Variation in frequency of plastid RNA editing within Adiantum implies rapid evolution in fern plastomes.

PREMISE: Recent studies of plant RNA editing have demonstrated that the number of editing sites can vary widely among large taxonomic groups (orders, families). Yet, very little is known about intrageneric variation in frequency of plant RNA editing, and no study has been conducted in ferns. METHODS: We determined plastid RNA-editing counts for two species of Adiantum (Pteridaceae), A. shastense and A. aleuticum, by implementing a pipeline that integrated read-mapping and SNP-calling software to identify RNA-editing sites. We then compared the edits found in A. aleuticum and A. shastense with previously published edits from A. capillus-veneris by generating alignments for each plastid gene. RESULTS: We found direct evidence for 505 plastid RNA-editing sites in A. aleuticum and 509 in A. shastense, compared with 350 sites in A. capillus-veneris. We observed striking variation in the number and location of the RNA-editing sites among the three species, with reverse (U-to-C) editing sites showing a higher degree of conservation than forward (C-to-U) sites. Additionally, sites involving start and stop codons were highly conserved. CONCLUSIONS: Variation in the frequency of RNA editing within Adiantum implies that RNA-editing sites can be rapidly gained or lost throughout evolution. However, varying degrees of conservation between both C-to-U and U-to-C sites and sites in start or stop codons, versus other codons, hints at the likely independent origin of both types of edits and a potential selective advantage conferred by RNA editing.

Seed size evolution and biogeography of Plukenetia (Euphorbiaceae), a pantropical genus with traditionally cultivated oilseed species.

BACKGROUND: Plukenetia is a small pantropical genus of lianas and vines with variably sized edible oil-rich seeds that presents an ideal system to investigate neotropical and pantropical diversification patterns and seed size evolution. We assessed the biogeography and seed evolution of Plukenetia through phylogenetic analyses of a 5069 character molecular dataset comprising five nuclear and two plastid markers for 86 terminals in subtribe Plukenetiinae (representing 20 of ~ 23 Plukenetia species). Two nuclear genes, KEA1 and TEB, were used for phylogenetic reconstruction for the first time. Our goals were: (1) produce a robust, time-dependent evolutionary framework for Plukenetia using BEAST; (2) reconstruct its biogeographical history with ancestral range estimation in BIOGEOBEARS; (3) define seed size categories; (4) identify patterns of seed size evolution using ancestral state estimation; and (5) conduct regression analyses with putative drivers of seed size using the threshold model. RESULTS: Plukenetia was resolved into two major groups, which we refer to as the pinnately- and palmately-veined clades. Our analyses suggest Plukenetia originated in the Amazon or Atlantic Forest of Brazil during the Oligocene (28.7 Mya) and migrated/dispersed between those regions and Central America/Mexico throughout the Miocene. Trans-oceanic dispersals explain the pantropical distribution of Plukenetia, including from the Amazon to Africa in the Early Miocene (17.4 Mya), followed by Africa to Madagascar and Africa to Southeast Asia in the Late Miocene (9.4 Mya) and Pliocene (4.5 Mya), respectively. We infer a single origin of large seeds in the ancestor of Plukenetia. Seed size fits a Brownian motion model of trait evolution and is moderately to strongly associated with plant size, fruit type/dispersal syndrome, and seedling ecology. Biome shifts were not drivers of seed size, although there was a weak association with a transition to fire prone semi-arid savannas. CONCLUSIONS: The major relationships among the species of Plukenetia are now well-resolved. Our biogeographical analyses support growing evidence that many pantropical distributions developed by periodic trans-oceanic dispersals throughout the Miocene and Pliocene. Selection on a combination of traits contributed to seed size variation, while movement between forest edge/light gap and canopy niches likely contributed to the seed size extremes in Plukenetia.

Overlapping Patterns of Gene Expression Between Gametophyte and Sporophyte Phases in the Fern Polypodium amorphum (Polypodiales).

Ferns are unique among land plants in having sporophyte and gametophyte phases that are both free living and fully independent. Here, we examine patterns of sporophytic and gametophytic gene expression in the fern Polypodium amorphum, a member of the homosporous polypod lineage that comprises 80% of extant fern diversity, to assess how expression of a common genome is partitioned between two morphologically, ecologically, and nutritionally independent phases. Using RNA-sequencing, we generated transcriptome profiles for three replicates of paired samples of sporophyte leaf tissue and whole gametophytes to identify genes with significant differences in expression between the two phases. We found a nearly 90% overlap in the identity and expression levels of the genes expressed in both sporophytes and gametophytes, with less than 3% of genes uniquely expressed in either phase. We compare our results to those from similar studies to establish how phase-specific gene expression varies among major land plant lineages. Notably, despite having greater similarity in the identity of gene families shared between P. amorphum and angiosperms, P. amorphum has phase-specific gene expression profiles that are more like bryophytes and lycophytes than seed plants. Our findings suggest that shared patterns of phase-specific gene expression among seed-free plants likely reflect having relatively large, photosynthetic gametophytes (compared to the gametophytes of seed plants that are highly reduced). Phylogenetic analyses were used to further investigate the evolution of phase-specific expression for the phototropin, terpene synthase, and MADS-box gene families.

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.

Homoeologous chromosome pairing across the eukaryote phylogeny.

During the past quarter century, molecular phylogenetic inferences have significantly resolved evolutionary relationships spanning the eukaryotic tree of life. With improved phylogenies in hand, the focus of systematics will continue to expand from estimating species relationships toward examining the evolution of specific, fundamental traits across the eukaryotic tree. Undoubtedly, this will expose knowledge gaps in the evolution of key traits, particularly with respect to non-model lineages. Here, we examine one such trait across eukaryotes-the regulation of homologous chromosome pairing during meiosis-as an illustrative example. Specifically, we present an overview of the breakdown of homologous chromosome pairing in model eukaryotes and provide a discussion of various meiotic aberrations that result in the failure of homolog recognition, with a particular focus on lineages with a history of hybridization and polyploidization, across major eukaryotic clades. We then explore what is known about these processes in natural and non-model eukaryotic taxa, thereby exposing disparities in our understanding of this key trait among non-model groups.

An Exploration into Fern Genome Space.

Ferns are one of the few remaining major clades of land plants for which a complete genome sequence is lacking. Knowledge of genome space in ferns will enable broad-scale comparative analyses of land plant genes and genomes, provide insights into genome evolution across green plants, and shed light on genetic and genomic features that characterize ferns, such as their high chromosome numbers and large genome sizes. As part of an initial exploration into fern genome space, we used a whole genome shotgun sequencing approach to obtain low-density coverage (∼0.4X to 2X) for six fern species from the Polypodiales (Ceratopteris, Pteridium, Polypodium, Cystopteris), Cyatheales (Plagiogyria), and Gleicheniales (Dipteris). We explore these data to characterize the proportion of the nuclear genome represented by repetitive sequences (including DNA transposons, retrotransposons, ribosomal DNA, and simple repeats) and protein-coding genes, and to extract chloroplast and mitochondrial genome sequences. Such initial sweeps of fern genomes can provide information useful for selecting a promising candidate fern species for whole genome sequencing. We also describe variation of genomic traits across our sample and highlight some differences and similarities in repeat structure between ferns and seed plants.

The evolutionary history of ferns inferred from 25 low-copy nuclear genes.

UNLABELLED: • PREMISE OF THE STUDY: Understanding fern (monilophyte) phylogeny and its evolutionary timescale is critical for broad investigations of the evolution of land plants, and for providing the point of comparison necessary for studying the evolution of the fern sister group, seed plants. Molecular phylogenetic investigations have revolutionized our understanding of fern phylogeny, however, to date, these studies have relied almost exclusively on plastid data.• METHODS: Here we take a curated phylogenomics approach to infer the first broad fern phylogeny from multiple nuclear loci, by combining broad taxon sampling (73 ferns and 12 outgroup species) with focused character sampling (25 loci comprising 35877 bp), along with rigorous alignment, orthology inference and model selection.• KEY RESULTS: Our phylogeny corroborates some earlier inferences and provides novel insights; in particular, we find strong support for Equisetales as sister to the rest of ferns, Marattiales as sister to leptosporangiate ferns, and Dennstaedtiaceae as sister to the eupolypods. Our divergence-time analyses reveal that divergences among the extant fern orders all occurred prior to ∼200 MYA. Finally, our species-tree inferences are congruent with analyses of concatenated data, but generally with lower support. Those cases where species-tree support values are higher than expected involve relationships that have been supported by smaller plastid datasets, suggesting that deep coalescence may be reducing support from the concatenated nuclear data.• CONCLUSIONS: Our study demonstrates the utility of a curated phylogenomics approach to inferring fern phylogeny, and highlights the need to consider underlying data characteristics, along with data quantity, in phylogenetic studies.

Between two fern genomes.

Ferns are the only major lineage of vascular plants not represented by a sequenced nuclear genome. This lack of genome sequence information significantly impedes our ability to understand and reconstruct genome evolution not only in ferns, but across all land plants. Azolla and Ceratopteris are ideal and complementary candidates to be the first ferns to have their nuclear genomes sequenced. They differ dramatically in genome size, life history, and habit, and thus represent the immense diversity of extant ferns. Together, this pair of genomes will facilitate myriad large-scale comparative analyses across ferns and all land plants. Here we review the unique biological characteristics of ferns and describe a number of outstanding questions in plant biology that will benefit from the addition of ferns to the set of taxa with sequenced nuclear genomes. We explain why the fern clade is pivotal for understanding genome evolution across land plants, and we provide a rationale for how knowledge of fern genomes will enable progress in research beyond the ferns themselves.

Evidence for reciprocal origins in Polypodium hesperium (Polypodiaceae): a fern model system for investigating how multiple origins shape allopolyploid genomes.

UNLABELLED: • PREMISE OF THE STUDY: Many polyploid species are composed of distinct lineages originating from multiple, independent polyploidization events. In the case of allopolyploids, reciprocal crosses between the same progenitor species can yield lineages with different uniparentally inherited plastid genomes. While likely common, there are few well-documented examples of such reciprocal origins. Here we examine a case of reciprocal allopolyploid origins in the fern Polypodium hesperium and present it as a natural model system for investigating the evolutionary potential of duplicated genomes.• METHODS: Using a combination of uniparentally inherited plastid and biparentally inherited nuclear sequence data, we investigated the distributions and relative ages of reciprocally formed lineages in Polypodium hesperium, an allotetraploid fern that is broadly distributed in western North America.• KEY RESULTS: The reciprocally derived plastid haplotypes of Polypodium hesperium are allopatric, with populations north and south of 42°N latitude having different plastid genomes. Incorporating biogeographic information and previously estimated ages for the diversification of its diploid progenitors, we estimate middle to late Pleistocene origins of P. hesperium.• CONCLUSIONS: Several features of Polypodium hesperium make it a particularly promising system for investigating the evolutionary consequences of allopolyploidy. These include reciprocally derived lineages with disjunct geographic distributions, recent time of origin, and extant diploid progenitors.

Origin of a novel regulatory module by duplication and degeneration of an ancient plant transcription factor.

It is commonly believed that gene duplications provide the raw material for morphological evolution. Both the number of genes and size of gene families have increased during the diversification of land plants. Several small proteins that regulate transcription factors have recently been identified in plants, including the LITTLE ZIPPER (ZPR) proteins. ZPRs are post-translational negative regulators, via heterodimerization, of class III Homeodomain Leucine Zipper (C3HDZ) proteins that play a key role in directing plant form and growth. We show that ZPR genes originated as a duplication of a C3HDZ transcription factor paralog in the common ancestor of euphyllophytes (ferns and seed plants). The ZPRs evolved by degenerative mutations resulting in loss all of the C3HDZ functional domains, except the leucine zipper that modulates dimerization. ZPRs represent a novel regulatory module of the C3HDZ network unique to the euphyllophyte lineage, and their origin correlates to a period of rapid morphological changes and increased complexity in land plants. The origin of the ZPRs illustrates the significance of gene duplications in creating developmental complexity during land plant evolution that likely led to morphological evolution.

Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns.

Ferns are well known for their shade-dwelling habits. Their ability to thrive under low-light conditions has been linked to the evolution of a novel chimeric photoreceptor--neochrome--that fuses red-sensing phytochrome and blue-sensing phototropin modules into a single gene, thereby optimizing phototropic responses. Despite being implicated in facilitating the diversification of modern ferns, the origin of neochrome has remained a mystery. We present evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in our large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 Mya, long after the split between the two plant lineages (at least 400 Mya). By analyzing the draft genome of the hornwort Anthoceros punctatus, we also discovered a previously unidentified phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was transferred horizontally to ferns, where it may have played a significant role in the diversification of modern ferns.

Transcriptome-mining for single-copy nuclear markers in ferns.

BACKGROUND: Molecular phylogenetic investigations have revolutionized our understanding of the evolutionary history of ferns-the second-most species-rich major group of vascular plants, and the sister clade to seed plants. The general absence of genomic resources available for this important group of plants, however, has resulted in the strong dependence of these studies on plastid data; nuclear or mitochondrial data have been rarely used. In this study, we utilize transcriptome data to design primers for nuclear markers for use in studies of fern evolutionary biology, and demonstrate the utility of these markers across the largest order of ferns, the Polypodiales. PRINCIPAL FINDINGS: We present 20 novel single-copy nuclear regions, across 10 distinct protein-coding genes: ApPEFP_C, cryptochrome 2, cryptochrome 4, DET1, gapCpSh, IBR3, pgiC, SQD1, TPLATE, and transducin. These loci, individually and in combination, show strong resolving power across the Polypodiales phylogeny, and are readily amplified and sequenced from our genomic DNA test set (from 15 diploid Polypodiales species). For each region, we also present transcriptome alignments of the focal locus and related paralogs-curated broadly across ferns-that will allow researchers to develop their own primer sets for fern taxa outside of the Polypodiales. Analyses of sequence data generated from our genomic DNA test set reveal strong effects of partitioning schemes on support levels and, to a much lesser extent, on topology. A model partitioned by codon position is strongly favored, and analyses of the combined data yield a Polypodiales phylogeny that is well-supported and consistent with earlier studies of this group. CONCLUSIONS: The 20 single-copy regions presented here more than triple the single-copy nuclear regions available for use in ferns. They provide a much-needed opportunity to assess plastid-derived hypotheses of relationships within the ferns, and increase our capacity to explore aspects of fern evolution previously unavailable to scientific investigation.