Chromosome-level reference genome of Tetrastigma hemsleyanum (Vitaceae) provides insights into genomic evolution and the biosynthesis of phenylpropanoids and flavonoids.

Here, we present a high-quality chromosome-scale genome assembly (2.19 Gb) and annotation of Tetrastigma hemsleyanum, a perennial herbaceous liana native to subtropical China with diverse medicinal applications. Approximately 73% of the genome was comprised of transposable elements (TEs), of which long terminal repeat retrotransposons (LTR-RTs) were a predominant group (69% of the genome). The genome size increase of T. hemsleyanum (relative to Vitis species) was mostly due to the proliferation of LTR-RTs. Of the different modes of gene duplication identified, transposed duplication (TRD) and dispersed duplication (DSD) were the predominant ones. Genes, particularly those involved in the phenylpropanoid-flavonoid (PF) pathway and those associated with therapeutic properties and environmental stress resistance, were significantly amplified through recent tandem duplications. We dated the divergence of two intraspecific lineages in Southwest (SW) versus Central-South-East (CSE) China to the late Miocene (approximately 5.2 million years ago). Of those, the former showed more upregulated genes and metabolites. Based on resequencing data of 38 individuals representing both lineages, we identified various candidate genes related to 'response to stimulus' and 'biosynthetic process', including ThFLS11, which is putatively involved in flavonoid accumulation. Overall, this study provides abundant genomic resources for future evolutionary, ecological, and functional genomics studies in T. hemsleyanum and related species.

An overlooked dispersal route of Cardueae (Asteraceae) from the Mediterranean to East Asia revealed by phylogenomic and biogeographical analyses of Atractylodes.

BACKGROUND AND AIMS: The East Asian-Tethyan disjunction pattern and its mechanisms of formation have long been of interest to researchers. Here, we studied the biogeographical history of Asteraceae tribe Cardueae, with a particular focus on the temperate East Asian genus Atractylodes DC., to understand the role of tectonic and climatic events in driving the diversification and disjunctions of the genus. METHODS: A total of 76 samples of Atractylodes from 36 locations were collected for RAD-sequencing. Three single nucleotide polymorphism (SNP) datasets based on different filtering strategies were used for phylogenetic analyses. Molecular dating and ancestral distribution reconstruction were performed using both chloroplast DNA sequences (127 Cardueae samples) and SNP (36 Atractylodes samples) datasets. KEY RESULTS: Six species of Atractylodes were well resolved as individually monophyletic, although some introgression was identified among accessions of A. chinensis, A. lancea and A. koreana. Dispersal of the subtribe Carlininae from the Mediterranean to East Asia occurred after divergence between Atractylodes and Carlina L. + Atractylis L. + Thevenotia DC. at ~31.57 Ma, resulting in an East Asian-Tethyan disjunction. Diversification of Atractylodes in East Asia mainly occurred from the Late Miocene to the Early Pleistocene. CONCLUSIONS: Aridification of Asia and the closure of the Turgai Strait in the Late Oligocene promoted the dispersal of Cardueae from the Mediterranean to East China. Subsequent uplift of the Qinghai-Tibet Plateau as well as changes in Asian monsoon systems resulted in an East Asian-Tethyan disjunction between Atractylodes and Carlina + Atractylis + Thevenotia. In addition, Late Miocene to Quaternary climates and sea level fluctuations played major roles in the diversification of Atractylodes. Through this study of different taxonomic levels using genomic data, we have revealed an overlooked dispersal route between the Mediterranean and far East Asia (Japan/Korea) via Central Asia and East China.

The Neuroprotective Effect of Tea Polyphenols on the Regulation of Intestinal Flora.

Tea polyphenols (TPs) are the general compounds of natural polyhydroxyphenols extracted in tea. Although a large number of studies have shown that TPs have obvious neuroprotective and neuro repair effects, they are limited due to the low bioavailability in vivo. However, TPs can act indirectly on the central nervous system by affecting the "microflora-gut-brain axis", in which the microbiota and its composition represent a factor that determines brain health. Bidirectional communication between the intestinal microflora and the brain (microbe-gut-brain axis) occurs through a variety of pathways, including the vagus nerve, immune system, neuroendocrine pathways, and bacteria-derived metabolites. This axis has been shown to influence neurotransmission and behavior, which is usually associated with neuropsychiatric disorders. In this review, we discuss that TPs and their metabolites may provide benefits by restoring the imbalance of intestinal microbiota and that TPs are metabolized by intestinal flora, to provide a new idea for TPs to play a neuroprotective role by regulating intestinal flora.

Genomic insights on the contribution of balancing selection and local adaptation to the long-term survival of a widespread living fossil tree, Cercidiphyllum japonicum.

'Living fossils' are testimonies of long-term sustained ecological success, but how demographic history and natural selection contributed to their survival, resilience, and persistence in the face of Quaternary climate fluctuations remains unclear. To better understand the interplay between demographic history and selection in shaping genomic diversity and evolution of such organisms, we assembled the whole genome of Cercidiphyllum japonicum, a widespread East Asian Tertiary relict tree, and resequenced 99 individuals of C. japonicum and its sister species, Cercidiphyllum magnificum (Central Japan). We dated this speciation event to the mid-Miocene, and the intraspecific lineage divergence of C. japonicum (China vs Japan) to the Early Pliocene. Throughout climatic upheavals of the late Tertiary/Quaternary, population bottlenecks greatly reduced the genetic diversity of C. japonicum. However, this polymorphism loss was likely counteracted by, first, long-term balancing selection at multiple chromosomal and heterozygous gene regions, potentially reflecting overdominance, and, second, selective sweeps at stress response and growth-related genes likely involved in local adaptation. Our findings contribute to a better understanding of how living fossils have survived climatic upheaval and maintained an extensive geographic range; that is, both types of selection could be major factors contributing to the species' survival, resilience, and persistence.

Genetic insights into the evolution of genera with the eastern Asia-eastern North America floristic disjunction: a transcriptomics analysis.

PREMISE: Large disjunctions in species distributions provide excellent opportunities to study processes that shape biogeographic patterns. One such disjunction is the eastern Asia-eastern North America (EA-ENA) floristic disjunction. For many genera with this disjunction, species richness is greater in EA than in ENA; this pattern has been attributed, in part, to higher rates of molecular evolution and speciation in EA. Longer branch lengths have been found in some EA clades, relative to their ENA sister clades, suggesting that the EA lineages have evolved at a higher rate, possibly due to environmental heterogeneity, potentially contributing to the species richness anomaly. METHODS: To evaluate whether rates of molecular evolution are elevated in EA relative to ENA, we used transcriptomes from species in 11 genera displaying this disjunction. Rates of molecular evolution were estimated for up to 385 orthologous nuclear loci per genus. RESULTS: No statistically significant differences were identified in pairwise comparisons between EA and ENA sister species, suggesting equal rates of molecular evolution for both species; the data also suggest similar selection pressures in both regions. For larger genera, evidence likewise argues against more species-rich clades having higher molecular evolutionary rates, regardless of region. Our results suggest that genes across multiple gene ontology categories are evolving at similar rates under purifying selection in species in both regions. CONCLUSIONS: Our data support the hypothesis that greater species richness in EA than ENA is due to factors other than an overall increase in rates of molecular evolution in EA.

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.

Chloroplast genome analyses and genomic resource development for epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae), using genome skimming data.

BACKGROUND: Epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae) have an epilithic habitat (rocky slopes) and a parapatric distribution in East Asia, which makes them an ideal model for a more comprehensive understanding of the demographic and divergence history and the influence of climate changes in East Asia. However, the genetic background and resources for these two genera are scarce. RESULTS: The complete chloroplast (cp) genomes of two Oresitrophe rupifraga and one Mukdenia rossii individuals were reconstructed and comparative analyses were conducted to examine the evolutionary pattern of chloroplast genomes in Saxifragaceae. The cp genomes ranged from 156,738 bp to 156,960 bp in length and had a typical quadripartite structure with a conserved genome arrangement. Comparative analysis revealed the intron of rpl2 has been lost in Heuchera parviflora, Tiarella polyphylla, M. rossii and O. rupifraga but presents in the reference genome of Penthorum chinense. Seven cp hotspot regions (trnH-psbA, trnR-atpA, atpI-rps2, rps2-rpoC2, petN-psbM, rps4-trnT and rpl33-rps18) were identified between Oresitrophe and Mukdenia, while four hotspots (trnQ-psbK, trnR-atpA, trnS-psbZ and rpl33-rps18) were identified within Oresitrophe. In addition, 24 polymorphic cpSSR loci were found between Oresitrophe and Mukdenia. Most importantly, we successfully developed 126 intergeneric polymorphic gSSR markers between Oresitrophe and Mukdenia, as well as 452 intrageneric ones within Oresitrophe. Twelve randomly selected intergeneric gSSRs have shown that these two genera exhibit a significant genetic structure. CONCLUSIONS: In this study, we conducted genome skimming for Oresitrophe rupifraga and Mukdenia rossii. Using these data, we were able to not only assemble their complete chloroplast genomes, but also develop abundant genetic resources (cp hotspots, cpSSRs, polymorphic gSSRs). The genomic patterns and genetic resources presented here will contribute to further studies on population genetics, phylogeny and conservation biology in Saxifragaceae.

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.

Evolutionary insights from comparative transcriptome and transcriptome-wide coalescence analyses in Tetrastigma hemsleyanum.

BACKGROUND: Tetrastigma hemsleyanum is of great medicinal importance and used as a model system to address the evolutionary history of warm-temperate evergreen (WTE) forest biomes in East Asia over Neogene time scales. However, further studies on the neutral and adaptive divergence processes of T. hemsleyanum are currently impeded by a lack of genomic resources. In this study, we de novo assembled and annotated a reference transcriptome for two cpDNA lineages (Central-South-East vs. Southwest) of T. hemsleyanum. We further used comparative genomic and multilocus coalescent approaches to investigate the tempo and mode of lineage diversification in T. hemsleyanum. RESULTS: A total of 52,838 and 65,197 unigenes with an N50 of 1,667 and 1,841 bp for Central-South-East (CSE) and Southwest (SW) lineages, respectively, were recovered, and 6,692 putative orthologs were identified between the two lineages. Estimation of Ka/Ks ratios for these orthologs revealed that ten genes had Ka/Ks values significantly greater than 0.5 (P < 0.05), whereas 2,099 (Ka/Ks < 0.5, P < 0.05) were inferred to be under purifying selection. Based on three bioinformatic strategies, we identified a total of 1,018 single-copy nuclear genes (SCNGs) from the orthologs. We successfully designed eight nuclear gene primer pairs with high intraspecific variation (e.g. hT = 0.923, πT = 1.68×10-3), when surveyed across a subset of T. hemsleyanum individuals. Concordant with the previous cpDNA data, the haplotype networks constructed for most nuclear gene loci clearly identified the two lineages. A multilocus coalescence analysis suggested that the separation between the two lineages appears to have occurred during the mid-Pliocene. Despite their ancient divergence, both lineages experienced expansion at rather localized scales and have continued to exchange genes at a low rate. CONCLUSIONS: This study demonstrated the utility of transcriptome sequencing as a basis for SCNG development in non-model species and the advantages of integrating multiple nuclear loci for phylogeographic and phylogenetic studies.

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.

Testing the monophyly of Aesculus L. and Billia Peyr., woody genera of tribe Hippocastaneae of the Sapindaceae.

Hippocastaneae is a well-supported clade in Sapindaceae that comprises 15+ species; 12+ in Aesculus, two in Billia, and one in Handeliodendron Rehder. The monophyly of Aesculus and Billia were widely assumed, but a recent molecular phylogenetic study of Sapindanceae used seven species of Aesculus and one each of Billia and Handeliodendron and showed that Billia and Handeliodendron were nested within Aesculus. Here, we tested the hypothesis that Aesculus and Billia are mutually monophyletic using phylogenetic analyses of seven molecular markers and 31 accessions of Hippocastaneae representing 14 species. We performed phylogenetic analyses using a dataset of concatenated genes as well as with coalescent method for constructing a species tree from individual gene trees. The analysis of seven concatenated markers and the species tree strongly supported the mutual monophyly of Aesculus and Billia. We also recovered support for the traditional arrangement of genera within Hippocastaneae: Aesculus and Billia comprising a clade that is sister to Handeliodendron. However, the relationships among the genera remain incompletely resolved.

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 ultrasonic-assisted antifungal film loaded with fermented walnut meal on Rosa roxburghii Tratt during near-freezing temperature storage.

Fermented walnut meal (FW) has antifungal activity against Penicillium victoriae, a fungus responsible for Rosa roxbughii Tratt spoilage. This study characterized and applied ultrasonic-assisted antifungal film loaded with FW to preserve R. roxbughii Tratt during near-freezing temperature (NFT). Results showed that O2 and CO2 transmission rates decreased by 80.02% and 29.05%, respectively, and antimicrobial properties were improved with ultrasound at 560 W for 5 min and 1% FW. Fourier transform infrared spectroscopy and X-ray diffraction results revealed ultrasound improved hydrogen bonds and inductive effect via ─NH, ─OH, and C═O bonds. The addition of FW led to the formation of CMCS-GL-FW polymer via C═O bond. Thermogravimetric analysis and transmission electron microscope results demonstrated thermal degradation process was decomposed by ultrasound, and the internal structure of P. victoriae was accelerated by the addition of FW. Compared to the U-CMCS/GL group, the vitamin C content, peroxidase, and catalase activities of U-CMCS/GL/FW were enhanced by 4.24%, 8.52%, and 14.3% during NFT (-0.8 to -0.4°C), respectively. Particularly, the fungal count of the U-CMCS/GL/FW group did not exceed 105 CFU g-1 at the end of storage, and the relative abundance of P. victoriae decreased to 0.007%. Our findings provide an effective route for agricultural waste as natural antifungal compounds in the active packaging industry. PRACTICAL APPLICATION: In this study, the barrier and antimicrobial properties of film were successfully improved by ultrasonic treatment and loaded fermented walnut meal. The ultrasonic-assisted antifungal film loaded with fermented walnut meal effectively delayed the degradation of nutrients and reduced microbial invasion of Rosa roxburghii Tratt. These results provide a theoretical basis for the application of agricultural waste in the food packaging industry.

Comparative phylogeography of the Smilax hispida group (Smilacaceae) in eastern Asia and North America--implications for allopatric speciation, causes of diversity disparity, and origins of temperate elements in Mexico.

The Smilax hispida group (Smilacaceae) exhibits a discontinuous distribution in eastern Asia, eastern and western United States, and Mexico. A broad scale phylogeographic analysis was conducted for this group to evaluate the hypotheses of accelerated allopatric divergence in eastern Asia and a northern origin of the temperate elements in Mexico. Phylogeny was inferred using seven plastid and nuclear DNA sequences. Species delineation was assessed using genealogical sorting indices (GSI). Lineage divergence time, haplotype diversification rates, and ancestral distributions were estimated using Bayesian methods. Phylogeographic patterns in eastern Asia and North America were compared by analyzing 539 individuals from 64 populations to assess allopatric diversification. Results strongly supported delineation of six allopatric species, the origin of this group from a Mexican ancestor around 11.42mya, and Mexican origins of the temperate species in Mexico. Significant geographic structure of haplotypes was found in eastern Asia, and greater haplotype diversification rate was observed for the North American lineage. Our data support allopatric speciation in eastern Asia but do not find evidence of an elevated diversification rate. Greater species diversity of the study system in eastern Asia may be due to a longer evolutionary history. Our results do not support northern origins of the Mexican temperate species.

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.

Phylogeography of North American herbaceous Smilax (Smilacaceae): Combined AFLP and cpDNA data support a northern refugium in the Driftless Area.

PREMISE OF THE STUDY: The genetic structure of North American herbaceous Smilax, a clade of closely related understory herbs that has a wide and disjunct distribution, was investigated to test the hypothesis of whether a northern refugium in the upper Midwest may have existed for these plants during the last glaciation. METHODS: We analyzed 33 populations sampled from California and throughout the eastern United States using AFLP and chloroplast DNA sequence data. KEY RESULTS: Individuals of S. jamesii from northern California formed a clade sister to the eastern North American species, and they likely diverged from each other during the Pleistocene. Among the eastern species, two lineages were found on opposite sides of the Appalachian Mountains. The populations near the "Driftless Area" contained most of the chlorotypes found in the Midwest, including a unique one endemic to this area, and the AFLP data indicated that one population from this area has the highest frequency-down-weighted-marker value. CONCLUSIONS: This study, and others that have focused on mammals, amphibians, and woody plants, provides further evidence for the debate over whether the upper Midwest's "Driftless Area" may have hosted a biologically diverse northern glacial refugium. Herbaceous species of Smilax from eastern North American exhibit an Appalachian discontinuity. They appear to have persisted in multiple refugia both east and west of the Appalachians, with several populations persisting in situ through the Last Glacial Maximum (LGM). We suggest that they experienced localized expansion after the LGM, possibly followed by subsequent contact between the Midwest and the East Coastal lineages.

Using landscape genomics to assess local adaptation and genomic vulnerability of a perennial herb Tetrastigma hemsleyanum (Vitaceae) in subtropical China.

Understanding adaptive genetic variation of plant populations and their vulnerabilities to climate change are critical to preserve biodiversity and subsequent management interventions. To this end, landscape genomics may represent a cost-efficient approach for investigating molecular signatures underlying local adaptation. Tetrastigma hemsleyanum is, in its native habitat, a widespread perennial herb of warm-temperate evergreen forest in subtropical China. Its ecological and medicinal values constitute a significant revenue for local human populations and ecosystem. Using 30,252 single nucleotide polymorphisms (SNPs) derived from reduced-representation genome sequencing in 156 samples from 24 sites, we conducted a landscape genomics study of the T. hemsleyanum to elucidate its genomic variation across multiple climate gradients and genomic vulnerability to future climate change. Multivariate methods identified that climatic variation explained more genomic variation than that of geographical distance, which implied that local adaptation to heterogeneous environment might represent an important source of genomic variation. Among these climate variables, winter precipitation was the strongest predictor of the contemporary genetic structure. F ST outlier tests and environment association analysis totally identified 275 candidate adaptive SNPs along the genetic and environmental gradients. SNP annotations of these putatively adaptive loci uncovered gene functions associated with modulating flowering time and regulating plant response to abiotic stresses, which have implications for breeding and other special agricultural aims on the basis of these selection signatures. Critically, modelling revealed that the high genomic vulnerability of our focal species via a mismatch between current and future genotype-environment relationships located in central-northern region of the T. hemsleyanum's range, where populations require proactive management efforts such as assistant adaptation to cope with ongoing climate change. Taken together, our results provide robust evidence of local climate adaption for T. hemsleyanum and further deepen our understanding of adaptation basis of herbs in subtropical China.