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.

Pollinator shift and reproductive performance of the qinghai-tibetan plateau endemic and endangered Swertia przewalskii (Gentianaceae)

Reproductive failure results in many plant species becoming endangered. However, little is known of how and to what extent pollinator shifts affect reproductive performance of endangered species as a result of the artificial introduction of alien insects. In this study we examined breeding systems, visitor species, visiting frequency and seed set coefficients of Swertia przewalskii in two years that had different dominant pollinator species (native vs. alien). Flowers of this species were protandrous and herkogamous and insects were needed for the production of seeds. The stigmatic receptivity of this species was shorter than for other gentians. No significant difference in seed set coefficient was found for hand-pollinated plants between the two years, indicating that pollinator shift only had a minor effect on this plant's breeding system. The commonest pollinators in 2002 were native bumblebees, alien honeybees and occasional solitary bees, however, only alien honeybees were observed in 2004. The flower visitation rate in both years was relatively high, although the total visit frequency decreased significantly in 2004. The control flowers without any treatment produced significantly fewer seed sets in 2004 than in 2002. In the past decade the seed production of this species may have partly decreased due to pollination by alien honeybees, however, we suggest that they might have acted as alternative pollinators ensuring seed production of S. przewalskii when native pollinators were unavailable. The main reason that this plant is endangered is probably the result of habitat destruction, but changes in land use, namely intensified agricultural practice and unfavorable animal husbandry have also contributed to its decline. We recommend that in-situ conservation, including the establishment of a protected area, is the best way to preserve this species effectively.

ISSR analysis of genetic diversity of the Qinghai-Tibet Plateau endemic, Rhodiola chrysanthemifolia (Crassulaceae)

Inter-simple sequence repeat markers (ISSR) were used to estimate genetic diversity within and among 10 populations of Rhodiola chrysanthemifolia along Nianqingtangula Mountains and Brahmaputra, a species endemic to the Qinghai-Tibet Plateau and an endangered medicinal plant. Of the 100 primers screened, 13 produced highly polymorphic DNA fragments. Using these primers, 116 discernible DNA fragments were generated of which 104 (89.7%) were polymorphic, indicating substantial genetic diversity at the species level. Genetic diversity measured by the percentage of polymorphic bands (PPB) at the population level ranged from 21.97% to 48.8%. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly among populations (77.3%), but no regional differentiation was discernible. Variance within populations was only 22.7%. The main factor responsible for this high level of differentiation among populations is probably the historical geographical and genetic isolation of populations in a harsh mountainous environment. Concerning the management of R. chrysanthemifolia, the high genetic differentiation of populations indicates the necessity of conserving the maximum possible number of populations. (c) 2006 Elsevier Ltd. All rights reserved.

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.

Patterns of genetic variation in Swertia przewalskii, an endangered endemic species of the Qinghai-Tibet Plateau

Swertia przewalskii Pissjauk. (Gentianaceae) is a critically endangered and endemic plant of the Qinghai-Tibet Plateau in China. RAPD and ISSR analyses were carried out on a total of 63 individuals to assess the extent of genetic variation in the remaining three populations. Percentage of polymorphic bands was 94% (156 bands) for RAPD and 96% (222 bands) for ISSR. A pairwise distance measure calculated from the RAPD and ISSR data was used as input for analysis of molecular variance (AMOVA). AMOVA indicated that a high proportion of the total genetic variation (52% for RAPD and 56% for ISSR) was found among populations; pairwise Phi(ST) comparisons showed that the three populations examined were significantly different (p < 0.001). Significant genetic differentiation was found based on different measures (AMOVA and Hickory theta(B)) in S. przewalskii (0.52 on RAPD and 0.56 on ISSR; 0.46 on RAPD and 0.45 on ISSR). The differentiation of the populations corresponded to low average gene flow (0.28 based on RAPD and 0.31 based on ISSR), whereas genetic distance-based clustering and coalescent-based assignment analyses revealed significant genetic isolation among populations. Our results indicate that genetic diversity is independent of population size. We conclude that although sexual reproduction and gene flow between populations of S. przewalskii are very limited, they have preserved high levels of genetic diversity. The main factors responsible for the high level of difference among populations are the isolation and recent fragmentation under human disturbance.

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.

Phylogeography of the Qinghai-Tibetan Plateau endemic Juniperus przewalskii (Cupressaceae) inferred from chloroplast DNA sequence variation

The vegetation of the northeast Qinghai-Tibetan Plateau is dominated by alpine meadow and desert-steppe with sparse forests scattered within it. To obtain a better understanding of the phylogeography of one constituent species of the forests in this region, we examined chloroplast trnT-trnF and trnS-trnG sequence variation within Juniperus przewalskii, a key endemic tree species. Sequence data were obtained from 392 trees in 20 populations covering the entire distribution range of the species. Six cpDNA haplotypes were identified. Significant population subdivision was detected (G(ST) = 0.772, N-ST = 0.834), suggesting low levels of recurrent gene flow among populations and significant phylogeographic structure (N-ST > G(ST), P < 0.05). Eight of the nine disjunct populations surveyed on the high-elevation northeast plateau were fixed for a single haplotype (A), while the remaining, more westerly population, contained the same haplotype at high frequency together with two low frequency haplotypes (C and F). In contrast, most populations that occurred at lower altitudes at the plateau edge were fixed or nearly fixed for one of two haplotypes, A or E. However, two plateau edge populations had haplotype compositions different from the rest. In one, four haplotypes (A, B, D and E) were present at approximately equivalent frequencies, which might reflect a larger refugium in the area of this population during the last glacial period. Phylogenetic analysis indicated that the most widely distributed haplotype A is not ancestral to other haplotypes. The contrasting phylogeographic structures of the haplotype-rich plateau edge area and the almost haplotype-uniform plateau platform region indicate that the plateau platform was recolonized by J. przewalskii during the most recent postglacial period. This is supported by the findings of a nested clade analysis, which inferred that postglacial range expansion from the plateau edge followed by recent fragmentation is largely responsible for the present-day spatial distribution of cpDNA haplotypes within the species.

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.

RAPD profiling in detecting genetic variation in endemic Coelonema (Brassicaceae) of Qinghai-Tibet Plateau of China

Random amplified polymorphic DNA ( RAPD) markers were used to measure genetic diversity of Coelonema draboides ( Brassicaceae), a genus endemic to the Qilian Mountains of the Qinghai-Tibet Plateau. We sampled 90 individuals in 30 populations of Coelonema draboides from Datong and Huzhu counties of Qinghai Province in P. R. China. A total of 186 amplified bands were scored from the 14 RAPD primers, with a mean of 13.3 amplified bands per primer, and 87% ( 161 bands) polymorphic bands (PPB) was found. Analysis of molecular variance (AMOVA) shows that a large proportion of genetic variation (84.2%) resides among individuals within populations, while only 15.8% resides among populations. The species shows higher genetic diversity between individuals than other endemic and endangered plants. The RAPDs provide a useful tool for assessing genetic diversity of rare, endemic species and for resolving relationships among populations. The results show that the genetic diversity of this species is high, possibly allowing it to adapt more easily to environmental variations. The main factor responsible for the high level of differentiation within populations and the low level of diversity among populations is probably the outcrossing and long-lived nature of this species. Some long-distance dispersal, even among far separated populations, is also a crucial determinant for the pattern of genetic variation in the species. This distributive pattern of genetic variation of C. draboides populations provides important baseline data for conservation and collection strategies for the species. It is suggested that only populations in different habitats should be studied and protected, not all populations, so as to retain as much genetic diversity as possible.

Molecular phylogeny and biogeography of the Qinghai-Tibet Plateau endemic Nannoglottis (Asteraceae)

All taxa endemic to the Qinghai-Tibet Plateau are hypothesized to have originated in situ or from immediately adjacent areas because of the relatively recent formation of the plateau since the Pliocene, followed by the large-scaled biota extinction and recession caused by the Quaternary ice sheet. However, identification of specific progenitors remains difficult for some endemics, especially some endemic genera. Nannoglottis, with about eight species endemic to this region, is one such genus. Past taxonomic treatments have suggested its relationships with four different tribes of Asteraceae. We intend to identify the closest relatives of Nannoglottis by evaluating the level of monophyly, tribal delimitation, and systematic position of the genus by using molecular data from ndhF gene, trnL-F, and ITS region sequences. We find that all sampled species of Nannoglottis are a well-defined monophyly. This supports all recent taxonomic treatments of Nannoglottis, in which all sampled species were placed in one broadly re-circumscribed genus. Nannoglottis is most closely related to the Astereae, but stands as an isolated genus as the first diverging lineage of the tribe, without close relatives. A tentative relationship was suggested for Nannoglottis and the next lineage of the tribe was based on the ITS topology, the "basal group," which consists of seven genera from the Southern Hemisphere. Such a relationship is supported by some commonly shared plesiomorphic morphological characters. Despite the very early divergence of Nannoglottis in the Astereae, the tribe must be regarded to have its origin in Southern Hemisphere rather than in Asia, because based on all morphological, molecular, biogeographical, and fossil data, the Asteraceae and its major lineages (tribes) are supposed to have originated in the former area. Long-distance dispersal using Southeast Asia as a steppingstone from Southern Hemisphere to the Qinghai-Tibet Plateau is the most likely explanation for this unusual biogeographic link of Nannoglottis. The 23-32-million-year divergence time between Nannoglottis and the other Astereae estimated by DNA sequences predated the formation of the plateau. This estimation is further favored by the fossil record of the Asteraceae and the possible time of origin of the Astereae. Nannoglottis seems to have reached the Qinghai-Tibet area in the Oligocene-Eocene and then re-diversified with the uplift of the plateau. The molecular infragenetic phylogeny of the genus identifies two distinct clades, which reject the earlier infrageneric classification based on the arrangement of the involucral bracts and the length of the ligules, but agree well with the habits and ecological preferences of its current species. The "alpine shrub" vs. "coniferous forest" divergence within Nannoglottis was estimated at about 3.4 million years ago when the plateau began its first large-scale uplifting and the coniferous vegetation began to appear. Most of the current species at the "coniferous forest" clade of the genus are estimated to have originated from 1.02 to 1.94 million years ago, when the second and third uprisings of the plateau occurred, the climate oscillated and the habitats were strongly changed. The assumed evolution, speciation diversity, and radiation of Nannoglottis based on molecular phylogeny and divergence times agree well with the known geological and paleobotanical histories of the Qinghai-Tibet Plateau. (C) 2002 Elsevier Science (USA). All rights reserved.