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.     

Assessing the roles of wolves and dogs in livestock predation with suggestions for mitigating human–wildlife conflict and conservation of wolves

Predation on livestock is a cause of serious and long-lasting conflict between farmers and wildlife, promoting negative public attitudes and endangering conservation of large carnivores. However, while large carnivores, especially the grey wolf (Canis lupus), are often blamed for killing sheep and other farm animals, free-ranging dogs may also act as predators. To develop appropriate measures for livestock protection, reliable methods for identifying predator species are critical. Identification of predators from visual examination of livestock wounds can be ambiguous and genetic analysis is strongly preferable for accurate species determination. To estimate the proportion of wolves and dogs implicated in sheep predation, we developed a sensitive genetic assay to distinguish between wolves and domestic dogs. A total of 183 predator saliva samples collected from killed sheep in Estonia were analysed. The assay identified the predator species in 143 cases (78%). Sheep were most often killed by wolves (81%); however, predation by dogs was substantial (15%). We compared the molecular results with field observations conducted by local environmental officials and recorded some disagreement, with the latter underestimating the role of dogs. As predator saliva samples collected from prey are often of poor quality, we suggest using mitochondrial DNA as a primary tool to maximise the number of successfully analysed samples. We also suggest adopting forensic DNA analysis more widely in livestock predation assessments as a legislative measure since misidentification that is biased against wolves can be counterproductive for conservation by enhancing conflict with society and leading to increased culling and poaching.     

Biogenic Methane Cycling in a Laboratory Model of an Abandoned Bituminous Coal Mine

Microbial communities in decommissioned coal mines have the potential to promote methane generation. Here, two 1 m x 10 cm diameter column bioreactors designed to mimic an abandoned coal mine were monitored for a year, with zones of methanogenesis in the bottom, saturated waters and aerobic coal degradation and methane oxidation at the top. The resilience of aerobic methanotrophs to survive periods with low methane and oxygen conditions suggests methanotrophs may be useful in decreasing atmospheric methane fugitive emissions from decommissioned mines. When biogenic methane production from coal did occur, the rate was slow, ≤ 0.073 nmol CH₄/g coal/day.     

The influence of hydrogeological disturbance and mining on coal seam microbial communities

The microbial communities present in two underground coal mines in the Bowen Basin, Queensland, Australia, were investigated to deduce the effect of pumping and mining on subsurface methanogens and methanotrophs. The micro‐organisms in pumped water from the actively mined areas, as well as, pre‐ and post‐mining formation waters were analyzed using 16S rRNA gene amplicon sequencing. The methane stable isotope composition of Bowen Basin coal seam indicates that methanogenesis has occurred in the geological past. More recently at the mine site, changing groundwater flow dynamics and the introduction of oxygen in the subsurface has increased microbial biomass and diversity. Consistent with microbial communities found in other coal seam environments, pumped coal mine waters from the subsurface were dominated by bacteria belonging to the genera Pseudomonas and the family Rhodocyclaceae. These environments and bacterial communities supported a methanogen population, including Methanobacteriaceae, Methanococcaceae and Methanosaeta. However, one of the most ubiquitous micro‐organisms in anoxic coal mine waters belonged to the family ‘Candidatus Methanoperedenaceae’. As the Archaeal family ‘Candidatus Methanoperedenaceae’ has not been extensively defined, the one studied species in the family is capable of anaerobic methane oxidation coupled to nitrate reduction. This introduces the possibility that a methane cycle between archaeal methanogenesis and methanotrophy may exist in the anoxic waters of the coal seam after hydrogeological disturbance.     

Molecular heterogeneity of XY sex reversal in horses

Male-to-female 64,XY sex reversal is a frequently reported chromosome abnormality in horses. Despite this, the molecular causes of the condition are as yet poorly understood. This is partially because only limited molecular information is available for the horse Y chromosome (ECAY). Here, we used the recently developed ECAY map and carried out the first comprehensive study of the Y chromosome in XY mares (n=18). The integrity of the ECAY in XY females was studied by FISH and PCR using markers evenly distributed along the euchromatic region. The results showed that the XY sex reversal condition in horses has two molecularly distinct forms: (i) a Y-linked form that is characterized by Y chromosome deletions and (ii) a non-Y-linked form where the Y chromosome of affected females is molecularly the same as in normal males. Further analysis of the Y-linked form (13 cases) showed that the condition is molecularly heterogeneous: the smallest deletions spanned about 21 kb, while the largest involved the entire euchromatic region. Regardless of the size, all deletions included the SRY gene. We show that the deletions were likely caused by inter-chromatid recombination events between repeated sequences in ECAY. Further, we hypothesize that the occurrence of SRY-negative XY females in some species (horse, human) but not in others (pig, dog) is because of differences in the organization of the Y chromosome. Finally, in contrast to the Y-linked SRY-negative form of equine XY sex reversal, the molecular causes of SRY-positive XY mares (5 cases) remain as yet undefined.     

A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.     

Mitochondrial phylogeny of the Chrysisignita (Hymenoptera: Chrysididae) species group based on simultaneous Bayesian alignment and phylogeny reconstruction

The ignita species group within the genus Chrysis includes over 100 cuckoo wasp species, which all lead a parasitic lifestyle and exhibit very similar morphology. The lack of robust, diagnostic morphological characters has hindered phylogenetic reconstructions and contributed to frequent misidentification and inconsistent interpretations of species in this group. Therefore, molecular phylogenetic analysis is the most suitable approach for resolving the phylogeny and taxonomy of this group. We present a well-resolved phylogeny of the Chrysis ignita species group based on mitochondrial sequence data from 41 ingroup and six outgroup taxa. Although our emphasis was on European taxa, we included samples from most of the distribution range of the C. ignita species group to test for monophyly. We used a continuous mitochondrial DNA sequence consisting of 16S rRNA, tRNA(Val), 12S rRNA and ND4. The location of the ND4 gene at the 3' end of this continuous sequence, following 12S rRNA, represents a novel mitochondrial gene arrangement for insects. Due to difficulties in aligning rRNA genes, two different Bayesian approaches were employed to reconstruct phylogeny: (1) using a reduced data matrix including only those positions that could be aligned with confidence; or (2) using the full sequence dataset while estimating alignment and phylogeny simultaneously. In addition maximum-parsimony and maximum-likelihood analyses were performed to test the robustness of the Bayesian approaches. Although all approaches yielded trees with similar topology, considerably more nodes were resolved with analyses using the full data matrix. Phylogenetic analysis supported the monophyly of the C. ignita species group and divided its species into well-supported clades. The resultant phylogeny was only partly in accordance with published subgroupings based on morphology. Our results suggest that several taxa currently treated as subspecies or names treated as synonyms may in fact constitute separate species. Our study provides a solid basis for further systematic investigations of this enigmatic insect group.     

Towards global patterns in the diversity and community structure of ectomycorrhizal fungi

Global species richness patterns of soil micro-organisms remain poorly understood compared to macro-organisms. We use a global analysis to disentangle the global determinants of diversity and community composition for ectomycorrhizal (EcM) fungi-microbial symbionts that play key roles in plant nutrition in most temperate and many tropical forest ecosystems. Host plant family has the strongest effect on the phylogenetic community composition of fungi, whereas temperature and precipitation mostly affect EcM fungal richness that peaks in the temperate and boreal forest biomes, contrasting with latitudinal patterns of macro-organisms. Tropical ecosystems experience rapid turnover of organic material and have weak soil stratification, suggesting that poor habitat conditions may contribute to the relatively low richness of EcM fungi, and perhaps other soil biota, in most tropical ecosystems. For EcM fungi, greater evolutionary age and larger total area of EcM host vegetation may also contribute to the higher diversity in temperate ecosystems. Our results provide useful biogeographic and ecological hypotheses for explaining the distribution of fungi that remain to be tested by involving next-generation sequencing techniques and relevant soil metadata.