）对3种植物生理生化特性在总体上表现为正效应。