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.

Ecophysiological differentiation between life stages in filmy ferns (Hymenophyllaceae).

Desiccation tolerance was a key trait that allowed plants to colonize land. However, little is known about the transition from desiccation tolerant non-vascular plants to desiccation sensitive vascular ones. Filmy ferns (Hymenophyllaceae) represent a useful system to investigate how water-stress strategies differ between non-vascular and vascular stages within a single organism because they have vascularized sporophytes and nonvascular gametophytes that are each capable of varying degrees of desiccation tolerance. To explore this, we surveyed sporophytes and gametophytes of 19 species (22 taxa including varieties) of filmy ferns on Moorea (French Polynesia) and used chlorophyll fluorescence to measure desiccation tolerance and light responses. We conducted phylogenetically informed analyses to identify differences in physiology between life stages and growth habits. Gametophytes had similar or less desiccation tolerance (ability to recover from 2 days desiccation at - 86 MPa) and lower photosynthetic optima (maximum electron transport rate of photosystem II and light level at 95% of that rate) than sporophytes. Epiphytes were more tolerant of desiccation than terrestrial species in both life stages. Despite their lack of greater physiological tolerances, gametophytes of several species occurred over a wider elevational range than conspecific sporophytes. Our results demonstrate that filmy fern gametophytes and sporophytes differ in their physiology and niche requirements, and point to the importance of microhabitat in shaping the evolution of water-use strategies in vascular plants.

Life in the canopy: community trait assessments reveal substantial functional diversity among fern epiphytes.

The expansion of angiosperm-dominated forests in the Cretaceous and early Cenozoic had a profound effect on terrestrial biota by creating novel ecological niches. The majority of modern fern lineages are hypothesized to have arisen in response to this expansion, particularly fern epiphytes that radiated into the canopy. Recent evidence, however, suggests that epiphytism does not correlate with increased diversification rates in ferns, calling into question the role of the canopy habitat in fern evolution. To understand the role of the canopy in structuring fern community diversity, we investigated functional traits of fern sporophytes and gametophytes across a broad phylogenetic sampling on the island of Moorea, French Polynesia, including > 120 species and representatives of multiple epiphytic radiations. While epiphytes showed convergence in small size and a higher frequency of noncordate gametophytes, they showed greater functional diversity at the community level relative to terrestrial ferns. These results suggest previously overlooked functional diversity among fern epiphytes, and raise the hypothesis that while the angiosperm canopy acted as a complex filter that restricted plant size, it also facilitated diversification into finely partitioned niches. Characterizing these niche axes and adaptations of epiphytic ferns occupying them should be a priority for future pteridological research.

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.

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.

A plastid phylogeny and character evolution of the Old World fern genus Pyrrosia (Polypodiaceae) with the description of a new genus: Hovenkampia (Polypodiaceae).

The Old World fern genus Pyrrosia (Polypodiaceae) offers a rare system in ferns to study morphological evolution because almost all species of this genus are well studied for their morphology, anatomy, and spore features, and various hypotheses have been proposed in terms of the phylogeny and evolution in this genus. However, the molecular phylogeny of the genus lags behind. The monophyly of the genus has been uncertain and a modern phylogenetic study of the genus based on molecular data has been lacking. In the present study, DNA sequences of five plastid markers of 220 accessions of Polypodiaceae representing two species of Drymoglossum, 14 species of Platycerium, 50 species of Pyrrosia, and the only species of Saxiglossum (subfamily Platycerioideae), and 12 species of other Polypodiaceae representing the remaining four subfamilies are used to infer a phylogeny of the genus. Major results and conclusions of this study include: (1) Pyrrosia as currently circumscribed is paraphyletic in relation to Platycerium and can be divided into two genera: Pyrrosia s.s. and Hovenkampia (gen. nov.), with Hovenkampia and Platycerium forming a strongly supported clade sister to Pyrrosia s.s.; (2) Subfamily Platycerioideae should contain three genera only, Hovenkampia, Platycerium, and Pyrrosia s.s.; (3) Based on the molecular phylogeny, macromorphology, anatomical features, and spore morphology, four major clades in the genus are identified and three of the four are further resolved into four, four, and six subclades, respectively; (4) Three species, P. angustissima, P. foveolata, and P. mannii, not assigned to any groups by Hovenkamp (1986) because of their unusual morphology, each form monospecific clades; (5) Drymoglossum is not monophyletic and those species previously assigned to this genus are resolved in two different subclades; (6) Saxiglossum is resolved as the first lineage in the Niphopsis clade; and (7) The evolution of ten major morphological characters in the subfamily is inferred based on the phylogeny and various morphological synapomorphies for various clades and subclades are identified.

Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna.

The cladoceran crustacean Daphnia exhibits phenotypic plasticity, a phenomenon that leads to diverse phenotypes from one genome. Alternative usage of gene isoforms has been considered a key gene regulation mechanism for controlling different phenotypes. However, to understand the phenotypic plasticity of Daphnia, gene isoforms have not been comprehensively analyzed. Here we identified 25,654 transcripts derived from the 9710 genes expressed during environmental sex determination of Daphnia magna using the long-read RNA-Seq with PacBio Iso-Seq. We found that 14,924 transcripts were previously unidentified and 5713 genes produced two or more isoforms. By a combination of Illumina short-read RNA-Seq, we detected 824 genes that implemented switching of the highest expressed isoform between females and males. Among the 824 genes, we found isoform switching of an ortholog of CREB-regulated transcription coactivator, a major regulator of carbohydrate metabolism in animals, and a correlation of this switching event with the sexually dimorphic expression of carbohydrate metabolic genes. These results suggest that a comprehensive catalog of isoforms may lead to understanding the molecular basis for environmental sex determination of Daphnia. We also infer the applicability of the full-length isoform analyses to the elucidation of phenotypic plasticity in Daphnia.

A survey of the fern gametophyte flora of Japan: Frequent independent occurrences of noncordiform gametophytes.

PREMISE OF THE STUDY: Ferns and lycophytes are the only extant land plants with two free-living generations (sporophytes and gametophytes); hence, a single species may have two different distributions. The distribution of the gametophytes of most fern species, which are much smaller in size than sporophytes, are almost unknown due to the difficulty of identifying gametophytes using morphological characters. METHODS: Twelve quadrats (1 m(2) or 0.25 m(2)), each subdivided into a grid of 100 (10 × 10) or 25 (5 × 5) squares, were used to survey gametophytes in the Japanese Archipelago, where distribution data of sporophytes and "DNA barcodes" for identification of gametophytes have fully been established in previous studies. Collected gametophytes were identified using the plastid rbcL-a region. KEY RESULTS: In total, gametophytes of 38 species in two broad morphological categories (28 cordiform and 10 noncordiform species) were identified among 407 collections. The cordiform gametophytes discovered are without exception accompanied by their conspecific sporophytes at the periphery of the quadrats. On the other hand, the sporophytic counterparts of the noncordiform gametophytes are often not found or are rare around the sites. CONCLUSIONS: This study demonstrates with a regional flora that fern gametophytes do not always co-occur with sporophytes of the same species. In particular, noncordiform gametophytes tended to occur independently of conspecific sporophytes. This pattern may be due to the capability for indeterminate growth and vegetative reproduction by gemmae in noncordiform gametophytes.

Identifying cryptic fern gametophytes using DNA barcoding: A review.

Ferns and lycophytes are unique among land plants in having sporophyte (diploid) and gametophyte (haploid) generations that can grow independently of each other. While most studies of fern ecology focus on the more visible sporophytic stage, the gametophyte is critically important, as it is the sexual phase of the life cycle. Yet, fern gametophytes have long been neglected in field studies due to their small size and cryptic morphology. DNA barcoding is a powerful method that can be used to identify field-collected gametophytes to species and allow for detailed study of their ecology. Here, we review the state of DNA barcoding as applied to fern gametophytes. First, we trace the history of DNA barcoding and how it has come to be applied to fern gametophytes. Next, we summarize case studies that show how DNA barcoding has been used to better understand fern species distributions, gametophyte ecology, and community ecology. Finally, we propose avenues for future research using this powerful tool, including next-generation DNA sequencing for in-field identification of cryptic gametophytes.

An open and continuously updated fern tree of life.

Ferns, with about 12,000 species, are the second most diverse lineage of vascular plants after angiosperms. They have been the subject of numerous molecular phylogenetic studies, resulting in the publication of trees for every major clade and DNA sequences from nearly half of all species. Global fern phylogenies have been published periodically, but as molecular systematics research continues at a rapid pace, these become quickly outdated. Here, we develop a mostly automated, reproducible, open pipeline to generate a continuously updated fern tree of life (FTOL) from DNA sequence data available in GenBank. Our tailored sampling strategy combines whole plastomes (few taxa, many loci) with commonly sequenced plastid regions (many taxa, few loci) to obtain a global, species-level fern phylogeny with high resolution along the backbone and maximal sampling across the tips. We use a curated reference taxonomy to resolve synonyms in general compliance with the community-driven Pteridophyte Phylogeny Group I classification. The current FTOL includes 5,582 species, an increase of ca. 40% relative to the most recently published global fern phylogeny. Using an updated and expanded list of 51 fern fossil constraints, we find estimated ages for most families and deeper clades to be considerably older than earlier studies. FTOL and its accompanying datasets, including the fossil list and taxonomic database, will be updated on a regular basis and are available via a web portal (https://fernphy.github.io) and R packages, enabling immediate access to the most up-to-date, comprehensively sampled fern phylogeny. FTOL will be useful for anyone studying this important group of plants over a wide range of taxonomic scales, from smaller clades to the entire tree. We anticipate FTOL will be particularly relevant for macroecological studies at regional to global scales and will inform future taxonomic systems with the most recent hypothesis of fern phylogeny.

Reticulate evolution in the Crepidomanes minutum species complex (Hymenophyllaceae).

PREMISE OF THE STUDY: Hybridization is an important mechanism of speciation in plants and often results in complexes that comprise multiple sexual diploids and their hybrid offspring. However, the intricacy of these systems has prevented a thorough understanding of many groups. The Crepidomanes minutum species complex (Hymenophyllaceae) is a widely distributed, morphologically variable fern species complex. Although prior reports of apogamy and polyploidy suggest hybridization, it has never been the focus of a phylogenetic study. METHODS: Morphology, nuclear (gapCp), and chloroplast (rbcL) DNA sequences, cytology, field observation, and spore counts were used to infer phylogeny and trace hybrid origins. KEY RESULTS: The C. minutum species complex is composed of at least three major clades: the African clade, clade 1 (East Asia and the Pacific), and clade 2 (Southeast Asia and the South Pacific). Clades 1 and 2 differ strikingly in morphological variation (uniform in clade 1 vs. highly variable in clade 2) and occurrence of hybrids (rare in clade 1 vs. frequent in clade 2). Apogamy and polyploidy were confirmed as likely mechanisms of hybrid stabilization in clade 2. Despite the large genetic distance between clades 1 and 2, several specimens were observed with gapCp sequences from both; diploid genome size and sexual reproduction indicate maintenance of genetic diversity via introgression or incomplete lineage sorting, rather than ongoing hybridization, in these specimens. CONCLUSIONS: The C. minutum species complex is a reticulate network including multiple diploid lineages and their stabilized hybrid crosses. Additional sampling focused on reproductive mode and ploidy level is needed to delimit diploid species and hybrids.

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.

Molecular species identification with rich floristic sampling: DNA barcoding the pteridophyte flora of Japan.

BACKGROUND: DNA barcoding is expected to be an effective identification tool for organisms with heteromorphic generations such as pteridophytes, which possess a morphologically simple gametophyte generation. Although a reference data set including complete coverage of the target local flora/fauna is necessary for accurate identification, DNA barcode studies including such rich taxonomic sampling on a countrywide scale are lacking. METHODOLOGY/PRINCIPAL FINDINGS: The Japanese pteridophyte flora (733 taxa including subspecies and varieties) was used to test the utility of two plastid DNA barcode regions (rbcL and trnH-psbA) with the intention of developing an identification system for native gametophytes. DNA sequences were obtained from each of 689 (94.0%) taxa for rbcL and 617 (84.2%) taxa for trnH-psbA. Mean interspecific divergence values across all taxon pairs (K2P genetic distances) did not reveal a significant difference in rate between trnH-psbA and rbcL, but mean K2P distances of each genus showed significant heterogeneity according to systematic position. The minimum fail rate of taxon discrimination in an identification test using BLAST (12.52%) was obtained when rbcL and trnH-psbA were combined, and became lower in datasets excluding infraspecific taxa or apogamous taxa, or including sexual diploids only. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the overall effectiveness of DNA barcodes for species identification in the Japanese pteridophyte flora. Although this flora is characterized by a high occurrence of apogamous taxa that pose a serious challenge to identification using DNA barcodes, such taxa are limited to a small number of genera, and only minimally detract from the overall success rate. In the case that a query sequence is matched to a known apogamous genus, routine species identification may not be possible. Otherwise, DNA barcoding is a practical tool for identification of most Japanese pteridophytes, and is especially anticipated to be helpful for identification of non-hybridizing gametophytes.