Potential refugium on the Qinghai-Tibet Plateau revealed by the chloroplast DNA phylogeography of the alpine species Metagentiana striata (Gentianaceae)

Metagentiana striata is an alpine annual herbaceous plant endemic to the east of the Qinghai-Tibet (Q-T) Plateau and adjacent areas. The phylogeography of M. striata was studied by sequencing the chloroplast DNA (cpDNA) trnS-trnG intergenic spacer. Ten haplotypes were identified from an investigation of 232 individuals of M. striata from 14 populations covering the entire geographical range of this species. The level of differentiation amongst populations was very high (G(ST) = 0.746; N-ST = 0.774) and a significant phylogeographical structure was observed (P < 0.05). An analysis of molecular variance found a high variation amongst populations (76%), with F-ST = 0.762 (highly significant, P < 0.001), indicating that little gene flow occurred amongst the different regions; this was explained by the isolation of populations by high mountains along the Q-T Plateau and adjacent areas (N-m = 0.156). Only one ancestral haplotype (A) was common and widespread throughout the distributional range of M. striata. The populations of the Hengduan Mountains region of the south-eastern Q-T Plateau showed high diversity and uniqueness of haplotypes. It is suggested that this region was the potential refugium of M. striata during the Quaternary glaciation, and that interglacial and postglacial range expansion occurred from this refugium. This scenario was in good agreement with the results of nested clade analysis, which inferred that the current spatial distribution of cpDNA haplotypes and populations resulted from range expansion, together with past allopatric fragmentation events. (c) 2008 The Linnean Society of London.

Changes in plant biomass and species composition of alpine Kobresia meadows along altitudinal gradient on the Qinghai-Tibetan Plateau

Alpine Kobresia meadows are major vegetation types on the Qinghai-Tibetan Plateau. There is growing concern over their relationships among biodiversity, productivity and environments. Despite the importance of species composition, species richness, the type of different growth forms, and plant biomass structure for Kobresia meadow ecosystems, few studies have been focused on the relationship between biomass and environmental gradient in the Kobresia meadow plant communities, particularly in relation to soil moisture and edaphic gradients. We measured the plant species composition, herbaceous litter, aboveground and belowground biomass in three Kobresia meadow plant communities in Haibei Alpine Meadow Ecosystem Research Station from 2001 to 2004. Community differences in plant species composition were reflected in biomass distribution. The total biomass showed a decrease from 13196.96 +/- 719.69 g/m(2) in the sedge-dominated K. tibetica swamp to 2869.58 +/- 147.52 g/m(2) in the forb and sedge dominated K. pygmaea meadow, and to 2153.08 +/- 141.95 g/m(2) in the forbs and grasses dominated K. humilis along with the increase of altitude. The vertical distribution of belowground biomass is distinct in the three meadow communities, and the belowground biomass at the depth of 0-10 cm in K. tibetica swamp meadow was significantly higher than that in K. humilis and K. pygmaea meadows (P < 0.01). The herbaceous litter in K. tibetica swamp was significantly higher than those in K. pygnaeca and K. humilis meadows. The effects of plant litter are enhanced when ground water and soil moisture levels are raised. The relative importance of litter and vegetation may vary with soil water availability. In the K. tibetica swamp, total biomass was negatively correlated to species richness (P < 0.05); aboveground biomass was positively correlated to soil organic matter, soil moisture, and plant cover (P < 0.05); belowground biomass was positively correlated with soil moisture (P < 0.05). However, in the K. pygnaeca and K. humilis meadow communities, aboveground biomass was positively correlated to soil organic matter and soil total nitrogen (P < 0.05). This suggests that the distribution of biomass coincided with soil moisture and edaphic gradient in alpine meadows.

Genetic variation in the endangered Anisodus tanguticus (Solanaceae), an alpine perennial endemic to the Qinghai-Tibetan Plateau

We used random amplified polymorphic DNA markers (RAPDs) to assess genetic variation between- and within-populations of Anisodus tanguticus (Solanaceae), an endangered perennial endemic to the Qinghai-Tibetan Plateau with important medicinal value. We recorded a total of 92 amplified bands, using 12 RAPD primers, 76 of which (P = 82.61%) were polymorphic, and calculated values of H-t and H-sp of 0.3015 and 0.4459, respectively, suggesting a remarkably high rate of genetic variation at the species level. The average within-population diversity also appeared to be high, with P, H-e and H-pop values of 55.11%, 0.1948 and 0.2918, respectively. Analyses of molecular variance (AMOVA) showed that among- and between-population genetic variation accounted for 67.02% and 32.98% of the total genetic variation, respectively. In addition, Nei's coefficient of differentiation (G(ST)) was found to be high (0.35), confirming the relatively high level of genetic differentiation among the populations. These differentiation coefficients are higher than mean corresponding coefficients for outbreeding species, but lower than reported coefficients for some rare species from this region. The genetic structure of A. tanguticus has probably been shaped by its breeding attributes, biogeographic history and human impact due to collection for medicinal purposes. The observed genetic variations suggest that as many populations as possible should be considered in any planned in situ or ex situ conservation programs for this species.

Effect of imitated global warming on Delta C-13 values in seven plant species growing in Tibet alpine meadows

Open-top chambers were used to estimate the possible effects of global warming on delta C-13 of seven plant species grown in alpine meadow ecosystem. The delta C-13 values of plant species were lower after long-term growth in open-top chambers. In the course of experiment, temperature significantly increased inside the chambers by 4 degrees C. Plant species grown at a lower elevation above sea level had higher delta C-13 values as compared to those grown at a higher elevation. This was in accordance with the effect of open-top chamber on delta C-13 values in plants. Greater availability of CO2 and lower water vapor as indicated by an increase in discrimination against (CO2)-C-13, probably result in more negative delta C-13 values of plants because higher stomatal conductance increases availability of CO2 and causes greater discrimination against (CO2)-C-13. The plant species studied could be the indicator species for testing global warming by the change in carbon isotope ratios at the two growth temperatures.

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.

Short-term effect of targeted placements of sheep excrement on grassland in inner mongolia on soil and plant parameters

Grazing animal excrement plays an important role in nutrient cycling and redistribution in grazing ecosystems, due to grazing in large areas and return in small areas. To elucidate the changes to the soil and pasture caused by sheep urine, fresh dung, and compost patches, a short- term field experiment using artificially placed pats was set up in the autumn of 2003 in the Inner Mongolian steppe. Urine application significantly increased soil pH during the first 32 days in soil layers at depths of both 0 - 5 cm and 5 - 15 cm. Rapid hydrolysis of urea gave large amounts of urine- nitrogen ( N) as ammonium ( NH4+) in soil extracts and was followed by apparent nitrification from day 2. Higher inorganic N content in the urine- treated soil was found throughout the experiment compared with the control. No significant effects of sheep excrement on soil microbial carbon ( C) and soil microbial N was found, but microbial activities significantly increased compared with the control after application of sheep excrement. Forty- six percent of dung- N and 27% of compost- N were transferred into vegetation after the experiment. The results from this study suggest that large amounts of nutrients have been lost from the returned excrement patches in the degraded grassland of Inner Mongolia, especially from sheep urine- N.

Defoliation, nitrogen, and competition: effects on plant growth and resource allocation of Cleistogenes squarrosa and Artemisia frigida

Two species, Artemisia frigida Willd. (C-3, semishrub, and dominant on overgrazed sites) and Cleistogenes squarrosa (Trin.) Keng (C-4, perennial bunchgrass, and dominant or codominant on moderately grazed sites) were studied to determine the effects of defoliation, nitrogen (N) availability, competition, and their interactions on growth, biomass, and N allocation in a greenhouse experiment. The main treatments were: two nitrogen levels (NO = 0 mg N pot(-1), N1 = 60 mg N pot(-1)), two defoliation intensities (removing 60% of total aboveground biomass and no defoliation), and three competitive replacement series (monocultures of each species and mixtures at 0.5:0.5). Our results were inconsistent with our hypothesis on the adaptive mechanisms of A. frigida regarding the interactive effects of herbivory, N, and competition in determining its dominant position on overgrazed sites. Cleistogenes squarrosa will be replaced by A. frigida on over-grazed sites, although C. squarrosa had higher tolerance to defoliation than did A. frigida. Total biomass and N yield and N-15 recovery of C. squarrosa in mixed culture were consistently lower than in monocultures, whereas those of A. frigida grown in mixtures were consistently higher than in monocultures, suggesting higher competitive ability of A. frigida. Our results suggest that interspecific competitive ability may be of equal or greater importance than herbivory tolerance in determining herbivore-induced species replacement in semi-arid Inner Mongolian steppe. In addition, the dominance of A. frigida on overgrazed sites has been attributed to its ability to shift plant-plant interactions through (lap colonization, root niche differentiation, and higher resistance to water stress.

Effects of altitude on plant-species diversity and productivity in an alpine meadow, Qinghai-Tibetan plateau

During the growing seasons of 2002 and 2003, biomass productivity and diversity were examined along an altitudinal transect on the south-western slope of Beishan Mountain, Maqin County (33 degrees 43'-35 degrees 16'N, 98 degrees 48'-100 degrees 55'E), Qinghai-Tibetan Plateau. Six altitudes were selected, between 3840 and 4435 m. Soil organic matter, soil available N and P and environmental factors significantly affected plant-species diversity and productivity of the alpine meadows. Aboveground biomass declined significantly with increasing altitude (P < 0.05) and it was positively and linearly related to late summer soil-surface temperature. Belowground biomass (0 - 10-cm depth) was significantly greater at the lowest and highest altitudes than at intermediate locations, associated with water and nutrient availabilities. At each site, the maximum belowground biomass values occurred at the beginning and the end of the growing seasons (P < 0.05). Soil organic matter content, and available N and P were negatively and closely related to plant diversity (species richness, Shannon-Wiener diversity index, and Pielou evenness index).

Phylogenetic origins of the Himalayan endemic Dolomiaea, Diplazoptilon and Xanthopappus (Asteraceae : Cardueae) based on three DNA regions

Background and Aims It is an enduring question as to the mechanisms leading to the high diversity and the processes producing endemics with unusual morphologies in the Himalayan alpine region. In the present study, the phylogenetic relationships and origins of three such endemic genera were analysed, Dolomiaea, Diplazoptilon and Xanthopappus, all in the tribe Cardueae of Asteraceae.Methods The nuclear rDNA internal transcribed spacer (ITS) and plastid trnL-F and psbA-trnH regions of these three genera were sequenced. The same regions for other related genera in Cardueae were also sequenced or downloaded from GenBank. Phylogenetic trees were constructed from individual and combined data sets of the three types of sequences using maximum parsimony, maximum likelihood and Bayesian analyses.Key Results The phylogenetic tree obtained allowed earlier hypotheses concerning the relationships of these three endemic genera based on gross morphology to be rejected. Frolovia and Saussurea costus were deeply nested within Dolomiaea, and the strong statistical support for the Dolomiaea-Frolovia clade suggested that circumscription of Dolomiaea should be more broadly redefined. Diplazoptilon was resolved as sister to Himalaiella, and these two together are sister to Lipschitziella. The clade comprising these three genera is sister to Jurinea, and together these four genera are sister to the Dolomiaea-Frolovia clade. Xanthopappus, previously hypothesized to be closely related to Carduus, was found to be nested within a well-supported but not fully resolved Onopordum group with Alfredia, Ancathia, Lamyropappus, Olgaea, Synurus and Syreitschikovia, rather than the Cardinis group. The crude dating based on ITS sequence divergence revealed that the divergence time of Dolomiaea-Frolovia from its sister group probably occurred 13.6-12.2 million years ago (Ma), and the divergence times of the other two genera, Xanthopappus and Diplazoptilon, from their close relatives around 5.7-4.7 Ma and 2.0-1.6 Ma, respectively.Conclusions The findings provide an improved understanding of the intergeneric relationships in Cardueae. The crude calibration of lineages indicates that the uplifts of the Qiinghai -Tibetan Plateau since the Miocene might have served as a continuous stimulus for the production of these morphologically aberrant endemic elements of the Himalayan flora.

Enrichment of stable carbon and nitrogen isotopes of plant populations and plateau pikas along altitudes

Stable carbon and nitrogen isotope ratios were measured in plant populations and plateau pikas to determine enrichment in stable isotopes of three alpine meadow ecosystems at different elevations in the Qinghai-Tibet Plateau. The results indicated that stable carbon isotope signatures of plant populations at the three locations showed significant variations, delta C-13 of plant populations showed an enrichment of 0.86 parts per thousand per 1000 in over the linear proportion of the altitudinal response, while stable nitrogen isotopes showed no apparent difference. The stable nitrogen isotopes of plateau pikas became significantly isotopically heavier along altitudinal gradients and showed an enrichment of 3.17 parts per thousand/km. Stable carbon isotopes showed no significance, however, and the enrichment of 0.448 parts per thousand/km. delta C-13 and delta N-15 in plateau pikas were not significantly correlated. There appeared to be segregation between the metabolism of stable carbon and nitrogen isotopes of plateau pikas. Variances in metabolic rate at lower water availability and temperatures are presumed to be the main cause of enrichment of stable nitrogen isotopes along altitudinal gradients. Attention should be paid to construct trophic positions and to trace food chain information based oil an isotopic enrichment model.

Molecular authentication of the traditional Tibetan medicinal plant Swertia mussotii

Swertia mussotii is an important species in Tibetan folk medicine. However, it is quite expensive and frequently adulterated, so reliable methods for authentication of putative specimens and preparations of the species are needed to protect consumers and to support conservation measures. We show here that the chloroplast (cp) DNA rpl16 intron has limited utility for differentiating S. mussotii from closely related species, since the cpDNA rpl16 sequences are identical in S. mussotii and two other species of Swertia. However, the rDNA internal transcribed spacer (ITS) sequences differ significantly between S. mussotii and all of 13 tested potential adulterants. Thus, the ITS region provides a robust molecular marker for differentiating the medicinal S. mussotii from related adulterants. Therefore, a pair of allele-specific diagnostic primers based on the divergent ITS region was designed to distinguish S. mussotii from the other species. Authentication by allele-specific diagnostic PCR using these primers is convenient, effective and both simpler and less time-consuming than sequencing the ITS region.

Response of chlorophyll fluorescence to dynamic light in three alpine species differing in plant architecture

A study was carried out to examine the effect of dynamic photosynthetically active photon flux density (PPFD) on photoinhibition and energy use in three herbaceous species, prostrate Saussurea superba, erect-leaved S. katochaete, and half-erect-leaved Gentiana straminea, from the Qinghai-Tibet Plateau. Chlorophyll fluorescence response was measured under each of three sets of high-low PPFD combinations: 1700-0, 1400-300, and 1200-500 mu mol m(-2) s(-1), illuminating in four dynamic frequencies: 1, 5, 15, and 60 cycles per 2 h. The total light exposure time was 2h and the integrated PPFD was the same in all treatments. The highest frequency of PPFD fluctuation resulted in the lowest photochemical activity, the highest level of non-photochemical quenching, and the greatest decrease of F-v/F-m (maximal photochemical efficiency of PSII). The 5 and 15 cycles per 2h treatments resulted in higher photochemical activity than the 1 cycle per 2h treatment. The 1700-0 PPFD combination led to the lowest photochemical activity and more serious photoinhibition in all species. S. superba usually exhibited the highest photochemical activity and CO2 uptake rate, the lowest reduction of F-v/F-m,F- and the smallest fraction of energy in thermal dissipation. With similar fractions of thermal dissipation, S. katochaete had relatively less photoinhibition than G. straminea owing to effective F-o quenching. The results suggest that high frequency of fluctuating PPFD generally results in photoinhibition, which is more serious under periods of irradiation with high light intensity. (c) 2005 Elsevier B.V. All rights reserved.

Distribution and species diversity of plant communities along transect on the Northeastern Tibetan plateau

The distribution and species diversity of plant communities along a 600 km transect through the northeastern Tibetan Plateau (32 degrees 42'-35 degrees 07' N, 101 degrees 02'-97 degrees 38' E) with altitudes from 3255 to 4460 m are described. The transect started from the Youyi Bridge of Banma through Dari, Maqin and Maduo to Zaling Lake. The data from 47 plots along the transect are summarized and analyzed. The mean annual temperature, the mean annual rainfall and the length of growing season decreases from 2.6 to -4.5 degrees C, from 767.2 to 240.1 mm, from 210 to 140 days, respectively, along the transect from the southeastern Banma to northwestern Zaling Lake. The number of vascular plant species recorded in 47 plots is 242 including 2 tree, 34 shrub, 206 herb species. Main vegetation types on the transect from southeast to northwest are: Sabina convallium forest, Picea likiangensis forest, Pyracantha fortuneana + Spiraea alpina shrub, Hippophae neurocarpu shrub, Sibiraea angustata + Polygonum viviparum shrub, Stellera chamaejasme herb meadow, Potentilla fruticosa + Salix obscura + Carex sp. Shrub, Kobresia capillifolia meadow, P. froticosa + Kobresia humilis shrub, Caragana jubata + S. obscura shrub, Kobresia tibetica meadow, Kobresia pygmaea meadow, K. pygmaea + Stipa purpurea steppe meadow, Stipa purpurea steppe. Plant richness and diversity index all showed a decreasing trend with increasing of elevation along transect from southeast to northwest. Detailed information on altitudinal ranges and distribution of the alpine vegetation, vascular flora and environments over the alpine zone at northeastern Tibetan Plateau provides baseline records relevant to future assessment of probable effects of global climate changes.

The effect of land management on plant community composition, species diversity, and productivity of alpine Kobersia steppe meadow

Grassland degradation is widespread and severe on the Tibet Plateau. To explore management approaches for sustainable development of degraded and restored ecosystems, we studied the effect of land degradation on species composition, species diversity, and vegetation productivity, and examined the relative influence of various rehabilitation practices (two seeding treatments and a non-seeded natural recovery treatment) on community structure and vegetation productivity in early secondary succession. The results showed: (1) All sedge and grass species of the natural steppe meadow had disappeared from the severely degraded land. The above-ground and root biomass of severely degraded land were only 38 and 14.7%, respectively, of those of the control. So, the original ecosystem has been dramatically altered by land degradation on alpine steppe meadow. (2) Seeding measures may promote above-ground biomass, particularly grass biomass, and ground cover. Except for the grasses seeded, however, other grass and sedge species did not occur after seeding treatments in the sixth year of seeding. Establishment of grasses during natural recovery treatment progressed slowly compared with during seeding treatments. Many annual forbs invaded and established during the 6 years of natural recovery. In addition, there was greater diversity after natural recovery treatment than after seeding treatments. (3) The above-ground biomass after seeding treatment and natural recovery treatment were 114 and 55%, respectively, of that of the control. No significant differences in root biomass occurred among the natural recovery and seeded treatments. Root biomass after rehabilitation treatment was 23-31% that of the control.

Origin of the Qinghai-Tibetan Plateau endemic Milula (Liliaceae): further insights from karyological comparisons with Allium

Milula, a monotypic genus endemic to the Qinghai-Tibetan Plateau, was found to be nested deeply within Allium by the molecular phylogeny despite the aberrant morphology. It remains unknown what had contributed to the rapid evolution of morphology and origin of this exceptional species. In contrast to a previous report of its karyotypes with 2n = 16 = 8M+8SM (2SAT), similar to most species of Allium, a rather different karyotype, 2n = 20 = 4M +10SM+6T (2SAT), was found in examined 31 individuals from 6 populations of M. spicata distributed in the central Tibet. Karyotypes of 7 Allium species occurring in the Qinghai-Tibetan Plateau were further reported. The basic number x = 8 was confirmed for all of them and their karyotypes consist mainly of metacentric and submetacentric chromosomes with rare subterminal and terminal chromosomes. The karyotype of M. spicata is distinctly different from that of most Allium species occurring in the plateau through a complete comparison of all available species in this region and adjacent areas. However, the same chromosome number and similar karyotypic structure were found in A. fasciculatum of Sect. Bromatorrhiza, indicating a possible close relationship between them. But this similarity is contradictory to the preliminary molecular phylogenetic analysis that Milula was closely related to A. cyathophorum of Sect. Bromatorrhiza with x=8, but the other species with x=10 and 11 in this section were clearly placed in the other clade. We therefore suggested that the paralleling evolution from x=8 to x=9, 10 and 11 with increasing asymmetry of karyotype possibly due to the chromosomal Robertsonian translocation might occur separately in the two recognized phylogenetic lineages of Allium. In addition to aneuploidy and following change of the chromosomal structures, the habitat isolation due to the recent uplift of the Qinghai-Tibetan Plateau and the Quaternary climatic oscillation, plays a greater role in origin of Milula and other endemic species (genera) with aberrant morphology from their progenitors.