Sigisfredo Garnica

Phylogenetic relationships in Cortinarius , section Calochroi , inferred from nuclear DNA sequences

BackgroundSection Calochroi is one of the most species-rich lineages in the genus Cortinarius (Agaricales, Basidiomycota) and is widely distributed across boreo-nemoral areas, with some extensions into meridional zones. Previous phylogenetic studies of Calochroi (incl. section Fulvi) have been geographically restricted; therefore, phylogenetic and biogeographic relationships within this lineage at a global scale have been largely unknown. In this study, we obtained DNA sequences from a nearly complete taxon sampling of known species from Europe, Central America and North America. We inferred intra- and interspecific phylogenetic relationships as well as major morphological evolutionary trends within section Calochroi based on 576 ITS sequences, 230 ITS + 5.8S + D1/D2 sequences, and a combined dataset of ITS + 5.8S + D1/D2 and RPB1 sequences of a representative subsampling of 58 species.ResultsMore than 100 species were identified by integrating DNA sequences with morphological, macrochemical and ecological data. Cortinarius section Calochroi was consistently resolved with high branch support into at least seven major lineages: Calochroi, Caroviolacei, Dibaphi, Elegantiores, Napi, Pseudoglaucopodes and Splendentes; whereas Rufoolivacei and Sulfurini appeared polyphyletic. A close relationship between Dibaphi, Elegantiores, Napi and Splendentes was consistently supported. Combinations of specific morphological, pigmentation and molecular characters appear useful in circumscribing clades.ConclusionOur analyses demonstrate that Calochroi is an exclusively northern hemispheric lineage, where species follow their host trees throughout their natural ranges within and across continents. Results of this study contribute substantially to defining European species in this group and will help to either identify or to name new species occurring across the northern hemisphere. Major groupings are in partial agreement with earlier morphology-based and molecular phylogenetic hypotheses, but some relationships were unexpected, based on external morphology. In such cases, their true affinities appear to have been obscured by the repeated appearance of similar features among distantly related species. Therefore, further taxonomic studies are needed to evaluate the consistency of species concepts and interpretations of morphological features in a more global context. Reconstruction of ancestral states yielded two major evolutionary trends within section Calochroi: (1) the development of bright pigments evolved independently multiple times, and (2) the evolution of abruptly marginate to flattened stipe bulbs represents an autapomorphy of the Calochroi clade.

Sebacinales Everywhere: Previously Overlooked Ubiquitous Fungal Endophytes

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.

THE EVOLUTIONARY HISTORY OF MYCORRHIZAL SPECIFICITY AMONG LADY'S SLIPPER ORCHIDS

Abstract Although coevolution is acknowledged to occur in nature, coevolutionary patterns in symbioses not involving species-to-species relationships are poorly understood. Mycorrhizal plants are thought to be too generalist to coevolve with their symbiotic fungi; yet some plants, including some orchids, exhibit strikingly narrow mycorrhizal specificity. Here, we assess the evolutionary history of mycorrhizal specificity in the ladys slipper orchid genus, Cypripedium. We sampled 90 populations of 15 taxa across three continents, using DNA methods to identify fungal symbionts and quantify mycorrhizal specificity. We assessed phylogenetic relationships among sampled Cypripedium taxa, onto which we mapped mycorrhizal specificity. Cypripedium taxa associated almost exclusively with fungi within family Tulasnellaceae. Ancestral specificity appears to have been narrow, followed by a broadening after the divergence of C. debile. Specificity then narrowed, resulting in strikingly narrow specificity in most of the taxa in this study, with no taxon rewidening to the same extant as basal members of the genus. Sympatric taxa generally associated with different sets of fungi, and most clades of Cypripedium-mycorrhizal fungi were found throughout much of the northern hemisphere, suggesting that these evolutionary patterns in specificity are not the result of biogeographic lack of opportunity to associate with potential partners. Mycorrhizal specificity in genus Cypripedium appears to be an evolvable trait, and associations with particular fungi are phylogenetically conserved.

The systematic relevance of conidiogenesis modes in the gilled Agaricales

Dikaryotic and haploid mycelia of more than 150 gilled species of euagarics were studied morphologically and by molecular phylogenetic methods. The morphological investigations revealed anamorphs in more than 90 species that were often specific at the genus or family level. Thallic conidiogenesis dominated and varied from fragmentation of normally branched hyphae to the formation of differentiated sympodially branched conidiophores. Secession modes, coiling of the conidiogenous hyphae or the swelling of the conidia were additional distinguishing features. Phylogenetic analysis of the D1-D3 domains of the nuclear gene for the ribosomal large subunit using a Bayesian Markov chain Monte Carlo approach resulted in several well-supported groups that are consistent with anamorph morphology. These results indicate that the anamorphs provide valuable characters for a natural classification of the Agaricales.

Atractiellomycetes belonging to the ‘rust’ lineage (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic neotropical orchids

Distinctive groups of fungi are involved in the diverse mycorrhizal associations of land plants. All previously known mycorrhiza-forming Basidiomycota associated with trees, ericads, liverworts or orchids are hosted in Agaricomycetes, Agaricomycotina. Here we demonstrate for the first time that Atractiellomycetes, members of the ‘rust’ lineage (Pucciniomycotina), are mycobionts of orchids. The mycobionts of 103 terrestrial and epiphytic orchid individuals, sampled in the tropical mountain rainforest of Southern Ecuador, were identified by sequencing the whole ITS1-5.8S-ITS2 region and part of 28S rDNA. Mycorrhizae of 13 orchid individuals were investigated by transmission electron microscopy. Simple septal pores and symplechosomes in the hyphal coils of mycorrhizae from four orchid individuals indicated members of Atractiellomycetes. Molecular phylogeny of sequences from mycobionts of 32 orchid individuals out of 103 samples confirmed Atractiellomycetes and the placement in Pucciniomycotina, previously known to comprise only parasitic and saprophytic fungi. Thus, our finding reveals these fungi, frequently associated to neotropical orchids, as the most basal living basidiomycetes involved in mycorrhizal associations of land plants.

Tracking the evolutionary history of Cortinarius species in section Calochroi, with transoceanic disjunct distributions.

BackgroundCortinarius species in section Calochroi display local, clinal and circumboreal patterns of distribution across the Northern Hemisphere where these ectomycorrhizal fungi occur with host trees throughout their geographical range within a continent, or have disjunct intercontinental distributions, the origins of which are not understood. We inferred evolutionary histories of four species, 1) C. arcuatorum, 2) C. aureofulvus, 3) C. elegantior and 4) C. napus, from populations distributed throughout the Old World, and portions of the New World (Central- and North America) based on genetic variation of 154 haplotype internal transcribed spacer (ITS) sequences from 83 population samples. By describing the population structure of these species across their geographical distribution, we attempt to identify their historical migration and patterns of diversification.ResultsModels of population structure from nested clade, demographic and coalescent-based analyses revealed genetically differentiated and geographically structured haplotypes in C. arcuatorum and C. elegantior, while C. aureofulvus showed considerably less population structure and C. napus lacked sufficient genetic differentiation to resolve any population structure. Disjunct populations within C. arcuatorum, C. aureofulvus and C. elegantior show little or no morphological differentiation, whereas in C. napus there is a high level of homoplasy and phenotypic plasticity for veil and lamellae colour. The ITS sequences of the type specimens of C. albobrunnoides and C. albobrunnoides var. violaceovelatus were identical to one another and are treated as one species with a wider range of geographic distribution under C. napus.ConclusionsOur results indicate that each of the Calochroi species has undergone a relatively independent evolutionary history, hypothesised as follows: 1) a widely distributed ancestral population of C. arcuatorum diverged into distinctive sympatric populations in the New World; 2) two divergent lineages in C. elegantior gave rise to the New World and Old World haplotypes, respectively; and 3) the low levels of genetic divergence within C. aureofulvus and C. napus may be the result of more recent demographic population expansions. The scenario of migration via the Bering Land Bridge provides the most probable explanation for contemporaneous disjunct geographic distributions of these species, but it does not offer an explanation for the low degree of genetic divergence between populations of C. aureofulvus and C. napus. Our findings are mostly consistent with the designation of New World allopatric populations as separate species from the European counterpart species C. arcuatorum and C. elegantior. We propose the synonymy of C. albobrunnoides, C. albobrunnoides var. violaceovelatus and C. subpurpureophyllus var. sulphureovelatus with C. napus. The results also reinforce previous observations that linked C. arcuatorum and C. aureofulvus displaying distributions in parts of North America and Europe. Interpretations of the population structure of these fungi suggest that host tree history has heavily influenced their modern distributions; however, the complex issues related to co-migration of these fungi with their tree hosts remain unclear at this time.

Phylogenetic relationships of European Phlegmacium species (Cortinarius, Agaricales)

Phylogenetic relationships of 54 European Phlegmacium species, including members of most of the sections of classical systematics, were studied, integrating macro-, micromorphological and chemical characters of the basidiomes, as well as molecular phylogenetic analysis of nuclear rDNA sequences. Microscopical structures of the basidiomes were studied by light microscopy. Basidiospore morphology was examined by scanning electron microscopy. Internal-transcribed spacers (ITS 1 and 2, including the 5.8S) and the D1/D2 (LSU) regions of nuclear rDNA were sequenced and analyzed with a Bayesian Markov chain Monte Carlo approach. Many subgroups detected by the molecular analysis are related to groups known from classical systematical concepts. Among others, these subgroups were significantly supported: i) a group containing most of the members of section Fulvi ss. Brandrud and the species Cortinarius arcuatorum, C. dibaphus and C. multiformis; ii) a group comprising taxa of section Calochroi ss. Brandrud and the species C. fulvocitrinus and C. osmophorus; iii) a group containing species of section Glaucopodes ss. Brandrud and C. caerulescens; iv) a group including members of section Phlegmacioides ss. Brandrud; v) a group that includes the species C. cephalixus, C. nanceiensis and C. mussivus. Stipe shape, color of flesh, pigment contents, KOH reaction on pileipellis and gelatinous layer, degree of development of a gelatinous layer on the pileipellis, and pileipellis structure were useful characters in delimiting subgroups in Phlegmacium, while basidiospore morphology was significant at species level. With the exception of C. glaucopus, C. infractus and C. scaurus, ITS and D1/D2 sequences obtained from collections of the same species from different geographical origins showed very little variation. Our molecular and morphological analyses suggest revisions of the traditional concepts of the subgenus Phlegmacium in Europe.

Phylogenetic diversity and structure of sebacinoid fungi associated with plant communities along an altitudinal gradient

To study the diversity and phylogenetic structure of Sebacinales communities from eight vegetation communities along an altitudinal gradient in the Bavarian Alps (Germany), we analysed 456 thalli or roots of plants. We detected 264 sebacinoid sequences, spanning the intergenic transcribed spacer region, 5.8S and D1/D2 regions of the nuclear rRNA gene, mostly using a nested PCR approach. Based on 97% sequence similarity, 73 Sebacinales molecular taxonomic units were found from 70 host species belonging to 44 plant families. Twenty-six molecular taxonomic units represented singletons, the most frequent of these being restricted exclusively to either wooded or grassland habitats. Although Sebacinales appear to occur in low abundance in plant roots, these microorganisms are phylogenetically diverse and widely spread in the ecosystems studied. Ordination analyses showed that land use, pH and humus content strongly influence the diversity and assembly of Sebacinales. In most cases, Sebacinales communities in ecosystems with extreme soil conditions or intensive land use exhibited significant phylogenetic clustering, whereas in undisturbed plant communities no trend was observed. These results suggest that ecosystem disturbance and environmental forces have an influence on the diversity and structure of Sebacinales community assembly over local and spatial scales.

Morphological and molecular phylogenetic studies in South American Cortinarius species

Thirty South American species of Cortinarius belonging to the subgenera Telamonia, Dermocybe, Myxacium, Phlegmacium, and Cystogenes were studied using an integrated approach that included morphological, anatomical, and ultrastructural data, and also molecular phylogenetic analysis of nuclear rDNA sequences. The micromorphology of the basidiomes was studied by light microscopy, and the principal structures were illustrated by line drawings. Basidiospore ornamentation was studied by scanning electron microscopy (SEM). Nuclear internal transcribed spacers (ITS, including the 5.8S gene) and the rDNA coding for the D1/D2 domains of the large ribosomal subunit (LSU) were sequenced and analysed using a Bayesian Markov chain Monte Carlo method to estimate phylogenetic relationships between the studied Cortinarius species. Morphology and anatomy of the pileus surface and basidiome pigmentation appeared to be the most useful characters to delimit some natural groups, whereas microcharacters related to the structure of pileus context, hymenophoral and stipe trama were of little taxonomic value. Basidiospore morphology and cheilocystidia seem to be taxonomically relevant at the species level. The following five infrageneric groups were supported by the morphological, chemical and molecular data: (1) Telamonia characterized by wide hyaline hyphae of the veil and by small basidiomes; (2) Dermocybe spp. with an epicutis as the most external layer of the pileus, and skyrin and hypericin pigments; (3) Dermocybe spp. with a thin viscid layer on the pileus, and endocrocin and dermolutein pigments; (4) Phlegmacium spp. characterized by a long and radicating stipe; and (5) Phlegmacium spp. that overlap in some macrocharacters with Telamonia species. Our analyses suggest that classification concepts based mainly on macromorphological characters are likely to lead to artificial grouping, whereas certain microscopical and chemical characters seem to be useful in constructing a more natural classification system for Cortinarius.

Morphology and molecules: the Sebacinales, a case study

Morphological and molecular discrepancies in the biodiversity of monophyletic groups are challenging. The intention of this study was to find out whether the high molecular diversity in Sebacinales can be verified by micromorphological characteristics. Therefore, we carried out molecular and morphological studies on all generic type species of Sebacinales and additional representative taxa. Our results encouraged us to disentangle some phylogenetic and taxonomic discrepancies and to improve sebacinalean classifications. This comprises generic circumscriptions and affiliations, as well as higher taxon groupings. At the family level, we redefined the Sebacinaceae, formerly the Sebacinales group A, and set it apart from the Sebacinales group B. For taxonomical purposes, it seems appropriate to refer Paulisebacina, Craterocolla, Chaetospermum, Globulisebacina, Tremelloscypha, and Sebacina to the Sebacinaceae and Piriformospora, and Serendipita to the Sebacinales group B. At the lower taxonomic level, we propose within the Sebacinaceae (1) to introduce Paulisebacina for Sebacina allantoidea, (2) to transfer Efibulobasidium rolleyi into a new monotypic genus, Globulisebacina, (3) to include Tremellostereum in Tremelloscypha, (4) to transfer Sebacina amesii into Tremelloscypha, (5) to combine S. helvelloides and S. concrescens in their own genus, Helvellosebacina, (6) to transfer Tremellodendron spp. into Sebacina, (7) to define S. epigaea s.str. without cystidia and flagelliform dikaryophyses, but with star-shaped resting spores, and (8) to separate S. cystidiata with simultaneously irregular germinating spores and inconspicuous cystidia, and S. flagelliformis with flagelliform dikaryophyses from S. epigaea s.str. Additional clades in Sebacina, based on molecular differences, cannot be distinguished morphologically at present.