Seed type and origin-dependent seedling emergence patterns in Danthonia californica, a species commonly used in grassland restoration.

Danthonia californica Bolander (Poaceae)is a native perennial bunchgrass commonly used in the restoration of prairie ecosystems in the western United States. Plants of this species simultaneously produce both chasmogamous (potentially outcrossed) and cleistogamous (obligately self-fertilized) seeds. Restoration practitioners almost exclusively use chasmogamous seeds for outplanting, which are predicted to perform better in novel environments due to their greater genetic diversity. Meanwhile, cleistogamous seeds may exhibit greater local adaptation to the conditions in which the maternal plant exists. We performed a common garden experiment at two sites in the Willamette Valley, Oregon, to assess the influence of seed type and source population (eight populations from a latitudinal gradient) on seedling emergence and found no evidence of local adaptation for either seed type. Cleistogamous seeds outperformed chasmogamous seeds, regardless of whether seeds were sourced directly from the common gardens (local seeds) or other populations (nonlocal seeds). Furthermore, average seed weight had a strong positive effect on seedling emergence, despite the fact that chasmogamous seeds had significantly greater mass than cleistogamous seeds. At one common garden, we observed that seeds of both types sourced from north of our planting site performed significantly better than local or southern-sourced seeds. We also found a significant seed type and distance-dependent interaction, with cleistogamous seedling emergence peaking approximately 125 km from the garden. These results suggest that cleistogamous seeds should be considered for greater use in D. californica restoration.

Climate warming threatens the persistence of a community of disturbance-adapted native annual plants.

With ongoing climate change, populations are expected to exhibit shifts in demographic performance that will alter where a species can persist. This presents unique challenges for managing plant populations and may require ongoing interventions, including in situ management or introduction into new locations. However, few studies have examined how climate change may affect plant demographic performance for a suite of species, or how effective management actions could be in mitigating climate change effects. Over the course of two experiments spanning 6 yr and four sites across a latitudinal gradient in the Pacific Northwest, United States, we manipulated temperature, precipitation, and disturbance intensity, and quantified effects on the demography of eight native annual prairie species. Each year we planted seeds and monitored germination, survival, and reproduction. We found that disturbance strongly influenced demographic performance and that seven of the eight species had increasingly poor performance with warmer conditions. Across species and sites, we observed 11% recruitment (the proportion of seeds planted that survived to reproduction) following high disturbance, but just 3.9% and 2.3% under intermediate and low disturbance, respectively. Moreover, mean seed production following high disturbance was often more than tenfold greater than under intermediate and low disturbance. Importantly, most species exhibited precipitous declines in their population growth rates (λ) under warmer-than-ambient experimental conditions and may require more frequent disturbance intervention to sustain populations. Aristida oligantha, a C4 grass, was the only species to have λ increase with warmer conditions. These results suggest that rising temperatures may cause many native annual plant species to decline, highlighting the urgency for adaptive management practices that facilitate their restoration or introduction to newly suitable locations. Frequent and intense disturbances are critical to reduce competitors and promote native annuals' persistence, but even such efforts may prove futile under future climate regimes.

The 'black box' of plant demography: how do seed type, climate and seed fungal communities affect grass seed germination?

Demographic studies measure drivers of plant fecundity including seed production and survival, but few address both abiotic and biotic drivers of germination such as variation in climate among sites, population density, maternal plants, seed type and fungal pathogen abundance. We examined germination and microbial communities of seeds of Danthonia californica, which are either chasmogamous (external, wind-pollinated) or cleistogamous (internal, self-fertilized) and Festuca roemeri, which are solely chasmogamous. Seed populations were sourced across environmental gradients. We tested germination and used high-throughput sequencing to characterize seed fungal community structure. For F. roemeri, maternal plants significantly influenced germination as did climate and pathogens; germination increased from wetter, cooler sites. For D. californica, the main drivers of germination were maternal plant, seed type and pathogens; on average, more chasmogamous seeds germinated. Fungal communities depended largely on seed type, with fewer fungi associated with cleistogamous seeds, but the communities also depended on site factors such as vapor pressure deficit, plant density and whether the seeds had germinated. Putative pathogens that were negatively correlated with germination were more abundant for both D. californica and F. roemeri chasmogamous seeds than D. californica cleistogamous seeds. In D. californica, cleistogamous and chasmogamous seeds contain vastly different fungal communities.

Mycena citrinomarginata is associated with roots of the perennial grass Festuca roemeri in Pacific Northwest prairies.

Prairies in the Pacific Northwest are dominated by perennial bunchgrasses. A Mycena in the citrinomarginata complex was observed to tightly co-occur with bunchgrasses at several prairie study sites. Mapping and spatial statistics showed that it was strongly and significantly associated with Festuca roemeri tussocks. We further found that this fungus is attached to F. roemeri roots (17/17 examined) and both specific primers and next-generation DNA sequencing established that the fungus is in the roots, suggesting that M. citrinomarginata may be endophytic or biotrophic in some contexts, and not simply saprotrophic. These results combined with a literature review indicate that Mycena species are often found as endophytes in grass roots. Given the importance of grasses and grasslands for humans, this ecological association deserves further study.

Latitudinal gradients in population growth do not reflect demographic responses to climate.

Spatial gradients in population growth, such as across latitudinal or elevational gradients, are often assumed to primarily be driven by variation in climate, and are frequently used to infer species' responses to climate change. Here, we use a novel demographic, mixed-model approach to dissect the contributions of climate variables vs. other latitudinal or local site effects on spatiotemporal variation in population performance in three perennial bunchgrasses. For all three species, we find that performance of local populations decreases with warmer and drier conditions, despite latitudinal trends of decreasing population growth toward the cooler and wetter northern portion of each species' range. Thus, latitudinal gradients in performance are not predictive of either local or species-wide responses to climate. This pattern could be common, as many environmental drivers, such as habitat quality or species' interactions, are likely to vary with latitude or elevation, and thus influence or oppose climate responses.

Double lives: transfer of fungal endophytes from leaves to woody substrates.

Fungal endophytes are a ubiquitous feature of plants, yet for many fungi the benefits of endophytism are still unknown. The Foraging Ascomycete (FA) hypothesis proposes that saprotrophic fungi can utilize leaves both as dispersal vehicles and as resource havens during times of scarcity. The presence of saprotrophs in leaf endophyte communities has been previously observed but their ability to transfer to non-foliar saprobic substrates has not been well investigated. To assess this ability, we conducted a culture study by placing surface-sterilized leaves from a single tropical angiosperm tree (Nectandra lineatifolia) directly onto sterile wood fragments and incubating them for 6 weeks. Fungi from the wood were subsequently isolated in culture and identified to the genus level by ITS sequences or morphology. Four-hundred and seventy-seven fungal isolates comprising 24 taxa were cultured from the wood. Of these, 70.8% of taxa (82.3% of isolates) belong to saprotrophic genera according to the FUNGuild database. Furthermore, 27% of OTUs (6% of isolates) were basidiomycetes, an unusually high proportion compared to typical endophyte communities. Xylaria flabelliformis, although absent in our original isolations, formed anamorphic fruiting structures on the woody substrates. We introduce the term viaphyte (literally, "by way of plant") to refer to fungi that undergo an interim stage as leaf endophytes and, after leaf senescence, colonize other woody substrates via hyphal growth. Our results support the FA hypothesis and suggest that viaphytism may play a significant role in fungal dispersal.

Prairie plant phenology driven more by temperature than moisture in climate manipulations across a latitudinal gradient in the Pacific Northwest, USA.

Plant phenology will likely shift with climate change, but how temperature and/or moisture regimes will control phenological responses is not well understood. This is particularly true in Mediterranean climate ecosystems where the warmest temperatures and greatest moisture availability are seasonally asynchronous. We examined plant phenological responses at both the population and community levels to four climate treatments (control, warming, drought, and warming plus additional precipitation) embedded within three prairies across a 520 km latitudinal Mediterranean climate gradient within the Pacific Northwest, USA. At the population level, we monitored flowering and abundances in spring 2017 of eight range-restricted focal species planted both within and north of their current ranges. At the community level, we used normalized difference vegetation index (NDVI) measured from fall 2016 to summer 2018 to estimate peak live biomass, senescence, seasonal patterns, and growing season length. We found that warming exerted a stronger control than our moisture manipulations on phenology at both the population and community levels. Warming advanced flowering regardless of whether a species was within or beyond its current range. Importantly, many of our focal species had low abundances, particularly in the south, suggesting that establishment, in addition to phenological shifts, may be a strong constraint on their future viability. At the community level, warming advanced the date of peak biomass regardless of site or year. The date of senescence advanced regardless of year for the southern and central sites but only in 2018 for the northern site. Growing season length contracted due to warming at the southern and central sites (~3 weeks) but was unaffected at the northern site. Our results emphasize that future temperature changes may exert strong influence on the timing of a variety of plant phenological events, especially those events that occur when temperature is most limiting, even in seasonally water-limited Mediterranean ecosystems.

Greenhouse gas emissions limited by low nitrogen and carbon availability in natural, restored, and agricultural Oregon seasonal wetlands.

Wetlands are the major natural source of the greenhouse gas methane (CH4) and are also potentially an important source of nitrous oxide (N2O), though there is considerable variability among wetland types with some of the greatest uncertainty in freshwater mineral-soil wetlands. In particular, trace gas emissions from seasonal wetlands have been very poorly studied. We measured fluxes of CH4, N2O, and CO2(carbon dioxide), soil nutrients, and net primary productivity over one year in natural, restored, and agricultural seasonal wetland prairies in the Willamette Valley, Oregon, USA. We found zero fluxes for CH4 and N2O, even during periods of extended waterlogging of the soil. To explore this lack of emissions, we performed a laboratory experiment to examine the controls over these gases. In a fully-factorial design, we amended anaerobic soils from all wetlands with nitrate, glucose, and NaOH (to neutralize pH) and measured production potentials of N2, N2O, CH4, and CO2. We found that denitrification and N2O production were co-limited by nitrate and carbon, with little difference between the three wetland types. This co-limitation suggests that low soil carbon availability will continue to constrain N2O emissions and denitrification in these systems even when receiving relatively high levels of nitrogen inputs. Contrary to the results for N2O, the amended wetland soils never produced significant amounts of CH4 under any treatment. We hypothesize that high concentrations of alternative electron acceptors exist in these soils so that methanogens are noncompetitive with other microbial groups. As a result, these wetlands do not appear to be a significant source or sink of soil carbon and thus have a near zero climate forcing effect. Future research should focus on determining if this is a generalizable result in other seasonal wetlands.

Disentangling visual and olfactory signals in mushroom-mimicking Dracula orchids using realistic three-dimensional printed flowers.

Flowers use olfactory and visual signals to communicate with pollinators. Disentangling the relative contributions and potential synergies between signals remains a challenge. Understanding the perceptual biases exploited by floral mimicry illuminates the evolution of these signals. Here, we disentangle the olfactory and visual components of Dracula lafleurii, which mimics mushrooms in size, shape, color and scent, and is pollinated by mushroom-associated flies. To decouple signals, we used three-dimensional printing to produce realistic artificial flower molds that were color matched and cast using scent-free surgical silicone, to which we could add scent. We used GC-MS to measure scents in co-occurring mushrooms, and related orchids, and used these scents in field experiments. By combining silicone flower parts with real floral organs, we created chimeras that identified the mushroom-like labellum as a source of volatile attraction. In addition, we showed remarkable overlap in the volatile chemistry between D. lafleurii and co-occurring mushrooms. The characters defining the genus Dracula - a mushroom-like, 'gilled' labellum and a showy, patterned calyx - enhance pollinator attraction by exploiting the visual and chemosensory perceptual biases of drosophilid flies. Our techniques for the manipulation of complex traits in a nonmodel system not conducive to gene silencing or selective breeding are useful for other systems.

The herbaceous landlord: integrating the effects of symbiont consortia within a single host.

Plants are typically infected by a consortium of internal fungal associates, including endophytes in their leaves, as well as arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) in their roots. It is logical that these organisms will interact with each other and the abiotic environment in addition to their host, but there has been little work to date examining the interactions of multiple symbionts within single plant hosts, or how the relationships among symbionts and their host change across environmental conditions. We examined the grass Agrostis capillaris in the context of a climate manipulation experiment in prairies in the Pacific Northwest, USA. Each plant was tested for presence of foliar endophytes in the genus Epichloë, and we measured percent root length colonized (PRLC) by AMF and DSE. We hypothesized that the symbionts in our system would be in competition for host resources, that the outcome of that competition could be driven by the benefit to the host, and that the host plants would be able to allocate carbon to the symbionts in such a way as to maximize fitness benefit within a particular environmental context. We found a correlation between DSE and AMF PRLC across climatic conditions; we also found a fitness cost to increasing DSE colonization, which was negated by presence of Epichloë endophytes. These results suggest that selective pressure on the host is likely to favor host/symbiont relationships that structure the community of symbionts in the most beneficial way possible for the host, not necessarily favoring the individual symbiont that is most beneficial to the host in isolation. These results highlight the need for a more integrative, systems approach to the study of host/symbiont consortia.

An exploration of hypotheses that explain herbivore and pathogen attack in restored plant communities.

Many hypotheses address the associations of plant community composition with natural enemies, including: (i) plant species diversity may reduce enemy attack, (ii) attack may increase as host abundance increases, (iii) enemy spillover may lead to increased attack on one host species due to transmission from another host species, or enemy dilution may lead to reduced attack on a host that would otherwise have more attack, (iv) physical characteristics of the plant community may influence attack, and (v) plant vigor may affect attack. Restoration experiments with replicated plant communities provide an exceptional opportunity to explore these hypotheses. To explore the relative predictive strengths of these related hypotheses and to investigate the potential effect of several restoration site preparation techniques, we surveyed arthropod herbivore and fungal pathogen attack on the six most common native plant species in a restoration experiment. Multi-model inference revealed a weak but consistent negative correlation with pathogen attack and host diversity across the plant community, and no correlation between herbivory and host diversity. Our analyses also revealed host species-specific relationships between attack and abundance of the target host species, other native plant species, introduced plant species, and physical community characteristics. We found no relationship between enemy attack and plant vigor. We found minimal differences in plant community composition among several diverse site preparation techniques, and limited effects of site preparation techniques on attack. The strongest associations of community characteristics with attack varied among plant species with no community-wide patterns, suggesting that no single hypothesis successfully predicts the dominant community-wide trends in enemy attack.

Grassland fires may favor native over introduced plants by reducing pathogen loads.

Grasslands have been lost and degraded in the United States since Euro-American settlement due to agriculture, development, introduced invasive species, and changes in fire regimes. Fire is frequently used in prairie restoration to control invasion by trees and shrubs, but may have additional consequences. For example, fire might reduce damage by herbivore and pathogen enemies by eliminating litter, which harbors eggs and spores. Less obviously, fire might influence enemy loads differently for native and introduced plant hosts. We used a controlled burn in a Willamette Valley (Oregon) prairie to examine these questions. We expected that, without fire, introduced host plants should have less damage than native host plants because the introduced species are likely to have left many of their enemies behind when they were transported to their new range (the enemy release hypothesis, or ERH). If the ERH holds, then fire, which should temporarily reduce enemies on all species, should give an advantage to the natives because they should see greater total reduction in damage by enemies. Prior to the burn, we censused herbivore and pathogen attack on eight plant species (five of nonnative origin: Bromus hordaceous, Cynosuros echinatus, Galium divaricatum, Schedonorus arundinaceus (= Festuca arundinacea), and Sherardia arvensis; and three natives: Danthonia californica, Epilobium minutum, and Lomatium nudicale). The same plots were monitored for two years post-fire. Prior to the burn, native plants had more kinds of damage and more pathogen damage than introduced plants, consistent with the ERH. Fire reduced pathogen damage relative to the controls more for the native than the introduced species, but the effects on herbivory were negligible. Pathogen attack was correlated with plant reproductive fitness, whereas herbivory was not. These results suggest that fire may be useful for promoting some native plants in prairies due to its negative effects on their pathogens.

Tall fescue is a potential spillover reservoir host for Alternaria species.

The spread of invasive species is complicated and multifaceted. Enemy spillover (i.e. the transfer of a natural enemy from a reservoir host to a novel host) is one mechanism that facilitates the spread of non-native species. The reservoir host is a species that harbors high abundance of the enemy with little cost to fitness. We asked whether Schedonorus arundinaceus (tall fescue), a highly invasive grass species in North America, is a potential reservoir host for the ubiquitous genus of fungi, Alternaria. We also asked whether spillover of Alternaria is possible among grasses that commonly occur with S. arundinaceus in grassland ecosystems. We performed a greenhouse cross inoculation of three isolates of Alternaria and six grass species (three native, three invasive, including S. arundinaceus). We determined that spillover is possible because the fungal isolates infected and caused disease symptoms on all six grasses and decreased biomass in two of the grass species. We also determined that the invasive grass species appear to be more competent hosts than the native species and that S. arundinaceus could be a likely reservoir host for Alternaria spp. because it can harbor the pathogen with no apparent fitness cost.

Testing assumptions of the enemy release hypothesis: generalist versus specialist enemies of the grass Brachypodium sylvaticum.

The enemy release hypothesis (ERH) suggests greater success of species in an invaded range due to release from natural enemies. The ERH assumes there will be more specialist enemies in the native range and that generalists will have an equal effect in both ranges. We tested these assumptions with the grass Brachypodium sylvaticum in the native range (Switzerland) and invaded range (Oregon, USA). We assessed all the kinds of damage present (caused by fungi, insects, mollusk and deer) on both leaves and seeds at 10 sites in each range and correlated damage with host fitness. Only two of the 20 fungi found on leaves were specialist pathogens, and these were more frequent in the native range. Conversely there was more insect herbivory on leaves in the invaded range. All fungi and insects found on seeds were generalists. More species of fungi were found on seeds in the native range, and a higher proportion of them were pathogenic than in the invaded range. There were more kinds of enemies in the native range, where the plants had lower fitness, in accordance with the ERH. However, contrary to assumptions of the ERH, generalists appear to be equally or more important than specialists in reducing host fitness.

Population regulation by enemies of the grass Brachypodium sylvaticum: demography in native and invaded ranges.

The enemy-release hypothesis (ERH) states that species become more successful in their introduced range than in their native range because they leave behind natural enemies in their native range and are thus "released" from enemy pressures in their introduced range. The ERH is popularly cited to explain the invasive properties of many species and is the underpinning of biological control. We tested the prediction that plant populations are more strongly regulated by natural enemies (herbivores and pathogens) in their native range than in their introduced range with enemy-removal experiments using pesticides. These experiments were replicated at multiple sites in both the native and invaded ranges of the grass Brachypodium sylvaticum. In support of the ERH, enemies consistently regulated populations in the native range. There were more tillers and more seeds produced in treated vs. untreated plots in the native range, and few seedlings survived in the native range. Contrary to the ERH, total measured leaf damage was similar in both ranges, though the enemies that caused it differed. There was more damage by generalist mollusks and pathogens in the native range, and more damage by generalist insect herbivores in the invaded range. Demographic analysis showed that population growth rates were lower in the native range than in the invaded range, and that sexually produced seedlings constituted a smaller fraction of the total in the native range. Our removal experiment showed that enemies regulate plant populations in their native range and suggest that generalist enemies, not just specialists, are important for population regulation.

Abiotic constraints on the competitive ability of exotic and native grasses in a Pacific Northwest prairie.

In prairie ecosystems, abiotic constraints on competition can structure plant communities; however, the extent to which competition between native and exotic plant species is constrained by environmental factors is still debated. The objective of our study was to use paired field and greenhouse experiments to evaluate the competitive dynamics between two native (Danthonia californica and Deschampsia cespitosa) and two exotic (Schedonorus arundinaceus and Lolium multiflorum) grass species under varying nutrient and moisture conditions in an upland prairie in the Willamette Valley, Oregon. We hypothesized the two invasive, exotic grasses would be more competitive under high-nutrient, moderate-moisture conditions, resulting in the displacement of native grasses from these environments. In the field, the experimental reduction of competition resulted in shorter, wider plants, but only the annual grass, Lolium multiflorum, produced more aboveground biomass when competition was reduced. In the greenhouse, the two exotic grasses produced more total biomass than the two native grasses. Competitive hierarchies were influenced by nutrient and/or moisture treatments for the two exotic grasses, but not for the two native grasses. L. multiflorum dominated competitive interactions with all other grasses across treatments. In general, S. arundinaceus dominated when in competition with native grasses, and D. cespitosa produced the most biomass in monoculture or under interspecific competition with the other native grass, D. californica. D. californica, D. cespitosa, and S. arundinaceus all produced more biomass in high-moisture, high-nutrient environments, and D. cespitosa, L. multiflorum, and S. arundinaceus allocated more biomass belowground in the low nutrient treatment. Taken together, these experiments suggest the competitive superiority of the exotic grasses, especially L. multiflorum, but, contrary to our hypothesis, the native grasses were not preferentially excluded from nutrient-rich, moderately wet environments.

Context-dependent pollinator behavior: an explanation for patterns of hybridization among three species of Indian paintbrush.

In some areas of sympatry, reproductively compatible plant species hybridize, but in other areas of sympatry, they do not and they remain reproductively isolated from one another. Explanations offered to explain patterns of hybridization that vary by population have usually focused on genetic or environmental factors. Instead, we examined whether different community contexts might change pollinator preference and constancy and thus influence the likelihood of hybridization among three Indian paintbrush species (Castilleja miniata, C. rhexifolia, and C. sulphurea). To determine whether visitation was context-dependent, we observed pollinator behavior in experimental arrays (constructed using flowering stems of the three Indian paintbrush species) in different contexts. Contexts were defined by which Castilleja species occurred in the immediate neighborhood of the arrays. Specifically, we asked, does visitation to particular species in the arrays depend on context? In general, each Castilleja species was preferred when it matched the surrounding community context, as is predicted by optimal foraging theory. More interestingly, pollinator constancy was weakened in the hybrid context (an area where the three species co-occurred with morphologically intermediate plants), which is likely to increase pollen flow among the species. Reduced pollinator constancy in hybrid zones could set up a positive feedback loop in which more flower diversity is created through hybridization, decreasing pollinator constancy, and leading to more hybridization. This self-reinforcing mechanism could lead to "hybridization hot spots" and to a patchy distribution of hybrid populations. We expect that this mechanism may be important in other animal-pollinated plant hybrid zones.

Insect-mediated reproduction of systemic infections by Puccinia arrhenatheri on Berberis vulgaris.

• Witches' brooms on Berberis vulgaris are induced by a systemically infecting rust fungus, Puccinia arrhenatheri. These witches' brooms bear yellow discolored leaves on which the fungus exposes its gametes in a sugary nectar. During the spermatial stage of the fungus the infected leaves emit a strong, flowery scent. • An exclusion-experiment was used to evaluate whether fungal reproductive success, defined by the ability of the fungus to produce aeciospores, depended on gamete transfer by insects. To determine whether insects were attracted to the infected leaves, and if so, why, visitation to infected and uninfected leaves was quantified and volatiles produced by leaves, infected leaves and flowers were analyzed. • The production of aeciospores was significantly higher on witches' brooms with insect visitation. Visitation rates were higher and visits were longer on witches' brooms than on uninfected branches. A wide diversity of visitors, mainly Diptera and Hymenoptera, was observed. The volatiles emitted by infected leaves were composed of sweet floral fragrances and insect pheromones. • Our results suggest that sexual reproduction of the pathogen requires out-crossing by insects and that infected leaves attract insects by floral mimicry (bright yellow color and the production of sugary nectar and volatiles).