Orchid-mycorrhizal fungi interactions reveal a duality in their network structure in two European regions differing in climate.

Network analysis is an effective tool to describe and quantify the ecological interactions between plants and root-associated fungi. Mycoheterotrophic plants, such as orchids, critically rely on mycorrhizal fungi for nutrients to survive, so investigating the structure of those intimate interactions brings new insights into the plant community assembly and coexistence. So far, there is little consensus on the structure of those interactions, described either as nested (generalist interactions), modular (highly specific interactions) or of both topologies. Biotic factors (e.g., mycorrhizal specificity) were shown to influence the network structure, while there is less evidence of abiotic factor effects. By using next-generation sequencing of the orchid mycorrhizal fungal (OMF) community associated to with plant individuals belonging to 17 orchid species, we assessed the structure of four orchid-OMF networks in two European regions under contrasting climatic conditions (Mediterranean vs. Continental). Each network contained four to 12 co-occurring orchid species, including six species shared among the regions. All four networks were both nested and modular, and fungal communities were different between co-occurring orchid species, despite multiple sharing of fungi across some orchids. Co-occurring orchid species growing in Mediterranean climate were associated with more dissimilar fungal communities, consistent with a more modular network structure compared to the Continental ones. OMF diversity was comparable among orchid species since most orchids were associated with multiple rarer fungi and with only a few highly dominant ones in the roots. Our results provide useful highlights into potential factors involved in structuring plant-mycorrhizal fungus interactions in different climatic conditions.

Variability in Nutrient Use by Orchid Mycorrhizal Fungi in Two Medium Types.

Orchid mycorrhizal fungi (OMF) from the rhizoctonia aggregate are generally considered to be soil saprotrophs, but their ability to utilize various nutrient sources has been studied in a limited number of isolates cultivated predominantly in liquid media, although rhizoctonia typically grow on the surface of solid substrates. Nine isolates representing the key OMF families (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae), sampled in Southern France and the Czech Republic, were tested for their ability to utilize carbon (C), nitrogen (N) and phosphorus (P) sources in vitro in both liquid and solid media. The isolates showed significant inter- and intra-familiar variability in nutrient utilization, most notably in N sources. Isolates produced generally larger amounts of dry biomass on solid medium than in liquid one, but some isolates showed no or limited biomass production on solid medium with particular nutrient sources. The largest amount of biomass was produced by isolates from the family Ceratobasidiaceae on most sources in both medium types. The biomass production of Tulasnellaceae isolates was affected by their phylogenetic relatedness on all sources and medium types. The ability of isolates to utilize particular nutrients in a liquid medium but not a solid one should be considered when optimizing solid media for symbiotic orchid seed germination and in understanding of OMF functional traits under in situ conditions.

Host age and surrounding vegetation affect the community and colonization rates of arbuscular mycorrhizal fungi in a temperate grassland.

Arbuscular mycorrhizal fungi (AMF) are important symbionts for the majority of terrestrial vascular plants, yet the drivers of the compositional variation in AMF communities need to be better understood. What effects does the ontogenetic stage of host plants have and do these effects differ between plant functional groups? Are the AMF communities modified by the properties of surrounding vegetation, such as the proportion of different functional groups or nonmycorrhizal plants ? We addressed these questions in a temperate grassland and studied AMF communities using next-generation sequencing and light microscopy, evaluating their composition, taxonomic, phylogenetic and functional diversity, functional traits and root colonization levels. We found important differences between AMF communities and their diversity between seedlings and adults which are larger than the differences among host species or between functional groups. The proportion of nonmycorrhizal plants in the surrounding affected AMF community composition and increased its richness. Our results highlight the need for further investigating the existence of a common mycelial networks. The decision to use seedlings for experimental work can affect the results more than the chosen host species.

Arbuscular mycorrhizal fungal communities of forbs and C3 grasses respond differently to cultivation and elevated nutrients.

Arbuscular mycorrhizal fungi (AMF) represent important players in the structure and function of many ecosystems. Yet, we learn about their roles mostly from greenhouse-based experiments, with results subjected to cultivation bias. This study explores multiple aspects of this bias and separates the effect of increased nutrient availability from other cultivation specifics. For 15 grassland plant species from two functional groups (C3 grasses vs dicotyledonous forbs), we compared AMF communities of adults collected from non-manipulated vegetation with those in plants grown in a greenhouse. Nutrient availability was comparable to field conditions or experimentally elevated. We evaluated changes in AMF community composition, diversity, root colonisation, and the averages of functional traits characterising hyphal soil exploration. Additionally, we use the data from the greenhouse experiment to propose a new plant functional trait-the change of AMF colonisation in response to nutrient surplus. The AMF community differed profoundly between field-collected and greenhouse-grown plants, with a larger change of its composition in grass species, and AMF community composition in grasses also responded more to fertilisation than in forbs. Taxonomic and phylogenetic diversity declined more in forbs under cultivation (particularly with elevated nutrients), because in their roots, the AMF taxa from families other than Glomeraceae largely disappeared. A decline in AMF colonisation was not caused by greenhouse cultivation itself but selectively by the elevation of nutrient availability, particularly in grass host species. We demonstrate that the extent of decrease in AMF colonisation with elevated nutrients is a useful plant functional trait explaining an observed response of the plant community to manipulation.

Altered rhizoctonia assemblages in grasslands on ex-arable land support germination of mycorrhizal generalist, not specialist orchids.

Species-rich seminatural grasslands in Central Europe have suffered a dramatic loss of biodiversity due to conversion to arable land, but vast areas are being restored. Population recovery of orchids, which depend on mycorrhizal fungi for germination, is however limited. We hypothesised that ploughing and fertilisation caused shifts in orchid mycorrhizal communities in soil and restricted orchid germination. We examined edaphic conditions in 60 restored and seminatural grasslands, and germination success in 10 restored grasslands. Using a newly designed primer, we screened the composition of rhizoctonias in soil, seedlings and roots of seven orchid species. Seminatural and restored grasslands differed significantly in nutrient amounts and rhizoctonia assemblages in soil. While Serendipitaceae prevailed in seminatural grasslands with a higher organic matter content, Ceratobasidiaceae were more frequent in phosphorus-rich restored grasslands with increased abundance on younger restored sites. Tulasnellaceae displayed no preference. Germination success in restored grasslands differed significantly between orchid species; two mycorrhizal generalist species germinated with a broad range of rhizoctonias at most restored grasslands, while germination success of specialists was low. Past agricultural practices have a long-lasting effect on soil conditions and orchid mycorrhizal communities. Altered mycorrhizal availability may be the main reason for low germination success of specialist orchid species.

Foraging speed and precision of arbuscular mycorrhizal fungi under field conditions: An experimental approach.

To better understand the ecology of arbuscular mycorrhizal (AM) symbiosis, we need to measure functional traits of individual fungal virtual taxa under field conditions. The efficiency of AM fungi in locating nutrient-rich patches in soil space is one of their central traits in this symbiotic relationship. We used plots of a long-term field experiment in grassland with manipulated functional group composition of host plant community to establish ingrowth patches with substrate free of roots and fungi and with varying nutrient availability. Comparison of the original AM fungal community before patch creation with that present 9 weeks after patch establishment enabled us to estimate relative hyphal foraging speed for 41 fungal taxa, and a comparison of the fungal community in neighbouring patches differing in nutrient availability provided estimates of hyphal foraging precision for 22 taxa. Members of two dominant fungal families, Glomeraceae and Claroideoglomeraceae, differed in their foraging speed and precision. Glomeraceae taxa responded more slowly, but with a higher focus on enriched patches. We further demonstrated the usefulness of the obtained fungal functional traits by testing the differences between grass and dicotyledonous plant hosts using a data set obtained in another experiment at the same plots. Grass species hosted AM fungal communities with higher foraging speed, but lower foraging precision than the dicotyledonous species. Our study results support the use of field experiments for measuring comparative characteristics of AM fungi, which are highly elusive (or misrepresented) under controlled conditions.

Contrasting effects of host identity, plant community, and local species pool on the composition and colonization levels of arbuscular mycorrhizal fungal community in a temperate grassland.

Arbuscular mycorrhizal fungi (AMFs) are important plant symbionts, but we know little about the effects of plant taxonomic identity or functional group on the AMF community composition. To examine the effects of the surrounding plant community, of the host, and of the AMF pool on the AMF community in plant roots, we manipulated plant community composition in a long-term field experiment. Within four types of manipulated grassland plots, seedlings of eight grassland plant species were planted for 12 wk, and AMFs in their roots were quantified. Additionally, we characterized the AMF community of individual plots (as their AMF pool) and quantified plot abiotic conditions. The largest determinant of AMF community composition was the pool of available AMFs, varying at metre scale due to changing soil conditions. The second strongest predictor was the host functional group. The differences between grasses and dicotyledonous forbs in AMF community variation and diversity were much larger than the differences among species within those groups. High cover of forbs in the surrounding plant community had a strong positive effect on AMF colonization intensity in grass hosts. Using a manipulative field experiment enabled us to demonstrate direct causal effects of plant host and surrounding vegetation.

Identification of drivers of landscape distribution of forest orchids using germination experiment and species distribution models.

The family of orchids involves a number of critically endangered species. Understanding of drivers of their landscape distribution could provide a valuable insight into their decline. Our objectives were to develop models predicting distribution of selected orchid species-four co-occurring forest orchid species, Cephalanthera rubra, Epipactis atrorubens, E. helleborine, and Neottia nidus-avis-at a landscape scale using a wide range of habitat characteristics. Subsequently, we compared the model predictions with species occurrence and the results of the field germination experiment while considering two germination stages-asymbiotic (early stage) and symbiotic. And finally, we attempted to identify possible drivers of species' landscape distribution (i.e., dispersal, availability of habitat patches, or fungal associates). We have discovered that different habitat characteristics determined the distribution of different orchids. The species also differed in terms of availability of suitable habitat patches and patch occupancy (the highest being E. atrorubens with 80%). Landscape distribution of the species was primarily restricted by the availability of fungal associates (the most important factor for C. rubra) and by the availability of suitable habitat patches (the most important in case of N. nidus-avis). Despite expected easy dispersal of spores, orchid distribution seems to be limited by the availability of fungal associates in the landscape. In contrast, the availability of orchid seeds does not seem to limit their distribution. These results can provide useful guidelines for conservation of the studied species.

Fungal root symbionts of high-altitude vascular plants in the Himalayas.

Arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) form symbiotic relationships with plants influencing their productivity, diversity and ecosystem functions. Only a few studies on these fungi, however, have been conducted in extreme elevations and none over 5500 m a.s.l., although vascular plants occur up to 6150 m a.s.l. in the Himalayas. We quantified AMF and DSE in roots of 62 plant species from contrasting habitats along an elevational gradient (3400-6150 m) in the Himalayas using a combination of optical microscopy and next generation sequencing. We linked AMF and DSE communities with host plant evolutionary history, ecological preferences (elevation and habitat type) and functional traits. We detected AMF in elevations up to 5800 m, indicating it is more constrained by extreme conditions than the host plants, which ascend up to 6150 m. In contrast, DSE were found across the entire gradient up to 6150 m. AMF diversity was unimodally related to elevation and positively related to the intensity of AMF colonization. Mid-elevation steppe and alpine plants hosted more diverse AMF communities than plants from deserts and the subnival zone. Our results bring novel insights to the abiotic and biotic filters structuring AMF and DSE communities in the Himalayas.

The Root-Associated Microbial Community of the World's Highest Growing Vascular Plants.

Upward migration of plants to barren subnival areas is occurring worldwide due to raising ambient temperatures and glacial recession. In summer 2012, the presence of six vascular plants, growing in a single patch, was recorded at an unprecedented elevation of 6150 m.a.s.l. close to the summit of Mount Shukule II in the Western Himalayas (Ladakh, India). Whilst showing multiple signs of stress, all plants have managed to establish stable growth and persist for several years. To learn about the role of microbes in the process of plant upward migration, we analysed the root-associated microbial community of the plants (three individuals from each) using microscopy and tagged amplicon sequencing. No mycorrhizae were found on the roots, implying they are of little importance to the establishment and early growth of the plants. However, all roots were associated with a complex bacterial community, with richness and diversity estimates similar or even higher than the surrounding bare soil. Both soil and root-associated communities were dominated by members of the orders Sphingomonadales and Sphingobacteriales, which are typical for hot desert soils, but were different from communities of temperate subnival soils and typical rhizosphere communities. Despite taxonomic similarity on the order level, the plants harboured a unique set of highly dominant operational taxonomic units which were not found in the bare soil. These bacteria have been likely transported with the dispersing seeds and became part of the root-associated community following germination. The results indicate that developing soils act not only as a source of inoculation to plant roots but also possibly as a sink for plant-associated bacteria.

Life History Traits Reflect Changes in Mediterranean Butterfly Communities Due to Forest Encroachment.

The biodiversity of the Southern Balkans, part of the Mediterranean global biodiversity hot-spot, is threatened by land use intensification and abandonment, the latter causing forest encroachment of formerly open habitats. We investigated the impact of forest encroachment on butterfly species richness, community species composition and the representation of life history traits by repeated seasonal visits of 150 one-hectare sites in five separate regions in three countries-Greece, Bulgaria, and the Republic of Macedonia (FYROM-the Former Yugoslav Republic of Macedonia)- 10 replicates for each habitat type of grasslands, open formations and scrub forest within each region. Grasslands and open formations sites hosted in average more species and more red-listed species than scrub forest, while no pattern was found for numbers of Mediterranean species. As shown by ordination analyses, each of the three habitat types hosted distinct butterfly communities, with Mediterranean species inclining either towards grasslands or open formations. Analysing the representation of life history traits revealed that successional development from grasslands and open formations towards scrub forest shifts the community composition towards species overwintering in earlier stages, having fewer generations per year, and inhabiting large European or Eurosiberian (e.g. northern) ranges; it decreases the representation of Mediterranean endemics. The loss of grasslands and semi-open formations due to forest encroachment thus threatens exactly the species that should be the focus of conservation attention in the Mediterranean region, and innovative conservation actions to prevent ongoing forest encroachment are badly needed.

Two widespread green Neottia species (Orchidaceae) show mycorrhizal preference for Sebacinales in various habitats and ontogenetic stages.

Plant dependence on fungal carbon (mycoheterotrophy) evolved repeatedly. In orchids, it is connected with a mycorrhizal shift from rhizoctonia to ectomycorrhizal fungi and a high natural (13)C and (15)N abundance. Some green relatives of mycoheterotrophic species show identical trends, but most of these remain unstudied, blurring our understanding of evolution to mycoheterotrophy. We analysed mycorrhizal associations and (13)C and (15)N biomass content in two green species, Neottia ovata and N. cordata (tribe Neottieae), from a genus comprising green and nongreen (mycoheterotrophic) species. Our study covered 41 European sites, including different meadow and forest habitats and orchid developmental stages. Fungal ITS barcoding and electron microscopy showed that both Neottia species associated mainly with nonectomycorrhizal Sebacinales Clade B, a group of rhizoctonia symbionts of green orchids, regardless of the habitat or growth stage. Few additional rhizoctonias from Ceratobasidiaceae and Tulasnellaceae, and ectomycorrhizal fungi were detected. Isotope abundances did not detect carbon gain from the ectomycorrhizal fungi, suggesting a usual nutrition of rhizoctonia-associated green orchids. Considering associations of related partially or fully mycoheterotrophic species such as Neottia camtschatea or N. nidus-avis with ectomycorrhizal Sebacinales Clade A, we propose that the genus Neottia displays a mycorrhizal preference for Sebacinales and that the association with nonectomycorrhizal Sebacinales Clade B is likely ancestral. Such a change in preference for mycorrhizal associates differing in ecology within the same fungal taxon is rare among orchids. Moreover, the existence of rhizoctonia-associated Neottia spp. challenges the shift to ectomycorrhizal fungi as an ancestral pre-adaptation to mycoheterotrophy in the whole Neottieae.