Seed macro- and micromorphology in Allium (Amaryllidaceae) and its phylogenetic significance.

BACKGROUND AND AIMS: Macro- and micromorphology of seeds are diagnostic characteristics of importance in delimiting taxa in Allium (Amaryllidaceae). However, there is no consensus on the phylogenetic significance of testa cell characteristics and whether they reflect the different evolutionary levels recognized in Allium. METHODS: Seeds of 95 species (98 samples) representing 14 subgenera and 58 sections of Allium were examined using scanning electron microscopy (SEM) for such traits as periclinal wall surface area of ten testa cells, distance between testa cells (macromorphology), testa cell shapes, and arrangement and structure of anticlinal and periclinal walls (micromorphology). The data matrix was subjected to cladistic analysis. The produced phylogenetic tree was examined against the molecular tree obtained from publically available ITS sequences. KEY RESULTS: The periclinal wall surface area of ten testa cells and the distance between them, examined for the first time, were found useful for delimitation of species in Allium. Based on seed macro- and micromorphology, we present a taxonomic key and a hypothetical reconstruction of the migration routes during the early stages of evolution of Allium. CONCLUSIONS: The ancestors of Allium originated in an area bounded by the Caucasus, Central Asia and Iran. The seed testa morphology-based evolutionary state of a species is determined by two parameters: the shape of the periclinal walls and curvature of the anticlinal walls.

Evolutionary history and biogeography of Mandragora L. (Solanaceae).

Mandragora L. (Solanaceae) is the only genus of the tribe Mandragoreae, one of the two tribes of the cosmopolitan nightshade family, which occur exclusively in Eurasia and northern Africa. The genus occurs discontinuously in the Mediterranean region, Turanian region, and on the Tibetan Plateau, representing a classical disjunction pattern in the biogeography of the Old World flora. In this study, we reconstructed the genus phylogeny using AFLP, eight plastid DNA regions and one nuclear (ITS) gene, and evaluated the taxonomic value of quantitative traits time to flowering, fruit and seed size. We also analyzed the evolutionary history of the genus based on a phylogenetic framework and dating inferred from a combined data set of seven plastid regions with one fossil calibration point. Our data suggest that Mandragora originated in the Eocene, apparently along the Tethyan coast in broadleaf deciduous mesophytic forests that covered most of the Mediterranean region at that time. The Mediterranean-Turanian clade diverged from the Tibetan Plateau clade about 20.5 million years ago (Ma) as a result of the plateau uplift which probably was enhanced by aridification in the interior of Eurasia. A second split within the genus occurred about 11.1 Ma and resulted in Western Mediterranean and Near East-Turanian clades. Mandragora turcomanica was found to have very closely related evolutionary history with plants from the Near East indicating a possible ancient human assisted migration from Israel to Persia in historic times. In the Tibetan Plateau area, the morphologically distinctive M. chinghaiensis is nested within the M. caulescens clade indicating a very recent diversification within this lineage.

Seed heteromorphism in Triticum dicoccoides: association between seed positions within a dispersal unit and dormancy.

Variation in seed size and dormancy can take the form of seed heteromorphism, i.e., production of different kinds of seeds by a single individual. In this paper, I tested for the effect of seed position within a spikelet on its germination over time, and the contribution of this effect to population differentiation along an aridity gradient in an annual grass, Triticum dicoccoides. The results show that the upper grain in a spikelet is larger than the bottom grain, and either germinates in the season following dispersal, or dies. In contrast, a substantial fraction of the bottom grains do not germinate in the first season, but remain dormant in the soil seed bank for 1 and, very rarely, 2 years. This pattern was observed in seeds of all origins, but the bottom grains from the most arid location had the lowest, and from the least arid location, the highest germination fraction in the 1st year and vice versa in the 2nd year. This difference in germination fraction was observed under controlled irrigation conditions but not in the field experiment. These mixed results suggest that seed dimorphism is a life history trait with a complicated evolutionary history and wide adaptive implications. Seed dimorphism in T. dicoccoides could initially be an adaptation for reducing competition in productive (i.e., high precipitation) environments. In addition to this, seed dimorphism under increasing aridity could become a bet-hedging trait allowing a population to survive periods of insufficient rainfall through dormancy.

Central Asia revealed as a key area in evolution of Eremurus (Asphodelaceae).

Eremurus was described at the beginning of the 19th century. However, due to limited sampling and the small number of gene markers to date, its phylogeny and evolution are largely unknown. In this study, we analyzed plastomes from 27 species belonging to 2 subgenera and 3 sections of Eremurus, which are found in Central Asia (its center of diversity) and China. We also analyzed nuclear DNA ITS of 33 species, encompassing all subgenera and sections of the genus in Central Asia, southwest Asia and China. Our findings revealed that the genus was monophyletic, although both subgenera Eremurus and Henningia were found to be paraphyletic. Both plastome and nrDNA-based phylogenetic trees had three clades that did not reflect the current taxonomy of the genus. Our biogeographical and time-calibrated trees suggest that Eremurus originated in the ancient Tethyan area in the second half of the Eocene. Diversification of Eremurus occurred from the early Oligocene to the late Miocene. Paratethys Sea retreat and several orogenetic events, such as the progressive uplift of the Qinghai-Tibet Plateau and surrounding mountain belts (Altai, Pamir, Tian Shan), caused serious topographic and climate (aridification) changes in Central Asia that may have triggered a split of clades and speciation. In this transformed Central Asia, speciation proceeded rapidly driven mainly by vicariance caused by numerous mountain chains and specialization to a variety of climatic, topographic and soil conditions that exist in this region.

Joint evolution of seed traits along an aridity gradient: seed size and dormancy are not two substitutable evolutionary traits in temporally heterogeneous environment.

Seed size and dormancy are reproductive traits that interact as adaptations to environmental conditions. Here, we explore the evolution of these traits in environments that differ in overall mean favorability and in the extent of temporal predictability. Our model simulates a population of annual plants living in a range of environments that differ in aridity, namely mean annual precipitation and inter-annual variation of this mean precipitation. The optimal fitness curve is investigated assuming density dependence, three alternative hypothetical relationships between seed mass and seed survival in the soil (negative, positive, and independent of mass), and three alternative relationships between survival in soil and precipitation (strong and intermediate negative relationships, and no relationship). Our results show that seed size and dormancy are not two substitutable evolutionary traits; that specific combinations of these two traits are selected in environments that differ in favorability and temporal predictability; that a certain degree of seed dormancy is advantageous not only in temporally unpredictable environments but also in temporally predictable environments with high competition; and that more than one combination of seed size and dormancy (defined in terms of germination fraction) can be optimal, even in spatially homogeneous environments, potentially allowing selection for more variation in these traits within and among species.

Genetic diversity and population structure: implications for conservation of wild soybean (Glycine soja Sieb. et Zucc) based on nuclear and chloroplast microsatellite variation.

Wild soybean (Glycine soja Sieb. et Zucc) is the most important germplasm resource for soybean breeding, and is currently subject to habitat loss, fragmentation and population decline. In order to develop successful conservation strategies, a total of 604 wild soybean accessions from 43 locations sampled across its range in China, Japan and Korea were analyzed using 20 nuclear (nSSRs) and five chloroplast microsatellite markers (cpSSRs) to reveal its genetic diversity and population structure. Relatively high nSSR diversity was found in wild soybean compared with other self-pollinated species, and the region of middle and lower reaches of Yangtze River (MDRY) was revealed to have the highest genetic diversity. However, cpSSRs suggested that Korea is a center of diversity. High genetic differentiation and low gene flow among populations were detected, which is consistent with the predominant self-pollination of wild soybean. Two main clusters were revealed by MCMC structure reconstruction and phylogenetic dendrogram, one formed by a group of populations from northwestern China (NWC) and north China (NC), and the other including northeastern China (NEC), Japan, Korea, MDRY, south China (SC) and southwestern China (SWC). Contrib analyses showed that southwestern China makes the greatest contribution to the total diversity and allelic richness, and is worthy of being given conservation priority.

Phylogenomics of Allium section Cepa (Amaryllidaceae) provides new insights on domestication of onion.

Allium sect. Cepa (Amaryllidaceae) comprises economically important plants, yet resolving the phylogenetic relationships within the section has been difficult as nuclear and chloroplast-based phylogenetic trees have been incongruent. Until now, phylogenetic studies of the section have been based on a few genes. In this study, we sequenced the complete chloroplast genome (plastomes) of four central Asian species of sect. Cepa: Allium oschaninii, A. praemixtum, A. pskemense and A. galanthum. Their chloroplast (cp) genomes included 114 unique genes of which 80 coded proteins. Seven protein-coding genes were highly variable and therefore promising for future phylogenetic and phylogeographic studies. Our plastome-based phylogenetic tree of Allium sect. Cepa revealed two separate clades: one comprising the central Asian species A. oschaninii, A. praemixtum, and A. pskemense, and another comprising A. galanthum, A. altaicum, and two cultivated species, A. cepa and A. fistulosum. These findings contradict previously reported phylogenies that relied on ITS and morphology. Possible explanations for this discrepancy are related to interspecific hybridization of species ancestral to A. galanthum and A. cepa followed by chloroplast capture; however, this is impossible to prove without additional data. Our results suggest that the central Asian Allium species did not play a role in the domestication of the common onion. Among the chloroplast genes, rpoC2 was identified as a gene of choice in further phylogeographical studies of the genus Allium.

Chloroplast phylogeny and phylogeography of Stellera chamaejasme on the Qinghai-Tibet Plateau and in adjacent regions.

Historic events such as the uplift of Qinghai-Tibet Plateau (Q-T Plateau) and climatic oscillations in the Quaternary period greatly affected the evolution and modern distribution of Sino-Tibetan flora. Stellera chamaejasme, a perennial herb with flower color polymorphism that is distributed from the mountainous southeastern Q-T Plateau (Hengduan Mountains, H-D Mountains) to the vast platform of the Q-T Plateau and the adjacent plain of northern China, provides an excellent model to explore the effects of historic events on the origination and variation of species. In this study, we conducted a phylogenetic and phylogeographical study using three chloroplast sequences (trnT-L, trnL-F and rpL16) in 26 populations of S. chamaejasme and 12 outgroups from the Thymeleaceae. Phylogenetic analysis and molecular clock estimation revealed that the monophyletic origin of S. chamaejasme occurred ca. 6.5892 Ma, which is consistent with the radical environment changes caused by the rapid uplift of the Q-T Plateau ca. 7 Ma. Intra-specific differentiation of S. chamaejasme is estimated to have occurred after ca. 2.1 Ma. Twelve haplotypes were revealed from combined trnL-F and rpL16 sequences. High genetic diversity (h(T)=0.834) and population differentiation (N(ST)=0.997 and G(ST)=0.982) imply restricted gene flow among populations and significant geographical or environmental isolation. All populations from the vast plain of northern China were dominated by one haplotype (H1), and the same haplotype was fixed in most populations from the high elevation platform of the western and northern Q-T Plateau. In contrast, the majority of the haplotypes were found in the relatively narrow area of the H-D Mountains, in the southeastern distribution of S. chamaejasme. The contrasting haplotype distribution patterns suggested that the H-D Mountains were either a refugium for S. chamaejasme during the Quaternary climatic oscillations or a diversification center of this species. The present wide distribution of this species on the Q-T Plateau platform and in northern China is likely to have resulted from a rapid post-glacial population expansion from the southeastern refugium involving founder effects, facilitated by the adjacent geographic range with a similar grassland habitat.

Dispersals of Hyoscyameae and Mandragoreae (Solanaceae) from the New World to Eurasia in the early Miocene and their biogeographic diversification within Eurasia.

The cosmopolitan Solanaceae contains 21 tribes and has the greatest diversity in South America. Hyoscyameae and Mandragoreae are the only tribes of this family distributed exclusively in Eurasia with two centers of diversity: the Mediterranean-Turanian (MT) region and the Tibetan Plateau (TP). In this study, we examined the origins and biogeographical diversifications of the two tribes based on the phylogenetic framework and chronogram inferred from a combined data set of six plastid DNA regions (the atpB gene, the ndhF gene, the rps16-trnK intergenic spacer, the rbcL gene, the trnC-psbM region and the psbA-trnH intergenic spacer) with two fossil calibration points. Our data suggest that Hyoscyameae and Mandragoreae each forms a monophyletic group independently derived from different New World lineages in the early Miocene. Phylogenetic relationships within both tribes are generally well resolved. All genera of Hyoscyameae are found to be monophyletic and they diversified in middle to late Miocene. At nearly the same time, Mandragoreae split into two clades, corresponding to the MT region and the TP region, respectively. Both the phylogenetic relationships and the estimated ages of Hyoscyameae and Mandragoreae support two independent dispersal events of their ancestors from the New World into Eurasia. After their arrivals in Eurasia, the two tribes diversified primarily in the MT region and in the TP region via multiple biogeographic processes including vicariance, dispersal, recolonization or being preserved as relicts, from the mid Miocene to the late Quaternary.

Conservation-oriented restoration - a two for one method to restore both threatened species and their habitats.

There is an urgent need for a new conservation approach as mere designation of protected areas, the primary approach to conserving biodiversity, revealed its low conservation efficiency and inability to cope with numerous challenges faced by nature in the Anthropocene. The paper discusses the new concept, which proposes that ecological restoration becomes an integral part of conservation planning and implementation, and is done using threatened plant species that are introduced not only into locations where they currently grow or grew in the recent past, but also into suitable locations within their potential distribution range. This new concept is called conservation-oriented restoration to distinguish it from the traditional restoration. Although the number of restoration projects focusing on recreation of once existing natural habitats is instantly growing, the majority of ecological restoration projects, in contrast to conservation-oriented restoration, have predominantly utilitarian goals, e.g. improvement or air quality, erosion control or soil replenishment. Conservation-oriented restoration should not be seen as an alternative either to the latter, or to the conservation dealing with particular threatened species (species-targeted conservation). These three conservation approaches, traditional ecological restoration, species-targeted conservation, and conservation-oriented restoration differ not only in broadly defined goals and attributes of their targets, but also in the types of ecosystems they are applicable to, and complement each other in combating global deterioration of the environment and biodiversity loss.

Translocation of Otostegia bucharica, a highly threatened narrowly distributed relict shrub.

Translocation is a recognized means of rescuing imperiled species but the evidence for the long-term success of translocations is limited. We report the successful translocation of reproductive individuals of a critically endangered shrub Otostegia bucharica from a site facing imminent habitat destruction into a nearby natural population of the species. The relocated plants were visited the year after planting and 13 years later to assess short- and long-term plant survival. Significant percentage of plants that survived transplanting shock and very dry spring following transplanting (around 36%), and further decrease of this number in the next 12 years by only 14%, indicated that O. bucharica is amenable to translocation using reproductive plants. Based on results of species distribution modeling, and failed attempts of ex situ cultivation, we propose introduction of this species into areas with suitable climatic and soil conditions. However, because there is currently no nature reserve in Uzbekistan having suitable conditions for the species under the present climate and that expected in the near future, and because all known habitats of O. bucharica are exposed to the very strong anthropogenic pressure, establishment of a new protected area, awareness building and involvement of local community in conservation activities are required to prevent extinction of this extremely rare species.

Conservation implications of population genetic structure in a threatened orchid Cypripedium tibeticum.

Cypripedium tibeticum is a threatened orchid which efficient conservation requires knowledge of its extent and structure of genetic variation. Using two chloroplast DNA fragments (rps16 and trnL-F), we analyzed 157 individuals from 9 populations representing the species range in China. Seven haplotypes were identified. C. tibeticum had high total genetic diversity (H T = 0.80) with major contribution to this diversity made by among-population component (G ST  = 0.64, Φ ST  = 0.86). However, despite high population differentiation there was no clear phylogeographic structure. The populations CY and DC made the greatest contribution to the total gene diversity as well as allelic richness. The possible mechanisms and implications of these findings for conservation of the species are discussed.

Alkaloid chemodiversity in Mandragora spp. is associated with loss-of-functionality of MoH6H, a hyoscyamine 6ß-hydroxylase gene.

Mandrakes (Mandragora spp., Solanaceae) are known to contain tropane alkaloids and have been used since antiquity in traditional medicine. Tropane alkaloids such as scopolamine and hyoscyamine are used in modern medicine to treat pain, motion sickness, as eye pupil dilators and antidotes against organo-phosphate poisoning. Hyoscyamine is converted to 6ß-hydroxyhyoscyamine (anisodamine) and scopolamine by hyoscyamine 6ß-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. We describe here a marked chemo-diversity in the tropane alkaloid content in Mandragora spp. M. officinarum and M. turcomanica lack anisodamine and scopolamine but display up to 10 fold higher hyoscyamine levels as compared with M. autumnalis. Transcriptomic analyses revealed that H6H is highly conserved among scopolamine-producing Solanaceae. MoH6H present in M. officinarum differs in several amino acid residues including a homozygotic mutation in the substrate binding region of the protein and its prevalence among accessions was confirmed by Cleaved-Amplified-Polymorphic-Sequence analyses. Functional expression revealed that MaH6H, a gene isolated from M. autumnalis encodes an active H6H enzyme while the MoH6H sequence isolated from M. officinarum was functionally inactive. A single G to T mutation in nucleotide 663 of MoH6H is associated with the lack of anisodamine and scopolamine in M. officinalis.

Securing a future for China's plant biodiversity through an integrated conservation approach.

The severely threatened Chinese flora urgently needs a new, well adapted to China and properly formulated conservation strategy. The present review provides a detailed conservation methodology that complements previously described guidelines for preservation of plant species with extremely small populations (PSESP) in China. This review adds to the above concept in several aspects, making it relevant to all threatened Chinese plant species. The proposed integral conservation strategy has the following crucial components:-ecoregional basis for conservation planning and implementation;-a unified scoring system that is used in regional systematic planning for reserve design, monitoring and assessment of efficiency of a reserve network, and creation of seed banks and living collections;-a focus on population demography and the presence of naturally occurring regeneration as the key criteria for defining the conservation status of a species and the appropriate major focus of the species recovery plan;-creation of multi-species living collections that preserve species genetic variation and provide material for in situ actions;-experimental translocation of threatened species into multiple locations within and outside their known range. Adopting and implementing these strategies successfully and more fully in China requires that the country changes PA legislation and improves PA management, the National Science Foundation of China (NSFC) re-prioritizes the type of research that receives research funds, and local scientists improve their approach toward information sharing.

Conservation utility of botanic garden living collections: Setting a strategy and appropriate methodology.

In the realities of the modern world, when the natural habitat is rapidly disappearing and the number of imperiled plants is constantly growing, ex situ conservation is gaining importance. To meet this challenge, botanic gardens need to revise both their strategic goals and their methodologies to achieve the new goals. This paper proposes a strategy for the management of threatened plants in living collections, which includes setting regional conservation priorities for the species, creation of genetically representative collections for the high priority species, and usage of these collections in in situ actions. In this strategy, the value of existing and future species living collections for conservation is determined by the species' conservation status and how well the accessions represent their natural genetic variation.

Complementarities of two existing intermediate conservation approaches.

The need for integration of ex situ and in situ approaches in conservation of plants has long been recognized. However, ex situ collections have numerous limitations that reduce their utility for conservation, necessitating the introduction of new, more appropriate, flexible and less costly approaches. Two new approaches that can be called "intermediate" between in situ and ex situ, and bridging them in some way have been proposed over the last two decades. In these approaches material collected in natural populations is planted and maintained outside the original location, but with a different purpose. While the purpose of the inter situs approach is reintroduction, the concern of the quasi in situ approach is long-term storage of species genetic diversity. I view these two approaches as complementary and necessary components of conservation-oriented restoration. In restoration of a degraded habitat using threatened species (i.e. inter situs), quasi in situ collections can serve an important role in providing long-term preservation of these species' genetic diversity and production of seeds needed for restoration.

Role of the Qinghai-Tibetan Plateau uplift in the Northern Hemisphere disjunction: evidence from two herbaceous genera of Rubiaceae.

To assess the role of the Qinghai-Tibetan Plateau uplift in shaping the intercontinental disjunction in Northern Hemisphere, we analyzed the origin and diversification within a geological timeframe for two relict herbaceous genera, Theligonum and Kelloggia (Rubiaceae). Phylogenetic relationships within and between Theligonum and Kelloggia as well as their relatives were inferred using five chloroplast markers with parsimony, Bayesian and maximum-likelihood approaches. Migration routes and evolution of these taxa were reconstructed using Bayesian relaxed molecular clock and ancestral area reconstruction. Our results suggest the monophyly of each Theligonum and Kelloggia. Eastern Asian and North American species of Kelloggia diverged at ca.18.52 Mya and the Mediterranean species of Theligonum diverged from eastern Asian taxa at ca.13.73 Mya. Both Kelloggia and Theligonum are Tethyan flora relicts, and their ancestors might have been occurred in warm tropical to subtropical environments along the Tethys coast. The Qinghai-Tibetan Plateau separated the eastern and western Tethyan area may contribute significantly to the disjunct distributions of Theligonum, and the North Atlantic migration appears to be the most likely pathway of expansion of Kelloggia to North America. Our results highlight the importance role of the QTP uplift together with corresponding geological and climatic events in shaping biodiversity and biogeographic distribution in the Northern Hemisphere.

How to conserve threatened Chinese plant species with extremely small populations?

The Chinese flora occupies a unique position in global plant diversity, but is severely threatened. Although biodiversity conservation in China has made significant progress over the past decades, many wild plant species have extremely small population sizes and therefore are in extreme danger of extinction. The concept of plant species with extremely small populations (PSESPs), recently adopted and widely accepted in China, lacks a detailed description of the methodology appropriate for conserving PSESPs. Strategies for seed sampling, reintroduction, protecting PSESP locations, managing interactions with the local human population, and other conservation aspects can substantially differ from those commonly applied to non-PSESPs. The present review is an attempt to provide a detailed conservation methodology with realistic and easy-to-follow guidelines for PSESPs in China.

Fine-scale spatial genetic structure in predominantly selfing plants with limited seed dispersal: A rule or exception?

Gene flow at a fine scale is still poorly understood despite its recognized importance for plant population demographic and genetic processes. We tested the hypothesis that intensity of gene flow will be lower and strength of spatial genetic structure (SGS) will be higher in more peripheral populations because of lower population density. The study was performed on the predominantly selfing Avena sterilis and included: (1) direct measurement of dispersal in a controlled environment; and (2) analyses of SGS in three natural populations, sampled in linear transects at fixed increasing inter-plant distances. We found that in A. sterilis major seed dispersal is by gravity in close (less than 2 m) vicinity of the mother plant, with a minor additional effect of wind. Analysis of SGS with six nuclear SSRs revealed a significant autocorrelation for the distance class of 1 m only in the most peripheral desert population, while in the two core populations with Mediterranean conditions, no genetic structure was found. Our results support the hypothesis that intensity of SGS increases from the species core to periphery as a result of decreased within-population gene flow related to low plant density. Our findings also show that predominant self-pollination and highly localized seed dispersal lead to SGS at a very fine scale, but only if plant density is not too high.