Kadri Põldmaa

Towards a unified paradigm for sequence‐based identification of fungi

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third‐party annotation effort. We introduce the term ‘species hypothesis’ (SH) for the taxa discovered in clustering on different similarity thresholds (97–99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web‐based sequence management system in UNITE.

Global diversity and geography of soil fungi

Introduction The kingdom Fungi is one of the most diverse groups of organisms on Earth, and they are integral ecosystem agents that govern soil carbon cycling, plant nutrition, and pathology. Fungi are widely distributed in all terrestrial ecosystems, but the distribution of species, phyla, and functional groups has been poorly documented. On the basis of 365 global soil samples from natural ecosystems, we determined the main drivers and biogeographic patterns of fungal diversity and community composition. Direct and indirect effects of climatic and edaphic variables on plant and fungal richness. Line thickness corresponds to the relative strength of the relationships between the variables that affect species richness. Dashed lines indicate negative relationships. MAP, mean annual precipitation; Fire, time since last fire; Dist. equator, distance from the equator; Ca, soil calcium concentration; P, soil phosphorus concentration; pH, soil pH. Rationale We identified soil-inhabiting fungi using 454 Life Sciences (Branford, CN) pyrosequencing and through comparison against taxonomically and functionally annotated sequence databases. Multiple regression models were used to disentangle the roles of climatic, spatial, edaphic, and floristic parameters on fungal diversity and community composition. Structural equation models were used to determine the direct and indirect effects of climate on fungal diversity, soil chemistry, and vegetation. We also examined whether fungal biogeographic patterns matched paradigms derived from plants and animals—namely, that species’ latitudinal ranges increase toward the poles (Rapoport’s rule) and diversity increases toward the equator. Last, we sought group-specific global biogeographic links among major biogeographic regions and biomes using a network approach and area-based clustering. Results Metabarcoding analysis of global soils revealed fungal richness estimates approaching the number of species recorded to date. Distance from equator and mean annual precipitation had the strongest effects on richness of fungi, including most fungal taxonomic and functional groups. Diversity of most fungal groups peaked in tropical ecosystems, but ectomycorrhizal fungi and several fungal classes were most diverse in temperate or boreal ecosystems, and many fungal groups exhibited distinct preferences for specific edaphic conditions (such as pH, calcium, or phosphorus). Consistent with Rapoport’s rule, the geographic range of fungal taxa increased toward the poles. Fungal endemicity was particularly strong in tropical regions, but multiple fungal taxa had cosmopolitan distribution. Conclusions Climatic factors, followed by edaphic and spatial patterning, are the best predictors of soil fungal richness and community composition at the global scale. Richness of all fungi and functional groups is causally unrelated to plant diversity, with the exception of ectomycorrhizal root symbionts, suggesting that plant-soil feedbacks do not influence the diversity of soil fungi at the global scale. The plant-to-fungi richness ratio declined exponentially toward the poles, indicating that current predictions—assuming globally constant ratios—overestimate fungal richness by 1.5- to 2.5-fold. Fungi follow similar biogeographic patterns as plants and animals, with the exception of several major taxonomic and functional groups that run counter to overall patterns. Strong biogeographic links among distant continents reflect relatively efficient long-distance dispersal compared with macro-organisms. Fungi play major roles in ecosystem processes, but the determinants of fungal diversity and biogeographic patterns remain poorly understood. Using DNA metabarcoding data from hundreds of globally distributed soil samples, we demonstrate that fungal richness is decoupled from plant diversity. The plant-to-fungus richness ratio declines exponentially toward the poles. Climatic factors, followed by edaphic and spatial variables, constitute the best predictors of fungal richness and community composition at the global scale. Fungi show similar latitudinal diversity gradients to other organisms, with several notable exceptions. These findings advance our understanding of global fungal diversity patterns and permit integration of fungi into a general macroecological framework. Global metagenomics detects hotspots of fungal diversity and macroecological patterns and indicates that plant and fungal diversity are uncoupled. [Also see Perspective by Wardle and Lindahl] Assessing fungal diversity worldwide Fungi are hyperdiverse but poorly known, despite their ecological and economic impacts. Tedersoo et al. collected nearly 15,000 topsoil samples from 365 sites worldwide and sequenced their genomes (see the Perspective by Wardle and Lindahl). Overall, they found a striking decline in fungal species richness with distance from the equator. For some specialist groups though, diversity depended more on the abundance of host plants than host diversity or geography. The findings reveal a huge gap between known and described species and the actual numbers of distinct fungi in the worlds soils. Science, this issue 10.1126/science.1256688; see also p. 1052

PlutoF—a Web Based Workbench for Ecological and Taxonomic Research, with an Online Implementation for Fungal ITS Sequences

DNA sequences accumulating in the International Nucleotide Sequence Databases (INSD) form a rich source of information for taxonomic and ecological meta-analyses. However, these databases include many erroneous entries, and the data itself is poorly annotated with metadata, making it difficult to target and extract entries of interest with any degree of precision. Here we describe the web-based workbench PlutoF, which is designed to bridge the gap between the needs of contemporary research in biology and the existing software resources and databases. Built on a relational database, PlutoF allows remote-access rapid submission, retrieval, and analysis of study, specimen, and sequence data in INSD as well as for private datasets though web-based thin clients. In contrast to INSD, PlutoF supports internationally standardized terminology to allow very specific annotation and linking of interacting specimens and species. The sequence analysis module is optimized for identification and analysis of environmental ITS sequences of fungi, but it can be modified to operate on any genetic marker and group of organisms. The workbench is available at http://plutof.ut.ee.

The phylogeny and taxonomy of genera Cystoderma and Cystodermella (Agaricales) based on nuclear ITS and LSU sequences

Species delimitation in Cystoderma and Cystodermella was evaluated based on ITS and LSU rDNA sequences as well as morphological data. Two species of Cystoderma are synonymised with C. carcharias and three species with C. jasonis, distinguishing the synonymised taxa as varieties of these accepted species. Analyses of partial LSU rDNA sequences revealed Cystoderma and Cystodermella as distinct monophyletic genera, with Ripartitella representing a well-supported sister group of the latter. Phaeolepiota aurea represents either an unsupported sister group or member of Cystoderma in the phylogenies based on LSU and ITS sequences rDNA data, respectively. The tribe Cystodermateae sensu Singer did not appear monophyletic according to analyses of LSU sequences. On the basis of these data, the phylogenetic relationships among most of the analyzed genera could not be resolved unequivocally.

Evidence for the polyphyly of Encoelia and Encoelioideae with reconsideration of respective families in Leotiomycetes

This study focuses on the genus Encoelia and the subfamily Encoelioideae in the morphologically and ecologically diverse Helotiales. The 28S and 18S rDNA as well as tef1, rpb1 and rpb2 were sequenced for 70 species. Phylogenetic analyses revealed Encoelia and Encoelioideae to be highly polyphyletic, with species distributed among eight major lineages. Encoelia fascicularis and E. pruinosa belonged to Sclerotiniaceae and were combined in a new genus, Sclerencoelia. Rutstroemiaceae comprised E. tiliacea and Dencoeliopsis johnstonii, both accepted in Rutstroemia. The type of Encoelia, E. furfuracea, was closely related to species of Velutarina, Cenangiopsis and Crumenulopsis. These species together with members of Hemiphacidiaceae formed a clade conforming to the emended concept of Cenangiaceae, introduced here. Another resurrected family, Cordieritidaceae, comprised E. fimbriata, E. heteromera and species of Ameghiniella, Cordierites, Diplocarpa and Ionomidotis, characterised by inamyloid asci and a positive ionomidotic reaction. Encoelia glauca showed closest affinities with Chlorociboria species in Chlorociboriaceae. A new genus, Xeropilidium, with sporodochial and pycnidial synanamorphs, was described for the distinct encoelioid member of the Chaetomellaceae, previously known as E. fuckelii. Morphological and ecological synapomorphies were distinguished from convergent characters to delimit monophyletic taxa including encoelioid fungi. Incorporation of public sequences from various biological samples in ITS rDNA analyses allowed identification of sequenced organisms at species, genus, or family level and added information on the ecology of seversal taxa. Members of Cenangiaceae appeared to be widespread as endophytes. Inclusion of encoelioid genera in Chaetomellaceae and Sclerotiniaceae added xylicolous saprotrophs to these families.

Species diversity in the Antrodia crassa group (Polyporales, Basidiomycota)

Antrodia is a polyphyletic genus, comprising brown-rot polypores with annual or short-lived perennial resupinate, dimitic basidiocarps. Here we focus on species that are closely related to Antrodia crassa, and investigate their phylogeny and species delimitation using geographic, ecological, morphological and molecular data (ITS and LSU rDNA, tef1). Phylogenetic analyses distinguished four clades within the monophyletic group of eleven conifer-inhabiting species (five described herein): (1)A. crassa s. str. (boreal Eurasia), Antrodia cincta sp. nova (North America) and Antrodia cretacea sp. nova (holarctic), all three being characterized by inamyloid skeletal hyphae that dissolve quickly in KOH solution; (2) Antrodia ignobilis sp. nova, Antrodia sitchensis and Antrodia sordida from North America, and Antrodia piceata sp. nova (previously considered conspecific with A. sitchensis) from Eurasia, possessing amyloid skeletal hyphae; (3) Antrodia ladiana sp. nova from the southern part of the USA, Antrodia pinea from East Asia, and Antrodia ferox - so far known from subtropical North America, but here reported also from Eurasia. These three species have inamyloid hyphae and narrow basidiospores; (4) the North American Antrodia pini-cubensis, sharing similar morphological characters with A. pinea, forming a separate clade. The habitat data indicate that several species are threatened by intensive forestry.

Bacterial Communities in Boreal Forest Mushrooms Are Shaped Both by Soil Parameters and Host Identity

Despite recent advances in understanding the microbiome of eukaryotes, little is known about microbial communities in fungi. Here we investigate the structure of bacterial communities in mushrooms, including common edible ones, with respect to biotic and abiotic factors in the boreal forest. Using a combination of culture-based and Illumina high-throughput sequencing, we characterized the bacterial communities in fruitbodies of fungi from eight genera spanning four orders of the class Agaricomycetes (Basidiomycota). Our results revealed that soil pH followed by fungal identity are the main determinants of the structure of bacterial communities in mushrooms. While almost half of fruitbody bacteria were also detected from soil, the abundance of several bacterial taxa differed considerably between the two environments. The effect of host identity was significant at the fungal genus and order level and could to some extent be ascribed to the distinct bacterial community of the chanterelle, representing Cantharellales—the earliest diverged group of mushroom-forming basidiomycetes. These data suggest that besides the substantial contribution of soil as a major taxa source of bacterial communities in mushrooms, the structure of these communities is also affected by the identity of the host. Thus, bacteria inhabiting fungal fruitbodies may be non-randomly selected from environment based on their symbiotic functions and/or habitat requirements.

Two new Neuratelia Rondani (Diptera, Mycetophilidae) species from Western Palaearctic: a case of limited congruence between morphology and DNA sequence data.

Abstract Two new Mycetophilidae species, Neuratelia jabalmoussae sp. n. and Neuratelia salmelai sp. n. are described on the basis of material collected from Lebanon, Estonia and Finland. Detailed figures of male terminalia and photographs of general facies are provided along with discussions of their morphological distinction from sibling species. For the first time molecular characters are used to distinguish new fungus gnat species. Molecular analysis relies on cytochrome oxidase subunit one (COI) but has additionally been corroborated by information from the 28S and ITS2 regions of nuclear ribosomal DNA. Situations where morphological and molecular data provide conflicting evidence for species delimitation are discussed. A new country record from Georgia is provided for Neuratelia caucasica.

The Mycetophilaruficollis Meigen (Diptera, Mycetophilidae) group in Europe: elucidating species delimitation with COI and ITS2 sequence data.

Abstract European species of the Mycetophila ruficollis group are compared on the basis of morphology and sequences of mitochondrial cytochrome oxidase subunit one (COI) and the ITS2 region of nuclear ribosomal DNA. The study represents the first evaluation of morphology-based species delimitation of closely related fungus gnat species by applying molecular information. Detailed descriptions and illustrations of the male terminalia are presented along with a key for the identification of all nine European species of the group. Phylogenetic analyses of molecular data generally supported the morphological species discrimination. The barcoding region of COI superseded ITS2 rDNA in resolving species. In the COI barcoding region interspecific differences ranged from 2.9 to 10.6% and the intraspecific distance from 0.08 to 0.8%. Only COI data distinguished between the similar and closely related Mycetophila ichneumonea and Mycetophila uninotata of which the latter was observed to include cryptic species. The host range of some species is suggested to be narrower than previously considered and to depend on the forest type. Presented evidence indicates the importance of analysing sequence data of morphologically very similar mycetophages reared from identified host fungi for elucidating species delimitation as well as their geographic and host ranges. New country records, viz. Estonia for Mycetophila evanida, Georgia for Mycetophila ichneumonea, Mycetophila idonea and Mycetophila ruficollis, and Norway for Mycetophila strobli, widen the known distribution ranges of these species.

Polyphagy on unpredictable resources does not exclude host specialization: insects feeding on mushrooms

The degree of ecological specialization plays a crucial role in shaping the structure and functioning of communities. However, comparing specialization within and among groups of organisms is complicated by both methodological issues and conceptual and terminological inconsistencies. Environmental predictability has been considered a key determinant of specialization though empirical evidence is still limited. Fungi and their insect consumers provide a poorly studied but promising system to measure host specialization and test the predictability hypothesis. In this study, we systematically sampled mushrooms in North European boreal forest, and reared total samples of fungivores colonizing the fruitbodies. Due to the unpredictable nature of mushrooms as a resource, low levels of host specialization can be predicted for these insects, which have indeed widely been considered polyphagous. Contrary to expectations, the majority of the studied fungus gnats were found not to exploit their host taxa indiscriminately. Not only were some mushroom taxa never colonized, the infestation rate of acceptable hosts also differed in most of these fungivores. Gnat species themselves formed continua with respect to the estimates of the degree of specialization, derived from parametric individual-based analyses of presence-absence data. In most cases, host use was best explained by models in which the hosts were classified at genus level, with limited support to specialization to particular host species, families, or orders. Indeed, most of the common fungivores appeared to preferentially use various species from one or a few mushroom genera while occasionally feeding on members of other host taxa. This pattern has likely evolved as a compromise between selective forces stemming from host unpredictability and taxon-specific chemical profiles of the mushrooms. Our study highlights the multidimensional nature of ecological specialization: a high number of acceptable hosts does not preclude considerable discrimination among members of the available resource pool. Such situations can only be revealed by individual-based analyses capable of capturing differences in partner-to-partner interaction intensities.