Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions.

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

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.

Cellular and gene expression patterns associated with root bifurcation in Selaginella.

The roots of lycophytes branch through dichotomy or bifurcation, during which the root apex splits into two daughter roots. This is morphologically distinct from lateral root (LR) branching in the extant euphyllophytes, with LRs developing along the root axis at different distances from the apex. Although the process of root bifurcation is poorly understood, such knowledge can be important, because it may represent an evolutionarily ancient strategy that roots recruited to form new stem cells or meristems. In this study, we examined root bifurcation in the lycophyte Selaginella moellendorffii. We characterized an in vitro developmental time frame based on repetitive apex bifurcations, allowing us to sample different stages of dichotomous root branching and analyze the root meristem and root branching in S. moellendorffii at the microscopic and transcriptomic level. Our results showed that, in contrast to previous assumptions, initial cells (ICs) in the root meristem are mostly not tetrahedral but rather show an irregular shape. Tracking down the early stages of root branching argues for the occurrence of a symmetric division of the single IC, resulting in two apical stem cells that initiate root meristem bifurcation. Moreover, we generated a S. moellendorffii root branching transcriptome that resulted in the delineation of a subset of core meristem genes. The occurrence of multiple putative orthologs of meristem genes in this dataset suggests the presence of conserved pathways in the control of meristem and root stem cell establishment or maintenance.

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.

Impacts of methyl jasmonate on Selaginella martensii: volatiles, transcriptomics, phytohormones, and gas exchange.

Methyl jasmonate (MeJA) induces various defence responses in seed plants, but for early plant lineages, information on the potential of jasmonates to elicit stress signalling and trigger physiological modifications is limited. The spikemoss Selaginella martensii was exposed to a range of MeJA concentrations (0, 10, 25, and 50 mM), and biogenic volatile organic compound (BVOC) emissions, photosynthetic rate (A), and stomatal conductance (gs) were continuously measured. In addition, changes in phytohormone concentrations and gene expression were studied. Enhancement of methanol, lipoxygenase pathway volatiles and linalool emissions, and reductions in A and gs, were MeJA dose-dependent. Before MeJA treatment, the concentration of 12-oxo-phytodienoic acid (OPDA) was 7-fold higher than jasmonic acid (JA). MeJA treatment rapidly increased OPDA and JA concentrations (within 30 min), with the latter more responsive. Some genes involved in BVOC biosynthesis and OPDA-specific response were up-regulated at 30 min after MeJA spraying, whereas those in the JA signalling pathway were not affected. Although JA was synthesized in S. martensii, OPDA was prioritized as a signalling molecule upon MeJA application. MeJA inhibited primary and enhanced secondary metabolism; we propose that fast-emitted linalool could serve as a marker of elicitation of stress-induced metabolism in lycophytes.

Expression Atlas of Selaginella moellendorffii Provides Insights into the Evolution of Vasculature, Secondary Metabolism, and Roots.

Selaginella moellendorffii is a representative of the lycophyte lineage that is studied to understand the evolution of land plant traits such as the vasculature, leaves, stems, roots, and secondary metabolism. However, only a few studies have investigated the expression and transcriptional coordination of Selaginella genes, precluding us from understanding the evolution of the transcriptional programs behind these traits. We present a gene expression atlas comprising all major organs, tissue types, and the diurnal gene expression profiles for S. moellendorffii We show that the transcriptional gene module responsible for the biosynthesis of lignocellulose evolved in the ancestor of vascular plants and pinpoint the duplication and subfunctionalization events that generated multiple gene modules involved in the biosynthesis of various cell wall types. We demonstrate how secondary metabolism is transcriptionally coordinated and integrated with other cellular pathways. Finally, we identify root-specific genes and show that the evolution of roots did not coincide with an increased appearance of gene families, suggesting that the development of new organs does not coincide with increased fixation of new gene functions. Our updated database at conekt.plant.tools represents a valuable resource for studying the evolution of genes, gene families, transcriptomes, and functional gene modules in the Archaeplastida kingdom.

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.

Plastome structure, evolution, and phylogeny of Selaginella.

As one of the earliest land plant lineages, Selaginella is important for studying land plant evolution. It is the largest genus of lycophytes containing 700-800 species. Some unique characters of Selaginella plastomes have been reported, but based only on 20 species. There have been no plastome phylogenies of Selaginella based on a relatively large sampling, and no efforts have been made to resolve the phylogeny of the enigmatic Sinensis group whose relationships have been unclear based on small datasets. Here we investigated the structures of 59 plastomes representing 51 species covering all six subgenera and 18 sections of Selaginella except two sections and including the intriguing Sinensis group for the first time. Our major results include: (1) the plastome size of Selaginella ranges tremendously from 78,492 bp to 187,632 bp; (2) there are numerous gene losses in Selaginella comparing with other lycophytes, Isoëtaceae and Lycopodiaceae; (3) the gene contents and plastome structures in Selaginella vary lineage-specifically and all infrageneric taxa are well supported in the plastome phylogeny; (4) the ndh gene family tends to lose or pseudogenize in those species with DR structure and without other short or medium repeats; (5) the short and medium repeat regions in SC mediate many conformations causing diverse and complex plastome structures, and six new conformations are discovered; (6) forty-eight species sampled have high GC content (>50%) but three species in the Sinensis group have âˆ¼ 30% GC content in plastomes, similar to most vascular plants; (7) the Sinensis group is monophyletic, includes at least two subgroups, and has the smallest plastomes in land plants except some parasitic plants, and their plastomes do not contain any tRNAs; (8) the younger lineages in Selaginella tend to have higher GC content, whereas the older lineages tend to have lower GC content; and (9) because of incomplete genomic data and abnormal structures or some unknown reasons, even the concatenated plastomes could not well resolve the phylogenetic relationships in Selaginella with confidence, highlighting the difficulty in resolving the phylogeny and evolution of this particularly important land plant lineage.

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.

All type II classic MADS-box genes in the lycophyte Selaginella moellendorffii are broadly yet discretely expressed in vegetative and reproductive tissues.

The MADS-box genes constitute a large transcription factor family that appear to have evolved by duplication and diversification of function. Two types of MADS-box genes are distinguished throughout eukaryotes, types I and II. Type II classic MADS-box genes, also known as MIKC-type, are key developmental regulators in flowering plants and are particularly well-studied for their role in floral organ specification. However, very little is known about the role that these genes might play outside of the flowering plants. We investigated the evolution of type II classic MADS-box genes across land plants by performing a maximum likelihood analysis with a particular focus on lycophytes. Here, we present the expression patterns of all three type II classic MADS-box homologs throughout plant development in the lycophyte Selaginella moellendorffii: SmMADS1, SmMADS3, and SmMADS6. We used scanning electron microscopy and histological analyses to define stages of sporangia development in S. moellendorffii. We performed phylogenetic analyses of this gene lineage across land plants and found that lycophyte sequences appeared before the multiple duplication events that gave rise to the major MADS-box gene lineages in seed plants. Our expression analyses by in situ hybridization show that all type II classic MADS-box genes in S. moellendorffii have broad but distinct patterns of expression in vegetative and reproductive tissues, where SmMADS1 and SmMADS6 only differ during late sporangia development. The broad expression during S. moellendorffii development suggests that MADS-box genes have undergone neofunctionalization and subfunctionalization after duplication events in seed plants.

Plastid Genomes of the Early Vascular Plant Genus Selaginella Have Unusual Direct Repeat Structures and Drastically Reduced Gene Numbers.

The early vascular plants in the genus Selaginella, which is the sole genus of the Selaginellaceae family, have an important place in evolutionary history, along with ferns, as such plants are valuable resources for deciphering plant evolution. In this study, we sequenced and assembled the plastid genome (plastome) sequences of two Selaginella tamariscina individuals, as well as Selaginella stauntoniana and Selaginella involvens. Unlike the inverted repeat (IR) structures typically found in plant plastomes, Selaginella species had direct repeat (DR) structures, which were confirmed by Oxford Nanopore long-read sequence assembly. Comparative analyses of 19 lycophytes, including two Huperzia and one Isoetes species, revealed unique phylogenetic relationships between Selaginella species and related lycophytes, reflected by structural rearrangements involving two rounds of large inversions that resulted in dynamic changes between IR and DR blocks in the plastome sequence. Furthermore, we present other uncommon characteristics, including a small genome size, drastic reductions in gene and intron numbers, a high GC content, and extensive RNA editing. Although the 16 Selaginella species examined may not fully represent the genus, our findings suggest that Selaginella plastomes have undergone unique evolutionary events yielding genomic features unparalleled in other lycophytes, ferns, or seed plants.

Biochemical Characterization and Function of Eight Microbial Type Terpene Synthases from Lycophyte Selaginella moellendorffii.

Selaginella moellendorffii is a lycophyte, a member of an ancient vascular plant lineage. Two distinct types of terpene synthase (TPS) genes were identified from this species, including S. moellendorffii TPS genes (SmTPSs) and S. moellendorffii microbial TPS-like genes (SmMTPSLs). The goal of this study was to investigate the biochemical functions of SmMTPSLs. Here, eight full-length SmMTPSL genes (SmMTPSL5, -15, -19, -23, -33, -37, -46, and -47) were functionally characterized from S. moellendorffii. Escherichia coli-expressed recombinant SmMTPSLs were tested for monoterpenes synthase and sesquiterpenes synthase activities. These enzymatic products were typical monoterpenes and sesquiterpenes that have been previous shown to be generated by typical plant TPSs when provided with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as the substrates. Meanwhile, SmMTPSL23, -33, and -37 were up-regulated when induced by alamethicin (ALA) and methyl jasmonate (MeJA), suggesting a role for these genes in plants response to abiotic stresses. Furthermore, this study pointed out that the terpenoids products of SmMTPSL23, -33, and -37 have an antibacterial effect on Pseudomonas syringae pv. tomato DC3000 and Staphylococcus aureus. Taken together, these results provide more information about the catalytic and biochemical function of SmMTPSLs in S. moellendorffii plants.

Comparative transcriptome analysis suggests convergent evolution of desiccation tolerance in Selaginella species.

BACKGROUND: Desiccation tolerant Selaginella species evolved to survive extreme environmental conditions. Studies to determine the mechanisms involved in the acquisition of desiccation tolerance (DT) have focused on only a few Selaginella species. Due to the large diversity in morphology and the wide range of responses to desiccation within the genus, the understanding of the molecular basis of DT in Selaginella species is still limited. RESULTS: Here we present a reference transcriptome for the desiccation tolerant species S. sellowii and the desiccation sensitive species S. denticulata. The analysis also included transcriptome data for the well-studied S. lepidophylla (desiccation tolerant), in order to identify DT mechanisms that are independent of morphological adaptations. We used a comparative approach to discriminate between DT responses and the common water loss response in Selaginella species. Predicted proteomes show strong homology, but most of the desiccation responsive genes differ between species. Despite such differences, functional analysis revealed that tolerant species with different morphologies employ similar mechanisms to survive desiccation. Significant functions involved in DT and shared by both tolerant species included induction of antioxidant systems, amino acid and secondary metabolism, whereas species-specific responses included cell wall modification and carbohydrate metabolism. CONCLUSIONS: Reference transcriptomes generated in this work represent a valuable resource to study Selaginella biology and plant evolution in relation to DT. Our results provide evidence of convergent evolution of S. sellowii and S. lepidophylla due to the different gene sets that underwent selection to acquire DT.

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.

Tracheophytes Contain Conserved Orthologs of a Basic Helix-Loop-Helix Transcription Factor That Modulate ROOT HAIR SPECIFIC Genes.

ROOT HAIR SPECIFIC (RHS) genes, which contain the root hair-specific cis-element (RHE) in their regulatory regions, function in root hair morphogenesis. Here, we demonstrate that an Arabidopsis thaliana basic helix-loop-helix transcription factor, ROOT HAIR DEFECTVE SIX-LIKE4 (RSL4), directly binds to the RHE in vitro and in vivo, upregulates RHS genes, and stimulates root hair formation in Arabidopsis. Orthologs of RSL4 from a eudicot (poplar [Populus trichocarpa]), a monocot (rice [Oryza sativa]), and a lycophyte (Selaginella moellendorffii) each restored root hair growth in the Arabidopsis rsl4 mutant. In addition, the rice and S. moellendorffii RSL4 orthologs bound to the RHE in in vitro and in vivo assays. The RSL4 orthologous genes contain RHEs in their promoter regions, and RSL4 was able to bind to its own RHEs in vivo and amplify its own expression. This process likely provides a positive feedback loop for sustainable root hair growth. When RSL4 and its orthologs were expressed in cells in non-root-hair positions, they induced ectopic root hair growth, indicating that these genes are sufficient to specify root hair formation. Our results suggest that RSL4 mediates root hair formation by regulating RHS genes and that this mechanism is conserved throughout the tracheophyte (vascular plant) lineage.

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.

Lycophyte plastid genomics: extreme variation in GC, gene and intron content and multiple inversions between a direct and inverted orientation of the rRNA repeat.

Lycophytes are a key group for understanding vascular plant evolution. Lycophyte plastomes are highly distinct, indicating a dynamic evolutionary history, but detailed evaluation is hindered by the limited availability of sequences. Eight diverse plastomes were sequenced to assess variation in structure and functional content across lycophytes. Lycopodiaceae plastomes have remained largely unchanged compared with the common ancestor of land plants, whereas plastome evolution in Isoetes and especially Selaginella is highly dynamic. Selaginella plastomes have the highest GC content and fewest genes and introns of any photosynthetic land plant. Uniquely, the canonical inverted repeat was converted into a direct repeat (DR) via large-scale inversion in some Selaginella species. Ancestral reconstruction identified additional putative transitions between an inverted and DR orientation in Selaginella and Isoetes plastomes. A DR orientation does not disrupt the activity of copy-dependent repair to suppress substitution rates within repeats. Lycophyte plastomes include the most archaic examples among vascular plants and the most reconfigured among land plants. These evolutionary trends correlate with the mitochondrial genome, suggesting shared underlying mechanisms. Copy-dependent repair for DR-localized genes indicates that recombination and gene conversion are not inhibited by the DR orientation. Gene relocation in lycophyte plastomes occurs via overlapping inversions rather than transposase/recombinase-mediated processes.

An update and reassessment of fern and lycophyte diversity data in the Japanese Archipelago.

The fern and lycophyte flora of Japan comprising 721 native taxa (including subspecies and varieties) plus 371 interspecific hybrids was reassessed using a nearly comprehensively sampled distribution map at 10 km resolution vouchered by 216,687 specimens, up-to-date cytotaxonomic information covering 74% of the taxa, and an rbcL sequence dataset covering 97.9% of the taxa. Spatial distribution of species richness and phylogenetic diversity was visualized. Apomixis was observed in 11.0% of the native taxa whose reproductive modes are known. The number of sexually reproducing polyploid taxa (n = 199) is less than sexual diploids (n = 241), and 30 of them are evidently allopolyploid, in contrast with the low number of possible autopolyploids (n = 4). Apomictic taxa were found to have smaller latitudinal ranges than sexual taxa or taxa with multiple reproductive modes. A morphological character dataset in Lucid format is provided for taxonomic identification of the native taxa.

Virtual issue: Ecology and evolution of pteridophytes in the era of molecular genetics.

The past quarter-century has witnessed a revolution in our understanding of the phylogenetics, systematics, and ecology of pteridophytes (ferns and lycophytes), particularly due to the rapid accumulation of plastid sequence data and a renewed interest in the ecology of the sexual phase of the life cycle. We here compile 19 papers recently published in the Journal of Plant Research dealing with the biology of pteridophytes, grouped into six categories: (1) breeding systems, (2) species complexes and polyploidization, (3) fossil taxa, (4) gametophyte ecology, (5) systematics, (6) biodiversity. We hope this collection of papers will be of value to researchers interested in this fascinating group of plants.

Candidate genes of flavonoid biosynthesis in Selaginella bryopteris (L.) Baker identified by RNA-Seq.

In the present study, de novo transcriptome analysis of Selaginella bryopteris in frond and root was performed to understand the regulation of flavonoid (FL) biosynthesis. High-quality data of 5.84 and 5.86 Gb was generated for frond and root, respectively, that assembled into 94,713 and 81,567 transcripts. A total of 87,471 and 73,395 unigenes were obtained from frond and root, respectively. A total of 41,267 and 31,048 CDS of frond and root, respectively, were annotated by BLASTX, which showed maximum hits against S. moellendorffii. Out of 11,285 differentially expressed genes, a total of 5639 genes were found to be down-regulated and 5628 genes up-regulated in frond as compared to those in root. In silico analysis of expression of genes in frond as compared to that in root was done for those related to phenylpropanoid (PP)/FL biosynthesis along with transcription factors (TFs) after DESeq and MapMan-based information. Results showed that genes of PP/FL biosynthesis pathway namely SbCHS, SbCHI, SbF3H, SbF3'H, SbDFR, SbUF3GT, SbCCOAMT, and SbCATOMT and TFs (SbMYB1, SbMYB2, SbMYB3, SbBHLH1, and SbWD40-5) were up-regulated in frond in comparison to those in root. Further, this in silico expression data was validated by RT-PCR analysis which showed predominant expression of most of these genes in frond and indicated their importance in the biosynthesis of flavonoids in S. bryopteris. A total of 9074 simple sequence repeats (SSRs) were also identified for frond and 3811 SSRs for root; these can be used for experimental validation.