Once more unto the breach, dear friends: Resolving the origins and relationships of the Pellaea wrightiana hybrid complex.

PREMISE: The taxonomic status of Wright's cliff brake fern, Pellaea wrightiana, has been in dispute ever since it was first described by Hooker in 1858. Previously published evidence suggested that this "taxon" may represent a polyploid complex rather than a single discrete species, a hypothesis tested here using a multifaceted analytical approach. METHODS: Data derived from cytogenetics, spore analyses, leaf morphometrics, enzyme electrophoresis, and phylogenetic analyses of plastid and nuclear DNA sequences are used to elucidate the origin, relationships, and taxonomic circumscription of P. wrightiana. RESULTS: Plants traditionally assigned to this taxon represent three distinct polyploids. The most widespread, P. wrightiana, is a fertile allotetraploid that arose through hybridization between two divergent diploid species, P. truncata and P. ternifolia. Sterile triploids commonly identified as P. wrightiana, were found to be backcross hybrids between this fertile tetraploid and diploid P. truncata. Relatively common across Arizona and New Mexico, they are here assigned to P. ×wagneri hyb. nov. In addition, occasional sterile tetraploid plants assigned to P. wrightiana are shown here to be hybrids between the fertile allotetraploid and the tetraploid P. ternifolia subsp. arizonica. These tetraploid hybrids originated independently in two regions of parental sympatry (southern Arizona and west Texas) and are here assigned to P. ×gooddingii hyb. nov. CONCLUSIONS: Weaving together data from a diversity of taxonomic approaches, we show that plants identified as P. wrightiana represent three morphologically distinguishable polyploids that have arisen through repeated hybridization events involving the divergent sexual taxa P. ternifolia and P. truncata.

A novel chloroplast gene reported for flagellate plants.

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.

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.

Adiantumshastense, a new species of maidenhair fern from California.

A new species of Adiantum is described from California. This species is endemic to northern California and is currently known only from Shasta County. We describe its discovery after first being collected over a century ago and distinguish it from Adiantumjordanii and Adiantumcapillus-veneris. It is evergreen and is sometimes, but not always, associated with limestone. The range of Adiantumshastense Huiet & A.R.Sm., sp. nov., is similar to several other Shasta County endemics that occur in the mesic forests of the Eastern Klamath Range, close to Shasta Lake, on limestone and metasedimentary substrates.

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.

DNA barcoding exposes a case of mistaken identity in the fern horticultural trade.

Using cheilanthoid ferns, we provide an example of how DNA barcoding approaches can be useful to the horticultural community for keeping plants in the trade accurately identified. We use plastid rbcL, atpA, and trnG-R sequence data to demonstrate that a fern marketed as Cheilanthes wrightii (endemic to the southwestern USA and northern Mexico) in the horticultural trade is, in fact, Cheilanthes distans (endemic to Australia and adjacent islands). Public and private (accessible with permission) databases contain a wealth of DNA sequence data that are linked to vouchered plant material. These data have uses beyond those for which they were originally generated, and they provide an important resource for fostering collaborations between the academic and horticultural communities. We strongly advocate the barcoding approach as a valuable new technology available to the horticulture industry to help correct plant identification errors in the international trade.

A molecular phylogeny of the fern family Pteridaceae: assessing overall relationships and the affinities of previously unsampled genera.

The monophyletic Pteridaceae accounts for roughly 10% of extant fern diversity and occupies an unusually broad range of ecological niches, including terrestrial, epiphytic, xeric-adapted rupestral, and even aquatic species. In this study, we present the results of the first broad-scale and multi-gene phylogenetic analyses of these ferns, and determine the affinities of several previously unsampled genera. Our analyses of two newly assembled data sets (including 169 newly obtained sequences) resolve five major clades within the Pteridaceae: cryptogrammoids, ceratopteridoids, pteridoids, adiantoids, and cheilanthoids. Although the composition of these clades is in general agreement with earlier phylogenetic studies, it is very much at odds with the most recent subfamilial classification. Of the previously unsampled genera, two (Neurocallis and Ochropteris) are nested within the genus Pteris; two others (Monogramma and Rheopteris) are early diverging vittarioid ferns, with Monogramma resolved as polyphyletic; the last previously unsampled genus (Adiantopsis) occupies a rather derived position among cheilanthoids. Interestingly, some clades resolved within the Pteridaceae can be characterized by their ecological preferences, suggesting that the initial diversification in this family was tied to ecological innovation and specialization. These processes may well be the basis for the diversity and success of the Pteridaceae today.