All tangled up: Unraveling phylogenetics and reticulate evolution in the vining ferns, Lygodium (Schizaeales).

PREMISE: Reticulate evolution, often accompanied by polyploidy, is prevalent in plants, and particularly in the ferns. Resolving the resulting non-bifurcating histories remains a major challenge for plant phylogenetics. Here, we present a phylogenomic investigation into the complex evolutionary history of the vining ferns, Lygodium (Lygodiaceae, Schizaeales). METHODS: Using a targeted enrichment approach with the GoFlag 408 flagellate land plant probe set, we generated large nuclear and plastid sequence datasets for nearly all taxa in the genus and constructed the most comprehensive phylogeny of the family to date using concatenated maximum likelihood and coalescence approaches. We integrated this phylogeny with cytological and spore data to explore karyotype evolution and generate hypotheses about the origins of putative polyploids and hybrids. RESULTS: Our data and analyses support the origins of several putative allopolyploids (e.g., L. cubense, L. heterodoxum) and hybrids (e.g., L. ×fayae) and also highlight the potential prevalence of autopolyploidy in this clade (e.g., L. articulatum, L. flexuosum, and L. longifolium). CONCLUSIONS: Our robust phylogenetic framework provides valuable insights into dynamic reticulate evolution in this clade and demonstrates the utility of target-capture data for resolving these complex relationships.

Phylogenomic evolutionary insights in the fern family Gleicheniaceae.

The pantropical fern family Gleicheniaceae comprises approximately 157 species. Seven genera are currently recognized in the family, although their monophyly is still uncertain due to low sampling in phylogenetic studies. We examined the monophyly of the genera through extended sampling, using the first phylogenomic inference of the family including data from both nuclear and plastid genomes. Seventy-six samples were sequenced (70 Gleicheniaceae species and six outgroups) using high throughput sequencing, including all seven currently recognized genera. Plastid and nuclear data were recovered and assembled; the nuclear data was phased to reduce paralogy as well as hybrid noise in the final recovered topology. Maximum likelihood trees were built for each locus, and a concatenated dataset was built for both datasets. A species tree based on a multispecies coalescent model was generated, and divergence time analyses performed. We here present the first genomic phylogenetic inferences concerning Gleicheniaceae, confirming the monophyly of most genera except Sticherus, which we recovered as paraphyletic. Although most of the extant genera of Gleicheniaceae originated during the Mesozoic, several genera show Neogene and even Quaternary diversifications, and our results suggest that reticulation and polyploidy may have played significant roles during this diversification. However, some genera, such as Rouxopteris and Stromatopteris, appear to represent evolutionary relicts.

Madagascar's extraordinary biodiversity: Evolution, distribution, and use.

Madagascar's biota is hyperdiverse and includes exceptional levels of endemicity. We review the current state of knowledge on Madagascar's past and current terrestrial and freshwater biodiversity by compiling and presenting comprehensive data on species diversity, endemism, and rates of species description and human uses, in addition to presenting an updated and simplified map of vegetation types. We report a substantial increase of records and species new to science in recent years; however, the diversity and evolution of many groups remain practically unknown (e.g., fungi and most invertebrates). Digitization efforts are increasing the resolution of species richness patterns and we highlight the crucial role of field- and collections-based research for advancing biodiversity knowledge and identifying gaps in our understanding, particularly as species richness corresponds closely to collection effort. Phylogenetic diversity patterns mirror that of species richness and endemism in most of the analyzed groups. We highlight humid forests as centers of diversity and endemism because of their role as refugia and centers of recent and rapid radiations. However, the distinct endemism of other areas, such as the grassland-woodland mosaic of the Central Highlands and the spiny forest of the southwest, is also biologically important despite lower species richness. The documented uses of Malagasy biodiversity are manifold, with much potential for the uncovering of new useful traits for food, medicine, and climate mitigation. The data presented here showcase Madagascar as a unique "living laboratory" for our understanding of evolution and the complex interactions between people and nature. The gathering and analysis of biodiversity data must continue and accelerate if we are to fully understand and safeguard this unique subset of Earth's biodiversity.

The relationship between chlorophyllous spores and mycorrhizal associations in ferns: evidence from an evolutionary approach.

PREMISE: Approximately 14% of all fern species have physiologically active chlorophyllous spores that are much more short-lived than the more common and dormant achlorophyllous spores. Most chlorophyllous-spored species (70%) are epiphytes and account for almost 37% of all epiphytic ferns. Chlorophyllous-spored ferns are also overrepresented among fern species in habitats with waterlogged soils, of which nearly 60% have chlorophyllous spores. Ferns in these disparate habitat types also have a low incidence of mycorrhizal associations. We therefore hypothesized that autotrophic chlorophyllous spores represent an adaptation of ferns to habitats with scarce mycorrhizal associations. METHODS: We evaluated the coevolution of chlorophyllous spores and mycorrhizal associations in ferns and their relation to habitat type using phylogenetic comparative methods. RESULTS: Although we did not find support for the coevolution of spore type and mycorrhizal associations, we did find that chlorophyllous spores and the absence of mycorrhizal associations have coevolved with epiphytic and waterlogged habitats. Transition rates to epiphytic and waterlogged habitats were significantly higher in species with chlorophyllous spores compared to achlorophyllous lineages. CONCLUSIONS: Spore type and mycorrhizal associations appear to play important roles in the radiation of ferns into different habitat types. Future work should focus on clarifying the functional significance of these associations.

Deep vicariance and frequent transoceanic dispersal shape the evolutionary history of a globally distributed fern family.

PREMISE: The historical biogeography of ferns is typically expected to be dominated by long-distance dispersal due to their minuscule spores. However, few studies have inferred the historical biogeography of a large and widely distributed group of ferns to test this hypothesis. Our aims were to determine the extent to which long-distance dispersal vs. vicariance have shaped the history of the fern family Blechnaceae, to explore ecological correlates of dispersal and diversification, and to determine whether these patterns differ between the northern and southern hemispheres. METHODS: We used sequence data for three chloroplast loci to infer a time-calibrated phylogeny for 154 of 265 species of Blechnaceae, including representatives of all genera in the family. This tree was used to conduct ancestral range reconstruction and stochastic character mapping, estimate diversification rates, and identify ecological correlates of diversification. RESULTS: Blechnaceae originated in Eurasia and began diversifying in the late Cretaceous. A lineage comprising most extant diversity diversified principally in the austral Pacific region around the Paleocene-Eocene Thermal Maximum. Land connections that existed near the poles during periods of warm climates likely facilitated migration of several lineages, with subsequent climate-mediated vicariance shaping current distributions. Long-distance dispersal is frequent and asymmetrical, with New Zealand/Pacific Islands, Australia, and tropical America being major source areas. CONCLUSIONS: Ancient vicariance and extensive long-distance dispersal have shaped the history of Blechnaceae in both the northern and southern hemispheres. The exceptional diversity in austral regions appears to reflect rapid speciation in these areas; mechanisms underlying this evolutionary success remain uncertain.

Hydathodes in ferns: their phylogenetic distribution, structure and function.

BACKGROUND AND AIMS: Ferns are the second largest group of vascular plants and are distributed nearly worldwide. Although ferns have been integrated into some comparative ecological studies focusing on hydathodes, there is a considerable gap in our understanding of the functional anatomy of these secretory tissues that are found on the vein endings of many fern leaves. In this study, we aimed to investigate the phylogenetic distribution, structure and function of fern hydathodes. METHODS: We performed a global review on fern hydathodes and their phylogenetic distribution, carried out an ancestral character state reconstruction, and studied the structure, guttation and elemental composition of salt residues of eight species, and the diurnal patterns of xylem pressure of two species. KEY RESULTS: Hydathodes are known from 1189 fern species, 92 genera and 19 families of 2 orders, Equisetales and Polypodiales. Stochastic character mapping indicated multiple gains and losses of hydathodes at the genus level, occurring especially during the last 50 million years of fern evolution. Hydathodes were located on the adaxial leaf surface and characterized by a cytoplasm-rich, pore-free epidermis, and became functional for several weeks after nearly complete leaf expansion. In two species, positive xylem pressure built up at night, potentially facilitating guttation. Guttation fluid was rich in Ca and often Si, but also contained P, Mg, Na and Al. CONCLUSIONS: Stochastic character mapping and the structural and functional diversity of hydathodes indicate multiple origins, and their presence/absence in closely related taxa implies secondary losses during fern evolution. Positive xylem pressure and high air humidity play an important role as drivers of guttation. Hydathodes may contribute to the regulation of leaf nutrient stoichiometry by the release of excessive compounds and minerals other than waste products, but the presence of essential chemical elements in salt residues also indicates possible leakage.

A permineralized Early Cretaceous lycopsid from China and the evolution of crown clubmosses.

Lycopodiaceae are one of three surviving families of lycopsids, a lineage of vascular plants with a fossil history dating to at least the Early Devonian or perhaps the Late Silurian (c. 415 Ma). Many fossils have been linked to crown Lycopodiaceae, but the lack of well-preserved material has hindered definitive recognition of this group in the paleobotanical record. New, exceptionally well-preserved permineralized lycopsid fossils from the Early Cretaceous (125.6 ± 1.0 Ma) of Inner Mongolia, China, were examined in detail using acetate peel and micro-computed tomography techniques. The anatomy of extant Lycopodiaceae was analyzed for comparison using fluorescence microscopy. Phylogenetic relationships of the new fossil to extant Lycopodiaceae were evaluated using parsimony and maximum likelihood analyses. Lycopodicaulis oellgaardii gen. et sp. nov. provides the earliest unequivocal and best-documented evidence of crown Lycopodiaceae and Lycopodioideae, based on anatomically-preserved fossil material. Recognition of Lycopodicaulis in Asia during the Early Cretaceous indicates the presence of crown Lycopodiaceae at this time, and striking similarities of stem anatomy with extant species provide a framework for the understanding of the interaction of branching and vascular anatomy in crown-group lycopsids.

A target enrichment probe set for resolving the flagellate land plant tree of life.

PREMISE: New sequencing technologies facilitate the generation of large-scale molecular data sets for constructing the plant tree of life. We describe a new probe set for target enrichment sequencing to generate nuclear sequence data to build phylogenetic trees with any flagellate land plants, including hornworts, liverworts, mosses, lycophytes, ferns, and all gymnosperms. METHODS: We leveraged existing transcriptome and genome sequence data to design the GoFlag 451 probes, a set of 56,989 probes for target enrichment sequencing of 451 exons that are found in 248 single-copy or low-copy nuclear genes across flagellate plant lineages. RESULTS: Our results indicate that target enrichment using the GoFlag451 probe set can provide large nuclear data sets that can be used to resolve relationships among both distantly and closely related taxa across the flagellate land plants. We also describe the GoFlag 408 probes, an optimized probe set covering 408 of the 451 exons from the GoFlag 451 probe set that is commercialized by RAPiD Genomics. CONCLUSIONS: A target enrichment approach using the new probe set provides a relatively low-cost solution to obtain large-scale nuclear sequence data for inferring phylogenetic relationships across flagellate land plants.

Pleistocene aridification underlies the evolutionary history of the Caribbean endemic, insular, giant Consolea (Opuntioideae).

PREMISE: The Caribbean islands are in the top five biodiversity hotspots on the planet; however, the biogeographic history of the seasonally dry tropical forest (SDTF) there is poorly studied. Consolea consists of nine species of dioecious, hummingbird-pollinated tree cacti endemic to the West Indies, which form a conspicuous element of the SDTF. Several species are threatened by anthropogenic disturbance, disease, sea-level rise, and invasive species and are of conservation concern. However, no comprehensive phylogeny yet exists for the clade. METHODS: We reconstructed the phylogeny of Consolea, sampling all species using plastomic data to determine relationships, understand the evolution of key morphological characters, and test their biogeographic history. We estimated divergence times to determine the role climate change may have played in shaping the current diversity of the clade. RESULTS: Consolea appears to have evolved very recently during the latter part of the Pleistocene on Cuba/Hispaniola likely from a South American ancestor and, from there, moved into the Bahamas, Jamaica, Puerto Rico, Florida, and the Lesser Antilles. The tree growth form is a synapomorphy of Consolea and likely aided in the establishment and diversification of the clade. CONCLUSIONS: Pleistocene aridification associated with glaciation likely played a role in shaping the current diversity of Consolea, and insular gigantism may have been a key innovation leading to the success of these species to invade the often-dense SDTF. This in-situ Caribbean radiation provides a window into the generation of species diversity and the complexity of the SDTF community within the Antilles.

Insights into the evolutionary history and widespread occurrence of antheridiogen systems in ferns.

Sex expression of homosporous ferns is controlled by multiple factors, one being the antheridiogen system. Antheridiogens are pheromones released by sexually mature female fern gametophytes, turning nearby asexual gametophytes precociously male. Nevertheless, not all species respond. It is still unknown how many fern species use antheridiogens, how the antheridiogen system evolved, and whether it is affected by polyploidy and/or apomixis. We tested the response of 68 fern species to antheridiogens in cultivation. These results were combined with a comprehensive review of literature to form the largest dataset of antheridiogen interactions to date. Analyzed species also were coded as apomictic or sexual and diploid or polyploid. Our final dataset contains a total of 498 interactions involving 208 species (c. 2% of all ferns). About 65% of studied species respond to antheridiogen. Multiple antheridiogen types were delimited and their evolution is discussed. Antheridiogen responsiveness was not significantly affected by apomixis or polyploidy. Antheridiogens are widely used by ferns to direct sex expression. The antheridiogen system likely evolved multiple times and provides homosporous ferns with the benefits often associated with heterospory, such as increased rates of outcrossing. Despite expectations, antheridiogens may be beneficial to polyploids and apomicts.

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.

The rise of the Andes promoted rapid diversification in Neotropical Phlegmariurus (Lycopodiaceae).

Tropical mountains are disproportionately biodiverse relative to their surface area, but the processes underlying their exceptional diversity require further study. Here, we use comparative phylogenetic methods to examine the impact of the Andean orogeny on the diversification of Neotropical Phlegmariurus, a species-rich lycophyte clade. We generated a time-calibrated phylogeny of 105 species of Neotropical Phlegmariurus and estimated lineage diversification rates. We tested for correlations between lineage diversification rates and species range size, niche breadth, elevational range amplitude, and mean elevation of occurrence. A recently developed macroevolutionary model was used to incorporate geological data and test for an association between diversification rates and the Andean uplift. Diversification rates of Neotropical Phlegmariurus are negatively correlated with species range size and positively correlated with mean elevation of species occurrence. The rise of the Andes is strongly associated with increased rates of diversification in Neotropical Phlegmariurus during the last 10 Myr. Our study demonstrates the importance of mountain-building events and geographical isolation of alpine populations as drivers of rapid diversification, even in spore-dispersed plants. This work also highlights the usefulness of combined phylogenetic, geological and ecological datasets, and the promise of comparative environment-dependent diversification models in better understanding the evolutionary origins of biodiversity.

Target sequence capture of nuclear-encoded genes for phylogenetic analysis in ferns.

PREMISE OF THE STUDY: Until recently, most phylogenetic studies of ferns were based on chloroplast genes. Evolutionary inferences based on these data can be incomplete because the characters are from a single linkage group and are uniparentally inherited. These limitations are particularly acute in studies of hybridization, which is prevalent in ferns; fern hybrids are common and ferns are able to hybridize across highly diverged lineages, up to 60 million years since divergence in one documented case. However, it not yet clear what effect such hybridization has on fern evolution, in part due to a paucity of available biparentally inherited (nuclear-encoded) markers. METHODS: We designed oligonucleotide baits to capture 25 targeted, low-copy nuclear markers from a sample of 24 species spanning extant fern diversity. RESULTS: Most loci were successfully sequenced from most accessions. Although the baits were designed from exon (transcript) data, we successfully captured intron sequences that should be useful for more focused phylogenetic studies. We present phylogenetic analyses of the new target sequence capture data and integrate these into a previous transcript-based data set. DISCUSSION: We make our bait sequences available to the community as a resource for further studies of fern phylogeny.

Are rates of species diversification and body size evolution coupled in the ferns?

PREMISE OF THE STUDY: Understanding the relationship between phenotypic evolution and lineage diversification is a central goal of evolutionary biology. To extend our understanding of the role morphological evolution plays in the diversification of plants, we examined the relationship between leaf size evolution and lineage diversification across ferns. METHODS: We tested for an association between body size evolution and lineage diversification using a comparative phylogenetic approach that combined a time-calibrated phylogeny and leaf size data set for 2654 fern species. Rates of leaf size change and lineage diversification were estimated using BAMM, and rate correlations were performed for rates obtained for all families and individual species. Rates and patterns of rate-rate correlation were also analyzed separately for terrestrial and epiphytic taxa. KEY RESULTS: We find no significant correlation between rates of leaf area change and lineage diversification, nor was there a difference in this pattern when growth habit is considered. Our results are consistent with the findings of an earlier study that reported decoupled rates of body size evolution and diversification in the Polypodiaceae, but conflict with a recent study that reported a positive correlation between body size evolution and lineage diversification rates in the tree fern family Cyatheaceae. CONCLUSIONS: Our findings indicate that lineage diversification in ferns is largely decoupled from shifts in body size, in contrast to several other groups of organisms. Speciation in ferns appears to be primarily driven by hybridization and isolation along elevational gradients, rather than adaptive radiations featuring prominent morphological restructuring. The exceptional diversity of leaf morphologies in ferns appears to reflect a combination of ecophysiological constraints and adaptations that are not key innovations.

Geometry, Allometry and Biomechanics of Fern Leaf Petioles: Their Significance for the Evolution of Functional and Ecological Diversity Within the Pteridaceae.

Herbaceous plants rely on a combination of turgor, ground tissues and geometry for mechanical support of leaves and stems. Unlike most angiosperms however, ferns employ a sub-dermal layer of fibers, known as a hypodermal sterome, for support of their leaves. The sterome is nearly ubiquitous in ferns, but nothing is known about its role in leaf biomechanics. The goal of this research was to characterize sterome attributes in ferns that experience a broad range of mechanical stresses, as imposed by their aquatic, xeric, epiphytic, and terrestrial niches. Members of the Pteridaceae meet this criteria well. The anatomical and functional morphometrics along with published values of tissue moduli were used to model petiole flexural rigidity and susceptibility to buckling in 20 species of the Pteridaceae. Strong allometric relationships were observed between sterome thickness and leaf size, with the sterome contributing over 97% to petiole flexural rigidity. Surprisingly, the small-statured cheilanthoid ferns allocated the highest fraction of their petiole to the sterome, while large leaves exploited aspects of geometry (second moment of area) to achieve bending resistance. This pattern also revealed an economy of function in which increasing sterome thickness was associated with decreasing fiber cell reinforcement, and fiber wall fraction. Lastly, strong petioles were associated with durable leaves, as approximated by specific leaf area. This study reveals meaningful patterns in fern leaf biomechanics that align with species leaf size, sterome attributes and life-history strategy.

Phylogenetic systematics, morphological evolution, and natural groups in neotropical Phlegmariurus (Lycopodiaceae).

The Neotropical clade of the lycophyte genus Phlegmariurus is comprised of an estimated 150 described species and exhibits exceptional morphological and ecological diversity. Because of their simple morphology, frequent convergent evolution, and the recentness of the group's diversification, the delimitation of species and species groups has remained challenging. Here, we present a robustly support phylogeny of Neotropical Phlegmariurus based on six chloroplast markers and ca. 70% of known species, and use ancestral character state reconstruction to investigate morphological evolution in the clade, and define natural species groups. The Neotropical species of Phlegmariurus form a clade that also includes a small number of Afro-Madagascan species. A morphologically and ecologically variable group of species from southeastern Brazil form a monophyletic group and represent a parallel radiation to principally Andean lineages. Species groups in Neotropical Phlegmariurus that were previously recognized based on morphology are not monophyletic. We find support for 11 morphologically cohesive and well-supported species groups. Morphological homoplasy is common in Phlegmariurus and complicates infrageneric classification of the Neotropical taxa. Our results provide a useful framework for identifying species groups and understanding patterns of morphological evolution in Neotropical Phlegmariurus. The radiation of the Brazilian species remains poorly understood and requires further study.

The evolution of aluminum accumulation in ferns and lycophytes.

PREMISE OF THE STUDY: This paper investigates the occurrence and evolution of aluminum (Al) accumulation within ferns and lycophytes, which is characterized by Al concentrations above 1000 mg·kg-1 in aboveground plant tissues. We hypothesize that this feature is more common in ferns than in angiosperms, and potentially correlated with growth form and other chemical elements. METHODS: Aluminum concentrations were obtained from novel analyses and literature for a total of 354 specimens and 307 species. Moreover, a semiquantitative aluminon test was applied for a subset of 105 species and validated against exact Al measurements. KEY RESULTS: Molecular phylogenetic analyses showed that the major Al-accumulating groups were primarily found in the Gleicheniales and Cyatheales, and largely absent in the Polypodiales. At the species and generic level, Al accumulation was typically either absent or present, and mixed results within a single species and genus were limited to less than 30% of the species and genera tested. Epiphytic ferns had significantly lower Al levels than terrestrial ferns, although this finding was not significant after phylogenetic correction. In addition, a significant, positive correlation was found between Al and iron, while Al was negatively correlated with phosphorus and potassium concentrations. CONCLUSIONS: Aluminum accumulation is most common outside of the Polypodiales and occurs in 38% of the species studied, indicating that this trait is indeed common within subtropical and tropical ferns, a finding that could be in line with their role as pioneer species on landslides and soils with high levels of soluble Al.

A 4000-species dataset provides new insight into the evolution of ferns.

Ferns are the second-most diverse lineage of vascular plants on Earth, yet the best-sampled time-calibrated phylogeny of the group to date includes fewer than 5% of global diversity and was published seven years ago. We present a time-calibrated phylogeny that includes nearly half of extant fern diversity. Our results are evaluated in the context of previous studies and the fossil record, and we develop new hypotheses about the radiation of leptosporangiate ferns. We used sequence data from six chloroplast regions for nearly 4000 species of ferns to generate the most comprehensive phylogeny of the group ever published. We calibrate the phylogeny with twenty-six fossils and use an array of phylogenetic methods to resolve phylogenetic relationships, estimate divergence times, and infer speciation, extinction, and net diversification rates. We infer a mid-late Silurian origin for ferns (including horsetails) and an early Carboniferous origin for leptosporangiate ferns. Most derived fern families appeared in the Cretaceous and persisted for millions of years before rapidly diversifying in the Cenozoic. We find no evidence of differential rates of diversification among terrestrial and epiphytic species. Our findings challenge previous hypotheses on the evolutionary history of ferns and present a new paradigm for their Cenozoic radiation. We estimate earlier divergences for most fern lineages than were reported in previous studies and provide evidence of extended persistence of major fern lineages prior to rapid diversification in the last fifty million years.

On the widespread capacity for, and functional significance of, extreme inbreeding in ferns.

Homosporous vascular plants utilize three different mating systems, one of which, gametophytic selfing, is an extreme form of inbreeding only possible in homosporous groups. This mating system results in complete homozygosity in all progeny and has important evolutionary and ecological implications. Ferns are the largest group of homosporous land plants, and the significance of extreme inbreeding for fern evolution has been a subject of debate for decades. We cultured gametophytes in the laboratory and quantified the relative frequencies of sporophyte production from isolated and paired gametophytes, and examined associations between breeding systems and several ecological and evolutionary traits. The majority of fern species studied show a capacity for gametophytic selfing, producing sporophytes from both isolated and paired gametophytes. While we did not follow sporophytes to maturity to investigate potential detrimental effects of homozygosity at later developmental stages, our results suggest that gametophytic selfing may have greater significance for fern evolution and diversification than has previously been realized. We present evidence from the largest study of mating behavior in ferns to date that the capacity for extreme inbreeding is prevalent in this lineage, and we discuss its implications and relevance and make recommendations for future studies of fern mating systems.