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BackgroundMycoheterotrophic plants are considered to associate very specifically with fungi. Mycoheterotrophic orchids are mostly associated with ectomycorrhizal fungi in temperate regions, or with saprobes or parasites in tropical regions. Although most mycoheterotrophic orchids occur in the tropics, few studies have been devoted to them, and the main conclusions about their specificity have hitherto been drawn from their association with ectomycorrhizal fungi in temperate regions.ResultsWe investigated three Asiatic Neottieae species from ectomycorrhizal forests in Thailand. We found that all were associated with ectomycorrhizal fungi, such as Thelephoraceae, Russulaceae and Sebacinales. Based on 13C enrichment of their biomass, they probably received their organic carbon from these fungi, as do mycoheterotrophic Neottieae from temperate regions. Moreover, 13C enrichment suggested that some nearby green orchids received part of their carbon from fungi too. Nevertheless, two of the three orchids presented a unique feature for mycoheterotrophic plants: they were not specifically associated with a narrow clade of fungi. Some orchid individuals were even associated with up to nine different fungi.ConclusionOur results demonstrate that some green and mycoheterotrophic orchids in tropical regions can receive carbon from ectomycorrhizal fungi, and thus from trees. Our results reveal the absence of specificity in two mycoheterotrophic orchid-fungus associations in tropical regions, in contrast to most previous studies of mycoheterotrophic plants, which have been mainly focused on temperate orchids.

Abstract. Some forest plants adapt to shade by mixotrophy, i.e., they obtain carbon both from photosynthesis and from their root mycorrhizal fungi. Fully achlorophyllous species using exclusively fungal carbon (the so-called mycoheterotrophic plants) have repeatedly evolved from such mixotrophic ancestors. However, adaptations for this evolutionary transition, and the reasons why it has happened a limited number of times, remain unknown. We investigated this using achlorophyllous variants (i.e., albinos) spontaneously occurring in Cephalanthera damasonium, a mixotrophic orchid. In two populations, we compared albinos with co-occurring green individuals in situ. We investigated vegetative traits, namely, shoot phenology, dormancy, CO 2 and H 2 O leaf exchange, mycorrhizal colonization, degree of mycoheterotrophy (using 13 C abundance as a proxy), and susceptibility to pathogens and herbivores. We monitored seed production (in natural or experimental crosses) and seed germination. Albinos displayed (1) more frequent shoot drying at fruiting, possibly due to stomatal dysfunctions, (2) lower basal metabolism, (3) increased sensitivity to pathogens and herbivores, (4) higher dormancy and maladapted sprouting, and, probably due to the previous differences, (5) fewer seeds, with lower germination capacity. Over the growing season, green shoots shifted from using fungal carbon to an increasingly efficient photosynthesis at time of fruiting, when fungal colonization reached its minimum. Conversely, the lack of photosynthesis in fruiting albinos may contribute to carbon limitation, and to the abovementioned trends. With a 10 3 3 fitness reduction, albinos failed a successful transition to mycoheterotrophy because some traits inherited from their green ancestors are maladaptive. Conversely, mycoheterotrophy requires at least degeneration of leaves and stomata, optimization of the temporal pattern of fungal colonization and shoot sprouting, and new defenses against pathogens and herbivores. Transition to mycoheterotrophy likely requires progressive, joint evolution of these traits, while a sudden loss of photosynthesis leads to unfit plants. We provide explanations for the evolutionary stability of mixotrophic nutrition and for the rarity of emergence of carbon sinks in mycorrhizal networks. More broadly, this may explain what prevents the emergence of fully heterotrophic taxa in the numerous other mixotrophic plant or algal lineages recently described.

Microscopic investigations revealed that thick rhizomes were densely colonized by fungi bearing clamp-connections and dolipores, i.e. basidiomycetes. Molecular analysis identified Inocybe species as exclusive symbionts of 75 % of the plants investigated and, more rarely, other basidiomycetes (Hebeloma, Xerocomus, Lactarius, Thelephora species). Additionally, ascomycetes, probably endophytes or parasites, were sometimes present. Although E. aphyllum associates with diverse species from Inocybe subgenera Mallocybe and Inocybe sensu stricto, no evidence for cryptic speciation in E. aphyllum was found. Since basidiomycetes colonizing the orchid are ectomycorrhizal, surrounding trees are probably the ultimate carbon source. Accordingly, in one population, ectomycorrhizae sampled around an individual orchid revealed the same fungus on 11.2 % of tree roots investigated. Conversely, long, thin stolons bearing bulbils indicated active asexual multiplication, but these propagules were not colonized by fungi. These findings are discussed in the framework of ecology and evolution of mycoheterotrophy.

Abstract• Introduction Millions of hectares of Quercus ilex forests dominate disturbed landscapes in the western part of the Mediterranean basin. Although these forests are very widespread, little is known about the composition and structure of their associated ectomycorrhizal fungal communities.• Results and discussion We examined seasonal patterns in ectomycorrhizal communities and their response to increased drought using a rainfall exclusion experiment established in a Q. ilex coppice since 2003. Ectomycorrhizae were sampled four times in [2007][2008][2009]. By sequencing fungal ITS, we identified 129 species in 1,147 sequenced ectomycorrhizal root tips. The fungal community in the surface organic horizon was well described by the logseries theoretical model, with 47.9% of singleton species. The composition of the community was strongly dominated by Basidiomycetes, with three families (Thelephoraceae, Russulaceae and Cortinariaceae) accounting for 72.9% of the root tips. Relative abundance of Russulaceae and Thelephoraceae showed pronounced seasonal shifts. Experimental reduction of rainfall resulted in significant shifts in community composition and seasonal fluctuations but had no effect on global richness of the community.• Conclusions Together, these results suggest that the predicted rainfall reduction in this region due to climate change will lead to shifts in species composition in ectomycorrhizal communities.

Summary Some forest understorey plants recover carbon (C) not only from their own photosynthesis, but also from mycorrhizal fungi colonizing their roots. How these mixotrophic plants use the resources obtained from mycorrhizal and photosynthetic sources remains unknown. We investigated C sources and allocation in mixotrophic perennial orchids from the genus Epipactis. Based on the assumption that fungal biomass has high δ13C and N content, while photosynthetic biomass has lower δ13C and N content, we indirectly estimated the respective contributions of these two resources to various organs, at various times over the growth season. Fully heterotrophic and fully autotrophic plants from the same sites were used as references for δ13C and N content of biomass purely issuing from fungi and photosynthesis, respectively. In four investigated populations, the biomass shifted from fully heterotrophic in young spring shoots to 80–100% autotrophic in leaves and fruits at fruiting time, suggesting that photosynthesis supported mostly fruiting costs. In addition, fungal colonization decreased in roots over this period. Based on δ13C and N content, below‐ground organs and young spring shoots from green (mixotrophic) individuals and spontaneous achlorophyllous variants (fully heterotrophic) displayed similar fungal C contributions. Similar fungal contributions were also found in shoots of individuals that were either sprouting (and thus partially photosynthetic) or dormant (and thus fully heterotrophic) in the previous years. Therefore, fungal C supported mostly young spring shoots and below‐ground organs. Although experimentally shaded plants had decreased contributions of photosynthetic C in shoots, experimentally defoliated plants showed no increase in fungal C contribution as compared with non‐defoliated controls. Strikingly, these defoliated plants maintained the same seed production: they likely compensated defoliation by increasing stem and fruit photosynthesis. Synthesis. We propose a falsifiable model of C resource allocation in mixotrophic orchids, where mycorrhizal fungi mostly support below‐ground organs and survival, while photosynthesis mostly supports above‐ground sexual reproduction, but not below‐ground reserves. We discuss how this allocation pattern, where seed production depends on photosynthesis, complicates the evolutionary route to full heterotrophy in mixotrophic orchids.

Infrageneric relations of the genetically diverse milkcap genus Lactifluus (Russulales, Basidiomycota) are poorly known. Currently used classification systems still largely reflect the traditional, mainly morphological, characters used for infrageneric delimitations of milkcaps. Increased sampling, combined with small-scale molecular studies, show that this genus is underexplored and in need of revision. For this study, we assembled an extensive dataset of the genus Lactifluus, comprising 80 % of all known species and 30 % of the type collections. To unravel the infrageneric relationships within this genus, we combined a multi-gene molecular phylogeny, based on nuclear ITS, LSU, RPB2 and RPB1, with a morphological study, focussing on five important characteristics (fruit body type, presence of a secondary velum, colour reaction of the latex/context, pileipellis type and presence of true cystidia). Lactifluus comprises four supported subgenera, each containing several supported clades. With extensive sampling, ten new clades and at least 17 new species were discovered, which highlight the high diversity in this genus. The traditional infrageneric classification is only partly maintained and nomenclatural changes are proposed. Our morphological study shows that the five featured characteristics are important at different evolutionary levels, but further characteristics need to be studied to find morphological support for each clade. This study paves the way for a more detailed investigation of biogeographical history and character evolution within Lactifluus.