Browsing by Author "Rothfels, CJ"

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Open Access
A novel chloroplast gene reported for flagellate plants.
(American journal of botany, 2018-01) Song, M; Kuo, L; Huiet, L; Pryer, KM; Rothfels, CJ; Li, F
PREMISE OF THE STUDY:Gene space in plant plastid genomes is well characterized and annotated, yet we discovered an unrecognized open reading frame (ORF) in the fern lineage that is conserved across flagellate plants. METHODS:We initially detected a putative uncharacterized ORF by the existence of a highly conserved region between rps16 and matK in a series of matK alignments of leptosporangiate ferns. We mined available plastid genomes for this ORF, which we now refer to as ycf94, to infer evolutionary selection pressures and assist in functional prediction. To further examine the transcription of ycf94, we assembled the plastid genome and sequenced the transcriptome of the leptosporangiate fern Adiantum shastense Huiet & A.R. Sm. KEY RESULTS:The ycf94 predicted protein has a distinct transmembrane domain but with no sequence homology to other proteins with known function. The nonsynonymous/synonymous substitution rate ratio of ycf94 is on par with other fern plastid protein-encoding genes, and additional homologs can be found in a few lycophyte, moss, hornwort, and liverwort plastid genomes. Homologs of ycf94 were not found in seed plants. In addition, we report a high level of RNA editing for ycf94 transcripts-a hallmark of protein-coding genes in fern plastomes. CONCLUSIONS:The degree of sequence conservation, together with the presence of a distinct transmembrane domain and RNA-editing sites, suggests that ycf94 is a protein-coding gene of functional significance in ferns and, potentially, bryophytes and lycophytes. However, the origin and exact function of this gene require further investigation.
Open Access
A plastid phylogeny of the cosmopolitan fern family cystopteridaceae (Polypodiopsida)
(Systematic Botany, 2013-06-01) Rothfels, CJ; Windham, MD; Pryer, KM
Among the novel results of recent molecular phylogenetic analyses are the unexpectedly close evolutionary relationships of the genera Acystopteris, Cystopteris, and Gymnocarpium, and the phylogenetic isolation of these genera from Woodsia. As a consequence, these three genera have been removed from Woodsiaceae and placed into their own family, the Cystopteridaceae. Despite the ubiquity of this family in rocky habitats across the northern hemisphere, and its cosmopolitan distribution (occurring on every continent except Antarctica), sampling of the Cystopteridaceae in phylogenetic studies to date has been sparse. Here we assemble a three-locus plastid dataset (matK, rbcL, trnG-R) that includes most recognized species in the family and multiple accessions of widespread taxa from across their geographic ranges. All three sampled genera are robustly supported as monophyletic, Cystopteris is strongly supported as sister to Acystopteris, and those two genera together are sister to Gymnocarpium. The Gymnocarpium phylogeny is deeply divided into three major clades, which we label the disjunctum clade, the robertianum clade, and core Gymnocarpium. The Cystopteris phylogeny, similarly, features four deeply diverged clades: C. montana, the sudetica clade, the bulbifera clade, and the fragilis complex. Acystopteris includes only three species, each of which is supported as monophyletic, with A. taiwaniana sister to the japonica/tenuisecta clade. Our results yield the first species-level phylogeny of the Cystopteridaceae and the first molecular phylogenetic evidence for species boundaries. These data provide an essential foundation for further investigations of complex patterns of geographic diversification, speciation, and reticulation in this family. © Copyright 2013 by the American Society of Plant Taxonomists.
Open Access
A revised classification for eupolypod II ferns (Polypodiales: Polypodiopsida)
(Taxon, 2012) Rothfels, CJ; Sundue, MA; Kato, M; Larsson, A; Kuo, LY; Schuettpelz, E; Pryer, KM
Open Access
A revised family-level classification for eupolypod II ferns (Polypodiidae: Polypodiales)
(Taxon, 2012-01-01) Rothfels, CJ; Sundue, MA; Kuo, L; Larsson, A; Kato, M; Schuettpelz, E; Pryer, KM
We present a family-level classification for the eupolypod II clade of leptosporangiate ferns, one of the two major lineages within the Eupolypods, and one of the few parts of the fern tree of life where family-level relationships were not well understood at the time of publication of the 2006 fern classification by Smith & al. Comprising over 2500 species, the composition and particularly the relationships among the major clades of this group have historically been contentious and defied phylogenetic resolution until very recently. Our classification reflects the most current available data, largely derived from published molecular phylogenetic studies. In comparison with the five-family (Aspleniaceae, Blechnaceae, Onocleaceae, Thelypteridaceae, Woodsiaceae) treatment of Smith & al., we recognize 10 families within the eupolypod II clade. Of these, Aspleniaceae, Thelypteridaceae, Blechnaceae, and Onocleaceae have the same composition as treated by Smith & al. Woodsiaceae, which Smith & al. acknowledged as possibly non-monophyletic in their treatment, is circumscribed here to include only Woodsia and its segregates; the other "woodsioid" taxa are divided among Athyriaceae, Cystopteridaceae, Diplaziopsidaceae, Rhachidosoraceae, and Hemidictyaceae. We provide circumscriptions for each family, which summarize their morphological, geographical, and ecological characters, as well as a dichotomous key to the eupolypod II families. Three of these families- Diplaziopsidaceae, Hemidictyaceae, and Rhachidosoraceae-were described in the past year based on molecular phylogenetic analyses; we provide here their first morphological treatment.
Open Access
A step-by-step protocol for meiotic chromosome counts in flowering plants: A powerful and economical technique revisited.
(Applications in plant sciences, 2020-04-23) Windham, MD; Pryer, KM; Poindexter, DB; Li, F; Rothfels, CJ; Beck, JB
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.
Open Access
An exploration into fern genome space
(Genome Biology and Evolution, 2015) Wolf, PG; Sessa, EB; Marchant, DB; Li, F; Rothfels, CJ; Sigel, EM; Gitzendanner, MA; Visger, CJ; Banks, JA; Soltis, DE
Open Access
An Exploration into Fern Genome Space.
(Genome Biol Evol, 2015-08-26) Wolf, PG; Sessa, EB; Marchant, DB; Li, F; Rothfels, CJ; Sigel, EM; Gitzendanner, MA; Visger, CJ; Banks, JA; Soltis, DE
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.
Open Access
Are there too many fern genera?
(Taxon, 2018-06-01) Schuettpelz, E; Rouhan, G; Pryer, KM; Rothfels, CJ; Prado, J; Sundue, MA; Windham, MD; Moran, RC; Smith, AR
Open Access
Genes Translocated into the Plastid Inverted Repeat Show Decelerated Substitution Rates and Elevated GC Content.
(Genome biology and evolution, 2016-08-25) Li, F; Kuo, L; Pryer, KM; Rothfels, CJ
Plant chloroplast genomes (plastomes) are characterized by an inverted repeat (IR) region and two larger single copy (SC) regions. Patterns of molecular evolution in the IR and SC regions differ, most notably by a reduced rate of nucleotide substitution in the IR compared to the SC region. In addition, the organization and structure of plastomes is fluid, and rearrangements through time have repeatedly shuffled genes into and out of the IR, providing recurrent natural experiments on how chloroplast genome structure can impact rates and patterns of molecular evolution. Here we examine four loci (psbA, ycf2, rps7, and rps12 exon 2-3) that were translocated from the SC into the IR during fern evolution. We use a model-based method, within a phylogenetic context, to test for substitution rate shifts. All four loci show a significant, 2- to 3-fold deceleration in their substitution rate following translocation into the IR, a phenomenon not observed in any other, nontranslocated plastid genes. Also, we show that after translocation, the GC content of the third codon position and of the noncoding regions is significantly increased, implying that gene conversion within the IR is GC-biased. Taken together, our results suggest that the IR region not only reduces substitution rates, but also impacts nucleotide composition. This finding highlights a potential vulnerability of correlating substitution rate heterogeneity with organismal life history traits without knowledge of the underlying genome structure.
Open Access
Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns.
(Proceedings of the National Academy of Sciences of the United States of America, 2014-05) Li, F; Villarreal, JC; Kelly, S; Rothfels, CJ; Melkonian, M; Frangedakis, E; Ruhsam, M; Sigel, EM; Der, JP; Pittermann, J
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.
Open Access
Infraspecific diversification of the star cloak fern (Notholaena standleyi) in the deserts of the United States and Mexico.
(American journal of botany, 2020-04-07) Kao, T; Rothfels, CJ; Melgoza‐Castillo, A; Pryer, KM; Windham, MD
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.
Open Access
Low-copy nuclear data confirm rampant allopolyploidy in the cystopteridaceae (Polypodiales)
(Taxon, 2014-10-01) Rothfels, CJ; Johnson, AK; Windham, MD; Pryer, KM
© International Association for Plant Taxonomy (IAPT) 2014. Here we present the first nuclear phylogeny for Cystopteridaceae (Polypodiales), using the single-copy locus gapCp “short”. This phylogeny corroborates broad results from plastid data in demonstrating strong support for the monophyly of the family’s three genera—Cystopteris, Acystopteris, and Gymnocarpium—and of the major groups within Cystopteris (C. montana, the sudetica and bulbifera clades, and the C. fragilis complex). In addition, it confirms the rampant hybridization (allopolyploidy) that has long been suspected within both Cystopteris and Gymnocarpium. In some cases, these data provide the first DNA-sequence-based evidence for previous hypotheses of polyploid species origins (such as the cosmopolitan G. dryopteris being an allotetraploid derivative of the diploids G. appalachianum and G. disjunctum). Most of the allopolyploids, however, have no formal taxonomic names. This pattern is particularly strong within the C. fragilis complex, where our results imply that the eight included accessions of “C. fragilis” represent at least six distinct allopolyploid taxa.
Open Access
Low-copy nuclear sequence data confirm complex patterns of farina evolution in notholaenid ferns (Pteridaceae).
(Molecular phylogenetics and evolution, 2019-09) Kao, T; Pryer, KM; Freund, FD; Windham, MD; Rothfels, CJ
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.
Open Access
Maidenhair ferns, adiantum, are indeed monophyletic and sister to shoestring ferns, vittarioids (Pteridaceae)
(Systematic Botany, 2016-01-01) Pryer, KM; Huiet, L; Li, F; Rothfels, CJ; Schuettpelz, E
© 2016 by the American Society of Plant Taxonomists. Across the tree of life, molecular phylogenetic studies often reveal surprising relationships between taxa with radically different morphologies that have long obscured their close affiliations. A spectacular botanical example is Rafflesia, a holoparasite that produces the largest flowers in the world, but that evolved from tiny-flowered ancestors within the Euphorbiaceae. Outside of parasitic lineages, such abrupt transformations are rarely seen. One exception involves the "maidenhair ferns" (Adiantum), which are quintessential ferns: beautifully dissected, terrestrial, and shade loving. The closely related "shoestring ferns" (vittarioids), in contrast, have an extremely simplified morphology, are canopy-dwelling epiphytes, and exhibit greatly accelerated rates of molecular evolution. While Adiantum and the vittarioids together have been shown to form a robust monophyletic group (adiantoids), there remain unanswered questions regarding the monophyly of Adiantum and the evolutionary history of the vittarioids. Here we review recent phylogenetic evidence suggesting support for the monophyly of Adiantum, and analyze new plastid data to confirm this result. We find that Adiantum is monophyletic and sister to the vittarioids. With this robust phylogenetic framework established for the broadest relationships in the adiantoid clade, we can now focus on understanding the evolutionary processes associated with the extreme morphological, ecological, and genetic transitions that took place within this lineage.
Open Access
Natural hybridization between genera that diverged from each other approximately 60 million years ago.
(Am Nat, 2015-03) Rothfels, CJ; Johnson, AK; Hovenkamp, PH; Swofford, DL; Roskam, HC; Fraser-Jenkins, CR; Windham, MD; Pryer, KM
A fern from the French Pyrenees-×Cystocarpium roskamianum-is a recently formed intergeneric hybrid between parental lineages that diverged from each other approximately 60 million years ago (mya; 95% highest posterior density: 40.2-76.2 mya). This is an extraordinarily deep hybridization event, roughly akin to an elephant hybridizing with a manatee or a human with a lemur. In the context of other reported deep hybrids, this finding suggests that populations of ferns, and other plants with abiotically mediated fertilization, may evolve reproductive incompatibilities more slowly, perhaps because they lack many of the premating isolation mechanisms that characterize most other groups of organisms. This conclusion implies that major features of Earth's biodiversity-such as the relatively small number of species of ferns compared to those of angiosperms-may be, in part, an indirect by-product of this slower "speciation clock" rather than a direct consequence of adaptive innovations by the more diverse lineages.
Open Access
Next-generation polyploid phylogenetics: rapid resolution of hybrid polyploid complexes using PacBio single-molecule sequencing.
(The New phytologist, 2017-01) Rothfels, CJ; Pryer, KM; Li, F
Difficulties in generating nuclear data for polyploids have impeded phylogenetic study of these groups. We describe a high-throughput protocol and an associated bioinformatics pipeline (Pipeline for Untangling Reticulate Complexes (Purc)) that is able to generate these data quickly and conveniently, and demonstrate its efficacy on accessions from the fern family Cystopteridaceae. We conclude with a demonstration of the downstream utility of these data by inferring a multi-labeled species tree for a subset of our accessions. We amplified four c. 1-kb-long nuclear loci and sequenced them in a parallel-tagged amplicon sequencing approach using the PacBio platform. Purc infers the final sequences from the raw reads via an iterative approach that corrects PCR and sequencing errors and removes PCR-mediated recombinant sequences (chimeras). We generated data for all gene copies (homeologs, paralogs, and segregating alleles) present in each of three sets of 50 mostly polyploid accessions, for four loci, in three PacBio runs (one run per set). From the raw sequencing reads, Purc was able to accurately infer the underlying sequences. This approach makes it easy and economical to study the phylogenetics of polyploids, and, in conjunction with recent analytical advances, facilitates investigation of broad patterns of polyploid evolution.
Open Access
Phytochrome diversity in green plants and the origin of canonical plant phytochromes
(Nature communications, 2015) Li, F; Melkonian, M; Rothfels, CJ; Villarreal, JC; Stevenson, DW; Graham, SW; Wong, GK; Pryer, KM; Mathews, S
Open Access
Phytochrome diversity in green plants and the origin of canonical plant phytochromes.
(Nat Commun, 2015-07-28) Melkonian, M; Pryer, KM; Rothfels, CJ; Graham, SW; Li, F; Villarreal, JC; Wong, GK; Stevenson, DW; Mathews, S
Phytochromes are red/far-red photoreceptors that play essential roles in diverse plant morphogenetic and physiological responses to light. Despite their functional significance, phytochrome diversity and evolution across photosynthetic eukaryotes remain poorly understood. Using newly available transcriptomic and genomic data we show that canonical plant phytochromes originated in a common ancestor of streptophytes (charophyte algae and land plants). Phytochromes in charophyte algae are structurally diverse, including canonical and non-canonical forms, whereas in land plants, phytochrome structure is highly conserved. Liverworts, hornworts and Selaginella apparently possess a single phytochrome, whereas independent gene duplications occurred within mosses, lycopods, ferns and seed plants, leading to diverse phytochrome families in these clades. Surprisingly, the phytochrome portions of algal and land plant neochromes, a chimera of phytochrome and phototropin, appear to share a common origin. Our results reveal novel phytochrome clades and establish the basis for understanding phytochrome functional evolution in land plants and their algal relatives.
Open Access
rbcL and matK earn two thumbs up as the core DNA barcode for ferns.
(PloS one, 2011-01) Li, F; Kuo, L; Rothfels, CJ; Ebihara, A; Chiou, W; Windham, MD; Pryer, KM
BACKGROUND: DNA barcoding will revolutionize our understanding of fern ecology, most especially because the accurate identification of the independent but cryptic gametophyte phase of the fern's life history--an endeavor previously impossible--will finally be feasible. In this study, we assess the discriminatory power of the core plant DNA barcode (rbcL and matK), as well as alternatively proposed fern barcodes (trnH-psbA and trnL-F), across all major fern lineages. We also present plastid barcode data for two genera in the hyperdiverse polypod clade--Deparia (Woodsiaceae) and the Cheilanthes marginata group (currently being segregated as a new genus of Pteridaceae)--to further evaluate the resolving power of these loci. PRINCIPAL FINDINGS: Our results clearly demonstrate the value of matK data, previously unavailable in ferns because of difficulties in amplification due to a major rearrangement of the plastid genome. With its high sequence variation, matK complements rbcL to provide a two-locus barcode with strong resolving power. With sequence variation comparable to matK, trnL-F appears to be a suitable alternative barcode region in ferns, and perhaps should be added to the core barcode region if universal primer development for matK fails. In contrast, trnH-psbA shows dramatically reduced sequence variation for the majority of ferns. This is likely due to the translocation of this segment of the plastid genome into the inverted repeat regions, which are known to have a highly constrained substitution rate. CONCLUSIONS: Our study provides the first endorsement of the two-locus barcode (rbcL+matK) in ferns, and favors trnL-F over trnH-psbA as a potential back-up locus. Future work should focus on gathering more fern matK sequence data to facilitate universal primer development.
Open Access
Revealing a cryptic fern distribution through DNA sequencing: Pityrogramma trifoliata in the Western Andes of Peru
(American Fern Journal, 2013-01-01) León, B; Rothfels, CJ; Arakaki, M; Young, KR; Pryer, KM
Fern identification usually requires the use of mature sporophytes, since attempts to identify juveniles using morphological traits often provides unsatisfactory results. Here we examined young sporophytes found among boulders in a river basin of a xeric valley in central Peru. Attempts to identify these sporophytes first pointed to four different genera, two in Pteridaceae (Anogramma and Pityrogramma), and the others in Aspleniaceae (Asplenium) and Cystopteridaceae (Cystopteris). Here, we resolved this puzzle combining morphology and sequences of DNA (rbcL and trnG-R) that point to Pityrogramma trifoliata of Pteridaceae.