Target Enrichment and Extensive Population Sampling Help Untangle the Recent, Rapid Radiation of Oenothera Sect. Calylophus.

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.].

Identifying predictors of translocation success in rare plant species.

The fundamental goal of a rare plant translocation is to create self-sustaining populations with the evolutionary resilience to persist in the long term. Yet, most plant translocation syntheses focus on a few factors influencing short-term benchmarks of success (e.g., survival and reproduction). Short-term benchmarks can be misleading when trying to infer future growth and viability because the factors that promote establishment may differ from those required for long-term persistence. We assembled a large (n = 275) and broadly representative data set of well-documented and monitored (7.9 years on average) at-risk plant translocations to identify the most important site attributes, management techniques, and species' traits for six life-cycle benchmarks and population metrics of translocation success. We used the random forest algorithm to quantify the relative importance of 29 predictor variables for each metric of success. Drivers of translocation outcomes varied across time frames and success metrics. Management techniques had the greatest relative influence on the attainment of life-cycle benchmarks and short-term population trends, whereas site attributes and species' traits were more important for population persistence and long-term trends. Specifically, large founder sizes increased the potential for reproduction and recruitment into the next generation, whereas declining habitat quality and the outplanting of species with low seed production led to increased extinction risks and a reduction in potential reproductive output in the long-term, respectively. We also detected novel interactions between some of the most important drivers, such as an increased probability of next-generation recruitment in species with greater seed production rates, but only when coupled with large founder sizes. Because most significant barriers to plant translocation success can be overcome by improving techniques or resolving site-level issues through early intervention and management, we suggest that by combining long-term monitoring with adaptive management, translocation programs can enhance the prospects of achieving long-term success.

Identificación de pronosticadores del éxito de reubicación en especies raras de plantas Resumen El objetivo fundamental de la reubicación de plantas raras es la creación de poblaciones autosuficientes con resiliencia evolutiva que persistan a la larga. De todas maneras, la mayoría de las síntesis de estas reubicaciones se enfocan en unos cuantos factores que influyen sobre los parámetros a corto plazo del éxito (supervivencia y reproducción). Los parámetros a corto plazo pueden ser engañosos si se intenta inferir el crecimiento y la viabilidad en el futuro ya que los factores que promueven el establecimiento pueden diferir de aquellos requeridos para la persistencia a largo plazo. Ensamblamos un conjunto grande de datos representativos en general (n = 275) de las reubicaciones de plantas en riesgo bien documentadas y monitoreadas (7.9 años en promedio) para identificar los atributos de sitio más importantes, las técnicas de manejo y los rasgos de las especies para seis parámetros de ciclos de vida y medidas poblacionales del éxito de reubicación. Usamos el algoritmo de bosque aleatorio para cuantificar la importancia relativa de las 29 variables de pronosticadores para cada medida del éxito. Los factores en los resultados de las reubicaciones variaron con los marcos temporales y las medidas de éxito. Las técnicas de manejo tuvieron la mayor influencia relativa sobre la obtención de parámetros de ciclos de vida y tendencias poblacionales a corto plazo, mientras que los atributos de sitio y los rasgos de la especie fueron más importantes para la persistencia poblacional y las tendencias a largo plazo. En específico, las grandes cantidades de fundadores incrementaron el potencial de reproducción y reclutamiento de la siguiente generación, mientras que la declinación de la calidad del hábitat incrementó el riesgo de extinción y el trasplante de especies con baja producción de semillas redujo el rendimiento del potencial reproductivo a la larga. También detectamos interacciones novedosas entre algunos de los factores más importantes, como el aumento en la probabilidad del reclutamiento en la siguiente generación en especies con tasas mayores de producción de semillas, pero sólo cuando se emparejó con grandes cantidades de fundadores. Ya que las barreras más significativas para el éxito de la reubicación de plantas pueden superarse al mejorar las técnicas o resolver los temas a nivel de sitio por medio de un manejo y una intervención temprana, sugerimos que con la combinación del monitoreo a largo plazo con el manejo adaptativo los programas de reubicación pueden aumentar el prospecto de lograr el éxito a largo plazo.

Reproductive strategies and their consequences for divergence, gene flow, and genetic diversity in three taxa of Clarkia.

Differences in reproductive strategies can have important implications for macro- and micro-evolutionary processes. We used a comparative approach through a population genetics lens to evaluate how three distinct reproductive strategies shape patterns of divergence among as well as gene flow and genetic diversity within three closely related taxa in the genus Clarkia. One taxon is a predominantly autonomous self-fertilizer and the other two taxa are predominantly outcrossing but vary in the primary pollinator they attract. In genotyping populations using genotyping-by-sequencing and comparing loci shared across taxa, our results suggest that differences in reproductive strategies in part promote evolutionary divergence among these closely related taxa. Contrary to expectations, we found that the selfing taxon had the highest levels of heterozygosity but a low rate of polymorphism. The high levels of fixed heterozygosity for a subset of loci suggests this pattern is driven by the presence of structural rearrangements in chromosomes common in other Clarkia taxa. In evaluating patterns within taxa, we found a complex interplay between reproductive strategy and geographic distribution. Differences in the mobility of primary pollinators did not translate to a difference in rates of genetic diversity and gene flow within taxa - a pattern likely due to one taxon having a patchier distribution and a less temporally and spatially reliable pollinator. Taken together, this work advances our understanding of the factors that shape gene flow and the distribution of genetic diversity within and among closely related taxa.

Hawkmoth and bee pollinators impact pollen dispersal at the landscape but not local scales in two species of Oenothera.

PREMISE: Animal pollinators play an important role in pollen dispersal. Here, we assessed differences in pollen and seed dispersal and the role of pollinator functional groups with different foraging behaviors in generating patterns of genetic diversity over similar geographic ranges for two closely related taxa. We focused on two members of Oenothera section Calylophus (Onagraceae) that co-occur on gypsum outcrops throughout the northern Chihuahuan Desert but differ in floral phenotype and primary pollinator: Oenothera gayleana (bee) and O. hartwegii subsp. filifolia (hawkmoth). METHODS: We measured breeding system and floral traits and studied gene flow and population differentiation at the local (<13 km; four populations) and landscape (60-440 km; five populations) scales using 10-11 nuclear (pollen dispersal) and three plastid (seed dispersal) microsatellite markers. RESULTS: Both taxa were self-incompatible and floral traits were consistent with expectations for different pollinators. Seed and pollen dispersal patterns were distinctly different for both species. We found no evidence of genetic structure at the local scale but did at the landscape scale; O. gayleana showed greater differentiation and significant isolation by distance than in O. hartwegii subsp. filifolia. The plastid data were consistent with gravity dispersal of seeds and suggest that pollen dispersal is the principal driver of genetic structure in both species. CONCLUSIONS: We demonstrated that pollinator functional groups can impact genetic differentiation in different and predictable ways. Hawkmoths, with larger foraging distances, can maintain gene flow across greater spatial scales than bees.

Genotype accounts for intraspecific variation in the timing and duration of multiple, sequential life-cycle events in a willow species.

PREMISE: Phenological variation among individuals within populations is common and has a variety of ecological and evolutionary consequences, including forming the basis for population-level responses to environmental change. Although the timing of life-cycle events has genetic underpinnings, whether intraspecific variation in the duration of life-cycle events reflects genetic differences among individuals is poorly understood. METHODS: We used a common garden experiment with 10 genotypes of Salix hookeriana (coastal willow) from northern California, United States to investigate the extent to which genetic variation explains intraspecific variation in the timing and duration of multiple, sequential life-cycle events: flowering, leaf budbreak, leaf expansion, fruiting, and fall leaf coloration. We used seven clones of each genotype, for a total of 70 individual trees. RESULTS: Genotype affected each sequential life-cycle event independently and explained on average 62% of the variation in the timing and duration of vegetative and reproductive life-cycle events. All events were significantly heritable. A single genotype tended to be "early" or "late" across life-cycle events, but for event durations, there was no consistent response within genotypes. CONCLUSIONS: This research demonstrates that genetic variation can be a major component underlying intraspecific variation in the timing and duration of life-cycle events. It is often assumed that the environment affects durations, but we show that genetic factors also play a role. Because the timing and duration of events are independent of one another, our results suggest that the effects of environmental change on one event will not necessarily cascade to subsequent events.

Differential gene expression associated with a floral scent polymorphism in the evening primrose Oenothera harringtonii (Onagraceae).

BACKGROUND: Plant 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. RESULTS: Three 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. CONCLUSIONS: By using a naturally occurring polymorphism and comparing different tissues, we were able to identify candidate genes putatively involved in the biosynthesis of (R)-(-)-linalool. Expression of these genes in linalool- plants, while low, 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.

Evolution of selfing syndrome and its influence on genetic diversity and inbreeding: A range-wide study in Oenothera primiveris.

PREMISE: 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. METHODS: We conducted a breeding system assessment, evaluated floral traits in the field and under controlled conditions, and measured population genetic parameters using RADseq data. RESULTS: 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. CONCLUSIONS: 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.

Incomplete reproductive isolation and low genetic differentiation despite floral divergence across varying geographic scales in Castilleja.

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.

Taxonomic similarity does not predict necessary sample size for ex situ conservation: a comparison among five genera.

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 in ex situ collections. We investigated this question for 11 taxa across five genera. In this first study analysing and optimizing ex situ genetic 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 (though Quercus was 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.

Applying the zoo model to conservation of threatened exceptional plant species.

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.

Aplicación del Modelo Zoológico a la Conservación de Especies Excepcionales de Plantas Amenazadas Resumen El mantenimiento de una colección de plantas vivas es el método más común para de conservación ex situ para especies de plantas que no pueden almacenarse en bancos de semillas (i. e., especies excepcionales). La viabilidad de las colecciones vivientes, junto con el valor que representan para los futuros esfuerzo de conservación, puede estar limitada si no existen esfuerzos coordinados para rastrear y manejar a los individuos entre las instituciones. Mediante una estrategia enfocada en el linaje, la comunidad de zoológicos ha establecido una infraestructura interinstitucional que respalda la viabilidad a largo plazo de las poblaciones de animales en cautiverio. Evaluamos la habilidad de esta infraestructura coordinada de metacolecciones para apoyar en la conservación de cuatro especies de plantas curadas en colecciones vivientes en varios jardines botánicos de todo el mundo. Las limitaciones de las prácticas contemporáneas incluyen la incapacidad de recopilar, compartir y analizar los datos de las colecciones de plantas a nivel individual, así como la dificultad de rastrear la procedencia original del material ex situ. El marco de trabajo de metacolecciones coordinadas que utilizan los zoológicos puede ser adoptado por la comunidad botánica para mejorar los resultados de conservación al minimizar la pérdida de la diversidad genética que ocurre en las colecciones. Sugerimos acciones que aumenten la conservación ex situ de las especies excepcionales de plantas. Estas acciones incluyen el desarrollo de una base de datos central para acumular datos y rastrear entre las instituciones a los individuos únicos de las especies amenazadas prioritarias y la adaptación de una herramienta de manejo poblacional basada en el linaje que incorpore los aspectos únicos de la historia de vida de las plantas. Si estas acciones se plantean colaborativamente a escala regional, nacional y global, podrían transformar la conservación ex situ de las especies amenazadas de plantas.

Pollinator identity and spatial isolation influence multiple paternity in an annual plant.

The occurrence and extent of multiple paternity is an important component of variation in plant mating dynamics. However, links between pollinator activity and multiple paternity are generally lacking, especially for plant species that attract functionally diverse floral visitors. In this study, we separated the influence of two functionally distinct floral visitors (hawkmoths and solitary bees) and characterized their impacts on multiple paternity in a self-incompatible, annual forb, Oenothera harringtonii (Onagraceae). We also situated pollinator-mediated effects in a spatial context by linking variation in multiple paternity to variation in plant spatial isolation. We documented pronounced differences in the number of paternal sires as function of pollinator identity: on average, the primary pollinator (hawkmoths) facilitated mating with nearly twice as many pollen donors relative to the secondary pollinator (solitary bees). This effect was consistent for both isolated and nonisolated individuals, but spatial isolation imposed pronounced reductions on multiple paternity regardless of pollinator identity. Considering that pollinator abundance and pollen dispersal distance did not vary significantly with pollinator identity, we attribute variation in realized mating dynamics primarily to differences in pollinator morphology and behaviour as opposed to pollinator abundance or mating incompatibility arising from underlying spatial genetic structure. Our findings demonstrate that functionally distinct pollinators can have strongly divergent effects on polyandry in plants and further suggest that both pollinator identity and spatial heterogeneity have important roles in plant mating dynamics.

Land-use change has no detectable effect on reproduction of a disturbance-adapted, hawkmoth-pollinated plant species.

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.

Examining the efficacy of a genotyping-by-sequencing technique for population genetic analysis of the mushroom Laccaria bicolor and evaluating whether a reference genome is necessary to assess homology.

Given the diversity and ecological importance of Fungi, there is a lack of population genetic research on these organisms. The reason for this can be explained in part by their cryptic nature and difficulty in identifying genets. In addition the difficulty (relative to plants and animals) in developing molecular markers for fungal population genetics contributes to the lack of research in this area. This study examines the ability of restriction-site associated DNA (RAD) sequencing to generate SNPs in Laccaria bicolor. Eighteen samples of morphologically identified L. bicolor from the United States and Europe were selected for this project. The RAD sequencing method produced anywhere from 290 000 to more than 3 000 000 reads. Mapping these reads to the genome of L. bicolor resulted in 84 000-940 000 unique reads from individual samples. Results indicate that incorporation of non-L. bicolor taxa into the analysis resulted in a precipitous drop in shared loci among samples, suggests the potential of these methods to identify cryptic species. F-statistics were easily calculated, although an observable "noise" was detected when using the "All Loci" treatment versus filtering loci to those present in at least 50% of the individuals. The data were analyzed with tests of Hardy-Weinburg equilibrium, population genetic statistics (FIS and FST), and population structure analysis using the program Structure. The results provide encouraging feedback regarding the potential utility of these methods and their data for population genetic analysis. We were unable to draw conclusions of life history of L. bicolor populations from this dataset, given the small sample size. The results of this study indicate the potential of these methods to address population genetics and general life history questions in the Agaricales. Further research is necessary to explore the specific application of these methods in the Agaricales or other fungal groups.

Local topography shapes fine-scale spatial genetic structure in the Arkansas Valley evening primrose, Oenothera harringtonii (Onagraceae).

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.

Conservation genomics assessment of Tharp's bluestar (Amsonia tharpii) with comparisons to widespread (A. longilora) and narrowly endemic (A. fugatei) congeners.

Land-use change and habitat fragmentation are threats to biodiversity. The decrease in available habitat, increase in isolation, and mating within populations can lead to elevated inbreeding, lower genetic diversity, and poor fitness. Here we investigate the genetics of two rare and threatened plant species, Amsonia tharpii and A. fugatei, and we compare them to a widespread congener A. longiflora. We also report the first phylogenetic study of the genus Amsonia (Apocynaceae), including 10 of the 17 taxa and multiple sampling locations, to understand species relationships. We used a double digest restriction-site associated DNA sequencing (ddRADseq) approach to investigate the genetic diversity and gene flow of each species and to create a maximum likelihood phylogeny. The ddRADseq data was mapped to a reference genome to separate out the chloroplast and nuclear markers for population genetic analysis. Our results show that genetic diversity and inbreeding were low across all three species. The chloroplast and nuclear dataset in A. tharpii were highly structured, whereas they showed no structure for A. fugatei, while A. longiflora lacked structure for nuclear data but not chloroplast. Phylogenetic results revealed that A. tharpii is distinct and sister to A. fugatei, and together they are distantly related to A. longiflora. Our results demonstrated that evolutionary history and contemporary ecological processes largely influences genetic diversity within Amsonia. Interestingly, we show that in A. tharpii there was significant structure despite being pollinated by large, bodied hawkmoths that are known to be able to carry pollen long distances, suggesting that other factors are contributing to the structure observed among A. tharpii populations. Conservation efforts should focus on protecting all of the A. tharpii populations, as they contain unique genetic diversity, and a protection plan for A. fugatei needs to be established due to its limited distribution.

Adaptive generalization in pollination systems: Hawkmoths increase fitness to long-tubed flowers, but secondary pollinators remain important.

Selection on floral traits by animal pollinators is important in the evolution of flowering plants, yet whether floral divergence requires specialized pollination remains uncertain. Longer floral tubes, a trait associated with long-tongued pollinators, can also exclude other pollinators from accessing rewards, a potential mechanism for specialization. Across most of its range, Castilleja sessiliflora displays much longer corollas than most Castilleja species, though tube length varies geographically and correlates partially with hawkmoth visitation. To assess whether long corolla tubes reflect adaptation to hawkmoth pollinators, we performed a day/night pollinator exclusion experiment in nine natural populations that varied in corolla length across the range of C. sessiliflora and short-tubed members of the parapatric C. purpurea complex. We compared the fitness contributions of nocturnal and diurnal visitors, revealing that long-tubed populations visited predominantly by hawkmoths experienced greater fruit set at night, in contrast with short-tubed populations or those visited mainly by diurnal pollinators. Next, leveraging a range-wide multiyear dataset of pollinator visitation to these species, we identify that hawkmoth visitation is associated with increased fitness in long-tubed populations overall, and that long tubes are associated with less diverse visitor assemblages. Thus, long corollas represent an adaptation to hawkmoth pollination at the exclusion of diverse pollinators. Nonetheless, while hawkmoths were scarce in the northern range, secondary diurnal pollinators contributed to fruit set across the range, providing reproductive assurance despite possible trait mismatch. This study illustrates adaptive generalization in pollination systems and that floral divergence may proceed along a continuum of generalized and specialized pollinator interactions.

Species-specific effects of production practices on genetic diversity in plant reintroduction programs.

Plant production practices can influence the genetic diversity of cultivated plant materials and, ultimately, their potential to adapt to a reintroduction site. A common step in the plant production process is the application of seed pretreatment to alleviate physiological seed dormancy and successfully germinate seeds. In production settings, the seeds that germinate more rapidly may be favored in order to fill plant quotas. In this study, we investigated how the application of cold-moist stratification treatments with different durations can lead to differences in the genetic diversity of the propagated plant materials. Specifically, we exposed seeds of three Viola species to two different cold stratification durations, and then we analyzed the genetic diversity of the resulting subpopulations through double-digestion restriction site-associated sequencing (ddRADseq). Our results show that, in two out of three species, utilizing a short stratification period will decrease the genetic diversity of neutral and expressed loci, likely due to the imposition of a genetic bottleneck and artificial selection. We conclude that, in some species, the use of minimal stratification practices in production may jeopardize the adaptive potential and long-term persistence of reintroduced populations and suggest that practitioners carefully consider the evolutionary implications of their production protocols. We highlight the need to consider the germination ecology of target species when selecting the length of dormancy-breaking pretreatments.

A phylogeny of the evening primrose family (Onagraceae) using a target enrichment approach with 303 nuclear loci.

BACKGROUND: The evening primrose family (Onagraceae) includes 664 species (803 taxa) with a center of diversity in the Americas, especially western North America. Ongoing research in Onagraceae includes exploring striking variation in floral morphology, scent composition, and breeding system, as well as the role of these traits in driving diversity among plants and their interacting pollinators and herbivores. However, these efforts are limited by the lack of a comprehensive, well-resolved phylogeny. Previous phylogenetic studies based on a few loci strongly support the monophyly of the family and the sister relationship of the two largest tribes but fail to resolve several key relationships. RESULTS: We used a target enrichment approach to reconstruct the phylogeny of Onagraceae using 303 highly conserved, low-copy nuclear loci. We present a phylogeny for Onagraceae with 169 individuals representing 152 taxa sampled across the family, including extensive sampling within the largest tribe, Onagreae. Deep splits within the family are strongly supported, whereas relationships among closely related genera and species are characterized by extensive conflict among individual gene trees. CONCLUSIONS: This phylogenetic resource will augment current research projects focused throughout the family in genomics, ecology, coevolutionary dynamics, biogeography, and the evolution of characters driving diversification in the family.

Microsatellite primers in Oenothera harringtonii (Onagraceae), an annual endemic to the shortgrass prairie of Colorado.

PREMISE OF THE STUDY: Microsatellite markers were developed in the annual herb, Oenothera harringtonii, to investigate patterns of genetic diversity, gene flow, and parentage within and among populations of this Colorado endemic. METHODS AND RESULTS: Ten polymorphic loci were identified in O. harringtonii and tested in four populations sampled across the range of the species. These loci contained trinucleotide repeats with 7-29 alleles per locus. Nine of the 10 loci also amplified in O. caespitosa subsp. macroglottis, O. caespitosa subsp. marginata, and O. caespitosa subsp. navajoensis. In addition, we optimized three markers developed for O. biennis and provide reports of their effectiveness in all four taxa. CONCLUSIONS: These results indicate the utility of these markers in O. harringtonii for future studies of genetic structure, gene flow, and parentage as well as their applicability in other members of the O. caespitosa species complex.

Anthropogenic fragmentation increases risk of genetic decline in the threatened orchid Platanthera leucophaea.

Protecting biodiversity requires an understanding of how anthropogenic changes impact the genetic processes associated with extinction risk. Studies of the genetic changes due to anthropogenic fragmentation have revealed conflicting results. This is likely due to the difficulty in isolating habitat loss and fragmentation, which can have opposing impacts on genetic parameters. The well-studied orchid, Platanthera leucophaea, provides a rich dataset to address this issue, allowing us to examine range-wide genetic changes. Midwestern and Northeastern United States. We sampled 35 populations of P. leucophaea that spanned the species' range and varied in patch composition, degree of patch isolation, and population size. From these populations we measured genetic parameters associated with increased extinction risk. Using this combined dataset, we modeled landscape variables and population metrics against genetic parameters to determine the best predictors of increased extinction risk. All genetic parameters were strongly associated with population size, while development and patch isolation showed an association with genetic diversity and genetic structure. Genetic diversity was lowest in populations with small census sizes, greater urbanization pressures (habitat loss), and small patch area. All populations showed moderate levels of inbreeding, regardless of size. Contrary to expectation, we found that critically small populations had negative inbreeding values, indicating non-random mating not typically observed in wild populations, which we attribute to selection for less inbred individuals. The once widespread orchid, Platanthera leucophaea, has suffered drastic declines and extant populations show changes in the genetic parameters associated with increased extinction risk, especially smaller populations. Due to the important correlation with risk and habitat loss, we advocate continued monitoring of population sizes by resource managers, while the critically small populations may need additional management to reverse genetic declines.