Polyploid goldback and silverback ferns (Pentagramma) occupy a wider, colder, and wetter bioclimatic niche than diploid counterparts.

PREMISE: The western North American fern genus Pentagramma (Pteridaceae) is characterized by complex patterns of ploidy variation, an understanding of which is critical to comprehending both the evolutionary processes within the genus and its current diversity. METHODS: We undertook a cytogeographic study across the range of the genus, using a combination of chromosome counts and flow cytometry to infer ploidy level. Bioclimatic variables and elevation were used to compare niches. RESULTS: We found that diploids and tetraploids are common and widespread, and triploids are rare and sporadic; in contrast with genome size inferences in earlier studies, no hexaploids were found. Diploids and tetraploids show different geographic ranges: only tetraploids were found in the northernmost portion of the range (Washington, Oregon, and British Columbia) and only diploids were found in the Sierra Nevada of California. Diploid, triploid, and tetraploid cytotypes were found to co-occur in relatively few localities: in the southern (San Diego County, California) and desert Southwest (Arizona) parts of the range, and along the Pacific Coast of California. CONCLUSIONS: Tetraploids occupy a wider bioclimatic niche than diploids both within P. triangularis and at the genus-wide scale. It is unknown whether the wider niche of tetraploids is due to their expansion upon the diploid niche, if diploids have contracted their niche due to competition or changing abiotic conditions, or if this wider niche occupancy is due to multiple origins of tetraploids.

The rapid radiation of Bomarea (Alstroemeriaceae: Liliales), driven by the rise of the Andes.

Geological events such as mountain uplift affect how, when, and where species diversify, but measuring those effects is a longstanding challenge. Andean orogeny impacted the evolution of regional biota by creating barriers to gene flow, opening new habitats, and changing local climate. B⁢o⁢m⁢a⁢r⁢e⁢a (Alstroemeriaceae) are tropical plants with (often) small, isolated ranges; in total, B⁢o⁢m⁢a⁢r⁢e⁢a species occur from central Mexico to central Chile. This genus appears to have evolved rapidly and quite recently, and rapid radiations are often challenging to resolve with traditional phylogenetic inference. In this study, we apply phylogenomics-with hundreds of loci, gene-tree-based data curation, and a multispecies-coalescent approach-to infer the phylogeny of B⁢o⁢m⁢a⁢r⁢e⁢a. We use this phylogeny to untangle the potential drivers of diversification and biogeographic history. In particular, we test if Andean orogeny contributed to the diversification of B⁢o⁢m⁢a⁢r⁢e⁢a. We find that B⁢o⁢m⁢a⁢r⁢e⁢a originated in the central Andes during the mid-Miocene, then spread north, following the trajectory of mountain uplift. Furthermore, Andean lineages diversified faster than non-Andean relatives. B⁢o⁢m⁢a⁢r⁢e⁢a thus demonstrates that-at least in some cases-geological change rather than environmental stability has driven high species diversity in a tropical biodiversity hotspot. These results also demonstrate the utility (and danger) of genome-scale data for making macroevolutionary inferences.

Diversification Models Conflate Likelihood and Prior, and Cannot be Compared Using Conventional Model-Comparison Tools.

Time-calibrated phylogenetic trees are a tremendously powerful tool for studying evolutionary, ecological, and epidemiological phenomena. Such trees are predominantly inferred in a Bayesian framework, with the phylogeny itself treated as a parameter with a prior distribution (a "tree prior"). However, we show that the tree "parameter" consists, in part, of data, in the form of taxon samples. Treating the tree as a parameter fails to account for these data and compromises our ability to compare among models using standard techniques (e.g., marginal likelihoods estimated using path-sampling and stepping-stone sampling algorithms). Since accuracy of the inferred phylogeny strongly depends on how well the tree prior approximates the true diversification process that gave rise to the tree, the inability to accurately compare competing tree priors has broad implications for applications based on time-calibrated trees. We outline potential remedies to this problem, and provide guidance for researchers interested in assessing the fit of tree models. [Bayes factors; Bayesian model comparison; birth-death models; divergence-time estimation; lineage diversification].

Phasing Gene Copies into Polyploid Subgenomes Using a Bayesian Phylogenetic Approach.

This chapter describes the usage of homologizer to phase gene copies into polyploid subgenomes. Allopolyploids contain multiple copies of each genetic locus, where each copy potentially belongs to a different subgenome with its own distinct evolutionary history. If gene copies across different loci are incorrectly phased (i.e., assigned to the wrong subgenome), then the bifurcating tree assumption underlying multilocus phylogenetic inference and related analyses will be violated, leading to unsound results. homologizer is a highly flexible Bayesian method that uses a phylogenetic framework to infer the posterior probabilities of the phasing of gene copies into subgenomes. We describe how to prepare input data and other considerations needed to perform homologizer analyses and demonstrate how to visualize and interpret the results. We first walk through a basic example using homologizer to phase gene copies into polyploid subgenomes and then demonstrate how homologizer can be used as a hypothesis-testing tool to detect non-homeologous sequences such as hidden paralogs or allelic variation through the tools of Bayesian model comparison.

PURC Provides Improved Sequence Inference for Polyploid Phylogenetics and Other Manifestations of the Multiple-Copy Problem.

Inferring the true biological sequences from amplicon mixtures remains a difficult bioinformatics problem. The traditional approach is to cluster sequencing reads by similarity thresholds and treat the consensus sequence of each cluster as an "operational taxonomic unit" (OTU). Recently, this approach has been improved by model-based methods that correct PCR and sequencing errors in order to infer "amplicon sequence variants" (ASVs). To date, ASV approaches have been used primarily in metagenomics, but they are also useful for determining homeologs in polyploid organisms. To facilitate the usage of ASV methods among polyploidy researchers, we incorporated ASV inference alongside OTU clustering in PURC v2.0, a major update to PURC (Pipeline for Untangling Reticulate Complexes). In addition, PURC v2.0 features faster demultiplexing than the original version and has been updated to be compatible with Python 3. In this chapter we present results indicating that using the ASV approach is more likely to infer the correct biological sequences in comparison to the earlier OTU-based PURC and describe how to prepare sequencing data, run PURC v2.0 under several different modes, and interpret the output.

Unearthing Modes of Climatic Adaptation in Underground Storage Organs Across Liliales.

Testing adaptive hypotheses about how continuous traits evolve in association with developmentally structured discrete traits, while accounting for the confounding influence of other, hidden, evolutionary forces, remains a challenge in evolutionary biology. For example, geophytes are herbaceous plants-with underground buds-that use underground storage organs (USOs) to survive extended periods of unfavorable conditions. Such plants have evolved multiple times independently across all major vascular plant lineages. Even within closely related lineages, however, geophytes show impressive variation in the morphological modifications and structures (i.e.,"types" of USOs) that allow them to survive underground. Despite the developmental and structural complexity of USOs, the prevailing hypothesis is that they represent convergent evolutionary "solutions" to a common ecological problem, though some recent research has drawn this conclusion into question. We extend existing phylogenetic comparative methods to test for links between the hierarchical discrete morphological traits associated with USOs and adaptation to environmental variables, using a phylogeny of 621 species in Liliales. We found that plants with different USO types do not differ in climatic niche more than expected by chance, with the exception of root morphology, where modified roots are associated with lower temperature seasonality. These findings suggest that root tubers may reflect adaptations to different climatic conditions than those represented by other types of USOs. Thus, the tissue type and developmental origin of the USO structure may influence the way it mediates ecological relationships, which draws into question the appropriateness of ascribing broad ecological patterns uniformly across geophytic taxa. This work provides a new framework for testing adaptive hypotheses and for linking ecological patterns across morphologically varying taxa while accounting for developmental (non-independent) relationships in morphological data. [Climatic niche evolution; geophytes; imperfect correspondence; macroevolution.].

Travel Tales of a Worldwide Weed: Genomic Signatures of Plantago major L. Reveal Distinct Genotypic Groups With Links to Colonial Trade Routes.

Retracing pathways of historical species introductions is fundamental to understanding the factors involved in the successful colonization and spread, centuries after a species' establishment in an introduced range. Numerous plants have been introduced to regions outside their native ranges both intentionally and accidentally by European voyagers and early colonists making transoceanic journeys; however, records are scarce to document this. We use genotyping-by-sequencing and genotype-likelihood methods on the selfing, global weed, Plantago major, collected from 50 populations worldwide to investigate how patterns of genomic diversity are distributed among populations of this global weed. Although genomic differentiation among populations is found to be low, we identify six unique genotype groups showing very little sign of admixture and low degree of outcrossing among them. We show that genotype groups are latitudinally restricted, and that more than one successful genotype colonized and spread into the introduced ranges. With the exception of New Zealand, only one genotype group is present in the Southern Hemisphere. Three of the most prevalent genotypes present in the native Eurasian range gave rise to introduced populations in the Americas, Africa, Australia, and New Zealand, which could lend support to the hypothesis that P. major was unknowlingly dispersed by early European colonists. Dispersal of multiple successful genotypes is a likely reason for success. Genomic signatures and phylogeographic methods can provide new perspectives on the drivers behind the historic introductions and the successful colonization of introduced species, contributing to our understanding of the role of genomic variation for successful establishment of introduced taxa.

Polyploidy and microbiome associations mediate similar responses to pathogens in Arabidopsis.

It has become increasingly clear that the microbiome plays a critical role in shaping the host organism's response to disease. There also exists mounting evidence that an organism's ploidy level is important in their response to pathogens and parasites. However, no study has determined whether or how these two factors influence one another. We investigate the effect of whole-genome duplication in Arabidopsis thaliana on the above-ground (phyllosphere) microbiome and determine the interacting impacts of ploidy and microbiome on disease outcome. Using seven independently derived synthetic autotetraploid Arabidopsis accessions and a synthetic leaf-associated bacterial community, we confirm that polyploids are generally more resistant to the model pathogen Pseudomonas syringae pv. Tomato DC3000. Polyploids fare better against the pathogen than diploids do, regardless of microbial inoculation, whereas diploids harboring an intact microbiome have lower pathogen densities than those without. In addition, diploids have elevated numbers of defense-related genes that are differentially expressed in the presence of their phyllosphere microbiota, whereas polyploids exhibit some constitutively activated defenses, regardless of colonization by the synthetic community. These results imply that whole-genome duplication can enhance immunity, resulting in a decreased dependence on the microbiome for protection against pathogens.

Inferring the Total-Evidence Timescale of Marattialean Fern Evolution in the Face of Model Sensitivity.

Phylogenetic divergence-time estimation has been revolutionized by two recent developments: 1) total-evidence dating (or "tip-dating") approaches that allow for the incorporation of fossils as tips in the analysis, with their phylogenetic and temporal relationships to the extant taxa inferred from the data and 2) the fossilized birth-death (FBD) class of tree models that capture the processes that produce the tree (speciation, extinction, and fossilization) and thus provide a coherent and biologically interpretable tree prior. To explore the behavior of these methods, we apply them to marattialean ferns, a group that was dominant in Carboniferous landscapes prior to declining to its modest extant diversity of slightly over 100 species. We show that tree models have a dramatic influence on estimates of both divergence times and topological relationships. This influence is driven by the strong, counter-intuitive informativeness of the uniform tree prior, and the inherent nonidentifiability of divergence-time models. In contrast to the strong influence of the tree models, we find minor effects of differing the morphological transition model or the morphological clock model. We compare the performance of a large pool of candidate models using a combination of posterior-predictive simulation and Bayes factors. Notably, an FBD model with epoch-specific speciation and extinction rates was strongly favored by Bayes factors. Our best-fitting model infers stem and crown divergences for the Marattiales in the mid-Devonian and Late Cretaceous, respectively, with elevated speciation rates in the Mississippian and elevated extinction rates in the Cisuralian leading to a peak diversity of ${\sim}$2800 species at the end of the Carboniferous, representing the heyday of the Psaroniaceae. This peak is followed by the rapid decline and ultimate extinction of the Psaroniaceae, with their descendants, the Marattiaceae, persisting at approximately stable levels of diversity until the present. This general diversification pattern appears to be insensitive to potential biases in the fossil record; despite the preponderance of available fossils being from Pennsylvanian coal balls, incorporating fossilization-rate variation does not improve model fit. In addition, by incorporating temporal data directly within the model and allowing for the inference of the phylogenetic position of the fossils, our study makes the surprising inference that the clade of extant Marattiales is relatively young, younger than any of the fossils historically thought to be congeneric with extant species. This result is a dramatic demonstration of the dangers of node-based approaches to divergence-time estimation, where the assignment of fossils to particular clades is made a priori (earlier node-based studies that constrained the minimum ages of extant genera based on these fossils resulted in much older age estimates than in our study) and of the utility of explicit models of morphological evolution and lineage diversification. [Bayesian model comparison; Carboniferous; divergence-time estimation; fossil record; fossilized birth-death; lineage diversification; Marattiales; models of morphological evolution; Psaronius; RevBayes.].

Polyploid phylogenetics.

Polyploidy is a dominant feature of extant plant diversity. However, major research questions, including whether polyploidy is important to long-term evolution or is just 'evolutionary noise', remain unresolved due to difficulties associated with the generation and analysis of data from polyploid lineages. Many of these difficulties have been recently overcome, such that it is now often relatively straightforward to infer the full and often reticulate phylogenetic history of groups with recently formed polyploids. This nascent field of 'polyploid phylogenetics' allows researchers to tackle long-standing questions of polyploid macroevolution, supplies the foundation for mechanistic models of ploidy change, and provides the opportunity to include a more complete and representative sample of plant taxa in our analyses in general.

Comparative transcriptomics of a monocotyledonous geophyte reveals shared molecular mechanisms of underground storage organ formation.

Many species from across the vascular plant tree-of-life have modified standard plant tissues into tubers, bulbs, corms, and other underground storage organs (USOs), unique innovations which allow these plants to retreat underground. Our ability to understand the developmental and evolutionary forces that shape these morphologies is limited by a lack of studies on certain USOs and plant clades. We take a comparative transcriptomics approach to characterizing the molecular mechanisms of tuberous root formation in Bomarea multiflora (Alstroemeriaceae) and compare these mechanisms to those identified in other USOs across diverse plant lineages; B. multiflora fills a key gap in our understanding of USO molecular development as the first monocot with tuberous roots to be the focus of this kind of research. We sequenced transcriptomes from the growing tip of four tissue types (aerial shoot, rhizome, fibrous root, and root tuber) of three individuals of B. multiflora. We identified differentially expressed isoforms between tuberous and non-tuberous roots and tested the expression of a priori candidate genes implicated in underground storage in other taxa. We identify 271 genes that are differentially expressed in root tubers versus non-tuberous roots, including genes implicated in cell wall modification, defense response, and starch biosynthesis. We also identify a phosphatidylethanolamine-binding protein, which has been implicated in tuberization signalling in other taxa and, through gene-tree analysis, place this copy in a phylogenetic context. These findings suggest that some similar molecular processes underlie the formation of USOs across flowering plants despite the long evolutionary distances among taxa and non-homologous morphologies (e.g., bulbs vs. tubers). (Plant development, tuberous roots, comparative transcriptomics, geophytes).

A step-by-step protocol for meiotic chromosome counts in flowering plants: A powerful and economical technique revisited.

PREMISE: Counting chromosomes is a fundamental botanical technique, yet it is often intimidating and increasingly sidestepped. Once mastered, the basic protocol can be applied to a broad range of taxa and research questions. It also reveals an aspect of the plant genome that is accessible with only the most basic of resources-access to a microscope with 1000× magnification is the most limiting factor. METHODS AND RESULTS: Here we provide a detailed protocol for choosing, staining, and squashing angiosperm pollen mother cells. The protocol is supplemented by figures and two demonstration videos. CONCLUSIONS: The protocol we provide will hopefully demystify and reinvigorate a powerful and once commonplace botanical technique that is available to researchers regardless of their location and resources.

A basic ddRADseq two-enzyme protocol performs well with herbarium and silica-dried tissues across four genera.

PREMISE: The ability to sequence genome-scale data from herbarium specimens would allow for the economical development of data sets with broad taxonomic and geographic sampling that would otherwise not be possible. Here, we evaluate the utility of a basic double-digest restriction site-associated DNA sequencing (ddRADseq) protocol using DNAs from four genera extracted from both silica-dried and herbarium tissue. METHODS: DNAs from Draba, Boechera, Solidago, and Ilex were processed with a ddRADseq protocol. The effects of DNA degradation, taxon, and specimen age were assessed. RESULTS: Although taxon, preservation method, and specimen age affected data recovery, large phylogenetically informative data sets were obtained from the majority of samples. DISCUSSION: These results suggest that herbarium samples can be incorporated into ddRADseq project designs, and that specimen age can be used as a rapid on-site guide for sample choice. The detailed protocol we provide will allow users to pursue herbarium-based ddRADseq projects that minimize the expenses associated with fieldwork and sample evaluation.

Infraspecific diversification of the star cloak fern (Notholaena standleyi) in the deserts of the United States and Mexico.

PREMISE: Not all ferns grow in moist and shaded habitats. One well-known example is Notholaena standleyi, a species that thrives in deserts of the southwestern United States and Mexico. This species exhibits several "chemotypes" that differ in farina (flavonoid exudates) color and chemistry. By integrating data from molecular phylogenetics, cytology, biochemistry, and biogeography, we circumscribed the major evolutionary lineages within N. standleyi and reconstructed their diversification histories. METHODS: Forty-eight samples were selected from across the geographic distribution of N. standleyi. Phylogenetic relationships were inferred using four plastid and five nuclear markers. Ploidy levels were inferred using spore sizes calibrated by chromosome counts, and farina chemistry was compared using thin-layer chromatography. RESULTS: Four clades are recognized, three of which roughly correspond to previously recognized chemotypes. The diploid clades G and Y are found in the Sonoran and Chihuahuan deserts, respectively; they are estimated to have diverged in the Pleistocene, congruent with the postulated timing of climatological events separating these two deserts. Clade P/YG is tetraploid and partially overlaps the distribution of clade Y in the eastern Chihuahuan Desert. It is apparently confined to limestone, a geologic substrate rarely occupied by members of the other clades. The cryptic (C) clade, a diploid group known only from southern Mexico and highly disjunct from the other three clades, is newly recognized here. CONCLUSIONS: Our results reveal a complex intraspecific diversification history of N. standleyi, traceable to a variety of evolutionary drivers including classic allopatry, parapatry with or without changes in geologic substrate, and sympatric divergence through polyploidization.

A global plastid phylogeny of the fern genus Asplenium (Aspleniaceae).

The infrageneric relationships and taxonomy of the largest fern genus, Asplenium (Aspleniaceae), have remained poorly understood. Previous studies have focused mainly on specific species complexes involving a few or dozens of species only, or have achieved a large taxon sampling but only one plastid marker was used. In the present study, DNA sequences from six plastid markers (atpB, rbcL, rps4, rps4-trnS, trnL and trnL-F) of 1030 accessions (616 of them newly sequenced here) representing c. 420 species of Asplenium (60% of estimated species diversity), 16 species of Hymenasplenium, three Diplaziopsidaceae, and four Rhachidosoraceae were used to produce the largest genus-level phylogeny yet for ferns. Our major results include: (i) Asplenium as broadly circumscribed is monophyletic based on our inclusion of representatives of 32 of 38 named segregate genera; (ii) 11 major clades in Asplenium are identified, and their relationships are mostly well-resolved and strongly supported; (iii) numerous species, unsampled in previous studies, suggest new relationships and numerous cryptic species and species complexes in Asplenium; and (iv) the accrued molecular evidence provides an essential foundation for further investigations of complex patterns of geographical diversification, speciation and reticulate evolution in this family.

Organellomic data sets confirm a cryptic consensus on (unrooted) land-plant relationships and provide new insights into bryophyte molecular evolution.

PREMISE: Phylogenetic trees of bryophytes provide important evolutionary context for land plants. However, published inferences of overall embryophyte relationships vary considerably. We performed phylogenomic analyses of bryophytes and relatives using both mitochondrial and plastid gene sets, and investigated bryophyte plastome evolution. METHODS: We employed diverse likelihood-based analyses to infer large-scale bryophyte phylogeny for mitochondrial and plastid data sets. We tested for changes in purifying selection in plastid genes of a mycoheterotrophic liverwort (Aneura mirabilis) and a putatively mycoheterotrophic moss (Buxbaumia), and compared 15 bryophyte plastomes for major structural rearrangements. RESULTS: Overall land-plant relationships conflict across analyses, generally weakly. However, an underlying (unrooted) four-taxon tree is consistent across most analyses and published studies. Despite gene coverage patchiness, relationships within mosses, liverworts, and hornworts are largely congruent with previous studies, with plastid results generally better supported. Exclusion of RNA edit sites restores cases of unexpected non-monophyly to monophyly for Takakia and two hornwort genera. Relaxed purifying selection affects multiple plastid genes in mycoheterotrophic Aneura but not Buxbaumia. Plastid genome structure is nearly invariant across bryophytes, but the tufA locus, presumed lost in embryophytes, is unexpectedly retained in several mosses. CONCLUSIONS: A common unrooted tree underlies embryophyte phylogeny, [(liverworts, mosses), (hornworts, vascular plants)]; rooting inconsistency across studies likely reflects substantial distance to algal outgroups. Analyses combining genomic and transcriptomic data may be misled locally for heavily RNA-edited taxa. The Buxbaumia plastome lacks hallmarks of relaxed selection found in mycoheterotrophic Aneura. Autotrophic bryophyte plastomes, including Buxbaumia, hardly vary in overall structure.

Modifications during Early Plant Development Promote the Evolution of Nature's Most Complex Woods.

Secondary growth is the developmental process by which woody plants grow radially. The most complex presentations of secondary growth are found in lianas (woody vines) as a result of the unique demand to maintain stems that can twist without breaking. The complex woody forms in lianas arise as non-circular stem outlines, aberrant tissue configurations, and/or shifts in the relative abundance of secondary tissues. Previous studies demonstrate that abnormal activity of the vascular cambium leads to variant secondary growth; however, the developmental and evolutionary basis for this shift is still largely unknown. Here, we adopt an integrative approach, leveraging techniques from historically distinct disciplines-developmental anatomy and phylogenetic comparative methods-to elucidate the evolution of development of the complex woody forms in a large lineage of tropical lianas, Paullinia L. (Sapindaceae). We find that all forms of variant secondary growth trace back to the same modification during early stem development, which results in young plants with lobed stem outlines and a discontinuous distribution of vascular bundles. By placing development in a phylogenetic context, we further show that the lobed primary plant bauplan is the evolutionary precursor to all complex woody forms. We find evidence for three evolutionary mechanisms that generate phenotypic novelty: exaptation and co-opting of the ancestral bauplan, the quasi-independence of the interfascicular and fascicular cambia, and the inclusion of additional developmental stages to the end of the ancestral ontogeny. Our study demonstrates the utility of integrating developmental data within a phylogenetic framework to investigate the evolution of complex traits.

Phylogeny of Paullinia L. (Paullinieae: Sapindaceae), a diverse genus of lianas with dynamic fruit evolution.

Paullinia L. is a genus of c. 220 mostly Neotropical forest-dwelling lianas that display a wide diversity of fruit morphologies. Paullinia resembles other members of the Paullinieae tribe in being a climber with stipulate compound leaves and paired inflorescence tendrils. However, it is distinct in having capsular fruits with woody, coriaceous, or crustaceous pericarps. While consistent in this basic plan, the pericarps of Paullinia fruits are otherwise highly variable-in some species they are winged, whereas in others they are without wings or covered with spines. With the exception of the water-dispersed indehiscent spiny fruits of some members of Paullinia sect. Castanella, all species are dehiscent, opening their capsules while they are still attached to the branch, to reveal arillate animal-dispersed seeds. Here we present a molecular phylogeny of Paullinia derived from 11 molecular markers, including nine newly-developed single-copy nuclear markers amplified by microfluidics PCR. This is the first broadly sampled molecular phylogeny for the genus. Paullinia is supported as monophyletic and is sister to Cardiospermum L., which together are sister to Serjania Mill + Urvillea Kunth. We apply this novel phylogenetic hypothesis to test previous infrageneric classifications and to infer that unwinged fruits represent the ancestral condition, from which there were repeated evolutionary transitions and reversals. However, because the seeds of both winged and unwinged fruits are dispersed by animals, we conclude that the repeated transitions in fruit morphology may relate to visual display strategies to attract animal dispersers, and do not represent transitions to wind dispersal.

Low-copy nuclear sequence data confirm complex patterns of farina evolution in notholaenid ferns (Pteridaceae).

Notholaenids are an unusual group of ferns that have adapted to, and diversified within, the deserts of Mexico and the southwestern United States. With approximately 40 species, this group is noted for being desiccation-tolerant and having "farina"-powdery exudates of lipophilic flavonoid aglycones-that occur on both the gametophytic and sporophytic phases of their life cycle. The most recent circumscription of notholaenids based on plastid markers surprisingly suggests that several morphological characters, including the expression of farina, are homoplasious. In a striking case of convergence, Notholaena standleyi appears to be distantly related to core Notholaena, with several taxa not before associated with Notholaena nested between them. Such conflicts can be due to morphological homoplasy resulting from adaptive convergence or, alternatively, the plastid phylogeny itself might be misleading, diverging from the true species tree due to incomplete lineage sorting, hybridization, or other factors. In this study, we present a species phylogeny for notholaenid ferns, using four low-copy nuclear loci and concatenated data from three plastid loci. A total of 61 individuals (49 notholaenids and 12 outgroup taxa) were sampled, including 31 out of 37 recognized notholaenid species. The homeologous/allelic nuclear sequences were retrieved using PacBio sequencing and the PURC bioinformatics pipeline. Each dataset was first analyzed individually using maximum likelihood and Bayesian inference, and the species phylogeny was inferred using *BEAST. Although we observed several incongruences between the nuclear and plastid phylogenies, our principal results are broadly congruent with previous inferences based on plastid data. By mapping the presence of farina and their biochemical constitutions on our consensus phylogenetic tree, we confirmed that the characters are indeed homoplastic and have complex evolutionary histories. Hybridization among recognized species of the notholaenid clade appears to be relatively rare compared to that observed in other well-studied fern genera.

Widespread co-occurrence of multiple ploidy levels in fragile ferns (Cystopteris fragilis complex; Cystopteridaceae) probably stems from similar ecology of cytotypes, their efficient dispersal and inter-ploidy hybridization.

BACKGROUND AND AIMS: Polyploidy has played an important role in the evolution of ferns. However, the dearth of data on cytotype diversity, cytotype distribution patterns and ecology in ferns is striking in comparison with angiosperms and prevents an assessment of whether cytotype coexistence and its mechanisms show similar patterns in both plant groups. Here, an attempt to fill this gap was made using the ploidy-variable and widely distributed Cystopteris fragilis complex. METHODS: Flow cytometry was used to assess DNA ploidy level and monoploid genome size (Cx value) of 5518 C. fragilis individuals from 449 populations collected over most of the species' global distributional range, supplemented with data from 405 individuals representing other related species from the complex. Ecological preferences of C. fragilis tetraploids and hexaploids were compared using field-recorded parameters and database-extracted climate data. KEY RESULTS: Altogether, five different ploidy levels (2x, 4x, 5x, 6x, 8x) were detected and three species exhibited intraspecific ploidy-level variation: C. fragilis, C. alpina and C. diaphana. Two predominant C. fragilis cytotypes, tetraploids and hexaploids, co-occur over most of Europe in a diffuse, mosaic-like pattern. Within this contact zone, 40 % of populations were mixed-ploidy and most also contained pentaploid hybrids. Environmental conditions had only a limited effect on the distribution of cytotypes. Differences were found in the Cx value of tetraploids and hexaploids: between-cytotype divergence was higher in uniform-ploidy than in mixed-ploidy populations. CONCLUSIONS: High ploidy-level diversity and widespread cytotype coexistence in the C. fragilis complex match the well-documented patterns in some angiosperms. While ploidy coexistence in C. fragilis is not driven by environmental factors, it could be facilitated by the perennial life-form of the species, its reproductive modes and efficient wind dispersal of spores. Independent origins of hexaploids and/or inter-ploidy gene flow may be expected in mixed-ploidy populations according to Cx value comparisons.