An efficient and effective RNA extraction protocol for ferns.

Premise: The extraction of high-quality RNA is the critical first step for the analysis of gene expression and gene space. This remains particularly challenging in plants, and especially in ferns, where the disruption of the cell wall and separation of organic compounds from nucleic acids is not trivial.

Methods: We developed a cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocol that consistently performs well across a large phylogenetic breadth of ferns-a lineage of plants high in secondary compounds-and in an array of tissue types.

A DNA barcode reference of Asian ferns with expert-identified voucher specimens and DNA samples.

Ferns belong to species-rich group of land plants, encompassing more than 11,000 extant species, and are crucial for reflecting terrestrial ecosystem changes. However, our understanding of their biodiversity hotspots, particularly in Southeast Asia, remains limited due to scarce genetic data. Despite harboring around one-third of the world's fern species, less than 6% of Southeast Asian ferns have been DNA-sequenced.

The role of deep hybridization in fern speciation: Examples from the Thelypteridaceae.

Premise: Hybridization is recognized as an important mechanism in fern speciation, with many allopolyploids known among congeners, as well as evidence of ancient genome duplications. Several contemporary instances of deep (intergeneric) hybridization have been noted, invariably resulting in sterile progeny. We chose the christelloid lineage of the family Thelypteridaceae, recognized for its high frequency of both intra- and intergeneric hybrids, to investigate recent hybrid speciation between deeply diverged lineages.

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.

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.

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.

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 ( and ) ~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 and the diploid .

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.

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

T.C.Hsu & Z.

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- "", "", and "".

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.

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.

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.

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.

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.

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.

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

Two East Asian (Lomariopsidaceae, Polypodiales) species, and , which were previously misidentified as , 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 (+ + ) showed that and each formed a well-supported monophyletic group which was distantly related to both and the other morphologically similar East Asian species, .

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.

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.

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.