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—Oenotherasect.Pachylophushas proven to be a valuable system in which to study plant-insect coevolution and the drivers of variation in floral morphology and scent. Current species circumscriptions based on morphological characteristics suggest that the section consists of five species, one of which is subdivided into five subspecies. Previous attempts to understand species (and subspecies) relationships at a molecular level have been largely unsuccessful due to high levels of incomplete lineage sorting and limited phylogenetic signal from slowly evolving gene regions. In the present study, target enrichment was used to sequence 322 conserved protein-coding nuclear genes from 50 individuals spanning the geographic range ofOenotherasect.Pachylophus, with species trees inferred using concatenation and coalescent-based methods. Our findings concur with previous research in suggesting thatO. psammophilaandO. harringtoniiare nested within a paraphyleticOenothera cespitosa. By contrast, our results show clearly that the two annual species (O. cavernaeandO. brandegeei) did not arise from theO. cespitosalineage, but rather from a common ancestor ofOenotherasect.Pachylophus. Budding speciation as a result of edaphic specialization appears to best explain the evolution of the narrow endemic speciesO. harringtoniiandO. psammophila. Complete understanding of possible introgression among subspecies ofO. cespitosawill require broader sampling across the full geographical and ecological ranges of these taxa.

Premise Divergence depends on the strength of selection and frequency of gene flow between taxa, while reproductive isolation relies on mating barriers and geographic distance. Less is known about how these processes interact at early stages of speciation. Here, we compared population-level differentiation in floral phenotype and genetic sequence variation among recently diverged Castilleja to explore patterns of diversification under different scenarios of reproductive isolation. Methods Using target enrichment enabled by the Angiosperms353 probe set, we assessed genetic distance among 50 populations of four Castilleja species. We investigated whether patterns of genetic divergence are explained by floral trait variation or geographic distance in two focal groups: the widespread C. sessiliflora and the more restricted C. purpurea species complex. Results We document that C. sessiliflora and the C. purpurea complex are characterized by high diversity in floral color across varying geographic scales. Despite phenotypic divergence, groups were not well supported in phylogenetic analyses, and little genetic differentiation was found across targeted Angiosperms353 loci. Nonetheless, a principal coordinate analysis of single nucleotide polymorphisms revealed differentiation within C. sessiliflora across floral morphs and geography and less differentiation among species of the C. purpurea complex. Conclusions Patterns of genetic distance in C. sessiliflora suggest species cohesion maintained over long distances despite variation in floral traits. In the C. purpurea complex, divergence in floral color across narrow geographic clines may be driven by recent selection on floral color. These contrasting patterns of floral and genetic differentiation reveal that divergence can arise via multiple eco-evolutionary paths.

Premise of research. Ex situ plant conservation can be improved through genetic analysis. One area of interest is the relative value of conserving smaller or larger populations and how sampling str...

Pollinator interactions are important in the evolution of floral traits, given that pollinators can directly influence plant fitness and mating patterns through interactions with flowers. However, geographic variation in both plant traits and floral visitors across multiple populations is largely understudied, despite being ubiquitous. This study explores whether a geographic mosaic of ecological interactions underlies observed patterns of floral divergence 1) among species of the Castilleja purpurea complex (C. purpurea, C. citrina, and C. lindheimeri) and the congener C. sessiliflora, as well as 2) within C. sessiliflora, across its wide geographic range. We sampled floral visitors and floral traits (morphology and color) at 23 populations across a 1900 km study area in 1-3 years, with reproductive fitness (fruit set) data for 18 of these populations.We documented a wide diversity of pollinator functional groups visiting the four focal species, including bees, butterflies, hawkmoths, and hummingbirds. Visitor assemblages varied among species and across geography in the composition and diversity of floral visitors. We found relationships between floral traits and visitation by certain pollinator groups, which often aligned with syndrome-associated predictions. Additionally, we found evidence that visitation from pollinators predicted via syndromes was associated with increased reproductive fitness for two species: the red-flowered C. lindheimeri and hummingbird visitors, and the long-floral-tubed C. sessiliflora and hawkmoths.Beyond these cases, we found that pollinator functional groups were not restricted to plant species, and instead pollinators were largely generalist in their foraging behavior, suggesting the likelihood for incomplete reproductive isolation and the potential for ongoing gene flow among plant species where their ranges overlap.This study provides a large-scale exploration of how variation in pollinator assemblages across distributions may underlie floral trait divergence within and among recently diverged species, even when characterized by largely generalized modes of pollination. Our extensive sampling of 23 populations over multiple years across a large geographic area highlights the value of range-wide studies for characterizing patterns of divergence and speciation mediated by ecological interactions.

Oenothera sect. Calylophus is a North American group of 13 recognized taxa in the evening primrose family (Onagraceae) with an evolutionary history that may include independent origins of bee pollination, edaphic endemism, and permanent translocation heterozygosity. Like other groups that radiated relatively recently and rapidly, taxon boundaries within Oenothera sect. Calylophus have remained challenging to circumscribe. In this study, we used target enrichment, flanking noncoding regions, gene tree/species tree methods, tests for gene flow modified for target-enrichment data, and morphometric analysis to reconstruct phylogenetic hypotheses, evaluate current taxon circumscriptions, and examine character evolution in Oenothera sect. Calylophus. Because sect. Calylophus comprises a clade with a relatively restricted geographic range, we were able to extensively sample across the range of geographic, edaphic, and morphological diversity in the group. We found that the combination of exons and flanking noncoding regions led to improved support for species relationships. We reconstructed potential hybrid origins of some accessions and note that if processes such as hybridization are not taken into account, the number of inferred evolutionary transitions may be artificially inflated. We recovered strong evidence for multiple evolutionary origins of bee pollination from ancestral hawkmoth pollination, edaphic specialization on gypsum, and permanent translocation heterozygosity. This study applies newly emerging techniques alongside dense infraspecific sampling and morphological analyses to effectively reconstruct the recalcitrant history of a rapid radiation. [Gypsum endemism; Oenothera sect. Calylophus; Onagraceae; phylogenomics; pollinator shift; recent radiation; target enrichment.]

Premise of research. To safeguard threatened plant species, best-practice guidelines and genetic modeling emphasize that ex situ collections should be composed of high numbers of maternal lines. Th...

The genetic diversity of germplasm used in reintroduction and restoration efforts can influence how resulting populations establish, reproduce, and evolve over time, particularly in disturbed and changing conditions. Regional admixture provenancing, mixing seeds derived from multiple populations within the same region as the target site, has been suggested to produce genetically diverse germplasm. Yet little empirical evidence shows how genetic diversity in germplasm resulting from this approach compares to source populations, or how it varies in restored populations. Here, we use neutral molecular markers to follow genetic diversity through production and use of germplasm when mixing multiple source populations in nursery production beds. Castilleja levisecta is a rare species experiencing inbreeding depression in remaining populations, with a federal recovery plan requiring the re‐establishment of populations in areas where it has been extirpated. Specifically, we track diversity from wild‐collected source populations through different production approaches and reintroductions using two propagule types. We show that measures of genetic diversity, inbreeding, and relatedness change during the production and use of material produced with a regional admixture provenancing approach, with the step at which source populations are mixed and germplasm type used influencing whether all source populations are equally represented. While genetic diversity increased throughout the process, inbreeding and relatedness increased in nursery production beds but decreased in reintroductions, with the lowest inbreeding and relatedness in populations restored using seeds rather than plugs. The results highlight the importance of taking an integrated approach informed by research when planning and implementing reintroductions with mixed‐source germplasm.

To avoid inbreeding depression, plants have evolved diverse breeding systems to favor outcrossing, such as self-incompatibility. However, changes in biotic and abiotic conditions can result in selective pressures that lead to a breakdown in self-incompatibility. The shift to increased selfing is commonly associated with reduced floral features, lower attractiveness to pollinators, and increased inbreeding. We tested the hypothesis that the loss of self-incompatibility, a shift to self-fertilization (autogamy), and concomitant evolution of the selfing syndrome (reduction in floral traits associated with cross-fertilization) will lead to increased inbreeding and population differentiation in Oenothera primiveris. Across its range, this species exhibits a shift in its breeding system and floral traits from a self-incompatible population with large flowers to self-compatible populations with smaller flowers.We conducted a breeding system assessment, evaluated floral traits in the field and under controlled conditions, and measured population genetic parameters using RADseq data.Our results reveal a bimodal transition to the selfing syndrome from the west to the east of the range of O. primiveris. This shift includes variation in the breeding system and the mating system, a reduction in floral traits (flower diameter, herkogamy, and scent production), a shift to greater autogamy, reduced genetic diversity, and increased inbreeding.The observed variation highlights the importance of range-wide studies to understand breeding system variation and the evolution of the selfing syndrome within populations and species.

Land-use change is among the top drivers of global biodiversity loss, which impacts the arrangement and distribution of suitable habitat for species. Population-level effects include increased isolation, decreased population size, and changes to mutualistic and antagonistic interactions. However, the extent to which species are impacted is determined by life history characteristics including dispersal. In plants, mating dynamics can be changed in ways that can negatively impact population persistence if dispersal of pollen and/or seed is disrupted. Long-distance dispersal has the potential to buffer species from the negative impacts of land-use change. Biotic vectors of long-distance dispersal have been less frequently studied, though specific taxa are known to travel great distances. Here, we describe population genetic diversity and structure in a sphingophilous species that is experiencing habitat fragmentation through land-use change, Oenothera harringtonii W. L. Wagner, Stockh. & W. M. Klein (Onagraceae). We use 12 nuclear and four plastid microsatellite markers and show that pollen dispersal by hawkmoths drives high gene flow and low population differentiation despite a range-wide gradient of land-use change and habitat fragmentation. By separating the contributions of pollen and seed dispersal to gene flow, we show that most of the genetic parameters are driven by hawkmoth-facilitated long-distance pollen dispersal, but populations with small, effective population sizes experience higher levels of relatedness and inbreeding. We discuss considerations for conservation efforts for this and other species that are pollinated by long-distance dispersers.

Genetic diversity is a critical resource for species’ survival during times of environmental change. Conserving and sustainably managing genetic diversity requires understanding the distribution and amount of genetic diversity (in situ and ex situ) across multiple species. This paper focuses on three emblematic and IUCN Red List threatened oaks (Quercus, Fagaceae), a highly speciose tree genus that contains numerous rare species and poses challenges for ex situ conservation. We compare the genetic diversity of three rare oak species—Quercus georgiana, Q. oglethorpensis, and Q. boyntonii—to common oaks, investigate the correlation of range size, population size, and the abiotic environment with genetic diversity within and among populations in situ, and test how well genetic diversity preserved in botanic gardens correlates with geographic range size. Our main findings are: (1) these three rare species generally have lower genetic diversity than more abundant oaks, (2) in some cases, small population size and geographic range correlate with genetic diversity and differentiation, and (3) genetic diversity currently protected in botanic gardens is inadequately predicted by geographic range size and number of samples preserved, suggesting non-random sampling of populations for conservation collections. Our results highlight that most populations of these three rare oaks have managed to avoid severe genetic erosion, but their small size will likely necessitate genetic management going forward.

Oenothera sect. Calylophus is a North American group of 13 recognized taxa in the evening primrose family (Onagraceae) with an evolutionary history that may include independent origins of bee pollination, edaphic endemism, and permanent translocation heterozygosity. Like other groups that radiated relatively recently and rapidly, taxon boundaries within Oenothera sect. Calylophus have remained challenging to circumscribe. In this study, we used target enrichment, flanking non-coding regions, summary coalescent methods, tests for gene flow modified for target-enrichment data, and morphometric analysis to reconstruct phylogenetic hypotheses, evaluate current taxon circumscriptions, and examine character evolution in Oenothera sect. Calylophus. Because sect. Calylophus comprises a clade with a relatively restricted geographic range, we were able to extensively sample across the range of geographic and morphological diversity in the group. We found that the combination of exons and flanking non-coding regions led to improved support for species relationships. We reconstructed potential hybrid origins of some accessions and note that if processes such as hybridization are not taken into account, the number of inferred evolutionary transitions may be artificially inflated. We recovered strong evidence for multiple origins of the evolution of bee pollination from ancestral hawkmoth pollination, the evolution of edaphic specialization on gypsum, and permanent translocation heterozygosity. This study applies newly emerging techniques alongside dense infraspecific sampling and morphological analyses to effectively address a relatively common but recalcitrant problem in evolutionary biology.

BackgroundPlant volatiles play an important role in both plant-pollinator and plant-herbivore interactions. Intraspecific polymorphisms in volatile production are ubiquitous, but studies that explore underlying differential gene expression are rare. Oenothera harringtonii populations are polymorphic in floral emission of the monoterpene (R)-(-)-linalool; some plants emit (R)-(-)-linalool (linalool+ plants) while others do not (linalool-plants). However, the genes associated with differential production of this floral volatile in Oenothera are unknown. We used RNA-Seq to broadly characterize differential gene expression involved in (R)-(-)-linalool biosynthesis. To identify genes that may be associated with the polymorphism for this trait, we used RNA-Seq to compare gene expression in six different Oenothera harringtonii tissues from each of three linalool+ and linalool-plants.ResultsThree clusters of differentially expressed genes were enriched for terpene synthase activity: two were characterized by tissue-specific upregulation and one by upregulation only in plants with flowers that produce (R)-(-)-linalool. A molecular phylogeny of all terpene synthases identified two putative (R)-(-)-linalool synthase transcripts in Oenothera harringtonii, a single allele of which is found exclusively in linalool+ plants.ConclusionsBy using a naturally occurring polymorphism and comparing different tissues, we were able to identify genes putatively involved in the biosynthesis of (R)-(-)-linalool. Expression of these genes in linalool-plants suggests a regulatory polymorphism, rather than a population-specific loss-of-function allele. Additional terpene biosynthesis-related genes that are up-regulated in plants that emit (R)-(-)-linalool may be associated with herbivore defense, suggesting a potential economy of scale between plant reproduction and defense.

Edaphic endemic species are considered high conservation priority, as they are a unique and critical component of the ecosystems and are often restricted to small fragmented habitats. Castilleja affinis ssp. neglecta is a serpentine endemic species, known from six sites within the San Francisco Bay Area and is the focus of active restoration efforts. It grows sympatrically with the subspecies, C. affinis ssp. affinis, which is more widespread but differs in floral color and soil preference. In this study, we used morphometric measurements (three bract, ten floral, and two leaf measurements) and microsatellite markers to determine (1) how the two subspecies differ, (2) if there is evidence of hybridization and (3) quantify the genetic structure of known populations of C. affinis ssp. neglecta. We found that all 15 morphological measurements differed significantly between the subspecies and neutral genetic markers show strong genetic differentiation. Overall we found C. affinis ssp. neglecta populations had similar levels of genetic diversity and genetic differentiation between populations but higher inbreeding than compared to its more common congener. Two populations of C. affinis ssp. neglecta showed lower genetic differentiation from the C. affinis ssp. affinis populations, with some individuals showing considerable overlap in genotypic diversity, hence we cannot rule out historic or low levels hybridization in those populations. Castilleja affinis ssp. neglecta is a federally endangered species that would benefit from restoration efforts that aims to maintain genetic diversity while minimizing inbreeding in reintroduction efforts.

Invasion biology research, often performed by scientists at relatively small spatial scales, provides experimental precision but may be limited in generalizability. Conversely, large-scale invasive species management represents a largely untapped wealth of information on invasion ecology and management, but such data are difficult to capture and synthesize. We developed a network (“PhragNet”) of individuals managing wetlands occupied by native and non-native lineages of the invasive wetland grass Phragmites australis (common reed). This network collected environmental and genetic samples, habitat data, and management information to identify environmental and plant community associations of Phragmites invasion and patterns of management responses. Fifty managers overseeing 209 Phragmites stands in 16 US states and ON, Canada participated. Participants represented federal agencies (26%), municipalities (20%), NGOs (20%), academia (14%), state agencies (12%), and private landowners (8%). Relative to the native lineage, non-native Phragmites occurred in areas with higher nitrate/nitrite and ammonium than non-native Phragmites. Stand interiors had higher soil electrical conductivity than nearby uninvaded areas, consistent with use of road salt promoting spread of Phragmites. Non-native Phragmites co-occurred with fewer plant species than native Phragmites and was actively targeted for management. Herbicide was applied to 51% of non-native stands; surprisingly, 11% of native stands were also treated with herbicide. This project demonstrates the utility of crowdsourcing standardized data from resource managers. We conclude by describing how this approach could be expanded into an adaptive management framework, strengthening connections between wetland management and research.

Isoetes butleri Engelm. (Butler’s Quillwort) is a lycopod endemic to the central and southeastern United States. It inhabits sparsely vegetated moist depressions within dolomite prairies that become ephemeral wetlands in the spring. Classified as endangered in portions of its range (Illinois and Kentucky), little is known about the ecology and genetics of I. butleri . In Illinois, the five known populations are disjunct from all other known populations, and there is concern that these populations could be vulnerable to a number of threats, including a lack of suitable microhabitats, displacement by other plant species, accumulation of plant litter, and low genetic diversity and limited gene flow. We evaluated these putative threats by analyzing demographic data collected by Plants of Concern, a citizen-science rare-plant monitoring program; performing experimental litter removals; and assessing population genetic structure. There were no significant differences in soil nutrient availability in topographically similar depressions with and without I. butleri ; however, areas inhabited by I. butleri had lower abundance of woody species and higher abundance of conservative (habitat specialist) species than uninhabited areas. Following experimental litter removal, I. butleri exhibited lower size but higher density and recruitment. We found that all populations had high genetic diversity. However, there was also evidence of high inbreeding, which may be due to spatially fine-scale genetic structure and poor dispersal ability. Our findings suggest that reductions of woody plant growth and accumulated litter—both of which can be achieved through prescribed fire—should be management priorities for I. butleri conservation.

Maintaining a living plant collection is the most common method of ex situ conservation for plant species that cannot be seed banked (i.e., exceptional species). Viability of living collections, and their value for future conservation efforts, can be limited without coordinated efforts to track and manage individuals across institutions. Using a pedigree‐focused approach, the zoological community has established an inter‐institutional infrastructure to support long‐term viability of captive animal populations. We assessed the ability of this coordinated metacollection infrastructure to support the conservation of 4 plant species curated in living collections at multiple botanic gardens around the world. Limitations in current practices include the inability to compile, share, and analyze plant collections data at the individual level, as well as difficulty in tracking original provenance of ex situ material. The coordinated metacollection framework used by zoos can be adopted by the botanical community to improve conservation outcomes by minimizing the loss of genetic diversity in collections. We suggest actions to improve ex situ conservation of exceptional plant species, including developing a central database to aggregate data and track unique individuals of priority threatened species among institutions and adapting a pedigree‐based population management tool that incorporates life‐history aspects unique to plants. If approached collaboratively across regional, national, and global scales, these actions could transform ex situ conservation of threatened plant species., Article Impact Statement: Using zoo‐style studbooks and pedigree management will be transformative for the conservation of exceptional plants like Brighamia insignis.

Premise of the study Land-use change is cited as a primary driver of global biodiversity loss, with myriad consequences for species, populations, and ecosystems. However, few studies have examined its impact on species interactions, particularly pollination. Furthermore, when the effects of land-use change on pollination have been studied, the focus has largely been on species pollinated by diurnal pollinators, namely, bees and butterflies. Here, we focus on Oenothera harringtonii, a night-flowering, disturbance-adapted species that has experienced a range-wide gradient of land-use change. We tested the hypothesis that the negative impacts of land-use change are mitigated by long-distance pollination. Methods Our study included both temporal (4 yr) and spatial (19 populations range-wide, and 1, 2, and 5 km from the population center) data, providing a comprehensive understanding of the role of land-use change on pollination biology and reproduction. Key results We first confirmed that O. harringtonii is self-incompatible and reliant on pollinators for reproduction. We then showed that hawkmoths (primarily Hyles lineata) are highly reliable and effective pollinators in both space and time. Unlike other studies, we did not detect an effect of population size, increased isolation, or a reduction in suitable habitat in areas with evidence of land-use change on pollination (visitation, pollen removal and deposition). Furthermore, the proportion of suitable habitat and other fragmentation metrics examined were not associated with population size or density in this plant species. Conclusions We conclude that nocturnal pollination of Oenothera harringtonii via hawkmoths is robust to the negative impacts of land-use change.

Aim A surprising finding of genetic studies of Phragmites australis is that native populations, whether in Europe or North America, are characterized by lower genetic diversity than non-native populations. What is not clear is whether higher diversity within invasive stands results from disturbance facilitating sexual reproduction or whether individuals with higher diversity have greater invasion potential. Location Upper Midwest, United States, North America. Methods To answer this question, we investigated genetic structure of native and non-native P. australis stands at multiple spatial scales and under differing levels of anthropogenic disturbance. Our goals were to assess the influence of habitat disturbance on genetic structure and determine whether patterns differed in native versus non-native stands. We also screened for hybrid genotypes, which have not been reported from this region. Results When controlling for disturbance, native and non-native P. australis exhibited relatively similar within-site genetic diversity and degrees of clonal growth, suggesting that their spread is comparably reliant on clonal versus sexual dispersal. In all cases, discrete clumps of P. australis were composed of multiple, closely related genotypes, indicating seed heads as the key dispersal units. The genetic structure of P. australis stands did not differ with disturbance; however, only non-native P. australis was found in highly disturbed anthropogenic habitats. Native P. australis stands exhibited lower genetic diversity overall, lower sexual reproduction in less disturbed sites and greater isolation by distance and differentiation among sites, which is consistent with its longer residence time in this region. We found no evidence of hybrid genotypes, despite native and non-native stands sometimes co-occurring. Main conclusions Our results suggest that invasion by non-native P. australis is sporadic, associated with disturbance and involves closely related individuals, likely derived from a single seed head. The ability of the non-native lineage to exploit a variety of anthropogenic habitats helps to explain its invasiveness.

Lepidospartum burgessii is a rare gypsophilic shrub with limited distribution in New Mexico and Texas. Most of the known plants are restricted to two large populations, with a few small, isolated populations scattered in the surrounding area. The low recruitment observed in the two largest populations may be due to low seed set resulting from high inbreeding and/or self-incompatibility. We used eight microsatellite loci to quantify diversity, relatedness, inbreeding, population structure, and frequency of clonal reproduction. Seed collections were made to quantify seed set and germination rates. Overall, there was a moderate level of clonal diversity within patches of L. burgessii indicating asexual growth is important for population persistence. Inbreeding coefficients were high both between and within populations. Most patches showed a significant level of relatedness between individuals. At a fine scale, patches within populations were significantly different from each other, however when all patches were combined, the two populations of L. burgessii were not genetically distinct. Compared to a population of its common congener, Lepidospartum latisquamum, L. burgessii populations had similar measures of diversity, more clonal reproduction, and lower germination rates. High relatedness and inbreeding may explain the low seed set and recruitment in L. burgessii, however factors such as insect herbivory and precipitation changes may further depress recruitment.

Populations of scarlet Indian paintbrush (Castilleja coccinea) in the Midwestern United States exhibit a bract color polymorphism, with each population having predominantly yellow or scarlet bracts. We investigated a possible mechanism for this maintenance of bract color polymorphism in C. coccinea by conducting hand-pollination experiments in two nearby populations, one predominantly yellow and one predominantly scarlet. The hand-pollination treatments were either self-pollination or cross pollination using pollen from within and between populations. Both color morphs were used as pollen donors for the within and between crosses. We found that both color morphs of C. coccinea were self-compatible. When the scarlet morph was the maternal plant it had higher seed set. When pollinators were excluded, the yellow morph outperformed the scarlet morph in fruit set and seed set. The apparent trade-offs between a higher reproductive output in the scarlet morph and a reproductive assurance advantage in the yellow morph may explain the maintenance of the polymorphism in C. coccinea. While many previous studies have provided evidence for pollinator preference playing a role in floral color polymorphism, the results of the current study indicate that reproductive assurance, which would be important for fluctuations in pollinator abundance or colonizing new areas, may act as a selective agent to maintain such polymorphisms.

Effectively conserving biodiversity with limited resources requires scientifically informed and efficient strategies. Guidance is particularly needed on how many living plants are necessary to conserve a threshold level of genetic diversity inex situcollections. We investigated this question for 11 taxa across five genera. In this first study analysing and optimizingex situgenetic diversity across multiple genera, we found that the percentage of extant genetic diversity currently conserved varies among taxa from 40% to 95%. Most taxa are well below genetic conservation targets. Resampling datasets showed that ideal collection sizes vary widely even within a genus: one taxon typically required at least 50% more individuals than another (thoughQuercuswas an exception). Still, across taxa, the minimum collection size to achieve genetic conservation goals is within one order of magnitude. Current collections are also suboptimal: they could remain the same size yet capture twice the genetic diversity with an improved sampling design. We term this deficiency the ‘genetic conservation gap’. Lastly, we show that minimum collection sizes are influenced by collection priorities regarding the genetic diversity target. In summary, current collections are insufficient (not reaching targets) and suboptimal (not efficiently designed), and we show how improvements can be made.

Forest resources face numerous threats that require costly management. Hence, there is an increasing need for data-informed strategies to guide conservation practices. The introduction of the emerald ash borer to North America has caused rapid declines in ash populations (Fraxinus spp. L.). Natural resource managers are faced with a choice of either allowing ash trees to die, risking forest degradation and reduced functional resilience, or investing in conserving trees to preserve ecosystem structure and standing genetic diversity. The information needed to guide these decisions is not always readily available. Therefore, to address this concern, we used eight microsatellites to genotype 352 white ash trees (Fraxinus americana L.) across 17 populations in the Allegheny National Forest; a subset of individuals sampled are part of an insecticide treatment regimen. Genetic diversity (number of alleles and He) was equivalent in treated and untreated trees, with little evidence of differentiation or inbreeding, suggesting current insecticidal treatment is conserving local, neutral genetic diversity. Using simulations, we demonstrated that best practice is treating more populations rather than more trees in fewer populations. Furthermore, through genetic screening, conservation practitioners can select highly diverse and unique populations to maximize diversity and reduce expenditures (by up to 21%). These findings will help practitioners develop cost-effective strategies to conserve genetic diversity.

Native plants that are able to persist and reproduce in highly disturbed habitats (i.e., “native winners”) may be useful to include in seed mixes when restoring similarly disturbed habitat. Establishing whether these plants produce viable seeds that germinate to a high degree under appropriate conditions is a first step to determining their utility as restoration species. We identified 10 potential native winners at sites degraded by cheatgrass (Bromus tectorum), an invasive annual grass ubiquitous in the Colorado Plateau. We assessed seed viability for each species to determine its potential to reproduce within a cheatgrass-invaded site, and conducted a series of germination and competition experiments to test how effective these species may be when restoring habitat invaded by cheatgrass. All species produced viable seed (ranging from 56% to 100% viability), and the seeds of many species had high germination under a range of fall conditions without cold stratification, which is thought to incr...

Ecological restoration aims to assist the recovery of degraded, damaged, or destroyed ecosystems. Restoration practitioners increasingly recognize the value of using ecologically appropriate and genetically diverse native plant material to support ecosystem recovery and long-term persistence in the face of unpredictable current and future conditions. Producing genetically diverse native plant material, however, can be incredibly challenging. Each step of production, from procuring raw material to installing produced material into a restoration site, has the potential to affect the genetic diversity of the produced material. Here we examine each of the production steps, from wildland seed collection through seed or seedling production. We outline each step where genetic diversity can be lost or gained, and describe 10 rules that can be used to maintain high genetic variability in native plant material throughout the production process.

For plant species important in ecological restoration, seed transfer zones have been developed to maximize the probability that sown seed will germinate, establish, persist, and reproduce without negatively impacting the genetic composition of remnant plant populations. However, empirically based seed transfer zones have not been developed for most species. In their absence, maps based on ecological or climatic variables have been suggested as proxies. In the United States, these maps typically include the Environmental Protection Agency's Levels III and IV Ecoregion maps and the US Forest Service's Provisional Seed Zones. Maps of different spatial scales represent a compromise between economic and ecological considerations; those that delineate larger seed transfer zones are less costly to implement but impose more risk of poor adaptation to local conditions. To test the relative suitability of each map in delineating seed transfer zones, we conducted common garden experiments using five forb s...

Proper sourcing of seed for ecological restoration has never been straightforward, and it is becoming even more challenging and complex as the climate changes. For decades, restoration practitioners have subscribed to the “local is best” tenet, even if the definition of “local” was often widely divergent between projects. However, given our increasing ability to characterize habitats, and rapid climate change, we can no longer assume that locally sourced seeds are always the best or even an appropriate option. We discuss how plants are responding to changing climates through plasticity, adaptation, and migration, and how this may influence seed sourcing decisions. We recommend focusing on developing adequate supplies of “workhorse” species, undertaking more focused collections in both “bad” years and “bad” sites to maximize the potential to be able to adapt to extreme conditions as well as overall genetic diversity, and increasing seed storage capacity to ensure we have seed available as we continue to conduct research to determine how best to deploy it in a changing climate.

A study was conducted to assess the effects of predicted climate change on seedling emergence, growth, and survival for the federally listed threatened species Cirsium pitcheri in the region proximal to Chicago, Illinois, U.S.A. Three geographically distant extant populations that could potentially serve as donors to re- introduced Illinois populations were tested to determine the influence of local adaptation on seed response to climate scenarios at two future time points. For all three populations, temperature was shown to be the most critical factor impacting future growth. All populations performed worst at the 2095 temperature for four measured growth metrics: length of longest adult leaf, number of adult leaves, dry shoot mass, and dry root mass. Growth performance at the 2095 conditions did not correlate with climate familiarity but did reflect the genetic diversity of the parent population. Predicted changes in average precipitation did not produce a significant effect on any growth metric, possibly reflecting a limitation in the study conditions. Seedling emergence and survival were tied to seed mass, a trait influenced by maternal effects and correlated with the level of inbreeding depression in the parent population. These results suggest managing populations to maintain high genetic diversity could be an important factor in tolerating temperature stress associated with climate change.

We investigated the role of genetic, reproductive and demographic factors in the decline of two co-occurring milkweeds, Asclepias lanuginosa and A. viridiflora, in fragmented populations in Illinois and Wisconsin. Asclepias lanuginosa flowers but does not set seed while seed set is regularly observed in A. viridiflora. We used microsatellite genotyping to determine the extent of clonal growth, genetic diversity, and genetic structure in nine populations of A. lanuginosa and five populations of A. viridiflora. Microsatellite genotyping revealed extremely high clonality in A. lanuginosa; only 32 multilocus genotypes occurred among more than 300 ramets, compared to 118 multilocus genotypes among 124 ramets for A. viridiflora. Four A. lanuginosa populations were monoclonal. While we found no evidence for inbreeding, A. lanuginosa had significantly lower expected heterozygosity and a lower mean number of effective alleles than A. viridiflora. Population viability analysis (PVA) conducted at one site indicated a high probability of persistence, although the population was comprised of only two clones. Because PVA does not distinguish between ramets and genets, results should be interpreted with caution when conducted on highly clonal species. A nearly complete shift in the mode of reproduction, from sexual to asexual, appears to be the most immediate threat to survival of A. lanuginosa in these fragmented prairie remnants. Conservation management strategies should include actions to increase clonal diversity in remnant populations.

Identifying factors that shape the spatial distribution of genetic variation is crucial to understanding many population- and landscape-level processes. In this study, we explore fine-scale spatial genetic structure in Oenothera harringtonii (Onagraceae), an insect-pollinated, gravity-dispersed herb endemic to the grasslands of south-central and southeastern Colorado, USA. We genotyped 315 individuals with 11 microsatellite markers and utilized a combination of spatial autocorrelation analyses and landscape genetic models to relate life history traits and landscape features to dispersal processes. Spatial genetic structure was consistent with theoretical expectations of isolation by distance, but this pattern was weak (Sp = 0.00374). Anisotropic analyses indicated that spatial genetic structure was markedly directional, in this case consistent with increased dispersal along prominent slopes. Landscape genetic models subsequently confirmed that spatial genetic variation was significantly influenced by local topographic heterogeneity, specifically that geographic distance, elevation and aspect were important predictors of spatial genetic structure. Among these variables, geographic distance was ~68% more important than elevation in describing spatial genetic variation, and elevation was ~42% more important than aspect after removing the effect of geographic distance. From these results, we infer a mechanism of hydrochorous seed dispersal along major drainages aided by seasonal monsoon rains. Our findings suggest that landscape features may shape microevolutionary processes at much finer spatial scales than typically considered, and stress the importance of considering how particular dispersal vectors are influenced by their environmental context. Subject areas: Reproductive strategies and kinship analysis; Population structure and phylogeography

Rapid spread of Phragmites australis (common reed) in North American wetlands is widely attributed to cryptic invasion by an introduced lineage. However, in the Midwestern U.S., the native subspecies (subsp. americanus) may also exhibit rapid expansion. Where both lineages occur, wetland managers are sometimes unsure whether they should limit management activities to the introduced lineage or control both. We conducted field studies to contrast the ecology of native and introduced Phragmites by pairing patches of each with native reference vegetation. We measured each lineage’s association with environmental conditions, their growth metrics (stem heights, stem densities, and plant cover), and their invasiveness as indicated by the diversity and composition of associated plant communities. Introduced Phragmites exhibited more robust growth than the native, and its growth was more positively correlated with increases in soil nutrient availability and salinity. Areas with introduced Phragmites had lower plant diversity and altered species composition relative to reference vegetation. We did not observe similar evidence of invasiveness in native Phragmites. We encourage wetland managers to differentiate populations by lineage and, unless there is compelling evidence to do otherwise, restrict control efforts to the introduced lineage.

The aim of any reintroduction is to provide sufficient genetic variability to buffer against changing selection pressures and ensure long-term survival. To date, few empirical studies have compared levels of genetic diversity in reintroduced and native plant populations. Using microsatellite markers, we measured the genetic diversity within reintroduced and native populations of the threatened Cirsium pitcher (Eaton) Torrey and Gray. We found that the use of local mixed source was successful in establishing populations with significantly higher genetic diversity (P < 0.005) than the native populations (allelic richness is 3.39 in reintroduced and 1.84 in native populations). However, the reintroduced populations had significantly higher inbreeding coefficients (P < 0.002) (FISis 0.405 and 0.213 in reintroduced and in native populations, respectively), despite having multiple genetic founders, population sizes equivalent to native populations and a positive growth rate. These results may be due to inbreeding or the Wahlund effect, driven by genetic substructuring. This suggests that the small population size of these reintroduced populations may lead to genetic issues in the future, given the low number of flowering individuals each year. This highlights the importance of considering not only the number of source individuals but the effective population size of the reintroduction.

With increasing urbanization and creation of novel habitat types, green roofs can provide habitable space for many species. To date, most research on green roofs has focused on minimizing environmental impacts of buildings and little is known about the ecological services they may provide. Previous research has found a deficiency of pollinating bees on green roofs, which could result in pollen limitation, poor seed production and reproductive failure of many plant species requiring bee pollination. This study aims to determine whether pollination services on modern green roofs are sufficient for these novel habitats to function sustainably. Nine native Illinois prairie plant species and their pollinator communities were studied on green roofs and ground-level locations in the Chicago area. Pan traps were used to assess pollinator communities and supplemental pollination treatments were used to evaluate pollen limitation. All species showed significantly reduced seed set when pollinators were excluded but few significant differences were observed between supplemental and open pollination treatments. Seed set differed by habitat type in that green roofs had a higher overall mean percent maximum seed set compared to ground-level sites. Our results support previous studies, showing lower numbers and diversity of bees on green roofs compared to the ground level. Together, these data suggest that although green roofs contain a smaller and less diverse community of pollinators, the insects that are present provide sufficient pollinator services for many native plants. This study therefore supports the use of biotically pollinated native forbs in future green roof design.

Due to loss of natural habitats, human-dominated green spaces are likely to increase in importance for biodiversity support. We assessed the potential value of urban “green roofs” for native pollinator conservation in the Chicago region, comparing them with reference habitats of tallgrass prairie natural areas and traditional city-park green spaces. We found that native bees are present on green roofs, though at lower abundance and diversity than in reference habitats. Green-roof and prairie bee communities were distinct from each other, while those in parks were intermediate and similar to the other two habitat types. Bee-community patterns were related to habitat characteristics at both the site and landscape scales. Overall, bee abundance and species richness increased with greater proportions of green space in the surrounding landscape. However, this relationship disappeared in cases where green space was dominated by turf grass. At the site scale, bees benefited from greater plant diversity, and bee and plant-community composition were significantly correlated. Green roofs are potentially valuable sites for bee conservation in urban areas, particularly if planted with diverse native forbs to provide foraging resources, and designed to accommodate bees with different nesting habits.

We studied the sole population of Opuntia fragilis in Illinois for four years, tracking plant and individual pad fates, and investigating flowering success. The population appears to be fairly stable in size from year to year. The fate of a given pad is strongly dependent upon the position of the pad within the plant; basal pads and transitional pads are likely to remain from year to year, while terminal pads experience substantially higher rates of death or fragmentation, and are much more likely to produce new growth and thus become transitional pads themselves. A fungal “black spot” disease, probably a result of winter damage, appears to negatively affect pad survival during some winters but not during other winters. While genetic diversity is not low, flowers do not produce viable seed, even when controlled cross-pollinations are performed, although they are visited by a variety of potential insect pollinators.