First Fossil Record of Trichomanes sensu lato (Hymenophyllaceae) from the Mid-Cretaceous Kachin Amber, Myanmar.

Hymenophyllaceae (filmy ferns), with ca. 430 species, are the most species-rich family of early diverging leptosporangiate ferns but have a poor fossil record dating back to the Late Triassic period. Traditionally, Hymenophyllaceae comprise two species-rich genera or clades: Hymenophyllum (hymenophylloids) and Trichomanes sensu lato (s.l.) (trichomanoids). Unequivocal fossils of Hymenophyllum have been reported from the Early Cretaceous of central Mongolia and the early Eocene of Okanogan Highlands, Washington, USA. However, despite being a highly diversified lineage with an estimated 184 extant species, Trichomanes s.l. lack a definitive fossil record, which severely affects the reliability of the molecular dating of this group. Here, we report the first unequivocal fossil record of Trichomanes s.l. as T. angustum comb. nov. on the basis of fertile material with tubular involucres and long exserted receptacles from the mid-Cretaceous Kachin amber, Myanmar. This species was previously tentatively assigned to Hymenophyllites due to a lack of fertile evidence. Inferred to be an epiphytic fern, T. angustum further enriches the species diversity of the epiphytic palaeocommunities in the mid-Cretaceous Kachin amber, which are mainly composed of Porellalean leafy liverworts and Dicranalean and Hypnodendralean mosses. Fossil records indicate that Hymenophyllaceae probably originated in the tropical Pangea at the latest in the Triassic when all continents were coalesced into a single landmass and had already accumulated some notable diversity in low-middle latitude areas of Laurasia by the mid-Cretaceous period.

Evolution of genome space occupation in ferns: linking genome diversity and species richness.

BACKGROUND AND AIMS: The dynamics of genome evolution caused by whole genome duplications and other processes are hypothesized to shape the diversification of plants and thus contribute to the astonishing variation in species richness among the main lineages of land plants. Ferns, the second most species-rich lineage of land plants, are highly suitable to test this hypothesis because of several unique features that distinguish fern genomes from those of seed plants. In this study, we tested the hypothesis that genome diversity and disparity shape fern species diversity by recording several parameters related to genome size and chromosome number. METHODS: We conducted de novo measurement of DNA C-values across the fern phylogeny to reconstruct the phylogenetic history of the genome space occupation in ferns by integrating genomic parameters such as genome size, chromosome number and average DNA amount per chromosome into a time-scaled phylogenetic framework. Using phylogenetic generalized least square methods, we determined correlations between chromosome number and genome size, species diversity and evolutionary rates of their transformation. KEY RESULTS: The measurements of DNA C-values for 233 species more than doubled the taxon coverage from ~2.2 % in previous studies to 5.3 % of extant diversity. The dataset not only documented substantial differences in the accumulation of genomic diversity and disparity among the major lineages of ferns but also supported the predicted correlation between species diversity and the dynamics of genome evolution. CONCLUSIONS: Our results demonstrated substantial genome disparity among different groups of ferns and supported the prediction that alterations of reproductive modes alter trends of genome evolution. Finally, we recovered evidence for a close link between the dynamics of genome evolution and species diversity in ferns for the first time.

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.

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.

Establishment of an allotetraploid fern species, Lepisorus yamaokae Seriz., between two highly niche-differentiated parental species.

PREMISE: The successful establishment of polyploid species is hypothesized to be promoted by niche differentiation from the parental species or by range shifts during climate oscillations. However, few studies have considered both of these factors simultaneously. We resolved the origin of a tetraploid fern, Lepisorus yamaokae, and explored a pattern of niche differentiation among the allotetraploid and parental species in past and current climates. METHODS: We reconstructed phylogenetic trees based on plastid marker and single-copy nuclear genes to resolve the allopolyploid origin of L. yamaokae. We also evaluated climatic niche differentiation among L. yamaokae and its two parental species using species distribution models in geographic space and principal component analysis. RESULTS: We infer that L. yamaokae had a single allotetraploid origin from L. annuifrons and L. uchiyamae. Climatic niche analyses show that the parental species currently occupy different niche spaces. The predicted distribution of the parental species at the Last Glacial Maximum (LGM) suggests more opportunities for hybridization during the LGM or during other recent temporary range shifts. Lepisorus yamaokae has a narrower niche than the additive niche of the parental species. We also observed niche conservatism in L. yamaokae. CONCLUSIONS: Range shifts of the parental species during climatic oscillations in the Quaternary likely facilitated the formation and establishment of L. yamaokae. Further, the genetic intermediacy of L. yamaokae may have enabled a niche shift in its microenvironment, resulting in its successful establishment without a macroclimatic niche shift in L. yamaokae.

Genomic insights into genetic diploidization in the homosporous fern Adiantum nelumboides.

Whole genome duplication has been recognized as a major process in speciation of land plants, especially in ferns. Whereas genome downsizing contributes greatly to the post-genome shock responses of polyploid flowering plants, diploidization of polyploid ferns diverges by maintaining most of the duplicated DNA and is thus expected to be dominated by genic processes. As a consequence, fern genomes provide excellent opportunities to study ecological speciation enforced by expansion of protein families via polyploidy. To test the key predictions of this hypothesis, we reported the de novo genome sequence of Adiantum nelumboides, a tetraploid homosporous fern. The obtained draft genome had a size of 6.27 Gb assembled into 11,767 scaffolds with the contig N50 of 1.37 Mb. Repetitive DNA sequences contributed with about 81.7%, a remarkably high proportion of the genome. With 69,568 the number of predicted protein-coding genes exceeded those reported in most other land plant genomes. Intragenomic synteny analyses recovered 443 blocks with the average block size of 1.29 Mb and the average gene content of 16 genes. The results are consistent with the hypothesis of high ancestral chromosome number, lack of substantial genome downsizing, and dominance of genic diploidization. As expected in the calciphilous plants, a notable number of detected genes were involved in calcium uptake and transport. In summary, the genome sequence of a tetraploid homosporous fern not only provides access to a genomic resource of a derived fern, but also supports the hypothesis of maintenance of high chromosome numbers and duplicated DNA in young polyploid ferns.

Reticulate evolution in the Pteris fauriei group (Pteridaceae).

The Pteris fauriei group (Pteridaceae) has a wide distribution in Eastern Asia and includes 18 species with similar but varied morphology. We collected more than 300 specimens of the P. fauriei group and determined ploidy by flow cytometry and inferred phylogenies by molecular analyses of chloroplast and nuclear DNA markers. Our results reveal a complicated reticulate evolution, consisting of seven parental taxa and 58 hybrids. The large number of hybrid taxa have added significant morphological complexity to the group leading to difficult taxonomic issues. The hybrids generally had broader ranges and more populations than their parental taxa. Genetic combination of different pairs of parental species created divergent phenotypes of hybrids, exhibited by both morphological characteristics and ecological fidelities. Niche novelty could facilitate hybrid speciation. Apogamy is common in this group and potentially contributes to the sustainability of the whole group. We propose that frequent hybridizations among members of the P. fauriei group generate and maintain genetic diversity, via novel genetic combinations, niche differentiation, and apogamy.

Spatial phylogenetics of Japanese ferns: Patterns, processes, and implications for conservation.

PREMISE: Biodiversity is often only measured with species richness; however, this metric ignores evolutionary history and is not sufficient for making conservation decisions. Here, we characterize multiple facets and drivers of biodiversity to understand how these relate to bioregions and conservation status in the ferns of Japan. METHODS: We compiled a community data set of 1239 grid cells (20 × 20 km each) including 672 taxa based on >300,000 specimen records. We combined the community data with a phylogeny and functional traits to analyze taxonomic, phylogenetic, and functional diversity and modeled biodiversity metrics in response to environmental factors and reproductive mode. Hierarchical clustering was used to delimit bioregions. Conservation status and threats were assessed by comparing the overlap of significantly diverse grid cells with conservation zones and range maps of native Japanese deer. RESULTS: Taxonomic richness was highest at mid-latitudes. Phylogenetic and functional diversity and phylogenetic endemism were highest in small southern islands. Relative phylogenetic and functional diversity were high at high and low latitudes, and low at mid-latitudes. Grid cells were grouped into three (phylogenetic) or four (taxonomic) major bioregions. Temperature and apomixis were identified as drivers of biodiversity patterns. Conservation status was generally high for grid cells with significantly high biodiversity, but the threat due to herbivory by deer was greater for taxonomic richness than other metrics. CONCLUSIONS: Our integrative approach reveals previously undetected patterns and drivers of biodiversity in the ferns of Japan. Future conservation efforts should recognize that threats can vary by biodiversity metric and consider multiple metrics when establishing conservation priorities.

A dormant resource for genome size estimation in ferns: C-value inference of the Ophioglossaceae using herbarium specimen spores.

PREMISE: The great variation of genome size (C-value) across land plants is linked to various adaptative features. Flow cytometry (FCM), the standard approach to estimating C-values, relies mostly on fresh materials, performing poorly when used with herbarium materials. No fern C-value reports have been derived from herbarium specimens; however, the herbarium spores of some ferns remain highly viable for decades and are thus promising for further investigation. To explore this possibility, we evaluated herbarium spore collections of Ophioglossaceae ferns using FCM. METHODS: Flow cytometry was conducted on 24 spore samples, representing eight of the 12 genera of the Ophioglossaceae, using specimens ranging in age from 2.6 to 111 years obtained from five herbaria. RESULTS: Regardless of the genus or the source herbarium, high-quality C-value data were generated from 17 samples, with the oldest being 26 years old. Estimates of the C-values from sporophytic tissues of known ploidy did not reveal any evidence of apomixis for the species surveyed here. We also detected a pronounced genome downsizing in Sceptridium polyploids. DISCUSSION: The recent success of FCM for C-value estimation using spores provides a much more convenient method of utilizing "dry" refrigerated materials. We demonstrate here that herbarium spores of some ferns are also promising for this use, even for older specimens.

Mating system evolution and genetic structure of diploid sexual populations of Cyrtomium falcatum in Japan.

Evolution of mating systems has become one of the most important research areas in evolutionary biology. Cyrtomium falcatum is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of C. falcatum are recognized: subsp. littorale and subsp. australe. Subspecies littorale shows intermediate selfing rates, while subsp. australe is an obligate outcrosser. We aimed to evaluate the process of mating system evolution and divergence for the two subspecies using restriction site associated DNA sequencing (RAD-seq). The results showed that subsp. littorale had lower genetic diversity and stronger genetic drift than subsp. australe. Fluctuations in the effective population size over time were evaluated by extended Bayesian skyline plot and Stairway plot analyses, both of which revealed a severe population bottleneck about 20,000 years ago in subsp. littorale. This bottleneck and the subsequent range expansion after the LGM appear to have played an important role in the divergence of the two subspecies and the evolution of selfing in subsp. littorale. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.

Two new species in the fern genus Lomariopsis (Lomariopsidaceae) from East Asia.

Two East Asian Lomariopsis (Lomariopsidaceae, Polypodiales) species, Lomariopsismoorei and Lomariopsislongini, which were previously misidentified as L.spectabilis, are here described as new species based on evidence from morphological characters and a molecular phylogeny. The two species differ from the three other described species in East Asia by their venation, pinna shapes, and perine morphology. A phylogeny based on a combined dataset of three chloroplast regions (rbcL+ rps4-trnS + trnL-L-F) showed that L.moorei and L.longini each formed a well-supported monophyletic group which was distantly related to both L.spectabilis and the other morphologically similar East Asian species, L.boninensis.

A taxonomic and molecular survey of the pteridophytes of the Nectandra Cloud Forest Reserve, Costa Rica.

Floristic surveys are crucial to the conservation of biodiversity, but the vast majority of such surveys are limited to listing species names, and few take into account the evolutionary history of species. Here, we combine classical taxonomic and molecular phylogenetic (DNA barcoding) approaches to catalog the biodiversity of pteridophytes (ferns and lycophytes) of the Nectandra Cloud Forest Reserve, Costa Rica. Surveys were carried out over three field seasons (2008, 2011, and 2013), resulting in 176 species representing 69 genera and 22 families of pteridophytes. Our literature survey of protected areas in Costa Rica shows that Nectandra has an exceptionally diverse pteridophyte flora for its size. Plastid rbcL was selected as a DNA barcode marker and obtained for >95% of pteridophyte taxa at this site. Combined molecular and morphological analyses revealed two previously undescribed taxa that appear to be of hybrid origin. The utility of rbcL for species identification was assessed by calculating minimum interspecific distances and found to have a failure rate of 18%. Finally we compared the distribution of minimum interspecific rbcL distances with two other areas that have been the focus of pteridophyte molecular surveys: Japan and Tahiti. The comparison shows that Nectandra is more similar to Japan than Tahiti, which may reflect the biogeographic history of these floras.

New Guinea has the world's richest island flora.

New Guinea is the world's largest tropical island and has fascinated naturalists for centuries1,2. Home to some of the best-preserved ecosystems on the planet3 and to intact ecological gradients-from mangroves to tropical alpine grasslands-that are unmatched in the Asia-Pacific region4,5, it is a globally recognized centre of biological and cultural diversity6,7. So far, however, there has been no attempt to critically catalogue the entire vascular plant diversity of New Guinea. Here we present the first, to our knowledge, expert-verified checklist of the vascular plants of mainland New Guinea and surrounding islands. Our publicly available checklist includes 13,634 species (68% endemic), 1,742 genera and 264 families-suggesting that New Guinea is the most floristically diverse island in the world. Expert knowledge is essential for building checklists in the digital era: reliance on online taxonomic resources alone would have inflated species counts by 22%. Species discovery shows no sign of levelling off, and we discuss steps to accelerate botanical research in the 'Last Unknown'8.

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.

Non-destructive DNA extraction from herbarium specimens: a method particularly suitable for plants with small and fragile leaves.

Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of morphological information from herbarium specimens, especially for small plants with few leaves, and reduces the voucher value of specimens. Here, we developed a new, non-destructive DNA extraction protocol (Protocol 1) that only needs a small piece of leaf (< 25 mm2) to obtain DNA suitable for DNA sequencing from fragile herbarium specimens. The protocol was very simple and rapid; an extraction buffer was placed on the leaf surface of an intact specimen for 30 min at room temperature (20 °C). The quality of extracted DNA was checked by PCR amplification of two standard plant DNA barcode regions, the maturase K gene (matK, ca. 850 bp) and the ribulose-1,5-bisphosphatecarboxylase/oxygenase gene (rbcL, ca. 550 bp), for 14 vascular plant species encompassing various taxonomic groups. The protocol retrieved sequences from 80.0% of specimens for matK and 46.2% of specimens for rbcL. Placing of the extraction buffer onto specimens did not cause any tears or deformation, but caused discoloration in some plants. To improve DNA yield for specimens incompatible with Protocol 1, we developed an alternative protocol for DNA extraction with minimally invasive destruction of specimens (Protocol 2). In this protocol, a cut leaf was immersed in the extraction buffer for 30 min and stored subsequently in a fragment pocket on the specimen sheet. This alternative method retrieved matK sequences from 80.0% of specimens and rbcL sequences from 92.8% of specimens. The combination of Protocols 1 and 2 enabled us to obtain matK sequences from 90.0% of specimens and rbcL sequences form 92.8% of specimens. The new protocols facilitate the use of museum specimens for use of DNA of museum specimens while still preserving morphological information.

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

In the original publication of this article, the affiliation of one of the authors was listed incorrectly as "Smithsonian Institute".

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.

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.

Fern gametophytes of Angiopteris lygodiifolia and Osmunda japonica harbor diverse Mucoromycotina fungi.

Mycorrhizal symbiosis between plants and fungi is ubiquitous, and has been played key roles in plant terrestrialization and diversification. Although arbuscular mycorrhizal (AM) symbioses with Glomeromycotina fungi have long been recognized as both ancient and widespread symbionts, recent studies showed that Mucoromycotina fungi were also ancestral symbionts and would thus be expected to co-exist with many land plants. To explore whether Mucoromycotina colonize fern gametophytes, we subjected fungal associations with gametophytes of two distantly related ferns, Angiopteris lygodiifolia (Marattiales) and Osmunda japonica (Osmundales), to molecular analysis. Direct PCR amplification from intracellular hyphal coils was also performed. We detected Mucoromycotina sequences in the gametophytes of A. lygodiifolia and O. japonica at rates of 41% (7/17) and 50% (49/98) of gametophytes, respectively, and assigned them to 10 operational taxonomic units of Endogonales lineages. In addition, we used AM fungal-specific primers and detected Glomeromycotina sequences in all individuals examined. The results suggest that Glomeromycotina and Mucoromycotina colonized fern gametophytes simultaneously. We found that Mucoromycotina were present in fern gametophytes of Marratiales and Osmundales, which implies that a variety of fern taxa have Mucoromycotina associations.