Transcriptomic analyses in the gametophytes of the apomictic fern Dryopteris affinis.

MAIN CONCLUSION: A novel genomic map of the apogamous gametophyte of the fern Dryopteris affinis unlocks oldest hindrance with this complex plant group, to gain insight into evo-devo approaches. The gametophyte of the fern Dryopteris affinis ssp. affinis represents a good model to explore the molecular basis of vegetative and reproductive development, as well as stress responses. Specifically, this fern reproduces asexually by apogamy, a peculiar case of apomixis whereby a sporophyte forms directly from a gametophytic cell without fertilization. Using RNA-sequencing approach, we have previously annotated more than 6000 transcripts. Here, we selected 100 of the inferred proteins homolog to those of Arabidopsis thaliana, which were particularly interesting for a detailed study of their potential functions, protein-protein interactions, and distance trees. As expected, a plethora of proteins associated with gametogenesis and embryogenesis in angiosperms, such as FERONIA (FER) and CHROMATING REMODELING 11 (CHR11) were identified, and more than a dozen candidates potentially involved in apomixis, such as ARGONAUTE family (AGO4, AGO9, and AGO 10), BABY BOOM (BBM), FASCIATED STEM4 (FAS4), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), and MATERNAL EFFECT EMBRYO ARREST29 (MEE29). In addition, proteins involved in the response to biotic and abiotic stresses were widely represented, as shown by the enrichment of heat-shock proteins. Using the String platform, the interactome revealed that most of the protein-protein interactions were predicted based on experimental, database, and text mining datasets, with MULTICOPY SUPPRESSOR OF IRA4 (MSI4) showing the highest number of interactions: 16. Lastly, some proteins were studied through distance trees by comparing alignments with respect to more distantly or closely related plant groups. This analysis identified DCL4 as the most distant protein to the predicted common ancestor. New genomic information in relation to gametophyte development, including apomictic reproduction, could expand our current vision of evo-devo approaches.

Proteome and Interactome Linked to Metabolism, Genetic Information Processing, and Abiotic Stress in Gametophytes of Two Woodferns.

Ferns and lycophytes have received scant molecular attention in comparison to angiosperms. The advent of high-throughput technologies allowed an advance towards a greater knowledge of their elusive genomes. In this work, proteomic analyses of heart-shaped gametophytes of two ferns were performed: the apomictic Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. In total, a set of 218 proteins shared by these two gametophytes were analyzed using the STRING database, and their proteome associated with metabolism, genetic information processing, and responses to abiotic stress is discussed. Specifically, we report proteins involved in the metabolism of carbohydrates, lipids, and nucleotides, the biosynthesis of amino acids and secondary compounds, energy, oxide-reduction, transcription, translation, protein folding, sorting and degradation, and responses to abiotic stresses. The interactome of this set of proteins represents a total network composed of 218 nodes and 1792 interactions, obtained mostly from databases and text mining. The interactions among the identified proteins of the ferns D. affinis and D. oreades, together with the description of their biological functions, might contribute to a better understanding of the function and development of ferns as well as fill knowledge gaps in plant evolution.

The Shared Proteome of the Apomictic Fern Dryopteris affinis ssp. affinis and Its Sexual Relative Dryopteris oreades.

Ferns are a diverse evolutionary lineage, sister to the seed plants, which is of great ecological importance and has a high biotechnological potential. Fern gametophytes represent one of the simplest autotrophic, multicellular plant forms and show several experimental advantages, including a simple and space-efficient in vitro culture system. However, the molecular basis of fern growth and development has hardly been studied. Here, we report on a proteomic study that identified 417 proteins shared by gametophytes of the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. Most proteins are predicted to localize to the cytoplasm, the chloroplast, or the nucleus, and are linked to enzymatic, binding, and structural activities. A subset of 145 proteins are involved in growth, reproduction, phytohormone signaling and biosynthesis, and gene expression, including homologs of SHEPHERD (SHD), HEAT SHOCK PROTEIN 90-5 (CR88), TRP4, BOBBER 1 (BOB1), FLAVONE 3'-O-METHYLTRANSFERASE 1 (OMT1), ZEAXANTHIN EPOXIDASE (ABA1), GLUTAMATE DESCARBOXYLASE 1 (GAD), and dsRNA-BINDING DOMAIN-LIKE SUPERFAMILY PROTEIN (HLY1). Nearly 25% of the annotated proteins are associated with responses to biotic and abiotic stimuli. As for biotic stress, the proteins PROTEIN SGT1 HOMOLOG B (SGT1B), SUPPRESSOR OF SA INSENSITIVE2 (SSI2), PHOSPHOLIPASE D ALPHA 1 (PLDALPHA1), SERINE/THREONINE-PROTEIN KINASE SRK2E (OST1), ACYL CARRIER PROTEIN 4 (ACP4), and NONHOST RESISTANCE TO P. S. PHASEOLICOLA1 (GLPK) are worth mentioning. Regarding abiotic stimuli, we found proteins associated with oxidative stress: SUPEROXIDE DISMUTASE[CU-ZN] 1 (CSD1), and GLUTATHIONE S-TRANSFERASE U19 (GSTU19), light intensity SERINE HYDROXYMETHYLTRANSFERASE 1 (SHM1) and UBIQUITIN-CONJUGATING ENZYME E2 35 (UBC35), salt and heavy metal stress included MITOCHONDRIAL PHOSPHATE CARRIER PROTEIN 3 (PHT3;1), as well as drought and thermotolerance: LEA7, DEAD-BOX ATP-DEPENDENT RNA HELICASE 38 (LOS4), and abundant heat-shock proteins and other chaperones. In addition, we identified interactomes using the STRING platform, revealing protein-protein associations obtained from co-expression, co-occurrence, text mining, homology, databases, and experimental datasets. By focusing on ferns, this proteomic study increases our knowledge on plant development and evolution, and may inspire future applications in crop species.

Integrated mRNA and miRNA Transcriptome Analysis Suggests a Regulatory Network for UV-B-Controlled Terpenoid Synthesis in Fragrant Woodfern (Dryopteris fragrans).

Fragrant woodfern (Dryopteris fragrans) is a medicinal plant rich in terpenoids. Ultraviolet-B (UV-B) light could increase concentration of terpenoids. The aim of this study was to analyze how UV-B regulates the terpenoid synthesis of the molecular regulatory mechanism in fragrant woodfern. In this study, compared with the control group, the content of the terpenes was significantly higher in fragrant woodfern leaves under UV-B treatment for 4 days (d). In order to identify how UV-B regulates the terpenoid metabolic mechanism in fragrant woodfern, we examined the mRNAs and small RNAs in fragrant woodfern leaves under UV-B treatment. mRNA and miRNA-seq identified 4533 DEGs and 17 DEMs in the control group compared with fragrant woodfern leaves under UV-B treatment for 4 d. mRNA-miRNA analysis identified miRNA target gene pairs consisting of 8 DEMs and 115 miRNAs. The target genes were subjected to GO and KEGG analyses. The results showed that the target genes were mainly enriched in diterpene biosynthesis, terpenoid backbone biosynthesis, plant hormone signal transduction, MEP pathway and MVA pathway, in which miR156 and miR160 regulate these pathways by targeting DfSPL and DfARF, respectively. The mRNA and miRNA datasets identified a subset of candidate genes. It provides the theoretical basis that UV-B regulates the terpenoid synthesis of the molecular regulatory mechanism in fragrant woodfern.

Transcriptomic Analysis and Specific Expression of Transcription Factor Genes in the Root and Sporophyll of Dryopteris fragrans (L.) Schott.

Background: Dryopteris fragrans, which is densely covered with glandular trichomes, is considered to be one of the ferns with the most medicinal potential. The transcriptomes from selected tissues of D. fragrans were collected and analyzed for functional and comparative genomic studies. The aim of this study was to determine the transcriptomic characteristics of wild D. fragrans sporangium in tissues from the SR (root), SL (sporophyll), and TRL (sporophyll with glandular trichomes removed). Results: Cluster analysis identified genes that were highly expressed in an organ-specific manner according to read mapping, feature counting, and normalization. The functional map identified gene clusters that can uniquely describe the function of each tissue. We identified a group of three tissue-specific transcription factors targeting the SL, SR, and TRL. In addition, highly expressed transcription factors (TFs) were found in each tissue-specific gene cluster, where ERF and bHLH transcription factors were the two types showing the most distinct expression patterns between the three different tissues. The specific expression of transcription factor genes varied between the different types of tissues. The numbers of transcription factors specifically expressed in the roots and sporophylls were 60 and 30, respectively, while only seven were found for the sporophylls with glandular trichomes removed. The expression of genes known to be associated with the development of glandular trichomes in flowering plants, including MIXTA, ATML1, and MYB106, were also validated and are discussed. In particular, a unigene encoding MIXTA was identified and exhibited the highest expression level in SL in D. fragrans. Conclusions: This study is the first report of global transcriptomic analysis in different tissues of D. fragrans, and the first to discuss these findings in the context of the development of homologous glandular trichomes. These results set the stage for further research on the development, stress resistance, and secondary metabolism of D. fragrans glandular trichomes.

Cloning and functional analysis of squalene synthase gene from Dryopteris fragrans (L.) Schott.

Dryopteris fragrans (L.) Schott is a traditional herbal medicine containing medicinal sterols and triterpenoids. Squalene synthase (SQS) is the first crucial enzyme in the biosynthesis pathway of sterols and triterpenoids. The full-length cDNA named DfSQS1 was isolated by RACE. It was predicted that DfSQS1 contained an open reading frame (ORF) of 1239 bp coding 412 amino acid residues with molecular weight of 46.6 kDa. It had 18 potential phosphorylation sites, 1 potential N-glycosylation site and 2 transmembrane domains. In neighbor-joining (NJ) phylogenetic tree, DfSQS1 was away from branch of gymnosperms and angiosperms. One hydrophobic domain at the C-terminal of DfSQS1 was deleted to express soluble recombinant enzyme. The truncated DfSQS1 (tDfSQS1) was expressed in Escherichia coli BL21 (DE3). Then, tDfSQS1 was obtained and incubated with farnesyl diphosphate (FPP) to identify its enzymatic activity. The result demontrated that squalene, the product of enzyme catalyzed reaction, was detected by HPLC. Quantitative real-time PCR (qRT-PCR) analysis revealed that the transcription level of DfSQS1 in D. fragrans was the highest in roots, followed by leaves and rhizomes. This work is the first report on cloning, characteration and expression of SQS from D. fragrans. It will be helpful to understand the regulatory role of SQS on the biosynthesis of triterpenoids in the fern.

Global transcriptome analysis and characterization of Dryopteris fragrans (L.) Schott sporangium in different developmental stages.

BACKGROUND: Dryopteris fragrans (D. fragrans) is a potential medicinal fern distributed in volcanic magmatic rock areas under tough environmental condition. Sporangia are important organs for fern reproduction. This study was designed to characterize the transcriptome characteristics of the wild D. fragrans sporangia in three stages (stage A, B, and C) with the aim of uncovering its molecular mechanism of growth and development. RESULTS: Using a HiSeq 4000, 79.81 Gb clean data (each sample is at least 7.95 GB) were obtained from nine samples, with three being supplied from each period, and assembled into 94,705 Unigenes, among which 44,006 Unigenes were annotated against public protein databases (NR, Swiss-Prot, KEGG, COG, KOG, GO, eggNOG and Pfam). Furthermore, we observed 7126 differentially expressed genes (DEG) (Fold Change > 4, FDR < 0.001), 349,885 SNP loci, and 10,584 SSRs. DEGs involved in DNA replication and homologous recombination were strongly expressed in stage A, and several DEGs involved in cutin, suberin and wax biosynthesis had undergone dramatic changes during development, which was consistent with morphological observations. DEGs responsible for secondary metabolism and plant hormone signal transduction changed clearly in the last two stages. DEGs homologous to those known genes associated with the development of reproductive organs of flowering plants have also been validated and discussed, such as AGL61, AGL62, ONAC010. In particular, a Unigene encoding TFL1, an important flower-development regulator in flowering plants, was identified and exhibited the highest expression level in stage B in D. fragrans sporangia. CONCLUSIONS: This study is the first report on global transcriptome analysis in the development of sporangia of wild D. fragrans. DEGs related to development and homologous to flower-seed development in flowering plants were discussed. All DEGs involved in DNA replication and homologous recombination were consistent with morphological observations of paraffin slices. The results of this study provide rare resources for further investigation of the D. fragrans sporangium development, stress resistance and secondary metabolism.

Into Africa: Molecular phylogenetics and historical biogeography of sub-Saharan African woodferns (Dryopteris).

PREMISE OF THE STUDY: Our goal was to infer the phylogenetic relationships and historical biogeography of the genus Dryopteris with a focus on taxa in sub-Saharan Africa and neighboring islands. In general, little is known about the relationships between African fern species and their congeners in other geographic regions, and our aim was to determine whether the sub-Saharan African species of Dryopteris are monophyletic and evolved within Africa or arrived there via repeated dispersals into Africa from other regions. METHODS: We obtained sequence data for five chloroplast markers from 214 species of Dryopteris and 18 outgroups. We performed phylogenetic and molecular dating analyses using a Bayesian relaxed clock method in BEAST with fossil and secondary calibration points and estimated ancestral ranges for the genus globally by comparing multiple models in BioGeoBEARS. KEY RESULTS: We found that 22 of 27 accessions of sub-Saharan African Dryopteris belong to a large clade of 31 accessions that also includes taxa from Indian and Atlantic Ocean islands. Additional accessions of taxa from our regions of interest have Asian, Hawaiian, European, or North American species as their closest relatives. CONCLUSIONS: The majority of sub-Saharan African Dryopteris species are descended from a shared common ancestor that dispersed to Africa from Asia approximately 10 Ma. There have been subsequent dispersal events from the African mainland to islands in the Atlantic and Indian Oceans, including Madagascar. Several additional species are estimated to have descended from ancestors that reached Africa via separate events over the last roughly 20 million years.

Between sexual and apomictic: unexpectedly variable sporogenesis and production of viable polyhaploids in the pentaploid fern of the Dryopteris affinis agg. (Dryopteridaceae).

BACKGROUND AND AIMS: In ferns, apomixis is an important mode of asexual reproduction. Although the mechanisms of fern reproduction have been studied thoroughly, most previous work has focused on cases in which ferns reproduce either exclusively sexually or exclusively asexually. Reproduction of ferns with potentially mixed systems and inheritance of apomixis remains largely unknown. This study addresses reproduction of the pentaploid Dryopteris × critica, a hybrid of triploid apomictic D. borreri and tetraploid sexual D. filix-mas. METHODS: Spore size, abortion percentage and number of spores per sporangium were examined in pentaploid plants of D. × critica grown in an experimental garden. The sporangial content of leaf segments was cultivated on an agar medium, and DNA ploidy levels were estimated by DAPI flow cytometry in 259 gametophytes or sporophytes arising from the F2 generation of the pentaploid hybrid. KEY RESULTS: The hybrid is partly fertile (89-94% of aborted spores) and shows unstable sporogenesis with sexual and apomictic reproduction combined. The number of spores per sporangium varied from approx. 31 to 64. Within a single sporangium it was possible to detect formation of either only aborted spores or various mixtures of aborted and well-developed reduced spores and unreduced diplospores. The spores germinated in viable gametophytes with two ploidy levels: pentaploid (5x, from unreduced spores) and half of that (approx. 2·5x, from reduced spores). Moreover, 2-15% of gametophytes (both 2·5x and 5x) formed a viable sporophyte of the same ploidy level due to apogamy. CONCLUSIONS: This study documents the mixed reproductive mode of a hybrid between apomictic and sexual ferns. Both sexual reduced and apomictic unreduced spores can be produced by a single individual, and even within a single sporangium. Both types of spores give rise to viable F2 generation gametophytes and sporophytes.

Identification and expression analysis of 4-Coumarate: Coenzyme A ligase gene family in Dryopteris Fragrans.

4-Coumarate: coenzyme A ligase (4CL) catalyzes the conversion of hydroxycinnamates into corresponding CoA esters for biosynthesis of flavonoids and lignin. It has been widely studied in seed plants; however, it is poorly characterized in ferns. In this study, we identified 4CLs genes in ferns D. fragrans (L.) Schott (Df4CL gene) by rapid amplification of cDNA ends (RACE), and then investigated the expressions of the genes by real-time PCR, and determined total flavonoids and lignin contents. The results showed that four members of the 4CL genes were found from this species, which named Df4CL 1, 2, 3, and 4 genes. Their full-length cDNA was sequenced. Also, our analyses showed that the amino acid sequences derived from these cDNAs exhibited similar conserved regions (Box I and Box II), and substrate-binding regions compared to 4CLs isolated from seed plants.At the same time, the developmental and stress-induced gene expression patterns showed that the changes on the expression levels of Df4CL genes affected the levels of flavonoids and lignin. In conclusion, we identified 4CLs genes in ferns D. fragrans and analyzed the expressions of these genes, and finally explored the relationship between the expressions of 4CLs and syntheses of flavonoids and lignin.

Identification and characterisation of DfCHS, a chalcone synthase gene regulated by temperature and ultraviolet in Dryopteris fragrans.

Chalcone synthase (CHS) is an enzyme that catalyzes the first committed step in flavonoid biosynthesis, and its transcription level is regulated by light conditions. By using homology cloning and rapid amplification of cDNA ends, we cloned a chalcone synthase gene (DfCHS) from Dryopteris fragrans (L.) Schott. The full-length cDNA of DfCHS is 1,737 bp, with an open reading frame (ORF) of 1,122 bp (deposited in GenBank under Accession Number KF530802) encoding a predicted protein of 373 amino acids. The calculated molecular mass of DfCHS is 41.3 kDa. We studied the expression of DfCHS and total flavonoid contents in tissue culture seedlings cultured under the low temperature at 4ºC, high temperature at 35ºC and UV conditions, respectively. The results show that the expression of DfCHS are not the same, but all present rising trends, then flavonoid contents were increased. Overall, our results imply that the expression of DfCHS gene provide a certain theory basis in the status of evolution among ferns.

Reticulate evolution in the apogamous Dryopteris varia complex (Dryopteridaceae, subg. Erythrovariae, sect. Variae) and its related sexual species in Japan.

Apogamous fern species are often difficult to distinguish from related species because of their continuous morphological variations. To clarify the genetic relationships among the members of the Dryopteris varia complex, we analyzed the nucleotide sequences of the plastid gene rbcL and the nuclear gene PgiC. We also analyzed the diploid sexual species D. caudipinna and D. chinensis, which have not been included in the complex, but were recently shown to be closely related to the complex in a molecular phylogenetic study. The PgiC sequences of the diploid sexual species, D. varia, D. saxifraga, D. sp. 'protobissetiana' (undescribed diploid sexual species), D. caudipinna, and D. chinensis, were well differentiated and hence designated A, B, C, D, and E, respectively. Thus, the PgiC constitution of apogamous species in the complex was as follows: D. bissetiana, B + C; D. kobayashii, B + C + E); D. pacifica, A + C, A + B + C, or A + C + D; D. sacrosancta, A + C + E; and D. saxifragivaria, B + C. These results suggest that these apogamous species are formed by hybridizations of species including not only the three diploid sexual species of the D. varia complex (A, B, and C) but also the two diploid sexual species D. caudipinna (D) and D. chinensis (E), which do not belong to the complex.

Unraveling reticulate evolution in North American Dryopteris (Dryopteridaceae).

BACKGROUND: The thirteen species of Dryopteris in North America have long been suspected of having undergone a complicated history of reticulate evolution via allopolyploid hybridization. Various explanations for the origins of the allopolyploid taxa have been suggested, and though most lines of evidence have supported the so-called "semicristata" hypothesis, contention over the group's history has continued in several recent, conflicting studies. RESULTS: Sequence data from nine plastid and two nuclear markers were collected from 73 accessions representing 35 species of Dryopteris. Sequences from each of the allopolyploids are most closely related to their progenitor species as predicted by the "semicristata" hypothesis. Allotetraploid D. campyloptera appears to be derived from a hybrid between diploid D. expansa and D. intermedia; D. celsa, from diploid D. ludoviciana x D. goldiana; and D. carthusiana and D. cristata, from diploid "D. semicristata" x D. intermedia and D. ludoviciana, respectively. Allohexaploid D. clintoniana appears to be derived from D. cristata x D.goldiana. The earliest estimated dates of formation of the allopolyploids, based on divergence time analyses, were within the last 6 Ma. We found no evidence for recurrent formation of any of the allopolyploids. The sexual allopolyploid taxa are derived from crosses between parents that show intermediate levels of genetic divergence relative to all pairs of potential progenitors. In addition, the four allotetraploids are transgressive with respect to geographic range relative to one or both of their parents (their ranges extend beyond those of the parents), suggesting that ecological advantages in novel habitats or regions may promote long-term regional coexistence of the hybrid taxa with their progenitors. CONCLUSIONS: This study provides the first thorough evaluation of the North American complex of woodferns using extensive sampling of taxa and genetic markers. Phylogenies produced from each of three datasets (one plastid and two nuclear) support the "semicristata" hypothesis, including the existence of a missing diploid progenitor, and allow us to reject all competing hypotheses. This study demonstrates the value of using multiple, biparentally inherited markers to evaluate reticulate complexes, assess the frequency of recurrent polyploidization, and determine the relative importance of introgression vs. hybridization in shaping the histories of such groups.

Molecular circumscription and major evolutionary lineages of the fern genus Dryopteris (Dryopteridaceae).

BACKGROUND: The fern genus Dryopteris (Dryopteridaceae) is among the most common and species rich fern genera in temperate forests in the northern hemisphere containing 225-300 species worldwide. The circumscription of Dryopteris has been controversial and various related genera have, over the time, been included in and excluded from Dryopteris. The infrageneric phylogeny has largely remained unclear, and the placement of the majority of the supraspecific taxa of Dryopteris has never been tested using molecular data. RESULTS: In this study, DNA sequences of four plastid loci (rbcL gene, rps4-trnS spacer, trnL intron, trnL-F spacer) were used to reconstruct the phylogeny of Dryopteris. A total of 122 accessions are sampled in our analysis and they represent 100 species of the expanded Dryopteris including Acrophorus, Acrorumohra, Diacalpe, Dryopsis, Nothoperanema, and Peranema. All four subgenera and 19 sections currently recognized in Dryopteris s.s. are included. One species each of Arachniodes, Leptorumohra, and Lithostegia of Dryopteridaceae are used as outgroups. Our study confirms the paraphyly of Dryopteris and provides the first strong molecular evidence on the monophyly of Acrophorus, Diacalpe, Dryopsis, Nothoperanema, and Peranema. However, all these monophyletic groups together with the paraphyletic Acrorumohra are suggested to be merged into Dryopteris based on both molecular and morphological evidence. Our analysis identified 13 well-supported monophyletic groups. Each of the 13 clades is additionally supported by morphological synapomophies and is inferred to represent a major evolutionary lineage in Dryopteris. In contrast, monophyly of the four subgenera and 15 out of 19 sections currently recognized in Dryopteris s.s is not supported by plastid data. CONCLUSIONS: The genera, Acrophorus, Acrorumohra, Diacalpe, Dryopsis, Nothoperanema, and Peranema, should all be merged into Dryopteris. Most species of these genera share a short rhizome and catadromic arrangement of frond segments, unlike the sister genus of Dryopteris s.l., Arachniodes, which has anadromic arrangement of frond segments. The non-monophyly of the 19 out of the 21 supraspecific taxa (sections, subgenera) in Dryopteris strongly suggests that the current taxonomy of this genus is in need of revision. The disagreement between the previous taxonomy and molecular results in Dryopteris may be due partly to interspecific hybridization and polyplodization. More morphological studies and molecular data, especially from the nuclear genome, are needed to thoroughly elucidate the evolutionary history of Dryopteris. The 13 well-supported clades identified based on our data represent 13 major evolutionary lineages in Dryopteris that are also supported by morphological synapomophies.

Reticulate evolution on a global scale: a nuclear phylogeny for New World Dryopteris (Dryopteridaceae).

Reticulate, or non-bifurcating, evolution is now recognized as an important phenomenon shaping the histories of many organisms. It appears to be particularly common in plants, especially in ferns, which have relatively few barriers to intra- and interspecific hybridization. Reticulate evolutionary patterns have been recognized in many fern groups, though very few have been studied rigorously using modern molecular phylogenetic techniques in order to determine the causes of the reticulate patterns. In the current study, we examine patterns of branching and reticulate evolution in the genus Dryopteris, the woodferns. The North American members of this group have long been recognized as a classic example of reticulate evolution in plants, and we extend analysis of the genus to all 30 species in the New World, as well as numerous taxa from other regions. We employ sequence data from the plastid and nuclear genomes and use maximum parsimony (MP), maximum likelihood (ML), Bayesian inference (BI), and divergence time analyses to explore the relationships of New World Dryopteris to other regions and to reconstruct the timing and events which may have led to taxa displaying reticulate rather than strictly branching histories. We find evidence for reticulation among both the North and Central/South American groups of species, and our data support a classic hypothesis for reticulate evolution via allopolyploid speciation in the North America taxa, including an extinct diploid progenitor in this group. In the Central and South American species, we find evidence of extensive reticulation involving unknown ancestors from Asia, and we reject deep coalescent processes such as incomplete lineage sorting in favor of more recent intercontinental hybridization and chloroplast capture as an explanation for the origin of the Latin American reticulate taxa.

Origin of Dryopteris shibipedis (Dryopteridaceae), a fern species extinct in the wild.

Dryopteris shibipedis was once treated as an extinct species in the Red List (2007) by the Japanese Ministry of the Environment, but 'rediscovered' in the Tsukuba Botanical Garden. To clarify its origin, using 'overlooked' cultivated stocks we analyzed nuclear PgiC intron sequences. As the PgiC genotype of D. shibipedis can be explained by a combination of alleles of D. kinkiensis and those of D. pacifica, the hypothesis of hybrid origin of the species is supported.

RNA editing restores critical domains of a group I intron in fern mitochondria.

In the leptosporangiate fern Osmunda regalis, cox1 gene is disrupted by a 1071-nucleotide-long group I intron that is homologous to the Marchantia polymorpha cox1 intron 4 (cox1i395g1). This intron, which shares 89% sequence identity with its bryophyte counterpart, lost the capacity to encode for a maturase due to insertion/deletion mutations. The cox1 coding region is interrupted by a stop codon in both exons. The cox1 transcript undergoes 58 C-to-U and 13 U-to-C conversions, including the suppression of two stop codons that result in the recovery of a functional cox1 ORF. Interestingly, 4 C-to-U conversions found in mRNA precursors showed that the O. regalis cox1i395g1 intron is efficiently edited. These modifications improved the sequence identity with the Marchantia cox1i395 intron. In particular, the RNA editing events affect regions involved in secondary and tertiary structures of the intron, restoring three base pairing in the structural P5a and P9 helices, and correcting a highly conserved U in the P7 helix that contributes to the catalytic core. Moreover, cox1 intron orthologous from three different fern species were found to be edited by both C-to-U and U-to-C conversions in P7 and P9. Thus, RNA editing helps to correct the conserved domains of group I introns in "true ferns", suggesting a possible link between editing and splicing. We present here the first experimental evidence of RNA editing concerning a group I intron in plant organelles.

Microsatellites reveal substantial among-population genetic differentiation and strong inbreeding in the relict fern Dryopteris aemula.

BACKGROUND AND AIMS: A previous study detected no allozyme diversity in Iberian populations of the buckler-fern Dryopteris aemula. The use of a more sensitive marker, such as microsatellites, was thus needed to reveal the genetic diversity, breeding system and spatial genetic structure of this species in natural populations. METHODS: Eight microsatellite loci for D. aemula were developed and their cross-amplification with other ferns was tested. Five polymorphic loci were used to characterize the amount and distribution of genetic diversity of D. aemula in three populations from the Iberian Peninsula and one population from the Azores. KEY RESULTS: Most microsatellite markers developed were transferable to taxa close to D. aemula. Overall genetic variation was low (H(T) = 0.447), but was higher in the Azorean population than in the Iberian populations of this species. Among-population genetic differentiation was high (F(ST) = 0.520). All loci strongly departed from Hardy-Weinberg equilibrium. In the population where genetic structure was studied, no spatial autocorrelation was found in any distance class. CONCLUSIONS: The higher genetic diversity observed in the Azorean population studied suggested a possible refugium in this region from which mainland Europe has been recolonized after the Pleistocene glaciations. High among-population genetic differentiation indicated restricted gene flow (i.e. lack of spore exchange) across the highly fragmented area occupied by D. aemula. The deviations from Hardy-Weinberg equilibrium reflected strong inbreeding in D. aemula, a trait rarely observed in homosporous ferns. The absence of spatial genetic structure indicated effective spore dispersal over short distances. Additionally, the cross-amplification of some D. aemula microsatellites makes them suitable for use in other Dryopteris taxa.

Differential gene expression profiling of one- and two-dimensional apogamous gametophytes of the fern Dryopteris affinis ssp. affinis.

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction.

Arg-type dihydroflavonol 4-reductase genes from the fern Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins.

Dihydroflavonol 4-reductase (DFR), a key enzyme involved in the biosynthesis of anthocyanins, has been cloned from various species. However, little research has been conducted on this enzyme in ferns, which occupy a unique evolutionary position. In this study, we isolated two novel DFR genes from the fern Dryopteris erythrosora. In vitro enzymatic analysis revealed that DeDFR1 and DeDFR2 enzymes can catalyze dihydrokaempferol and dihydroquercetin but cannot catalyze dihydromyricetin. Amino acid sequence analysis showed that DeDFR1 and DeDFR2 have an arginine at the same substrate-specificity-determining site as that in the ferns Salvinia cucullata and Azolla filiculoides. Thus, we speculate that the Arg-type DFR is a new DFR functional type. To further verify the substrate preferences of the Arg-type DFR, an amino acid substitution assay was conducted. When N133 was mutated to R133, Arabidopsis DFR protein completely lost its catalytic activity for dihydromyricetin, as observed for DeDFR1 and DeDFR2. Additionally, heterologous expression of DeDFR2 in the Arabidopsis tt3-1 mutant resulted in increasing anthocyanin accumulation. In summary, DeDFR1 and DeDFR2 are considered to be a new type of DFR with unique structures and functions. The discovery of the Arg-type DFR provides new insights into the anthocyanin biosynthesis pathway in ferns.