Novel microsatellite markers for evaluation of genetic diversity in the tetraploid flame azalea, Rhododendron calendulaceum (Ericaceae).

Microsatellite markers have become a popular and useful tool for investigating evolutionary processes at shallow taxonomic scales such as within a species or between extremely closely related species. Rhododendron sect. Pentanthera is a closely related group of deciduous azaleas that demonstrate both naturally occurring and horticulturally derived hybridization. Two species, flame azalea and Cumberland azalea, represent a particularly recalcitrant evolutionary problem, which will benefit from the development of rapidly evolving molecular markers. Microsatellite markers were specifically developed for Rhododendron calendulaceum, the flame azalea, for use in studies of genetic structure and potential hybridization with its close relative Rhododendron cumberlandense, the Cumberland azalea. Forty-eight primer pairs designed from paired-end Illumina MiSeq data were screened for robust amplification. Sixteen of these pairs were PCR-amplified in the presence of fluorescently labeled primers and genotyped in 66 flame azalea individuals from three geographically dispersed populations. Fifteen primer pairs were both reliable and polymorphic and exhibit ample variability for use in downstream population-level investigations. Cross-amplification in all other members of Rhododendron sect. Pentanthera was highly successful, suggesting broad utility across the entire clade. The novel microsatellite markers presented here functioned well within the target species and amplified with high success in the remaining members of the clade. They represent a significant improvement to the genetic toolkit available for Rhododendron sect. Pentanthera, and particularly for the flame/Cumberland azalea evolutionary problem.

Authentic Research in the Classroom Increases Appreciation for Plants in Undergraduate Biology Students.

Engaging students in authentic research increases student knowledge, develops STEM skills, such as data analysis and scientific communication, and builds community. Creating authentic research opportunities in plant biology might be particularly crucial in addressing plant awareness disparity (PAD) (formerly known as plant blindness), producing graduates with botanical literacy, and preparing students for plant-focused careers. Our consortium created four CUREs (course-based undergraduate research experiences) focused on dual themes of plant biology and global change, designed to be utilized by early and late-career undergraduates across a variety of educational settings. We implemented these CURES for four semesters, in a total of 15 courses, at four institutions. Pre- and post-course assessments used the Affective Elements of Science Learning Questionnaire and parts of a "plant blindness" instrument to quantify changes in scientific self-efficacy, science values, scientific identity, and plant awareness or knowledge. The qualitative assessment also queried self-efficacy, science values, and scientific identity. Data revealed significant and positive shifts in awareness of and interest in plants across institutions. However, quantitative gains in self-efficacy and scientific identity were only found at two of four institutions tested. This project demonstrates that implementing plant CUREs can produce affective and cognitive gains across institutional types and course levels. Focusing on real-world research questions that capture students' imaginations and connect to their sense of place could create plant awareness while anchoring students in scientific identities. While simple interventions can alleviate PAD, implementing multiple CUREs per course, or focusing more on final CURE products, could promote larger and more consistent gains in student affect across institutions.

Microsatellite markers for the biogeographically enigmatic sandmyrtle (Kalmia buxifolia, Phyllodoceae: Ericaceae).

PREMISE: Microsatellite markers were developed for sandmyrtle, Kalmia buxifolia (Ericaceae), to facilitate phylogeographic studies in this taxon and possibly many of its close relatives. METHODS AND RESULTS: Forty-eight primer pairs designed from paired-end Illumina MiSeq data were screened for robust amplification. Sixteen pairs were amplified again, but with fluorescently labeled primers to facilitate genotyping. Resulting chromatograms were evaluated for variability using three populations from Tennessee, North Carolina, and New Jersey, USA. Eleven primer pairs were reliable and polymorphic (mean 3.92 alleles), one was reliable but monomorphic, and four were not reliable. The markers exhibited lower heterozygosity (mean 0.246) than expected (mean 0.464). Cross-amplification in the remaining nine Kalmia species exhibited a phylogenetic pattern, suggesting broad applicability of the markers across the genus. CONCLUSIONS: These microsatellite markers will be useful in population genetics and species boundaries studies of K. buxifolia, K. procumbens, and likely all other Kalmia species.

Fifteen microsatellite markers for the Appalachian rockcap fern, Polypodium appalachianum (Polypodiaceae), and its relatives.

PREMISE OF THE STUDY: Microsatellite markers were developed for Polypodium appalachianum (Polypodiaceae) to facilitate investigation of species boundaries between P. appalachianum and its putative hybrid, P. virginianum, and potentially among other members of the Miocene-age P. vulgare species complex. METHODS AND RESULTS: Forty-eight primer pairs were designed from Illumina data and screened for successful amplification. Sixteen pairs were genotyped and evaluated for variability within and among three populations in North Carolina, Vermont, and New Hampshire. Twelve of these primer pairs were reliable and polymorphic, exhibiting one to 10 alleles per locus. Cross-species amplification experiments were conducted for P. virginianum and four additional close relatives from the P. vulgare complex in order to maximize information about likely utility within a phylogenetic context. CONCLUSIONS: These microsatellite markers will be useful in population genetics and species boundaries studies of P. appalachianum and P. virginianum, and likely in other species within the P. vulgare complex.

Fourteen polymorphic microsatellite markers for a widespread limestone endemic, Carex eburnea (Cyperaceae: Carex sect. Albae).

PREMISE OF THE STUDY: Microsatellite primers were developed for a widespread limestone endemic sedge, Carex eburnea, to facilitate investigation of the genetic diversity and phylogeography of this taxon and its closest relative, C. mckittrickensis. METHODS AND RESULTS: Forty-eight primer pairs were designed from Illumina sequence data and screened for suitability. Fourteen of these primer pairs were polymorphic and generated one to seven alleles per locus. Cross-species amplifications were conducted for all four members of Carex sect. Albae. CONCLUSIONS: These primer pairs can be used to assess the genetic diversity and population structure in future studies of C. eburnea and C. mckittrickensis, and likely in other members of Carex sect. Albae.

Sixteen polymorphic microsatellite markers for a federally threatened species, Hexastylis naniflora (Aristolochiaceae), and co-occurring congeners.

PREMISE OF THE STUDY: Twenty microsatellite loci were developed for the federally threatened species Hexastylis naniflora (Aristolochiaceae) to examine genetic diversity and to distinguish this species from co-occurring congeners, H. heterophylla and H. minor. METHODS AND RESULTS: Next-generation sequencing approaches were used to identify microsatellite loci and design primers. One hundred fifty-two primer pairs were screened for repeatability, and 20 of these were further characterized for polymorphism. In H. naniflora, the number of alleles identified for polymorphic loci ranged from two to 23 (mean ∼8.8), with a mean heterozygosity of 0.39. CONCLUSIONS: These 16 polymorphic primers for H. naniflora will be useful tools in species identification and quantifying genetic diversity within the genus.

Digitization workflows for flat sheets and packets of plants, algae, and fungi.

Effective workflows are essential components in the digitization of biodiversity specimen collections. To date, no comprehensive, community-vetted workflows have been published for digitizing flat sheets and packets of plants, algae, and fungi, even though latest estimates suggest that only 33% of herbarium specimens have been digitally transcribed, 54% of herbaria use a specimen database, and 24% are imaging specimens. In 2012, iDigBio, the U.S. National Science Foundation's (NSF) coordinating center and national resource for the digitization of public, nonfederal U.S. collections, launched several working groups to address this deficiency. Here, we report the development of 14 workflow modules with 7-36 tasks each. These workflows represent the combined work of approximately 35 curators, directors, and collections managers representing more than 30 herbaria, including 15 NSF-supported plant-related Thematic Collections Networks and collaboratives. The workflows are provided for download as Portable Document Format (PDF) and Microsoft Word files. Customization of these workflows for specific institutional implementation is encouraged.

The US Virtual Herbarium: working with individual herbaria to build a national resource.

The goal of the US Virtual Herbarium (USVH) project is to digitize (database, image, georeference) all specimens in all US herbaria, enabling them to be made available through a single portal. Herbaria house specimens of plants, fungi, and algae, so USVH will offer a rich portrait of biodiversity in the US and in the other countries represented in US herbaria. Equally importantly, working towards this goal will engage people with herbaria and the organisms they house, expanding their appreciation of both the power of biodiversity informatics and the demands that it places on data providers while developing improved communication among those working in and with herbaria. The project is not funded but has strong support among those working in herbaria. It works through regional herbarium networks, some of which existed prior to the USVH project, while others are still in gestation. It differs from most digitization projects in its emphasis on helping those involved with herbaria become part of a national enterprise, an aspect that is seen as critical to creating the resources needed to develop and sustain the project. In this paper, we present some of the lessons we have learned and the difficulties we have encountered during the first few years of the project.

The notes from nature tool for unlocking biodiversity records from museum records through citizen science.

Legacy data from natural history collections contain invaluable and irreplaceable information about biodiversity in the recent past, providing a baseline for detecting change and forecasting the future of biodiversity on a human-dominated planet. However, these data are often not available in formats that facilitate use and synthesis. New approaches are needed to enhance the rates of digitization and data quality improvement. Notes from Nature provides one such novel approach by asking citizen scientists to help with transcription tasks. The initial web-based prototype of Notes from Nature is soon widely available and was developed collaboratively by biodiversity scientists, natural history collections staff, and experts in citizen science project development, programming and visualization. This project brings together digital images representing different types of biodiversity records including ledgers , herbarium sheets and pinned insects from multiple projects and natural history collections. Experts in developing web-based citizen science applications then designed and built a platform for transcribing textual data and metadata from these images. The end product is a fully open source web transcription tool built using the latest web technologies. The platform keeps volunteers engaged by initially explaining the scientific importance of the work via a short orientation, and then providing transcription "missions" of well defined scope, along with dynamic feedback, interactivity and rewards. Transcribed records, along with record-level and process metadata, are provided back to the institutions.  While the tool is being developed with new users in mind, it can serve a broad range of needs from novice to trained museum specialist. Notes from Nature has the potential to speed the rate of biodiversity data being made available to a broad community of users.