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Premise:Equisetum is a distinctive vascular plant genus with 15 extant species worldwide. Species identification is complicated by morphological plasticity and frequent hybridization events, leading to a disproportionately high number of misidentified specimens. These may be correctly identified by applying appropriate computer vision tools. Methods:We hypothesize that aerial stem nodes can provide enough information to distinguish among Equisetum hyemale, E. laevigatum, and E . ×ferrissii, the latter being a hybrid between the other two. An object detector was trained to find nodes on a given image and to distinguish E. hyemale nodes from those of E. laevigatum. A classifier then took statistics from the detection results and classified the given image into one of the three taxa. Both detector and classifier were trained and tested on expert manually annotated images. Results:In our exploratory test set of 30 images, our detector/classifier combination identified all 10 E. laevigatum images correctly, as well as nine out of 10 E. hyemale images, and eight out of 10 E. ×ferrissii images, for a 90% classification accuracy. Discussion:Our results support the notion that computer vision may help with the identification of herbarium specimens once enough manual annotations become available.

PremiseThe 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.MethodsDNAs from Draba, Boechera, Solidago, and Ilex were processed with a ddRADseq protocol. The effects of DNA degradation, taxon, and specimen age were assessed.ResultsAlthough taxon, preservation method, and specimen age affected data recovery, large phylogenetically informative data sets were obtained from the majority of samples.DiscussionThese 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.

PremiseCounting 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 resultsHere we provide a detailed protocol for choosing, staining, and squashing angiosperm pollen mother cells. The protocol is supplemented by figures and two demonstration videos.ConclusionsThe 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.

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.

BackgroundDNA 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 findingsOur 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.ConclusionsOur 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.

Anthropogenic climate change is projected to have an especially negative impact on the survival of plants that are dependent on limited microclimatic refugia or that already reside at their climatic extreme. Gymnocarpium appalachianum is a narrowly endemic fern restricted to cold mountaintops and algific vents in the central and southern Appalachian region of eastern North America. It is the much rarer of the two documented diploid parents of the circumboreal allotetraploid G. dryopteris––one of the most widespread fern species on the planet. Gymnocarpium appalachianum is a good case study for forecasting how evolutionarily significant, but rare, species might survive on a warming planet. We utilize an ecological niche modeling approach (MaxEnt) to explore the projected distribution of G. appalachianum under past (Last Glacial Maximum) and future climate models. All known verified herbarium records of G. appalachianum were georeferenced, for a total of 70 occurrence points. Nineteen standard bioclimatic variables extracted from WorldClim were used to model near-current climate projections; representative concentration pathways (RCPs 2.6 and 8.5) were used for future climate projections (2070). The temperature annual range, mean temperature of warmest quarter, precipitation of driest month, precipitation of coldest quarter, and mean diurnal range were identified as the key variables for shaping the distribution of G. appalachianum. An unanticipated result from our analyses is that G. appalachianum has past and current projected habitat suitability in Alaska. Because this overlaps with the current range of G. disjunctum, the other diploid parent of G. dryopteris, it suggests a possible region of origin for this circumboreal tetraploid descendent of G. appalachianum––a research avenue to be pursued in the future. Our study envisions a dire fate for G. appalachianum; its survival will likely require an urgent contingency plan that includes human-mediated population relocation to cooler, northern locations. Understanding the long-term sustainability of narrowly endemic plants such as G. appalachianum is critical in decisions about their management and conservation. [ABSTRACT FROM AUTHOR]

Premise: Previously published evidence suggests that Draba maguirei, a mustard endemic to a few localities in the Bear River, Wellsville, and Wasatch Mountains of northern Utah, may represent a cryptic species complex rather than a single species. Conservation concerns prompted an in‐depth systematic study of this taxon and its putative relatives. Methods: Sampling most known populations of D. maguirei s.l. (D. maguirei var. maguirei and D. maguirei var. burkei), we integrate data from geography, ecology, morphology, cytogenetics and pollen, enzyme electrophoresis, and the phylogenetic analysis of nuclear internal transcribed spacer sequences to explore potential taxonomic diversity in the species complex. Results: Draba maguirei var. burkei is shown here to be a distinct species (D. burkei) most closely related to D. globosa, rather than to D. maguirei. Within D. maguirei s.s., the northern (high elevation) and southern (low elevation) population clusters are genetically isolated and morphologically distinguishable, leading to the recognition here of the southern taxon as D. maguirei subsp. stonei. Conclusions: Our study reveals that plants traditionally assigned to D. maguirei comprise three genetically divergent lineages (D. burkei and two newly recognized subspecies of D. maguirei), each exhibiting a different chromosome number and occupying a discrete portion of the geographic range. Although previously overlooked and underappreciated taxonomically, the three taxa are morphologically recognizable based on the distribution and types of trichomes present on the leaves, stems, and fruit. Our clarification of the diversity and distribution of these taxa provides an improved framework for conservation efforts. [ABSTRACT FROM AUTHOR]