BioFun: Biodiversity in the dark: High-throughput sequence analyses of arctic fungal communities
Tedersoo L, Liiv I, Kivistik PA, Anslan S, Kõljalg U, Bahram M. 2016. Genomics and metagenomics technologies to recover ribosomal DNA and single-copy genes from old fruitbody and ectomycorrhiza specimens. MycoKeys 13: 1-20.
Abstract. High-throughput sequencing (HTS) has become a standard technique for genomics, metagenomics and taxonomy, but these analyses typically require large amounts of high-quality DNA that is difficult to obtain from uncultivable organisms including fungi with no living culture or fruit-body representatives. By using 1 ng DNA and low coverage Illumina HiSeq HTS, we evaluated the usefulness of genomics and metagenomics tools to recover fungal barcoding genes from old and problematic specimens of fruit-bodies and ectomycorrhizal (EcM) root tips. Ribosomal DNA and single-copy genes were successfully recovered from both fruit-body and EcM specimens typically <10 years old (maximum, 17 years). Samples with maximum obtained DNA concentration <0.2 ng µl-1 were sequenced poorly. Fungal rDNA molecules assembled from complex mock community and soil revealed a large proportion of chimeras and artefactual consensus sequences of closely related taxa. Genomics and metagenomics tools enable recovery of fungal genomes from very low initial amounts of DNA from fruit-bodies and ectomycorrhizas, but these genomes include a large proportion of prokaryote and other eukaryote DNA. Nonetheless, the recovered scaffolds provide an important source for phylogenetic and phylogenomic analyses and mining of functional genes. PDF
Tedersoo L, Anslan S, Bahram M, Põlme S, Riit T, Liiv I, Kõljalg U, Kisand V, Nilsson RH, Bork P, Hildebrand F, Abarenkov K. 2015. Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi. MycoKeys 10: 1-43.
Abstract. Rapid development of high-throughput (HTS) molecular identification methods has revolutionized our knowledge about taxonomic diversity and ecology of fungi. However, PCR-based methods exhibit multiple technical shortcomings that may bias our understanding of the fungal kingdom. This study was initiated to quantify potential biases in fungal community ecology by comparing the relative performance of amplicon-free shotgun metagenomics and amplicons of nine primer pairs over seven nuclear ribosomal DNA (rDNA) regions often used in metabarcoding analyses. The internal transcribed spacer (ITS) barcodes ITS1 and ITS2 provided greater taxonomic and functional resolution and richness of operational taxonomic units (OTUs) at the 97% similarity threshold compared to barcodes located within the ribosomal small subunit (SSU) and large subunit (LSU) genes. All barcode-primer pair combinations provided consistent results in ranking taxonomic richness and recovering the importance of floristic variables in driving fungal community composition in soils of Papua New Guinea. The choice of forward primer explained up to 2.0% of the variation in OTU-level analysis of the ITS1 and ITS2 barcode data sets. Across the whole data set, barcode-primer pair combination explained 37.6–38.1% of the variation, which surpassed any environmental signal. Overall, the metagenomics data set recovered a similar taxonomic overview, but resulted in much lower fungal rDNA sequencing depth, inability to infer OTUs, and high uncertainty in identification. We recommend the use of ITS2 or the whole ITS region for metabarcoding and we advocate careful choice of primer pairs in consideration of the relative proportion of fungal DNA and expected dominant groups. PDF
Tedersoo L, Bahram M, Põlme S, Anslan S, Riit T, Kõljalg U, Nilsson RH, Hildebrand F, Abarenkov K. 2015. Response to comment on “Global diversity and geography of soil fungi”: Analytical biases in microbial diversity studies. Science 359: 936.
Abstract. Schadt and Rosling (Technical Comment, 26 June 2015, p. 1438) argue that primer-template mismatches neglected the fungal class Archaeorhizomycetes in a global soil survey. Amplicon-based metabarcoding of nine barcode-primer pair combinations and polymerase chain reaction (PCR)–free shotgun metagenomics revealed that barcode and primer choice and PCR bias drive the diversity and composition of microorganisms in general, but the Archaeorhizomycetes were little affected in the global study. We urge that careful choice of DNA markers and primers is essential for ecological studies using high-throughput sequencing for identification. PDF
Tedersoo L, Ramirez KS, Nilsson RH, Kaljuvee A, Kõljalg U, Abarenkov K. 2015. Standardizing metadata and taxonomic identification in metabarcoding studies. GigaScience 4:34.
Abstract. High-throughput sequencing-based metabarcoding studies produce vast amounts of ecological data, but a lack of consensus on standardization of metadata and how to refer to the species recovered severely hampers reanalysis and comparisons among studies. Here we propose an automated workflow covering data submission, compression, storage and public access to allow easy data retrieval and inter-study communication. Such standardized and readily accessible datasets facilitate data management, taxonomic comparisons and compilation of global metastudies. PDF
Mundra S, Halvorsen R, Kauserud H, Müller E, Vik U, Eidesen P. 2015. Arctic fungal communities associated with roots of Bistorta vivipara do not respond to the same fine-scale edaphic gradients as the aboveground vegetation. New Phytol. 205: 1587–1597.
Abstract. Soil conditions and microclimate are important determinants of the fine-scale distribution of plant species in the Arctic, creating locally heterogeneous vegetation. We hypothesize that root-associated fungal (RAF) communities respond to the same fine-scale environmental gradients as the aboveground vegetation, creating a coherent pattern between aboveground vegetation and RAF. We explored how RAF communities of the ectomycorrhizal (ECM) plant Bistorta vivipara and aboveground vegetation structure of arctic plants were affected by biotic and abiotic variables at 0.3–3.0-m scales. RAF communities were determined using pyrosequencing. Composition and spatial structure of RAF and aboveground vegetation in relation to collected biotic and abiotic variables were analysed by ordination and semi-variance analyses. The vegetation was spatially structured along soil C and N gradients, whereas RAF lakked significant spatial structure. A weak relationship between RAF community composition and the cover of two ECM plants, B. vivipara and S. polaris, was found, and RAF richness increased with host root length and root weight. Results suggest that the fine-scale spatial structure of RAF communities of B. vivipara and the aboveground vegetation are driven by different factors. At fine spatial scales, neighbouring ECM plants may affect RAF community composition, whereas soil nutrients gradients structure the vegetation. PDF
Davey M, Blaalid R, Vik U, Carlsen T, Kauserud H, Bronken Eidesen P. 2015. Primary succession of Bistorta vivipara (L.) Delabre (Polygonaceae) root associated fungi mirrors plant succession in two glacial chronosequences. Environmental Microbiology, online early.
Abstract. Glacier chronosequences are important sites for primary succession studies and have yielded well-defined primary succession models for plants that identify environmental resistance as an important determinant of the successional trajectory. Whether plant-associated fungal communities follow those same successional trajectories and also respond to environmental resistance is an open question. In this study, 454 amplicon pyrosequencing was used to compare the root-associated fungal communities of the ectomycorrhizal (ECM) herb Bistorta vivipara along two primary succession gradients with different environmental resistance (alpine versus arctic) and different successional trajectories in the vascular plant communities (directional replacement versus directional non-replacement). At both sites, the root-associated fungal communities were dominated by ectomycorrhizal basidiomycetes and community composition shifted with increasing time since deglaciation. However, the fungal community's successional trajectory mirrored the pattern observed in the surrounding plant community at both sites: the alpine site displayed a directional-replacement successional trajectory, and the arctic site displayed a directional-non-replacement successional trajectory. This suggests that, like in plant communities, environmental resistance is key in determining succession patterns in root-associated fungi. The need for further replicated study, including in other host species, is emphasized. PDF
Tedersoo L, Bahram M, Põlme S, Kõljalg U, Yorou NS, Wijesundera R, Villarreal-Ruiz L, Vasco-Palacios A, Quang Thu P, Suija A, Smith ME, Sharp C, Saluveer E, Saitta A, Ratkowsky D, Pritsch K, Riit T, Põldmaa K, Piepenbring M, Phosri C, Peterson M, Parts K, Pärtel K, Otsing E, Nouhra E, Njouonkou AL, Nilsson RH, Morgado LN, Mayor J, May TW, Kohout P, Hosaka K, Hiiesalu I, Henkel TW, Harend H, Guo L, Greslebin A, Grelet G, Geml J, Gates G, Dunstan W, Dunk C, Drenkhan R, Dearnaley J, De Kesel A, Dang T, Chen X, Buegger F, Brearley FQ, Bonito G, Anslan S, Abell S, Abarenkov K. 2014. Global diversity and geography of soil fungi. Science 346: 1078.
Abstract. 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. PDF
Tedersoo L, Bahram M, Ryberg M, Otsing E, Kõljalg U, Abarenkov K. 2014. Global biogeography of the ectomycorrhizal /sebacina lineage (Fungi, Sebacinales) as revealed from comparative phylogenetics analyses. Mol. Ecol. 23: 4168–4183.
Abstract. Compared with plants and animals, large-scale biogeographic patterns of microbes including fungi are poorly understood. By the use of a comparative phylogenetic approach and ancestral state reconstructions, we addressed the global biogeography, rate of evolution and evolutionary origin of the widely distributed ectomycorrhizal (EcM) /sebacina lineage that forms a large proportion of the Sebacinales order. We downloaded all publicly available internal transcribed spacer (ITS) sequences and metadata and supplemented sequence information from three genes to construct dated phylogenies and test biogeographic hypotheses. The /sebacina lineage evolved 45–57 Myr ago that groups it with relatively young EcM taxa in other studies. The most parsimonious origin for /sebacina is inferred to be North American temperate coniferous forests. Among biogeographic traits, region and biome exhibited stronger phylogenetic signal than host family. Consistent with the resource availability (environmental energy) hypothesis, the ITS region is evolving at a faster rate in tropical than nontropical regions. Most biogeographic regions exhibited substantial phylogenetic clustering suggesting a strong impact of dispersal limitation over a large geographic scale. In northern Holarctic regions, however, phylogenetic distances and phylogenetic grouping of isolates indicate multiple recent dispersal events. PDF