Striking genetic homogeneity in the widespread South American bracken.

PREMISE: Bracken (Pteridium, Dennstaedtiaceae) is a cosmopolitan genus of aggressive disturbance colonizers that are toxic to agricultural livestock. The taxonomy of Pteridium has been treated in multiple schemes, ranging from one to six species worldwide, with numerous subspecies and varieties. Recent work has focused on the worldwide distribution and systematics of the bracken fern, but South America has been poorly represented. We present the first continent-wide sampling and analysis of Pteridium esculentum, a Southern Hemisphere diploid species. METHODS: Within South America, P. esculentum has several morphotypes, distinguished into subspecies by variation in indument and lamina architecture. We used double digest restriction site-associated DNA sequencing (ddRADSeq) to assess the phylogenetic relationships of P. esculentum subspecies. RESULTS: We found a striking genetic homogeneity in the species, being able to support only two morphotypes from molecular data: P. e. arachnoideum and P. e. campestre. We had high confidence for shallow and deep phylogenetic relationships, but less support for relationships among crown groups. CONCLUSIONS: We describe an east-west geographic pattern that would explain the relationships between populations; and, in contrast to previous studies, we detected differences with P. esculentum from Australia. These results will lay the foundations for studying variations in this species' behavior as a weed, as well as its impact on the production of agricultural livestock in South America.

Cryptic diversity in the model fern genus Ceratopteris (Pteridaceae).

Cryptic species are present throughout the tree of life. They are especially prevalent in ferns, because of processes such hybridization, polyploidy, and reticulate evolution. In addition, the simple morphology of ferns limits phenotypic variation and makes it difficult to detect cryptic species. The model fern genus Ceratopteris has long been suspected to harbor cryptic diversity, in particular within the highly polymorphic C. thalictroides. Yet no studies have included samples from throughout its pan-tropical range or utilized genomic sequencing, making it difficult to assess the full extent of cryptic variation within this genus. Here, we present the first multilocus phylogeny of the genus using reduced representation genomic sequencing (RADseq) and examine population structure, phylogenetic relationships, and ploidy level variation. We recover similar species relationships found in previous studies, find support for the cryptic species C. gaudichaudii as genetically distinct, and identify novel genomic variation within two of the mostly broadly distributed species in the genus, C. thalictroides and C. cornuta. Finally, we detail the utility of our approach for working on cryptic, reticulate groups of ferns. Specifically, it does not require a reference genome, of which there are very few available for ferns. RADseq is a cost-effective way to work with study groups lacking genomic resources, and to obtain the thousands of nuclear markers needed to untangle species complexes.

Worldwide relationships in the fern genus Pteridium (bracken) based on nuclear genome markers.

PREMISE: Spore-bearing plants are capable of dispersing very long distances. However, it is not known if gene flow can prevent genetic divergence in widely distributed taxa. Here we address this issue, and examine systematic relationships at a global geographic scale for the fern genus Pteridium. METHODS: We sampled plants from 100 localities worldwide, and generated nucleotide data from four nuclear genes and two plastid regions. We also examined 2801 single nucleotide polymorphisms detected by a restriction site-associated DNA approach. RESULTS: We found evidence for two distinct diploid species and two allotetraploids between them. The "northern" species (Pteridium aquilinum) has distinct groups at the continental scale (Europe, Asia, Africa, and North America). The northern European subspecies pinetorum appears to involve admixture among all of these. A sample from the Hawaiian Islands contained elements of both North American and Asian P. aquilinum. The "southern" species, P. esculentum, shows little genetic differentiation between South American and Australian samples. Components of African genotypes are detected on all continents. CONCLUSIONS: We find evidence of distinct continental-scale genetic differentiation in Pteridium. However, on top of this is a clear signal of recent hybridization. Thus, spore-bearing plants are clearly capable of extensive long-distance gene flow; yet appear to have differentiated genetically at the continental scale. Either gene flow in the past was at a reduced level, or vicariance is possible even in the face of long-distance gene flow.

Admixture, evolution, and variation in reproductive isolation in the Boechera puberula clade.

BACKGROUND: Hybridization is very common in plants, and the incorporation of new alleles into existing lineages (i.e. admixture) can blur species boundaries. However, admixture also has the potential to increase standing genetic variation. With new sequencing methods, we can now study admixture and reproductive isolation at a much finer scale than in the past. The genus Boechera is an extraordinary example of admixture, with over 400 hybrid derivates of varying ploidy levels. Yet, few studies have assessed admixture in this genus on a genomic scale. RESULTS: In this study, we used Genotyping-by-Sequencing (GBS) to clarify the evolution of the Boechera puberula clade, whose six members are scattered across the western United States. We further assessed patterns of admixture and reproductive isolation within the group, including two additional species (B. stricta and B. retrofracta) that are widespread across North America. Based on 14,815 common genetic variants, we found evidence for some cases of hybridization. We find evidence of both recent and more ancient admixture, and that levels of admixture vary across species. CONCLUSIONS: We present evidence for a monophyletic origin of the B. puberula group, and a split of B. puberula into two subspecies. Further, when inferring reproductive isolation on the basis of presence and absence of admixture, we found that the accumulation of reproductive isolation between species does not seem to occur linearly with time since divergence in this system. We discuss our results in the context of sexuality and asexuality in Boechera.

Evolutionary Conservation of ABA Signaling for Stomatal Closure.

Abscisic acid (ABA)-driven stomatal regulation reportedly evolved after the divergence of ferns, during the early evolution of seed plants approximately 360 million years ago. This hypothesis is based on the observation that the stomata of certain fern species are unresponsive to ABA, but exhibit passive hydraulic control. However, ABA-induced stomatal closure was detected in some mosses and lycophytes. Here, we observed that a number of ABA signaling and membrane transporter protein families diversified over the evolutionary history of land plants. The aquatic ferns Azolla filiculoides and Salvinia cucullata have representatives of 23 families of proteins orthologous to those of Arabidopsis (Arabidopsis thaliana) and all other land plant species studied. Phylogenetic analysis of the key ABA signaling proteins indicates an evolutionarily conserved stomatal response to ABA. Moreover, comparative transcriptomic analysis has identified a suite of ABA-responsive genes that differentially expressed in a terrestrial fern species, Polystichum proliferum These genes encode proteins associated with ABA biosynthesis, transport, reception, transcription, signaling, and ion and sugar transport, which fit the general ABA signaling pathway constructed from Arabidopsis and Hordeum vulgare The retention of these key ABA-responsive genes could have had a profound effect on the adaptation of ferns to dry conditions. Furthermore, stomatal assays have shown the primary evidence for ABA-induced closure of stomata in two terrestrial fern species Pproliferum and Nephrolepis exaltata In summary, we report, to our knowledge, new molecular and physiological evidence for the presence of active stomatal control in ferns.

The biology of C. richardii as a tool to understand plant evolution.

The fern Ceratopteris richardii has been studied as a model organism for over 50 years because it is easy to grow and has a short life cycle. In particular, as the first homosporous vascular plant for which genomic resources were developed, C. richardii has been an important system for studying plant evolution. However, we know relatively little about the natural history of C. richardii. In this article, we summarize what is known about this aspect of C. richardii, and discuss how learning more about its natural history could greatly increase our understanding of the evolution of land plants.

Why Do Heterosporous Plants Have So Few Chromosomes?

The mechanisms controlling chromosome number, size, and shape, and the relationship of these traits to genome size, remain some of the least understood aspects of genome evolution. Across vascular plants, there is a striking disparity in chromosome number between homosporous and heterosporous lineages. Homosporous plants (comprising most ferns and some lycophytes) have high chromosome numbers compared to heterosporous lineages (some ferns and lycophytes and all seed plants). Many studies have investigated why homosporous plants have so many chromosomes. However, homospory is the ancestral condition from which heterospory has been derived several times. Following this phylogenetic perspective, a more appropriate question to ask is why heterosporous plants have so few chromosomes. Here, we review life history differences between heterosporous and homosporous plants, previous work on chromosome number and genome size in each lineage, known mechanisms of genome downsizing and chromosomal rearrangements, and conclude with future prospects for comparative research.

Dynamic genome evolution in a model fern.

The large size and complexity of most fern genomes have hampered efforts to elucidate fundamental aspects of fern biology and land plant evolution through genome-enabled research. Here we present a chromosomal genome assembly and associated methylome, transcriptome and metabolome analyses for the model fern species Ceratopteris richardii. The assembly reveals a history of remarkably dynamic genome evolution including rapid changes in genome content and structure following the most recent whole-genome duplication approximately 60 million years ago. These changes include massive gene loss, rampant tandem duplications and multiple horizontal gene transfers from bacteria, contributing to the diversification of defence-related gene families. The insertion of transposable elements into introns has led to the large size of the Ceratopteris genome and to exceptionally long genes relative to other plants. Gene family analyses indicate that genes directing seed development were co-opted from those controlling the development of fern sporangia, providing insights into seed plant evolution. Our findings and annotated genome assembly extend the utility of Ceratopteris as a model for investigating and teaching plant biology.

De novo characterization of the gametophyte transcriptome in bracken fern, Pteridium aquilinum.

BACKGROUND: Because of their phylogenetic position and unique characteristics of their biology and life cycle, ferns represent an important lineage for studying the evolution of land plants. Large and complex genomes in ferns combined with the absence of economically important species have been a barrier to the development of genomic resources. However, high throughput sequencing technologies are now being widely applied to non-model species. We leveraged the Roche 454 GS-FLX Titanium pyrosequencing platform in sequencing the gametophyte transcriptome of bracken fern (Pteridium aquilinum) to develop genomic resources for evolutionary studies. RESULTS: 681,722 quality and adapter trimmed reads totaling 254 Mbp were assembled de novo into 56,256 unique sequences (i.e. unigenes) with a mean length of 547.2 bp and a total assembly size of 30.8 Mbp with an average read-depth coverage of 7.0×. We estimate that 87% of the complete transcriptome has been sequenced and that all transcripts have been tagged. 61.8% of the unigenes had blastx hits in the NCBI nr protein database, representing 22,596 unique best hits. The longest open reading frame in 52.2% of the unigenes had positive domain matches in InterProScan searches. We assigned 46.2% of the unigenes with a GO functional annotation and 16.0% with an enzyme code annotation. Enzyme codes were used to retrieve and color KEGG pathway maps. A comparative genomics approach revealed a substantial proportion of genes expressed in bracken gametophytes to be shared across the genomes of Arabidopsis, Selaginella and Physcomitrella, and identified a substantial number of potentially novel fern genes. By comparing the list of Arabidopsis genes identified by blast with a list of gametophyte-specific Arabidopsis genes taken from the literature, we identified a set of potentially conserved gametophyte specific genes. We screened unigenes for repetitive sequences to identify 548 potentially-amplifiable simple sequence repeat loci and 689 expressed transposable elements. CONCLUSIONS: This study is the first comprehensive transcriptome analysis for a fern and represents an important scientific resource for comparative evolutionary and functional genomics studies in land plants. We demonstrate the utility of high-throughput sequencing of a normalized cDNA library for de novo transcriptome characterization and gene discovery in a non-model plant.

The evolution of chloroplast genes and genomes in ferns.

Most of the publicly available data on chloroplast (plastid) genes and genomes come from seed plants, with relatively little information from their sister group, the ferns. Here we describe several broad evolutionary patterns and processes in fern plastid genomes (plastomes), and we include some new plastome sequence data. We review what we know about the evolutionary history of plastome structure across the fern phylogeny and we compare plastome organization and patterns of evolution in ferns to those in seed plants. A large clade of ferns is characterized by a plastome that has been reorganized with respect to the ancestral gene order (a similar order that is ancestral in seed plants). We review the sequence of inversions that gave rise to this organization. We also explore global nucleotide substitution patterns in ferns versus those found in seed plants across plastid genes, and we review the high levels of RNA editing observed in fern plastomes.

Complete plastome sequences of Equisetum arvense and Isoetes flaccida: implications for phylogeny and plastid genome evolution of early land plant lineages.

BACKGROUND: Despite considerable progress in our understanding of land plant phylogeny, several nodes in the green tree of life remain poorly resolved. Furthermore, the bulk of currently available data come from only a subset of major land plant clades. Here we examine early land plant evolution using complete plastome sequences including two previously unexamined and phylogenetically critical lineages. To better understand the evolution of land plants and their plastomes, we examined aligned nucleotide sequences, indels, gene and nucleotide composition, inversions, and gene order at the boundaries of the inverted repeats. RESULTS: We present the plastome sequences of Equisetum arvense, a horsetail, and of Isoetes flaccida, a heterosporous lycophyte. Phylogenetic analysis of aligned nucleotides from 49 plastome genes from 43 taxa supported monophyly for the following clades: embryophytes (land plants), lycophytes, monilophytes (leptosporangiate ferns + Angiopteris evecta + Psilotum nudum + Equisetum arvense), and seed plants. Resolution among the four monilophyte lineages remained moderate, although nucleotide analyses suggested that P. nudum and E. arvense form a clade sister to A. evecta + leptosporangiate ferns. Results from phylogenetic analyses of nucleotides were consistent with the distribution of plastome gene rearrangements and with analysis of sequence gaps resulting from insertions and deletions (indels). We found one new indel and an inversion of a block of genes that unites the monilophytes. CONCLUSIONS: Monophyly of monilophytes has been disputed on the basis of morphological and fossil evidence. In the context of a broad sampling of land plant data we find several new pieces of evidence for monilophyte monophyly. Results from this study demonstrate resolution among the four monilophytes lineages, albeit with moderate support; we posit a clade consisting of Equisetaceae and Psilotaceae that is sister to the "true ferns," including Marattiaceae.

Chloroplast genome sequence of the moss Tortula ruralis: gene content, polymorphism, and structural arrangement relative to other green plant chloroplast genomes.

BACKGROUND: Tortula ruralis, a widely distributed species in the moss family Pottiaceae, is increasingly used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of T. ruralis, only the second published chloroplast genome for a moss, and the first for a vegetatively desiccation-tolerant plant. RESULTS: The Tortula chloroplast genome is approximately 123,500 bp, and differs in a number of ways from that of Physcomitrella patens, the first published moss chloroplast genome. For example, Tortula lacks the approximately 71 kb inversion found in the large single copy region of the Physcomitrella genome and other members of the Funariales. Also, the Tortula chloroplast genome lacks petN, a gene found in all known land plant plastid genomes. In addition, an unusual case of nucleotide polymorphism was discovered. CONCLUSIONS: Although the chloroplast genome of Tortula ruralis differs from that of the only other sequenced moss, Physcomitrella patens, we have yet to determine the biological significance of the differences. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for mosses) of the generation of DNA markers for fine-level phylogenetic studies, or to investigate individual variation within populations.

The evolution of chloroplast genome structure in ferns.

The plastid genome (plastome) is a rich source of phylogenetic and other comparative data in plants. Most land plants possess a plastome of similar structure. However, in a major group of plants, the ferns, a unique plastome structure has evolved. The gene order in ferns has been explained by a series of genomic inversions relative to the plastome organization of seed plants. Here, we examine for the first time the structure of the plastome across fern phylogeny. We used a PCR-based strategy to map and partially sequence plastomes. We found that a pair of partially overlapping inversions in the region of the inverted repeat occurred in the common ancestor of most ferns. However, the ancestral (seed plant) structure is still found in early diverging branches leading to the osmundoid and filmy fern lineages. We found that a second pair of overlapping inversions occurred on a branch leading to the core leptosporangiates. We also found that the unique placement of the gene matK in ferns (lacking a flanking intron) is not a result of a large-scale inversion, as previously thought. This is because the intron loss maps to an earlier point on the phylogeny than the nearby inversion. We speculate on why inversions may occur in pairs and what this may mean for the dynamics of plastome evolution.

How Many Tree Species of Birch Are in Alaska? Implications for Wetland Designations.

Wetland areas are critical habitats, especially in northern regions of North America. Wetland classifications are based on several factors, including the presence of certain plant species and assemblages of species, of which trees play a significant role. Here we examined wetland species of birch (Betula) in North America, with a focus on Alaska, and the use of birche tree species in wetland delineation. We sampled over 200 trees from sites, including Alaska, Alberta, Minnesota, and New Hampshire. We used genetic data from over 3000 loci detected by restriction site associated DNA analysis. We used an indirect estimate of ploidy based on allelic ratios and we also examined population genetic structure. We find that inferred ploidy is strongly associated with genetic groupings. We find two main distinct groups; one found throughout most of Alaska, extending into Alberta. This group is probably attributable to Betula kenaica, Betula neoalaskana, or both. This group has a diploid genetic pattern although this could easily be a function of allopolyploidy. The second major genetic group appears to extend from Eastern North America into parts of southeastern Alaska. This group represents Betula papyrifera, and is not diploid based on allelic ratios. Published chromosome counts indicate pentaploidy. Because B. papyrifera is the only one of the above species that is distinctly associated with wetland habitats, our findings indicate that tree species of birch found in most parts of Alaska are not reliable indicators of wetland habitats. These results help to support stronger wetland ratings assigned to the tree species of birch for delineation purposes.

Functional gene losses occur with minimal size reduction in the plastid genome of the parasitic liverwort Aneura mirabilis.

Aneura mirabilis is a parasitic liverwort that exploits an existing mycorrhizal association between a basidiomycete and a host tree. This unusual liverwort is the only known parasitic seedless land plant with a completely nonphotosynthetic life history. The complete plastid genome of A. mirabilis was sequenced to examine the effect of its nonphotosynthetic life history on plastid genome content. Using a partial genomic fosmid library approach, the genome was sequenced and shown to be 108,007 bp with a structure typical of green plant plastids. Comparisons were made with the plastid genome of Marchantia polymorpha, the only other liverwort plastid sequence available. All ndh genes are either absent or pseudogenes. Five of 15 psb genes are pseudogenes, as are 2 of 6 psa genes and 2 of 6 pet genes. Pseudogenes of cysA, cysT, ccsA, and ycf3 were also detected. The remaining complement of genes present in M. polymorpha is present in the plastid of A. mirabilis with intact open reading frames. All pseudogenes and gene losses co-occur with losses detected in the plastid of the parasitic angiosperm Epifagus virginiana, though the latter has functional gene losses not found in A. mirabilis. The plastid genome sequence of A. mirabilis represents only the second liverwort, and first mycoheterotroph, to have its plastid genome sequenced. We observed a pattern of genome evolution congruent with functional gene losses in parasitic angiosperms but suggest that its plastid genome represents a genome in the early stages of decay following the relaxation of selection pressures.

ReFernment: An R package for annotating RNA editing in plastid genomes.

PREMISE OF THE STUDY: In the absence of cDNA, the annotation of RNA editing in plastomes must be done manually, representing a significant time cost to those studying the organellar genomes of ferns and hornworts. METHODS AND RESULTS: We developed an R package to automatically annotate apparent nonsense mutations in plastid genomes. The software successfully annotates such sites and results in no false positives for data with no sequencing or assembly errors. CONCLUSIONS: Compared to manual annotation, ReFernment offers greater speed and accuracy for annotating RNA editing sites. This software should be especially useful for researchers generating large numbers of plastome sequences for taxa with high levels of RNA editing.

The C-Fern (Ceratopteris richardii) genome: insights into plant genome evolution with the first partial homosporous fern genome assembly.

Ferns are notorious for possessing large genomes and numerous chromosomes. Despite decades of speculation, the processes underlying the expansive genomes of ferns are unclear, largely due to the absence of a sequenced homosporous fern genome. The lack of this crucial resource has not only hindered investigations of evolutionary processes responsible for the unusual genome characteristics of homosporous ferns, but also impeded synthesis of genome evolution across land plants. Here, we used the model fern species Ceratopteris richardii to address the processes (e.g., polyploidy, spread of repeat elements) by which the large genomes and high chromosome numbers typical of homosporous ferns may have evolved and have been maintained. We directly compared repeat compositions in species spanning the green plant tree of life and a diversity of genome sizes, as well as both short- and long-read-based assemblies of Ceratopteris. We found evidence consistent with a single ancient polyploidy event in the evolutionary history of Ceratopteris based on both genomic and cytogenetic data, and on repeat proportions similar to those found in large flowering plant genomes. This study provides a major stepping-stone in the understanding of land plant evolutionary genomics by providing the first homosporous fern reference genome, as well as insights into the processes underlying the formation of these massive genomes.

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.

Mobile Elements Shape Plastome Evolution in Ferns.

Plastid genomes display remarkable organizational stability over evolutionary time. From green algae to angiosperms, most plastid genomes are largely collinear, with only a few cases of inversion, gene loss, or, in extremely rare cases, gene addition. These plastome insertions are mostly clade-specific and are typically of nuclear or mitochondrial origin. Here, we expand on these findings and present the first family-level survey of plastome evolution in ferns, revealing a novel suite of dynamic mobile elements. Comparative plastome analyses of the Pteridaceae expose several mobile open reading frames that vary in sequence length, insertion site, and configuration among sampled taxa. Even between close relatives, the presence and location of these elements is widely variable when viewed in a phylogenetic context. We characterize these elements and refer to them collectively as Mobile Open Reading Frames in Fern Organelles (MORFFO). We further note that the presence of MORFFO is not restricted to Pteridaceae, but is found across ferns and other plant clades. MORFFO elements are regularly associated with inversions, intergenic expansions, and changes to the inverted repeats. They likewise appear to be present in mitochondrial and nuclear genomes of ferns, indicating that they can move between genomic compartments with relative ease. The origins and functions of these mobile elements are unknown, but MORFFO appears to be a major driver of structural genome evolution in the plastomes of ferns, and possibly other groups of plants.

Fern genomes elucidate land plant evolution and cyanobacterial symbioses.

Ferns are the closest sister group to all seed plants, yet little is known about their genomes other than that they are generally colossal. Here, we report on the genomes of Azolla filiculoides and Salvinia cucullata (Salviniales) and present evidence for episodic whole-genome duplication in ferns-one at the base of 'core leptosporangiates' and one specific to Azolla. One fern-specific gene that we identified, recently shown to confer high insect resistance, seems to have been derived from bacteria through horizontal gene transfer. Azolla coexists in a unique symbiosis with N2-fixing cyanobacteria, and we demonstrate a clear pattern of cospeciation between the two partners. Furthermore, the Azolla genome lacks genes that are common to arbuscular mycorrhizal and root nodule symbioses, and we identify several putative transporter genes specific to Azolla-cyanobacterial symbiosis. These genomic resources will help in exploring the biotechnological potential of Azolla and address fundamental questions in the evolution of plant life.