Defining Populations and Predicting Future Suitable Niche Space in the Geographically Disjunct, Narrowly Endemic Leafy Prairie-Clover (Dalea foliosa; Fabaceae).

Conservation actions for rare species are often based on estimates of population size and number, which are challenging to capture in natural systems. Instead, many definitions of populations rely on arbitrarily defined distances between occurrences, which is not necessarily biologically meaningful despite having utility from a conservation management perspective. Here, we introduce a case study using the narrowly endemic and highly geographically disjunct leafy prairie-clover (Dalea foliosa), for which we use nuclear microsatellite loci to assess the current delimitations of populations and management units across its entire known range. We model future potential suitable niche space for the species to assess how currently defined populations could fare under predicted changes in climate over the next 50 years. Our results indicate that genetic variation within the species is extremely limited, particularly so in the distal portions of its range (Illinois and Alabama). Within the core of its range (Tennessee), genetic structure is not consistent with populations as currently defined. Our models indicate that predicted suitable niche space may only marginally overlap with the geology associated with this species (limestone glades and dolomite prairies) by 2070. Additional studies are needed to evaluate the extent to which populations are ecologically adapted to local environments and what role this could play in future translocation efforts.

Collections Education: The Extended Specimen and Data Acumen.

Biodiversity scientists must be fluent across disciplines; they must possess the quantitative, computational, and data skills necessary for working with large, complex data sets, and they must have foundational skills and content knowledge from ecology, evolution, taxonomy, and systematics. To effectively train the emerging workforce, we must teach science as we conduct science and embrace emerging concepts of data acumen alongside the knowledge, tools, and techniques foundational to organismal biology. We present an open education resource that updates the traditional plant collection exercise to incorporate best practices in twenty-first century collecting and to contextualize the activities that build data acumen. Students exposed to this resource gained skills and content knowledge in plant taxonomy and systematics, as well as a nuanced understanding of collections-based data resources. We discuss the importance of the extended specimen in fostering scientific discovery and reinforcing foundational concepts in biodiversity science, taxonomy, and systematics.

From Then to now: Diversity, Equity, and Inclusion in the Association of Southeastern Biologists.

The Association of Southeastern Biologists was founded in 1937 with the goal of increasing the contact and collaboration between scientists in the southeastern United States (US). With the exception of two years during World War II and one year during the COVID-19 pandemic, the Association has met annually to promote research and education in the biological sciences by providing a student-friendly networking environment. In recent years, the Association has placed an increased focus on diversity, equity, and inclusion among elected and appointed leaders, among participants in the annual meeting, and in the development of funding and other opportunities for students. This work prompted us to review the history of our Association, including periods of racial segregation and inequity, and focus on our current efforts to promote access and inclusion by students and scientists from myriad underrepresented groups. In so doing, the past provides us with the opportunity to cast a vision for the future of the Association. In this paper, we seek to share the journey of the Association of Southeastern Biologists in this regard so that we may be transparent, exposing the missteps and amplifying the successes of our organization. We envision this work as a first step toward creating a more open and inclusive scientific community for the future.

Estimating herbarium specimen digitization rates: Accounting for human experience.

PREMISE: Herbaria are invaluable sources for understanding the natural world, and in recent years there has been a concerted effort to digitize these collections. To organize such efforts, a method for estimating the necessary labor is desired. This work analyzes digitization productivity reports of 105 participants from eight herbaria, deriving generalized labor estimates that account for human experience. METHODS AND RESULTS: Individuals' rates of digitization were grouped based on cumulative time performing each task and then used to estimate a series of generalized labor projection models. In most cases, productivity was shown to improve with experience, suggesting longer technician retention can reduce labor requirements by 20%. CONCLUSIONS: Using student labor is a common tactic for digitization efforts, and the resulting outreach exposes future professionals to natural history collections. However, overcoming the learning curve should be considered when estimating the labor necessary to digitize a collection.

Small herbaria contribute unique biogeographic records to county, locality, and temporal scales.

PREMISE: With digitization and data sharing initiatives underway over the last 15 years, an important need has been prioritizing specimens to digitize. Because duplicate specimens are shared among herbaria in exchange and gift programs, we investigated the extent to which unique biogeographic data are held in small herbaria vs. these data being redundant with those held by larger institutions. We evaluated the unique specimen contributions that small herbaria make to biogeographic understanding at county, locality, and temporal scales. METHODS: We sampled herbarium specimens of 40 plant taxa from each of eight states of the United States of America in four broad status categories: extremely rare, very rare, common native, and introduced. We gathered geographic information from specimens held by large (≥100,000 specimens) and small (<100,000 specimens) herbaria. We built generalized linear mixed models to assess which features of the collections may best predict unique contributions of herbaria and used an Akaike information criterion-based information-theoretic approach for our model selection to choose the best model for each scale. RESULTS: Small herbaria contributed unique specimens at all scales in proportion with their contribution of specimens to our data set. The best models for all scales were the full models that included the factors of species status and herbarium size when accounting for state as a random variable. CONCLUSIONS: We demonstrated that small herbaria contribute unique information for research. It is clear that unique contributions cannot be predicted based on herbarium size alone. We must prioritize digitization and data sharing from herbaria of all sizes.

Digitization protocol for scoring reproductive phenology from herbarium specimens of seed plants.

PREMISE OF THE STUDY: Herbarium specimens provide a robust record of historical plant phenology (the timing of seasonal events such as flowering or fruiting). However, the difficulty of aggregating phenological data from specimens arises from a lack of standardized scoring methods and definitions for phenological states across the collections community. METHODS AND RESULTS: To address this problem, we report on a consensus reached by an iDigBio working group of curators, researchers, and data standards experts regarding an efficient scoring protocol and a data-sharing protocol for reproductive traits available from herbarium specimens of seed plants. The phenological data sets generated can be shared via Darwin Core Archives using the Extended MeasurementOrFact extension. CONCLUSIONS: Our hope is that curators and others interested in collecting phenological trait data from specimens will use the recommendations presented here in current and future scoring efforts. New tools for scoring specimens are reviewed.

Markers in time and space: A review of the last decade of plant phylogeographic approaches.

Plant studies comprise a relatively small proportion of the phylogeographic literature, likely as a consequence of the fundamental challenges posed by the complex genomic structures and life history strategies of these organisms. Comparative plastomics (i.e., comparisons of mutation rates within and among regions of the chloroplast genome) across plant lineages has led to an increased understanding of which markers are likely to provide the most information at low taxonomic levels. However, the extent to which the results of such work have influenced the literature has not been fully assessed, nor has the extent to which plant phylogeographers explicitly analyse markers in time and space, both of which are integral components of the field. Here, we reviewed more than 400 publications from the last decade of plant phylogeography to specifically address the following questions: (i) What is the phylogenetic breadth of studies to date? (ii) What molecular markers have been used, and why were they chosen? (iii) What kinds of markers are most frequently used and in what combinations? (iv) How frequently are divergence time estimation and ecological niche modelling used in plant phylogeography? Our results indicate that chloroplast DNA sequence data remain the primary tool of choice, followed distantly by nuclear DNA sequences and microsatellites. Less than half (42%) of all studies use divergence time estimation, while even fewer use ecological niche modelling (14%). We discuss the implications of our findings, as well as the need for community standards on data reporting.

Isolated coastal populations of Tilia americana var. caroliniana persist long-term through vegetative growth.

PREMISE OF THE STUDY: Sprouting in woody plant species allows for the long-term persistence of small, isolated populations experiencing changing environments and can preserve genetic diversity in these populations despite the infrequent recruitment of sexually produced individuals. We examined demographic data collected over a 10-yr period for Tilia americana var. caroliniana populations in the context of genetic structure as an empirical case study of this concept. METHODS: Two back-barrier islands on the Georgia coast of the United States were completely censused for Tilia americana var. caroliniana. Recruitment, growth, and mortality of all stems were tracked over 10 yr. All genets were genotyped using eight nuclear microsatellite loci to assess population genetic structure among sampled stems and among populations in the region. KEY RESULTS: The two island populations differed in their ability to establish seedlings despite having similar patterns in flowering frequency. Seedling mortality was high throughout the 10 yr, and cycling of ramets within genets was common. Long-term recruitment in this system appears to be primarily a result of vegetative growth via basal sprouts. Genetic structure was limited, both between islands and among populations in the region. CONCLUSIONS: Long-lived woody species that persist by vegetative reproduction may unexpectedly influence regional forest responses to climate change, particularly on the trailing edge of a species' distribution.

Idiosyncratic responses of evergreen broad-leaved forest constituents in China to the late Quaternary climate changes.

Subtropical evergreen broad-leaved forest (EBLF) is one of the most important vegetation types in China. Inferences from palaeo-biome reconstruction (PBR) and phylogeography regarding range shift history of EBLF during the late Quaternary are controversial and should be reconciled. We compared phylogeographic patterns of three EBLF constituents in China, Castanopsis tibetana, Machilus thunbergii and Schima superba. Contrary to a chorus of previous phylogeographic studies and the results of species distribution modelling (SDM) of this study (in situ survival during the LGM), the three species displayed three different phylogeographic patterns that conform to either an in situ survival model or an expansion-contraction model. These results are partially congruent with the inference of PBR that EBLF was absent to the north of 24° N at the LGM. This study suggests that the constituents of EBLF could have responded idiosyncratically to climate changes during the Late Quaternary. The community assemblages of EBLF could have been changing over time, resulting in no palaeo-analogs to modern-day EBLF, which may be the main reason responsible for the failure of PBR to detect the occurrence of EBLF north of 24° N at the LGM.

Development and characterization of microsatellite primers in the federally endangered Astragalus bibullatus (Fabaceae).

PREMISE OF THE STUDY: Microsatellites were developed for Astragalus bibullatus (Fabaceae), a federally endangered narrow endemic, to investigate reproductive ecology and species boundaries among closely related taxa. METHODS AND RESULTS: Next-generation sequencing was used to develop 12 nuclear microsatellite loci that amplify in A. bibullatus, as well as in A. crassicarpus var. trichocalyx, A. gypsodes, and A. tennesseensis. Identified loci were di- and trinucleotide repeats, with 1-15 alleles per locus. Observed and expected heterozygosities ranged from 0.000-0.938 and 0.000-0.860, respectively. Cross-amplification of three loci previously published in A. michauxii was also confirmed for the taxa included here. CONCLUSIONS: These data indicate the utility of novel microsatellite loci for conservation genetics and reproductive ecology in closely related Astragalus species.

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.

Chloroplast DNA sequence utility for the lowest phylogenetic and phylogeographic inferences in angiosperms: the tortoise and the hare IV.

PREMISE OF THE STUDY: Noncoding chloroplast DNA (NC-cpDNA) sequences are the staple data source of low-level phylogeographic and phylogenetic studies of angiosperms. We followed up on previous papers (tortoise and hare II and III) that sought to identify the most consistently variable regions of NC-cpDNA. We used an exhaustive literature review and newly available whole plastome data to assess applicability of previous conclusions at low taxonomic levels. METHODS: We aligned complete plastomes of 25 species pairs from across angiosperms, comparing the number of genetic differences found in 107 NC-cpDNA regions and matK. We surveyed Web of Science for the plant phylogeographic literature between 2007 and 2013 to assess how NC-cpDNA has been used at the intraspecific level. KEY RESULTS: Several regions are consistently the most variable across angiosperm lineages: ndhF-rpl32, rpl32-trnL((UAG)), ndhC-trnV((UAC)), 5'rps16-trnQ((UUG)), psbE-petL, trnT((GGU))-psbD, petA-psbJ, and rpl16 intron. However, there is no universally best region. The average number of regions applied to low-level studies is ∼2.5, which may be too little to access the full discriminating power of this genome. CONCLUSIONS: Plastome sequences have been used successfully at lower and lower taxonomic levels. Our findings corroborate earlier works, suggesting that there are regions that are most likely to be the most variable. However, while NC-cpDNA sequences are commonly used in plant phylogeographic studies, few of the most variable regions are applied in that context. Furthermore, it appears that in most studies too few NC-cpDNAs are used to access the discriminating power of the cpDNA genome.

Mature beech trees (Fagus grandifolia; Fagaceae) are persistently clonal in coves and beech gaps in the Great Smoky Mountains.

PREMISE OF THE STUDY: Angiosperms frequently have mixed sexual and asexual reproductive strategies, which can have significant consequences for population and community structure. Many tree species respond to disturbance by vegetative sprouting over sexual reproduction, but the ability to do so varies within and among species and is poorly studied. We tested the hypothesis that root sprouting in Fagus grandifolia is more important in high-elevation beech forests (extreme environmental conditions), relative to lower-elevation cove hardwood forests (optimal environmental conditions), in Great Smoky Mountains National Park. METHODS: Four cove hardwood and two high-elevation beech gap forests were sampled. Fagus grandifolia trees were tagged, mapped, and genotyped at each site using six nuclear microsatellite loci. A subset of trees was cored to determine minimum ages of identified clonal lineages. Relationships between measures of clonal richness, diameter, forest type, and elevation were compared by nonparametric analyses. KEY RESULTS: Five of six sites were highly dependent on sprouting; one site was entirely dependent on reproduction by seed. Clonal richness did not differ significantly by forest type or elevation, but differed significantly from previously published work. Tree diameter was significant by elevation and significant between the present study and previously published work. CONCLUSIONS: The results of this study in the context of previously published work suggest that relatively recent recruits may be more likely to reflect both sexual and asexual strategies, whereas mature F. grandifolia may be more likely to be the result of persistent clonal lineages.

Clonal structure of wild populations and origins of horticultural stocks of Illicium parviflorum (Illiciaceae).

PREMISE OF THE STUDY: Habitat fragmentation is often assumed to result in limited genetic diversity across impacted plant communities. Central Florida has undergone extensive anthropogenic changes, while also harboring large numbers of endemic species. In this study, we assessed genetic structure and dependence on clonality in a central Florida endemic, Illicium parviflorum (Illiciaceae), as well as evaluated genetic diversity of this species in horticultural stocks. • METHODS: Six sites were sampled across the geographic range of I. parviflorum. A PCR-based assay using intersimple sequence repeats (ISSRs) was used to assess genetic structure. • KEY RESULTS: Results, based on 26 ISSR loci, suggest that clonal structure plays a role in all populations, with PD values ranging from 0.25 to 0.50. Only two populations exhibited unique genotypes, while the remaining four populations shared genotypes. Horticultural samples all shared one genotype, which can be traced back to a single natural population. • CONCLUSIONS: Clonal reproduction is an important factor in the maintenance of natural populations of I. parviflorum, although the degree to which this is true varies by population. Horticultural samples likely represent a single or very few collection events, indicating the need for greater genetic diversity within horticultural stocks. Further analyses using microsatellites are planned to confirm these results.

Phylogeographical structure and temporal complexity in American sweetgum (Liquidambar styraciflua; Altingiaceae).

Eastern North American plant biogeography has traditionally focused on two primary issues: (i) the location of temperate Pleistocene refugia and their proximity to the southern margin of the ice sheet during the last glacial maximum, and (ii) the origin of the temperate element of northern Latin America. While numerous population genetic and phylogeographical studies have focused on the first issue, few (if any) have considered the second. We addressed these issues by surveying 117 individuals from 24 populations of Liquidambar styraciflua (American sweetgum; Altingiaceae) across the southeastern USA, eastern Mexico, and Guatemala, using more than 2200 bp of chloroplast DNA sequence data. To specifically address the issue of timing, we estimated intraspecific divergence times on the basis of multiple fossil-based calibration points, using taxa from Altingiaceae (Liquidambar and Altingia) and Hammamelidaceae (Hamamelis) as outgroups. More than half of the sampled localities exhibited multiple haplotypes. Remarkably, the greatest variation was observed within the USA, with Mexico and Guatemala sharing widespread haplotypes with Texas, Mississippi, Kentucky, Ohio, and northern Virginia. This lack of differentiation suggests shared ancestral polymorphisms, and that the genetic signal we observed is older than the disjunction itself. Our data provide support for previously proposed hypotheses of Pleistocene refugia in peninsular Florida and along the eastern Atlantic, but also for deeper divergences (approximately 8 million years ago) within the USA. These patterns reflect a dynamic biogeographical history for eastern North American trees, and emphasize the importance of the inclusion of a temporal component in any phylogeographical study.

Comparative phylogeography of unglaciated eastern North America.

Regional phylogeographical studies involving co-distributed animal and plant species have been conducted for several areas, most notably for Europe and the Pacific Northwest of North America. Until recently, phylogeographical studies in unglaciated eastern North America have been largely limited to animals. As more studies emerge for diverse lineages (including plants), it seems timely to assess the phylogeography across this region: (i) comparing and contrasting the patterns seen in plants and animals; (ii) assessing the extent of pseudocongruence; and (iii) discussing the potential applications of regional phylogeography to issues in ecology, such as response to climatic change. Unglaciated eastern North America is a large, geologically and topographically complex area with the species examined having diverse distributions. Nonetheless, some recurrent patterns emerge: (i) maritime - Atlantic vs. Gulf Coast; (ii) Apalachicola River discontinuity; (iii) Tombigbee River discontinuity; (iv) the Appalachian Mountain discontinuity; (v) the Mississippi River discontinuity; and (vi) the Apalachicola River and Mississippi River discontinuities. Although initially documented in animals, most of these patterns are also apparent in plants, providing support for phylogeographical generalizations. These patterns may generally be attributable to isolation and differentiation during Pleistocene glaciation, but in some cases may be older (Pliocene). Molecular studies sometimes agree with longstanding hypotheses of glacial refugia, but also suggest additional possible refugia, such as the southern Appalachian Mountains and areas close to the Laurentide Ice Sheet. Many species exhibit distinct patterns that reflect the unique, rather than the shared, aspects of species' phylogeographical histories. Furthermore, similar modern phylogeographical patterns can result from different underlying causal factors operating at different times (i.e. pseudocongruence). One underemphasized component of pseudocongruence may result from the efforts of researchers to categorize patterns visually - similar patterns may, in fact, not fully coincide, and inferring agreement may obscure the actual patterns and lead to erroneous conclusions. Our modelling analyses indicate no clear spatial patterning and support the hypothesis that phylogeographical structure in diverse temperate taxa is complex and was not shaped by just a few barriers.

Stratified analysis of the soil seed bank in the cedar glade endemic Astragalus bibullatus: evidence for historical changes in genetic structure.

Persistent seed banks may provide information on historical changes in the genetic composition of populations. We used stratified sampling of the soil seed bank of Astragalus bibullatus (Pyne's ground plum) to assess levels of temporal variation in population genetic structure and to infer historical changes in the levels of inbreeding and relative gene flow. This species has an extremely limited distribution in the Central Basin of Tennessee, where it is found in open areas and along the edges of cedar glades. Protein electrophoresis was conducted on seedlings grown from seeds that had been recovered from three successive 1 cm thick layers of soil sampled from six sites. Analyses of seven polymorphic allozyme loci indicated that there were substantial levels of genetic differentiation among soil layers and sites. Higher levels of genetic diversity were found in seed than in vegetative populations that had been sampled in a previous study. Seed populations from the uppermost soil layer had higher heterozygote deficiencies, displayed higher levels of differentiation among sites, and had higher private allele frequencies than seed populations from the lower two layers. The change in heterozygosity and distribution of genetic variation among sites for the youngest soil layer is consistent with a pattern of increased selfing, sib mating, and decreased gene flow among populations. These changes in inbreeding and relative levels of gene flow are corroborated by information on historical land use practices in the region and support the hypothesis that loss of appropriate habitat has led to smaller population sizes and a more fragmented distribution of this cedar glade endemic.