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Background Gymnosperms represent five of the six lineages of seed plants. However, most sequenced plant mitochondrial genomes (mitogenomes) have been generated for angiosperms, whereas mitogenomic sequences have been generated for only six gymnosperms. In particular, complete mitogenomes are available for all major seed plant lineages except Conifer II (non-Pinaceae conifers or Cupressophyta), an important lineage including six families, which impedes a comprehensive understanding of the mitogenomic diversity and evolution in gymnosperms. Results Here, we report the complete mitogenome of Taxus cuspidata in Conifer II. In comparison with previously released gymnosperm mitogenomes, we found that the mitogenomes of Taxus and Welwitschia have lost many genes individually, whereas all genes were identified in the mitogenomes of Cycas, Ginkgo and Pinaceae. Multiple tRNA genes and introns also have been lost in some lineages of gymnosperms, similar to the pattern observed in angiosperms. In general, gene clusters could be less conserved in gymnosperms than in angiosperms. Moreover, fewer RNA editing sites were identified in the Taxus and Welwitschia mitogenomes than in other mitogenomes, which could be correlated with fewer introns and frequent gene losses in these two species. Conclusions We have sequenced the Taxus cuspidata mitogenome, and compared it with mitogenomes from the other four gymnosperm lineages. The results revealed the diversity in size, structure, gene and intron contents, foreign sequences, and mutation rates of gymnosperm mitogenomes, which are different from angiosperm mitogenomes.

After the merger of the former Taxodiaceae and Cupressaceae s.s., currently the conifer family Cupressaceae (sensu lato) comprises seven subfamilies and 32 genera, most of which are important components of temperate and mountainous forests. With the exception of a recently published genus-level phylogeny of gymnosperms inferred from sequence analysis of 790 orthologs, previous phylogenetic studies of Cupressaceae were based mainly on morphological characters or a few molecular markers, and did not completely resolve the intergeneric relationships. In this study, we reconstructed a robust and well-resolved phylogeny of Cupressaceae represented by all 32 genera, using 1944 genes (Orthogroups) generated from transcriptome sequencing. Reticulate evolution analyses detected a possible ancient hybridization that occurred between ancestors of two subclades of Cupressoideae, including Microbiota-Platycladus-Tetraclinis (MPT) and Juniperus-Cupressus-Hesperocyparis-Callitropsis-Xanthocyparis (JCHCX), although both concatenation and coalescent trees are highly supported. Moreover, divergence time estimation and ancestral area reconstruction indicate that Cupressaceae very likely originated in Asia in the Triassic, and geographic isolation caused by continental separation drove the vicariant evolution of the two subfamilies Cupressoideae and Callitroideae in the northern and southern hemispheres, respectively. Evolutionary analyses of some morphological characters suggest that helically arranged linear-acicular leaves and imbricate bract-scale complexes represent ancestral states, and the shift from linear-acicular leaves to scale-like leaves was associated with the shift from helical to decussate arrangement. Our study sheds new light on phylogeny and evolutionary history of Cupressaceae, and strongly suggests that both dichotomous phylogenetic and reticulate evolution analyses be conducted in phylogenomic studies.

China is one of the diversity centers of gymnosperms. Nearly one-fifth (195 species) of gymnosperms are located in China, but 69 species are threatened. To date, the conservation status of gymnosperms, especially threatened gymnosperms in China, remains largely unknown, which seriously restricts the comprehensive protection of gymnosperms. Understanding the distribution pattern of species richness and exploring the relationships between species richness and environmental factors are key steps for their protection. In this study, we first constructed a database for the 69 threatened species of gymnosperms with 13 270 distribution records. It is found that 31 of the grid cells (50 × 50 km) cover all threatened gymnosperm species in China, and the grid cells of threatened gymnosperms are mainly distributed in the southern area of the Yellow River, with a distribution center in the Western Sichuan Plateau. Then, we evaluated the conservation status of threatened gymnosperms, and the results indicate that 9 (13%) threatened gymnosperms are distributed outside of nature reserves. Therefore, there are still conservation gaps in the protection of threatened gymnosperms in China. We should give more attention to unprotected threatened gymnosperms and conduct taxonomic studies on the species without detailed distribution records. Finally, conservation priority areas and priority conservation levels of threatened gymnosperms in China were proposed. The Western Sichuan Plateau is the most important conservation priority area of threatened gymnosperms. This study will shed light on plant protection and forest management in China.

Evolutionary radiation is a widely recognized mode of species diversification, but its underlying mechanisms have not been unambiguously resolved for species-rich cosmopolitan plant genera. In particular, it remains largely unknown how biological and environmental factors have jointly driven its occurrence in specific regions. Here, we use Rhododendron, the largest genus of woody plants in the Northern Hemisphere, to investigate how geographic and climatic factors, as well as functional traits, worked together to trigger plant evolutionary radiations and shape the global patterns of species richness based on a solid species phylogeny. Using 3,437 orthologous nuclear genes, we reconstructed the first highly supported and dated backbone phylogeny of Rhododendron comprising 200 species that represent all subgenera, sections, and nearly all multispecies subsections, and found that most extant species originated by evolutionary radiations when the genus migrated southward from circumboreal areas to tropical/subtropical mountains, showing rapid increases of both net diversification rate and evolutionary rate of environmental factors in the Miocene. We also found that the geographically uneven diversification of Rhododendron led to a much higher diversity in Asia than in other continents, which was mainly driven by two environmental variables, that is, elevation range and annual precipitation, and were further strengthened by the adaptation of leaf functional traits. Our study provides a good example of integrating phylogenomic and ecological analyses in deciphering the mechanisms of plant evolutionary radiations, and sheds new light on how the intensification of the Asian monsoon has driven evolutionary radiations in large plant genera of the Himalaya-Hengduan Mountains.

Cycads represent one of the most ancient lineages of living seed plants. Identifying genomic features uniquely shared by cycads and other extant seed plants, but not non-seed-producing plants, may shed light on the origin of key innovations, as well as the early diversification of seed plants. Here, we report the 10.5-Gb reference genome of Cycas panzhihuaensis, complemented by the transcriptomes of 339 cycad species. Nuclear and plastid phylogenomic analyses strongly suggest that cycads and Ginkgo form a clade sister to all other living gymnosperms, in contrast to mitochondrial data, which place cycads alone in this position. We found evidence for an ancient whole-genome duplication in the common ancestor of extant gymnosperms. The Cycas genome contains four homologues of the fitD gene family that were likely acquired via horizontal gene transfer from fungi, and these genes confer herbivore resistance in cycads. The male-specific region of the Y chromosome of C. panzhihuaensis contains a MADS-box transcription factor expressed exclusively in male cones that is similar to a system reported in Ginkgo, suggesting that a sex determination mechanism controlled by MADS-box genes may have originated in the common ancestor of cycads and Ginkgo. The C. panzhihuaensis genome provides an important new resource of broad utility for biologists. The study assembled a chromosome-level genome of Cycas panzhihuaensis, the last major lineage of seed plants for which a high-quality genome assembly was lacking. The study closes an important gap in our understanding of genome structure and evolution in seed plants.

Brassica juncea is an important vegetable and condiment crop widely grown in Asia, and the yield and quality of its product organs are affected by flowering time. AGAMOUS-LIKE18-1 (AGL18-1) belongs to a member of MADS-domain transcription factors, which play vital roles in flowering time control, but the biological role of AGL18-1 in B. juncea (BjuAGL18-1) has not been thoroughly revealed in flowering regulatory network. In this study, BjuAGL18-1 expressed highly in inflorescence and flower, but slightly in root, stem and leaf. The sense and anti-sense transgenic lines of BjuAGL18-1 were generated and showed that BjuAGL18-1 functioned as a flowering inhibitor and depressed growth of lateral branching. During the vegetative phase, BjuAGL18-1 induced another flowering repressor AGAMOUS-LIKE15 (BjuAGL15) but inhibited the flowering signal integrator of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BjuSOC1) in Brassica juncea. Whereas, during the flower developmental phase, both SOC1 and AGAMOUS-LIKE24 (AGL24) were down-regulated by BjuAGL18-1. By contrast, AGL15 was promoted by BjuAGL18-1, while SHORT VEGETATIVE PHASE (SVP) was independent of BjuAGL18-1. Additionally, HISTONE DEACETYLASE 9 (HDA9) was highly induced by BjuAGL18-1. These results will provide valuable information for clarifying the molecular mechanism of BjuAGL18-1 in mediating flowering time.

Significance Understanding what determines the distribution of biodiversity remains one of the great challenges in evolutionary biology and ecology. The Northern Hemisphere conifers do not follow a latitudinal diversity gradient and show a unique distribution pattern, that is most species are distributed at middle latitudes. To reveal the mechanisms underlying this pattern, we investigate the spatiotemporal evolution of the largest conifer genus Pinus. We find that the midlatitude region has been an evolutionary museum for Pinus, and particularly, topography has played the most important role in pine diversification. Moreover, our results indicate that pine’s preference of warm and somewhat drier habitats could be helpful for their adaptation to the Anthropocene climate warming, which is important for biodiversity conservation and forest management., How coniferous forests evolved in the Northern Hemisphere remains largely unknown. Unlike most groups of organisms that generally follow a latitudinal diversity gradient, most conifer species in the Northern Hemisphere are distributed in mountainous areas at middle latitudes. It is of great interest to know whether the midlatitude region has been an evolutionary cradle or museum for conifers and how evolutionary and ecological factors have driven their spatiotemporal evolution. Here, we investigated the macroevolution of Pinus, the largest conifer genus and characteristic of northern temperate coniferous forests, based on nearly complete species sampling. Using 1,662 genes from transcriptome sequences, we reconstructed a robust species phylogeny and reestimated divergence times of global pines. We found that ∼90% of extant pine species originated in the Miocene in sharp contrast to the ancient origin of Pinus, indicating a Neogene rediversification. Surprisingly, species at middle latitudes are much older than those at other latitudes. This finding, coupled with net diversification rate analysis, indicates that the midlatitude region has provided an evolutionary museum for global pines. Analyses of 31 environmental variables, together with a comparison of evolutionary rates of niche and phenotypic traits with a net diversification rate, found that topography played a primary role in pine diversification, and the aridity index was decisive for the niche rate shift. Moreover, fire has forced diversification and adaptive evolution of Pinus. Our study highlights the importance of integrating phylogenomic and ecological approaches to address evolution of biological groups at the global scale.

Floristic composition within a geographic area is driven by a wide array of factors from local biotic interactions to biogeographical processes. Subtropical East Asia is a key biodiversity hotspot of the world, and harbors the most families of extant gymnosperms and a large number of endemic genera with ancient origins, but rare phylogenetic studies explored whether it served as a diversification center for gymnosperms. Here, we investigated the evolutionary and biogeographical history of subtropical East Asian white pines using an integrative approach that combines phylotranscriptomic and ecological analyses. Using 2,606 orthologous nuclear genes, we reconstructed a fully resolved and dated phylogeny of these species. Two main clades first diverged in the early Miocene, and by the late Miocene, all species appeared. Two white pines endemic to Taiwan Island experienced independent colonization events and regional extinction, which resulted in the present disjunctive distribution from mainland China. Ecological and biogeographical analyses indicate that the monsoon-driven assembly of evergreen broadleaved forests (EBLFs) might have significantly affected the diversification of subtropical East Asian white pines. Our study highlights the interactions of biotic and abiotic forces in the diversification and speciation of subtropical East Asian white pines. These findings indicate that subtropical East Asia is not only a floristic museum, but also a diversification center for gymnosperms. Our study also demonstrates the importance of phylotranscriptomics on species delimitation and biodiversity conservation, particularly for closely related species.

The gymnosperm Welwitschia mirabilis belongs to the ancient, enigmatic gnetophyte lineage. It is a unique desert plant with extreme longevity and two ever-elongating leaves. We present a chromosome-level assembly of its genome (6.8 Gb/1 C) together with methylome and transcriptome data to explore its astonishing biology. We also present a refined, high-quality assembly of Gnetum montanum to enhance our understanding of gnetophyte genome evolution. The Welwitschia genome has been shaped by a lineage-specific ancient, whole genome duplication (~86 million years ago) and more recently (1-2 million years) by bursts of retrotransposon activity. High levels of cytosine methylation (particularly at CHH motifs) are associated with retrotransposons, whilst long-term deamination has resulted in an exceptionally GC-poor genome. Changes in copy number and/or expression of gene families and transcription factors (e.g. R2R3MYB, SAUR) controlling cell growth, differentiation and metabolism underpin the plant’s longevity and tolerance to temperature, nutrient and water stress., Welwitschia mirabilis is a unique plant that only has two leaves, but it can survive in hostile conditions of the African desert. Here, the authors report its chromosome-level genome assembly and discuss how gene function and regulation have given rise to its unique morphology and environmental adaptions.

How large cosmopolitan plant genera survived great environmental changes and rediversified remains largely unknown. Here we investigated mechanisms underlying the rediversification of Rhododendron, the largest genus of woody plants in the Northern Hemisphere. Using 3437 orthologous nuclear genes, we reconstructed the first completely resolved and dated phylogeny of Rhododendron. We found that most extant species of Rhododendron originated by Neogene rediversification from Paleogene relicts during southern migration. The geographically uneven rediversification of Rhododendron led to a much higher diversity in Asia than in other continents, which was driven by two main environmental variables, i.e., habitat heterogeneity represented by elevation range and annual precipitation related to the Asian monsoons, and can be explained by leaf functional traits that show strong phylogenetic signals and correspond well with leaf-forms and geographical regions. Our study highlights the importance of integrating phylogenomic and ecological analyses in revealing the spatiotemporal evolution of species-rich cosmopolitan plant genera.

The evolutionary dynamics of polyploid genomes and consequences of polyploidy have been studied extensively in angiosperms but very rarely in gymnosperms. The gymnospermous genus Ephedra is characterized by a high frequency of polyploidy, and thus provides an ideal system to investigate the evolutionary mode of allopolyploid genomes and test whether subgenome dominance has occurred in gymnosperms. Here, we sequenced transcriptomes of two allotetraploid species of Ephedra and their putative diploid progenitors, identified expressed homeologs, and analyzed alternative splicing and homeolog expression based on PacBio Iso-Seq and Illumina RNA-seq data. We found that the two subgenomes of the allotetraploids had similar numbers of expressed homeologs, similar percentages of homeologs with dominant expression, and approximately equal numbers of isoforms with alternative splicing, showing an unbiased subgenome evolution as in a few polyploid angiosperms, with a divergence of the two subgenomes at ∼8 Ma. In addition, the nuclear DNA content of the allotetraploid species is almost equal to the sum of two putative progenitors, suggesting limited genome restructuring after allotetraploid speciation. The allopolyploid species of Ephedra might have undergone slow diploidization, and the unbiased subgenome evolution implies that the formation of large genomes in gymnosperms could be attributed to even and slow fractionation following polyploidization.

Background Leaves have highly diverse morphologies. However, with an evolutionary history of approximately 200 million years, leaves of the pine family are relatively monotonous and often collectively called “needles”, although they vary in length, width and cross-section shapes. It would be of great interest to determine whether Pinaceae leaves share similar morpho-physiological features and even consistent developmental and adaptive mechanisms. Results Based on a detailed morpho-anatomical study of leaves from all 11 Pinaceae genera, we particularly investigated the expression patterns of adaxial-abaxial polarity genes in two types of leaves (needlelike and flattened) and compared their photosynthetic capacities. We found that the two types of leaves share conserved spatial patterning of vasculatures and genetic networks for adaxial-abaxial polarity, although they display different anatomical structures in the mesophyll tissue differentiation and distribution direction. In addition, the species with needlelike leaves exhibited better photosynthetic capacity than the species with flattened leaves. Conclusions Our study provides the first evidence for the existence of a conserved genetic module controlling adaxial-abaxial polarity in the development of different Pinaceae leaves.

Phylogenies of an increasing number of taxa have been resolved with the development of phylogenomics. However, the intergeneric relationships of Podocarpaceae, the second largest family of conifers comprising 19 genera and approximately 187 species mainly distributed in the Southern Hemisphere, have not been well disentangled in previous studies, even when genome-scale data sets were used. Here we used 993 nuclear orthologous groups (OGs) and 54 chloroplast OGs (genes), which were generated from 47 transcriptomes of Podocarpaceae and its sister group Araucariaceae, to reconstruct the phylogeny of Podocarpaceae. Our study completely resolved the intergeneric relationships of Podocarpaceae represented by all extant genera and revealed that topological conflicts among phylogenetic trees could be attributed to synonymous substitutions. Moreover, we found that two morphological traits, fleshy seed cones and flattened leaves, might be important for Podocarpaceae to adapt to angiosperm-dominated forests and thus could have promoted its species diversification. In addition, our results indicate that Podocarpaceae originated in Gondwana in the late Triassic and both vicariance and dispersal have contributed to its current biogeographic patterns. Our study provides the first robust transcriptome-based phylogeny of Podocarpaceae, an evolutionary framework important for future studies of this family.

Phylogenomics has shown great potential in resolving evolutionary relationships at different taxonomical levels. However, it remains controversial whether all orthologous genes under different selective pressures can be concatenated for phylogenomic reconstruction. Here we used sect. Cyathophora of Pedicularis, one of the most species-rich genera of angiosperms in the alpine and arctic regions of the Northern Hemisphere, as a model to investigate the efficiency of RNA-seq in resolving relationships of closely related congeneric species. Flower transcriptomes were sequenced for all species of sect. Cyathophora and two outgroup species. Forty-one highly conserved single-/low-copy nuclear genes and 1553 orthologous groups (OGs) were identified and concatenated into five datasets based on gene copy numbers and Ka/Ks values to reconstruct the phylogeny of section Cyathophora. We also tested how many genes minimally can resolve the interspecific relationships, and reconstructed the evolutionary history of some floral characters in sect. Cyathophora. The results showed that the five different datasets consistently resolved the interspecific relationships of sect. Cyathophora, and the interspecific relationships can be robustly reconstructed with maximal support when ⩾20 single-/low-copy nuclear genes or 25 OGs are used. Our study suggests that all OGs under different selective pressures can be concatenated for phylogenomic reconstruction, and provides a successful and efficient use of RNA-seq in reconstructing interspecific relationships of a non-model plant group with recent radiations.

The disjunct distribution between East Asia and North America is one of the best established biogeographic patterns. A robust phylogeny is fundamental for understanding the biogeographic histories of taxa with this distribution pattern. Tsuga (hemlock) is a genus of Pinaceae with a typical intercontinental disjunct distribution in East Asia and eastern and western North America, and its phylogeny has not been completely reconstructed in previous studies. In this study, we reconstructed a highly resolved phylogeny of Tsuga using 881 nuclear genes, 60 chloroplast genes and 23 mitochondrial genes and explored its biogeographic and reticulate evolutionary history. The results of phylogenetic analysis, molecular dating and ancestral area reconstruction indicate that Tsuga very likely originated from North America in the late Oligocene and dispersed from America to East Asia via the Bering Land Bridge during the middle Miocene. In particular, we found complex reticulate evolutionary pattern among the East Asian hemlock species. T. sieboldii possibly originated from hybridization with the ancestor of T. chinensis from mainland China and T. forrestii as the paternal donor and the ancestor of T. diversifolia and T. ulleungensis as the maternal donor. T. chinensis (Taiwan) could have originated by hybridization together with T. sieboldii and then evolved independently after dispersal to the Taiwan Island, subsequently experiencing mitochondrial DNA introgression with T. chinensis from mainland China. Moreover, our study found that T. chinensis from western China is more closely related to T. forrestii than to T. chinensis from eastern China. The nonmonophyletic T. chinensis needs taxonomic reconsideration.

We reconstructed a phylogenetic tree of Chinese vascular plants (Tracheophyta) using sequences of the chloroplast genes atpB, matK, ndhF, and rbcL and mitochondrial matR. We produced a matrix comprising 6098 species and including 13 695 DNA sequences, of which 1803 were newly generated. Our taxonomic sampling spanned 3114 genera representing 323 families of Chinese vascular plants, covering more than 93% of all genera known from China. The comprehensive large phylogeny supports most relationships among and within families recognized by recent molecular phylogenetic studies for lycophytes, ferns (monilophytes), gymnosperms, and angiosperms. For angiosperms, most families in Angiosperm Phylogeny Group IV are supported as monophyletic, except for a paraphyletic Dipterocarpaceae and Santalaceae. The infrafamilial relationships of several large families and monophyly of some large genera are well supported by our dense taxonomic sampling. Our results showed that two species of Eberhardtia are sister to a clade formed by all other taxa of Sapotaceae, except Sarcosperma. We have made our phylogeny of Chinese vascular plants publically available for the creation of subtrees via SoTree (http://www.darwintree.cn/flora/index.shtml), an automated phylogeny assembly tool for ecologists.

Understanding the factors that contribute to population genetic structures and ecological niche divergence is crucial for elucidating the biogeographical history and speciation of plants. To disentangle the present phylogeographical patterns and evolutionary history of two closely related parapatric species from Ephedra (Ephedra sinica and E. intermedia), we systematically profiled their population genetic structures, niche divergence and potential speciation processes based on sequence variation of four chloroplast DNA fragments and climate-based ecological niche modelling (ENM). Phylogenetic analyses suggested that E. sinica formed a monophyly which was nested in E. intermedia. The latter species formed two paraphyletic subclades, corresponding to Western and Eastern lineages. Furthermore, the phylogeographical analysis results indicated very significant inter- and intra-species genetic differentiation, and BEAST inference suggested that E. sinica diverged from E. intermedia at 3.20 million years ago (Ma; 95 % HPD: 2.05–8.10 Ma), while the Western lineage of E. intermedia diverged much earlier at 6.10 Ma (95 % HPD: 4.10–11.82 Ma). In addition, niche identity tests performed using MaxEnt strongly supported the hypothesis that the two sister species had significantly diverged on their ecological niches. Isolation by geographical and environment distance (IBD and IBE) tests suggested that the overall divergence of the populations from the two species were more influenced by environmental factors. Our data provide new insight into the ecological speciation of Ephedra and also provides molecular evidence for the identification and genetic conservation of these important pharmaceutical plants.

Biogeographic history of plants is much more complex in the Northern Hemisphere than in the Southern Hemisphere due to that both the Bering and the North Atlantic land bridges contributed to floristic exchanges in the Cenozoic, which led to hybridization between congeneric species from different continents. It would be interesting to know how intercontinental gene flow and introgression have affected plant phylogenetic reconstruction and biogeographic inference. In this study, we reinvestigated the phylogenetic and biogeographic history of Picea, a main component of the Northern Hemisphere forest with many species that originated from recent radiation, using two chloroplast (cp), one mitochondrial (mt) and three single-copy nuclear gene markers. The generated gene trees are topologically highly discordant and the geographically closely related species generally show a close affinity of mtDNA rather than cp- or nuclear DNA, suggesting that inter- and intra-continental gene flow and mtDNA introgression might have occurred commonly. However, all gene trees resolved Picea breweriana as the basal-most lineage, which, together with fossil evidence, supports the North American origin hypothesis for the genus. Both dispersal and vicariance have played important roles in the evolution of Picea, and the Bering Land Bridge could have mediated the “North America to Eurasia” dispersal at least two times during the Miocene and Pliocene. Our study again demonstrates the importance of applying data from three genomes for a clear understanding of evolutionary histories in the pine family. Any markers from a single genome alone will not reveal a clear picture of the phylogenetic relationships among closely related congeneric species. In particular, mtDNA markers should be cautiously used, considering that introgression of the maternally inherited mtDNA with a lower rate of gene flow (by seeds) could have occurred much more frequently than that of the paternally inherited cpDNA with a higher rate of gene flow (by pollen) in Pinaceae.

A laborious and difficult task in current tree of life reconstruction is to resolve evolutionary relationships of closely related congeneric species that originated from recent radiations. This is particularly difficult for forest species with long generation times and large effective population sizes such as conifers. The Qinghai-Tibetan Plateau (QTP) and adjacent areas are considered a species diversity center of Picea, harboring 11 species (including 5 varieties) of this genus, but evolutionary relationships of these species are far from being resolved due to recent radiations, morphological convergence, and frequent interspecific gene flow. In this study, we use these spruce species to test whether phylotranscriptomic analysis, combined with population genetic analysis, can disentangle their evolutionary relationships, and to explore whether reticulate evolution has occurred among them. Phylogenomic analyses indicate that all spruce species in the QTP and neighboring areas, except P. asperata and P. crassifolia, cluster together, and in particular, nearly all taxa (including varieties) reflect reciprocally monophyletic lineages, although the two species P. likiangensis and P. brachytyla are not monophyletic. We found that, compared to herbaceous plants, many more genes (a minimum of 600 OGs for Picea) are required to resolve interspecific relationships of conifers. Contrary to previous studies, our data do not support a hybrid origin of P. purpurea, but suggests a hybrid origin for P. brachytyla var. brachytyla and P. likiangensis var. rubescens. We emphasize that the species or species complex used for population genetic and phylogeographical studies should be monophyletic.

A robust phylogeny is prerequisite to understand the evolution and biogeography of organisms. However, ancient and recent evolutionary radiations occurred in many plant lineages, which pose great challenges for phylogenetic analysis, especially for conifers characterized by large effective population sizes and long generation times. Picea is an important component of the dark coniferous forests in the Northern Hemisphere. Previous studies improved our understanding of its evolutionary history, but its interspecific relationships and biogeographic history remain largely unresolved. In the present study, we reconstructed a well-resolved phylogeny of Picea by comparative transcriptomic analysis based on a complete species sampling. The phylogenetic analysis, together with molecular dating and ancestral area reconstruction, further supports the North American origin hypothesis for Picea, and indicates that this genus experienced multiple out-of-North America dispersals by the Bering Land Bridge. We also found that spruces in the Japanese Archipelago have multiple origins, and P. morrisonicola from the Taiwan Island has a close relationship with species from the Qinghai-Tibetan Plateau and adjacent regions. Our study provides the first complete phylogeny of Picea at the genomic level, which is important for future studies of this genus.

Conifers are the largest and ecologically and economically most important component group of the gymnosperms. Despite their slow rate of molecular evolution, rapid and recent diversification was unexpectedly prevalent in this ancient group in the Hengduan Mountains, a world’s biodiversity hotspot and gymnosperm diversity center in Southwest China. In this study, we investigated the underlying mechanisms and disentangled the interactions of geography and ecology in speciation and evolution in Pinus armandii, an important forest tree species endemic to China, by integrating analyses of population transcriptomics, population genetics and ecological niche modeling. Many lines of evidence suggest that cryptic speciation has occurred in P. armandii. During the process, geologically induced formation of Mount Gongga and other massive peaks might trigger the initial vicariance isolation of the northern and southern subdivisions, and ecologically based selection then reinforced their differentiation and local adaptation. Our ecological niche analysis and earlier reciprocal transplant experiments in P. armandii provided convincing evidences for the critical role of ecology in the process of speciation. These findings suggest that both geography and ecology contributed significantly to the abundance of very recent and rapid species divergences, which promoted the rising of the extremely high conifer diversity in the Hengduan Mountains.

Pinaceae comprises 11 genera, and represents the largest family of conifers with an extensive wild distribution in the Northern Hemisphere. Intergeneric relationships of Pinaceae have been investigated using many morphological characters and molecular markers, but phylogenetic positions of four genera, including Cathaya, Cedrus, Nothotsuga and Pseudolarix, remain controversial or have not been completely resolved. To completely resolve the intergeneric relationships of Pinaceae, we conducted a comparative transcriptomic study of 14 species representing all Pinaceae genera. Multiple data sets, containing up to 6,369,681 sites across 4676 loci, were analyzed using concatenation and coalescent methods. Our study generated a robust topology, which divides Pinaceae into two clades, one (pinoid) including Cathaya, Laric, Picea, Pinus, and Pseudotsuga, and the other (abietoid) including Abies, Cedrus, Keteleeria, Nothotsuga, Pseudolarix, and Tsuga. Cathaya and Pinus form a Glade sister to Picea; Cedrus is sister to the remaining abietoid genera, and the two genera Nothotsuga and Tsuga form a Glade sister to Pseudolarix. The discordant positions of Cathaya, Cedrus and Pseudolarix in different gene trees could be explained by ancient radiation and/or molecular homoplastic evolution. The hybrid origin hypothesis of Nothotsuga is not supported. Based on molecular dating, extant Pinaceae genera diverged since about 206 Mya, earlier than the break-up of Pangea, and the divergence among the pinoid genera occurred earlier than the split among the abietoid genera. Moreover, our study indicates that two radiation events occurred in the evolution of Pinaceae genera, and some important morphological characters evolved multiple times based on ancestral state reconstruction.

Floral nectar is thought to be one of the most important rewards that attract pollinators in Pedicularis; however, few studies have examined variation of nectary structure and/or nectar secretion in the genus, particularly among closely related species. Here we investigated nectary morphology, nectar quality, and nectar production dynamics in flowers of Pedicularis section Cyathophora. We found a conical floral nectary at the base of the ovary in species of the rex-thamnophila clade. Stomata were found on the surface of the nectary, and copious starch grains were detected in the nectary tissues. In contrast, a semi-annular nectary was found in flowers of the species of the superba clade. Only a few starch grains were observed in tissues of the semi-annular nectary, and the nectar sugar concentration in these flowers was much lower than that in the flowers of the rex-thamnophila clade. Our results indicate that the floral nectary has experienced considerable morphological, structural, and functional differentiation among closely related species of Pedicularis. This could have affected nectar production, leading to a shift of the pollination mode. Our results also imply that variation of the nectary morphology and nectar production may have played an important role in the speciation of sect. Cyathophora.

Climatic changes and tectonic events in the Cenozoic have greatly influenced the evolution and geographic distribution of the temperate flora. Such consequences should be most evident in plant groups that are ancient, widespread, and diverse. As one of the most widespread genera of trees, Pinus provides a good model for investigating the history of species diversification and biogeographic disjunction in the Northern Hemisphere. In this study, we reconstructed the phylogeny and investigated the evolutionary and biogeographic history of sect. Quinquefoliae (Pinus), a species-rich lineage disjunctly distributed in Asia, Europe and North America, based on complete taxon sampling and by using nine DNA fragments from chloroplast (cp), mitochondrial (mt) and nuclear genomes. The monophyly of the three subsections, Krempfianae, Gerardianae, and Strobus, is well-supported by cpDNA and nuclear gene phylogenies. However, neither subsect. Gerardianae nor subsect. Strobus forms a monophyletic group in the mtDNA phylogeny, in which sect. Quinquefoliae was divided into two major clades, one consisting of the North American and northeastern Asian species as well as the European P. peuce of subsect. Strobus, and the other comprising the remaining Eurasian species belonging to three subsections. The significant topological incongruence among the gene trees, in conjunction with divergence time estimation and ancestral area reconstruction, indicates that both ancient and relatively recent introgressive hybridization events occurred in the evolution of sect. Quinquefoliae, particularly in northeastern Asia and northwestern North America. In addition, the phylogenetic analysis suggests that the species of subsect. Strobus from subtropical eastern Asia and neighboring areas may have a single origin, although species non-monophyly is very widespread in the nuclear gene trees. Moreover, our study seems to support a Tethyan origin of sect. Quinquefoliae given the distributions and phylogenetic positions of subsects. Krempfianae and Gerardianae, and also highlights the importance of active mountain buildings and climatic changes during the Late Neogene in shaping the species diversity and geographic distribution of Pinus.

South-East Asia covers four of the world's biodiversity hotspots, showing high species diversity and endemism. Owing to the successive expansion and contraction of distribution and the fragmentation by geographical barriers, the tropical flora greatly diversified in this region during the Tertiary, but the evolutionary tempo and mode of species diversity remain poorly investigated. Paphiopedilum, the largest genus of slipper orchids comprising nearly 100 species, is mainly distributed in South-East Asia, providing an ideal system for exploring how plant species diversity was shaped in this region. Here, we investigated the evolutionary history of this genus with eight cpDNA regions and four low-copy nuclear genes. Discordance between gene trees and network analysis indicates that reticulate evolution occurred in the genus. Ancestral area reconstruction suggests that vicariance and long-distance dispersal together led to its current distribution. Diversification rate variation was detected and strongly correlated with the species diversity in subg. Paphiopedilum (~80 species). The shift of speciation rate in subg. Paphiopedilum was coincident with sea-level fluctuations in the late Cenozoic, which could have provided ecological opportunities for speciation and created bridges or barriers for gene flow. Moreover, some other factors (e.g. sympatric distribution, incomplete reproductive barriers and clonal propagation) might also be advantageous for the formation and reproduction of hybrid species. In conclusion, our study suggests that the interplay of reticulate evolution and sea-level fluctuations has promoted the diversification of the genus Paphiopedilum and sheds light into the evolution of Orchidaceae and the historical processes of plant species diversification in South-East Asia.

The Qinghai–Tibet Plateau (QTP) harbors the highest species diversity of alpine plants in the world, with a spectacular diversity of flower colors. Among these QTP plants, the genus Meconopsis comprises more than 50 species, for which flower color is a key diagnostic character. However, the mechanisms underlying flower color variation have rarely been investigated. In the present study, we used three chloroplast (cp) DNA fragments and two anthocyanin biosynthesis genes (F3H and F3′H) for phylogenetic reconstruction of Meconopsis. We revealed the presence of three well-supported clades and/or subclades in the cpDNA and nuclear gene trees; further, flower color transition occurred in each lineage. The results of selection tests and preliminary expression analyses of the anthocyanin biosynthesis genes indicate that the pigment pathway leading to cyanidin is active in blue and red flowers of Meconopsis; further, a blue–red color shift is not attributable to an on/off switching of the anthocyanin biosynthetic pathway (ABP) branches. Together with the results of previous flower pigment analyses, our findings suggest that blue–red flower color transitions in Meconopsis are attributable to modification of cyanidin. Our molecular dating results indicate that the lineage diversification in Meconopsis is closely related to the QTP uplift; thus, it is likely that environmental changes arising from the QTP uplift have played important roles in driving the diversification of flavonoids, through which species of Meconopsis have adapted physiologically to diverse habitats.

Angiosperms and gymnosperms are two major groups of extant seed plants. It has been suggested that gymnosperms lack FLOWERING LOCUS T (FT), a key integrator at the core of flowering pathways in angiosperms. Taking advantage of newly released gymnosperm genomes, we revisited the evolutionary history of the plant phosphatidylethanolamine-binding protein (PEBP) gene family through phylogenetic reconstruction. Expression patterns in three gymnosperm taxa and heterologous expression in Arabidopsis were studied to investigate the functions of gymnosperm FT-like and TERMINAL FLOWER 1 (TFL1)-like genes. Phylogenetic reconstruction suggests that an ancient gene duplication predating the divergence of seed plants gave rise to the FT and TFL1 genes. Expression patterns indicate that gymnosperm TFL1-like genes play a role in the reproductive development process, while GymFT1 and GymFT2, the FT-like genes resulting from a duplication event in the common ancestor of gymnosperms, function in both growth rhythm and sexual development pathways. When expressed in Arabidopsis, both spruce FT-like and TFL1-like genes repressed flowering. Our study demonstrates that gymnosperms do have FT-like and TFL1-like genes. Frequent gene and genome duplications contributed significantly to the expansion of the plant PEBP gene family. The expression patterns of gymnosperm PEBP genes provide novel insight into the functional evolution of this gene family.