Chemical and Genetic Comparative Analysis of Gentiana crassicaulis and Gentiana macrophylla

Gentiana crassicaulis Duthie ex Burk . and Gentiana macrophylla Pall . are two main sources of Radix Gentianae Macrophyllae (Qinjiao) available in markets, which has a wide range of anti‐inflammatory effects and has been extensively used for fighting rheumatoid arthritis. However, they vary in terms of chemical compositions, pharmacological activities, and biomass. In this study, a combined chemical and genetic (HPLC and DNA barcoding) approach was used to compare these two plants. Four predominant bioactive compounds, namely, gentiopicroside, loganic acid, swertiamarin, and sweroside, were used to assess the chemical variations. Based on chemical variations, 15 samples were clustered into two groups through PCA analyses. DNA barcoding utilizing the variable nuclear ITS2 regions were sequenced, aligned, and compared. Together with 61 sequences collected from GenBank, 76 batches of Qinjiao were clustered in two groups according to species origin. The genetic relationships indicated by the ITS2‐based NJ tree were consistent with the chemical variations. Thus, the chemical profiles determined by HPLC and DNA profiles obtained from ITS2 region could be applied for the quality control of Qinjiao.     

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。     

Multiple autopolyploidizations and range expansion of Allium przewalskianum Regel. (Alliaceae) in the Qinghai‐Tibetan Plateau

We used the Allium przewalskianum diploid–tetraploid complex on the Qinghai‐Tibetan Plateau (QTP) as a model to examine how this complex responded to the Quaternary climatic oscillations, and whether multiple autopolyploidizations have occurred. We sequenced five chloroplast DNA (cpDNA) fragments (accD‐psaI, trnH‐psbA, trnL‐trnF, trnS‐trnG and rpl16‐intron) in 306 individuals (all of known ploidy level) from 48 populations across the distribution of this species complex. We identified a total of 32 haplotypes—11 in diploids only, 13 in tetraploids only, and 8 found in both cytotypes. This, plus network analyses, indicated that tetraploids have arisen independently from diploids at least eight times. Most populations in the eastern QTP contained multiple haplotypes, but only a single haplotype was found for 17 tetraploid populations on the western QTP, suggesting a recent colonization of the western QTP. We further found that this species complex underwent an earlier range expansion around 5–150 thousand years ago (kya), after the largest glacial period (800–170 kya) in the QTP. In addition, the high frequencies of tetraploids in the QTP suggested that the tetraploid A. przewalskianum cytotype has evolutionary advantages over diploids in colonizing and/or surviving the arid habitats of the QTP.     

Demographic history of the Tibetan antelope Pantholops hodgsoni (chiru)

Abstract The Tibetan antelope (chiru, Pantholops hodgsoni), a heavily poached species and symbol of the Qinghai‐Tibetan Plateau (QTP), is noted worldwide for its special calving migration. This species originated in the early Quaternary and it is interesting to know how the following climatic oscillations affected its demographic dynamics in the climate‐sensitive QTP. In this study, we analyzed the mitochondrial D‐loop region from 312 individuals sampled in all of the six major populations. We found high rates of gene flow and little genetic differentiation between populations, suggesting that the calving migration may have homogenized the genetic pool of this species. Both mismatch distribution analyses and coalescent simulations suggested that this species experienced a demographic expansion approximately 600–200 Kyr following the retreat of the large glaciers developed in the QTP at 800–600 Kyr, rather than at the end of the last glacial age, as previously suggested, based on a limited sample size. In addition, we found evidence of a chiru population decrease probably related to the human settings at the QTP during the middle Holocene.     

Phylogeography of the genus Dasiphora (Rosaceae) in the Qinghai‐Tibetan Plateau: divergence blurred by expansion

Plateau uprisings and climatic oscillations are considered to have caused extensive allopatric divergences that account for the rich species diversity of the Qinghai‐Tibetan Plateau (QTP). However, secondary contact during range shifts in the Quaternary glacial cycles or inter‐uplift stages may have restored the gene flow between species and so counteracted these divergences, particularly in rapidly‐adapting dominant elements. We tested this hypothesis by determining the phylogeographical history of Dasiphora (Rosaceae), a genus of two species that are widely distributed on the QTP and co‐exist in numerous localities. We sequenced two chloroplast DNA fragments (rbcL, trnT‐L) for 559 individuals from 87 populations. Bayesian methods were used to identify phylogenetic relationships and to estimate divergence times. Demographic histories were inferred using neutrality tests, mismatch distribution analysis, and coalescent simulation. A total of 112 haplotypes that clustered into three major groups were identified. The formation of these groups and their subgroups was dated to between the Pliocene and the late Pleistocene. In addition, we found that some groups underwent multiple extensive expansions. Species‐specific haplotypes were identified for each species, although these haplotypes phylogenetically intermixed. These results suggest that recent plateau uplifts and climatic oscillations might have caused the deep divergences observed within this genus. However, later range expansions probably blurred these divergences and possible species boundaries. Our results shed new light on the complex evolutionary history of the QTP alpine plants. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 777–788.     

The Pleistocene demography of an alpine juniper of the Qinghai‐Tibetan Plateau: tabula rasa,cryptic refugia or something else?

Aim Numerous palaeoecological and genetic studies have shown that different tree species responded in very different ways to Pleistocene climatic oscillations. Some were forced into small refugia far from their current range, while others were able to survive in small refugia close to, or even within, their current natural range. In this study we examine the Pleistocene demography of a juniper species (Juniperus przewalskii, Cupressaceae) from the Qinghai‐Tibetan Plateau. Location The Qinghai‐Tibetan Plateau (QTP). Methods Eight nuclear loci were sequenced in 141 individuals from 20 natural populations distributed across the entire natural range of J. przewalskii, and coalescent analysis was used to test demographic hypotheses. Results The overall nucleotide diversity in the sample was low (π_sil = 0.0029), with few rare alleles and pronounced population genetic structure (F_ST = 0.181). We detected a division previously found using chloroplast DNA markers: all segregating sites in populations from the central part of the QTP appear to be a subset of those found around the edge of the plateau, confirming the relatively young age of the former. In contrast to the middle Pleistocene bottlenecks detected in boreal tree species, the coalescent‐based analyses failed to reject the standard neutral model for the juniper species considered here. Main conclusions Juniperus przewalskii did not undergo marked changes in population sizes during the Pleistocene, although this species seems to have experienced recent, post‐glacial expansion. This finding is largely consistent with the limited number of previous studies on conifer species of the QTP, but contradicts findings of studies on boreal species. These findings have wide implications for understanding plant species’ responses to past climatic oscillations on the high‐elevation QTP.     

Allopatric divergence and phylogeographic structure of the plateau zokor (Eospalax baileyi), a fossorial rodent endemic to the Qinghai–Tibetan Plateau

Aim Most species of temperate regions are believed to have shifted to lower latitudes or elevations during the glacial periods of the Quaternary. In this study we test whether this phylogeographic assumption is also true for the plateau zokor (Eospalax baileyi), a fossorial rodent endemic to the climate-sensitive Qinghai–Tibetan Plateau (QTP), which ranges in elevation from 2600 to 4600 m. Location The QTP of western China. Methods Phylogeographic analyses were conducted based on the mitochondrial cytochrome b gene sequences of 193 individuals from 20 populations over the entire range of the species. Results A total of 54 haplotypes identified in the present study clustered into four geographically correlated clades located in the interior of the QTP (clade A) and at the plateau edge (B, C and D). Molecular calibrations suggest that the interior plateau (A) and plateau-edge (B–D) clades diverged at 1.2 Ma and that the three plateau-edge clades diverged between 0.85 and 0.80 Ma. These estimates are concordant with diastrophism and glaciation events in the QTP. Coalescent tests rejected both the hypothesis that all current populations originated from a single refugium at a low elevation during the Last Glacial Maximum (LGM) and the hypothesis that the two lineages diverged during the LGM. The tests instead supported the hypothesis that there were four refugia during the LGM, and that the four clades diverged prior to the late Pleistocene. Main conclusions Our results suggest that Quaternary diastrophisms and glaciations repeatedly promoted allopatric divergence of the plateau zokor into geographical clades, and that these regional clades subsequently persisted at high elevations, rather than migrating to the low-elevation plateau edge during subsequent glacial ages.     

Extensive population expansion of Pedicularis longiflora (Orobanchaceae) on the Qinghai‐Tibetan Plateau and its correlation with the Quaternary climate change

The Qinghai‐Tibetan Plateau (QTP) is thought to be more strongly affected by the Quaternary glaciations than most other regions of the same latitude. It would be of great interest to investigate the population genetic structure of organisms distributed on the platform and its correlation with the Quaternary climatic oscillations. Here we used the chloroplast (cp)DNA trnT‐trnF sequence to study genetic variation and phylogeography of Pedicularis longiflora, an alpine herb with extensive distribution on the QTP. Based on a range‐wide sampling comprising 41 populations and 910 individuals, we detected 30 cpDNA haplotypes that were divided into five clades by phylogenetic and network analyses and a strong phylogeographical structure. All haplotypes but one in the three basal clades occur exclusively in the southeast QTP, whereas haplotypes in the young clade V occupy almost the whole species range. In particular, the young haplotype H18 occurs in 420 individuals, even at a frequency of 100% in some QTP platform populations and the Altai population. The haplotype distribution pattern, together with molecular clock estimation and mismatch distribution analysis, suggests that the southeast QTP was either a refuge for P. longiflora during the Quaternary climatic change or is the place of origin of the species. The present wide distribution of the species on the QTP platform has resulted from recent population expansions which could be dated back to 120 000–17 000 years ago, a period mostly before the last glacial maximum. The possible relationships among geographic genetic structure, climatic change and species diversification in Pedicularis are also discussed.     

Quaternary climate change and habitat preference shaped the genetic differentiation and phylogeography of Rhodiola sect. Prainia in the southern Qinghai–Tibetan Plateau

There are two long‐standing biogeographic hypotheses regarding the glacial survival of plant species in the Qinghai–Tibetan Plateau (QTP): the in situ survival hypothesis and the tabula rasa hypothesis. We tested these two hypotheses in a phylogeographic study of Rhodiola sect. Prainia, a monophyletic section with ecologically divergent lineages. Molecular data from the nuclear internal transcribed spacer, six plastid markers and 13 nuclear microsatellite loci were analyzed for 240 individuals from 19 populations of this section. Environmental data were used to analyze the niches of major phylogenetic lineages within this section and to model changes in their distributions since the Last Glacial Maximum (LGM). We found that Rhodiola sect. Prainia consists of three evolutionary lineages: all populations of R. stapfii, R. prainii populations at the southern edge of the QTP, and R. prainii populations in the interior part of the QTP. During the LGM, the survival of R. prainii in the interior part of the QTP corresponded with the in situ survival hypothesis, while R. stapfii most probably survived the LGM in a manner corresponding with the tabula rasa hypothesis. The evolutionary history of different lineages of this section was shaped by topography, climate change, and lineage‐specific habitat preferences.     

Induction of <Emphasis Type="Italic">Gentiana cruciata</Emphasis> hairy roots and their secondary metabolites

Gentiana cruciata L. (cross gentian) is a medicinal and ornamental plant. The root extracts of this species are known to exhibit many curative properties. The natural Gentiana populations are exposed to great danger because of their uncontrolled usage. In this study, hairy roots from Gentiana cruciata L. stem and leaf explants belonging to three different clones were induced by inoculation with four different Agrobacterium rhizogenes wild strains namely A4, 15834, 8196 and R1000. Induction of the root transformation was significantly dependent on the explant type used. On the other hand, the genotype and bacterial strain had no significant effect on hairy root formation. Hairy root formation percentages of the explants varied between 5.6–33.3% in the stem explants, and between 0.0–6.7% in the leaf explants. Transformations of the hairy roots were confirmed by PCR using rolC specific primers, and revealed the absence of contaminating A. rhizogenes with virC primers. Total of twelve hairy root clones were obtained, and their secondary metabolite content was also analyzed by HPLC. Quantitative results exhibited that gentiopicroside was the most abundant compound in all root samples. Furthermore, metabolites such as loganic acid, swertiamarin, and sweroside were also identified and quantified in the samples.     

Responses of plants to changes in Qinghai–Tibetan Plateau and glaciations: Evidence from phylogeography of a Sibiraea (Rosaceae) complex

The genetic structure and phylogeographical history of the alpine shrubs Sibiraea angustata (Rosaceae) and Sibiraea laevigata from the Qinghai–Tibetan Plateau (QTP) were investigated to identify alpine plant responses to changes in the QTP and glaciations. Fifty-five populations were analyzed using four chloroplast DNA (cpDNA) regions and (nuclear ribosomal internal transcribed spacer) nrITS sequence data. In all, 21 cpDNA haplotypes and 13 nrITS sequence types were detected. Analyses of the genetic diversity and phylogenetic relationships detected two rarely reported glacial refugia. One was the Yushu–Nangqian area, and the other consisted of the area from the Songpan Plateau to the southeastern margin of the QTP. Sibiraea species populations experienced divergent evolution and founder effects when they recolonized the QTP platform and adjacent high-altitude regions following glaciations. The divergence times of the main lineages and haplotypes were in the range of 1.60–2.58 Ma. The population size of Sibiraea species in the QTP decreased approximately 23-fold during the last 0.12 Ma, indicating that Sibiraea species were significantly affected by environmental changes in the QTP. Therefore, the rapid uplift of the QTP and subsequent glaciations likely played an important role in driving genetic divergence and population size changes of Sibiraea species in the QTP.     

History and evolution of alpine plants endemic to the Qinghai-Tibetan Plateau: Aconitum gymnandrum (Ranunculaceae)

How Quaternary climatic oscillations affected range distributions and intraspecific divergence of alpine plants on the Qinghai‐Tibetan Plateau (QTP) remains largely unknown. Here, we report a survey of chloroplast DNA (cpDNA) and nuclear ribosomal internal transcribed spacer (ITS) DNA variation aimed at exploring the phylogeographical history of the QTP alpine endemic Aconitum gymnandrum. We sequenced three cpDNA fragments (rpl20–rps12 intergenic spacer, the trnV intron and psbA‐trnH spacer) and also the nuclear (ITS) region in 245 individuals from 23 populations sampled throughout the species’ range. Two distinct lineages, with eastern and western geographical distributions respectively, were identified from a phylogenetic analysis of ITS sequence variation. Based on a fast substitution rate, these were estimated to have diverged from each other in the early Pleistocene approximately 1.45 Ma. The analysis of cpDNA variation identified nine chlorotypes that clustered into two major clades that were broadly congruent in geographical distribution with the two ITS lineages. The east–west split of cpDNA divergence was supported by an amova which partitioned approximately half of the total variance between these two groups of populations. Analysis of the spatial distribution of chlorotypes showed that each clade was subdivided into two groups of populations such that a total of four population groups existed in the species. It is suggested that these different groups derive from four independent glacial refugia that existed during the Last Glacial Maximum (LGM), and that three of these refugia were located at high altitude on the QTP platform itself at that time. Coalescent simulation of chlorotype genealogies supported both an early Pleistocene origin of the two main cpDNA clades and also the ‘four‐refugia’ hypothesis during the LGM. Two previous phylogeographical studies of QTP alpine plants indicated that such plants retreated to refugia at the eastern/south‐eastern plateau edge during the LGM and/or previous glacial maxima. However, the results for A. gymnandrum suggest that at least some of these cold‐tolerant species may have also survived centrally on the QTP platform throughout the Quaternary.     

红外光谱技术在滇龙胆栽培方式选择上的应用

该研究采用傅里叶变换红外光谱结合化学计量学,对条播、撒播、剪根后移栽、扦插和剪枝后移栽的滇龙胆进行了分析,以筛选滇龙胆的最佳栽培方式。结果表明:(1)不同栽培方式的滇龙胆原始谱图在峰形、峰位和峰强上有一定差异;用小波去噪法对光谱进行优化处理并进行偏最小二乘判别分析(Partial least squares discriminant analysis,PLS-DA),能较好地区分不同栽培方式的滇龙胆样品,PLS-DA二维得分图显示同一栽培方式的样品聚在一起,表明相同栽培方式的滇龙胆化学组成和含量差异较小;播种滇龙胆样品(条播和撒播)距离较近,移栽滇龙胆样品(剪根、扦插和剪枝)距离较近,而播种和移栽滇龙胆样品距离较远,表明栽培方式对滇龙胆化学成分的积累有影响。(2)滇龙胆四种主要成分总含量大小依次是剪枝剪根撒播条播扦插,除剪根后移栽,剪枝后移栽滇龙胆中四种主要成分总含量显著高于其他栽培方式下的滇龙胆(P0.05),剪枝后移栽滇龙胆质量最佳。(3)以液相数据为参考值,采用正交信号校正—偏最小二乘回归模型预测不同栽培模式滇龙胆中龙胆苦苷、马钱苷酸、獐牙菜苦苷和当药苷的含量。校正集和验证集的决定系数(R2)均大于0.90,校正均方根误差、交叉验证均方差和预测均方根误差均小于1.65,模型相关性和预测效果好,该方法对红外光谱分析在中药领域的推广应用提供了参考。     

不同生长季节下藏药麻花秦艽活性成分含量研究

采用高效液相色谱法测定了野生与栽培藏药麻花秦艽（Gentiana straminea）根中龙胆苦苷、落干酸、獐牙菜苦苷和獐牙菜苷四种环烯醚萜苷类化学成分的含量及其在不同生长季节的变化趋势。结果表明,4种环烯醚萜苷类成分的含量随植物的生长季节而波动,其活性成分含量在野生种与栽培种之间发生了一定的差异。但是,栽培根中的龙胆苦甙已达到药典的标准,可供药用。     

Simultaneous determination of four iridoid and secoiridoid glycosides and comparative analysis of Radix Gentianae Macrophyllae and their related substitutes by HPLC

Introduction – Radix Gentianae Macrophyllae, a traditional Chinese medicine, has been frequently used to dispel rheumatism and ease pain. There are four species of Gentiana (G. macrophylla, G. straminea, G. dahurica and G. crassicaulis) recorded as herbal drugs in the Chinese Pharmacopoeia and two other Gentiana species (G. officinalis and G. siphonantha) are often used as substitutes. Currently, the LC fingerprint comparison among different species and evidence for the equivalent application of these herbs are lacking. Objective – To develop an HPLC method for the simultaneous determination of four iridoid and secoiridoid glycosides and a comparative study of six species of Gentiana. Methodology – HPLC analysis was performed on a C₁₈ column (Phenomenex, 150 × 4.6 mm, 5 µm particle size) with gradient elution using 0.4% aqueous phosphoric acid and methanol at 242 nm. Results – The proposed method was precise, accurate and sensitive enough for simultaneous quantitative evaluation of four iridoid and secoiridoid glycosides (loganic acid, swertiamarin, gentiopicroside and sweroside) in the six species of Gentiana. Contents of the four marker compounds varied from each other even among the samples from the same species and the LC chromatograms of the six species of Gentiana showed high similarities. Conslusion – The close similarity of LC chromatograms and chemical composition of the four genuine Gentiana species explain their popular usage as Radix Gentianae Macrophyllae in Chinese medicine. By comparing the four genuine Gentiana species, it is suggested that the two substitutes could be used as Radix Gentianae Macrophyllae to relieve the scarcity of resources. Copyright © 2010 John Wiley & Sons, Ltd.     

River islands,refugia and genetic structuring in the endemic brown frog Rana kukunoris (Anura,Ranidae) of the Qinghai‐Tibetan Plateau

Frequently, Pleistocene climatic cycling has been found to be the diver of genetic structuring in populations, even in areas that did not have continental ice sheets, such as on the Qinghai‐Tibetan Plateau (QTP). Typically, species distributed on the plateau have been hypothesized to re‐treat to south‐eastern refugia, especially during the Last Glacial Maximum (LGM). We evaluated sequence variation in the mitochondrial DNA gene Cytb and the nuclear DNA gene RAG‐1 in Rana kukunoris, a species endemic to the QTP. Two major lineages, N and S, were identified, and lineage N was further subdivided into N1 and N2. The geographical distribution and genealogical divergences supported the hypothesis of multiple refugia. However, major lineages and sublineages diverged prior to the LGM. Demographical expansion was detected only in lineage S and sublineage N2. Sublineage N1 might have survived several glacial cycles in situ and did not expand after the LGM because of the absence of suitable habitat; it survived in river islands. Genetic analysis and environment modelling suggested that the north‐eastern edge of QTP contained a major refugium for R. kukunoris. From here, lineage S dispersed southwards after the LGM. Two microrefugia in northern Qilian Mountains greatly contributed to current level of intraspecific genetic diversity. These results were found to have important implications for the habitat conservation in Northwest China.     

HPLC Determination of Contents of Four Active Constituents in Tibetan Medicine Gentiana straminea ( Gentianaceae) During Different Growing Period

Contents of four iridoid glycosides viz. gentiopicroside, longanic acid, swertiamarin and sweroside of wild and cultivated Gentiana straminea roots gathered in different seasons were analyzed by means of HPLC determination. The results indicated that contents of the four iridoid glycosides varied greatly with different seasons between wild and cultivated Gentiana straminea. And according to the criterion of pharmacopoeia of China, after cultivation content of gentiopicroside in Gentiana str aminea root had accorded with the regulation and can preliminarily replaced the wild species to use as a plant medicine. The mechanism causing the content changes of the four iridoid glycosides may be due to the integrated effects of many factors, such as the ecological factors, the genetic factors.     

农林复合系统滇龙胆茎叶化学计量特征研究

以农林复合系统种植的滇龙胆(Gentiana rigescens Franch.ex Hemsl.)为材料,采用高效液相色谱法建立不同栽培系统滇龙胆茎、叶的色谱指纹图谱,并测定其主要活性成分马钱苷酸、獐牙菜苦苷、龙胆苦苷和当药苷含量,研究不同栽培系统滇龙胆茎、叶化学计量特征。采用相关性分析、指纹图谱相似度分析、偏最小二乘判别分析(PLS-DA)、变量投影重要性准则(VIP)等方法进行化学数据分析。结果显示,滇龙胆主要活性成分马钱苷酸含量为(1.85±0.92)mg/g~(7.43±7.64)mg/g,獐牙菜苦苷含量为(1.03±0.17)mg/g~(1.58±0.50)mg/g,龙胆苦苷含量为(15.28±11.34)mg/g~(24.59±7.84)mg/g,当药苷含量为(4.10±1.64)mg/g~(31.67±22.70)mg/g,且叶片中4种活性成分的总含量高于茎;不同栽培系统中,与尼泊尔桤木间作的滇龙胆茎、叶活性成分总含量最高,而与核桃间作的滇龙胆茎、叶活性成分总含量最低。相关性分析显示,植株相同部位和不同部位间的环烯醚萜和裂环烯醚萜含量呈显著(P0.05)或极显著(P0.01)正相关。指纹图谱相似度分析表明,不同栽培系统滇龙胆茎指纹图谱相似度介于0.989~0.992之间、叶指纹图谱相似度为0.988~0.996,相同部位样品化学成分种类相似。PLS-DA分析结果表明,茎和叶片整体化学计量特征具有明显差异;单作及林药间作的样品被区分为不同类群,不同间作模式下滇龙胆茎、叶化学成分具显著差异,叶片高效液相色谱指纹图谱可用于区分不同栽培系统滇龙胆样品。本研究结果可为农林复合系统滇龙胆有效成分含量研究及滇龙胆资源的合理开发利用提供科学依据。     

Phylogeographic structure of Hippophae tibetana (Elaeagnaceae) highlights the highest microrefugia and the rapid uplift of the Qinghai‐Tibetan Plateau

The uplift of the Qinghai‐Tibetan Plateau (QTP) dramatically changed the topography and climate of Asia and affected the biodiversity of the plateau and its adjacent areas. However, the effects of the uplift on the dispersal, differentiation and adaptation of plants remain a puzzle when the date and processes of the uplift cannot be determined with certainty and the impacts of the Quaternary glaciations on plants on the QTP are unknown. To clarify the relationships among plants on the QTP with the plateau uplift and the Quaternary glaciations, the cpDNA trnT‐trnF regions of 891 individuals from 37 populations of Hippophae tibetana, endemic to the QTP, were sequenced in the present study. A total of 50 haplotypes were found and a strong phylogeographic structure was revealed (N_ST = 0.854, G_ST = 0.611, N_ST > G_ST, P < 0.01). The results show that three main lineages of the present populations of H. tibetana occupy the western, the middle, and the eastern geographical range, respectively, and their divergence time dates back to 3.15 Ma before present. Of 50 haplotypes, 33 (66%) are private haplotypes, which are restricted to single populations. These private haplotypes are scattered throughout the present geographical range of H. tibetana and originated from multiple differentiations in many lineages during more than 1.0 Ma period, strongly suggesting that multiple microrefugia of H. tibetana existed throughout the present geographical range during the last glacial maximum (LGM) and even earlier glaciations. Additionally, the average elevation of present populations is over 4500 m in the west and the equilibrium‐line of glaciers in the LGM was 500–300 m lower than present in the major interior part of the plateau suggesting that at most sites in the west, LGM microrefugia of H. tibetana may have been above 4000 m above sea level, the highest of all known refugia. Moreover, the divergence times among and within the three lineages and their distinct distributions as well as dispersal barriers support the theory of the recent and rapid uplift of the QTP. The rapid uplift of the plateau within the last 3.4 Ma and the associated environmental changes may have affected the dispersal and differentiation of H .tibetana and shaped its phylogeographic structure.