The first homosporous lycophyte genome revealed the association between the recent dynamic accumulation of LTR-RTs and genome size variation.

The contrasting genome size between homosporous and heterosporous plants is fascinating. Different from the heterosporous seed plants and mainly homosporous ferns, the lycophytes are either heterosporous (Isoetales and Selaginellales) or homosporous (Lycopodiales). Many lycophytes are the resource plants of Huperzine A (HupA) which is invaluable for treating Alzheimer's disease. For the seed-free vascular plants, several high-quality genomes of heterosporous Selaginella, homosporous ferns (maidenhair fern, monkey spider tree fern), and heterosporous ferns (Azolla) have been published and provided important insights into the origin and evolution of early land plants. However, the homosporous lycophyte genome has not been decoded. Here, we assembled the first homosporous lycophyte genome and conducted comparative genomic analyses by applying a reformed pipeline for filtering out non-plant sequences. The obtained genome size of Lycopodium clavatum is 2.30 Gb, distinguished in more than 85% repetitive elements of which 62% is long terminal repeat (LTR). This study disclosed a high birth rate and a low death rate of the LTR-RTs in homosporous lycophytes, but the opposite occurs in heterosporous lycophytes. we propose that the recent activity of LTR-RT is responsible for the immense genome size variation between homosporous and heterosporous lycophytes. By combing Ks analysis with a phylogenetic approach, we discovered two whole genome duplications (WGD). Morover, we identified all the five recognized key enzymes for the HupA biosynthetic pathway in the L. clavatum genome, but found this pathway incomplete in other major lineages of land plants. Overall, this study is of great importance for the medicinal utilization of lycophytes and the decoded genome data will be a key cornerstone to elucidate the evolution and biology of early vascular land plants.

Mitogenome-based phylogenomics provides insights into the positions of the enigmatic sinensis group and the sanguinolenta group in Selaginellaceae (Lycophyte).

Spikemoss (Selaginellaceae) is one of the basal lineages of vascular plants. This family has a single genus Selaginella which consists of about 750 extant species. The phylogeny of Selaginellaceae has been extensively studied mainly based on plastid DNA and a few nuclear sequences. However, the placement of the enigmatic sinensis group is a long-term controversy because of the long branch in the plastid DNA phylogeny. The sanguinolenta group is also a phylogenetically problematic clade owing to two alternative positions resulted from different datasets. Here, we newly sequenced 34 mitochondrial genomes (mitogenomes) of individuals representing all seven subgenera and major clades in Selaginellaceae. We assembled the draft mitogenomes and annotated the genes and performed phylogenetic analyses based on the shared 17 mitochondrial genes. Our major results include: (1) all the assembled mitogenomes have complicated structures, unparalleled high GC content and a small gene content set, and the positive correlations among GC content, substitution rates and the number of RNA editing sites hold; (2) the sinensis group was well supported as a member of subg. Stachygynandrum; (3) the sanguinolenta group was strongly resolved as sister to all other Selaginella species except for subg. Selaginella. This study demonstrates the potential of mitogenome data in providing novel insights into phylogenetically recalcitrant problems.

The evolution of extremely diverged plastomes in Selaginellaceae (lycophyte) is driven by repeat patterns and the underlying DNA maintenance machinery.

Two factors are proposed to account for the unusual features of organellar genomes: the disruptions of organelle-targeted DNA replication, repair, and recombination (DNA-RRR) systems in the nuclear genome and repetitive elements in organellar genomes. Little is known about how these factors affect organellar genome evolution. The deep-branching vascular plant family Selaginellaceae is known to have a deficient DNA-RRR system and convergently evolved organellar genomes. However, we found that the plastid genome (plastome) of Selaginella sinensis has extremely accelerated substitution rates, a low GC content, pervasive repeat elements, a dynamic network structure, and it lacks direct or inverted repeats. Unexpectedly, its organelle DNA-RRR system is short of a plastid-targeted Recombinase A1 (RecA1) and a mitochondrion-targeted RecA3, in line with other explored Selaginella species. The plastome contains a large collection of short- and medium-sized repeats. Given the absence of RecA1 surveillance, we propose that these repeats trigger illegitimate recombination, accelerated mutation rates, and structural instability. The correlations between repeat quantity and architectural complexity in the Selaginella plastomes support these conclusions. We, therefore, hypothesize that the interplay of the deficient DNA-RRR system and the high repeat content has led to the extraordinary divergence of the S. sinensis plastome. Our study not only sheds new light on the mechanism of plastome divergence by emphasizing the power of cytonuclear integration, but it also reconciles the longstanding contradiction on the effects of DNA-RRR system disruption on genome structure evolution.

Plastid phylogenomic analyses of the Selaginella sanguinolenta group (Selaginellaceae) reveal conflict signatures resulting from sequence types, outlier genes, and pervasive RNA editing.

Different from the generally conserved plastomes (plastid genomes) of most land plants, the Selaginellaceae plastomes exhibit dynamic structure, high GC content and high substitution rates. Previous plastome analyses identified strong conflict on several clades in Selaginella, however the factors causing the conflictions and the impact on the phylogenetic inference have not been sufficiently investigated. Here, we dissect the distribution of phylogenetic signals and conflicts in Selaginella sanguinolenta group, the plastome of which is DR (direct repeats) structure and with genome-wide RNA editing. We analyzed the data sets including 22 plastomes representing all species of the S. sanguinolenta group, covering the entire geographical distribution from the Himalayas to Siberia and the Russian Far East regions. We recovered four different topologies by applying multispecies coalescent (ASTRAL) and concatenation methods (IQ-TREE and RAxML) on four data sets of PC (protein-coding genes), NC (non-coding sequences), PCN (the concatenated PC and NC), and RC (predicted RNA editing sites "C" were corrected by "T"), respectively. Six monophyletic clades, S. nummularifolia clade, S. rossii clade, S. sajanensis clade, S. sanguinolenta I clade, S. sanguinolenta II clade, and S. sanguinolenta III clade, were consistently resolved and supported by the characteristics of GC content, RNA editing frequency, and gene content. However, the relationships among these clades varied across the four topologies. To explore the underlying causes of the uncertainty, we compared the phylogenetic signals of the four topologies. We identified that the sequence types (coding versus non-coding), outlier genes (genes with extremely high |ΔGLS| values), and C-to-U RNA editing frequency in the protein-coding genes were responsible for the unstable phylogenomic relationship. We further revealed a significant positive correlation between the |ΔGLS| values and the variation coefficient of the RNA editing number. Our results demonstrated that the coalescent method performed better than the concatenation method in overcoming the problems caused by outlier genes and extreme RNA editing events. Our study particularly focused on the importance of exploring the plastid phylogenomic conflicts and suggested conducting concatenated analyses cautiously when adopting organelle genome data.

Fire-prone Rhamnaceae with South African affinities in Cretaceous Myanmar amber.

The rapid Cretaceous diversification of flowering plants remains Darwin's 'abominable mystery' despite numerous fossil flowers discovered in recent years. Wildfires were frequent in the Cretaceous and many such early flower fossils are represented by charcoalified fragments, lacking complete delicate structures and surface textures, making their similarity to living forms difficult to discern. Furthermore, scarcity of information about the ecology of early angiosperms makes it difficult to test hypotheses about the drivers of their diversification, including the role of fire in shaping flowering plant evolution. We report the discovery of two exquisitely preserved fossil flower species, one identical to the inflorescences of the extant crown-eudicot genus Phylica and the other recovered as a sister group to Phylica, both preserved as inclusions together with burned plant remains in Cretaceous amber from northern Myanmar (~99 million years ago). These specialized flower species, named Phylica piloburmensis sp. nov. and Eophylica priscastellata gen. et sp. nov., exhibit traits identical to those of modern taxa in fire-prone ecosystems such as the fynbos of South Africa, and provide evidence of fire adaptation in angiosperms.

Ellipinema and ×Ellipisorus? Just Lepisorus (Polypodiaceae)!

The establishment of a segregate lepisoroid fern genus Ellipinema was mainly to accommodate the isolated position of Lepisorus jakonensis (Polypodiaceae) recovered in plastid gene tree. Using newly obtained nuclear data, we recovered that Ellipinema and allied genera, such as Lepidomicrosorium, Lemmaphyllum, Neolepisorus, Paragramma, Tricholepidium and Weatherbya are deeply nested within Lepisorus. The nuclear phylogeny showing incongruent phylogenetic placement in comparison with plastid results perhaps indicated ancient hybridization events. The diagnostic morphology characterizing Ellipinema - elliptic scale-like paraphyses, which is shared by all the taxa of sect. Lepisorus and sect. Hymenophyton - falls within the range of continuous variation in the type species Ellipinema jakonense (=Lepisorus jakonensis). Our study, which integrated molecular and morphological data, demonstrates that the segregation of Ellipinema and ×Ellipisorus (= ×Lepinema Li Bing Zhang & Liang Zhang, nom. illeg.) from Lepisorus should be rejected.

Distinctive evolutionary pattern of organelle genomes linked to the nuclear genome in Selaginellaceae.

Plastids and mitochondria are endosymbiotic organelles that store genetic information. The genomes of these organelles generally exhibit contrasting patterns regarding genome architecture and genetic content. However, they have similar genetic features in Selaginellaceae, and little is known about what causes parallel evolution. Here, we document the multipartite plastid genomes (plastomes) and the highly divergent mitochondrial genomes (mitogenomes) from spikemoss obtained by combining short- and long-reads. The 188-kb multipartite plastome has three ribosomal operon copies in the master genomic conformation, creating the alternative subgenomic conformation composed of 110- and 78-kb subgenomes. The long-read data indicated that the two different genomic conformations were present in almost equal proportions in the plastomes of Selaginella nipponica. The mitogenome of S. nipponica was assembled into 27 contigs with a total size of 110 kb. All contigs contained directly arranged repeats at both ends, which introduced multiple conformations. Our results showed that plastomes and mitogenomes share high tRNA losses, GC-biased nucleotides, elevated substitution rates and complicated organization. The exploration of nuclear-encoded organelle DNA replication, recombination and repair proteins indicated that, several single-targeted proteins, particularly plastid-targeted recombinase A1, have been lost in Selaginellaceae; conversely, the dual-targeted proteins remain intact. According to the reported function of recombinase A1, we propose that the plastomes of spikemoss often fail to pair homologous sequences during recombination, and the dual-targeted proteins play a key role in the convergent genetic features of plastomes and mitogenomes. Our results provide a distinctive evolutionary pattern of the organelle genomes in Selaginellaceae and evidence of their convergent evolution.

Plastome-based phylogenomics resolves the placement of the sanguinolenta group in the spikemoss of lycophyte (Selaginellaceae).

Selaginellaceae have been shown to be monophyletic in previous studies, and include only the single genus Selaginella. However, the two most recent classifications of the genus disagree in terms of the number of subgenera recognized, and the position of problematic clades such as the "sanguinolenta" group, which has been resolved in quite different positions in different studies. Here, we performed a plastid-genome based phylogenomic analysis of Selaginellaceae to address this problem. The sanguinolenta group, represented here by three species, was resolved as sister to the remaining members of subg. Stachygynandrum. Additionally, subg. Exaltatae, subg. Ericetorum, and subg. Gymnogynum in clade A clustered into a well supported monophyletic clade but with conflicting topology between subgenera inside, which is possibly attributed to the early divergence among them. We uncovered substantial variation in both synonymous (dS) and nonsynonymous (dN) substitution rate, and GC content in plastomes of Selaginellaceae. The values of dS, dN, and GC content were significantly higher than those of other lycophytes (Isoetaceae and Lycopodiaceae). We observed a significant positive correlation between the high GC content, and the elevated dS and dN rates. In addition, the dS and dN values inferred among branches of Selaginellaceae were extremely variable. Our data indicate that this unevenly distributed substitution rate likely reflected relaxed or intensified selection among different lineages, which is possibly related to the inconsistency of the subgeneric phylogenetic topologies of Selaginellaceae.

Backbone phylogeny of Lepisorus (Polypodiaceae) and a novel infrageneric classification based on the total evidence from plastid and morphological data.

The fern genus Lepisorus represents one of the most complicated and controversial lineages in Polypodiaceae, with about 80 species which have been classified into several separate genera, and is notorious for its taxonomic difficulty. Despite progress in recent phylogenetic studies of the family Polypodiaceae involving Lepisorus and its allies, the deep phylogenetic relationship within this group of ferns is still unresolved, and no formal infrageneric classification has been proposed. This contribution presents the most comprehensive phylogenetic analysis of the genus, with 72% species sampled, using a total-evidence approach based on eight plastid markers and ≤25 morphological characters for each species. The analyses resolve the backbone relationship and yield the most robust phylogenetic framework to date. Congruent with previous studies but with new findings, the results herein show that Lepisorus is monophyletic when Neolepisorus, Lemmaphyllum, Tricholepidium, Neocheiropteris and Lepidomicrosorium are included, as well as Lepisorus jakonensis and Paragramma. Furthermore, 17 well-resolved clades are found in the phylogenetic topology, which can be characterized by morphological synapomorphies from traits of rhizome scales, laminae, sori and paraphyses. Based on molecular and morphological evidence, a new infrageneric classification system of Lepisorus is proposed which subdivided Lepisorus into 17 sections.

Correction to: Directed Repeats Co-occur with Few Short-Dispersed Repeats in Plastid Genome of a Spikemoss, Selaginella vardei (Selaginellaceae, Lycopodiopsida).

Following the publication of this article [1], the authors reported that the Fig. 2 described in the article had a mistake that two grey blocks in S. moellendorffii was not placed as background color, and in the Fig. 2 legend, chlL-chlN was wrongly written into chlL-chlL. They have therefore provided the following alternative Fig. 2 in this Correction article in order to show the accurate information.

Directed Repeats Co-occur with Few Short-Dispersed Repeats in Plastid Genome of a Spikemoss, Selaginella vardei (Selaginellaceae, Lycopodiopsida).

BACKGROUND: It is hypothesized that the highly conserved inverted repeats (IR) structure of land plant plastid genomes (plastomes) is beneficial for stabilizing plastome organization, whereas the mechanism of the occurrence and stability maintenance of the recently reported direct repeats (DR) structure is yet awaiting further exploration. Here we describe the DR structure of the Selaginella vardei (Selaginellaceae) plastome, to elucidate the mechanism of DR occurrence and stability maintenance. RESULTS: The plastome of S. vardei is 121,254 bp in length and encodes 76 genes, of which 62 encode proteins, 10 encode tRNAs, and four encode rRNAs. Unexpectedly, the two identical rRNA gene regions (13,893 bp) are arranged in a direct orientation (DR), rather than inverted. Comparing to the IR organization in Isoetes flaccida (Isoetaceae, Lycopodiopsida) plastome, a ca. 50-kb trnN-trnF inversion that spans one DR copy was found in the plastome of S. vardei, which might cause the orientation change. In addition, we find extremely rare short dispersed repeats (SDRs) in the plastomes of S. vardei and its closely related species S. indica. CONCLUSIONS: We suggest that the ca. 50-kb inversion resulted in the DR structure, and the reduction in SDRs plays a key role in maintaining the stability of plastomes with DR structure by avoiding potential secondary recombination. We further confirmed the presence of homologous recombination between DR regions, which are able to generate subgenomes and form diverse multimers. Our study deepens the understanding of Selaginella plastomes and provides new insights into the diverse plastome structures in land plants.

The Unique Evolutionary Trajectory and Dynamic Conformations of DR and IR/DR-Coexisting Plastomes of the Early Vascular Plant Selaginellaceae (Lycophyte).

Both direct repeats (DR) and inverted repeats (IR) are documented in the published plastomes of Selaginella species indicating the unusual and diverse plastome structure in the family Selaginellaceae. In this study, we newly sequenced complete plastomes of seven species from five main lineages of Selaginellaceae and also resequenced three species (Selaginella tamariscina, Selaginella uncinata, and Selaginella moellendorffii) to explore the evolutionary trajectory of Selaginellaceae plastomes. Our results showed that the plastomes of Selaginellaceae vary remarkably in size, gene contents, gene order, and GC contents. Notably, both DR and IR structures existed in the plastomes of Selaginellaceae with DR structure being an ancestral state. The occurrence of DR structure was at ∼257 Ma and remained in most subgenera of Selaginellaceae, whereas IR structure only reoccurred in Selaginella sect. Lepidophyllae (∼143 Ma) and Selaginella subg. Heterostachys (∼19 Ma). The presence of a pair of large repeats psbK-trnQ, together with DR/IR region in Selaginella bisulcata, Selaginella pennata, S. uncinata, and Selaginella hainanensis, could frequently mediate diverse homologous recombination and create approximately equal stoichiometric isomers (IR/DR-coexisting) and subgenomes. High proportion of repeats is presumably responsible for the dynamic IR/DR-coexisting plastomes, which possess a lower synonymous substitution rate (dS) compared with DR-possessing and IR-possessing plastomes. We propose that the occurrence of DR structure, together with few repeats, is possibly selected to keep the stability of plastomes and the IR/DR-coexisting plastomes also reached an equilibrium in plastome organization through highly efficient homologous recombination to maintain stability.

Plastid Phylogenomics Resolve Deep Relationships among Eupolypod II Ferns with Rapid Radiation and Rate Heterogeneity.

The eupolypods II ferns represent a classic case of evolutionary radiation and, simultaneously, exhibit high substitution rate heterogeneity. These factors have been proposed to contribute to the contentious resolutions among clades within this fern group in multilocus phylogenetic studies. We investigated the deep phylogenetic relationships of eupolypod II ferns by sampling all major families and using 40 plastid genomes, or plastomes, of which 33 were newly sequenced with next-generation sequencing technology. We performed model-based analyses to evaluate the diversity of molecular evolutionary rates for these ferns. Our plastome data, with more than 26,000 informative characters, yielded good resolution for deep relationships within eupolypods II and unambiguously clarified the position of Rhachidosoraceae and the monophyly of Athyriaceae. Results of rate heterogeneity analysis revealed approximately 33 significant rate shifts in eupolypod II ferns, with the most heterogeneous rates (both accelerations and decelerations) occurring in two phylogenetically difficult lineages, that is, the Rhachidosoraceae-Aspleniaceae and Athyriaceae clades. These observations support the hypothesis that rate heterogeneity has previously constrained the deep phylogenetic resolution in eupolypods II. According to the plastome data, we propose that 14 chloroplast markers are particularly phylogenetically informative for eupolypods II both at the familial and generic levels. Our study demonstrates the power of a character-rich plastome data set and high-throughput sequencing for resolving the recalcitrant lineages, which have undergone rapid evolutionary radiation and dramatic changes in substitution rates.

Elevation Shift in Abies Mill. (Pinaceae) of Subtropical and Temperate China and Vietnam-Corroborative Evidence from Cytoplasmic DNA and Ecological Niche Modeling.

The "elevational shift" scenario has been proposed as a model to explain the response of cold-adapted organisms to Quaternary climatic fluctuations in Europe and North America. However, the elevational shift model has not been well-explored in eastern Asia, which is more topographically complex than the other Northern Hemisphere biogeographic regions. Here, we evaluated the role of elevational shift in the closely related firs, or Abies Mill., of subtropical and temperate China. These firs are typical alpine trees with sensitivity to climate change. We tested the elevational shift hypothesis in firs of China using phylogeographic methods and ecological niche models. Our phylogeographic analyses comprised mitochondrial and chloroplast polymorphisms surveyed across 479 individuals from 43 populations representing 11 species. M1 of the 11 mitotypes and C1 of the 25 chlorotypes were inferred as the ancestral haplotype, and they had the widest distribution. The results of our phylogeographic survey revealed multiple centers of genetic diversity in distinct geographic regions and no latitudinal trend. Moreover, our results showed range expansions for seven taxa during the last glacial (64.9-18.2 or 32.5-9.1 kya), and this was consistent with the Quaternary fossil record of Abies in China. Taken together, our findings support a historical biogeographic pattern in firs of glacial expansions, probably through corridors at lower elevation, and interglacial fragmentations, through isolation at higher elevation peaks. Therefore, Abies in China probably undergoes elevational shift in response to climate change. Facing the forecasting global warming, the risk of several critically endangered firs was further enhanced as these species would have little escape space in situ to higher altitudes. According to our ENMs, we proposed an ex situ conservation strategy in the southern Hengduan Mountains region of south western China.

Isolation and characterization of microsatellite markers in the Lepisorus clathratus complex (Polypodiaceae).

PREMISE OF THE STUDY: Microsatellites were designed and characterized in the Sino-Himalayan fern Lepisorus clathratus complex (Polypodiaceae) to further study the phylogeography and reproductive ecology of this species. METHODS AND RESULTS: From a genomic library obtained by next-generation sequencing, 10 polymorphic and six monomorphic microsatellite loci were developed. In one population of L. clathratus from Taibaishan in central China, the number of alleles observed for these microsatellites ranged from seven to 29, and observed and expected heterozygosity ranged from 0.463 to 0.919 and from 0.797 to 0.947, respectively. Cross-amplification in other taxa within this complex was successful, but cross-amplification was poor for other congeneric species. CONCLUSIONS: This set of newly developed microsatellite markers will be useful for assessing genetic diversity, population structure, and mating system, and to infer polyploid origin in the L. clathratus complex.

Phylogenetic relationships, possible ancient hybridization, and biogeographic history of Abies (Pinaceae) based on data from nuclear, plastid, and mitochondrial genomes.

Abies, the second largest genus of Pinaceae, consists of approximately 48 species occurring in the north temperate region. Previous molecular phylogenetic studies improved our understanding of relationships within the genus, but were limited by relying on only DNA sequence data from single genome and low taxonomic sampling. Here we use DNA data from three genomes (sequences of internal transcribed spacer of nrITS, three chloroplast DNA intergenic spacers, and two mitochondrial intergenic spacers) from 42 species to elucidate species relationships and construct the biogeographic history of Abies. We further estimated the divergence times of intercontinental disjunction using a relaxed molecular clock calibrated with three macro-fossils. Our phylogenetic analyses recovered six robust clades largely consistent with previous classifications of sections. A sister relationship between the eastern Asian and Europe-Mediterranean clades was highly supported. The monophyly of section Balsamea, disjunct in Far East and western North America, is supported by the nrITS data but not by the cpDNA data. Discordance on placement of section Balsamea between the paternally inherited cpDNA and maternally inherited mtDNA trees was also observed. The data suggested that ancient hybridization was likely involved in the origin of sect. Balsamea. Results from biogeographic analyses and divergence time estimation suggested an origin and early diversification of Abies in an area of high latitude around the Pacific during the Eocene. The present disjunction in eastern Asia and Europe-Mediterranean area of Abies was likely the result of southward migration and isolation by the Turgai Strait in the Late Eocene. An 'out-of-America' migration, for the origin of an eastern Asian and western North American disjunct species pairs in section Amabilis was supported. The results suggested a western North American origin of the section with subsequent dispersal across the Bering Land Bridge (BLB) to Japan during the Middle Miocene.

The rise of the Himalaya enforced the diversification of SE Asian ferns by altering the monsoon regimes.

BACKGROUND: The rise of high mountain chains is widely seen as one of the factors driving rapid diversification of land plants and the formation of biodiversity hotspots. Supporting evidence was reported for the impact of the rapid rise of the Andean mountains but this hypothesis has so far been less explored for the impact of the "roof of the world". The formation of the Himalaya, and especially the rise of the Qinghai-Tibetan Plateau in the recent 20 million years, altered the monsoon regimes that dominate the current climates of South East Asia. Here, we infer the hypothesis that the rise of Himalaya had a strong impact on the plant diversity in the biodiversity hotspot of the Southwest Chinese Mountains. RESULTS: Our analyses of the diversification pattern of the derived fern genus Lepisorus recovered evidence for changes in plant diversity that correlated with the strengthening of South East Asian monsoon. Southwest China or Southwest China and Japan was recovered as the putative area of origin of Lepisorus and enhancing monsoon regime were found to shape the early diversification of the genus as well as subsequent radiations during the late Miocene and Pliocene. CONCLUSIONS: We report new evidence for a coincidence of plant diversification and changes of the climate caused by the uplift of the Himalaya. These results are discussed in the context of the impact of incomplete taxon sampling, uncertainty of divergence time estimates, and limitations of current methods used to assess diversification rates.

Phylogeography of the Sino-Himalayan fern Lepisorus clathratus on "the roof of the world".

BACKGROUND: The Qinghai-Tibetan Plateau (QTP) and its southern and southeastern mountain ranges, Himalaya-Hengduan Mountains (HHM), are one of the most extensive habitats for alpine plants in the world. How ferns occurring in QTP and HHM changed their distribution ranges in response to Quaternary climatic oscillations remains almost unknown. METHODOLOGY AND RESULTS: We employed sequences of two chloroplast DNA regions, rps4-trnS and trnL-trnF, to reconstruct phylogeography of the Sino-Himalayan fern Lepisorus clathratus, occurring mainly in the QTP and HHM. Individuals of this species have either dehiscent or indehiscent sporangia with the latter evolved from the plesiomorphic dehiscent forms. Based on a range-wide sampling, we detected 27 cpDNA haplotypes that were divided into five groups by network analyses. Populations in the Hengduan Mountains possess the highest genetic diversity, while a single haplogroup is detected across the north-central region. A distinct phylogeographical subdivision was detected between the Hengduan Mountains and north-central region by AMOVA analysis. The haplogroup distribution pattern, coalescence and AMOVA analysis suggest that a long term survival area (refugia) of the species was located in the Hengduan Mountains during glaciations, with probable range expansions into north-central regions during interglacial periods. Populations with indehiscent sporangium can carry private haplotypes and are inclined to maintain genetic homogeneity. One group with indehiscent sporangia most likely survived in situ on the QTP during glaciations. CONCLUSIONS/SIGNIFICANCE: This study for the first time sheds light on the response of alpine ferns in the QTP and HHM to the Quaternary climatic oscillations.

Resolving and dating the phylogeny of Cornales--Effects of taxon sampling, data partitions, and fossil calibrations.

The order Cornales descends from the earliest split in the Asterid clade of flowering plants. Despite a few phylogenetic studies, relationships among families within Cornales remain unclear. In the present study, we increased taxon and character sampling to further resolve the relationships and to date the early diversification events of the order. We conducted phylogenetic analyses of sequence data from 26S rDNA and six chloroplast DNA (cpDNA) regions using parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) methods with different partition models and different data sets. We employed relaxed, uncorrelated molecular clocks on BEAST to date the phylogeny and examined the effects of different taxon sampling, fossil calibration, and data partitions. Our results from ML and BI analyses of the combined cpDNA sequences and combined cpDNA and 26S rDNA data suggested the monophyly of each family and the following familial relationships ((Cornaceae-Alangiaceae)-(Curtisiaceae-Grubbiaceae))-(((Nyssaceae-Davidiaceae)-Mastixiaceae)-((Hydrostachyaceae-(Hydrangeaceae-Loasaceae))). These relationships were strongly supported by posterior probability and bootstrap values, except for the sister relationship between the N-D-M and H-H-L clades. The 26S rDNA data and some MP trees from cpDNA and total evidence suggested some alternative alignments for Hydrostachyaceae within Cornales, but results of SH tests indicated that these trees were significantly worse explanations of the total data. Phylogenetic dating with simultaneous calibration of multiple nodes suggested that the crown group of Cornales originated around the middle Cretaceous and rapidly radiated into several major clades. The origins of most families dated back to the late Cretaceous except for Curtisiaceae and Grubbiaceae which may have diverged in the very early Tertiary. We found that reducing sampling density within families and analyzing partitioned data sets from coding and noncoding cpDNA, 26S rDNA, and combined data sets produced congruent estimation of divergence times, but reducing the number and changing positions of calibration points resulted in very different estimations.

Phylogeny of the paleotropical fern genus Lepisorus (Polypodiaceae, Polypodiopsida) inferred from four chloroplast DNA regions.

Phylogenetic relationships within the paleotropical genus Lepisorus (Polypodiaceae) were investigated using plastid DNA sequences from four regions: rbcL, rps4 and rps4-trnS IGS, trnL intron plus trnL-F IGS, rbcL-atpB IGS. Over 4000 nucleotides were sequenced for 77 specimens belonging to 54 species. Each cpDNA region was analyzed separately and combined into a single dataset. All phylogenetic analyses, maximum parsimony, maximum likelihood and Bayesian Inference of phylogeny, revealed the paraphyly of Lepisorus with the monotypic Drymotaenium miyoshianum and of the paleotropical genus Belvisia nested within the Lepisorus clade. Nine well-supported major clades were found. The phylogenetic results provided new evidence for the sectional classification of Lepisorus. The evolution of three morphological characters, clathrateness of rhizome scales, margin of rhizome scales and defoliated leaves, and the evolution of the karyotype, were reconstructed to identify lineage specific phenotypic character states or combination of characters. Unique character combinations, rather than synapomorphies, were found to be of systematic value in sectional delimitation. The variation of chromosome numbers is largely due to a single aneuploidy event instead of a stepwise reduction during the evolutionary history of this genus.