Honeybees affect floral microbiome composition in a central food source for wild pollinators in boreal ecosystems.

Basic knowledge on dispersal of microbes in pollinator networks is essential for plant, insect, and microbial ecology. Thorough understanding of the ecological consequences of honeybee farming on these complex plant-pollinator-microbe interactions is a prerequisite for sustainable honeybee keeping. Most research on plant-pollinator-microbe interactions have focused on temperate agricultural systems. Therefore, information on a wild plant that is a seasonal bottleneck for pollinators in cold climate such as Salix phylicifolia is of specific importance. We investigated how floral visitation by insects influences the community structure of bacteria and fungi in Salix phylicifolia inflorescences under natural conditions. Insect visitors were experimentally excluded with net bags. We analyzed the microbiome and measured pollen removal in open and bagged inflorescences in sites where honeybees were foraging and in sites without honeybees. Site and plant individual explained most of the variation in floral microbial communities. Insect visitation and honeybees had a smaller but significant effect on the community composition of microbes. Honeybees had a specific effect on the inflorescence microbiome and, e.g., increased the relative abundance of operational taxonomic units (OTUs) from the bacterial order Lactobacillales. Site had a significant effect on the amount of pollen removed from inflorescences but this was not due to honeybees. Insect visitors increased bacterial and especially fungal OTU richness in the inflorescences. Pollinator visits explained 38% variation in fungal richness, but only 10% in bacterial richness. Our work shows that honeybee farming affects the floral microbiome in a wild plant in rural boreal ecosystems.

Arbuscular mycorrhizal fungi influence host infection during epidemics in a wild plant pathosystem.

While pathogenic and mutualistic microbes are ubiquitous across ecosystems and often co-occur within hosts, how they interact to determine patterns of disease in genetically diverse wild populations is unknown. To test whether microbial mutualists provide protection against pathogens, and whether this varies among host genotypes, we conducted a field experiment in three naturally occurring epidemics of a fungal pathogen, Podosphaera plantaginis, infecting a host plant, Plantago lanceolata, in the Åland Islands, Finland. In each population, we collected epidemiological data on experimental plants from six allopatric populations that had been inoculated with a mixture of mutualistic arbuscular mycorrhizal fungi or a nonmycorrhizal control. Inoculation with arbuscular mycorrhizal fungi increased growth in plants from every population, but also increased host infection rate. Mycorrhizal effects on disease severity varied among host genotypes and strengthened over time during the epidemic. Host genotypes that were more susceptible to the pathogen received stronger protective effects from inoculation. Our results show that arbuscular mycorrhizal fungi introduce both benefits and risks to host plants, and shift patterns of infection in host populations under pathogen attack. Understanding how mutualists alter host susceptibility to disease will be important for predicting infection outcomes in ecological communities and in agriculture.

Geranium sylvaticum increases pollination probability by sexually dimorphic flowers.

Sexual dimorphism is expressed as different morphologies between the sexes of a species. Dimorphism is pronounced in gynodioecious populations which consist of female and hermaphrodite individuals. The small size of female flowers in gynodioecious species is often explained by resource re-allocation to seed production instead of large flowers. However, pollinator attraction is critical to female fitness, and factors other than resource savings are needed to explain the small size of female flowers. We hypothesized that the floral size dimorphism in the perennial gynodioecious Geranium sylvaticum (L.) is adaptive in terms of pollination. To test this "pollination hypothesis," we video recorded the small female and large hermaphrodite G. sylvaticum flowers. We parameterized floral visitor behavior when visiting a flower and calculated pollination probabilities by a floral visitor as the probability of touching anther and stigma with the same body part. Pollination probability differed in terms of flower sex and pollinator species. Bumblebees had the highest pollination probability. The small female flowers were more likely to receive pollen via several pollinator groups than the large hermaphrodite flowers. The pollen display of hermaphrodites matched poorly with the stigma display of hermaphrodites, but well with that of females. Although the small size of female flowers is commonly explained by resource re-allocation, we show that sexual dimorphism in flower size may increase the main reproductive functions of the females and hermaphrodites. Dimorphism increases pollination probability in females and fathering probability of the hermaphrodites likely driving G. sylvaticum populations towards dioecy.

Soil legacy determines arbuscular mycorrhizal spore bank and plant performance in the low Arctic.

Human impact is rapidly changing vegetation globally. The effect of plant cover that no longer exists in a site may still affect the development of future vegetation. We focused on a little studied factor-arbuscular mycorrhizal (AM) fungus spore bank-and its effect on three test plant species. In a low Arctic field site, plots were maintained for 6 years, devoid of any vegetation or with a Solidago virgaurea monoculture cover. We analysed the AM fungal morphospecies composition and identified 21 morphospecies in the field plots. The AM morphospecies community was dominated by members of Acaulosporaceae. Monoculturing under low Arctic field conditions changed the soil AM spore community, which became dominated by Glomus hoi. We tested the soil feedback in the greenhouse and grew Solidago virgaurea, Potentilla crantzii and Anthoxanthum odoratum in the field soils from the plots without plant cover, covered with Solidago virgaurea or with intact vegetation. Our results suggest that monoculturing resulted in improved N acquisition by the monocultured plant species Solidago virgaurea which may be related to the AM fungus community. Our results show that a rich community of AM fungus spores may remain viable under field conditions for 6 years in the low Arctic. Spore longevity in field soil in the absence of any host plants differed among AM fungus species. We suggest that AM fungus spore longevity be considered an AM fungal life-history trait.

Cryptogams signify key transitions of bacteria and fungi in Arctic sand dune succession.

Primary succession models focus on aboveground vascular plants. However, the prevalence of mosses and lichens, that is cryptogams, suggests they play a role in soil successions. Here, we explore whether effects of cryptogams on belowground microbes can facilitate progressive shifts in sand dune succession. We linked aboveground vegetation, belowground bacterial and fungal communities, and soil chemical properties in six successional stages in Arctic inland sand dunes: bare sand, grass, moss, lichen, ericoid heath and mountain birch forest. Compared with the bare sand and grass stages, microbial biomass and the proportion of fungi increased in the moss stage, and later stage microbial groups appeared despite the absence of their host plants. Microbial communities of the lichen stage resembled the communities in the vascular plant stages. Bacterial communities correlated better with soil chemical variables than with vegetation and vice versa for fungal communities. The correlation of fungi with vegetation increased with vascular vegetation. Distinct bacterial and fungal patterns of biomass, richness and plant-microbe interactions showed that the aboveground vegetation change structured the bacterial and fungal community differently. The asynchrony of aboveground vs belowground changes suggests that cryptogams can drive succession towards vascular plant dominance through microbially mediated facilitation in eroded Arctic soil.

Change in dominance determines herbivore effects on plant biodiversity.

Herbivores alter plant biodiversity (species richness) in many of the world's ecosystems, but the magnitude and the direction of herbivore effects on biodiversity vary widely within and among ecosystems. One current theory predicts that herbivores enhance plant biodiversity at high productivity but have the opposite effect at low productivity. Yet, empirical support for the importance of site productivity as a mediator of these herbivore impacts is equivocal. Here, we synthesize data from 252 large-herbivore exclusion studies, spanning a 20-fold range in site productivity, to test an alternative hypothesis-that herbivore-induced changes in the competitive environment determine the response of plant biodiversity to herbivory irrespective of productivity. Under this hypothesis, when herbivores reduce the abundance (biomass, cover) of dominant species (for example, because the dominant plant is palatable), additional resources become available to support new species, thereby increasing biodiversity. By contrast, if herbivores promote high dominance by increasing the abundance of herbivory-resistant, unpalatable species, then resource availability for other species decreases reducing biodiversity. We show that herbivore-induced change in dominance, independent of site productivity or precipitation (a proxy for productivity), is the best predictor of herbivore effects on biodiversity in grassland and savannah sites. Given that most herbaceous ecosystems are dominated by one or a few species, altering the competitive environment via herbivores or by other means may be an effective strategy for conserving biodiversity in grasslands and savannahs globally.

Native arbuscular mycorrhizal symbiosis alters foliar bacterial community composition.

The effects of arbuscular mycorrhizal (AM) fungi on plant-associated microbes are poorly known. We tested the hypothesis that colonization by an AM fungus affects microbial species richness and microbial community composition of host plant tissues. We grew the grass, Deschampsia flexuosa in a greenhouse with or without the native AM fungus, Claroideoglomus etunicatum. We divided clonally produced tillers into two parts: one inoculated with AM fungus spores and one without AM fungus inoculation (non-mycorrhizal, NM). We characterized bacterial (16S rRNA gene) and fungal communities (internal transcribed spacer region) in surface-sterilized leaf and root plant compartments. AM fungus inoculation did not affect microbial species richness or diversity indices in leaves or roots, but the AM fungus inoculation significantly affected bacterial community composition in leaves. A total of three OTUs in leaves belonging to the phylum Firmicutes positively responded to the presence of the AM fungus in roots. Another six OTUs belonging to the Proteobacteria (Alpha, Beta, and Gamma) and Bacteroidetes were significantly more abundant in NM plants when compared to AM fungus-inoculated plants. Further, there was a significant correlation between plant dry weight and leaf microbial community compositional shift. Also, there was a significant correlation between leaf bacterial community compositional shift and foliar nitrogen content changes due to AM fungus inoculation. The results suggest that AM fungus colonization in roots has a profound effect on plant physiology that is reflected in leaf bacterial community composition.

Microbial community composition but not diversity changes along succession in arctic sand dunes.

The generality of increasing diversity of fungi and bacteria across arctic sand dune succession was tested. Microbial communities were examined by high-throughput sequencing of 16S rRNA genes (bacteria) and internal transcribed spacer (ITS) regions (fungi). We studied four microbial compartments (inside leaf, inside root, rhizosphere and bulk soil) and characterized microbes associated with a single plant species (Deschampsia flexuosa) across two sand dune successional stages (early and late). Bacterial richness increased across succession in bulk soil and leaf endosphere. In contrast, soil fungal richness remained constant while root endosphere fungal richness increased across succession. There was, however, no significant difference in Shannon diversity indices between early and late successional stage in any compartment. There was a significant difference in the composition of microbial communities between early and late successional stage in all compartments, although the major microbial OTUs were shared between early and late successional stage. Co-occurrence network analysis revealed successional stage-specific microbial groups. There were more co-occurring modules in early successional stage than in late stage. Altogether, these results emphasize that succession strongly affects distribution of microbial species, but not microbial diversity in arctic sand dune ecosystem and that fungi and bacteria may not follow the same successional trajectories.

Light availability affects sex lability in a gynodioecious plant.

PREMISE OF THE STUDY: Sex lability (i.e., gender diphasy) in plants is classically linked to the larger resource needs associated with the female sexual function (i.e., seed production) compared to the male function (i.e., pollen production). Sex lability in response to the environment is extensively documented in dioecious species, but has been largely overlooked in gynodioecious plants. METHODS: Here, we tested whether environmental conditions induce sex lability in the gynodioecious Geranium sylvaticum. We conducted a transplantation experiment in the field where plants with different sex expression were reciprocally transplanted between high light and low light habitats. We measured plants' reproductive output and sex expression over four years. KEY RESULTS: Our results show that sex expression was labile over the study period. The light level at the destination habitat had a significant effect on sexual expression and reproductive output, because plants decreased their reproductive output when transplanted to the low light habitat. Transplantation origin did not affect any parameter measured. CONCLUSIONS: This study shows that sex expression in Geranium sylvaticum is labile and related to light availability. Sexually labile plants did not produce more seeds or pollen, and thus, there was no apparent fitness gain in sexually labile individuals. Sex lability in gynodioecious plants may be more common than previously believed because detection of sex lability necessitates data on the same individuals over time, which is rare in sexually dimorphic herbaceous plants.

Absence of sex differential plasticity to light availability during seed maturation in Geranium sylvaticum.

Sex-differential plasticity (SDP) hypothesis suggests that since hermaphrodites gain fitness through both pollen and seed production they may have evolved a higher degree of plasticity in their reproductive strategy compared to females which achieve fitness only through seed production. SDP may explain the difference in seed production observed between sexes in gynodioecious species in response to resource (nutrients or water) availability. In harsh environments, hermaphrodites decrease seed production whereas females keep it relatively similar regardless of the environmental conditions. Light availability can be also a limiting resource and thus could theoretically affect differently female and hermaphrodite seed output even though this ecological factor has been largely overlooked. We tested whether the two sexes in the gynodioecious species Geranium sylvaticum differ in their tolerance to light limitation during seed maturation in the field. We used a fully factorial block experiment exposing female and hermaphrodite plants to two different light environments (control and shade) after their peak flowering period. Specifically, we measured fruit and seed production in response to decreased light availability and compared it between the sexes. Shading reduced the number of fruits and seeds produced, but the decrease was similar between the sexes. Furthermore, shading delayed seed production by three days in both sexes, but did not affect seed mass, seed P content, or the probability of re-flowering the following year. Our results give no evidence for reproductive SDP in response to light during seed maturation.

Culturable endophytic microbial communities in the circumpolar grass, Deschampsia flexuosa in a sub-Arctic inland primary succession are habitat and growth stage specific.

Little is known about endophytic microbes in cold climate plants and how their communities are formed.We compared culturable putative endophytic bacteria and fungi in the ecologically important circumpolargrass, Deschampsia flexuosa growing in two successional stages of subarctic sand dune (68°29'N).Sequence analyses of partial 16S rRNA and internal transcribed spacer (ITS) sequences of culturable endophytes showed that diverse bacteria and fungi inhabit different tissues of D. flexuosa. A total of 178 bacterial isolates representing seven taxonomic divisions, Alpha, Beta and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Acidobacteria, and 30 fungal isolates representing the phylum Ascomycota were identified. Several endophytes were affiliated with specific plant tissues or successional stages. This first report of bacterial endophytes in D. flexuosa revealed that the genus Pseudomonas is tightly associated with D. flexuosa, and encompassed 39% of the bacterial isolates, and 58% of seed isolates. Based on 16S rRNA and ITS sequence data, most of the D. flexuosa endophytes were closely related to microbes from other cold environments. The majority of seed endophytic bacterial isolates were able to solubilize organic form of phosphate suggesting that these endophytes could play a role in resource mobilization in germinating seeds in nutrient-poor habitat.

Arctic arbuscular mycorrhizal spore community and viability after storage in cold conditions.

Arbuscular mycorrhizal fungi (AMF) form probably the most widespread symbiosis on earth and are found across all ecosystems including the Arctic regions. In the Arctic, the prevalent harsh cold conditions experienced by both host plants and fungi may have selected for AMF species with long-surviving spores, the principal means for dispersal and survival. However, basic knowledge about their viability is lacking. AMF spore assembly from two Arctic sites was examined in soil samples collected across an 11-year period and stored at -20 °C for up to 10 years. AMF spore viability and ability to colonize plants were investigated in the greenhouse using Plantago lanceolata. It was predicted that Arctic AMF spores would survive in cold conditions for several years, with an expected decrease in viability over time as suggested by other experiments with temperate material. Results show that even though the two study sites differed in AMF spore density, the relative abundance of spore morphotypes was rather similar across sites and years. Furthermore, spore viability over time was site-dependent as it decreased only in one site. Although spores were viable, only a very small proportion of hosts and roots became colonized in the greenhouse even 21 months after inoculation. Taken together, these results suggest a certain site-dependent variability in AMF spore communities and the ability of Arctic AMF spores to remain viable after a long-term storage in cold conditions. The lack of host colonization in the greenhouse may be related to the inability to overcome spore dormancy under these conditions.

Arbuscular mycorrhizal fungal community divergence within a common host plant in two different soils in a subarctic Aeolian sand area.

There is rising awareness that different arbuscular mycorrhizal (AM) fungi have different autoecology and occupy different soil niches and that the benefits they provide to the host plant are dependent on plant-AM fungus combination. However, the role and community composition of AM fungi in succession are not well known and the northern latitudes remain poorly investigated ecosystems. We studied AM fungal communities in the roots of the grass Deschampsia flexuosa in two different, closely located, successional stages in a northern Aeolian sand area. The AM fungal taxa richness in planta was estimated by cloning and sequencing small subunit ribosomal RNA genes. AM colonization, shoot δ (13)C signature, and %N and %C were measured. Soil microbial community structure and AM fungal mycelium abundance were estimated using phospholipid (PLFA) and neutral lipid (NLFA) analyses. The two successional stages were characterized by distinct plant, microbial, and fungal communities. AM fungal species richness was very low in both the early and late successional stages. AM frequency in D. flexuosa roots was higher in the early successional stage than in the late one. The AM fungal taxa retrieved belonged to the genera generally adapted to Arctic or extreme environments. AM fungi seemed to be important in the early stage of the succession, suggesting that AM fungi may help plants to better cope with the harsh environmental conditions, especially in an early successional stage with more extreme environmental fluctuations.

Transgenerational effects of plant sex and arbuscular mycorrhizal symbiosis.

In gynodioecious plants, females are predicted to produce more and/or better offspring than hermaphrodites in order to be maintained in the same population. In the field, the roots of both sexes are usually colonized by arbuscular mycorrhizal (AM) fungi. Transgenerational effects of mycorrhizal symbiosis are largely unknown, although theoretically expected. We examined the maternal and paternal effects of AM fungal symbiosis and host sex on seed production and posterior seedling performance in Geranium sylvaticum, a gynodioecious plant. We hand-pollinated cloned females and hermaphrodites in symbiosis with AM fungi or in nonmycorrhizal conditions and measured seed number and mass, and seedling survival and growth in a glasshouse experiment. Females produced more seeds than hermaphrodites, but the seeds did not germinate, survive or grow better. Mycorrhizal plants were larger, but did not produce more seeds than nonmycorrhizal plants. Transgenerational parental effects of AM fungi were verified in seedling performance. This is the first study to show transgenerational mycorrhiza-mediated parental effects in a gynodioecious species. Mycorrhizal symbiosis affects plant fitness mainly through female functions with enduring effects on the next generation.

Nectar sugar production across floral phases in the Gynodioecious Protandrous Plant Geranium sylvaticum [corrected].

Many zoophilous plants attract their pollinators by offering nectar as a reward. In gynodioecious plants (i.e. populations are composed of female and hermaphrodite individuals) nectar production has been repeatedly reported to be larger in hermaphrodite compared to female flowers even though nectar production across the different floral phases in dichogamous plants (i.e. plants with time separation of pollen dispersal and stigma receptivity) has rarely been examined. In this study, sugar production in nectar standing crop and secretion rate were investigated in Geranium sylvaticum, a gynodioecious plant species with protandry (i.e. with hermaphrodite flowers releasing their pollen before the stigma is receptive). We found that flowers from hermaphrodites produced more nectar than female flowers in terms of total nectar sugar content. In addition, differences in nectar production among floral phases were found in hermaphrodite flowers but not in female flowers. In hermaphrodite flowers, maximum sugar content coincided with pollen presentation and declined slightly towards the female phase, indicating nectar reabsorption, whereas in female flowers sugar content did not differ between the floral phases. These differences in floral reward are discussed in relation to visitation patterns by pollinators and seed production in this species.

Dioecious species and arbuscular mycorrhizal symbioses: the case of Antennaria dioica.

Sex-specific interactions with herbivores and pollinators have been observed in female and male plants of dioecious species. However, only a limited number of studies have revised sex-specific patterns in mycorrhizal symbiosis. To test whether female and male plants of Antennaria dioica differ in their relationship with arbuscular mycorrhizal (AM) fungi, we examined the temporal and spatial variation in AM fungi in female, male and non-reproductive A. dioica plants in three natural populations in Finland during flowering and after seed production. Our results are consistent with previous studies both under greenhouse and field conditions with the same species showing differences in AM colonization between the sexes linked with allocation to reproduction. Taken together, the results indicate that there is a sex-specific interaction between A. dioica and AM fungi. Overall, females have a greater investment in AM fungi, likely to enhance their uptake of soil nutrients and support the reproduction by seed.

Differential competitive ability between sexes in the dioecious Antennaria dioica (Asteraceae).

BACKGROUND AND AIMS: Differences in competitive ability between the sexes of dioecious plants are expected as a result of allocation trade-offs associated with sex-differential reproductive costs. However, the available data on competitive ability in dioecious plants are scarce and contradictory. In this study sexual competition was evaluated using the dioecious plant Antennaria dioica in a common garden transplantation experiment. METHODS: Male and female plants were grown for 3 years either in isolation, or in competition with a plant of the same sex or the opposite sex. Flowering phenology, sexual and asexual reproduction, plant growth, nutrient content and arbuscular mycorrhizal colonization in the roots were assessed. KEY RESULTS: Our results showed little evidence of sexual differences in competitive ability. Both sexes suffered similarly from competition, and competitive effects were manifested in some traits related to fitness but not in others. Survival was unaffected by competition, but competing plants reduced their vegetative growth and reproductive investment compared with non-competing plants. In addition, differences in sexual competitive ability were observed in relation to flowering frequency, an important life history trait not reported in previous studies. CONCLUSIONS: The findings indicate that female and male A. dioica plants possess similar intersexual competitive abilities which may be related to the similar costs of reproduction between sexes in this species. Nevertheless, intrasexual competition is higher in females, giving support for asymmetric niche segregation between the sexes.

Mycorrhizal benefit differs among the sexes in a gynodioecious species.

Both plant sex and arbuscular mycorrhizal (AM) symbiosis influence resource acquisition and allocation in plants, but the interaction between these two components is not well established. As the different plant sexes differ in their resource needs and allocation patterns, it is logical to presume that they might differ in their relationship with AM as well. We investigate whether the association with AM symbiosis is different according to the host plant sex in the gynodioecious Geranium sylvaticum, of which, besides female and hermaphrodite plants, intermediate plants are also recognized. Specifically, we examine the effects of two different AM fungi in plant mass allocation and phosphorus acquisition using a factorial greenhouse/common garden experiment. Cloned G. sylvaticum material was grown in symbiosis with AM fungi or in non-mycorrhizal condition. We evaluated both the symbiotic plant benefit in terms of plant mass and plant P content and the fungal benefit in terms of AM colonization intensity in the plant roots and spore production. Our results suggest that G. sylvaticum plants benefit from the symbiosis with both AM fungal species tested but that the benefits gained from the symbiosis depend on the sex of the plant and on the trait investigated. Hermaphrodites suffered most from the lack of AM symbiosis as the proportion of flowering plants was dramatically reduced by the absence of AM fungi. However, females and intermediates benefited from the symbiosis relatively more than hermaphrodites in terms of higher P acquisition. The two AM fungal species differed in the amount of resources accumulated, and the fungal benefit was also dependent on the sex of the host plant. This study provides the first evidence of sex-specific benefits from mycorrhizal symbiosis in a gynodioecious plant species.

Sex-specific responses to mycorrhiza in a dioecious species.

In most studies about dioecious plants, the role of arbuscular mycorrhizae (AM) and the potential sex-specific differences between the plant hosts have been overlooked. Because plant sexes frequently differ in drought tolerance and AM fungal colonization provides higher resistance to drought, we investigated whether the relation of mycorrhizal fungi with either male or female Antennaria dioica plants differs using a factorial experiment. We hypothesized that because AM usually increase growth rate and male plants usually grow larger than females, males should gain more benefit from the mycorrhizal symbiosis in terms of mineral nutrition and water supply. Because of higher demands of carbohydrates (C) in males, we expected males to allocate less C resources to the mycorrhizal fungus so that the associated fungi should benefit less of the association with males. In contrast to our initial hypothesis, the male plants, although faster growing under drought, did not gain more symbiosis-mediated benefits than did the females, and both sexes seemed to provide resources equally to their fungal symbiont. Therefore, we conclude that the two plant sexual morphs provide equal amounts of C to their fungal root symbionts and that they can gain specific benefits from the symbiosis, which, however, depend on soil water availability.

Diversity and persistence of arbuscular mycorrhizas in a low-Arctic meadow habitat.

Little is known about the ecology and diversity of arbuscular mycorrhizal (AM) fungi in Arctic ecosystems. Here, the diversity and composition of the AM fungal community and its response to host plant community composition were studied in a low-Arctic meadow habitat. The natural vegetation in two low-Arctic meadow sites was manipulated. Plots with natural vegetation, monoculture and no vegetation were established. Seeds of Solidago virgaurea were sown into the plots and the AM fungal community in the seedling roots was analysed using the terminal restriction fragment length polymorphism (T-RFLP) method. The vegetation manipulation treatments affected the community composition but not the diversity of AM fungi found in S. virgaurea roots. The diversity of AM fungi was higher in S. virgaurea roots in the site with naturally higher plant species diversity. These results show that AM fungi in low-Arctic meadows are able to survive for a period of 2 yr without a host plant. This ability buffers the AM fungal community against short-term changes in host plant community composition and diversity.