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Interaction between plants and their microbiota is a central theme to understand adaptation of plants to their environment. Considering herbaria as repositories of holobionts that preserved traces of ancient plant-associated microbial communities, we propose to explore these historical collections to evaluate the impact of long-lasting global changes on plant-microbiota interactions.

Background and AimsEpiphytism has evolved repeatedly in plants and has resulted in a considerable number of species with original characteristics. Succulent forms in particular are thought to have evolved as an adaptation to the epiphytic environment, because the water supply is generally erratic compared to soils’. However, succulent organs also exist in terrestrial plants, and the question of the concomitant evolution of epiphytism and succulence has received little attention, not even in the epidendroid orchids, which account for 68% of vascular epiphytes.MethodsWe reconstructed a new time-calibrated phylogenetic tree of Epidendroideae with 203 genera treated in Genera Orchidacearum, from which we reconstructed the evolution of epiphytism and other traits including stem and leaf succulence, while testing the correlated evolution between lifestyle and morphological traits. Furthermore, we reconstructed the ancestral geographic ranges to interpret major character changes during the Cenozoic.Key ResultsEpiphytism evolved at least 7.1 My ago in the neotropical Sobralieae, 11.5 My ago in the Arethuseae in Southeast Asia and Australia, and 39.0 My ago in the common ancestor of the Dendrobieae and Cymbidieae in the three previous areas, and was notably lost in the Malaxideae, Collabieae, Calypsoeae, Bletiinae, and Eulophiinae. Stem succulence is inferred to have evolved once, in a terrestrial ancestor 43.1 My ago, thus preceding the evolution of epiphytism by at least 4.1 My. If lost, stem succulence was almost systematically replaced by leaf succulence in epiphytic lineages.ConclusionsEpiphytism probably evolved from terrestrial orchids already possessing succulent stems, which appeared during Eocene climatic cooling. Both epiphytic and secondary terrestrial Epidendroideae may have appeared in seasonally-dry forests. Thus, we believe that the emergence of stem succulence in early epidendroids was a key innovation in the evolution of orchids, facilitating the colonisation of epiphytic environments that led to the greatest diversification of orchids.

Whether interactions between species are conserved on evolutionary time-scales has spurred the development of both correlative and process-based approaches for testing phylogenetic signal in interspecific interactions: do closely related species interact with similar partners? Here we use simulations to test the statistical performances of the two approaches that are the most widely used in the field: Mantel tests and the Phylogenetic Bipartite Linear Model (PBLM). Mantel tests investigate the correlation between phylogenetic distances and dissimilarities in sets of interacting partners, while PBLM is a process-based approach that relies on strong assumptions about how interactions evolve. We find that PBLM often detects a phylogenetic signal when it should not. Simple Mantel tests instead have infrequent false positives and moderate statistical power; however, they often artifactually detect that closely related species interact with dissimilar partners. Partial Mantel tests, which are used to partial out the phylogenetic signal in the number of partners, actually fail at correcting for this confounding effect, and we instead recommend evaluating the significance of Mantel tests with network permutations constraining the number of partners. We also explore the ability of simple Mantel tests to analyze clade-specific phylogenetic signals. We provide general guidelines and an application on an interaction network between orchids and mycorrhizal fungi.

International audience; Approximately 10% of vascular plants are epiphytes and, even though this has long been ignored in past research, are able to interact with a variety of fungi, including mycorrhizal taxa. However, the structure of fungal communities on bark, as well as their relationship with epiphytic plants, is largely unknown.To fill this gap, we conducted environmental metabarcoding of the ITS-2 region to understand the spatial structure of fungal communities of the bark of tropical trees, with a focus on epiphytic orchid mycorrhizal fungi, and tested the influence of root proximity.For all guilds, including orchid mycorrhizal fungi, fungal communities were more similar when spatially close on bark (i.e. they displayed positive spatial autocorrelation). They also showed distance decay of similarity with respect to epiphytic roots, meaning that their composition on bark increasingly differed, compared to roots, with distance from roots.We first showed that all of the investigated fungal guilds exhibited spatial structure at very small scales. This spatial structure was influenced by the roots of epiphytic plants, suggesting the existence of an epiphytic rhizosphere. Finally, we showed that orchid mycorrhizal fungi were aggregated around them, possibly as a result of reciprocal influence between the mycorrhizal partners.

Analysing diversification dynamics is key to understanding the past evolutionary history of clades that led to present-day biodiversity patterns. While such analyses are widespread in well-characterized groups of species, they are much more challenging in groups for which diversity is mostly known through molecular techniques. Here, we use the largest global database on the small subunit (SSU) rRNA gene of Glomeromycotina, a subphylum of microscopic arbuscular mycorrhizal fungi that provide mineral nutrients to most land plants by forming one of the oldest terrestrial symbioses, to analyse the diversification dynamics of this clade in the past 500 million years. We perform a range of sensitivity analyses and simulations to control for potential biases linked to the nature of the data. We find that Glomeromycotina tend to have low speciation rates compared to other eukaryotes. After a peak of speciations between 200 and 100 million years ago, they experienced an important decline in speciation rates toward the present. Such a decline could be at least partially related to a shrinking of their mycorrhizal niches and to their limited ability to colonize new niches. Our analyses identify patterns of diversification in a group of obligate symbionts of major ecological and evolutionary importance and illustrate that short molecular markers combined with intensive sensitivity analyses can be useful for studying diversification dynamics in microbial groups.

We present a taxonomic revision of the genus Jumellea (Angraecinae; Orchidaceae) in the Mascarenes based on morphological study and field observations. We examined 328 specimens mostly from the MAU, P and REU, and recognise nine species. We provide a key, morphological descriptions, distributions, habitats, phenologies and conservation assessments following the Red List categories and criteria (IUCN). All nine species are present on Réunion with two being endemic there, and five have been confirmed on Mauritius, none of them endemic. The single species recorded for Rodrigues is also the only one found on all three islands of the Mascarenes and Madagascar. Five species are endemic to the Mascarene Archipelago, whereas the other four also occur in Madagascar. All nine species qualify as threatened with extinction on at least one of the islands of the Mascarenes. Two species are probably extinct on one island, and only two species on Réunion are not threatened.

The peer review history for this article is available at https://publons.com/publon/10.1111/1365-2745.13986.Raw data have been deposited in the Sequence Read Archive (SRA) under the BioProject accession no. PRJNA692353.; International audience; In soils, plants and fungi can form complex mycorrhizal networks allowing nutrient transfers between plant individuals and species. It is less clear, however, whether such networks exist on the bark of trees where epiphytic plant communities thrive in rainforests. Previous work showed that tropical epiphytic orchids especially, harbour symbiotic fungi in their roots, but the structure and determinants of the resulting networks remain unknown at the tree scale.We tested the hypothesis that epiphytic orchids rooted in the same area on the bark share mycorrhizal fungi, regardless of their species (i.e. spatial determinant). For this purpose, we selected the trunk of six trees of two common species in a rainforest and sampled orchid roots, protocorms and surrounding bark. We identified mycorrhizal fungi including Tulasnellaceae using high-throughput sequencing of the ITS2 marker, and reconstructed orchid-fungus bipartite networks for each tree to analyse their structure and the spatial turnover of this symbiosis.We found that epiphytic orchid communities form antinested and highly modular networks with mycorrhizal fungi spread on the bark. As expected, modules of interactions are explained by their spatial structure, with nearby roots sharing fungi, but also by the orchid species involved. These results reveal the presence of shared mycelial networks in epiphytic habitats, whose roles in the resilience and facilitation of epiphytic plant communities need to be assessed.Synthesis. Tropical tree barks are densely colonized by certain mycorrhizal fungi that can form symbioses in nearby adult and young orchids simultaneously. These mycorrhizal networks may allow water and nutrient transfers to alleviate the stressful conditions of the epiphytic habitats.

International audience; Angraecoid orchids present a remarkable diversity of chromosome numbers, which makes them a highly suitable system for exploring the impact of karyotypic changes on cladogenesis, diversification and morphological differentiation. We compiled an annotated cytotaxonomic checklist for 126 species of Angraecinae, which was utilised to reconstruct chromosomal evolution using a newly-produced, near-comprehensive phylogenetic tree that includes 245 angraecoid taxa. In tandem with this improved phylogenetic framework, using combined Bayesian, maximum likelihood and parsimony approaches on ITS-1 and five plastid markers, we propose a new cladistic nomenclature for the angraecoids, and we estimate a new timeframe for angraecoid radiation based on a secondary calibration, and calculate diversification rates using a Bayesian approach. Coincident divergence dates between clades with identical geographical distributions in the angraecoids and the pantropical orchid genus Bulbophyllum suggest that the same events may have intervened in the dispersal of these two epiphytic groups between Asia, continental Africa, Madagascar and the Neotropics. The major angraecoid lineages probably began to differentiate in the Middle Miocene, and most genera and species emerged respectively around the Late Miocene-Pliocene boundary and the Pleistocene. Ancestral state reconstruction using maximum likelihood estimation revealed an eventful karyotypic history dominated by descending dysploidy. Karyotypic shifts seem to have paralleled cladogenesis in continental tropical Africa, where approximately 90% of the species have descended from at least one inferred transition from n = 17–18 to n = 25 during the Middle Miocene Climatic Transition, followed by some clade-specific descending and ascending dysploidy from the Late Miocene to the Pleistocene. Conversely, detected polyploidy is restricted to a few species lineages mostly originating during the Pleistocene. No increases in net diversification could be related to chromosome number changes, and the apparent net diversification was found to be highest in Madagascar, where karyotypic stasis predominates. Finally, shifts in chromosome number appear to have paralleled the evolution of rostellum structure, leaflessness, and conspicuous changes in floral colour.

International audience; Phylogenetic relationships within the Orchideae sensu Pridgeon et al, remain one of the biggest unresolved issues in our understanding of the taxonomy of the orchids. Members of the Orchideae are numerous and widespread in Africa but remain poorly represented in phylogenetic research. In this study we included a broad sampling of African taxa for which we sequenced three plastid (rbcl, matK and trnL + trnL-F) and two nuclear regions (ITS and 18S). We used 368 sequences representing 278 species and 49 genera to infer relationships using the Bayesian Inference and Maximum Likelihood method. Our results show strong support for three clades, two of which almost entirely match the historical circumscription of Orchidinae and Habenariinae, and the third, Bartholininae, sister to the former two, includes the genera Holothrix and Bartholina. Stenoglottis should be assigned to Orchidinae and not to Habenariinae. Several genera such as Habenaria, Cynorkis and Benthamia are shown to be para- or polyphyletic: Bonatea, Centrostigma, Platycoryne and Roeperocharis are all embedded in Habenaria; Physoceras, Arnottia and part of Benthamia are embedded in Cynorkis. We propose a subdivision of Orchideae sensu lato into nine subtribes, but refrain from making generic re-arrangements until more extensive or more in-depth studies have been done.

Little is known about the soil factors influencing root-associated fungal communities in Orchidaceae. Limited evidence suggests that soil nutrients may modulate the association with orchid mycorrhizal fungi (OMF), but their influence on non-mycorrhizal fungi remains unexplored. To study how nutrient availability affects mycorrhizal and non-mycorrhizal fungi associated with the orchid Bipinnula fimbriata, we conducted a metagenomic investigation within a large population with variable soil conditions. Additionally, we tested the effect of phosphorus (P) addition on fungal communities and mycorrhizal colonization. Soil P negatively correlated with the abundance of OMF, but not with the abundance of non-mycorrhizal fungi. After fertilization, increments in soil P negatively affected mycorrhizal colonization; however, they had no effect on OMF richness or composition. The abundance and richness of pathotrophs were negatively related to mycorrhizal colonization and then, after fertilization, the decrease in mycorrhizal colonization correlated with an increase in pathogen richness. Our results suggest that OMF are affected by soil conditions differently from non-mycorrhizal fungi. Bipinnula fimbriata responds to fertilization by altering mycorrhizal colonization rather than by switching OMF partners in the short term, and the influence of nutrients on OMF is coupled with indirect effects on the whole fungal community and potentially on plant's health.

The mistletoe Bakerella hoyifolia subsp. bojeri (Baker) Balle was presumed extinct from Reunion Island, where it is native, for over 50 years. We recently found B. hoyifolia plants in a well-preserved forest stand in the southeastern part of the island. The small population consists of six individuals, distributed in two groups, ca. 50 m apart, growing on five host plant species from different families, suggesting a low host specificity. Each group is structured vertically with a large mistletoe established on an emerging tree while smaller mistletoes are seen on the trees below. Moreover, we observed the Reunion grey white-eye, Zosterops borbonicus (J. R. Forster, 1781) visiting flowers of these mistletoes found on Reunion. Further investigations are required in order to clarify the genetic isolation and taxonomic position of this population relative to those in Mauritius and Madagascar, and the mechanisms that have caused its decline on Reunion. Two possible mechanisms are discussed. The loss of most frugivorous vertebrates on Reunion Island soon after human settlement, may have driven this mistletoe to extinction. Among rapid conservation actions, propagation from seeds should be carried out in order to prevent the extinction of this native plant population.

International audience; Although mutualistic interactions are widespread and essential in ecosystem functioning, the emergence of uncooperative cheaters threatens their stability, unless there are some physiological or ecological mechanisms limiting interactions with cheaters.In this framework, we investigated the patterns of specialization and phylogenetic distribution of mycoheterotrophic cheaters vs noncheating autotrophic plants and their respective fungi, in a global arbuscular mycorrhizal network with> 25 000 interactions.We show that mycoheterotrophy evolved repeatedly among vascular plants, suggesting low phylogenetic constraints for plants. However, mycoheterotrophic plants are significantly more specialized than autotrophic plants, and they tend to be associated with specialized and closely related fungi. These results raise new hypotheses about the mechanisms (e.g. sanctions, or habitat filtering) that actually limit the interaction of mycoheterotrophic plants and their associated fungi with the rest of the autotrophic plants.Beyond mycorrhizal symbiosis, this unprecedented comparison of mycoheterotrophic vs autotrophic plants provides a network and phylogenetic framework to assess the presence of constraints upon cheating emergences in mutualisms.

International audience; Following the global trend of deforestation and degradation, tropical dry forests in the Mascarenes archipelago on Reunion has undergone harsh reduction and fragmentation within 3 centuries of human occupation. We investigated the genetic diversity, mating system, and gene flow in fragmented populations of the native tree Foetidia mauritiana (Lecythidaceae) on Reunion, using microsatellite genotyping of adults (in- and ex situ) and seed progenies (in situ only). To test genetic isolation between the Mascarene islands, we also genotyped conspecific adults on Mauritius, and trees of Foetidia rodriguesiana on Rodrigues. We found a high genetic diversity among the trees on Reunion, but no population structure (⁠G′ST⁠: 0.039–0.090), and an increase of the fixation index (FIS) from adults to progenies. A subsequent analysis of mating systems from progeny arrays revealed selfing rates >50% in fragmented populations and close to 100% in lone trees. A paternity analysis revealed pollen flow ranging from 15.6 to 296.1 m within fragments. At broader scale, the populations of F. mauritiana on Reunion and Mauritius are genetically differentiated. The morphologically allied taxa F. rodriguesiana and F. mauritiana are clearly isolated. Therefore, this case study shows that genetic diversity may persist after deforestation, especially in long-lived tree species, but the reproductive features may be deeply altered during this process. This would explain the low seed production and the absence of recruitment in F. mauritiana. Restoration programs should take into account these features, as well as the importance that trees ex situ represent in restoring and conserving diversity.

While mutualisms are widespread and essential in ecosystem functioning, the emergence of uncooperative cheaters threatens their stability, unless there are functional or evolutionary mechanisms limiting cheaters interactions.Here, we evaluated the constraints upon mycoheterotrophic (MH) cheating plants in the mutualistic interaction network of autotrophic (AT) plants and arbuscular mycorrhizal fungi. For this purpose, we assembled a world-scale network of >25,000 interactions in order to investigate(i)the specialization and(ii)the phylogenetic distribution of MH or AT plants and their respective fungal partners.We show that MH cheating repeatedly evolved in the vascular flora, suggesting low phylogenetic constraint for plants. However, MH cheaters are significantly more specialized than AT plants, and their fungi also appear more specialized and more closely related than fungi of AT plants, which suggest that cheaters are specifically isolated into modules by functional constraintsThis unprecedented comparison of MH vs. AT plants thus reveals that MH cheating is most likely constrained by the specialization of phylogenetically conserved cheating-susceptible fungi, which suggests mechanisms for avoidance of these fungi. Beyond the mycorrhizal symbiosis, our approach highlights an empirical multiple-partners mutualistic system illustrating that the overall persistence of mutualism can be linked to functional constraints upon cheating emergences.

International audience; Despite significant progress made in recent years toward developing an infrafamilial classification of Orchidaceae, our understanding of relationships among and within tribal and subtribal groups of epidendroid orchids remains incomplete. To reassess generic delimitation among one group of these epidendroids, the African angraecoids, phylogenetic relationships were inferred from DNA sequence data from three regions, ITS, matK, and the trnL-trnF intergenic spacer, obtained from a broadly representative sample of taxa. Parsimony and Bayesian analyses yielded highly resolved trees that are in clear agreement and show significant support for many key clades within subtribe Angraecinae s.l. Angraecoid orchids comprise two well-supported clades: an African/American group and an Indian Ocean group. Molecular results also support many previously proposed relationships among genera, but also reveal some unexpected relationships. The genera Aerangis, Ancistrorhynchus, Bolusiella, Campylocentrum, Cyrtorchis, Dendrophylax, Eurychone, Microcoelia, Nephrangis, Podangis and Solenangis are all shown to be monophyletic, but Angraecopsis, Diaphananthe and Margelliantha are polyphyletic. Diaphananthe forms three well-supported clades, one of which might represent a new genus, and Rhipidoglossum is paraphyletic with respect to Cribbia and Rhaesteria, and also includes taxa currently assigned to Margelliantha. Tridactyle too is paraphyletic as Eggelingia is embedded within it. The large genus Angraecum is confirmed to be polyphyletic and several groups will have to be recognized as separate genera, including sections Dolabrifolia and Hadrangis. The recently segregated genus Pectinariella (previously recognized as A. sect. Pectinaria) is polyphyletic and its Continental African species will have to be removed. Similarly, some of the species recently transferred to Angraecoides that were previously placed in Angraecum sects. Afrangraecum and Conchoglossum will have to be moved and described as a new genus.

In Zambia, wild edible terrestrial orchids are used to produce a local delicacy called chikanda, which has become increasingly popular throughout the country. Commercialization puts orchid populations in Zambia and neighbouring countries at risk of overharvesting. Hitherto, no study has documented which orchid species are traded on local markets, as orchid tubers are difficult to identify morphologically. In this study, the core land-plant DNA barcoding markers rbcL and matK were used in combination with nrITS to determine which species were sold in Zambian markets. Eighty-two interviews were conducted to determine harvesting areas, as well as possible sustainability concerns. By using nrITS DNA barcoding, a total of 16 orchid species in six different genera could be identified. Both rbcL and matK proved suitable to identify the tubers up to the genus or family level. Disa robusta, Platycoryne crocea and Satyrium buchananii were identified most frequently and three previously undocumented species were encountered on the market. Few orchid species are currently listed on the global International Union for the Conservation of Nature (IUCN) Red List. Local orchid populations and endemic species could be at risk of overharvesting due to the intensive and indiscriminate harvesting of chikanda orchids, and we therefore encourage increased conservation assessment of terrestrial African orchids.

Source at https://doi.org/10.1007/s10531-017-1343-7 . Chikanda is a traditional dish made with wild-harvested ground orchid tubers belonging to three orchidioid genera, Disa, Satyrium and Habenaria, all of which are CITES appendix II-listed. Identification of collected orchid tubers is very difficult and documentation of constituent species in prepared chikanda has hitherto been impossible. Here amplicon metabarcoding was used in samples of six prepared chikanda cakes to study genetic sequence diversity and species diversity in this product. Molecular operational taxonomic unit identification using similarity-matching reveals that species of all three genera were present in the chikanda samples studied. Disa was present in all of the samples, Satyrium in five out of six and Habenaria in one of the samples, as well as a number of other plants. The fact that each sample contained orchids and the presence of a wide variety of species from all genera in this traditional dish raise serious concerns about the sustainability of this trade and the future of wild orchid populations in the main harvest areas. This proof-of-concept study shows that Ion-Torrent PGM is a cost-effective scalable platform for metabarcoding using the relatively long nrITS1 and nrITS2 regions. Furthermore, nrITS metabarcoding can be successfully used for the detection of specific ingredients in a highly-processed food product at genus level, and this makes it a useful tool in the detection of possible conservation issues arising from commercialized trade or processed plant products.

Fungi from the Ceratobasidiaceae family have important ecological roles as pathogens, saprotrophs, non-mycorrhizal endophytes, orchid mycorrhizal and ectomycorrhizal symbionts, but little is known about the distribution and evolution of these nutritional modes. All public ITS sequences of Ceratobasidiaceae were downloaded from databases, annotated with ecological and taxonomic metadata, and tested for the non-random phylogenetic distribution of nutritional modes. Phylogenetic analysis revealed six main clades within Ceratobasidiaceae and a poor correlation between molecular phylogeny and morphological–cytological characters traditionally used for taxonomy. Sequences derived from soil (representing putative saprotrophs) and orchid mycorrhiza clustered together, but remained distinct from pathogens. All nutritional modes were phylogenetically conserved in the Ceratobasidiaceae based on at least one index. Our analyses suggest that in general, autotrophic orchids form root symbiosis with available Ceratobasidiaceae isolates in soil. Ectomycorrhiza-forming capability has evolved twice within the Ceratobasidiaceae and it had a strong influence on the evolution of mycoheterotrophy and host specificity in certain orchid taxa.

International audience; Premise of the study: Polymorphic markers were required for a native tree of the Mascarene Islands, Foetidia mauritiana (Lecythidaceae), to investigate the effects of fragmentation of lowland tropical habitats on tree mating systems and on gene flow.Methods and Results: Using microsatellite enrichment and next‐generation sequencing, we identified 13 microsatellite loci (dinucleotide repeats). They were highly polymorphic in 121 trees sampled in the largest three populations on Réunion, revealing 2–17 different alleles per locus. Furthermore, they were found to be polymorphic in conspecific populations on Mauritius and in F. rodriguesiana from Rodrigues.Conclusions: These results indicate the utility of these markers to investigate genetic diversity, mating systems, and gene flow in a genus native to the biodiversity hotspot of Madagascar and the Indian Ocean islands.

Characterizing the architecture of bipartite networks is increasingly used as a framework to study biotic interactions within their ecological context and to assess the extent to which evolutionary constraint shape them. Orchid mycorrhizal symbioses are particularly interesting as they are viewed as more beneficial for plants than for fungi, a situation expected to result in an asymmetry of biological constraint. This study addressed the architecture and phylogenetic constraint in these associations in tropical context. We identified a bipartite network including 73 orchid species and 95 taxonomic units of mycorrhizal fungi across the natural habitats of Reunion Island. Unlike some recent evidence for nestedness in mycorrhizal symbioses, we found a highly modular architecture that largely reflected an ecological barrier between epiphytic and terrestrial subnetworks. By testing for phylogenetic signal, the overall signal was stronger for both partners in the epiphytic subnetwork. Moreover, in the subnetwork of epiphytic angraecoid orchids, the signal in orchid phylogeny was stronger than the signal in fungal phylogeny. Epiphytic associations are therefore more conservative and may co-evolve more than terrestrial ones. We suggest that such tighter phylogenetic specialization may have been driven by stressful life conditions in the epiphytic niches. In addition to paralleling recent insights into mycorrhizal networks, this study furthermore provides support for epiphytism as a major factor affecting ecological assemblage and evolutionary constraint in tropical mycorrhizal symbioses.

The roots of orchids associate with mycorrhizal fungi, the rhizoctonias, which are considered to exchange mineral nutrients against plant carbon. The recent discovery that rhizoctonias grow endophytically in non-orchid plants raises the possibility that they provide carbon to orchids, explaining why some orchids differ in isotopic abundances from autotrophic plants.

Population studies have revealed that the fungal ectomycorrhizal morphospecies Tricholoma scalpturatum consists of at least two genetically distinct groups that occur sympatrically in several geographical areas. This discovery prompted us to examine species boundaries and relationships between members formerly assigned to T. scalpturatum and allied taxa using phylogenetic analyses. Sequence data were obtained from three nuclear DNA regions [internal transcribed spacer (ITS), gpd and tef], from 101 carpophores collected over a large geographical range in Western Europe, and some reference sequences from public databases. The ITS was also tested for its applicability as DNA barcode for species delimitation. Four highly supported phylogenetic clades were detected. The two previously detected genetic groups of T. scalpturatum were assigned to the phylospecies Tricholoma argyraceum and T. scalpturatum. The two remaining clades were referred to as Tricholoma cingulatum and Tricholoma inocybeoides. Unexpectedly, T. cingulatum showed an accelerated rate of evolution that we attributed to narrow host specialization. This study also reveals recombinant ITS sequences in T. inocybeoides, suggesting a hybrid origin. The ITS was a useful tool for the determination of species boundaries: the mean value of intraspecific genetic distances in the entire ITS region (including 5.8S rDNA) was

Mycoheterotrophic plants are achlorophyllous plants that obtain carbon from their mycorrhizal fungi. They are usually considered to associate with fungi that are (1) specific of each mycoheterotrophic species and (2) mycorrhizal on surrounding green plants, which are the ultimate carbon source of the entire system. Here we review recent works revealing that some mycoheterotrophic plants are not fungal-specific, and that some mycoheterotrophic orchids associate with saprophytic fungi. A re-examination of earlier data suggests that lower specificity may be less rare than supposed in mycoheterotrophic plants. Association between mycoheterotrophic orchids and saprophytic fungi arose several times in the evolution of the two partners. We speculate that this indirectly illustrates why transition from saprotrophy to mycorrhizal status is common in fungal evolution. Moreover, some unexpected fungi occasionally encountered in plant roots should not be discounted as ‘molecular scraps’, since these facultatively biotrophic encounters may evolve into mycorrhizal symbionts in some other plants.

Many plant species attract insect pollinators through chemical mimicry of their oviposition sites, often detaining them in a trap chamber that ensures pollen transfer. These plant mimics are considered to be unspecialized at the pollinator species level, yet field observations of a mycoheterotrophic rainforest orchid (Gastrodia similis), which emits an odour reminiscent of rotting fruit, indicate that it is pollinated by a single drosophilid fly species (Scaptodrosophila bangi). We investigated the roles of floral volatiles and the dimensions of the trap chamber in enforcing this specialization, using gas chromatography-mass spectrometry analyses, bioassays and scanning electron microscopy. We showed that G. similis flowers predominantly emit three fatty-acid esters (ethyl acetate, ethyl isobutyrate and methyl isobutyrate) that were shown in experiments to attract only Scaptodrosophila flies. We additionally showed that the trap chamber, which flies enter into via a touch-sensitive 'trapdoor', closely matches the body size of the pollinator species S. bangi and plays a key role in pollen transfer. Our study demonstrates that specialization in oviposition site mimicry is due primarily to volatile chemistry and is reflected in the dimensions of the trapping apparatus. It also indicates that mycoheterotrophic plants can be specialized both on mycorrhizal fungi and insect pollinators.

Identifying factors that promote population differentiation is of interest for understanding the early stages of speciation. Gene flow among populations inhabiting different environments can be reduced by geographical distance (isolation-by-distance) or by divergent selection resulting from local adaptation (isolation-by-ecology). Few studies have investigated the influence of these factors in small oceanic islands where the influence of geographic distance is expected to be null but where habitat diversity could have a strong effect on population differentiation. In this study, we tested for the spatial divergence of phenotypes (floral morphology and floral scent) and genotypes (microsatellites) among ten populations of Jumellea rossii, an epiphytic orchid endemic to Reunion growing in three different habitats. We found a significant genetic differentiation between populations that is structured by habitat heterogeneity rather than by geographic distance between populations. These results suggest that ecological factors might reduce gene flow among populations located in different habitats. This pattern of isolation-by-habitat may be the result of both isolation-by-ecology by habitat filtering and asynchrony in flowering phenology. Furthermore, data on floral morphology match these findings, with multivariate analysis grouping populations by habitat type but could be only due to phenotypic plasticity. Indeed floral scent compounds were not significantly different between populations indicating that specific plant-pollinator mutualism does not seem to play a major role in the population differentiation of J. rossii. In conclusion, the results from our study emphasize the importance of habitat diversity of small oceanic islands as a factor of population differentiation.

Characterizing the architecture of bipartite networks is increasingly used as a framework to study biotic interactions within their ecological context and to assess the extent to which evolutionary constraint shape them. Orchid mycorrhizal symbioses are particularly interesting as they are viewed as more beneficial for plants than for fungi, a situation expected to result in an asymmetry of biological constraint. This study addressed the architecture and phylogenetic constraint in these associations in tropical context. We identified a bipartite network including 73 orchid species and 95 taxonomic units of mycorrhizal fungi across the natural habitats of Reunion Island. Unlike some recent evidence for nestedness in mycorrhizal symbioses, we found a highly modular architecture that largely reflected an ecological barrier between epiphytic and terrestrial subnetworks. By testing for phylogenetic signal, the overall signal was stronger for both partners in the epiphytic subnetwork. Moreover, in the subnetwork of epiphytic angraecoid orchids, the signal in orchid phylogeny was stronger than the signal in fungal phylogeny. Epiphytic associations are therefore more conservative and may co-evolve more than terrestrial ones. We suggest that such tighter phylogenetic specialization may have been driven by stressful life conditions in the epiphytic niches. In addition to paralleling recent insights into mycorrhizal networks, this study furthermore provides support for epiphytism as a major factor affecting ecological assemblage and evolutionary constraint in tropical mycorrhizal symbioses.

International audience; Inconspicuous basidiomycetes from the order Sebacinales are known to be involved in a puzzling variety of mutualistic plant-fungal symbioses (mycorrhizae), which presumably involve transport of mineral nutrients. Recently a few members of this fungal order not fitting this definition and commonly referred to as ‘endophytes’ have raised considerable interest by their ability to enhance plant growth and to increase resistance of their host plants against abiotic stress factors and fungal pathogens. Using DNA-based detection and electron microscopy, we show that Sebacinales are not only extremely versatile in their mycorrhizal associations, but are also almost universally present as symptomless endophytes. They occurred in field specimens of bryophytes, pteridophytes and all families of herbaceous angiosperms we investigated, including liverworts, wheat, maize, and the non-mycorrhizal model plant Arabidopsis thaliana. They were present in all habitats we studied on four continents. We even detected these fungi in herbarium specimens originating from pioneering field trips to North Africa in the 1830s/40s. No geographical or host patterns were detected. Our data suggest that the multitude of mycorrhizal interactions in Sebacinales may have arisen from an ancestral endophytic habit by specialization. Considering their proven beneficial influence on plant growth and their ubiquity, endophytic Sebacinales may be a previously unrecognized universal hidden force in plant ecosystems.

Population studies have revealed that the fungal ectomycorrhizal morphospecies Tricholoma scalpturatum consists of at least two genetically distinct groups that occur sympatrically in several geographical areas. This discovery prompted us to examine species boundaries and relationships between members formerly assigned to T. scalpturatum and allied taxa using phylogenetic analyses. Sequence data were obtained from three nuclear DNA regions [internal transcribed spacer (ITS), gpd and tef], from 101 carpophores collected over a large geographical range in Western Europe, and some reference sequences from public databases. The ITS was also tested for its applicability as DNA barcode for species delimitation. Four highly supported phylogenetic clades were detected. The two previously detected genetic groups of T. scalpturatum were assigned to the phylospecies Tricholoma argyraceum and T. scalpturatum. The two remaining clades were referred to as Tricholoma cingulatum and Tricholoma inocybeoides. Unexpectedly, T. cingulatum showed an accelerated rate of evolution that we attributed to narrow host specialization. This study also reveals recombinant ITS sequences in T. inocybeoides, suggesting a hybrid origin. The ITS was a useful tool for the determination of species boundaries: the mean value of intraspecific genetic distances in the entire ITS region (including 5.8S rDNA) was0.2%, whereas interspecific divergence estimates ranged from 1.78% to 4.22%. Apart from giving insights into the evolution of the T. scalpturatum complex, this study contributes to the establishment of a library of taxonomically verified voucher specimens, an a posteriori correlation between phenotype and genotype, and DNA barcoding of ectomycorrhizal fungi.

† Background and Aims Nutritional changes associated with the evolution of achlorophyllous, mycoheterotrophic plants have not previously been inferred with robust phylogenetic hypotheses. Variations in heterotrophy in accordance with the evolution of leaflessness were examined using a chlorophyllous‐achlorophyllous species pair in Cymbidium (Orchidaceae), within a well studied phylogenetic background. † Methods To estimate the level of mycoheterotrophy in chlorophyllous and achlorophyllous Cymbidium, natural 13 Ca nd 15 N contents (a proxy for the level of heterotrophy) were measured in four Cymbidium species and co-existing autotrophic and mycoheterotrophic plants and ectomycorrhizal fungi from two Japanese sites. † Key Results d 13 C and d 15 N values of the achlorophyllous C. macrorhizon and C. aberrans indicated that they are full mycoheterotrophs. d 13 C and d 15 N values of the chlorophyllous C. lancifolium and C. goeringii were intermediate between those of reference autotrophic and mycoheterotrophic plants; thus, they probably gain 30 ‐50 % of their carbon resources from fungi. These data suggest that some chlorophyllous Cymbidium exhibit partial mycoheterotrophy (¼ mixotrophy). † Conclusions It is demonstrated for the first time that mycoheterotrophy evolved after the establishment of mixotrophy rather than through direct shifts from autotrophy to mycoheterotrophy. This may be one of the principal patterns in the evolution of mycoheterotrophy. The results also suggest that the establishment of symbiosis with ectomycorrhizal fungi in the lineage leading to mixotrophic Cymbidium served as pre-adaptation to the evolution of the mycoheterotrophic species. Similar processes of nutritional innovations probably occurred in several independent orchid groups, allowing niche expansion and radiation in Orchidaceae, probably the largest plant family.

International audience; Mycoheterotrophic orchids have adapted to shaded forest understory by shifting to achlorophylly and receiving carbon from their mycorrhizal fungi. In temperate forests, they associate in a highly specific way with fungi forming ectomycorrhizas on nearby trees, and exploiting tree photosynthates. However, many rainforests lack ectomycorrhizal fungi, and there is evidence that some tropical Asiatic species associate with saprotrophic fungi. To investigate this in different geographic and phylogenetic contexts, we identified the mycorrhizal fungi supporting two tropical mycoheterotrophic orchids from Mascarene (Indian Ocean) and Caribbean islands. We tested their possible carbon sources by measuring natural nitrogen (15N) and carbon (13C) abundances. Saprotrophic basidiomycetes were found: Gastrodia similis associates with a wood-decaying Resinicium (Hymenochaetales); Wullschlaegelia aphylla associates with both litter-decaying Gymnopus and Mycena species, whose rhizomorphs link orchid roots to leaf litter. The 15N and 13C abundances make plausible food chains from dead wood to G. similis and from dead leaves to W. aphylla. We propose that temperature and moisture in rainforests, but not in most temperate forests, may favour sufficient saprotrophic activity to support development of mycoheterotrophs. By enlarging the spectrum of mycorrhizal fungi and the level of specificity in mycoheterotrophic orchids, this study provides new insights on orchid and mycorrhizal biology in the tropics. New Phytologist (2009)doi: 10.1111/j.1469-8137.2009.02987.x

Gastrodia madagascariensis, a leafless achlorophyllous orchid, is described and illustrated here. The epithet was originally coined by Perrier de la Bâthie in 1939 for fruiting material found on the eastern coastal plain of Madagascar more than a century ago, but the name was never validly published. This new species is closely related to G. similis from Reunion Island, from which it can be distinguished by the perianth tube spreading towards the apex, the shape of the column and stigma, and the flower colour. The achlorophyllous genus Gastrodia currently comprises five species in the tropical parts of the Afro-Madagascan region, one of which, G. africana, is possibly extinct. We provide an artificial key to distinguish them. In addition, there is also an extratropical species in continental Africa, the introduced G. sesamoides (very local near Cape Town, South Africa).

Phylogenetic relationships were inferred for the African-centred orchid subtribe Eulophiinae, which includes the large genus Eulophia and three closely related genera, Oeceoclades, Pteroglossaspis and Acrolophia. We sequenced one nuclear (ITS) and four chloroplast (rbcL, trnK-matK, rpl32­trnL, trnQ­5' rps16) markers for 86 samples, representing 58 species (62 taxa) and one outgroup. The combined sequence matrix had 6525 characters and was analysed using parsimony and Bayesian criteria. Our results show that Acrolophia and Oeceoclades are monophyletic but are nested in Eulophia as currently circumscribed, thus rendering it paraphyletic. We argue for retaining Acrolophia and Oeceoclades, and splitting Eulophia into two genera on the basis of evidence that each of these four clades is genetically and morphologically distinct. We propose to reinstate the genus name Orthochilus for one of the Eulophia clades and transfer 35 taxa, including those assigned to Pteroglossaspis, to Orthochilus. Thirty-two of these are new combinations.

A plethora of recent studies have characterized the composition and functional role of microbial communities hosted by animals and plants, called microbiota. The overall goal of my PhD is to advance our understanding of how microbiota evolve with their host species, using data comprised of the phylogenetic relationships between host species and metabarcoding microbial sequences characterizing their microbial communities. We developed new quantitative tools, collected data, and performed a series of analyses, all directed to this common overarching goal. We considered both microbiota-animal and microbiota-plant systems, with a specific focus on mycorrhizal interactions. In Chapter I, we study the evolution of the microbiota during the diversification of host clades. We develop a quantitative approach for inferring the modes of microbial inheritance as host clades diversify. Given a host phylogeny and the microbiota of present-day species, each characterized by a list of short DNA sequences clustered into operational taxonomic units (OTUs), our approach uses nucleotide variability within OTUs to detect OTUs that are vertically transmitted. We apply this approach to two distinct systems, the gut microbiota of primates and a clade of Hawaiian spiders. We find that >5% of bacteria in primate guts are vertically transmitted, whereas there is no evidence of vertical transmission in spiders, confirming that host-microbiota evolutionary dynamics are highly heterogeneous across the animal kingdom. Finally, we compare the performances of our model to other available approaches and find that it is less prone to false-positives when the nucleotide variability within OTUs is low. In Chapter II, we examine the interplay between the evolutionary history of host and host-associated microbial clades. We focus on two specific questions: “To what extent does evolutionary history influence which microbial species interact with which host species?” and “How does the evolutionary history of hosts influence the diversification of host-associated microbial clades?”. The first question leads us to compare different methods for estimating phylogenetic signals in host-microbiota interactions, i.e. whether closely related species share similar sets of partners, with an application on plant-mycorrhizal interactions. We find that one of the most widely used approaches often detects phylogenetic signals when it should not and that Mantel tests perform best. We explore the second question by studying the diversification of the arbuscular mycorrhizal fungi (Glomeromycotina) in the past 500 million years and evaluating how land plants might have affected the diversification of these obligate mycorrhizal symbionts. Our analyses support that these fungi have experienced a recent diversification slowdown that might be linked to the shrinkage of their mycorrhizal niches as plant lineages evolve alternative symbiotic strategies. In Chapter III, we focus on the evolution of cheating in host-microbiota mutualisms, by taking the mycorrhizal symbiosis as a case study. We explore constraints on the evolutionary emergence of cheating in plants (mycoheterotrophy) by analyzing the patterns of plant-mycorrhizal fungus interactions at the global scale. Next, we investigate whether similar constraints are found in local mycorrhizal networks including initially mycoheterotrophic plants (Lycopodiaceae) that we have sampled in La Réunion island. We conclude that there are overall strong constraints limiting the emergence of cheaters in the mycorrhizal symbiosis, but these constraints might be relaxed in the local communities where cheating occurs. Overall, my thesis illustrates how new or recent computational tools, in combination with metabarcoding sequencing data, allow studying how microbiota evolve with their hosts. We discuss the challenges and promise of this comparative approach to host-microbiota evolution., De nombreuses études récentes ont permis de caractériser la composition des communautés microbiennes, appelées microbiotes, hébergées par plantes et animaux. Le but de ma thèse est de faire avancer notre compréhension de l’évolution des microbiotes associés aux espèces hôtes animales ou végétales, en utilisant comme données les arbres phylogénétiques des hôtes et des séquences d’ADN microbien de metabarcoding caractérisant leurs microbiotes. Pour cela, nous avons développé de nouvelles méthodes quantitatives, collecté des données ainsi que réalisé une série d’analyses. Nous avons considéré à la fois les microbes des animaux et des plantes, et tout particulièrement les interactions mycorhizennes. Dans le chapitre I, nous nous sommes intéressés à l’évolution du microbiote au cours de la diversification d’un clade d’hôtes. Nous avons développé une approche quantitative afin d’inférer les microbes transmis. À partir de la phylogénie des hôtes et de séquences d’ADN de leurs microbiotes, regroupées en unité taxonomique opérationnelle (OTU), notre approche utilise la variation nucléotidique au sein des OTUs pour détecter ceux qui sont transmis lors de la diversification des hôtes. Appliquée aux microbiotes de primates et araignées, nous avons trouvé que >5% des bactéries intestinales des primates étaient transmises verticalement, tandis qu’il n’y a vraisemblablement pas de transmission chez les araignées, confirmant l’hétérogénéité de l’évolution des interactions hôtes-microbes chez les animaux. Enfin, nous avons comparé les performances de notre modèle à celles d’autres approches existantes et montré que notre modèle est moins enclin aux faux-positifs lorsque la variation nucléotidique intra-OTU est faible. Dans le chapitre II, nous avons examiné les liens entre les histoires évolutives des hôtes et de leurs microbes associés. Nous avons plus particulièrement cherché à répondre à deux questions : « Dans quelle mesure les patrons d’interactions hôtes-microbiotes sont influencés par leurs histoires évolutives ? » et « comment l’histoire évolutive des hôtes influence-t-elle la diversification de leurs microbes associés ? ». La première question nous a amené à comparer les méthodes disponibles pour estimer le signal phylogénétique dans les réseaux d’interactions, afin de déterminer par exemple si des espèces de plantes proches ont tendance à interagir avec les mêmes champignons mycorhiziens. Nous avons trouvé qu’une approche fréquemment utilisée génère beaucoup de faux-positifs et qu’à l’inverse, les tests de Mantel donnent des résultats assez satisfaisants. Nous avons enfin exploré la seconde question en évaluant comment les plantes ont pu affecter la diversification des champignons endomycorhiziens (Glomeromycotina). Nos analyses suggèrent que ces symbiontes obligatoires ont récemment subit un ralentissement de leur diversification, qui peut être lié à l’évolution, chez de nombreuses plantes, de stratégies alternatives à l’endomycorhize. Dans le chapitre III, nous nous sommes focalisés sur l’évolution de la tricherie dans le mutualisme hôte-microbiote, et plus particulièrement dans la symbiose mycorhizienne. Nous avons exploré les contraintes limitant l’émergence de la tricherie chez les plantes (mycohétérotrophie) en analysant les patrons d’interactions endomycorhizennes à l’échelle mondiale. Nous avons ensuite étudié si des contraintes similaires s’appliquaient dans les communautés locales où vivent des plantes initialement mycohétérotrophes (les lycopodes), échantillonnées sur l’île de la Réunion. Nous en avons déduit qu’il existe généralement de fortes contraintes limitant la tricherie dans cette symbiose, mais que ces contraintes peuvent être relâchées au sein des communautés où la tricherie a lieu. Ainsi, ma thèse illustre que l’utilisation de méthodes quantitatives combinées à des données de metabarcoding, malgré leurs limites respectives, permet de mieux caractériser l’évolution des interactions hôtes-microbiotes.