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.

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.

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.

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.

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.

Systematics and Plastome Evolution in Schizaeaceae.

While the family Schizaeaceae (Schizaeales) represents only about 0.4% of the extant fern species diversity, it differs from other ferns greatly in gross morphologies, niche preferences, and life histories. One of the most notable features in this family is its mycoheterotrophic life style in the gametophytic stage, which appears to be associated with extensive losses of plastid genes. However, the limited number of sequenced plastomes, and the lack of a well-resolved phylogenetic framework of Schizaeaceae, makes it difficult to gain any further insight. Here, with a comprehensive sampling of ~77% of the species diversity of this family, we first inferred a plastid phylogeny of Schizaeaceae using three DNA regions. To resolve the deep relationships within this family, we then reconstructed a plastome-based phylogeny focusing on a selection of representatives that covered all the major clades. From this phylogenomic backbone, we traced the evolutionary histories of plastid genes and examined whether gene losses were associated with the evolution of gametophytic mycoheterotrophy. Our results reveal that extant Schizaeaceae is comprised of four major clades-Microschizaea, Actinostachys, Schizaea, and Schizaea pusilla. The loss of all plastid NADH-like dehydrogenase (ndh) genes was confirmed to have occurred in the ancestor of extant Schizaeaceae, which coincides with the evolution of mycoheterotrophy in this family. For chlorophyll biosynthesis genes (chl), the losses were interpreted as convergent in Schizaeaceae, and found not only in Actinostachys, a clade producing achlorophyllous gametophytes, but also in S. pusilla with chlorophyllous gametophytes. In addition, we discovered a previously undescribed but phylogenetically distinct species hidden in the Schizaea dichotoma complex and provided a taxonomic treatment and morphological diagnostics for this new species-Schizaea medusa. Finally, our phylogenetic results suggest that the current PPG I circumscription of Schizaea is non-monophyletic, and we therefore proposed a three-genus classification moving a subset of Schizaea species sensu PPG I to a third genus-Microschizaea.

Structural Variation of Plastomes Provides Key Insight Into the Deep Phylogeny of Ferns.

Structural variation of plastid genomes (plastomes), particularly large inversions and gene losses, can provide key evidence for the deep phylogeny of plants. In this study, we investigated the structural variation of fern plastomes in a phylogenetic context. A total of 127 plastomes representing all 50 recognized families and 11 orders of ferns were sampled, making it the most comprehensive plastomic analysis of fern lineages to date. The samples included 42 novel plastomes of 15 families with a focus on Hymenophyllales and Gleicheniales. We reconstructed a well-supported phylogeny of all extant fern families, detected significant structural synapomorphies, including 9 large inversions, 7 invert repeat region (IR) boundary shifts, 10 protein-coding gene losses, 7 tRNA gene losses or anticodon changes, and 19 codon indels (insertions or deletions) across the deep phylogeny of ferns, particularly on the backbone nodes. The newly identified inversion V5, together with the newly inferred expansion of the IR boundary R5, can be identified as a synapomorphy of a clade composed of Dipteridaceae, Matoniaceae, Schizaeales, and the core leptosporangiates, while a unique inversion V4, together with an expansion of the IR boundary R4, was verified as a synapomorphy of Gleicheniaceae. This structural evidence is in support of our phylogenetic inference, thus providing key insight into the paraphyly of Gleicheniales. The inversions of V5 and V7 together filled the crucial gap regarding how the "reversed" gene orientation in the IR region characterized by most extant ferns (Schizaeales and the core leptosporangiates) evolved from the inferred ancestral type as retained in Equisetales and Osmundales. The tRNA genes trnR-ACG and trnM-CAU were assumed to be relicts of the early-divergent fern lineages but intact in most Polypodiales, particularly in eupolypods; and the loss of the tRNA genes trnR-CCG, trnV-UAC, and trnR-UCU in fern plastomes was much more prevalent than previously thought. We also identified several codon indels in protein-coding genes within the core leptosporangiates, which may be identified as synapomorphies of specific families or higher ranks. This study provides an empirical case of integrating structural and sequence information of plastomes to resolve deep phylogeny of plants.

Mycobiome detection from a single subterranean gametophyte using metabarcoding techniques.

Premise: Several ferns and lycophytes produce subterranean gametophytes, including the Ophioglossaceae, Psilotaceae, and some members of the Schizaeaceae, Gleicheniaceae, and Lycopodiaceae. Despite the surge in plant-microbiome research, which has been particularly boosted by high-throughput sequencing techniques, the microbiomes of these inconspicuous fern gametophytes have rarely been examined. The subterranean gametophytes are peculiar due to their achlorophyllous nature, which makes them rely on fungi to obtain nutrients. Furthermore, the factors that shape the fungal communities (mycobiomes) of fern gametophytes have not been examined in depth. Methods and Results: We present a workflow to study the mycobiome of the achlorophyllous gametophytes of Ophioderma pendulum using a high-throughput metabarcoding approach. Simultaneously, each gametophyte was investigated microscopically to detect fungal structures. Two primer sets of the nuclear ITS sequence targeting general fungi were applied, in addition to a primer set that specifically targets the nuclear small subunit ribosomal rDNA region of arbuscular mycorrhizal fungi. Both the microscopic and metabarcoding approaches revealed many diverse fungi inhabiting a single gametophyte of O. pendulum. Discussion: This study provides methodological details and discusses precautions for the mycobiome investigation of achlorophyllous gametophytes. This research is also the first to uncover the mycobiome assembly of an achlorophyllous gametophyte of an epiphytic fern.

Providing the missing links in fern life history: Insights from a phenological survey of the gametophyte stage.

Premise: The entire life cycle of ferns has been documented, yet their life histories are still poorly understood. In particular, the phenology of fern gametophytes remains largely unknown. To address this issue, we demonstrated a new ecological approach to explore the phenological link between spore release and gametophyte maturation within the life history of a tree fern species. Methods: We conducted a serial survey of Alsophila podophylla gametophyte abundance in the field, and recorded the time of its spore release. Every two months for one year, all terrestrial fern gametophytes in an unsampled subplot were collected and identified using tissue-direct PCR. Results: We found temporal differences in gametophyte abundances, with a sevenfold difference between the highest and lowest months. The number of spores released was linked to the gametophyte abundance two months later. The switch from gametophyte to juvenile sporophyte was found to be most correlated with precipitation. Discussion: The observed fluctuation in gametophyte abundance and population structure was likely associated with the phenology of spore release and environmental factors. Importantly, these findings provide the first evidence of phenological links between different developmental stages in a fern's life history.

Integrating tissue-direct PCR into genetic identification: An upgraded molecular ecology approach to survey fern gametophytes in the field.

Premise: The gametophytes of different fern species collected in the field can be difficult to distinguish because of their morphological similarities. Nonetheless, emerging molecular ecology techniques are starting to be used to tackle such limitations. Here, using case studies and a detailed protocol, we demonstrate a convenient methodology, tissue-direct PCR (TD-PCR), that foregoes a traditional DNA extraction and facilitates the identification of fern gametophytes, as well as enabling the elucidation of their natural distribution. Methods: Based on updated plastome information, we designed a universal primer set targeting the trnL-L-F region, which is effective across extant ferns. We used this primer set to perform TD-PCR on the case-studied populations of Taiwanese Lomariopsis gametophytes, using the generated sequences for their identification. In the case study concerning the microhabitat preference of Vaginularia junghuhnii, we designed and used a taxon-specific primer set. Results: Compared with approaches requiring DNA extraction, the use of TD-PCR with either universal or taxon-specific primers could save significant time, money, labor, and research materials in the genetic identification of fern gametophytes. Discussion: The use of modern genetic tools can aid in the identification of fern gametophytes. An updated TD-PCR strategy not only facilitates the DNA-based identification of gametophytes, but also promotes new avenues of research for investigating these plants in the field.

The flying spider-monkey tree fern genome provides insights into fern evolution and arborescence.

To date, little is known about the evolution of fern genomes, with only two small genomes published from the heterosporous Salviniales. Here we assembled the genome of Alsophila spinulosa, known as the flying spider-monkey tree fern, onto 69 pseudochromosomes. The remarkable preservation of synteny, despite resulting from an ancient whole-genome duplication over 100 million years ago, is unprecedented in plants and probably speaks to the uniqueness of tree ferns. Our detailed investigations into stem anatomy and lignin biosynthesis shed new light on the evolution of stem formation in tree ferns. We identified a phenolic compound, alsophilin, that is abundant in xylem, and we provided the molecular basis for its biosynthesis. Finally, analysis of demographic history revealed two genetic bottlenecks, resulting in rapid demographic declines of A. spinulosa. The A. spinulosa genome fills a crucial gap in the plant genomic landscape and helps elucidate many unique aspects of tree fern biology.

Hymenophyllumchamaecyparicola (Hymenophyllaceae), a new filmy fern species from Taiwan.

Hymenophyllumchamaecyparicola T.C.Hsu & Z.X.Chang, a new filmy fern species (Hymenophyllaceae) has been described from Taiwan and illustrated based on morphological and phylogenetic evidence. Although the new species resembles members in the subgenus Mecodium, namely H.wrightii, our plastid phylogeny has revealed that it is genetically distant from H.wrightii and forms a clade nested within subg. Hymenophyllum. The most notable characteristic to differentiate H.chamaecyparicola from related species is the presence of minute spathulate hairs on the surface of the rachis and veins. Hymenophyllumchamaecyparicola is currently only known from a small area in northern Taiwan, and endemic to that country.

Fern Spores-"Ready-to-Use" Standards for Plant Genome Size Estimation Using a Flow Cytometric Approach.

Spores and pollen of plants were used as flow cytometric materials to efficiently infer genome sizes. Given this advantage, they hold great potential for various flow cytometric applications, particularly as plant genome size standards. To develop such novel standards, we investigated conditions of pretreatment (bead vortex), buffer, and reliable genome sizes of three fern spore collections-Cibotium taiwanense "Kuo4395", Sphaeropteris lepifera "Tang0001", and Alsophila metteniana "Lee s.n.". Additionally, up to 30 year-old spore collections were obtained from herbarium specimens and from samples stored at 4 °C; their spore nuclei were extracted, and the quality and quantity of these nucleus extractions through storage ages were examined. Nuclear extractions with a longer bead vortex duration or lower spore/bead ratio generally resulted in a higher recovered quantity but a lower quality or purity. For each spore standard, the protocol optimization was determined by their performance in bead vortex conditions, and a 1C genome size was further inferred by linear regression (C. taiwanense "Kuo4395" = 5.058 pg; S. lepifera "Tang0001" = 7.117 pg; and A. metteniana "Lee s.n." = 19.379 pg). Spore nucleus quality and quantity are significantly negatively correlated with storage ages. Nuclear extractions of 10-year-old refrigerated spores remained qualified as a genome size standard; however, none of the herbarium spore collections fit such criteria. Our study is the first to develop and apply dried and refrigerated spores for genome size standards. These standards are ready to use, easy to manipulate, and feature long-term storage in comparison with traditionally used standards of fresh leaves.

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.

Underwater CAM photosynthesis elucidated by Isoetes genome.

To conserve water in arid environments, numerous plant lineages have independently evolved Crassulacean Acid Metabolism (CAM). Interestingly, Isoetes, an aquatic lycophyte, can also perform CAM as an adaptation to low CO2 availability underwater. However, little is known about the evolution of CAM in aquatic plants and the lack of genomic data has hindered comparison between aquatic and terrestrial CAM. Here, we investigate underwater CAM in Isoetes taiwanensis by generating a high-quality genome assembly and RNA-seq time course. Despite broad similarities between CAM in Isoetes and terrestrial angiosperms, we identify several key differences. Notably, Isoetes may have recruited the lesser-known 'bacterial-type' PEPC, along with the 'plant-type' exclusively used in other CAM and C4 plants for carboxylation of PEP. Furthermore, we find that circadian control of key CAM pathway genes has diverged considerably in Isoetes relative to flowering plants. This suggests the existence of more evolutionary paths to CAM than previously recognized.

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.