A hemizygous supergene controls homomorphic and heteromorphic self-incompatibility systems in Oleaceae.

Self-incompatibility (SI) has evolved independently multiple times and prevents self-fertilization in hermaphrodite angiosperms. Several groups of Oleaceae such as jasmines exhibit distylous flowers, with two compatibility groups each associated with a specific floral morph.1 Other Oleaceae species in the olive tribe have two compatibility groups without associated morphological variation.2,3,4,5 The genetic basis of both homomorphic and dimorphic SI systems in Oleaceae is unknown. By comparing genomic sequences of three olive subspecies (Olea europaea) belonging to the two compatibility groups, we first locate the genetic determinants of SI within a 700-kb hemizygous region present only in one compatibility group. We then demonstrate that the homologous hemizygous region also controls distyly in jasmine. Phylogenetic analyses support a common origin of both systems, following a segmental genomic duplication in a common ancestor. Examination of the gene content of the hemizygous region in different jasmine and olive species suggests that the mechanisms determining compatibility groups and floral phenotypes (whether homomorphic or dimorphic) in Oleaceae rely on the presence/absence of two genes involved in gibberellin and brassinosteroid regulation.

What determines Alnus-associated ectomycorrhizal community diversity and specificity? A comparison of host and habitat effects at a regional scale.

· Global-scale analyses of ectomycorrhizal (ECM) fungi communities emphasize host plant families as the main drivers of diversity. This study aims to test, on Alnus-ECM communities, which fungi are said to be 'host-specific', to what extent host species, habitat and distance explain their alpha and beta diversity variations, and their specificity. · In France, ECM communities associated with two subgenera and five species of Alnus, were sampled on 165 trees from 39 lowland to subalpine sites. In all, 1178 internal transcribed spacer (ITS) sequences of ECM fungi clustered in 86 molecular operational taxonomic units (MOTUs). · The species richness was low but still variable, and the evenness of communities was lower on organic soils and in Corsica. Similarity between communities was influenced both by host, soil parameters, altitude and longitude, but not by climate and distance. A large majority of 'specific' fungi were shared between host species within a subgenus, and showed habitat preferences within the subgenus distribution range. · Our study confirms that Alnus ECM communities are low in diversity, highly conserved at a regional scale, and partly shared between congeneric host species. A large part of alpha and beta diversity variations remained unexplained, and other processes may shape these communities.

An efficient protocol for isolating melanised chaetothyrialean anamorphic fungi associated with plant-ants.

Because of their ecological characteristics, slow growth rates and the presence of contaminants, Chaetothyriales fungi associated with structures built by tropical plant-ants can be difficult to isolate with standard procedures. Here, we describe an easy-to-use protocol for obtaining pure cultures by using cotton as a first substrate. We have further found by means of fluorescent stains that nuclei concentrate either in young hyphae or in the tips of the hyphae.

Mutualistic interactions between ants and fungi: A review.

The large amount of dead plant biomass caused by the final extinction events triggered a fungi proliferation that mostly differentiated into saprophytes degrading organic matter; others became parasites, predators, likely commensals, and mutualists. Among the last, many have relationships with ants, the most emblematic seen in the Neotropical myrmicine Attina that cultivate Basidiomycota for food. Among them, leaf-cutting, fungus-growing species illustrate an ecological innovation because they grow fungal gardens from fresh plant material rather than arthropod frass and plant debris. Myrmecophytes shelter "plant-ants" in hollow structures, the domatia, whose inner walls are lined with thin-walled Ascomycota hyphae that, in certain cases, are eaten by the ants, showing a form of convergence. Typically, these Ascomycota have antibacterial properties illustrating cases of farming for protection. Ant gardens, or mutualistic associations between certain ant species and epiphytes, shelter endophytic fungi that promote the growth of the epiphytes. Because the cell walls of certain Ascomycota hyphae remain sturdy after the death of the mycelium, they form resistant fibers used by ants to reinforce their constructions (e.g., galleries, shelters for tended hemipterans, and carton nests). Thus, we saw cases of "true" fungal agriculture involving planting, cultivating, and harvesting Basidiomycota for food with Attina. A convergence with "plant-ants" feeding on Ascomycota whose antibacterial activity is generally exploited (i.e., farming for protection). The growth of epiphytes was promoted by endophytic fungi in ant gardens. Finally, farming for structural materials occurred with, in one case, a leaf-cutting, fungus-growing ant using Ascomycota fibers to reinforce its nests.

Specific, non-nutritional association between an ascomycete fungus and Allomerus plant-ants.

Ant-fungus associations are well known from attine ants, whose nutrition is based on a symbiosis with basidiomycete fungi. Otherwise, only a few non-nutritional ant-fungus associations have been recorded to date. Here we focus on one of these associations involving Allomerus plant-ants that build galleried structures on their myrmecophytic hosts in order to ambush prey. We show that this association is not opportunistic because the ants select from a monophyletic group of closely related fungal haplotypes of an ascomycete species from the order Chaetothyriales that consistently grows on and has been isolated from the galleries. Both the ants' behaviour and an analysis of the genetic population structure of the ants and the fungus argue for host specificity in this interaction. The ants' behaviour reveals a major investment in manipulating, growing and cleaning the fungus. A molecular analysis of the fungus demonstrates the widespread occurrence of one haplotype and many other haplotypes with a lower occurrence, as well as significant variation in the presence of these fungal haplotypes between areas and ant species. Altogether, these results suggest that such an interaction might represent an as-yet undescribed type of specific association between ants and fungus in which the ants cultivate fungal mycelia to strengthen their hunting galleries.

Phylogenetic species delimitation in ectomycorrhizal fungi and implications for barcoding: the case of the Tricholoma scalpturatum complex (Basidiomycota).

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 <0.2%, 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.

Genotypes of the aquatic plant Myriophyllum spicatum with different growth strategies show contrasting sensitivities to copper contamination.

Genotypic variability has been considered for years as a key attribute in species adaptation to new environments. It has been extensively studied in a context of chemical resistance, but remains poorly studied in response to chemical exposure in a context of global change. As aquatic ecosystems are particularly affected by environmental changes, we aimed to study how genotypic variability could inflect the sensitivity of aquatic plants to chemicals. Seven genotypes of Myriophyllum spicatum were exposed to three copper concentrations at 0, 0.15 and 0.5 mg/L. The sensitivity of the different genotypes was assessed through several endpoints such as relative growth rate (RGR) and morphological traits, as well as physiological markers, such as plant biomacromolecular composition. Our results showed that genotypes exhibited significant differences in their life-history traits in absence of chemical contamination. Some trait syndromes were observed, and three growth strategies were identified: (1) biomass production and main shoot elongation, (2) dry matter storage with denser whorls to promote resource conservation and (3) lateral shoot production. An up to eightfold difference in sensitivity for growth-related endpoints was observed among genotypes. Differences in sensitivity were partly attributed to morphological life-history traits. Our results confirm that genotypic variability can significantly affect M. spicatum sensitivity to Cu, and may influence the outcomes of laboratory testing based on the study of one single genotype. We recommend including genotypic variation as an assessment factor in ecological risk assessment and to study this source of variability more in depth as a possible driver of ecosystem resilience.

Do Host Plant and Associated Ant Species Affect Microbial Communities in Myrmecophytes?

Ant-associated microorganisms can play crucial and often overlooked roles, and given the diversity of interactions that ants have developed, the study of the associated microbiomes is of interest. We focused here on specialist plant-ant species of the genus Allomerus that grow a fungus to build galleries on their host-plant stems. Allomerus-inhabited domatia, thus, might be a rich arena for microbes associated with the ants, the plant, and the fungus. We investigated the microbial communities present in domatia colonised by four arboreal ants: Allomerus decemarticulatus, A. octoarticulatus, A. octoarticulatus var. demerarae, and the non-fungus growing plant-ant Azteca sp. cf. depilis, inhabiting Hirtella physophora or Cordia nodosa in French Guiana. We hypothesized that the microbial community will differ among these species. We isolated microorganisms from five colonies of each species, sequenced the 16S rRNA or Internal TranscribedSpacer (ITS) regions, and described both the alpha and beta diversities. We identified 69 microbial taxa, which belong to five bacterial and two fungal phyla. The most diverse phyla were Proteobacteria and Actinobacteria. The microbial community of Azteca cf. depilis and Allomerus spp. differed in composition and richness. Geographical distance affected microbial communities and richness but plant species did not. Actinobacteria were only associated with Allomerus spp.

Does intraspecific variability matter in ecological risk assessment? Investigation of genotypic variations in three macrophyte species exposed to copper.

To limit anthropogenic impact on ecosystems, regulations have been implemented along with global awareness that human activities are harmful to the environment. Ecological risk assessment (ERA) is the main procedure which allows to assess potential impacts of stressors on the environment as a result of human activities. ERA is typically implemented through different steps of laboratory testing. The approaches taken for ERA evolve along with scientific knowledge, to improve predictions on ecological risks for ecosystems. We here address the importance of intraspecific variability as a potential source of error in the laboratory evaluation of pollutants. To answer this question, three aquatic macrophyte species with different life-history traits but with their leaves directly in contact with the water were chosen; Lemna minor and Myriophyllum spicatum, two OECD model species, and Ceratophyllum demersum. For each species, three or four genotypes were exposed to 7-8 copper concentrations (up to 1.9 mg/L, 2 mg/L or 36 mg/L for C. demersum, L. minor and M. spicatum, respectively). To assess species sensitivity, growth-related endpoints such as Relative Growth Rate (RGR), based either on biomass production or on length/frond production, and chlorophyll fluorescence Fv/Fm, were measured. For each endpoint, the effective concentration 50% (EC50) was calculated. Almost all endpoints were affected by Cu exposure, except Fv/Fm of M. spicatum, and resulted in significant differences among genotypes for Cu sensitivity. Genotypes of L. minor exhibited up to 35% of variation in EC50 values based on Fv/Fm, showing differential sensivity among genotypes. Significant differences in EC50 values were found for RGR based on length for M. spicatum, with up to 72% of variation. Finally, C. demersum demonstrated significant sensitivity differences among genotypes with up to 78% variation for EC50 based on length. Overall, interspecific variation was higher than intraspecific variation, and explained 77% of the variation found among genotypes for RGR based on biomass, and 99% of the variation found for Fv/Fm. Our results highlight that depending on the endpoint, sensitivity can vary greatly within a species, and that pollutant- and species-specific endpoints should be considered in ERA.

Molecular evolution of mitochondrial ribosomal DNA in the fungal genus Tricholoma: barcoding implications.

The molecular evolution of the V6 and V9 domains of the mitochondrial SSU-rDNA was investigated to evaluate the use of these sequences for DNA barcodes in the Basidiomycota division. The PCR products from 27 isolates belonging to 11 Tricholoma species were sequenced. Both domains in the isolates belonging to the same species had identical sequences. All the species possess distinctive V9 sequences due to point mutations and insertion/deletion events. Secondary structures revealed that the insertion-deletion events occurred in regions not directly involved in the maintenance of the standard SSU-rRNA structure. The inserted sequences possess conserved motifs that enable their alignment among phylogenetically distant species. Hence, the V9 domain by displaying identical sequences within species, an adequate divergence level, easy amplification, and alignment represents an alternative molecular marker for the Basidiomycota division and opens the way for this sequence to be used as specific molecular markers of the fungal kingdom.

High sexual reproduction and limited contemporary dispersal in the ectomycorrhizal fungus Tricholoma scalpturatum: new insights from population genetics and spatial autocorrelation analysis.

Dispersal and establishment are fundamental processes influencing the response of species to environmental changes, and the long-term persistence of populations. A previous study on the symbiotic ectomycorrhizal fungus Tricholoma scalpturatum revealed strong genetic differentiations between populations in Western Europe, suggesting restricted dispersal for this wind-dispersed cosmopolitan fungus. Two distinct genetic groups (genetic groups 1 and 2), co-occurring in some locations, were also identified and could correspond to cryptic species. In the present work, we examine the reproductive strategy and dispersal biology of the two T. scalpturatum's genetic groups. Variable molecular markers (intersimple sequence repeats and intergenic spacer 2-restriction fragment length polymorphisms) and spatial autocorrelation analyses were used to examine fine-scale patterns (< 140 m) of genetic structure, in an effort to determine the physical scale at which genetic structure exists. A total of 473 fruit bodies were mapped and collected over 3 years from two plots located in the south of France, including 219 and 254 samples from group 1 and group 2, respectively. High genetic diversity and the presence of numerous small genets were observed in both groups. Autocorrelation analyses revealed significant positive spatial genetic structures of genets at close distances (up to few metres for both groups). Mantel tests confirmed this isolation-by-distance pattern. These results clearly demonstrate high sexual reproduction and spatial structuring of genets at very small geographical scales in this wind-dispersed ectomycorrhizal fungal species, a pattern consistent with restricted contemporary dispersal of spores.

Alder and the Golden Fleece: high diversity of Frankia and ectomycorrhizal fungi revealed from Alnus glutinosa subsp. barbata roots close to a Tertiary and glacial refugium.

BACKGROUND: Recent climatic history has strongly impacted plant populations, but little is known about its effect on microbes. Alders, which host few and specific symbionts, have high genetic diversity in glacial refugia. Here, we tested the prediction that communities of root symbionts survived in refugia with their host populations. We expected to detect endemic symbionts and a higher species richness in refugia as compared to recolonized areas. METHODS: We sampled ectomycorrhizal (EM) root tips and the nitrogen-fixing actinomycete Frankia communities in eight sites colonized by Alnus glutinosa subsp. barbata close to the Caucasus in Georgia. Three sites were located in the Colchis, one major Eurasian climatic refugia for Arcto-Tertiary flora and alders, and five sites were located in the recolonized zone. Endemic symbionts and plant ITS variants were detected by comparing sequences to published data from Europe and another Tertiary refugium, the Hyrcanian forest. Species richness and community structure were compared between sites from refugia and recolonized areas for each symbionts. RESULTS: For both symbionts, most MOTUs present in Georgia had been found previously elsewhere in Europe. Three endemic Frankia strains were detected in the Colchis vs two in the recolonized zone, and the five endemic EM fungi were detected only in the recolonized zone. Frankia species richness was higher in the Colchis while the contrary was observed for EM fungi. Moreover, the genetic diversity of one alder specialist Alnicola xanthophylla was particularly high in the recolonized zone. The EM communities occurring in the Colchis and the Hyrcanian forests shared closely related endemic species. DISCUSSION: The Colchis did not have the highest alpha diversity and more endemic species, suggesting that our hypothesis based on alder biogeography may not apply to alder's symbionts. Our study in the Caucasus brings new clues to understand symbioses biogeography and their survival in Tertiary and ice-age refugia, and reveals that isolated host populations could be of interest for symbiont diversity conservation.

Paxillus rubicundulus (Boletales, Paxillaceae) and two new alder-specific ectomycorrhizal species, Paxillus olivellus and Paxillus adelphus, from Europe and North Africa.

Paxillus rubicundulus P.D. Orton has been shown to be a complex of at least three ectomycorrhizal taxa strictly associated with alders (Alnus) in Europe, P. rubicundulus s. str., and two undescribed clades. To assess the taxonomic status of these three clades and their phylogenetic relationships, phylogenetic analyses of two independent gene regions (ITS and gpd), combined with macro- and micromorphological comparisons of genetically identified specimens, were carried out. A total of 85 sequences were successfully obtained from basidiomata and alder mycorrhizae collected in France and Algeria and combined with GenBank and UNITE sequences. The phylogenetic results and estimates of genetic diversity confirmed that the three clades are distinct species, often found in sympatry. As a result, P. rubicundulus s. str. was redefined based on the revision of type material, and Paxillus adelphus and Paxillus olivellus are introduced as new Linnaean names. The often used name Paxillus filamentosus is rejected since it could not be applied to any of the new species. The three species are distinguished micromorphologically by spore size and shape. They are widely distributed in Europe, North Africa and western Asia; P. rubicundulus is rare, and all species have a limited host range.

The Paxillus involutus (Boletales, Paxillaceae) complex in Europe: genetic diversity and morphological description of the new species Paxillus cuprinus, typification of P. involutus s.s., and synthesis of species boundaries.

Paxillus involutus is a model species for ecological or physiological studies of ectomycorrhizal agaricomycetes. Three to six groups or species linked to it have been ecologically and morphologically distinguished. Phylogenetic studies have revealed the existence of four species in Europe: Paxillus ammoniavirescens, Paxillus obscurisporus, P. involutus, and a fourth as yet not described species. We studied 47 collections from 24 French and Italian locations, supplemented with GenBank data, in order to genetically and taxonomically delineate these species. Phylogenetic analyses of three nuclear DNA regions (rDNA internal transcribed spacer (ITS), tef1-α, and gpd) confirmed the four European species. Morphology, culture, and ecology features allowed us to delineate species boundaries and to describe the fourth species we named Paxillus cuprinus since it turns coppery with age. As there is no existing original herbarium specimen for P. involutus, one of our collections was chosen as the epitype. The low genetic diversity found in P. cuprinus correlates with stable morphological traits (basidiome colour, ovoid-amygdaliform spores with an apical constriction) and with ecological preferences (association with Betulaceae in open and temperate areas). In contrast, P. ammoniavirescens is characterized by a high genetic diversity and a high variation of its morphological and ecological features.

Population genetics of ectomycorrhizal fungi: from current knowledge to emerging directions.

Ectomycorrhizal (EM) fungi are major microbial components of boreal, temperate and Mediterranean forests, as well as some tropical forest ecosystems. Nearly two decades of studies have clarified many aspects of their population biology, based on several model species from diverse lineages of fungi where the EM symbiosis evolved, i.e. among Hymenomycetes and, to a lesser extent, among Ascomycetes. In this review, we show how tools for individual recognition have changed, shifting from the use of somatic incompatibility reactions to dominant and non-specific markers (such as random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP)) and, more recently, to co-dominant and specific markers (such as microsatellites and single nucleotide polymorphisms (SNPs)). At the same time, the theoretical focus has also changed. In earlier studies, a major aim was the description of genet size and popul/ation strategy. For example, we show how some studies supported or challenged the simple, classical model of colonization of new forest stands by ruderal (R) species, propagating by spores and forming small genets, progressively replaced in older forests by more competitive (C) species, propagating by mycelial growth and forming larger genets. By contrast, more recent studies give insights into some genetic traits, such as partners' assortment (allo- versus autogamy), genetic structure of populations and gene flow that turn out to depend both on distance and on whether spores are animal- or wind-dispersed. We discuss the rising awareness that (i) many morphospecies contain cryptic biological species (often sympatric) and (ii) trans- and inter-continental species may often contain several biological species isolated by distance. Finally, we show the emergence of biogeographic approaches and call for some aspects to be developed, such as fine-scale and long-term population monitoring, analyses of subterranean populations of extra-radical mycelia, or more model species from the tropics, as well as from the Ascomycetes (whose genetic idiosyncrasies are discussed). With the rise of the '-omics' sciences, analysis of population structure for non-neutral genes is expected to develop, and forest management and conservation biology will probably profit from published and expected work.

Population evidence of cryptic species and geographical structure in the cosmopolitan ectomycorrhizal fungus, Tricholoma scalpturatum.

Tricholoma scalpturatum is an ectomycorrhizal fungus that forms symbioses with roots of diverse trees and shrubs. It is commonly encountered in a wide range of habitats, across temperate ecosystems. A previous study has revealed a high genetic diversity at a local scale, and ruderal abilities. To examine genetic structure at a large geographical scale, a total of 164 basidiocarps were collected from 30 populations located in Western Europe, from Spain to Scandinavia. These samples were analyzed by three molecular methods with different levels of resolution: inter-simple sequence repeats (ISSRs), restriction fragment length polymorphisms (RFLPs) in the rDNA internal transcribed spacer (ITS), and ITS sequence analysis. Considerable genetic variation was found, and the morphospecies was separated into two genetic groups that were distinct from each other. The ISSR data and the relatively low percentage value (96%) of shared sequence polymorphisms in the ITS between isolates from the two groups, strongly suggest cryptic species and long-lasting separation. No geographical exclusion was detected for these two widely distributed taxa. However, high estimates of population differentiation were observed in each group, including between populations less than a few kilometers apart. This result provides evidence for limited gene flow and/or founding effects. It also indicates that T. scalpturatum does not constitute a random mating population, and the hypothesis of endemism cannot be excluded for this cosmopolitan wind-dispersed fungus.

Population dynamics of the ectomycorrhizal fungal species Tricholoma populinum and Tricholoma scalpturatum associated with black poplar under differing environmental conditions.

Fungi combine sexual reproduction and clonal propagation. The balance between these two reproductive modes affects establishment dynamics, and ultimately the evolutionary potential of populations. The pattern of colonization was studied in two species of ectomycorrhizal fungi: Tricholoma populinum and Tricholoma scalpturatum. The former is considered to be a host specialist whereas T. scalpturatum is a generalist taxon. Fruit bodies of both basidiomycete species were mapped and collected over several years from a black poplar (Populus nigra) stand, at two different sites. Multilocus genotypes (= genets) were identified based on the analysis of random amplified polymorphic DNA (RAPD) patterns, inter-simple sequence repeat (ISSR) patterns and restriction fragment length polymorphisms (RFLPs) in the ribosomal DNA intergenic spacer (rDNA IGS). The genetic analyses revealed differences in local population dynamics between the two species. Tricholoma scalpturatum tended to capture new space through sexual spores whereas T. populinum did this by clonal growth, suggesting trade-offs in allocation of resources at the genet level. Genet numbers and sizes strongly differ between the two study sites, perhaps as a result of abiotic disturbance on mycelial establishment and genet behaviour.