We know very little about the reproductive microbiomes of plants. Microbes may play important roles in shaping pollination, fertilization, and seed production – processes which are important evolutionarily, ecologically, and agriculturally. Through a series of field and laboratory experiments, we show that the stigmatic microbiome in milkweeds influences the success of pollination. Isolation of individual bacterial and fungal taxa from stigmatic secretions allowed us to experimentally test their effects on pollen germination. These experiments demonstrate that individual taxa impact pollen differently, with many microbial taxa being neutral, but some being deleterious. Through isolation of microbes from the legs of pollinator insects we found that pollinators are a likely source for pollen-harmful bacterial taxa. Next, by utilizing a natural hybrid zone, we demonstrate species-specific responses to the stigmatic microbiome that be driving asymmetric patterns of gene-flow between species – with Asclepias exaltata being a better pollen host than A. syriaca . This study demonstrates that the reproductive microbiome is an underappreciated player in sexual reproduction of plants. Significance Statement The results presented here demonstrate an important but previously unappreciated role of stigmatic microbes in plant sexual reproduction. This study demonstrates that the microbial taxa living in stigmatic secretions in milkweed impact pollen germination. We found that filtering out the microbes from stigmatic secretions of milkweed flowers dramatically increases pollen germination. Through isolating microbial taxa from both stigmatic secretions, and pollinator legs, we found that individual microbial taxa impact pollen differently, with many taxa being neutral, but some being deleterious. Finally, by utilizing a naturally occurring milkweed hybrid zone we demonstrated that microbial taxa in stigmatic secretions may be acting as asymmetric prezygotic barrier. ### Competing Interest Statement The authors have declared no competing interest. William & Mary, Arts & Sciences Faculty Research Seed Grant Garden Club of America Garden Club of Virginia Graduate Fellowship Blandy Experimental Farms Graduate Research Fellowship W&M Plumeri Award

Herbivory is among the most well-studied biotic interactions, yet most studies do not incorporate effects on both sexual and clonal plant reproduction or the consequences of different amounts of tissue lost, i.e., herbivory severity. We address both of these gaps using a novel extension of an Integral Projection Model of Asclepias syriaca that uses both plant size and herbivory severity as continuous predictors of ramet population growth rate. Herbivory severity was a significant predictor of survival, growth, as well as sexual and clonal reproduction. We saw these effects using both observational data from across seven sites and five years as well as an experimental approach where we removed plant tissue. Increases in all three aspects of herbivory (probability of herbivory, and the mean and the variation among individuals in herbivory severity) led to decreases in population growth. Population growth rate decreased with herbivory largely due to negative effects of herbivory on clonal reproduction. Our approach to IPMs offers a powerful way to understand the individual-level effects of several aspects of herbivory on plant population growth.

The seedling stage is a critical point in the life history of plants. The survival and growth of tree seedlings can be affected by both the abiotic and biotic environment. Seedlings of both tropical and temperate forests have been shown to experience conspecific negative density dependence (CNDD) and positive density dependence (facilitation). American chestnut was once a common species throughout eastern North America that was decimated by an introduced pathogen in the early 1900 s. Here we present an analysis of chestnut seedling ecology in a unique, sexually reproducing population of American chestnut in central Maine, USA, to understand how both conspecific and heterospecific canopy trees along with size and age influence the survival and growth of understory chestnut trees. Using both a cohort analysis and a static life table approach, we found that seedling survival was lower in the first year of life, and those trees that survive their first and second years had consistently high survival. Our data did not support facilitation as none of the neighborhood indices were positive. We found evidence for CNDD for American chestnuts in the first year of life, but we found no evidence for continued effects beyond the first year. Heterospecifics decreased growth and survival of understory chestnuts beyond the first year of life. Enhanced understanding of American chestnut ecology, particularly at the critical seedling stage, can guide management strategies that support reintroduction efforts.

In intimate ecological interactions, the interdependency of species may result in correlated demographic histories. For species of conservation concern, understanding the long-term dynamics of such interactions may shed light on the drivers of population decline. Here, we address the demographic history of the monarch butterfly, Danaus plexippus, and its dominant host plant, the common milkweed Asclepias syriaca (A. syriaca), using broad-scale sampling and genomic inference. Because genetic resources for milkweed have lagged behind those for monarchs, we first release a chromosome-level genome assembly and annotation for common milkweed. Next, we show that despite its enormous geographic range across eastern North America, A. syriaca is best characterized as a single, roughly panmictic population. Using approximate Bayesian computation with random forests (ABC-RF), a machine learning method for reconstructing demo-graphic histories, we show that both monarchs and milkweed experienced population expansion during the most recent recession of North American glaciers 10,000-20,000 years ago. Our data also identify concurrent population expansions in both species during the large-scale clearing of eastern forests (-200 years ago). Finally, we find no evidence that either species experienced a reduction in effective population size over the past 75 years. Thus, the well-documented decline of monarch abundance over the past 40 years is not visible in our genomic dataset, reflecting a possible mismatch of the overwintering census population to effective population size in this species.

Ospreys (Pandion haliaetus) are obligate piscivores and their nesting success depends on sufficient amounts of fish delivered to the nests during the breeding season. Nests are considered successful when pairs raise a minimum of one young to fledging or near-fledging age. Through web cameras and online broadcasts of Osprey nests, citizen scientists quantified daily number of fish deliveries, nest survival, and nest success. We received and analyzed curated data (one to seven seasons, 2014-2020) from citizen scientist groups representing 19 Osprey web cameras from four countries in North America and Europe. We compared the average and the coefficient of variation of the number of fish delivered per day within the early breeding season between the failed and successful nests using a Wilcoxon rank-sum test. We also analyzed the effects of the average and the coefficient of variation of the number of fish delivered per day on the number of days of nest survival and whether a nest was successful or not using generalized linear mixed models. Successful and failed nests had significant differences in the average number of fish delivered per day and the failed nests had a higher variation in the number of fish deliveries. Moreover, the variation and average number of fish delivered per day had strong associations with whether a nest would fail or succeed. The global effort and manner in which these data were collected are novel and can further our understanding of this charismatic species. The combination of citizen science and technology is a powerful modern tool that can provide insights and has the potential to advance raptor research worldwide.

In intimate ecological interactions, the interdependency of species may result in correlated demographic histories. For species of conservation concern, understanding the long-term dynamics of such interactions may shed light on the drivers of population decline. Here we address the demographic history of the monarch butterfly, Danaus plexippus , and its dominant host plant, the common milkweed Asclepias syriaca , using broad-scale sampling and genomic inference. Because genetic resources for milkweed have lagged behind those for monarchs, we first release a chromosome-level genome assembly and annotation for common milkweed. Next, we show that despite its enormous geographic range across eastern North America, A. syriaca is best characterized as a single, roughly panmictic population. Using Approximate Bayesian Computation via Random Forests (ABC-RF), a machine learning method for reconstructing demographic histories, we show that both monarchs and milkweed experienced concurrent range expansion during the most recent recession of North American glaciers ∼12,000 years ago. Our data identify an expansion of milkweed during the large-scale clearing of eastern forests (∼200 years ago) but was inconclusive as to expansion or contraction of the monarch butterfly population during this time. Finally, our results indicate that neither species experienced a population contraction over the past 75 years. Thus, the well-documented decline of monarch abundance over the past 40 years is not visible in our genomic dataset, reflecting a possible mismatch of the overwintering census population to effective population size in this species. ### Competing Interest Statement The authors have declared no competing interest.

Herbivory can alter plant fitness directly through changing reproductive allocation and indirectly through changing pollinator identity or behavior. Common milkweed is a plant of conservation concern with an inducible chemical defense that is also an important nectar resource. In this study, we aim to understand how herbivory severity and plant traits, including morphology and nectar chemistry, interact to affect insect visitation and pod production in common milkweed. We conducted pollinator watches on plants with experimentally varied herbivory severity and quantified insect frequency and visit length as a response to nectar chemistry, ramet height, number of inflorescences, number of flowers per inflorescence and percent tissue removed. We also quantified pollinator effectiveness and importance. Increased herbivory severity reduced floral displays, including fewer inflorescences and fewer flowers per inflorescence. A reduced floral display was correlated with reduced sucrose, fructose and glucose and resulted in a reduced number and species richness of insect visitors. Fewer flowers per inflorescence reduced the frequency of bumble bee and fly visitors, which were two important pollinators. Although honeybees, flies, small bees, soldier beetles and bumble bees were equally effective pollinators, only bumble bee frequency was positively correlated with pod production. The differences in pollinator visitation have the potential to create diversifying selection on plant floral traits, many of which are also affected by herbivores. This research demonstrates potentially conflicting selection pressures between native and non-native pollinators as well as non-native herbivores.

PREMISE:Leaf economic spectrum (LES) theory has historically been employed to inform vegetation models of ecosystem processes, but largely neglects intraspecific variation and biotic interactions. We attempt to integrate across environment-plant trait-herbivore interactions within a species at a range-wide scale.METHODS:We measured traits in 53 populations spanning the range of common milkweed (Asclepias syriaca) and used a common garden to determine the role of environment in driving patterns of intraspecific variation. We used a feeding trial to determine the role of plant traits in monarch (Danaus plexippus) larval development.RESULTS:Trait-trait relationships largely followed interspecific patterns in LES theory and persisted in a common garden when individual traits change. Common milkweed showed intraspecific variation and biogeographic clines in traits. Clines did not persist in a common garden. Larvae ate more and grew larger when fed plants with more nitrogen. A longitudinal environmental gradient in precipitation corresponded to a resource gradient in plant nitrogen, which produces a gradient in larval performance.CONCLUSIONS:Biogeographic patterns in common milkweed traits can sometimes be predicted from LES, are largely driven by environmental conditions, and have consequences for monarch larval performance. Changes to nutrient dynamics of landscapes with common milkweed could potentially influence monarch population dynamics. We show how biogeographic trends in intraspecific variation can influence key ecological interactions, especially in common species with large distributions.

Recent studies have explored how nut weevils (Curculio and Conotrachelus spp. (Coleoptera: Curculionidae) prey on the fruits (acorns) of oak (Quercus spp.). However, few, if any, have examined these interactions over both an extensive geographic area and over several years. Here, we observed patterns of infestation in acorns of both red oak (Quercus rubra) and white oak (Quercus alba) over an eight-year period along a latitudinal transect, extending as far as 900km, across much of the shared range of these two oak species. Although weevil prevalence did not differ significantly between the two oak species, in red oak, infestation prevalence increased significantly with latitude. In contrast, an opposite pattern was evident in white oak, with the highest infestation prevalence occurring at lower latitudes. One controlled measure of cotyledon damage was significantly lower in acorns of red oak than those of white oak, which may in part be due to larger acorn size at the lower latitudes. Future investigations in this system should focus on the distribution of weevil species (with DNA barcoding) across this range and geographic variation in chemical gradients that likely determine patterns of weevil damage in individual acorns.

•Only 14 % of ramets belonged to the same clone; most clones were represented by only two ramets.•Ramets from the same clone were not more similar than ramets from different clones.•Despite prolific resprouting, sexual reproduction plays a prominent role.•It would not possible to assign ramets to clones based upon phenotype or proximity.

Competition among plants within populations affects plant size, nutrient status and allocation to defenses. Herbivory places additional stress on plant allocation of resources. When resources are limited due to intraspecific competition, induced defenses may reduce costs of defense responses and trade-offs between allocation to growth or defense. We hypothesized that increased intraspecific competition would result in a decrease in plant size and leaf tissue nutrient quality, and that both intraspecific competition and leaf damage severity would affect inducibility of leaf defensive traits. We tested these hypotheses in common milkweed (Asclepias syriaca) using greenhouse experiments that manipulated plant density and damage severity treatments. We measured a suite of leaf traits generally related with herbivore performance, including size; nitrogen, carbon, lignin, and fiber concentrations; and latex production. Increased density decreased plant size and leaf nutrient quality, but increased lignin levels. Damage severity increased leaf lignin levels and latex production. There were no density–damage severity interactions. We additionally addressed the question of whether plants respond differently to simulated or natural herbivory and hypothesized that insect herbivores and mechanical plant tissue removal would similarly affect induced defensive responses. Leaf fiber and lignin increased in response to damage, but the response was greater on plants subjected to simulated, compared with caterpillar herbivory. Other plant traits responded similarly to either damage type. Our findings suggest that intraspecific competition has the potential to generate feedbacks among plants and herbivores as plants respond to herbivory.

Proper hypothesis generation, data handling, graphing, and communication are essential skills that undergraduate majors in biology are expected to master. However, students rarely get hands-on practice that helps them to effectively develop these skills. The purpose of this lesson is to provide students with the opportunity to practice scientific techniques in the context of exploring how the timing of fire disturbance shapes plant community structure in the tallgrass prairie ecosystem, which provides an excellent model system for exploring how disturbance influences species composition. Over the course of four lab sessions, advanced undergraduate students read primary literature, work in teams to form testable and falsifiable hypotheses, replicate a published sampling design at a local field site, and graph, analyze, and interpret their own data. At each step in the scientific process, students complete short written assignments that provide opportunities for assessment and feedback. At the end of this lab module, student groups are given real-world scenarios, asked to form management decisions that integrate the content of their own results with social, economic, and political constraints outlined in their scenario, and then present and defend their proposed solution to the class. This four-week lab module allows students to engage in the process of science and emphasizes the development of quantitative reasoning skills. Student learning is assessed using in-class formative assessments and written summative assessments. Primary image: A restored tallgrass prairie at Prophetstown State Park (West Lafayette, Indiana) in mid-summer (photo by NC Emery).

Although dispersal is critical to plant life history, the relationships between seed traits and dispersal success in animal-dispersed plants remain unclear due to complex interactions among the effects of seed traits, habitat structure, and disperser behavior. We propose that in plants dispersed by scatter-hoarding granivores, seed trait evolution may have been driven by selective pressures that arise from interactions between seedling shade intolerance and predator-mediated caching behavior. Using an optimal foraging model that accounts for cache concealment, hoarder memory, and perceived predation risk, we show that hoarders can obtain cache-recovery advantages by placing caches in moderately risky locations that force potential pilferers to engage in high levels of vigilance. Our model also demonstrates that the level of risk needed to optimally protect a cache increases with the value of the cached food item. If hoarders perceive less sheltered, high-light conditions to be more risky and use this information to protect their caches, then shade-intolerant plants may increase their fitness by producing seeds with traits valued by hoarders. Consistent with this hypothesis, shade tolerance in scatter-hoarded tree species is inversely related to the value of their seeds as perceived by a scatter-hoarding rodent.

Monarch butterfly ( Danaus plexippus ) decline over the past 25 years has received considerable public and scientific attention, in large part because its decline, and that of its milkweed ( Asclepias spp.) host plant, have been linked to genetically modified (GM) crops and associated herbicide use. Here, we use museum and herbaria specimens to extend our knowledge of the dynamics of both monarchs and milkweeds in the United States to more than a century, from 1900 to 2016. We show that both monarchs and milkweeds increased during the early 20th century and that recent declines are actually part of a much longer-term decline in both monarchs and milkweed beginning around 1950. Herbicide-resistant crops, therefore, are clearly not the only culprit and, likely, not even the primary culprit: Not only did monarch and milkweed declines begin decades before GM crops were introduced, but other variables, particularly a decline in the number of farms, predict common milkweed trends more strongly over the period studied here.

We are pleased to see Wepprich (1) and Ries et al. (2) engaging with the museum records data presented in our original study (3). One of the strengths of digitized specimen data is that its portability allows precisely this kind of reanalysis. The main point of our original study was that genetically modified (GM) crops were unlikely to be an important factor in monarch declines because milkweed and monarchs began their declines decades before the introduction of GM crops (3). Both Wepprich (1) and Ries et al. (2) propose alternative methodologies for using museum data to calculate monarch butterfly abundance over time, and both suggest that the currently available data are insufficient to estimate the true monarch abundance trends over the past century. If Wepprich (1) and Ries et al. (2) are correct that monarch abundance over the 20th century is still unknown (we discuss the specifics of their proposals below), this does not change our primary conclusion. Farming of GM crops has been proposed to impact monarch populations by … [↵][1]1To whom correspondence may be addressed. Email: jrpuzey{at}wm.edu. [1]: #xref-corresp-1-1

Quantitatively linking individual variation in functional traits to demography is a necessary step to advance our understanding of trait-based ecological processes. We constructed a population model for Asclepias syriaca to identify how functional traits affect vital rates and population growth and whether trade-offs in chemical defence and demography alter population growth. Plants with higher foliar cardenolides had lower fibre, cellulose and lignin levels, as well as decreased sexual and clonal reproduction. Average cardenolide concentrations had the strongest effect on population growth. In both the sexual and clonal pathway, the trade-off between reproduction and defence affected population growth. We found that both increasing the mean of the distribution of individual plant values for cardenolides and herbivory decreased population growth. However, increasing the variance in both defence and herbivory increased population growth. Functional traits can impact population growth and quantifying individual-level variation in traits should be included in assessments of population-level processes.

Both seed predators and herbivores can have profound effects on individual plant growth, reproduction and survival, but their population-level effects are less well understood. While most plants interact with a suite of seed predators and herbivores over their life cycle, few studies incorporate the effects of multiple interacting partners and multiple life stages on plant population growth. We constructed a matrix model using 6years of data from a rare, seed-producing population of American chestnut (Castanea dentata). We combined field demographic data with published experimental results on the effects of pre-dispersal seed predators (weevils) and post-dispersal seed predators (scatter-hoarding vertebrates) and incorporated the effect of vertebrate herbivores estimated from the field data. We explored the impact of these three different animal interactions for short-term (transient) and long-term (asymptotic) tree population growth. In addition, we used the model to explore the conditions under which scatter hoarding would function as a mutualism. Seed predators had greater effect on both short- and long-term population growth than herbivores. Although weevil infestation can greatly reduce the probability of germination, pre-dispersal seed predators had smaller effects on both short- or long-term population growth than post-dispersal predators. The elasticities of weevil-related parameters were also small. The effect of browsers on both the short- and long-term population growth rate were the smallest of the effects studied. Post-dispersal seed predation affected population growth the most in the interactions studied. The probability of seed removal was among the largest elasticities, similar in magnitude to survival of large trees.Synthesis. Our results indicate that neither weevils nor the intensity of browse damage observed at our study site are likely to hinder tree regeneration or reintroduction, although both reduced population growth. Although researchers and forest managers often assume that seeds are unimportant for long-lived tree populations, our test of this assumption shows that scatterhoarders and other post-dispersal seed consumers can significantly limit natural regeneration. Forest management that alters scatterhoarder behaviour could have significant effects on tree population dynamics that are largely unexplored.

The conditional mutualism between scatterhoarders and trees varies on a continuum from mutualism to antagonism and can change across time and space, and among species. We examined 4 tree species (red oak [Quercus rubra], white oak [Quercus alba], American chestnut [Castanea dentata] and hybrid chestnut [C. dentata × Castanea mollissima) across 5 sites and 3 years to quantify the variability in this conditional mutualism. We used a published model to compare the rates of seed emergence with and without burial to the probability that seeds will be cached and left uneaten by scatterhoarders to quantify variation in the conditional mutualism that can be explained by environmental variation among sites, years, species, and seed provenance within species. All species tested had increased emergence when buried. However, comparing benefits of burial to the probability of caching by scatterhoarders indicated a mutualism in red oak, while white oak was nearly always antagonistic. Chestnut was variable around the boundary between mutualism and antagonism, indicating a high degree of context dependence in the relationship with scatterhoarders. We found that different seed provenances did not vary in their potential for mutualism. Temperature did not explain microsite differences in seed emergence in any of the species tested. In hybrid chestnut only, emergence on the surface declined with soil moisture in the fall. By quantifying the variation in the conditional mutualism that was not caused by changes in scatterhoarder behavior, we show that environmental conditions and seed traits are an important and underappreciated component of the variation in the relationship between trees and scatterhoarders.

PREMISE OF THE STUDYThe tallgrass prairie ecosystem has experienced a dramatic reduction over the past 150 yr. This reduction has impacted the abundance of native grassland species, including milkweeds (Asclepias).METHODSWe used two long-term (27 yr) data sets to examine how fire, grazing, and nutrient addition shape milkweed abundance in tallgrass prairie. We compared these results to those of a greenhouse experiment that varied nutrient levels in the absence of competition, herbivory, and mutualistic relationships.KEY RESULTSAsclepias species exhibited broad patterns in response to burning regimes that did not include grazing, but experienced more species-specific patterns in other combinations. Asclepias syriaca was the only species to increase in abundance in plots that included burning and nutrient addition. In the greenhouse we found that nitrogen significantly increased biomass, while no effect of phosphorus was detected.CONCLUSIONSThese results indicate that A. syriaca will do best in settings with high nutrient loads, low competition, and no grazers. These characteristics define a small portion of the tallgrass prairie but exemplify modern agricultural settings, which have replaced prairies. However, other milkweeds examined did not share this pattern, which indicates that milkweed species will respond differently when exposed to agricultural settings, with some less able to cope with land conversion to pasture or row-crop agriculture.

Abstract In the eastern United States, American chestnut (Castanea dentata) was historically a major component of forest communities, but was functionally extirpated in the early 20th century by an introduced pathogen, chestnut blight (Cryphonectria parasitica). Because chestnut is fast‐growing, long‐lived, and resistant to decay, restoration of American chestnut using blight‐resistant stock could have the potential to increase carbon sequestration or storage in forested landscapes. However, carbon dynamics are also affected by interspecific competition, succession, natural disturbance, and forest management activities, and it is unknown how chestnut restoration might interact with these other processes. We used the PnET‐Succession extension of the LANDIS‐II forest landscape model to study the implications of chestnut restoration on forest composition and carbon storage in the context of other disturbances, including timber harvest and insect pest outbreaks. Our results imply that it could take a millennium or more for chestnut to fully occupy landscapes without aggressive restoration efforts. When successful, chestnut restoration activities displaced other species approximately in proportion to their abundance on the landscape, rather than replacing a single species or genus (e.g., Quercus). Insect pests increased the rate of chestnut colonization by reducing the abundance of competitors, and also had a dominant effect on carbon dynamics. Although chestnut is fast‐growing, moderately shade‐tolerant, and decomposes very slowly, our results suggest that it can only modestly increase the carbon storage potential of eastern forests. However, our results also demonstrate that compositional changes in forest communities can have noticeable effects on biomass accumulation, even with the large uncertainties introduced by invasive pests.