Phylogenomic conflict resulting from ancient introgression following species diversification in Stewartia s.l. (Theaceae).

Phylogenetic analyses using diverse datasets can yield conflicting inference of evolutionary history. Phylogenetic conflicts observed in both animal and plant systems have often been explained by two competing (but not mutually exclusive) hypotheses, i.e., hybridization vs. incomplete lineage sorting (ILS). The likelihood of either process contributing to phylogenetic conflict in a given group is context-dependent, involving attributes of life history, distribution, and phylogeny, among others. Here we explore phylogenetic conflict in Stewartia s.l., a genus with ca. 20 species of trees and shrubs from the tea family (Theaceae) disjunctly distributed between eastern Asia (EAS) and eastern North America (ENA). We use both restriction-site associated DNA sequencing (RAD-seq) and complete plastome sequence data to reconstruct the phylogeny of the group using concatenation and coalescence approaches. Our results indicate strong conflicts between the topologies reconstructed using nuclear and plastid data. Four-taxon D-statistic (ABBA-BABA) tests detected prevailing signals of introgression. Bayesian Analysis of Macro-evolutionary Mixtures (BAMM) inferred that species diversification occurred in the middle to late Miocene. Ancestral range reconstructions indicated co-distribution of ancestral species (represented by internal nodes) for both the Hartia clade (in southern China) and the EAS Stewartia s.s. clade (Japan Archipelago and the Yangtze Valley of China). The latter clade experienced multiple events of dispersal and vicariance during its diversification history. Ancient introgressive hybridization following species diversification in the mid- to late-Miocene likely caused diverging histories in the nuclear and plastid genomes, leading to phylogenetic conflict in Stewartia s.l. Our study indicates that species diversification driven by both the intensification of the East Asian summer monsoon since the late Miocene and reduced risks of extinction due to frequent dispersal possibly via East China Sea Land Bridge impacted the anomalous species richness between EAS and ENA. Our study highlights the importance of using data from different genomes while reconstructing deep and shallow phylogenies of organisms.

Ginkgo biloba's footprint of dynamic Pleistocene history dates back only 390,000 years ago.

BACKGROUND: At the end of the Pliocene and the beginning of Pleistocene glaciation and deglaciation cycles Ginkgo biloba went extinct all over the world, and only few populations remained in China in relict areas serving as sanctuary for Tertiary relict trees. Yet the status of these regions as refuge areas with naturally existing populations has been proven not earlier than one decade ago. Herein we elaborated the hypothesis that during the Pleistocene cooling periods G. biloba expanded its distribution range in China repeatedly. Whole plastid genomes were sequenced, assembled and annotated, and sequence data was analyzed in a phylogenetic framework of the entire gymnosperms to establish a robust spatio-temporal framework for gymnosperms and in particular for G. biloba Pleistocene evolutionary history. RESULTS: Using a phylogenetic approach, we identified that Ginkgoatae stem group age is about 325 million years, whereas crown group radiation of extant Ginkgo started not earlier than 390,000 years ago. During repeated warming phases, Gingko populations were separated and isolated by contraction of distribution range and retreated into mountainous regions serving as refuge for warm-temperate deciduous forests. Diversification and phylogenetic splits correlate with the onset of cooling phases when Ginkgo expanded its distribution range and gene pools merged. CONCLUSIONS: Analysis of whole plastid genome sequence data representing the entire spatio-temporal genetic variation of wild extant Ginkgo populations revealed the deepest temporal footprint dating back to approximately 390,000 years ago. Present-day directional West-East admixture of genetic diversity is shown to be the result of pronounced effects of the last cooling period. Our evolutionary framework will serve as a conceptual roadmap for forthcoming genomic sequence data, which can then provide deep insights into the demographic history of Ginkgo.

Unique parallel radiations of high-mountainous species of the genus Sedum (Crassulaceae) on the continental island of Taiwan.

We explored the temporal and spatial diversification of the plant genus Sedum L. (Crassulaceae) in Taiwan based on molecular analysis of nrITS and cpDNA sequences from East Asian Sedum members. Our phylogenetic and ancestral area reconstruction analysis showed that Taiwanese Sedum comprised two lineages that independently migrated from Japan and Eastern China. Furthermore, the genetic distances among species in these two clades were smaller than those of other East Asian Sedum clades, and the Taiwanese members of each clade occupy extremely varied habitats with similar niches in high-mountain regions. These data indicate that species diversification occurred in parallel in the two Taiwanese Sedum lineages, and that these parallel radiations could have occurred within the small continental island of Taiwan. Moreover, the estimated time of divergence for Taiwanese Sedum indicates that the two radiations might have been correlated to the formation of mountains in Taiwan during the early Pleistocene. We suggest that these parallel radiations may be attributable to the geographical dynamics of Taiwan and specific biological features of Sedum that allow them to adapt to new ecological niches.

Molecular phylogeography and ecological niche modelling of a widespread herbaceous climber, Tetrastigma hemsleyanum (Vitaceae): insights into Plio-Pleistocene range dynamics of evergreen forest in subtropical China.

Warm-temperate evergreen (WTE) forest represents the typical vegetation type of subtropical China, but how its component species responded to past environmental change remains largely unknown. Here, we reconstruct the evolutionary history of Tetrastigma hemsleyanum, an herbaceous climber restricted to the WTE forest. Twenty populations were genotyped using chloroplast DNA sequences and nuclear microsatellite loci to assess population structure and diversity, supplemented by phylogenetic dating, ancestral area reconstructions and ecological niche modeling (ENM) of the species distributions during the Last Glacial Maximum (LGM) and at present. Lineages in Southwest vs Central-South-East China diverged through climate/tectonic-induced vicariance of an ancestral southern range during the early Pliocene. Long-term stability in the Southwest contrasts with latitudinal range shifts in the Central-South-East region during the early-to-mid-Pleistocene. Genetic and ENM data strongly suggest refugial persistence in situ at the LGM. Pre-Quaternary environmental changes appear to have had a persistent influence on the population genetic structure of this subtropical WTE forest species. Our findings suggest relative demographic stability of this biome in China over the last glacial-interglacial cycle, in contrast with palaeobiome reconstructions showing that this forest biome retreated to areas of today's tropical South China during the LGM.

Understanding the formation of Mediterranean-African-Asian disjunctions: evidence for Miocene climate-driven vicariance and recent long-distance dispersal in the Tertiary relict Smilax aspera (Smilacaceae).

Tethyan plant disjunctions, including Mediterranean-African-Asian disjunctions, are thought to be vicariant, but their temporal origin and underlying causes remain largely unknown. To address this issue, we reconstructed the evolutionary history of Smilax aspera, a hypothesized component of the European Tertiary laurel forest flora. Thirty-eight populations and herbarium specimens representing 57 locations across the species range were sequenced at seven plastid regions and the nuclear ribosomal internal transcribed spacer region. Time-calibrated phylogenetic and phylogeographic inferences were used to trace ancestral areas and biogeographical events. The deep intraspecific split between Mediterranean and African-Asian lineages is attributable to range fragmentation of a southern Tethyan ancestor, as colder and more arid climates developed shortly after the mid-Miocene. In the Mediterranean, climate-induced vicariance has shaped regional population structure since the Late Miocene/Early Pliocene. At around the same time, East African and South Asian lineages split by vicariance, with one shared haplotype reflecting long-distance dispersal. Our results support the idea that geographic range formation and divergence of Tertiary relict species are more or less gradual (mostly vicariant) processes over long time spans, rather than point events in history. They also highlight the importance of the Mediterranean Basin as a centre of intraspecific divergence for Tertiary relict plants.

Understanding population structure and historical demography in a conservation context: population genetics of the endangered Kirengeshoma palmata (Hydrangeaceae).

PREMISE OF THE STUDY: Both historical and contemporary microevolutionary processes greatly influence the genetic patterns of East Asian plant endemics, but the spatial and temporal contexts of these processes remain poorly understood. Here, we investigate the relative influences of historical and contemporary gene flow and drift on the population genetic structure of Kirengeshoma palmata, a perennial herb from East China and South Japan. METHODS: We used data from nine polymorphic microsatellite loci to assess the levels of genetic diversity, effective population size, and contemporary and historical gene flow for six of the seven known populations. KEY RESULTS: We found high levels of inbreeding and allelic diversity within populations. Both contemporary and historical migration rates among populations were low, and a test of alternate models of population history strongly favored a model of long-term drift-migration equilibrium. We inferred declines in population size ca. 10,000-100,000 yr ago, but failed to detect recent declines. Bayesian clustering divided K. palmata populations into three genetic clusters, two of which were consistent with a glacial refugium hypothesis for two mountain ranges in East China. CONCLUSIONS: These results suggest that anthropogenic fragmentation has had little effect on the genetic characteristics of Chinese K. palmata. Rather, past decline in population size due to Late Pleistocene climate change as well as restricted pollen and seed dispersal may have contributed to low levels of both historical and contemporary gene flow, resulting in high genetic differentiation between adjacent mountain ranges due to genetic drift and inbreeding.

Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants.

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH-psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1-92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9-79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

Characterization of the sequence and expression pattern of LFY homologues from dogwood species (Cornus) with divergent inflorescence architectures.

BACKGROUND AND AIMS: LFY homologues encode transcription factors that regulate the transition from vegetative to reproductive growth in flowering plants and have been shown to control inflorescence patterning in model species. This study investigated the expression patterns of LFY homologues within the diverse inflorescence types (head-like, umbel-like and inflorescences with elongated internodes) in closely related lineages in the dogwood genus (Cornus s.l.). The study sought to determine whether LFY homologues in Cornus species are expressed during floral and inflorescence development and if the pattern of expression is consistent with a function in regulating floral development and inflorescence architectures in the genus. METHODS: Total RNAs were extracted using the CTAB method and the first-strand cDNA was synthesized using the SuperScript III first-strand synthesis system kit (Invitrogen). Expression of CorLFY was investigated by RT-PCR and RNA in situ hybridization. Phylogenetic analyses were conducted using the maximum likelihood methods implemented in RAxML-HPC v7.2.8. KEY RESULTS: cDNA clones of LFY homologues (designated CorLFY) were isolated from six Cornus species bearing different types of inflorescence. CorLFY cDNAs were predicted to encode proteins of approximately 375 amino acids. The detection of CorLFY expression patterns using in situ RNA hybridization demonstrated the expression of CorLFY within the inflorescence meristems, inflorescence branch meristems, floral meristems and developing floral organ primordia. PCR analyses for cDNA libraries derived from reverse transcription of total RNAs showed that CorLFY was also expressed during the late-stage development of flowers and inflorescences, as well as in bracts and developing leaves. Consistent differences in the CorLFY expression patterns were not detected among the distinct inflorescence types. CONCLUSIONS: The results suggest a role for CorLFY genes during floral and inflorescence development in dogwoods. However, the failure to detect expression differences between the inflorescence types in the Cornus species analysed suggests that the evolutionary shift between major inflorescence types in the genus is not controlled by dramatic alterations in the levels of CorLFY gene transcript accumulation. However, due to spatial, temporal and quantitative limitations of the expression data, it cannot be ruled out that subtle differences in the level or location of CorLFY transcripts may underlie the different inflorescence architectures that are observed across these species. Alternatively, differences in CorLFY protein function or the expression or function of other regulators (e.g. TFL1 and UFO homologues) may support the divergent developmental trajectories.

Phylogenetic diversity of eastern Asia-eastern North America disjunct plants is mainly associated with divergence time.

The underlying causes of biodiversity disparities among geographic regions have long been a fundamental theme in ecology and evolution. However, the patterns of phylogenetic diversity (PD) and phylogenetic beta diversity (PBD) of congeners that are disjunctly distributed between eastern Asia-eastern North America (EA-ENA disjuncts) and their associated factors remain unknown. Here we investigated the standardized effect size of PD (SES-PD), PBD, and potentially associated factors in 11 natural mixed forest sites (five in EA and six in ENA) where abundant EA-ENA disjuncts occur. We found that the disjuncts in ENA possessed higher SES-PD than those in EA at the continental scale (1.96 vs -1.12), even though the number of disjunct species in ENA is much lower than in EA (128 vs 263). SES-PD of the EA-ENA disjuncts tended to decrease with increasing latitude in 11 sites. The latitudinal diversity gradient of SES-PD was stronger in EA sites than in ENA sites. Based on the unweighted unique fraction metric (UniFrac) distance and the phylogenetic community dissimilarity, PBD showed that the two northern sites in EA were more similar to the six-site ENA group than to the remaining southern EA sites. Based on the standardized effect size of mean pairwise distances (SES-MPD), nine of eleven studied sites showed a neutral community structure (-1.96 ≤ SES-MPD ≤ 1.96). Both Pearson's r and structural equation modeling suggested that SES-PD of the EA-ENA disjuncts was mostly associated with mean divergence time. Moreover, SES-PD of the EA-ENA disjuncts was positively correlated with temperature-related climatic factors, although negatively correlated with mean diversification rate and community structure. By applying approaches from phylogenetics and community ecology, our work sheds light on historical patterns of the EA-ENA disjunction and paves the way for further research.

Complete cpDNA genome sequence of Smilax china and phylogenetic placement of Liliales--influences of gene partitions and taxon sampling.

The complete nucleotide sequence of the chloroplast genome (cpDNA) of Smilax china L. (Smilacaceae) is reported. It is the first complete cp genome sequence in Liliales. Genomic analyses were conducted to examine the rate and pattern of cpDNA genome evolution in Smilax relative to other major lineages of monocots. The cpDNA genomic sequences were combined with those available for Lilium to evaluate the phylogenetic position of Liliales and to investigate the influence of taxon sampling, gene sampling, gene function, natural selection, and substitution rate on phylogenetic inference in monocots. Phylogenetic analyses using sequence data of gene groups partitioned according to gene function, selection force, and total substitution rate demonstrated evident impacts of these factors on phylogenetic inference of monocots and the placement of Liliales, suggesting potential evolutionary convergence or adaptation of some cpDNA genes in monocots. Our study also demonstrated that reduced taxon sampling reduced the bootstrap support for the placement of Liliales in the cpDNA phylogenomic analysis. Analyses of sequences of 77 protein genes with some missing data and sequences of 81 genes (all protein genes plus the rRNA genes) support a sister relationship of Liliales to the commelinids-Asparagales clade, consistent with the APG III system. Analyses of 63 cpDNA protein genes for 32 taxa with few missing data, however, support a sister relationship of Liliales (represented by Smilax and Lilium) to Dioscoreales-Pandanales. Topology tests indicated that these two alignments do not significantly differ given any of these three cpDNA genomic sequence data sets. Furthermore, we found no saturation effect of the data, suggesting that the cpDNA genomic sequence data used in the study are appropriate for monocot phylogenetic study and long-branch attraction is unlikely to be the cause to explain the result of two well-supported, conflict placements of Liliales. Further analyses using sufficient nuclear data remain necessary to evaluate these two phylogenetic hypotheses regarding the position of Liliales and to address the causes of signal conflict among genes and partitions.

Development of microsatellite markers for the dove tree, Davidia involucrata (Nyssaceae), a rare endemic from China.

PREMISE OF THE STUDY: Microsatellite primers were developed for the endangered Davidia involucrata to assess the population genetics and infer its evolutionary history. METHODS AND RESULTS: Using both the modified magnetic bead hybridization method and the dual-suppression PCR method, we isolated and characterized 12 polymorphic microsatellite loci using 134 individuals from five populations in southwestern China. The number of alleles per locus ranged from six to 21 (mean = 10.8). The expected heterozygosity per locus ranged from 0.404 to 0.918 and observed heterozygosity ranged from 0.015 to 0.821. CONCLUSIONS: All of the 12 microsatellite markers developed for D. involucrata are polymorphic, and lay a solid foundation for further studies of the population genetics of this famous tree.

Isolation and characterization of polymorphic microsatellite markers from the Chinese medicinal herb Atractylodes macrocephala (Asteraceae).

Atractylodes macrocephala Koidz. (Asteraceae) is an economically important Chinese medicinal herb. In this study, 15 polymorphic microsatellite markers were developed from A. macrocephala using the compound microsatellite marker technique. Levels of polymorphism within the 15 markers were assessed using 83 individuals from two wild and two cultivated populations in China. The number of alleles per locus ranged from 2 to 20, with an average of 9.9 alleles. Observed and expected heterozygosities ranged from 0.083 to 1.000 and from 0.097 to 0.938, respectively. These markers will be valuable for germplasm classification and identification, as well as for assessing the genetic diversity and spatial genetic structure among wild and cultivated populations of A. macrocephala.

Plio-Pleistocene climatic change drives allopatric speciation and population divergence within the Scrophularia incisa complex (Scrophulariaceae) of desert and steppe subshrubs in Northwest China.

Numerous temperate plants and animals on the Qinghai-Tibet Plateau (QTP) are hypothesized to have differentiated due to vicariant allopatric speciation associated with the geologic uplifts. However, this hypothesis has rarely been tested through a phylogeographic study of relative species in a broader geographic context, including the QTP, Tianshan Mountains, Mongolian Plateau, and surrounding regions. To understand the speciation and diversification process of plants across this wide area, phylogeographic analysis were examined from Scrophularia incisa and two other closely relative species comprising S. kiriloviana and S. dentata. Thirty-two populations of the three close relatives were genotyped using chloroplast DNA fragments and nuclear microsatellite loci to assess population structure and diversity, supplemented by phylogenetic dating, ancestral area reconstructions and species distribution modelings, as well as niche identity tests. Our chloroplast DNA (cpDNA) phylogeny showed that this monophyletic group of desert and steppe semi-shrub is derived from a Middle Pliocene ancestor of the Central Asia. Lineages in Central Asia vs. China diverged through climate/tectonic-induced vicariance during Middle Pliocene. Genetic and ENM data in conjunction with niche differentiation analyses support that the divergence of S. incisa, S. dentata and S. kiriloviana in China lineage proceeded through allopatric speciation, might triggered by early Pleistocene climate change of increase of aridification and enlargement of deserts, while subsequent climate-induced cycles of range contractions/expansions enhanced the geographical isolation and habit fragmentation of these taxa. These findings highlight the importance of the Plio-Pleistocene climate change in shaping genetic diversity and driving speciation in temperate steppes and deserts of Northwestern China.

Isolation of compound microsatellite markers for the common Mediterranean shrub Smilax aspera (Smilacaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the common Mediterranean shrub Smilax aspera to study the population genetics of this species. METHODS AND RESULTS: Using the compound microsatellite marker technique, a total of 14 pairs of primers were developed for S. aspera, of which 11 were polymorphic, and three were monomorphic. Levels of polymorphism in the 11 markers were checked in 43 individuals collected from two populations in Greece and Italy. The number of alleles per locus ranged from 10 to 26, with an average of 15.55 alleles per locus. All of these primers also could be amplified from a second species, S. hispida. CONCLUSIONS: These microsatellite markers can be used for future studies of genetic diversity in S. aspera, as well as in other related taxa, and will help us to improve our understanding of the microevolutionary processes of this species in the Mediterranean region.

Isolation and characterization of microsatellite markers for Dipteronia dyerana (Sapindaceae), an endangered endemic species in China.

PREMISE OF THE STUDY: Microsatellite markers were developed for Dipteronia dyerana, an endangered endemic species in China, to study the population genetics of this species. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, 12 microsatellite markers that were successfully amplified showed polymorphism when tested on 31 individuals from two populations in the counties of Wenshan and Mengzi. Overall, the number of alleles per locus ranged between seven and 25, with an average of 12.3. Nine of these markers were able to be amplified in D. sinensis. CONCLUSIONS: These microsatellite markers should facilitate further studies on the population genetics and the reproductive ecology of Dipteronia.

Molecular phylogeography of East Asian Kirengeshoma (Hydrangeaceae) in relation to quaternary climate change and landbridge configurations.

Kirengeshoma comprises two species inhabiting warm temperate-deciduous forests in East China/South Japan (Kirengeshoma palmata) and South Korea (Kirengeshoma koreana). A survey of chloroplast (cp) DNA and inter-simple sequence repeats (ISSRs) variation in Kirengeshoma was carried out to determine the population history of a plant taxon around the East China Sea (ECS). CpDNA and ISSRs revealed lower genetic divergence between China and Japan relative to the other contrasts, in line with intrageneric classification. Molecular dating suggests that K. koreana diverged at the Plio-Pleistocene boundary from the Japanese populations, whereas the latter migrated into China during the early-to-mid Pleistocene via the ECS basin. Vicariant segregation of Chinese and Japanese populations likely occurred during the mid-Pleistocene. Mismatch distributions and neutrality tests indicated that Chinese populations expanded their range during the Late Pleistocene, probably during a cold period, whereas those from Japan showed no significant population growth. We conclude that the current distribution and differentiation of components of presently isolated warm temperate-deciduous forests in China, Japan and Korea likely resulted from a combination of relatively ancient vicariant and immigration events, and those from Japan were particularly sensitive to range fragmentation and long-term refugial isolation throughout the Late Pleistocene.

Did glacials and/or interglacials promote allopatric incipient speciation in East Asian temperate plants? Phylogeographic and coalescent analyses on refugial isolation and divergence in Dysosma versipellis.

To explore the evolutionary consequences of climate-induced fluctuations in presently fragmented temperate forest habitats in continental East Asia we investigated the phylogeography and demographic history of the temperate-deciduous forest endemic Dysosma versipellis from disjunct montane sites in Central-Southeast China. Based on a survey of chloroplast (cp) DNA sequence variation, our analyses show that this perennial herb consists of morphologically indistinguishable western and central/eastern cpDNA lineages. Coalescent analyses under the 'isolation with migration' (IM) model support an ancient (Mid-Pleistocene) divergence between these lineages, with the western lineage having persisted without significant population growth in a long-term refuge just east of the Tibetan (Qinghai-Xizang) Plateau. In contrast, for the central/eastern lineage, we found strong evidence for population expansion from a refuge located south of the middle and lower reaches of the Yangtze River, and likely coinciding with the last or penultimate interglacial, followed by considerable population isolation and divergence in situ over (at least) the latest glacial-interglacial cycle. In line with recent evidence from palaeomodeling of East Asian forest biomes, our results suggest that the same vicariance factor, i.e. climate-induced eco-geographic isolation through (a)biotic displacement of temperate-deciduous forested habitats, promoted the divergence of D. versipellis lineages and populations at different spatial-temporal scales and over glacial and interglacial periods. Thus, there is no evidence that populations of D. versipellis merged at lower elevations during the last glacial(s). As such, D. versipellis accords with the premise that Late Quaternary refugial isolation is likely to have enhanced allopatric (incipient) species formation of temperate plants in East Asia.

Natural selection and repeated patterns of molecular evolution following allopatric divergence.

Although geographic isolation is a leading driver of speciation, the tempo and pattern of divergence at the genomic level remain unclear. We examine genome-wide divergence of putatively single-copy orthologous genes (POGs) in 20 allopatric species/variety pairs from diverse angiosperm clades, with 16 pairs reflecting the classic eastern Asia-eastern North America floristic disjunction. In each pair, >90% of POGs are under purifying selection, and <10% are under positive selection. A set of POGs are under strong positive selection, 14 of which are shared by 10-15 pairs, and one shared by all pairs; 15 POGs are annotated to biological processes responding to various stimuli. The relative abundance of POGs under different selective forces exhibits a repeated pattern among pairs despite an ~10 million-year difference in divergence time. Species divergence times are positively correlated with abundance of POGs under moderate purifying selection, but negatively correlated with abundance of POGs under strong purifying selection.

Resequencing 545 ginkgo genomes across the world reveals the evolutionary history of the living fossil.

As Charles Darwin anticipated, living fossils provide excellent opportunities to study evolutionary questions related to extinction, competition, and adaptation. Ginkgo (Ginkgo biloba L.) is one of the oldest living plants and a fascinating example of how people have saved a species from extinction and assisted its resurgence. By resequencing 545 genomes of ginkgo trees sampled from 51 populations across the world, we identify three refugia in China and detect multiple cycles of population expansion and reduction along with glacial admixture between relict populations in the southwestern and southern refugia. We demonstrate multiple anthropogenic introductions of ginkgo from eastern China into different continents. Further analyses reveal bioclimatic variables that have affected the geographic distribution of ginkgo and the role of natural selection in ginkgo's adaptation and resilience. These investigations provide insights into the evolutionary history of ginkgo trees and valuable genomic resources for further addressing various questions involving living fossil species.

Anti-proliferative effect of clitocine from the mushroom Leucopaxillus giganteus on human cervical cancer HeLa cells by inducing apoptosis.

Clitocine, a natural biologically active substance isolated from the mushroom Leucopaxillus giganteus, possesses several bioactivities including antitumor. Here, for the first time, we studied the molecular mechanism of clitocine-induced apoptosis in human cervical cancer cells (HeLa). Clitocine-induced cell death was characterized with the changes in cell morphology, DNA fragmentation, activation of caspase-3, -8, and -9 (like) activities, poly(ADP-ribose) polymerase (PARP) cleavage, release of cytochrome c (cyt c) into cytosol, and increase of Bax:Bcl-2 ratio. These results indicated that the induction of apoptosis by clitocine involved the multiple pathway including death receptor and mitochondrial pathways, and strongly suggested that the mitochondrial pathways were mediated by down-regulation of Bcl-2 and up-regulation of Bax, release of cytochrome c and subsequent activation of caspase-3 followed by down stream events leading to apoptotic mode of cell death.