Parasitism causes changes in caterpillar odours and associated bacterial communities with consequences for host-location by a hyperparasitoid.

Microorganisms living in and on macroorganisms may produce microbial volatile compounds (mVOCs) that characterise organismal odours. The mVOCs might thereby provide a reliable cue to carnivorous enemies in locating their host or prey. Parasitism by parasitoid wasps might alter the microbiome of their caterpillar host, affecting organismal odours and interactions with insects of higher trophic levels such as hyperparasitoids. Hyperparasitoids parasitise larvae or pupae of parasitoids, which are often concealed or inconspicuous. Odours of parasitised caterpillars aid them to locate their host, but the origin of these odours and its relationship to the caterpillar microbiome are unknown. Here, we analysed the odours and microbiome of the large cabbage white caterpillar Pieris brassicae in relation to parasitism by its endoparasitoid Cotesia glomerata. We identified how bacterial presence in and on the caterpillars is correlated with caterpillar odours and tested the attractiveness of parasitised and unparasitised caterpillars to the hyperparasitoid Baryscapus galactopus. We manipulated the presence of the external microbiome and the transient internal microbiome of caterpillars to identify the microbial origin of odours. We found that parasitism by C. glomerata led to the production of five characteristic volatile products and significantly affected the internal and external microbiome of the caterpillar, which were both found to have a significant correlation with caterpillar odours. The preference of the hyperparasitoid was correlated with the presence of the external microbiome. Likely, the changes in external microbiome and body odour after parasitism were driven by the resident internal microbiome of caterpillars, where the bacterium Wolbachia sp. was only present after parasitism. Micro-injection of Wolbachia in unparasitised caterpillars increased hyperparasitoid attraction to the caterpillars compared to untreated caterpillars, while no differences were found compared to parasitised caterpillars. In conclusion, our results indicate that host-parasite interactions can affect multi-trophic interactions and hyperparasitoid olfaction through alterations of the microbiome.

Arbuscular mycorrhizal fungal diversity and potential association networks among African tropical forest trees.

Tropical forests represent one of the most diverse and productive ecosystems on Earth. High productivity is sustained by efficient and rapid cycling of nutrients, which is in large part made possible by symbiotic associations between plants and mycorrhizal fungi. In these associations, an individual plant typically associates simultaneously with multiple fungi and the fungi associate with multiple plants, creating complex networks among fungi and plants. However, there are few studies that have investigated mycorrhizal fungal composition and diversity in tropical forest trees, particularly in Africa, or that assessed the structure of the network of associations among fungi and trees. In this study, we collected root and soil samples from Ise Forest Reserve (Southwest Nigeria) and used a metabarcoding approach to identify the dominant arbuscular mycorrhizal (AM) fungal taxa in the soil and associating with ten co-occurring tree species to assess variation in AM communities. Network analysis was used to elucidate the architecture of the network of associations between fungi and tree species. A total of 194 Operational Taxonomic Units (OTUs) belonging to six AM fungal families were identified, with 68% of all OTUs belonging to Glomeraceae. While AM fungal diversity did not differ among tree species, AM fungal community composition did. Network analyses showed that the network of associations was not significantly nested and showed a relatively low level of specialization (H2 = 0.43) and modularity (M = 0.44). We conclude that, although there were some differences in AM fungal community composition, the studied tree species associate with a large number of AM fungi. Similarly, most AM fungi had great host breadth and were detected in most tree species, thereby potentially working as interaction network hubs.

Plastome phylogenomics and morphological traits analyses provide new insights into the phylogenetic position, species delimitation and speciation of Triplostegia (Caprifoliaceae).

BACKGROUND: The genus Triplostegia contains two recognized species, T. glandulifera and T. grandiflora, but its phylogenetic position and species delimitation remain controversial. In this study, we assembled plastid genomes and nuclear ribosomal DNA (nrDNA) cistrons sampled from 22 wild Triplostegia individuals, each from a separate population, and examined these with 11 recently published Triplostegia plastomes. Morphological traits were measured from herbarium specimens and wild material, and ecological niche models were constructed. RESULTS: Triplostegia is a monophyletic genus within the subfamily Dipsacoideae comprising three monophyletic species, T. glandulifera, T. grandiflora, and an unrecognized species Triplostegia sp. A, which occupies much higher altitude than the other two. The new species had previously been misidentified as T. glandulifera, but differs in taproot, leaf, and other characters. Triplotegia is an old genus, with stem age 39.96 Ma, and within it T. glandulifera diverged 7.94 Ma. Triplostegia grandiflora and sp. A diverged 1.05 Ma, perhaps in response to Quaternary climate fluctuations. Niche overlap between Triplostegia species was positively correlated with their phylogenetic relatedness. CONCLUSIONS: Our results provide new insights into the species delimitation of Triplostegia, and indicate that a taxonomic revision of Triplostegia is needed. We also identified that either rpoB-trnC or ycf1 could serve as a DNA barcode for Triplostegia.

Metabarcoding read abundances of orchid mycorrhizal fungi are correlated to copy numbers estimated using ddPCR.

Quantifying the abundances of fungi is key to understanding natural variation in mycorrhizal communities in relation to plant ecophysiology and environmental heterogeneity. High-throughput metabarcoding approaches have transformed our ability to characterize and compare complex mycorrhizal communities. However, it remains unclear how well metabarcoding read counts correlate with actual read abundances in the sample, potentially limiting their use as a proxy for species abundances. Here, we use droplet digital PCR (ddPCR) to evaluate the reliability of ITS2 metabarcoding data for quantitative assessments of mycorrhizal communities in the orchid species Neottia ovata sampled at multiple sites. We performed specific ddPCR assays for eight families of orchid mycorrhizal fungi and compared the results with read counts obtained from metabarcoding. Our results demonstrate a significant correlation between DNA copy numbers measured by ddPCR assays and metabarcoding read counts of major mycorrhizal partners of N. ovata, highlighting the usefulness of metabarcoding for quantifying the abundance of orchid mycorrhizal fungi. Yet, the levels of correlation between the two methods and the numbers of false zero values varied across fungal families, which warrants cautious evaluation of the reliability of low-abundance families. This study underscores the potential of metabarcoding data for more quantitative analyses of mycorrhizal communities and presents practical workflows for metabarcoding and ddPCR to achieve a more comprehensive understanding of orchid mycorrhizal communities.

Historical biogeography and local adaptation explain population genetic structure in a widespread terrestrial orchid.

BACKGROUND AND AIMS: Historical changes in environmental conditions and colonization-extinction dynamics have a direct impact on the genetic structure of plant populations. However, understanding how past environmental conditions influenced the evolution of species with high gene flow is challenging when signals for genetic isolation and adaptation are swamped by gene flow. We investigated the spatial distribution and genetic structure of the widespread terrestrial orchid Epipactis helleborine to identify glacial refugia, characterize postglacial population dynamics and assess its adaptive potential. METHODS: Ecological niche modelling was used to locate possible glacial refugia and postglacial recolonization opportunities of E. helleborine. A large single-nucleotide polymorphism (SNP) dataset obtained through genotyping by sequencing was used to define population genetic diversity and structure and to identify sources of postglacial gene flow. Outlier analyses were used to elucidate how adaptation to the local environment contributed to population divergence. KEY RESULTS: The distribution of climatically suitable areas was restricted during the Last Glacial Maximum to the Mediterranean, south-western Europe and small areas in the Alps and Carpathians. Within-population genetic diversity was high in E. helleborine (mean expected heterozygosity, 0.373 ± 0.006; observed heterozygosity, 0.571 ± 0.012; allelic richness, 1.387 ± 0.007). Italy and central Europe are likely to have acted as important genetic sources during postglacial recolonization. Adaptive SNPs were associated with temperature, elevation and precipitation. CONCLUSIONS: Forests in the Mediterranean and Carpathians are likely to have acted as glacial refugia for Epipactis helleborine. Postglacial migration northwards and to higher elevations resulted in the dispersal and diversification of E. helleborine in central Europe and Italy, and to geographical isolation and divergent adaptation in Greek and Italian populations. Distinguishing adaptive from neutral genetic diversity allowed us to conclude that E. helleborine has a high adaptive potential to climate change and demonstrates that signals of adaptation and historical isolation can be identified even in species with high gene flow.

Phylogenomic analysis of the genus Rosenbergiella and description of Rosenbergiella gaditana sp. nov., Rosenbergiella metrosideri sp. nov., Rosenbergiella epipactidis subsp. epipactidis subsp. nov., Rosenbergiella epipactidis subsp. californiensis subsp. nov., Rosenbergiella epipactidis subsp. japonicus subsp. nov., Rosenbergiella nectarea subsp. nectarea subsp. nov. and Rosenbergiella nectarea subsp. apis subsp. nov., isolated from floral nectar and insects.

The genus Rosenbergiella is one of the most frequent bacterial inhabitants of flowers and a usual member of the insect microbiota worldwide. To date, there is only one publicly available Rosenbergiella genome, corresponding to the type strain of Rosenbergiella nectarea (8N4T), which precludes a detailed analysis of intra-genus phylogenetic relationships. In this study, we obtained draft genomes of the type strains of the other Rosenbergiella species validly published to date (R. australiborealis, R. collisarenosi and R. epipactidis) and 23 additional isolates of flower and insect origin. Isolate S61T, retrieved from the nectar of an Antirrhinum sp. flower collected in southern Spain, displayed low average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values when compared with other Rosenbergiella members (≤86.5 and ≤29.8 %, respectively). Similarly, isolate JB07T, which was obtained from the floral nectar of Metrosideros polymorpha plants in Hawaii (USA) had ≤95.7 % ANI and ≤64.1 % isDDH with other Rosenbergiella isolates. Therefore, our results support the description of two new Rosenbergiella species for which we propose the names Rosenbergiella gaditana sp. nov. (type strain: S61T=NCCB 100789T=DSM 111181T) and Rosenbergiella metrosideri sp. nov. (JB07T=NCCB 100888T=LMG 32616T). Additionally, some R. epipactidis and R. nectarea isolates showed isDDH values<79 % with other conspecific isolates, which suggests that these species include subspecies for which we propose the names Rosenbergiella epipactidis subsp. epipactidis subsp. nov. (S256T=CECT 8502T=LMG 27956T), Rosenbergiella epipactidis subsp. californiensis subsp. nov. (FR72T=NCCB 100898T=LMG 32786T), Rosenbergiella epipactidis subsp. japonicus subsp. nov. (K24T=NCCB 100924T=LMG 32785T), Rosenbergiella nectarea subsp. nectarea subsp. nov. (8N4T = DSM 24150T = LMG 26121T) and Rosenbergiella nectarea subsp. apis subsp. nov. (B1AT=NCCB 100810T= DSM 111763T), respectively. Finally, we present the first phylogenomic analysis of the genus Rosenbergiella and update the formal description of the species R. australiborealis, R. collisarenosi, R. epipactidis and R. nectarea based on new genomic and phenotypic information.

Sugar Concentration, Nitrogen Availability, and Phylogenetic Factors Determine the Ability of Acinetobacter spp. and Rosenbergiella spp. to Grow in Floral Nectar.

The floral nectar of angiosperms harbors a variety of microorganisms that depend predominantly on animal visitors for their dispersal. Although some members of the genus Acinetobacter and all currently known species of Rosenbergiella are thought to be adapted to thrive in nectar, there is limited information about the response of these bacteria to variation in the chemical characteristics of floral nectar. We investigated the growth performance of a diverse collection of Acinetobacter (n = 43) and Rosenbergiella (n = 45) isolates obtained from floral nectar and the digestive tract of flower-visiting bees in a set of 12 artificial nectars differing in sugar content (15% w/v or 50% w/v), nitrogen content (3.48/1.67 ppm or 348/167 ppm of total nitrogen/amino nitrogen), and sugar composition (only sucrose, 1/3 sucrose + 1/3 glucose + 1/3 fructose, or 1/2 glucose + 1/2 fructose). Growth was only observed in four of the 12 artificial nectars. Those containing elevated sugar concentration (50% w/v) and low nitrogen content (3.48/1.67 ppm) were limiting for bacterial growth. Furthermore, phylogenetic analyses revealed that the ability of the bacteria to grow in different types of nectar is highly conserved between closely related isolates and genotypes, but this conservatism rapidly vanishes deeper in phylogeny. Overall, these results demonstrate that the ability of Acinetobacter spp. and Rosenbergiella spp. to grow in floral nectar largely depends on nectar chemistry and bacterial phylogeny.

Mycorrhizal diversity and community composition in co-occurring Cypripedium species.

Orchids commonly rely on mycorrhizal fungi to obtain the necessary resources for seed germination and growth. Whereas most photosynthetic orchids typically associate with so-called rhizoctonia fungi to complete their life cycle, there is increasing evidence that other fungi may be involved as well and that the mycorrhizal communities associated with orchids may be more diverse. Coexisting orchid species also tend to associate with different fungi to reduce competition for similar resources and to increase long-term population viability. However, few studies have related the mycorrhizal communities in the rhizosphere to communities found in the roots of closely related coexisting orchid species. In this study, we used high-throughput sequencing to investigate the diversity and community composition of orchid mycorrhizal fungi in the roots and the rhizosphere of four Cypripedium species growing in forests in Northeast China. The results showed that the investigated Cypripedium species associated with a wide variety of fungi including members of Tulasnellaceae, Psathyrellaceae, and Herpotrichiellaceae, whereas members of Russulaceae, Cortinariaceae, Thelephoraceae, and Herpotrichiellaceae showed high abundance in rhizosphere soils. The diversity of fungi detected in the rhizosphere soil was much higher than that in the roots. The observed variation in fungal communities in Cypripedium roots was not related to forest site or orchid species. On the other hand, variation in mycorrhizal communities of rhizosphere soil was significantly related to sampling site. These results indicate that orchid mycorrhizal communities in the rhizosphere display considerable variation among sites and that orchids use only a subset of the locally available fungi. Future studies focusing on the fine-scale spatial distribution of orchid mycorrhizal fungi and more detailed assessments of local environmental conditions will provide novel insights into the mechanisms explaining variation of fungal communities in both orchid roots and the rhizosphere.

The Waiting Room Hypothesis revisited by orchids: were orchid mycorrhizal fungi recruited among root endophytes?

BACKGROUND: As in most land plants, the roots of orchids (Orchidaceae) associate with soil fungi. Recent studies have highlighted the diversity of the fungal partners involved, mostly within Basidiomycotas. The association with a polyphyletic group of fungi collectively called rhizoctonias (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae) is the most frequent. Yet, several orchid species target other fungal taxa that differ from rhizoctonias by their phylogenetic position and/or ecological traits related to their nutrition out of the orchid roots (e.g. soil saprobic or ectomycorrhizal fungi). We offer an evolutionary framework for these symbiotic associations. SCOPE: Our view is based on the 'Waiting Room Hypothesis', an evolutionary scenario stating that mycorrhizal fungi of land flora were recruited from ancestors that initially colonized roots as endophytes. Endophytes biotrophically colonize tissues in a diffuse way, contrasting with mycorrhizae by the absence of morphological differentiation and of contribution to the plant's nutrition. The association with rhizoctonias is probably the ancestral symbiosis that persists in most extant orchids, while during orchid evolution numerous secondary transitions occurred to other fungal taxa. We suggest that both the rhizoctonia partners and the secondarily acquired ones are from fungal taxa that have broad endophytic ability, as exemplified in non-orchid roots. We review evidence that endophytism in non-orchid plants is the current ecology of many rhizoctonias, which suggests that their ancestors may have been endophytic in orchid ancestors. This also applies to the non-rhizoctonia fungi that were secondarily recruited by several orchid lineages as mycorrhizal partners. Indeed, from our review of the published literature, they are often detected, probably as endophytes, in extant rhizoctonia-associated orchids. CONCLUSION: The orchid family offers one of the best documented examples of the 'Waiting Room Hypothesis': their mycorrhizal symbioses support the idea that extant mycorrhizal fungi have been recruited among endophytic fungi that colonized orchid ancestors.

The effect of DNA methylation on bumblebee colony development.

BACKGROUND: Although around 1% of cytosines in bees' genomes are known to be methylated, less is known about methylation's effect on bee behavior and fitness. Chemically altered DNA methylation levels have shown clear changes in the dominance and reproductive behavior of workers in queen-less colonies, but the global effect of DNA methylation on caste determination and colony development remains unclear, mainly because of difficulties in controlling for genetic differences among experimental subjects in the parental line. Here, we investigated the effect of the methylation altering agent decitabine on the developmental rate of full bumblebee colonies. Whole genome bisulfite sequencing was used to assess differences in methylation status. RESULTS: Our results showed fewer methylated loci in the control group. A total of 22 CpG loci were identified as significantly differentially methylated between treated and control workers with a change in methylation levels of 10% or more. Loci that were methylated differentially between groups participated in pathways including neuron function, oocyte regulation and metabolic processes. Treated colonies tended to develop faster, and therefore more workers were found at a given developmental stage. However, male production followed the opposite trend and it tended to be higher in control colonies. CONCLUSION: Overall, our results indicate that altered methylation patterns resulted in an improved cooperation between workers, while there were no signs of abnormal worker dominance or caste determination.

Temporal turnover in mycorrhizal interactions: a proof of concept with orchids.

Temporal turnover events in biotic interactions involving plants are rarely assessed, although such changes might afford a considerable acclimation potential to the plant. This could enable fairly rapid responses to short-term fluctuations in growth conditions as well as lasting responses to long-term climatic trends. Here, we present a classification of temporal turnover encompassing 11 possible scenarios. Using orchid mycorrhiza as a study model, we show that temporal changes are common, and discuss under which conditions temporal turnover of fungal symbiont is expected. We provide six research questions and identify technical challenges that we deem most important for future studies. Finally, we discuss how the same framework can be applied to other types of biotic interactions.

Symbiont switching and trophic mode shifts in Orchidaceae.

Mycorrhizal fungi are central to the biology of land plants. However, to what extent mycorrhizal shifts - broad evolutionary transitions in root-associated fungal symbionts - are related to changes in plant trophic modes remains poorly understood. We built a comprehensive DNA dataset of Orchidaceae fungal symbionts and a dated plant molecular phylogeny to test the hypothesis that shifts in orchid trophic modes follow a stepwise pattern, from autotrophy over partial mycoheterotrophy (mixotrophy) to full mycoheterotrophy, and that these shifts are accompanied by switches in fungal symbionts. We estimate that at least 17 independent shifts from autotrophy towards full mycoheterotrophy occurred in orchids, mostly through an intermediate state of partial mycoheterotrophy. A wide range of fungal partners was inferred to occur in the roots of the common ancestor of this family, including 'rhizoctonias', ectomycorrhizal, and wood- or litter-decaying saprotrophic fungi. Phylogenetic hypothesis tests further show that associations with ectomycorrhizal or saprotrophic fungi were most likely a prerequisite for evolutionary shifts towards full mycoheterotrophy. We show that shifts in trophic mode often coincided with switches in fungal symbionts, suggesting that the loss of photosynthesis selects for different fungal communities in orchids. We conclude that changes in symbiotic associations and ecophysiological traits are tightly correlated throughout the diversification of orchids.

Nitrogen Assimilation Varies Among Clades of Nectar- and Insect-Associated Acinetobacters.

Floral nectar is commonly colonized by yeasts and bacteria, whose growth largely depends on their capacity to assimilate nutrient resources, withstand high osmotic pressures, and cope with unbalanced carbon-to-nitrogen ratios. Although the basis of the ecological success of these microbes in the harsh environment of nectar is still poorly understood, it is reasonable to assume that they are efficient nitrogen scavengers that can consume a wide range of nitrogen sources in nectar. Furthermore, it can be hypothesized that phylogenetically closely related strains have more similar phenotypic characteristics than distant relatives. We tested these hypotheses by investigating the growth performance on different nitrogen-rich substrates of a collection of 82 acinetobacters isolated from nectar and honeybees, representing members of five species (Acinetobacter nectaris, A. boissieri, A. apis, and the recently described taxa A. bareti and A. pollinis). We also analyzed possible links between growth performance and phylogenetic affiliation of the isolates, while taking into account their geographical origin. Results demonstrated that the studied isolates could utilize a wide variety of nitrogen sources, including common metabolic by-products of yeasts (e.g., ammonium and urea), and that phylogenetic relatedness was associated with the variation in nitrogen assimilation among the studied acinetobacters. Finally, nutrient source and the origin (sample type and country) of isolates also predicted the ability of the acinetobacters to assimilate nitrogen-rich compounds. Overall, these results demonstrate inter-clade variation in the potential of the acinetobacters as nitrogen scavengers and suggest that nutritional dependences might influence interactions between bacteria and yeasts in floral nectar.

Effects of pollen and nectar inoculation by yeasts, bacteria or both on bumblebee colony development.

It is increasingly recognized that gut microbiota have a major effect on the physiology, biology, ecology and evolution of their animal hosts. Because in social insects, the gut microbiota is acquired through the diet and by contact with nest provisions, it can be hypothesized that regular supplementation of microorganisms to the diet will have an impact on the fitness of the consumer and on the development of the whole colony. To test this hypothesis, we investigated how supplementation of bacteria, yeasts, and combinations of the two to either pollen or nectar affected colony development in the social bumblebee Bombus terrestris. Three yeasts and three bacterial species that live at the flower-insect interface were used in the experiments and the development of bumblebee colonies was monitored over a period of 10 weeks. The results showed that administration of microbes via pollen had a stronger positive impact on colony development than when provided via sugar water. Supplementation of bacteria led, in general, to a faster egg laying, higher brood size and increased production of workers during the first weeks, whereas yeasts or a combination of yeasts and bacteria had less impact on colony development. However, the results differed between microbial species, with Wickerhamiella bombiphila and Rosenbergiella nectarea showing the strongest increase in colony development. Torulaspora delbrueckii induced early male production, which is likely a fitness cost. We conclude that the tested bacteria-yeast consortia did not result in better colony development than the interacting species alone.

The Pupal Parasitoid Trichopria drosophilae Is Attracted to the Same Yeast Volatiles as Its Adult Host.

There is increasing evidence that microorganisms, particularly fungi and bacteria, emit volatile compounds that mediate the foraging behaviour of insects and therefore have the potential to affect key ecological relationships. However, to what extent microbial volatiles affect the olfactory response of insects across different trophic levels remains unclear. Adult parasitoids use a variety of chemical stimuli to locate potential hosts, including those emitted by the host's habitat, the host itself, and microorganisms associated with the host. Given the great capacity of parasitoids to utilize and learn odours to increase foraging success, parasitoids of eggs, larvae, or pupae may respond to the same volatiles the adult stage of their hosts use when locating their resources, but compelling evidence is still scarce. In this study, using Saccharomyces cerevisiae we show that Trichopria drosophilae, a pupal parasitoid of Drosophila species, is attracted to the same yeast volatiles as their hosts in the adult stage, i.e. acetate esters. Parasitoids significantly preferred the odour of S. cerevisiae over the blank medium in a Y-tube olfactometer. Deletion of the yeast ATF1 gene, encoding a key acetate ester synthase, decreased attraction of T. drosophilae, while the addition of synthetic acetate esters to the fermentation medium restored parasitoid attraction. Bioassays with individual compounds revealed that the esters alone were not as attractive as the volatile blend of S. cerevisiae, suggesting that other volatile compounds also contribute to the attraction of T. drosophilae. Altogether, our results indicate that pupal parasitoids respond to the same volatiles as the adult stage of their hosts, which may aid them in locating oviposition sites.

Partner turnover and changes in ectomycorrhizal fungal communities during the early life stages of European beech (Fagus sylvatica L.).

The first life stages of a tree are subject to strong environmental stresses and competition, limiting their chances of survival. Establishing a mutualistic relationship with mycorrhizal fungi during early life stages may increase growth and survival rates of trees, but how mycorrhizal communities assemble during these stages remains unclear. Here, we studied variation in the ectomycorrhizal (EcM) fungal communities in the soil and roots of Fagus sylvatica seedlings and saplings. Fungal DNA was extracted from the soil and seedling and sapling roots collected in 156 plots across the beech-dominated Sonian forest (Belgium) and community composition was determined through metabarcoding. EcM fungal community composition significantly differed between soil, seedlings and saplings. Russula, Amanita and Inocybe were most abundant in soil, while Lactarius and Scleroderma were more abundant in seedling and sapling roots and Xerocomellus and Laccaria were most abundant in sapling roots. Our results provide evidence of partner turnover in EcM fungal community composition with increasing age in the early life stages of F. sylvatica.

Diversity and community structure of ericoid mycorrhizal fungi in European bogs and heathlands across a gradient of nitrogen deposition.

Despite the ecological significance of ericoid mycorrhizal fungi, little is known about the abiotic and biotic factors driving their diversity and community composition. To determine the relative importance of abiotic and biotic filtering in structuring ericoid mycorrhizal fungal communities, we established 156 sampling plots in two highly contrasting environments but dominated by the same Ericaceae plant species: waterlogged bogs and dry heathlands. Plots were located across 25 bogs and 27 dry heathlands in seven European countries covering a gradient in nitrogen deposition and phosphorus availability. Putatively ericoid mycorrhizal fungal communities in the roots of 10 different Ericaceae species were characterized using high-throughput amplicon sequencing. Variation in ericoid mycorrhizal fungal communities was attributed to both habitat and soil variables on the one hand and host plant identity on the other. Communities differed significantly between bogs and heathlands and, in a given habitat, communities differed significantly among host plant species. Fungal richness was negatively related to nitrogen deposition in bogs and phosphorus availability in bogs and heathlands. Our results demonstrate that both abiotic and biotic filtering shapes ericoid mycorrhizal fungal communities and advocate an environmental policy minimizing excess nutrient input in these nutrient-poor ecosystems to avoid loss of ericoid mycorrhizal fungal taxa.

Impact of mating system on range size and niche breadth in Epipactis (Orchidaceae).

BACKGROUND AND AIMS: The geographical distribution of plant species is linked fundamentally not only to environmental variables, but also to key traits that affect the dispersal, establishment and evolutionary potential of a species. One of the key plant traits that can be expected to affect standing genetic variation, speed of adaptation and the capacity to colonize and establish in new habitats, and therefore niche breadth and range size, is the plant mating system. However, the precise role of the mating system in shaping range size and niche breadth of plant species remains unclear, and different studies have provided contrasting results. In this study, we tested the hypothesis that range size and niche breadth differed with mating system in the orchid genus Epipactis. METHODS: We modelled the ecological niches of 14 Epipactis species in Europe using occurrence records and environmental satellite data in Maxent. Niche breadth and niche overlap in both geographic and environmental space were calculated from the resulting habitat suitability maps using ENMTools, and geographic range was estimated using α-hull range definition. Habitat suitability, environmental variable contributions and niche metrics were compared among species with different mating systems. KEY RESULTS: We did not detect significant differences in niche breadth, occurrence probability or geographical range between autogamous and allogamous Epipactis species, although autogamous species demonstrated notably low variation in niche parameters. We also found no significant differences in niche overlap between species with the same mating system or different mating systems. For all Epipactis species, occurrence was strongly associated with land cover, particularly broad-leafed and coniferous forests, and with limestone bedrock. CONCLUSIONS: These results suggest that the mating system does not necessarily contribute to niche breadth and differentiation, and that other factors (e.g. mycorrhizal specificity) may be more important drivers of range size and niche breadth in Epipactis and orchids in general.

Lack of strong selection pressures maintains wide variation in floral traits in a food-deceptive orchid.

BACKGROUND AND AIMS: Angiosperms vary remarkably in traits such as colour, size and shape of flowers, yet such variation generally tends to be low within species. In deceptive orchids, however, large variation in floral traits has been described, not only between but also within populations. Nonetheless, the factors driving variation in floral traits in deceptive orchids remain largely unclear. METHODS: To identify determinants of variation in floral traits, we investigated patterns of fruit set and selection gradients in the food-deceptive orchid Orchis purpurea, which typically presents large within-population variation in the colour and size of the flowers. Using long-term data, fruit set was quantified in two populations over 16 consecutive years (2004-2019). Artificial hand pollination was performed to test the hypothesis that fruit set was pollinator-limited and that selfing led to decreased seed set and viability. Annual variation (2016-2019) in selection gradients was calculated for three colour traits (brightness, contrast and the number of spots on the labellum), flower size (spur length, labellum length and width) and plant size (number of flowers, plant height). KEY RESULTS: Fruit set was, on average, low (~12 %) and severely pollinator-limited. Opportunities for selection varied strongly across years, but we found only weak evidence for selection on floral traits. In contrast, there was strong and consistent positive selection on floral display. Selfing led to reduced production of viable seeds and hence severe inbreeding depression (δ = 0.38). CONCLUSION: Overall, these results demonstrate that the large variation in flower colour and size that is regularly observed in natural O. purpurea populations is maintained by the consistent lack of strong selection pressures on these traits through time.

Do fungal associates of co-occurring orchids promote seed germination of the widespread orchid species Gymnadenia conopsea?

Interactions with mycorrhizal fungi have been increasingly recognized as one of the most important ecological factors determining the distribution and local abundance of orchids. While some orchid species may interact with a variety of fungal associates, others are more specific in their choice of mycorrhizal partners. Moreover, orchids that co-occur at a given site, often associate with different partners, possibly to avoid competition and to allow stable coexistence. However, whether differences in mycorrhizal partners directly affect seed germination and subsequent protocorm formation remains largely unknown. In this research, we used in vitro germination experiments to investigate to what extent seed germination and protocorm formation of Gymnadenia conopsea was affected by the origin and identity of fungal associates. Fungi were isolated from G. conopsea and three other co-occurring orchid species (Dactylorhiza viridis (Coeloglossum viride), Herminium monorchis, and Platanthera chlorantha). In total, eight fungal associates, belonging to Tulasnellaceae, Ceratobasidiaceae, and Serendipitaceae, were successfully isolated and cultured. While all eight fungal strains were able to promote early germination of G. conopsea seeds, only fungal strain GS2, a member of the Ceratobasidiaceae isolated from G. conopsea itself, was able to promote protocorm formation and subsequent growth to a seedling. Two other fungal strains isolated from G. conopsea only supported seed germination until the protocorm formation stage. The other five fungal strains isolated from the co-occurring orchid species did not support seed germination beyond the protocorm stage. We conclude that, although G. conopsea is considered a mycorrhizal generalist that associates with a wide range of fungi during its adult life, it requires specific fungi to promote protocorm formation and growth to a seedling.