Extraordinary preservation of gene collinearity over three hundred million years revealed in homosporous lycophytes.

Homosporous lycophytes (Lycopodiaceae) are a deeply diverged lineage in the plant tree of life, having split from heterosporous lycophytes (Selaginella and Isoetes) ~400 Mya. Compared to the heterosporous lineage, Lycopodiaceae has markedly larger genome sizes and remains the last major plant clade for which no chromosome-level assembly has been available. Here, we present chromosomal genome assemblies for two homosporous lycophyte species, the allotetraploid Huperzia asiatica and the diploid Diphasiastrum complanatum. Remarkably, despite that the two species diverged ~350 Mya, around 30% of the genes are still in syntenic blocks. Furthermore, both genomes had undergone independent whole genome duplications, and the resulting intragenomic syntenies have likewise been preserved relatively well. Such slow genome evolution over deep time is in stark contrast to heterosporous lycophytes and is correlated with a decelerated rate of nucleotide substitution. Together, the genomes of H. asiatica and D. complanatum not only fill a crucial gap in the plant genomic landscape but also highlight a potentially meaningful genomic contrast between homosporous and heterosporous species.

Systematics and evolutionary dynamics of insect-fern interactions in the specialized fern-spore feeding Cuprininae (Lepidoptera, Stathmopodidae).

Fern-spore-feeding (FSF) is rare and found in only four families of Lepidoptera. Stathmopodidae is the most speciose family that contains FSF species, and its subfamily Cuprininae exclusively specializes on FSF. However, three species of Stathmopodinae also specialize on FSF. To better understand the evolutionary history of FSF and, more generally, the significance of specialization on a peculiar host, a phylogenetic and taxonomic revision for this group is necessary. We reconstructed the most comprehensive molecular phylogeny, including one mitochondrial and four nuclear genes, of Stathmopodidae to date, including 137 samples representing 62 species, with a particular focus on the FSF subfamily, Cuprininae, including 33 species (41% of named species) from 6 of the 7 Cuprininae genera. Species from two other subfamilies, Stathmopodinae and Atkinsoniinae, were also included. We found that FSF evolved only once in Stathmopodidae and that the previous hypothesis of multiple origins of FSF was misled by inadequate taxonomy. Moreover, we showed that (1) speciation/extinction rates do not differ significantly between FSF and non-FSF groups and that (2) oligophage is the ancestral character state in Cuprininae. We further revealed that a faster rate of accumulating specialists over time, and thus a higher number of specialists, was achieved by a higher transition rate from oligophagages to specialists compared to the transition rate in the opposite direction. We finish by describing three new genera, Trigonodagen. nov., Petalagen. nov., and Pediformisgen. nov., and revalidating five genera: Cuprina, Calicotis, Thylacosceles, Actinoscelis, Thylacosceloides in Cuprininae, and we provide an updated taxonomic key to genera and a revised global checklist of Cuprininae.

Prevalent arbuscular mycorrhizae in roots and highly variable mycobiome in leaves of epiphytic subtropical fern Ophioderma pendulum.

PREMISE: Endophytic and mycorrhizal fungi are crucial in facilitating plant nutrition acquisition and stress tolerance. In epiphytic habitats, plants face nutrition and water stress, but their roots are mostly nonmycorrhizal and especially lacking in arbuscular mycorrhizal associations. Ophioderma pendulum is an epiphytic fern with a partially mycoheterotrophic lifestyle, likely heavily reliant on symbiotic fungi. To characterize fungal associations in the sporophyte of O. pendulum, we focused on leaves and roots of O. pendulum, seeking to reveal the fungal communities in these organs. METHODS: Roots and leaves from O. pendulum in a subtropical forest were examined microscopically to observe the morphology of fungal structures and determine the percentage of various fungal structures in host tissues. Fungal composition was profiled using metabarcoding techniques that targeted ITS2 of the nuclear ribosomal DNA. RESULTS: Roots were consistently colonized by arbuscular mycorrhizal fungi (Glomeromycota), especially Acaulospora. Unlike previous findings on epiphytic ferns, dark septate endophytes were rare in O. pendulum roots. Leaves were predominantly colonized by Ascomycota fungi, specifically the classes Dothideomycetes (46.88%), Eurotiomycetes (11.51%), Sordariomycetes (6.23%), and Leotiomycetes (6.14%). Across sampling sites, fungal community compositions were similar in the roots but differed significantly in the leaves. CONCLUSIONS: Ophioderma pendulum maintains stable, single-taxon-dominant communities in the roots, primarily featuring arbuscular mycorrhizal fungi, whereas the leaves may harbor opportunistic fungal colonizers. Our study underlines the significance of mycorrhizal fungi in the adaptation of epiphytic ferns.

Plastome phylogenomic analysis reveals evolutionary divergences of Polypodiales suborder Dennstaedtiineae.

BACKGROUND: Polypodiales suborder Dennstaedtiineae contain a single family Dennstaedtiaceae, eleven genera, and about 270 species, and include some groups that were previously placed in Dennstaedtiaceae, Hypolepidaceae, Monachosoraceae, and Pteridaceae. The classification and phylogenetic relationships among these eleven genera have been poorly understood. To explore the deep relationships within suborder Dennstaedtiineae and estimate the early diversification of this morphologically heterogeneous group, we analyzed complete plastomes of 57 samples representing all eleven genera of suborder Dennstaedtiineae using maximum likelihood and Bayesian inference. RESULTS: The phylogenetic relationships of all the lineages in the bracken fern family Dennstaedtiaceae were well resolved with strong support values. All six genera of Hypolepidoideae were recovered as forming a monophyletic group with full support, and Pteridium was fully supported as sister to all the other genera in Hypolepidoideae. Dennstaedtioideae (Dennstaedtia s.l.) fell into four clades with full support: the Microlepia clade, the northern Dennstaedtia clade, the Dennstaedtia globulifera clade, and the Dennstaedtia s.s. clade. Monachosorum was strongly resolved as sister to all the remaining genera of suborder Dennstaedtiineae. Based on the well resolved relationships among genera, the divergence between Monachosorum and other groups of suborder Dennstaedtiineae was estimated to have occurred in the Early Cretaceous, and all extant genera (and clades) in Dennstaedtiineae, were inferred to have diversified since the Late Oligocene. CONCLUSION: This study supports reinstating a previously published family Monachosoraceae as a segregate from Dennstaedtiaceae, based on unique morphological evidence, the shady habitat, and the deep evolutionary divergence from its closest relatives.

Simultaneous diversification of Polypodiales and angiosperms in the Mesozoic.

Comprising about 82% of the extant fern species diversity, Polypodiales are generally believed to have diversified in the Late Cretaceous. We estimated the divergence times of Polypodiales using both penalized likelihood and Bayesian methods, based on a dataset consisting of 208 plastomes representing all 28 families and 14 fossil constraints reflecting current interpretations of fossil record. Our plastome phylogeny recovered the same six major lineages as a recent nuclear phylogeny, but the position of Dennstaedtiineae was different. The present phylogeny showed high resolution of relationships among the families of Polypodiales, especially among those forming the Aspleniineae. The divergence time estimates supported the most recent common ancestor of Polypodiales and its closest relative dating back to the Triassic, establishment of the major lineages in the Jurassic, and a likely accelerated radiation during the late Jurassic and the Early Cretaceous. The estimated divergence patterns of Polypodiales and angiosperms converge to a scenario in which their main lineages were established simultaneously shortly before the onset of the Cretaceous Terrestrial Revolution, and further suggest a pre-Cretaceous hidden history for both lineages. The pattern of simultaneous diversifications shown here elucidate an important gap in our understanding of the Terrestrial Revolution that shaped today's ecosystems.

A global phylogeny of Stegnogramma ferns (Thelypteridaceae): generic and sectional revision, historical biogeography and evolution of leaf architecture.

The thelypteroid fern genus Stegnogramma s.l. contains around 18-35 species and has a global, cross-continental distribution ranging from tropical to temperate regions. Several genera and infrageneric sections have been recognized previously in Stegnogramma s.l., but their phylogenetic relationships are still unclear. In this study, we present a global phylogeny of Stegnogramma s.l. with the most comprehensive sampling to date and aim to pinpoint the phylogenetic positions of biogeographically and taxonomically important taxa. Based on the reconstructed historical biogeography and character evolution, we propose a new (infra)generic classification and discuss the diversification of Stegnogramma s.l. in a biogeographical context. New names or combinations are made for 12 (infra)species, including transferring the monotypic species of Craspedosorus to Leptogramma. Finally, we discuss a possible link between leaf architecture and ecological adaptation, and hypothesize that the increase in leaf dissection and free-vein proportion is an adaptive feature to cool climates in Stegnogramma s.l.

A well-resolved fern nuclear phylogeny reveals the evolution history of numerous transcription factor families.

Ferns account for 80% of nonflowering vascular plant species and are the sister lineage of seed plants. Recent molecular phylogenetics have greatly advanced understanding of fern tree of life, but relationships among some major lineages remain unclear. To better resolve the phylogenetic relationships of ferns, we generated transcriptomes from 125 ferns and two lycophytes, with three additional public datasets, to represent all 11 orders and 85% of families of ferns. Our nuclear phylogeny provides strong supports for the monophyly of all four subclasses and nearly all orders and families, and for relationships among these lineages. The only exception is Gleicheniales, which was highly supported as being paraphyletic with Dipteridaceae sister to a clade with Gleicheniaceae + Hymenophyllales. In addition, new and strongly supported phylogenetic relationships are found for suborders and families in Polypodiales. We provide the first dated fern phylogenomic tree using many nuclear genes from a large majority of families, with an estimate for separation of the ancestors of ferns and seed plants in early Devonian at ∼400 Mya and subsequent gradual divergences of fern orders from ∼380 to 200 Mya. Moreover, the newly obtained fern phylogeny provides a framework for gene family analyses, which indicate that the vast majority of transcription factor families found in seed plants were already present in the common ancestor of extant vascular plants. In addition, fern transcription factor genes show similar duplication patterns to those in seed plants, with some showing stable copy number and others displaying independent expansions in both ferns and seed plants. This study provides a robust phylogenetic and gene family evolution framework, as well as rich molecular resources for understanding the morphological and functional evolution in ferns.

Morphological characterization of infra-generic lineages in Deparia (Athyriaceae: Polypodiales).

Deparia, including the previously recognized genera Lunathyrium, Dryoathyrium (=Parathyrium), Athyriopsis, Triblemma, and Dictyodroma, is a fern genus comprising about 70 species in Athyriaceae. In this study, we inferred a robust Deparia phylogeny based on a comprehensive taxon sampling (~81% of species) that captures the morphological diversity displayed in the genus. All Deparia species formed a highly supported monophyletic group. Within Deparia, seven major clades were identified, and most of them were characterized by inferring synapomorphies using 14 morphological characters including leaf architecture, petiole base, rhizome type, soral characters, spore perine, and leaf indument. These results provided the morphological basis for an infra-generic taxonomic revision of Deparia.

Order-level fern plastome phylogenomics: new insights from Hymenophyllales.

PREMISE OF THE STUDY: Filmy ferns (Hymenophyllales) are a highly specialized lineage, having mesophyll one-cell layer thick and inhabiting particularly shaded and humid environments. The phylogenetic placement of Hymenophyllales has been inconclusive, and while over 87 whole fern plastomes have been published, none was from Hymenophyllales. To better understand the evolutionary history of filmy ferns, we sequenced the first complete plastome for this order. METHODS: We compiled a phylogenomic plastome data set encompassing all 11 fern orders, and reconstructed phylogenies using different data types (nucleotides, codons, and amino acids) and partition schemes (codon positions and loci). To infer the evolution of fern plastome organization, we coded plastome features, including inversions, inverted repeat boundary shifts, gene losses, and tRNA anticodon sequences as characters, and reconstructed the ancestral states for these characters. KEY RESULTS: We discovered a suite of novel, Hymenophyllales-specific plastome structures that likely resulted from repeated expansions and contractions of the inverted repeat regions. Our phylogenetic analyses reveal that Hymenophyllales is highly supported as either sister to Gleicheniales or to Gleicheniales + the remaining non-Osmundales leptosporangiates, depending on the data type and partition scheme. CONCLUSIONS: Although our analyses could not confidently resolve the phylogenetic position of Hymenophyalles, the results here highlight the danger of drawing conclusions from "all-in" phylogenomic data set without exploring potential inconsistencies in the data. Finally, our first order-level reconstruction of fern plastome structural evolution provides a useful framework for future plastome research.

A novel chloroplast gene reported for flagellate plants.

PREMISE OF THE STUDY: Gene space in plant plastid genomes is well characterized and annotated, yet we discovered an unrecognized open reading frame (ORF) in the fern lineage that is conserved across flagellate plants. METHODS: We initially detected a putative uncharacterized ORF by the existence of a highly conserved region between rps16 and matK in a series of matK alignments of leptosporangiate ferns. We mined available plastid genomes for this ORF, which we now refer to as ycf94, to infer evolutionary selection pressures and assist in functional prediction. To further examine the transcription of ycf94, we assembled the plastid genome and sequenced the transcriptome of the leptosporangiate fern Adiantum shastense Huiet & A.R. Sm. KEY RESULTS: The ycf94 predicted protein has a distinct transmembrane domain but with no sequence homology to other proteins with known function. The nonsynonymous/synonymous substitution rate ratio of ycf94 is on par with other fern plastid protein-encoding genes, and additional homologs can be found in a few lycophyte, moss, hornwort, and liverwort plastid genomes. Homologs of ycf94 were not found in seed plants. In addition, we report a high level of RNA editing for ycf94 transcripts-a hallmark of protein-coding genes in fern plastomes. CONCLUSIONS: The degree of sequence conservation, together with the presence of a distinct transmembrane domain and RNA-editing sites, suggests that ycf94 is a protein-coding gene of functional significance in ferns and, potentially, bryophytes and lycophytes. However, the origin and exact function of this gene require further investigation.

Evaluating the spore genome sizes of ferns and lycophytes: a flow cytometry approach.

Ferns and lycophytes produce spores to initiate the gametophyte stage for sexual reproduction. Approximately 10% of these seedless vascular plants are apomictic, and produce genomic unreduced spores. Genome size comparisons between spores and leaves are a reliable, and potentially easier way to determine their reproductive mode compared to traditional approaches. However, estimation of the spore genome sizes of these plants has not been attempted. We attempted to evaluate the spore genome sizes of ferns and lycophytes using flow cytometry, collected spores from selected species representing different spore physical properties and taxonomic groups, and sought to optimize bead-vortexing conditions. By evaluating the spore and sporophyte genome sizes, we examined whether reproductive modes could be ascertained from these flow cytometry results. We proposed two separate sets of optimized bead-vortexing conditions for the nuclear extraction of green and nongreen spores. We further successfully extracted spore nuclei of 19 families covering most orders, and the qualities and quantities of these extractions satisfied the C-value criteria. These evaluated genome sizes further supported the reproductive modes reported previously. In the current study, flow cytometry was used for the first time to evaluate the spore genome sizes of ferns and lycophytes. This use of spore flow cytometry provides a new, efficient approach to ascertaining the reproductive modes of these plants.

Detection of chlorophylls in spores of seven ferns.

Fern spores were traditionally classified into chlorophyllous (green) and nonchlorophyllous (nongreen) types based on the color visible to the naked eye. Recently, a third type, "cryptochlorophyllous spores", is recognized, and these spores are nongreen under white light but contain chlorophylls. Epifluorescence microscopy was previously used to detect chlorophylls in cryptochlorophyllous spores. In addition to epifluorescence microscopy, current study performed some other approaches, including spore-squash epifluorescence, absorption spectra, laser-induced fluorescence emission spectra, thin layer chromatography (TLC), and ultra-high performance liquid chromatography with ultraviolet and mass spectrometric detection (UHPLC-UV-MS) in order to detect chlorophylls of spores of seven ferns (Sphaeropteris lepifera, Ceratopteris thalictroides, Leptochilus wrightii, Leptochilus pothifolius, Lepidomicrosorum buergerianum, Osmunda banksiifolia, and Platycerium grande). Destructive methods, such as TLC and UHPLC-UV-MS, successfully detected chlorophylls inside the spores when their signals of red fluorescence under epifluorescence microscope were masked by spore wall. Although UHPLC-UV-MS analysis was the most sensitive and reliable for determining the chlorophylls of spores, spore-squash epifluorescence is not only reliable but also cost- and time-effective one among our study methods. In addition, we first confirmed that Lepidomicrosorium buergerianum, Leptochilus pothifolius, Leptochilus wrightii, and Platycerium grande, produce cryptochlorophyllous spores.

Not only in the temperate zone: independent gametophytes of two vittarioid ferns (Pteridaceae, Polypodiales) in East Asian subtropics.

Independent gametophyte ferns are unique among vascular plants because they are sporophyteless and reproduce asexually to maintain their populations in the gametophyte generation. Such ferns had been primarily discovered in temperate zone, and usually hypothesized with (sub)tropical origins and subsequent extinction of sporophyte due to climate change during glaciations. Presumably, independent fern gametophytes are unlikely to be distributed in tropics and subtropics because of relatively stable climates which are less affected by glaciations. Nonetheless, the current study presents cases of two independent gametophyte fern species in subtropic East Asia. In this study, we applied plastid DNA sequences (trnL-L-F and matK + ndhF + chlL datasets) and comprehensive sampling (~80%) of congeneric species for molecular identification and divergence time estimation of these independent fern gametophytes. The two independent gametophyte ferns were found belonging to genus Haplopteris (vittarioids, Pteridaceae) and no genetic identical sporophyte species in East Asia. For one species, divergence times between its populations imply recent oversea dispersal(s) by spores occurred during Pleistocene. By examining their ex situ and in situ fertility, prezygotic sterility was found in these two Haplopteris, in which gametangia were not or very seldom observed, and this prezygotic sterility might attribute to their lacks of functional sporophytes. Our field observation and survey on their habitats suggest microhabitat conditions might attribute to this prezygotic sterility. These findings point to consideration of whether recent climate change during the Pleistocene glaciation resulted in ecophysiological maladaptation of non-temperate independent gametophyte ferns. In addition, we provided a new definition to classify fern gametophyte independences at the population level. We expect that continued investigations into tropical and subtropical fern gametophyte floras will further illustrate the biogeographic significance of non-temperate fern gametophyte independence.

Historical biogeography of the fern genus Deparia (Athyriaceae) and its relation with polyploidy.

The wide geographical distribution of many fern species is related to their high dispersal ability. However, very limited studies surveyed biological traits that could contribute to colonization success after dispersal. In this study, we applied phylogenetic approaches to infer historical biogeography of the fern genus Deparia (Athyriaceae, Eupolypods II). Because polyploids are suggested to have better colonization abilities and are abundant in Deparia, we also examined whether polyploidy could be correlated to long-distance dispersal events and whether polyploidy could play a role in these dispersals/establishment and range expansion. Maximum likelihood and Bayesian phylogenetic reconstructions were based on a four-region combined cpDNA dataset (rps16-matK IGS, trnL-L-F, matK and rbcL; a total of 4252 characters) generated from 50 ingroup (ca. 80% of the species diversity) and 13 outgroup taxa. Using the same sequence alignment and maximum likelihood trees, we carried out molecular dating analyses. The resulting chronogram was used to reconstruct ancestral distribution using the DEC model and ancestral ploidy level using ChromEvol. We found that Deparia originated around 27.7Ma in continental Asia/East Asia. A vicariant speciation might account for the disjunctive distribution of East Asia-northeast North America. There were multiple independent long-distance dispersals to Africa/Madagascar (at least once), Southeast Asia (at least once), south Pacific islands (at least twice), Australia/New Guinea/New Zealand (at least once), and the Hawaiian Islands (at least once). In particular, the long-distance dispersal to the Hawaiian Islands was associated with polyploidization, and the dispersal rate was slightly higher in the polyploids than in diploids. Moreover, we found five species showing recent infraspecific range expansions, all of which took place concurrently with polyploidization. In conclusion, our study provides the first investigation using phylogenetic and biogeographic analyses trying to explore the link between historical biogeography and ploidy evolution in a fern genus and our results imply that polyploids might be better colonizers than diploids.

Overcoming deep roots, fast rates, and short internodes to resolve the ancient rapid radiation of eupolypod II ferns.

Backbone relationships within the large eupolypod II clade, which includes nearly a third of extant fern species, have resisted elucidation by both molecular and morphological data. Earlier studies suggest that much of the phylogenetic intractability of this group is due to three factors: (i) a long root that reduces apparent levels of support in the ingroup; (ii) long ingroup branches subtended by a series of very short backbone internodes (the "ancient rapid radiation" model); and (iii) significantly heterogeneous lineage-specific rates of substitution. To resolve the eupolypod II phylogeny, with a particular emphasis on the backbone internodes, we assembled a data set of five plastid loci (atpA, atpB, matK, rbcL, and trnG-R) from a sample of 81 accessions selected to capture the deepest divergences in the clade. We then evaluated our phylogenetic hypothesis against potential confounding factors, including those induced by rooting, ancient rapid radiation, rate heterogeneity, and the Bayesian star-tree paradox artifact. While the strong support we inferred for the backbone relationships proved robust to these potential problems, their investigation revealed unexpected model-mediated impacts of outgroup composition, divergent effects of methods for countering the star-tree paradox artifact, and gave no support to concerns about the applicability of the unrooted model to data sets with heterogeneous lineage-specific rates of substitution. This study is among few to investigate these factors with empirical data, and the first to compare the performance of the two primary methods for overcoming the Bayesian star-tree paradox artifact. Among the significant phylogenetic results is the near-complete support along the eupolypod II backbone, the demonstrated paraphyly of Woodsiaceae as currently circumscribed, and the well-supported placement of the enigmatic genera Homalosorus, Diplaziopsis, and Woodsia.

trnL-F is a powerful marker for DNA identification of field vittarioid gametophytes (Pteridaceae).

BACKGROUND AND AIMS: The gametophyte phase of ferns plays an important role in habitat selection, dispersal, adaptation and evolution. However, ecological studies on fern gametophytes have been impeded due to the difficulty of species identification of free-living gametophytes. DNA barcoding provides an alternative approach to identifying fern gametophytes but is rarely applied to field studies. In this study, an example of field vittarioid gametophyte identification using DNA barcoding, which has not been done before, is given. METHODS: A combination of distance-based and tree-based approaches was performed to evaluate the discriminating power of three candidate barcodes (matK, rbcL and trnL-F) on 16 vittarioid sporophytes. Sequences of the trnL-F region were generated from 15 fern gametophyte populations by tissue-direct PCR and were compared against the sporophyte dataset, using BLAST. KEY RESULTS: trnL-F earns highest primer universality and discriminatory ability scores, whereas PCR success rates were very low for matK and rbcL regions (10·8 % and 41·3 %, respectively). BLAST analyses showed that all the sampled field gametophytes could be successfully identified to species level. Three gametophyte populations were also discovered to be living beyond the known occurrence of their sporophyte counterparts. CONCLUSIONS: This study demonstrates that DNA barcoding (i.e. reference databasing, tissue-direct PCR and molecular analysis), especially the trnL-F region, is an efficient tool to identify field gametophytes, and has considerable potential in exploring the ecology of fern gametophytes.

Modified CTAB protocols for high-molecular-weight DNA extractions from ferns.

Premise: Efficient protocols for extracting high-molecular-weight (HMW) DNA from ferns facilitate the long-read sequencing of their large and complex genomes. Here, we perform two cetyltrimethylammonium bromide (CTAB)-based protocols to extract HMW DNA and evaluate their applicability in diverse fern taxa for the first time. Methods and Results: We describe two modified CTAB protocols, with key adjustments to minimize mechanical disruption during lysis to prevent DNA shearing. One of these protocols uses a small amount of fresh tissue but yields a considerable quantity of HMW DNA with high efficiency. The other accommodates a large amount of input tissue, adopts an initial step of nuclei isolation, and thus ensures a high yield in a short period of time. Both methods were proven to be robust and effective in obtaining HMW DNA from diverse fern lineages, including 33 species in 19 families. The DNA extractions mostly had high DNA integrity, with mean sizes larger than 50 kbp, as well as high purity (A260/A230 and A260/A280 > 1.8). Conclusions: This study provides HMW DNA extraction protocols for ferns in the hope of facilitating further attempts to sequence their genomes, which will bridge our genomic understanding of land plant diversity.

Organellar phylogenomics of Ophioglossaceae fern genera.

Previous phylogenies showed conflicting relationships among the subfamilies and genera within the fern family Ophioglossaceae. However, their classification remains unsettled where contrasting classifications recognize four to 15 genera. Since these treatments are mostly based on phylogenetic evidence using limited, plastid-only loci, a phylogenomic understanding is actually necessary to provide conclusive insight into the systematics of the genera. In this study, we have therefore compiled datasets with the broadest sampling of Ophioglossaceae genera to date, including all fifteen currently recognized genera, especially for the first time the South African endemic genus Rhizoglossum. Notably, our comprehensive phylogenomic matrix is based on both plastome and mitogenome genes. Inferred from the coding sequences of 83 plastid and 37 mitochondrial genes, a strongly supported topology for these subfamilies is presented, and is established by analyses using different partitioning approaches and substitution models. At the generic level, most relationships are well resolved except for few within the subfamily Ophioglossoideae. With this new phylogenomic scheme, key morphological and genomic changes were further identified along this backbone. In addition, we confirmed numerous horizontally transferred (HGT) genes in the genera Botrypus, Helminthostachys, Mankyua, Sahashia, and Sceptridium. These HGT genes are most likely located in mitogenomes and are predominately donated from angiosperm Santalales or non-Ophioglossaceae ferns. By our in-depth searches of the organellar genomes, we also provided phylogenetic overviews for the plastid and mitochondrial MORFFO genes found in these Ophioglossaceae ferns.

Development of PCR primer sets for intron 1 of the low-copy gene LEAFY in Davalliaceae.

PREMISE OF THE STUDY: Primers were designed for amplifying intron 1 of the single-copy nuclear LEAFY gene for species of Davalliaceae. METHODS AND RESULTS: New primer sets were designed and successfully amplified for intron 1 of the LEAFY gene in 13 species representing the five genera of Davalliaceae. The orthology of these sequences was further confirmed by phylogenetic analyses. Site variation in LEAFY intron 1 sequences across genera of the Davalliaceae and among accessions of the Humata repens complex were 18% and 8%, respectively. Such variation was greater than that for the cpDNA atpB-rbcL intergenic spacer region across the same taxa and accessions. CONCLUSIONS: Using our newly designed primers, intron 1 of the LEAFY gene could be amplified for all species tested. In addition, this single-copy, biparentally inherited, and quickly evolving region showed considerable potential for addressing infraspecific-level questions.