Isolation and characterization of microsatellite DNA primers in Juniperus przewalskii Kom (Cupressaceae)

Juniperus przewalskii (Cupressaceae) is a dominant tree species endemic to the northeast Qinghai-Tibetan Plateau. This species plays an important role in maintaining the arid ecosystem in this region. However, natural distributions of this species have been declined. In order to develop effective conservation methods, it is important to know the distribution of the genetic diversity within and among populations. In this study, we developed nine new microsatellite loci for this species. We used the combining biotin capture method to enrich AG/CT/AC/GGT microsatellites. The polymorphisms of each locus were further assessed in 12 individuals from four geographically distant populations. The number of alleles per locus varied from three to six and expected heterozygosity ranged from 0.58 to 0.70. These loci together provide a useful tool to investigate the genetic diversity of this species. In addition, all markers have been crossly checked in the other four congeneric species.

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.

基于叶绿体DNA trnT-trnF序列研究祁连圆柏的谱系地理学

由于青藏高原的地貌效应,第四纪冰期气候的反复变化应对现今该地区生物的地理分布及其居群遗传结构产生重大影响.本文对这一地区特有分布物种祁连圆柏Juniperus przewalskii Kom.整个分布区内20居群392个个体的trnT-trnF序列变化进行了研究;共发现3种单倍型(haplotype),构成两种地理区域:高原台面上的居群主要固定HapA,而HapA、HapB和HapC在高原边缘居群均有分布.所有居群总的遗传多样性HT=0.511,GST=0.847.在低海拔的高原边缘,Hap A、Hap B和Hap C高频率固定在不同的居群中,表明可能存在多个不同的避难所,居群反复缩小和扩张的瓶颈效应造成了遗传多样性的丢失.而边缘的一个居群含有两种相似单倍型频率则可能是冰期后迁移融合而成或者该居群在冰期经受的瓶颈作用更弱.高原台面东部间断分布的居群只固定Hap A,表明它们可能经历了冰期后共同的回迁过程和由此产生的奠基者效应.我们的研究结果表明祁连圆柏在冰期可能存在多个避难所,瓶颈效应和奠基者效应造成了这些居群现在的遗传多样性分布式样.

Przewalskin A: A new C-23 terpenoid with a 6/6/7 carbon ring skeleton from Salvia przewalskii maxim

Przewalskin A (1), a novel C-23 terpenoid with a 6/6/7 carbon ring skeleton, was isolated from Salvia przewalskii. Its structure was determined by comprehensive 1D NMR, 2D NMR, and MS spectroscopic analysis and subsequently confirmed by a single-crystal X

Przewalskin B, a novel diterpenoid with an unprecedented skeleton from Salvia przewalskii maxim

[GRAPHIC] Przewalskin B (1), a novel diterpenoid possessing a unique skeleton, was isolated from a Chinese medicinal plant Salvia przewalskii. Its structure and relative stereochemistry were elucidated by extensive NMR analysis and a single-crystal X-ray

Hydroacoustic study of spatial and temporal distribution of Gymnocypris przewalskii (Kessler, 1876) in Qinghai Lake, China

Gymnocypris przewalskii (Kessler 1876) is an endangered and state-protected rare fish species in Qinghai Lake, China. To further understand the life history and distribution of this fish, five surveys were carried out in Qinghai Lake between 2002-2006. Results of these surveys indicate that fishes were predominantly distributed about 2 m under the surface. In July, significant differences in fish density were found between surface and bottom layers (P = 0.001), and/or between middle and bottom layers (P = 0.025). Fish density was the greatest in the surface layer. In August and October, no significant differences were found between the different layers, but the bottom layer had a greater fish density. Furthermore, there were very large differences among different zones in fish distribution density. Differences in horizontal distribution were not significantly correlated to factors such as water depth and inshore distance, possibly because of very low and uniform fish density. Feeding, changes in water temperature, over-wintering and spawning appeared to influence fish distribution. Hydroacoustic estimates of G. przewalskii biomass in Qinghai Lake increased significantly between 2002 and 2006. We attribute this increase to the management measures put in place to protect this species.

Origin of Gymnocypris przewalskii and phylogenetic history of Gymnocypris eckloni (Teleostei : Cyprinidae)

The origins and phylogenetic patterns were assessed for G. przewalskii and G. eckloni by analyzing the complete mtDNA cytochrome b gene sequence (1140bp). Phylogenetic analyses further supported that there were three mtDNA lineages (A-C) identified in G. przewalskii and G. eckloni, demonstrating that outer rakers of the first gill have little significance in the phylogeny of the Gymnocypris fishes. The network established showed that G. eckloni of the Yellow River specific haplotype A1 was a founder and it radiated all haplotypes of G. przewalskii which suggested G. przewalskii might only originate from one of two maternals of G. eckloni from the Yellow River. Fs test and mismatch analysis showed at least two expansion events in the population of G. przewalskii about 0.2734 Ma and 0.0658 Ma, while G. eckloni from Qaidam Basin could have experienced severe bottleneck effect about 0.0693 Ma. The population expansion was detected in subclades A1 and A21 with the most recent common ancestor (TMRCA) about 0.2308 +/- 0.01 Ma and 0.1319 +/- 0.015 Ma, respectively, which were within the geological age range of "Gonghe Movement" event that caused the separation of Lake Qinghai from the upper Yellow River. These results suggested the effect of the fish diversification by rapid uplift of the Qinghai-Tibetan Plateau in the Late Pleistocene.

Amplified fragment length polymorphism analysis to identify the genetic structure of the Gymnocypris przewalskii (Kessler, 1876) population from the Qinghai Basin, China

Amplified fragment length polymorphism (AFLP) was used to analyse the genetic structure of 45 individuals of Gymnocypris przewalskii (Kessler, 1876), an endangered and state-protected rare fish species, from three areas [the Heima (HM), Buha (BH) and Shaliu rivers (SL), all draining into Qinghai Lake]. A total of 563 polymorphic loci were detected. The HM, BH and SL populations have 435, 433 and 391 loci, respectively (Zhu and Wu, 1975), which account for 77.26%, 76.91% and 69.45% of the total number of polymorphic loci of each population, respectively. The Nei indices of genetic diversities (H) of the three populations were calculated to be 0.2869 (HM), 0.2884 (BH) and 0.2663 (SL), respectively. Their Shannon informative indices are 0.4244, 0.4251 and 0.3915, respectively. Research results show that the mean genetic distance between HM and BH is the smallest (0.0511), between BH and SL is the second shortest (0.0608), and between HM and SL is the largest (0.0713), with the mean genetic distance among the three populations being over 0.05. Data mentioned above indicate that the three populations have a certain genetic differentiation. The total genetic diversity (H-t = 0.3045) and the mean value of genetic diversity within the population (H-s = 0.2786) indicate that the variations have mainly come from within the population.

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.

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.

Geographic variation in the leaf essential oils of Juniperus sabina L. and J-sablina var. arenaria (E.H. Wilson) Farjon

The composition of the leaf oils from seven populations of J. sabina L., one population of Juniperus sabina var. arenaria (E. H. Wilson) Farjon were examined for their geographic variation. In addition, the leaf oils of J. chinensis L. and J. davurica Pall. were compared to J. sabina. Juniperus sabina var. arenarla, the sand loving juniper, oil was found to be very similar to that of J. davurica, Mongolia, and J. sabina, on sand dunes in Mongolia. This suggests that J. sabina var. arenaria might be conspecific with J. davurica. Farjon's move (2001) of J. sabina var. arenaria out of J. chinensis is supported. Considerable differentiation was found in populations of J. sabina from the Iberian peninsula. Cedrol, citronellol, safrole, trans-sabinyl acetate, terpinen-4-ol and beta-thujone were found to be polymorphic in several populations.