Floral organs act as environmental filters and interact with pollinators to structure the yellow monkeyflower (Mimulus guttatus) floral microbiome.

Assembly of microbial communities is the result of neutral and selective processes. However, the relative importance of these processes is still debated. Microbial communities of flowers, in particular, have gained recent attention because of their potential impact to plant fitness and plant-pollinator interactions. However, the role of selection and dispersal in the assembly of these communities remains poorly understood. Here, we evaluated the role of pollinator-mediated dispersal on the contribution of neutral and selective processes in the assembly of floral microbiomes of the yellow monkeyflower (Mimulus guttatus). We sampled floral organs from flowers in the presence and absence of pollinators within five different serpentine seeps in CA and obtained 16S amplicon data on the epiphytic bacterial communities. Consistent with strong microenvironment selection within flowers we observed significant differences in community composition across floral organs and only a small effect of geographic distance. Pollinator exposure affected the contribution of environmental selection and depended on the rate and intimacy of interactions with flower visitors. This study provides evidence of the importance of dispersal and within-flower heterogeneity in shaping epiphytic bacterial communities of flowers, and highlights the complex interplay between pollinator behaviour, environmental selection and additional abiotic factors in shaping the epiphytic bacterial communities of flowers.

Contrasting effects of yeasts and bacteria on floral nectar traits.

Background and Aims: Flowers can be highly variable in nectar volume and chemical composition, even within the same plant, but the causes of this variation are not fully understood. One potential cause is nectar-colonizing bacteria and yeasts, but experimental tests isolating their effects on wildflowers are largely lacking. This study examines the effects of dominant species of yeasts and bacteria on the hummingbird-pollinated shrub, Mimulus aurantiacus, in California. Methods: Wildflowers were inoculated with field-relevant titres of either the yeast Metschnikowia reukaufii or the bacterium Neokomagataea sp. (formerly Gluconobacter sp.), both isolated from M. aurantiacus nectar. Newly opened flowers were bagged, inoculated, harvested after 3 d and analysed for microbial abundance, nectar volume, and sugar and amino acid concentration and composition. Key Results: Yeast inoculation reduced amino acid concentration and altered amino acid composition, but had no significant effect on nectar volume or sugar composition. In contrast, bacterial inoculation increased amino acid concentration, enhanced the proportion of nectar sugars comprised by monosaccharides, and reduced nectar volume. Conclusions: The results presented suggest that microbial inhabitants of floral nectar can make nectar characteristics variable among flowers through divergent effects of yeasts and bacteria on nectar chemistry and availability, probably modifying plant-pollinator interactions.

Testing the utility of fluorescent proteins in Mimulus lewisii by an Agrobacterium-mediated transient assay.

KEY MESSAGE: The Agrobacterium -mediated transient expression assay by leaf infiltration in Mimulus lewisii is robust. Fluorescent proteins EGFP, EYFP and DsRed give bright fluorescence signals in the infiltrated tissue. Mimulus lewisii is an emerging developmental genetic model system. Recently developed genomic and genetic resources and a stable transformation protocol have greatly facilitated the identification and functional characterization of genes controlling the development of ecologically important floral traits using this species. To further expedite gene and protein function analyses in M. lewisii, we adopted and simplified the Agrobacterium-mediated transient gene expression method routinely used in tobacco plants. With the validated transient assay, we examined the performance of fluorescent proteins EGFP, EYFP and DsRed in M. lewisii. All three proteins gave bright fluorescence signals when transiently expressed in agroinfiltrated leaves. Furthermore, we demonstrated the utility of fluorescent proteins in M. lewisii by showing the nuclear localization of Reduced Carotenoid Pigmentation 1 (RCP1), a recently discovered R2R3-MYB transcription factor that regulates carotenoid pigmentation during flower development. Both the transient assay and the fluorescent proteins are valuable additions to the M. lewisii toolbox, making this emerging genetic and developmental model system even more powerful.

Environmental variability counteracts priority effects to facilitate species coexistence: evidence from nectar microbes.

The order of species arrival during community assembly can greatly affect species coexistence, but the strength of these effects, known as priority effects, appears highly variable across species and ecosystems. Furthermore, the causes of this variation remain unclear despite their fundamental importance in understanding species coexistence. Here, we show that one potential cause is environmental variability. In laboratory experiments using nectar-inhabiting microorganisms as a model system, we manipulated spatial and temporal variability of temperature, and examined consequences for priority effects. If species arrived sequentially, multiple species coexisted under variable temperature, but not under constant temperature. Temperature variability prevented extinction of late-arriving species that would have been excluded owing to priority effects if temperature had been constant. By contrast, if species arrived simultaneously, species coexisted under both variable and constant temperatures. We propose possible mechanisms underlying these results using a mathematical model that incorporates contrasting effects of microbial species on nectar pH and amino acids. Overall, our findings suggest that understanding consequences of priority effects for species coexistence requires explicit consideration of environmental variability.

Historical contingency in species interactions: towards niche-based predictions.

The way species affect one another in ecological communities often depends on the order of species arrival. The magnitude of such historical contingency, known as priority effects, varies across species and environments, but this variation has proven difficult to predict, presenting a major challenge in understanding species interactions and consequences for community structure and function. Here, we argue that improved predictions can be achieved by decomposing species' niches into three components: overlap, impact and requirement. Based on classic theories of community assembly, three hypotheses that emphasise related, but distinct influences of the niche components are proposed: priority effects are stronger among species with higher resource use overlap; species that impact the environment to a greater extent exert stronger priority effects; and species whose growth rate is more sensitive to changes in the environment experience stronger priority effects. Using nectar-inhabiting microorganisms as a model system, we present evidence that these hypotheses complement the conventional hypothesis that focuses on the role of environmental harshness, and show that niches can be twice as predictive when separated into components. Taken together, our hypotheses provide a basis for developing a general framework within which the magnitude of historical contingency in species interactions can be predicted.

Nectar bacteria, but not yeast, weaken a plant-pollinator mutualism.

Mutualistic interactions are often subject to exploitation by species that are not directly involved in the mutualism. Understanding which organisms act as such 'third-party' species and how they do so is a major challenge in the current study of mutualistic interactions. Here, we show that even species that appear ecologically similar can have contrasting effects as third-party species. We experimentally compared the effects of nectar-inhabiting bacteria and yeasts on the strength of a mutualism between a hummingbird-pollinated shrub, Mimulus aurantiacus, and its pollinators. We found that the common bacterium Gluconobacter sp., but not the common yeast Metschnikowia reukaufii, reduced pollination success, seed set and nectar consumption by pollinators, thereby weakening the plant-pollinator mutualism. We also found that the bacteria reduced nectar pH and total sugar concentration more greatly than the yeasts did and that the bacteria decreased glucose concentration and increased fructose concentration whereas the yeasts affected neither. These distinct changes to nectar chemistry may underlie the microbes' contrasting effects on the mutualism. Our results suggest that it is necessary to understand the determinants of microbial species composition in nectar and their differential modification of floral rewards to explain the mutual benefits that plants and pollinators gain from each other.

Linking symbiont community structures in a model arbuscular mycorrhizal system.

• The influence of plant communities on symbiotic arbuscular mycorrhizal fungal (AMF) communities is difficult to study in situ as both symbionts are strongly influenced by some of the same soil and environmental conditions, and thus we have a poor understanding of the potential links in community composition and structure between host and fungal communities. • AMF were characterized in colonized roots of thermal soil Mimulus guttatus in both isolated plants supporting AMF for only a few months of the growing season and plants growing in mixed plant communities composed of annual and perennial hosts. Cluster and discriminant analysis were used to compare competing models based on either communities or soil conditions. • Mimulus guttatus in adjacent contrasting plant community situations harbored distinct AMF communities with few fungal taxa occurring in both community types. Isolated plants harbored communities of fewer fungal taxa with lower diversity than plants in mixed communities. Host community type was more indicative than pH of AMF community structure. • Our results support an inherent relationship between host plant and AMF community structures, although pH-based models were also statistically supported.

Flowers as islands: spatial distribution of nectar-inhabiting microfungi among plants of Mimulus aurantiacus, a hummingbird-pollinated shrub.

Microfungi that inhabit floral nectar offer unique opportunities for the study of microbial distribution and the role that dispersal limitation may play in generating distribution patterns. Flowers are well-replicated habitat islands, among which the microbes disperse via pollinators. This metapopulation system allows for investigation of microbial distribution at multiple spatial scales. We examined the distribution of the yeast, Metschnikowia reukaufii, and other fungal species found in the floral nectar of the sticky monkey flower, Mimulus aurantiacus, a hummingbird-pollinated shrub, at a California site. We found that the frequency of nectar-inhabiting microfungi on a given host plant was not significantly correlated with light availability, nectar volume, or the percent cover of M. aurantiacus around the plant, but was significantly correlated with the location of the host plant and loosely correlated with the density of flowers on the plant. These results suggest that dispersal limitation caused by spatially nonrandom foraging by pollinators may be a primary factor driving the observed distribution pattern.

Arbuscular mycorrhizal fungi ameliorate temperature stress in thermophilic plants.

Biotic interactions can affect the distribution of species across environmental gradients, and as air and soil temperatures increase, plant community response may depend on interactions with symbionts. We measured the effect of elevated soil temperatures on mycorrhizal function and on the response of both plant and fungal symbionts, using fungal inoculum isolated from either high-temperature thermal or nonthermal grassland soils. Our source for thermal soils was Yellowstone National Park, USA, where plants experience rooting zone temperatures of 45 degrees C or more. In the greenhouse, we grew three plant species (Dichanthelium lanuginosum, Agrostis scabra, and Mimulus guttatus) with three arbuscular mycorrhizal fungal (AMF) treatments (no AMF, nonthermal AMF, thermal AMF) and two soil temperatures (ambient, elevated). Biomass of the facultative thermal plants Agrostis scabra and Mimulus guttatus decreased by 50% in elevated-temperature soils, and AMF had no effect on measured plant traits. In contrast, the biomass and total root length of the obligate thermal plant Dichanthelium lanuginosum were greater at elevated soil temperatures, but only when mycorrhizal. Both mycorrhizal colonization levels and length of extraradical hyphae (ERH) increased with soil temperature across all host species. The source of the AMF inoculum, on the other hand, did not affect colonization level, ERH length, host plant biomass, or flowering for all host species in either temperature treatment, suggesting that AMF from thermal soils are not specifically adapted to higher temperatures. In the field we collected soil cores to measure in situ depth distributions of D. lanuginosum roots and ERH, and to determine which AMF species were active in plants growing in thermal soils. Roots were limited to soils with an average temperature < or =30 degrees C, while ERH existed in the hottest soils we sampled, averaging 35 degrees C. Molecular analyses of roots indicated that thermal AMF communities were composed of both generalist and possibly unique fungal species. The increase in host plant allocation to AMF, apparent lack of temperature adaptation by AMF, and differential host response to AMF suggest that AMF could be significant drivers of plant community response to increased soil temperature associated with global change.