A systematic survey of regional multi-taxon biodiversity: evaluating strategies and coverage.

BACKGROUND: In light of the biodiversity crisis and our limited ability to explain variation in biodiversity, tools to quantify spatial and temporal variation in biodiversity and its underlying drivers are critically needed. Inspired by the recently published ecospace framework, we developed and tested a sampling design for environmental and biotic mapping. We selected 130 study sites (40 × 40 m) across Denmark using stratified random sampling along the major environmental gradients underlying biotic variation. Using standardized methods, we collected site species data on vascular plants, bryophytes, macrofungi, lichens, gastropods and arthropods. To evaluate sampling efficiency, we calculated regional coverage (relative to the known species number per taxonomic group), and site scale coverage (i.e., sample completeness per taxonomic group at each site). To extend taxonomic coverage to organisms that are difficult to sample by classical inventories (e.g., nematodes and non-fruiting fungi), we collected soil for metabarcoding. Finally, to assess site conditions, we mapped abiotic conditions, biotic resources and habitat continuity. RESULTS: Despite the 130 study sites only covering a minute fraction (0.0005%) of the total Danish terrestrial area, we found 1774 species of macrofungi (54% of the Danish fungal species pool), 663 vascular plant species (42%), 254 bryophyte species (41%) and 200 lichen species (19%). For arthropods, we observed 330 spider species (58%), 123 carabid beetle species (37%) and 99 hoverfly species (33%). Overall, sample coverage was remarkably high across taxonomic groups and sufficient to capture substantial spatial variation in biodiversity across Denmark. This inventory is nationally unprecedented in detail and resulted in the discovery of 143 species with no previous record for Denmark. Comparison between plant OTUs detected in soil DNA and observed plant species confirmed the usefulness of carefully curated environmental DNA-data. Correlations among species richness for taxonomic groups were predominantly positive, but did not correlate well among all taxa suggesting differential and complex biotic responses to environmental variation. CONCLUSIONS: We successfully and adequately sampled a wide range of diverse taxa along key environmental gradients across Denmark using an approach that includes multi-taxon biodiversity assessment and ecospace mapping. Our approach is applicable to assessments of biodiversity in other regions and biomes where species are structured along environmental gradient.

Resurrection of Cortinarius coalescens: taxonomy, chemistry, and ecology.

Cortinarius coalescens Kärcher & Seibt is a rare European species of the subgenus Phlegmacium, section Phlegmacioides, neglected in recent molecular studies. New primers (CortF and CortR) designed for species in the section Phlegmacioides allowed to obtain ITS rDNA sequence data from the holotype collection of C. coalescens; according to the results, this epithet has priority over C. crassorum Rob. Henry ex Rob. Henry, C. pardinus Reumaux, and C. parargutus Bidaud, Moënne-Locc. & Reumaux. Morphological and ecological observations on recent collections of C. coalescens from the Czech Republic in comparison with the co-occurring C. largus are discussed. Nomenclatural and taxonomic comments on C. tomentosus Rob. Henry, C. balteatotomentosus Rob. Henry, and C. subtomentosus Reumaux are also provided. So far, C. coalescens is known with certainty from Germany, France, and the Czech Republic, where it grows in deciduous forests on acid to neutral soils. Arsenic and its compounds were determined in C. coalescens and related species of the section Phlegmacioides: C. largus, C. pseudodaulnoyae, and C. variecolor. Total arsenic concentrations were in the range 3.6-30.2 mg kg-1 (dry matter) and arsenobetaine was the major arsenic compound.

Unexpectedly High Beta-Diversity of Root-Associated Fungal Communities in the Bolivian Andes.

Bolivia is one of the most biologically diverse countries on the planet. Between the Andes and the Amazon drainage basin spans the Yungas, a vast forested region shown to be extremely species rich in macro-organisms. However, it remains unclear whether this high diversity is also reflected in microbial diversity. Here we assess the genetic, taxonomic and functional diversity of root-associated fungi surrounding Cinchona calisaya trees, a typical element of the intermediate altitudes of the Bolivian Yungas. We determine the relative effects of edaphic properties, climate, and geography in regulating fungal community assembly. We show that α-diversity for these fungal communities was similar to temperate and arid ecosystems, averaging 90.1 operational taxonomic units (OTUs) per sample, with reads predominantly assigned to the Ascomycota phylum and with a saprotrophic lifestyle. ß-diversity was calculated as the distance-decay rate, and in contrast to α-diversity, was exceptionally high with a rate of -0.407. Soil properties (pH and P) principally regulated fungal community assembly in an analogous manner to temperate environments, with pH and phosphorus explaining 7.8 and 7.2% of community variation respectively. Surprisingly, altitude does not influence community formation, and there is limited evidence that climate (precipitation and temperature) play a role. Our results suggest that sampling should be performed over a wide geographical and environmental range in order to capture the full root-associated fungal diversity in subtropical regions. This study sheds further light on the diversity and distribution of the world's "hidden biodiversity."

A three-gene phylogeny of the Mycena pura complex reveals 11 phylogenetic species and shows ITS to be unreliable for species identification.

Phylogenetic analyses of Mycena sect. Calodontes using ITS previously suggested ten cryptic monophyletic ITS lineages within the Mycena pura morphospecies. Here, we compare ITS data (645 bp incl. gaps) from 46 different fruit bodies that represent the previously described ITS diversity with partial tEF-1-α (423 bp) and RNA polymerase II (RPB1) (492 bp) sequence data to test the genealogical concordance. While neither of the markers were in complete topological agreement, the branches differing between the tEF and RPB1 trees had a low bootstrap (<50) support, and the partition homogeneity incongruence length difference (ILD) tests were not significant. ILD tests revealed significant discordances between ITS and the tEF and RPB1 markers in several lineages. And our analyses suggested recombination between ITS1 and ITS2, most pronounced in one phylospecies that was identical in tEF and RPB1. Based on the agreement between tEF and RPB1, we defined 11 mutually concordant terminal clades as phylospecies inside the M. pura morphospecies; most of them cryptic. While neither of the markers showed an unequivocal barcoding gap between inter- and intraspecific diversity, the overlap was most pronounced for ITS (intraspecific diversity 0-3.5 %, interspecific diversity 0.4 %-8.8 %). A clustering analysis on tEF separated at a 1.5 % level returned all phylogenetic species as Operational Taxonomic Units (OTUs), while ITS at both a 1.5 % level and at a 3 % threshold level not only underestimated diversity as found by the tEF and RPB1, but also identified an OTU which was not a phylogenetic species. Thus, our investigation does not support the universal suitability of ITS for species recognition in particular, and emphasises the general limitation of single gene analyses combined with single percentage separation values.

Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples.

We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFℓSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput.

A simple method for validation and verification of pipettes mounted on automated liquid handlers.

We have implemented a simple, inexpensive, and fast procedure for validation and verification of the performance of pipettes mounted on automated liquid handlers (ALHs) as necessary for laboratories accredited under ISO 17025. A six- or seven-step serial dilution of OrangeG was prepared in quadruplicates in a flat-bottom 96-well microtiter plate, manually using calibrated pipettes. Each pipette of the liquid handler (1-8) dispensed a selected volume (1-200 µL) of OrangeG eight times into the wells of the microtiter plate. All wells contained a total of 200 µL liquid. The absorbance was read, and the dispensed volume of each pipette was calculated based on a plot of volume and absorbance of a known set of OrangeG dilutions. Finally, the percent inaccuracy (%d) and the imprecision (%CV) of each pipette were calculated. Using predefined acceptance criteria, each pipette was then either approved or failed. Failed pipettes were either repaired or the volume deviation was compensated for by applying a calibration curve in the liquid-handler software. We have implemented the procedure on a Sias Xantus, an MWGt TheONYX, four Tecan Freedom EVO, a Biomek NX Span-8, and four Biomek 3000 robots, and the methods are freely available. In conclusion, we have set up a simple, inexpensive, and fast solution for the continuous validation of ALHs used for accredited work according to the ISO 17025 standard. The method is easy to use for aqueous solutions but requires a spectrophotometer that can read microtiter plates.

Seven new calochroid and fulvoid species of Cortinarius.

We describe seven new European species of Cortinarius. All species are based on analyses of morphological and DNA sequence data. They all belong to a well-supported clade comprising most species traditionally treated in Cortinarius subgenus Phlegmacium sections Fulvi and Calochroi (i.e. the/Calochroi clade). All taxa are either fulvoid (containing anthraquinoid pigments) or calochroid (without these pigments). Morphological and ecological data are presented for all species and compared with similar species. A dichotomous key is presented for C. calochrous and similar species, including all six newly described calochroid species. The calochroid species C. albertii, C. chailluzii, C. cisticola, C. sancti-felicis, C. selandicus and C. vesterholtii spp. nov., and the fulvoid species C. langeorum sp. nov. are described.

Phylogenetic relationships of Termitomyces and related taxa.

Phylogenetic relationships of termitophilic fungi were estimated with Bayesian as well as other phylogenetic methods from partial sequences of the nuclear encoded large subunit ribosomal DNA (nLSU-rDNA) and the mitochondrial encoded small subunit ribosomal DNA (mtSSU-rDNA). Sequences were obtained from basidiomes covering the morphological, taxonomical, and geographical span of termitophilic mushroom-forming fungi, and analysed together with sequences from termite nests and termite guts from most known genera of fungus growing termites from geographically diverse regions. Topologies of trees resulting from the combined analyses of the two ribosomal genes generally show no positive conflicts with those obtained from separate analyses. We show that termitophilic fungi constitute a strongly supported monophyletic group within lyophylloid species. The genera Sinotermitomyces and Podabrella are derived within Termitomyces, and do not form monophyletic groups. Identical sequences were frequently found among samples of basidiomes from the same continents and among fungi utilized by termites from the same continent. However, only two sequences were identical between basidiome samples and termite nest/gut samples suggesting fruiting species do not form a representative sample of termitophilic fungi. No sequences were identical between samples from Asia and Africa indicating some geographic differentiation between these continents.