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.

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.

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.

Plant and fungal species interactions differ between aboveground and belowground habitats in mountain forests of eastern China.

Plant and fungal species interactions drive many essential ecosystem properties and processes; however, how these interactions differ between aboveground and belowground habitats remains unclear at large spatial scales. Here, we surveyed 494 pairwise fungal communities in leaves and soils by Illumina sequencing, which were associated with 55 woody plant species across more than 2,000-km span of mountain forests in eastern China. The relative contributions of plant, climate, soil and space to the variation of fungal communities were assessed, and the plant-fungus network topologies were inferred. Plant phylogeny was the strongest predictor for fungal community composition in leaves, accounting for 19.1% of the variation. In soils, plant phylogeny, climatic factors and soil properties explained 9.2%, 9.0% and 8.7% of the variation in soil fungal community, respectively. The plant-fungus networks in leaves exhibited significantly higher specialization, modularity and robustness (resistance to node loss), but less complicated topology (e.g., significantly lower linkage density and mean number of links) than those in soils. In addition, host/fungus preference combinations and key species, such as hubs and connectors, in bipartite networks differed strikingly between aboveground and belowground samples. The findings provide novel insights into cross-kingdom (plant-fungus) species co-occurrence at large spatial scales. The data further suggest that community shifts of trees due to climate change or human activities will impair aboveground and belowground forest fungal diversity in different ways.

Comparative and Phylogenetic Analyses of Complete Chloroplast Genomes of Scrophularia incisa Complex (Scrophulariaceae).

The Scrophularia incisa complex is a group of closely related desert and steppe subshrubs that includes S. incisa, S. kiriloviana and S. dentata, which are the only S. sect. Caninae components found in Northwest China. Based on earlier molecular evidence, the species boundaries and phylogenetic relationships within this complex remain poorly resolved. Here, we characterized seven complete chloroplast genomes encompassing the representatives of the three taxa in the complex and one closely related species, S. integrifolia, as well as three other species of Scrophularia. Comparative genomic analyses indicated that the genomic structure, gene order and content were highly conserved among these eleven plastomes. Highly variable plastid regions and simple sequence repeats (SSRs) were identified. The robust and consistent phylogenetic relationships of the S. incisa complex were firstly constructed based on a total of 26 plastid genomes from Scrophulariaceae. Within the monophyletic complex, a S. kiriloviana individual from Pamirs Plateau was identified as the earliest diverging clade, followed by S. dentata from Tibet, while the remaining individuals of S. kiriloviana from the Tianshan Mountains and S. incisa from Qinghai-Gansu were clustered into sister clades. Our results evidently demonstrate the capability of plastid genomes to improve phylogenetic resolution and species delimitation, particularly among closely related species, and will promote the understanding of plastome evolution in Scrophularia.

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.

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.

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.

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.

Comparisons of Natural and Cultivated Populations of Corydalis yanhusuo Indicate Divergent Patterns of Genetic and Epigenetic Variation.

Epigenetic variation may contribute to traits that are important in domestication, but how patterns of genetic and epigenetic variation differ between cultivated and wild plants remains poorly understood. In particular, we know little about how selection may shape epigenetic variation in natural and cultivated populations. In this study, we investigated 11 natural populations and 6 major cultivated populations using amplified fragment length polymorphism (AFLP) and methylation-sensitive AFLP (MS-AFLP or MSAP) markers to identify patterns of genetic and epigenetic diversity among Corydalis yanhusuo populations. We further explored correlations among genetic, epigenetic, alkaloidal, and climatic factors in natural and cultivated C. yanhusuo. We found support for a single origin for all cultivated populations, from a natural population which was differentiated from the other natural populations. The magnitude of F ST based on AFLP was significantly correlated with that for MSAP in pairwise comparisons in both natural and cultivated populations, suggesting a relationship between genetic and epigenetic variation in C. yanhusuo. This relationship was further supported by dbRDA (distance-based redundancy analyses) where some of the epigenetic variation could be explained by genetic variation in natural and cultivated populations. Genetic variation was slightly higher in natural than cultivated populations, and exceeded epigenetic variation in both types of populations. However, epigenetic differentiation exceeded that of genetic differentiation among cultivated populations, while the reverse was observed among natural populations. The differences between wild and cultivated plants may be partly due to processes inherent to cultivation and in particular the differences in mode of reproduction. The importance of epigenetic compared to genetic modifications is thought to vary depending on reproductive strategies, and C. yanhusuo usually reproduces sexually in natural environments, while the cultivated C. yanhusuo are propagated clonally. In addition, alkaloid content of C. yanhusuo varied across cultivated populations, and alkaloid content was significantly correlated to climatic variation, but also to genetic (6.89%) and even more so to epigenetic (14.09%) variation in cultivated populations. Our study demonstrates that epigenetic variation could be important in cultivation of C. yanhusuo and serve as a source of variation for response to environmental conditions.

De novo Transcriptome Assembly, Gene Annotation and SSR Marker Development in the Moon Seed Genus Menispermum (Menispermaceae).

The moonseed genus Menispermum L. (Menispermaceae) is disjunctly distributed in East Asia and eastern North America. Although Menispermum has important medicinal value, genetic and genomic information is scarce, with very few available molecular markers. In the current study, we used Illumina transcriptome sequencing and de novo assembly of the two Menispermum species to obtain in-depth genetic knowledge. From de novo assembly, 53,712 and 78,921 unigenes were generated for M. canadense and M. dauricum, with 37,527 (69.87%) and 55,211 (69.96%) showing significant similarities against the six functional databases, respectively. Moreover, 521 polymorphic EST-SSRs were identified. Of them, 23 polymorphic EST-SSR markers were selected to investigate the population genetic diversity within the genus. The newly developed EST-SSR markers also revealed high transferability among the three examined Menispermaceae species. Overall, we provide the very first transcriptomic analyses of this important medicinal genus. In addition, the novel microsatellite markers developed here will aid future studies on the population genetics and phylogeographic patterns of Menispermum at the intercontinental geographical scale.

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.

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.

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.

Phylogenetic imprint of woody plants on the soil mycobiome in natural mountain forests of eastern China.

Recent studies have detected strong phylogenetic signals in tree-fungus associations for diseased leaves and mycorrhizal symbioses. However, the extent of plant phylogenetic constraints on the free-living soil mycobiome remains unknown, especially at broad geographic scales. Here, 343 soil samples were collected adjacent to individual tree trunks, representing 58 woody plant species located in five mountain forests of eastern China. Integrating plant species identity and phylogenetic information, we aimed to unravel the relative contributions of phylogenetic relationships among tree species, abiotic environmental filtering, and geographic isolation to the geographic distribution of soil mycobiome. We found that the community dissimilarities of total fungi and each dominant guild (viz. saprotrophs, plant pathogens, and ectomycorrhizal fungi) significantly increased with increasing plant phylogenetic distance. Plant phylogenetic eigenvectors explained 11.4% of the variation in community composition, whereas environmental and spatial factors explained 24.1% and 7.2% of the variation, respectively. The communities of ectomycorrhizal fungi and plant pathogens were relatively more strongly affected by plant phylogeny than those of saprotrophs (13.7% and 10.4% vs. 8.5%). Overall, our results demonstrate how plant phylogeny, environment, and geographic space contribute to forest soil fungal distributions and suggest that the influence of plant phylogeny on fungal association may differ by guilds.

Genomic effects of population collapse in a critically endangered ironwood tree Ostrya rehderiana.

Increased human activity and climate change are driving numerous tree species to endangered status, and in the worst cases extinction. Here we examine the genomic signatures of the critically endangered ironwood tree Ostrya rehderiana and its widespread congener O. chinensis. Both species have similar demographic histories prior to the Last Glacial Maximum (LGM); however, the effective population size of O. rehderiana continued to decrease through the last 10,000 years, whereas O. chinensis recovered to Pre-LGM numbers. O. rehderiana accumulated more deleterious mutations, but purged more severely deleterious recessive variations than in O. chinensis. This purging and the gradually reduced inbreeding depression together may have mitigated extinction and contributed to the possible future survival of the outcrossing O. rehderiana. Our findings provide critical insights into the evolutionary history of population collapse and the potential for future recovery of the endangered trees.

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.

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.