Biotic colonization of subtropical East Asian caves through time.

Caves are home to unique and fragile biotas with high levels of endemism. However, little is known about how the biotic colonization of caves has developed over time, especially in caves from middle and low latitudes. Subtropical East Asia holds the world's largest karst landform with numerous ancient caves, which harbor a high diversity of cave-dwelling organisms and are regarded as a biodiversity hotspot. Here, we assess the temporal dynamics of biotic colonization of subtropical East Asian caves through a multi-taxon analysis with representatives of green plants, animals, and fungi. We then investigate the consequences of paleonviromental changes on the colonization dynamics of these caves in combination with reconstructions of vegetation, temperature, and precipitation. We discover that 88% of cave colonization events occurred after the Oligocene-Miocene boundary, and organisms from the surrounding forest were a major source for subtropical East Asian cave biodiversity. Biotic colonization of subtropical East Asian caves during the Neogene was subject to periods of acceleration and decrease, in conjunction with large-scale, seasonal climatic changes and evolution of local forests. This study highlights the long-term evolutionary interaction between surface and cave biotas; our climate-vegetation-relict model proposed for the subtropical East Asian cave biota may help explain the evolutionary origins of other mid-latitude subterranean biotas.

Plastome phylogenomics and morphological traits analyses provide new insights into the phylogenetic position, species delimitation and speciation of Triplostegia (Caprifoliaceae).

BACKGROUND: The genus Triplostegia contains two recognized species, T. glandulifera and T. grandiflora, but its phylogenetic position and species delimitation remain controversial. In this study, we assembled plastid genomes and nuclear ribosomal DNA (nrDNA) cistrons sampled from 22 wild Triplostegia individuals, each from a separate population, and examined these with 11 recently published Triplostegia plastomes. Morphological traits were measured from herbarium specimens and wild material, and ecological niche models were constructed. RESULTS: Triplostegia is a monophyletic genus within the subfamily Dipsacoideae comprising three monophyletic species, T. glandulifera, T. grandiflora, and an unrecognized species Triplostegia sp. A, which occupies much higher altitude than the other two. The new species had previously been misidentified as T. glandulifera, but differs in taproot, leaf, and other characters. Triplotegia is an old genus, with stem age 39.96 Ma, and within it T. glandulifera diverged 7.94 Ma. Triplostegia grandiflora and sp. A diverged 1.05 Ma, perhaps in response to Quaternary climate fluctuations. Niche overlap between Triplostegia species was positively correlated with their phylogenetic relatedness. CONCLUSIONS: Our results provide new insights into the species delimitation of Triplostegia, and indicate that a taxonomic revision of Triplostegia is needed. We also identified that either rpoB-trnC or ycf1 could serve as a DNA barcode for Triplostegia.

Phylogenomic insights into the origin and evolutionary history of evergreen broadleaved forests in East Asia under Cenozoic climate change.

The evergreen versus deciduous leaf habit is an important functional trait for adaptation of forest trees and has been hypothesized to be related to the evolutionary processes of the component species under paleoclimatic change, and potentially reflected in the dynamic history of evergreen broadleaved forests (EBLFs) in East Asia. However, knowledge about the shift of evergreen versus deciduous leaf with the impact of paleoclimatic change using genomic data remains rare. Here, we focus on the Litsea complex (Lauraceae), a key lineage with dominant species of EBLFs, to gain insights into how evergreen versus deciduous trait shifted, providing insights into the origin and historical dynamics of EBLFs in East Asia under Cenozoic climate change. We reconstructed a robust phylogeny of the Litsea complex using genome-wide single-nucleotide variants (SNVs) with eight clades resolved. Fossil-calibrated analyses, diversification rate shifts, ancestral habit, ecological niche modelling and climate niche reconstruction were employed to estimate its origin and diversification pattern. Taking into account studies on other plant lineages dominating EBLFs of East Asia, it was revealed that the prototype of EBLFs in East Asia probably emerged in the Early Eocene (55-50 million years ago [Ma]), facilitated by the greenhouse warming. As a response to the cooling and drying climate in the Middle to Late Eocene (48-38 Ma), deciduous habits were evolved in the dominant lineages of the EBLFs in East Asia. Up to the Early Miocene (23 Ma), the prevailing of East Asian monsoon increased the extreme seasonal precipitation and accelerated the emergence of evergreen habits of the dominant lineages, and ultimately shaped the vegetation resembling that of today.

Species richness disparity in tropical terrestrial herbaceous floras: Evolutionary insight from Collabieae (Orchidaceae).

Species richness is spatially heterogeneous even in the hyperdiverse tropical floras. The main cause of uneven species richness among the four tropical regions are hot debated. To date, higher net diversification rates and/or longer colonization time have been usually proposed to contribute to this pattern. However, there are few studies to clarify the species richness patterns in tropical terrestrial floras. The terrestrial tribe Collabieae (Orchidaceae) unevenly distributes in the tropical regions with a diverse and endemic center in Asia. Twenty-one genera 127 species of Collabieae and 26 DNA regions were used to reconstruct the phylogeny and infer the biogeographical processes. We compared the topologies, diversification rates and niche evolutionary rates of Collabieae and regional lineages on empirical samplings and different simulated samplings fractions respectively. Our results suggested that the Collabieae originated in Asia at the earliest Oligocene, and then independently spread to Africa, Central America, and Oceania since the Miocene via long-distance dispersal. These results based on empirical data and simulated data were similar. BAMM, GeoSSE and niche analyses inferred that the Asian lineages had higher net diversification and niche evolutionary rates than those of Oceanian and African lineages on the empirical and simulated analyses. Precipitation is the most important factor for Collabieae, and the Asian lineage has experienced more stable and humid climate, which may promote the higher net diversification rate. Besides, the longer colonization time may also be associated with the Asian lineages' diversity. These findings provided a better understanding of the regional diversity heterogeneity in tropical terrestrial herbaceous floras.

Immigration dynamics of tropical and subtropical Southeast Asian limestone karst floras.

Ex situ origins and dispersal of taxa have played important roles in the assembly of island-like biodiversity hotspots. Insular limestone karsts in Southeast Asia are hotspots of biodiversity and endemism, but the immigration processes of their unique floras are still poorly known. Here, we used Gesneriaceae as a proxy to investigate the immigration dynamics of tropical and subtropical Southeast Asian karst floras. We present the most comprehensive phylogenetic analysis of the Old World gesneriads to date based on twelve loci. By estimating divergence times and reconstructing ancestral states (habitat, soil type and range), we found that immigration into subtropical Southeast Asian karst floras first occurred in the Early Miocene, with two peaks in the Early-Middle Miocene and the Pliocene-Early Pleistocene, whereas immigration into tropical Southeast Asian karsts initiated in the Late Eocene, with two peaks in the Late Oligocene and the Late Miocene. We also discover that Southeast Asian karst biodiversity comprises immigrant pre-adapted lineages and descendants from local acid soil ancestors, although niche shift from acid soil to karst in tropical Southeast Asian islands was lacking. This study advances our understanding of the historical assembly of Southeast Asian karst floras.

Assembly dynamics of East Asian subtropical evergreen broadleaved forests: New insights from the dominant Fagaceae trees.

The dominant species of a biome can be regarded as its genuine indicator. Evergreen broadleaved forests (EBLFs) in subtropical East Asia harbor high levels of species biodiversity and endemism and are vital to regional carbon storage and cycling. However, the historical assembly of this unique biome is still controversial. Fagaceae is the most essential family in East Asian subtropical EBLFs and its dominant species are vital for the existence of this biome. Here, we used the dominant Fagaceae species to shed light on the dynamic process of East Asian subtropical EBLFs over time. Our results indicate high precipitation in summer and low temperature in winter are the most influential climatic factors for the distribution of East Asian subtropical EBLFs. Modern East Asian subtropical EBLFs did not begin to appear until 23 Ma, subsequently experienced a long-lasting development in the Miocene and markedly deteriorated at about 4 Ma, driven jointly by orogenesis and paleoclimate. We also document that there is a lag time between when one clade invaded the region and when its members become dominant species within the region. This study may improve our ability to predict and mitigate the threats to biodiversity of East Asian subtropical EBLFs and points to a new path for future studies involving multidisciplinary methods to explore the assembly of regional biomes.

Biogeographic diversification of Mahonia (Berberidaceae): Implications for the origin and evolution of East Asian subtropical evergreen broadleaved forests.

The subtropical evergreen broadleaved forests (EBLFs) inhabit large areas of East Asia and harbor rich biodiversity and high endemism. However, the origin and evolution of biodiversity of East Asian subtropical EBLFs remain poorly understood. Here, we used Mahonia (Berberidaceae), an eastern Asian-western North American disjunct evergreen genus, to obtain new insights into the historical assembly of this biome. We present the most comprehensive phylogenetic analysis of Mahonia do date based on six nuclear and plastid loci. Using the phylogenetic framework, we estimated divergence times, reconstructed ancestral ranges, inferred evolutionary shift of habitats, and estimated diversification rates. Mahonia and each of its two groups (Orientales and Occidentales) are strongly supported as monophyletic. Mahonia originated in western North America during the late Eocene (c. 40.41 Ma) and subsequently dispersed into East Asia prior to the early Oligocene (c. 32.65 Ma). The North Atlantic Land Bridge might have played an important role in population exchanges of Mahonia between East Asia and western North America. The western North American Occidentales began to diversify in summer-dry climates and open landscapes in the early Miocene, whereas the eastern Asian Orientales began to diversify in subtropical EBLFs in the early Miocene and furthermore had a rapid lineage accumulation since the late Miocene. The net diversification rate of Mahonia in eastern Asia appeared to be higher than that in western North America, which is ascribed to lower extinction rates and ecological opportunity. Our findings suggest that western North America is a source of biodiversity of East Asian subtropical EBLFs. This biome in eastern Asia began to rise in the early Miocene and further diversified in the late Miocene, driven by the intensifying East Asian summer monsoon during these two periods.

Phylogeny and biogeography of Pachygoneae (Menispermaceae), with consideration of the boreotropical flora hypothesis and resurrection of the genera Cebatha and Nephroia.

The tribe Pachygoneae consists of four genera with about 40 species, primarily distributed in tropical and subtropical Asia and America, also in Australasia and Africa. This tribe presents an ideal model to investigate the origin of the tropical and subtropical amphi-Pacific disjunction pattern. More specifically, it allows us to test whether the tropical lineages diverged earlier than the subtropical ones during the fragmentation of the boreotropical flora. In this study, we reconstructed the phylogeny of Pachygoneae using five plastid (rbcL, atpB, matK, ndhF, trnL-F) and one nuclear (26S rDNA) DNA regions. Our results indicate that Pachygoneae is not monophyletic unless Cocculus pendulus and Cocculus balfourii are excluded. We resurrected the genus Cebatha to include these two species and established a new tribe for this genus. Within Pachygoneae, the species of Cocculus are distributed in three different clades, among which two are recognized as two distinct genera, Cocculus s.str. and Nephroia resurrected, and one species is transferred into Pachygone. Our molecular dating and ancestral area reconstruction analyses suggest that Pachygoneae began to diversify in tropical Asia around the early-middle Eocene boundary (c. 48 Ma) and expanded into the New World by c. 44 Ma. In the New World, tropical Hyperbaena originated in the late Eocene (c. 40 Ma), whereas the subtropical Cocculus carolinus and Cocculus diversifolius originated later, in the early Oligocene (c. 32  Ma). These two timings correspond with the two climatic cooling intervals, which suggests that the formation and breakup of the boreotropical floral may have been responsible for the amphi-Pacific disjunct distribution within Pachygoneae. One overland migration event from Asia into Australasia appears to have occurred in the early to late Miocene.

A dated phylogeny of Lardizabalaceae reveals an unusual long-distance dispersal across the Pacific Ocean and the rapid rise of East Asian subtropical evergreen broadleaved forests in the late Miocene.

Temperate South American-Asian disjunct distributions are the most unusual in organisms, and challenging to explain. Here, we address the origin of this unusual disjunction in Lardizabalaceae using explicit models and molecular data. The family (c.40 species distributed in ten genera) also provides an opportunity to explore the historical assembly of East Asian subtropical evergreen broadleaved forests, a typical and luxuriant vegetation in East Asia. DNA sequences of five plastid loci of 42 accessions representing 23 species of Lardizabalaceae (c. 57.5% of estimated species diversity), and 19 species from the six other families of Ranunculales, were used to perform phylogenetic analyses. By dating the branching events and reconstructing ancestral ranges, we infer that extant Lardizabalaceae dated to the Upper Cretaceous of East Asia and that the temperate South American lineage might have split from its East Asian sister group at c. 24.4 Ma. A trans-Pacific dispersal possibly by birds from East Asia to South America is plausible to explain the establishment of the temperate South American-East Asian disjunction in Lardizabalaceae. Diversification rate analyses indicate that net diversification rates of Lardizabalaceae experienced a significant increase around c. 7.5 Ma. Our findings suggest that the rapid rise of East Asian subtropical evergreen broadleaved forests occurred in the late Miocene, associated with the uplift of the Tibetan Plateau and the intensified East Asian monsoon, as well as the higher winter temperature and atmospheric CO2 levels.

Biogeography of Coptis Salisb. (Ranunculales, Ranunculaceae, Coptidoideae), an Eastern Asian and North American genus.

BACKGROUND: Numerous studies have favored dispersal (colonization) over vicariance (past fragmentation) events to explain eastern Asian-North American distribution patterns. In plants, however the disjunction between eastern Asia and western North America has been rarely examined using the integration of phylogenetic, molecular dating, and biogeographical methods. Meanwhile, the biogeographic patterns within eastern Asia remain poorly understood. The goldthread genus Coptis Salisb. includes 15 species disjunctly distributed in North America, Japan, mainland China, and Taiwan. We present a dated phylogeny for Coptis under the optimal clock model and infer its historical biogeography by comparing different biogeographic models. RESULTS: The split of Coptis and Xanthorhiza Marshall occurred in the middle Miocene (ca. 15.47 Ma). Coptis started their diversification in the early late Miocene (ca. 9.55 Ma). A late Miocene vicariance event resulted in the eastern Asian and western North American disjunction in the genus. Within eastern Asia, dispersals from mainland Asia to Japan and from Japan to Taiwan occurred at ca. 4.85 Ma and at ca. 1.34 Ma, respectively. CONCLUSIONS: Our analyses provide evidence that both vicariance and dispersal events have played important roles in shaping the current distribution and endemism of Coptis, likely resulting from eustatic sea-level changes, mountain formation processes and an increasing drier and cooler climate from the middle Miocene onwards.

New insights into the phylogeny of Burasaieae (Menispermaceae) with the recognition of a new genus and emphasis on the southern Taiwanese and mainland Chinese disjunction.

Taiwan is a continental island lying at the boundary between the Eurasian and the Philippine tectonic plates and possesses high biodiversity. Southern Taiwan, viz. Hengchun Peninsula, is notably floristically different from northern Taiwan. The floristic origin and relationships of the Hengchun Peninsula have been rarely investigated in a phylogenetic context. In this study, data from six plastid and nuclear sequences were used to reconstruct phylogenetic relationships within Burasaieae (Menispermaceae), which mainly inhabits tropical rainforests. The tree-based comparisons indicate that the position of Tinospora sensu stricto conflicts significantly between the cpDNA and ITS trees. However, alternative hypothesis tests from the ITS data did not reject the result of the cpDNA data, which suggests that tree-based comparisons might sometimes generate an artificial incongruence, especially when markers with high homoplasy are used. Based on the combined cpDNA and ITS data, we present an inter-generic phylogenetic framework for Burasaieae. Sampled species of Tinospora are placed in three different clades, including Tinospora dentata from southern Taiwan and T. sagittata from mainland China in an unresolved position alongside six lineages of Burasaieae. By integrating lines of evidence from molecular phylogeny, divergence times, and morphology, we recognize the three Tinospora clades as three different genera, including Tinospora sensu stricto, a new genus (Paratinospora) for T. dentata and T. sagittata, and Hyalosepalum resurrected. Tinospora dentata, now endemic to the Hengchun Peninsula, originated from the Late Eocene (ca. 39Ma), greatly predating the formation of Taiwan. Our study suggests that the flora of the Hengchun Peninsula contains some ancient components that might have migrated from mainland China.

A phylogenetic analysis of molecular and morphological characters of Herminium (Orchidaceae, Orchideae): evolutionary relationships, taxonomy, and patterns of character evolution.

The first comprehensive phylogenetic study of the orchid genus Herminium and its allies is presented, based on seven molecular markers (nuclear internal transcribed spacer, Xdh, chloroplast matK, psaB, psbA-trnH, rbcL and trnL-F) and 37 morphological characters. Phylogenetic analyses indicate that Herminium as currently delimited is paraphyletic and that several genera are deeply nested within it. Based on parsimony analysis of total evidence, the generic circumscription of Herminium is expanded to include Androcorys, Bhutanthera, Frigidorchis and Porolabium. Apomorphic and plesiomorphic character states are identified for various clades recovered in this study. A few species currently wrongly assigned to Peristylus and Platanthera are here included in Herminium. All necessary new combinations are made.

Molecular systematics of subtribe Orchidinae and Asian taxa of Habenariinae (Orchideae, Orchidaceae) based on plastid matK, rbcL and nuclear ITS.

The subtribe Orchidinae, distributed predominantly in Eastern Asia and the Mediterranean, presents some of the most intricate taxonomic problems in the family Orchidaceae with respect to generic delimitation. Based on three DNA markers (plastid matK, rbcL, and nuclear ITS), morphological characters, and a broad sampling of Orchidinae and selected Habenariinae mainly from Asia (a total of 153 accessions of 145 species in 31 genera), generic delimitation and phylogenetic relationships within the subtribe Orchidinae and Habenariinae from Asia were assessed. Orchidinae and Asian Habenariinae are monophyletic, and Orchidinae is divided into distinct superclades. Many genera, such as Amitostigma, Habenaria, Hemipilia, Herminium, Platanthera, Peristylus and Ponerorchis, are not monophyletic. Habenaria is subdivided into two distantly related groups, while Platanthera is subdivided into three even more disparate groups. Many previously undetected phylogenetic relationships, such as clades formed by the Amitostigma-Neottianthe-Ponerorchis complex, Platanthera latilabris group, Ponerorchis chrysea, Sirindhornia, and Tsaiorchis, are well supported by both molecular and morphological evidence. We propose to combine Hemipiliopsis with Hemipilia, Amitostigma and Neottianthe with Ponerorchis, Smithorchis with Platanthera, and Aceratorchis and Neolindleya with Galearis, and to establish a new genus to accommodate Ponerorchis chrysea. Tsaiorchis and Sirindhornia are two distinctive genera supported by both molecular data and morphological characters. A new genus, Hsenhsua, and 41 new combinations are proposed based on these findings.

Molecular systematics of Dendrobium (Orchidaceae, Dendrobieae) from mainland Asia based on plastid and nuclear sequences.

Dendrobium is one of the three largest genera and presents some of the most intricate taxonomic problems in the family Orchidaceae. Based on five DNA markers and a broad sampling of Dendrobium and its relatives from mainland Asia (109 species), our results indicate that mainland Asia Dendrobium is divided into eight clades (with two unplaced species) that form polytomies along the spine of the cladogram. Both Dendrobium and Epigeneium are well supported as monophyletic, whereas sect. Dendrobium, sect. Densiflora, sect. Breviflores, sect. Holochrysa, are paraphyletic/polyphyletic. Many ignored phylogenetic relationships, such as the one of major clades formed by D. jenkinsii and D. lindleyi (two members of sect. Densiflora), the Aphyllum group, the Devonianum group, the Catenatum group, the Crepidatum group, and the Dendrobium moniliforme complex are well supported by both molecular and morphological evidence. Based on our data, we propose to broaden sect. Dendrobium to include sect. Stuposa, sect. Breviflores, and sect. Holochrysa and to establish a new section to accommodate D. jenkinsii and D. lindleyi. Our results indicated that it is preferable to use a broad generic concept of Dendrobium and to pursue an improved infrageneric classification at sectional level, taking into account both morphology and current molecular findings.

Asymmetric migration dynamics of the tropical Asian and Australasian floras.

The tropical Asian and Australasian floras have a close relationship, and is a vital distribution pattern of seed plants worldwide. As estimated, more than 81 families and 225 genera of seed plants distributed between tropical Asia and Australasia. However, the evolutionary dynamics of two floras were still vague. Here, a total of 29 plant lineages, represented the main clades of seed plants and different habits, were selected to investigate the biotic interchange between tropical Asia and Australasia by integrated dated phylogenies, biogeography, and ancestral state reconstructions. Our statistics indicated that 68 migrations have occurred between tropical Asia and Australasia since the middle Eocene except terminal migrations, and the migration events from tropical Asia to Australasia is more than 2 times of the reverse. Only 12 migrations occurred before 15 Ma, whereas the remaining 56 migrations occurred after 15 Ma. Maximal number of potential dispersal events (MDE) analysis also shows obvious asymmetry, with southward migration as the main feature, and indicates the climax of bi-directional migrations occurred after 15 Ma. We speculate that the formation of island chains after the Australian-Sundaland collision and climate changes have driven seed plant migrations since the middle Miocene. Furthermore, biotic dispersal and stable habitat may be crucial for floristic interchange between tropical Asia and Australasia.

Field plus lab experiments help identify freezing tolerance and associated genes in subtropical evergreen broadleaf trees: A case study of Camellia oleifera.

The molecular mechanisms of freezing tolerance are unresolved in the perennial trees that can survive under much lower freezing temperatures than annual herbs. Since natural conditions involve many factors and temperature usually cannot be controlled, field experiments alone cannot directly identify the effects of freezing stress. Lab experiments are insufficient for trees to complete cold acclimation and cannot reflect natural freezing-stress responses. In this study, a new method was proposed using field plus lab experiments to identify freezing tolerance and associated genes in subtropical evergreen broadleaf trees using Camellia oleifera as a case. Cultivated C. oleifera is the dominant woody oil crop in China. Wild C. oleifera at the high-elevation site in Lu Mountain could survive below -30°C, providing a valuable genetic resource for the breeding of freezing tolerance. In the field experiment, air temperature was monitored from autumn to winter on wild C. oleifera at the high-elevation site in Lu Mountain. Leave samples were taken from wild C. oleifera before cold acclimation, during cold acclimation and under freezing temperature. Leaf transcriptome analyses indicated that the gene functions and expression patterns were very different during cold acclimation and under freezing temperature. In the lab experiments, leaves samples from wild C. oleifera after cold acclimation were placed under -10°C in climate chambers. A cultivated C. oleifera variety "Ganwu 1" was used as a control. According to relative conductivity changes of leaves, wild C. oleifera showed more freezing-tolerant than cultivated C. oleifera. Leaf transcriptome analyses showed that the gene expression patterns were very different between wild and cultivated C. oleifera in the lab experiment. Combing transcriptome results in both of the field and lab experiments, the common genes associated with freezing-stress responses were identified. Key genes of the flg22, Ca2+ and gibberellin signal transduction pathways and the lignin biosynthesis pathway may be involved in the freezing-stress responses. Most of the genes had the highest expression levels under freezing temperature in the field experiment and showed higher expression in wild C. oleifera with stronger freezing tolerance in the lab experiment. Our study may help identify freezing tolerance and underlying molecular mechanisms in trees.

Plastome evolution and phylogenomics of Trichosporeae (Gesneriaceae) with its morphological characters appraisal.

Trichosporeae is the largest and most taxonomically difficult tribe of Gesneriaceae due to its diverse morphology. Previous studies have not clarified the phylogenetic relationships within this tribe on several DNA markers, including the generic relationships within subtribes. Recently, plastid phylogenomics have been successfully employed to resolve the phylogenetic relationships at different taxonomic levels. In this study, plastid phylogenomics were used to explore the relationships within Trichosporeae. Eleven plastomes of Hemiboea were newly reported. Comparative analyses, phylogeny and morphological character evolution within Trichosporeae were conducted on 79 species representing seven subtribes. The Hemiboea plastomes range from 152,742 bp to 153,695 bp in length. Within Trichosporeae, the sampled plastomes range from 152,196 bp to 156,614 bp and GC content from 37.2% to 37.8%. A total of 121-133 genes were annotated in each species, including 80-91 protein-coding genes, 34-37 tRNA genes, and 8 rRNA genes. The contraction and expansion of IR borders were not detected, and gene rearrangements and inversions did not occur. The 13 hypervariable regions were proposed as the potential molecular markers for species identification. A total of 24,299 SNPs and 3,378 indels were inferred, and most of the SNPs were functionally missense and silent variations. There were 1968 SSRs, 2055 tandem repeats and 2802 dispersed repeats. The RSCU and ENC values indicated that the codon usage pattern was conserved in Trichosporeae. Both the phylogenetic frameworks based on the whole plastome and 80 CDSs were basically concordant. The sister relationships between Loxocarpinae and Didymocarpinae were confirmed, and Oreocharis was a sister group of Hemiboea with high support. The morphological characters showed a complex evolutionary pattern of Trichosporeae. Our findings may contribute to future research on genetic diversity, morphological evolutionary patterns, and conservation of the tribe Trichosporeae.

Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary.

• Modern tropical rainforests have the highest biodiversity of terrestrial biomes and are restricted to three low-latitude areas. However, the actual timeframe during which tropical rainforests began to appear on a global scale has been intensely disputed. Here, we used the moonseed family (Menispermaceae), an important physiognomic and structural component of tropical rainforests on a worldwide basis, to obtain new insights into the diversification of this biome. • We integrated phylogenetic, biogeographic and molecular dating methods to analyse temporal and spatial patterns of global diversification in Menispermaceae. • Importantly, a burst of moonseed diversification occurred in a narrow window of time, which coincides with the Cretaceous-Paleogene (K-Pg) boundary. Our data also suggest multiple independent migrations from a putative ancestral area of Indo-Malay into other tropical regions. • Our data for Menispermaceae suggest that modern tropical rainforests may have appeared almost synchronously throughout the three major tropical land areas close to, or immediately following, the K-Pg mass extinction.

High-throughput sequencing-based microsatellite genotyping for polyploids to resolve allele dosage uncertainty and improve analyses of genetic diversity, structure and differentiation: A case study of the hexaploid Camellia oleifera.

Conventional microsatellite (simple sequence repeat, SSR) genotyping methods cannot accurately identify polyploid genotypes leading to allele dosage uncertainty, introducing biases in population genetic analysis. Here, a new SSR genotyping method was developed to directly infer accurate polyploid genotypes. The frequency distribution of SSR sequences was obtained based on deep-coverage high-throughput sequencing data. Corrections were performed accounting for the "stutter peak" and amplification efficiency of SSR sequences. Perl scripts and an online SSR genotyping tool "SSRSeq" were provided to process the sequencing data and output genotypes with corrected allele dosages. Hexaploid Camellia oleifera is the dominant woody oilseed crop in China. Understanding the geographical pattern of genetic variation in wild C. oleifera is essential for the conservation and utilization of genetic resources. Six wild C. oleifera populations were sampled across geographical ranges in subtropical evergreen broadleaf forests of China. Using 35 SSR markers, the high-throughput sequencing-based SSRSeq method was applied to obtain accurate hexaploid genotypes of wild C. oleifera. The results demonstrated that the new method could resolve allele dosage uncertainty and considerably improve genetic diversity, structure and differentiation analyses for polyploids. The genetic variation patterns of wild C. oleifera across geographical ranges agree with the "central-marginal hypothesis", stating that genetic diversity is high in the central population and declines from the central to the peripheral populations, and genetic differentiation increases from the centre to the periphery. This method and findings can facilitate the utilization of wild C. oleifera genetic resources for the breeding of cultivated C. oleifera.

Characterization of the complete chloroplast genome of Pterocarya macroptera var. delavayi (Juglandaceae).

In this study, the complete chloroplast genome sequence of Pterocarya macroptera var. delavayi was reported and characterized. The chloroplast genome is 160,168 bp in length, and consists the typical quadripartite structure, a pair of inverted repeats (IRs, 26,007 bp) separated by a large single-copy region (89,701 bp) and a small single-copy region (18,453 bp). A total of 136 unique genes were predicted, including 88 protein-coding genes, 40 tRNA genes, and 8 rRNA genes. The GC content of the chloroplast genome is 36.2%. Phylogenetic analysis confirmed the close relationship between Pterocarya and Juglans.