A high frequency of allopolyploid speciation in the gymnospermous genus Ephedra and its possible association with some biological and ecological features.

The origin and evolution of polyploids have been studied extensively in angiosperms and ferns but very rarely in gymnosperms. With the exception of three species of conifers, all natural polyploid species of gymnosperms belong to Ephedra, in which more than half of the species show polyploid cytotypes. Here, we investigated the origin and evolution of polyploids of Ephedra distributed in the Qinghai-Tibetan Plateau (QTP) and neighbouring areas. Flow cytometry (FCM) was used to measure the ploidy levels of the sampled species that are represented by multiple individuals from different populations, and then, two single-copy nuclear genes (LFY and DDB2) and two chloroplast DNA fragments were used to unravel the possible origins and maternal donors of the polyploids. The results indicate that the studied polyploid species are allopolyploids, and suggest that allotetraploidy is a dominant mode of speciation in Ephedra. The high percentage of polyploids in the genus could be related to some of its biological attributes such as vegetative propagation, a relatively high rate of unreduced gamete formation, and a small genome size relative to most other gymnosperms. Significant ecological divergences between allotetraploids and their putative progenitors were detected by PCAs and anova and Tukey's tests, with the exception of E. saxatilis. The overlap of geographical distributions and ecological niches of some diploid species could have provided opportunities for interspecific hybridization and allopolyploid speciation.

Effects of mixed substrates of different agricultural and forestry residues on the cutting seedlings of Thuja sutchuenensis.

To screen environment-friendly seedling cultivation substrates which could replace peat and with less cost, we compared the effects of different agricultural and forestry residue mixed substrates on cutting propagation of Thuja sutchuenensis, in an experiment following randomized block design. There were five types of mixed substrates, including peat + vermiculite + perlite (T1), edible mushroom residue (EMR) + vermiculite + perlite (T2), carbo-nized rice husk (CRH) + vermiculite + perlite (T3), EMR + slag + sawdust (T4) and CRH + EMR + slag (T5). The results showed that the bulk density of T3 was the lowest, followed by T2, which significantly differed from other mixed substrates. The non-capillary porosity of T2 was significantly greater than that of T1, while the capillary porosity and the total porosity of T2 was lower than T1 and T3, respectively. T2 had the highest contents of total nitrogen, total phosphorus, total potassium, alkali-hydrolyzed nitrogen, available phosphorus, substrate moisture and the highest pH, which differed significantly from other mixed substrates in most chemical indicators. The membership function values of rooting rate and growth indicators of cuttings with different mixed substrates were in order of T2 > T3 > T1> T5 > T4. Most indicators with larger grey relation values were physical indicators. The top five indicators were capillary water capacity, total potassium, field water capacity, maximum water capacity, and total porosity, with both capillary water capacity and total potassium content ranking first. In general, the physicochemical properties, rooting rate, and growth characteristics of cuttings under T2 were better than those of other mixed substrates. The capillary water capacity and total potassium were the main factors affecting rooting and growth of cuttings. At the early stage of cutting, the physical properties of mixed substrate had greater effect on rooting rate and growth of cuttings than the chemical properties. Overall, our results suggested that T2 should be preferred in the cutting propagation of T. sutchuenensis.

Population Structure, Genetic Diversity and Candidate Genes for the Adaptation to Environmental Stress in Picea koraiensis.

Picea koraiensis is major silvicultural and timber species in northeast China, and its distribution area is an important transition zone for genus spruce migration. The degree of intraspecific differentiation of P. koraiensis is high, but population structure and differentiation mechanisms are not clear. In this study, 523,761 single nucleotide polymorphisms (SNPs) were identified in 113 individuals from 9 populations of P. koraiensis by genotyping-by-sequencing (GBS). Population genomic analysis showed that P. koraiensis was divided into three geoclimatic regions: Great Khingan Mountains climatic region, Lesser Khingan Mountains climatic region, and Changbai Mountain climatic region. Mengkeshan (MKS) population on the northern edge of the distribution area and Wuyiling (WYL) population located in the mining area are two highly differentiated groups. Selective sweep analysis showed that MKS and WYL populations had 645 and 1126 selected genes, respectively. Genes selected in the MKS population were associated with flowering and photomorphogenesis, cellular response to water deficit, and glycerophospholipid metabolism; genes selected in the WYL population were associated with metal ion transport, biosynthesis of macromolecules, and DNA repair. Climatic factors and heavy metal stress drives divergence in MKS and WYL populations, respectively. Our findings provide insights into adaptive divergence mechanisms in Picea and will contribute to molecular breeding studies.

Interspecific Gene Flow and Selective Sweeps in Picea wilsonii, P. neoveitchii and P. likiangensis.

Genome-wide single nucleotide polymorphism (SNP) markers were obtained by genotyping-by-sequencing (GBS) technology to study the genetic relationships, population structure, gene flow and selective sweeps during species differentiation of Picea wilsonii, P. neoveitchii and P. likiangensis from a genome-wide perspective. We used P. jezoensis and P. pungens as outgroups, and three evolutionary branches were obtained: P. likiangensis was located on one branch, two P. wilsonii populations were grouped onto a second branch, and two P. neoveitchii populations were grouped onto a third branch. The relationship of P. wilsonii with P. likiangensis was closer than that with P. neoveitchii. ABBA-BABA analysis revealed that the gene flow between P. neoveitchii and P. wilsonii was greater than that between P. neoveitchii and P. likiangensis. Compared with the background population of P. neoveitchii, the genes that were selected in the P. wilsonii population were mainly related to plant stress resistance, stomatal regulation, plant morphology and flowering. The genes selected in the P. likiangensis population were mainly related to plant stress resistance, leaf morphology and flowering. Selective sweeps were beneficial for improving the adaptability of spruce species to different habitats as well as to accelerate species differentiation. The frequent gene flow between spruce species makes their evolutionary relationships complicated. Insight into gene flow and selection pressure in spruce species will help us further understand their phylogenetic relationships and provide a scientific basis for their introduction, domestication and genetic improvement.

Allometric equations for estimating above-ground biomass of Nitraria sibirica Pall. in Gobi Desert of Mongolia.

Nitraria sibirica Pall. is a shrub species belonging to the family of Nitrariaceae. It plays pivotal role in arid ecosystems since it is tolerant to high salinity and drought. This species is widely distributed throughout Mongolia and it is mostly found in arid ecosystems of Mongolian Gobi Desert. In this study, we developed allometric equations for estimating above-ground biomass of N. sibirica using various structural descriptors and pinpointed the best models. Variables that precisely predicted above-ground biomass were a combination of basal diameter, crown area, and height. The allometric growth equation constructed is not merely helpful to achieve accurate estimations of the above-ground biomass in shrub vegetation in the Gobi Desert of Mongolia, but also can provide a reference for the above-ground biomass of Nitraria species growing in analogous habitats worldwide. Therefore, our research purposes an important advance for biomass estimation in Gobi ecosystems and complements previous studies of shrub biomass worldwide. This study provides reasonable estimates of biomass of N. sibirica, which will be valuable in evaluations of biological resources, especially for quantifying the main summer diet of Gobi bears, and also can be an alternative tool for assessing carbon cycling in Gobi Desert.

Development of microsatellite markers for the Japanese endemic conifer Thuja standishii and transfer to other East Asian species.

OBJECTIVE: Design polymorphic microsatellite loci that will be useful for studies of the genetic diversity, gene-flow and reproduction in the Japanese endemic conifer Thuja standishii and test the transferability of these loci to the two other East Asian species, T. sutchuenensis and T. koraiensis. RESULTS: Fifteen loci were developed which displayed 3 to 21 alleles per locus (average = 9.2) among 97 samples from three populations of T. standishii. Observed heterozygosity for all samples varied between 0.33 and 0.75 (average = 0.54) while expected heterozygosity values were higher with an average over the 15 loci of 0.62 (0.37-0.91). Low multi-locus probability of identity values (< 0.00002) indicate that these markers will be effective for identifying individuals derived from clonal reproduction. All 15 loci amplified in 13 samples of T. sutchuenensis, the sister species of T. standishii, with 1 to 11 alleles per locus (average = 4.33) while 13 loci amplified in four samples of the more distantly related T. koraiensis with 1 to 5 alleles per locus (average = 2.15).

Long-term responses of riparian plants' composition to water level fluctuation in China's Three Gorges Reservoir.

The water level fluctuation zone (WLFZ) has experienced a novel hydrological regime due to the anti-seasonal operation of China's Three Gorges Reservoir. Overall, hydrological change can significantly influence the riparian environment and shift the riparian vegetation. Although numerous studies have investigated the short-term responses of riparian plants to water level fluctuation in this zone, few have addressed long-term effects. In this study, four permanent plots in the WLFZ of the canyon landform area were chosen to evaluate the long-term responses of riparian plants to water level fluctuation from 2008 to 2015 and to screen candidate plants for ecological restoration. We recorded 146 species in 2008, 110 species in 2009, 68 species in 2012 and 69 species in 2015, indicating a conspicuous loss in riparian plants. Most of the remnant plants were annual and perennial herbs. Of the native species present in 2008, 82, 22 and 8 had disappeared in 2009, 2012 and 2015, respectively. Simultaneously, 45, 15 and 11 non-native species were first found, respectively. Additionally, over half of the native and the non-native species were not found after being subjected to a water level fluctuation. From 2008 to 2015, only 27 native species always presented; however, not all of them were chosen as candidates for ecological restoration because of their decreased importance values. In contrast, the importance value of Cynodon dactylon increased over time, suggesting its high tolerance to long-term winter flooding. We concluded that riparian plants' composition of the canyon landform area dramatically declined after long-term water level fluctuation and their presence was determined by the novel hydrological condition. Our results also suggested that Cynodon dactylon or its combination with other species (i.e. Digitaria chrysoblephara, Setaria glauca, Setaria viridis) is a better candidate for ecological restoration in the WLFZ.

Phylogeographic evidence for a link of species divergence of Ephedra in the Qinghai-Tibetan Plateau and adjacent regions to the Miocene Asian aridification.

The Qinghai-Tibetan Plateau (QTP) has become one of the hotspots for phylogeographical studies due to its high species diversity. However, most previous studies have focused on the effects of the Quaternary glaciations on phylogeographical structures and the locations of glacial refugia, and little is known about the effects of the aridization of interior Asia on plant population structure and speciation. Here the chloroplast DNA (cpDNA) trnT-trnF and trnS-trnfM sequences were used to investigate the differentiation and phylogeographical history of 14 Ephedra species from the QTP and northern China, based on a sampling of 107 populations. The phylogeographical analysis, together with phylogenetic reconstruction based on combined four cpDNA fragments (rbcL, rpl16, rps4, and trnS-trnfM), supports three main lineages (eastern QTP, southern QTP, and northern China) of these Ephedra species. Divergence of each lineage could be dated to the Middle or Late Miocene, and was very likely linked to the uplift of the QTP and the Asian aridification, given the high drought and/or cold tolerance of Ephedra. Most of the Ephedra species had low intraspecific variation and lacked a strong phylogeographical structure, which could be partially attributed to clonal reproduction and a relatively recent origin. In addition, ten of the detected 25 cpDNA haplotypes are shared among species, suggesting that a wide sampling of species is helpful to investigate the origin of observed haplotypes and make reliable phylogeographical inference. Moreover, the systematic positions of some Ephedra species are discussed.

Great genetic differentiation among populations of Meconopsis integrifolia and its implication for plant speciation in the Qinghai-Tibetan Plateau.

The complex tectonic events and climatic oscillations in the Qinghai-Tibetan Plateau (QTP), the largest and highest plateau in the world, are thought to have had great effects on the evolutionary history of the native plants. Of great interest is to investigate plant population genetic divergence in the QTP and its correlation with the geologic and climatic changes. We conducted a range-wide phylogeographical analysis of M. integrifolia based on the chloroplast DNA (cpDNA) trnL-trnF and trnfM-trnS regions, and defined 26 haplotypes that were phylogenetically divided into six clades dated to the late Tertiary. The six clades correspond, respectively, to highly differentiated population groups that do not overlap in geographic distribution, implying that the mountain ranges acting as corridors or barriers greatly affected the evolutionary history of the QTP plants. The older clade of M. integrifolia only occurs in the southwest of the species' range, whereas the distributions of younger clades extend northeastward in the eastern QTP, suggesting that climatic divergence resulting from the uplift of the QTP triggered the initial divergence of M. integrifolia native to the plateau. Also, the nrDNA ITS region was used to clarify the unexpected phylogenetic relationships of cpDNA haplotypes between M. integrifolia and M. betonicifolia. The topological incongruence between the two phylogenies suggests an ancestral hybridization between the two species. Our study indicates that geographic isolation and hybridization are two important mechanisms responsible for the population differentiation and speciation of Meconopsis, a species-rich genus with complex polyploids.