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High-throughput metabarcoding studies on fungi and other eukaryotic microorganisms are rapidly becoming more frequent and more complex, requiring researchers to handle ever increasing amounts of raw sequence data. Here, we provide a flexible pipeline for pruning and analyzing fungal barcode (ITS rDNA) data generated as paired-end reads on Illumina MiSeq sequencers. The pipeline presented includes specific steps fine-tuned for ITS, that are mostly missing from pipelines developed for prokaryotes. It (1) employs state of the art programs and follows best practices in fungal high-throughput metabarcoding; (2) consists of modules and scripts easily modifiable by the user to ensure maximum flexibility with regard to specific needs of a project or future methodological developments; and (3) is straightforward to use, also in classroom settings. We provide detailed descriptions and revision techniques for each step, thus giving the user maximum control over data treatment and avoiding a black-box approach. Employing this pipeline will improve and speed up the tedious and error-prone process of cleaning fungal Illumina metabarcoding data.
Smut fungi are well-suited to investigate the ecology and evolution of plant pathogens, as they are strictly biotrophic, yet cultivable on media. Here we report the genome sequence of Melanopsichium pennsylvanicum, closely related to Ustilago maydis and other Poaceae-infecting smuts, but parasitic to a dicot plant. To explore the evolutionary patterns resulting from host adaptation after this huge host jump, the genome of M. pennsylvanicum was sequenced and compared to the genomes of Ustilago maydis, Sporisorium reilianum, and Ustilago hordei. While all four genomes had a similar completeness in CEGMA analyses, gene absence was highest in M. pennsylvanicum, and most pronounced in putative secreted proteins, which are often considered as effector candidates. In contrast, the amount of private genes was similar among the species, highlighting that gene loss rather than gene gain is the hallmark of adaptation after the host jump to the dicot host. Our analyses revealed a trend of putative effectors to be next to another putative effector, but the majority of these are not in clusters and thus the focus on pathogenicity clusters might not be appropriate for all smut genomes. Positive selection studies revealed that M. pennsylvanicum has the highest number and proportion of genes under positive selection. In general, putative effectors showed a higher proportion of positively selected genes than non-effector candidates. The 248 putative secreted effectors found in all four smut genomes might constitute a core set needed for pathogenicity, while those 92 that are found in all grass-parasitic smuts, but have no ortholog in M. pennsylvanicum might constitute a set of effectors important for successful colonization of grass hosts.
Ceraceosorus bombacis is an early-diverging lineage of smut fungi and a pathogen of cotton trees (Bombax ceiba). To study the evolutionary genomics of smut fungi in comparison with other fungal and oomycete pathogens, the genome of C. bombacis was sequenced and comparative genomic analyses were performed. The genome of 26.09 Mb encodes for 8,024 proteins, of which 576 are putative-secreted effector proteins (PSEPs). Orthology analysis revealed 30 ortholog PSEPs among six Ustilaginomycotina genomes, the largest groups of which are lytic enzymes, such as aspartic peptidase and glycoside hydrolase. Positive selection analyses revealed the highest percentage of positively selected PSEPs in C. bombacis compared with other Ustilaginomycotina genomes. Metabolic pathway analyses revealed the absence of genes encoding for nitrite and nitrate reductase in the genome of the human skin pathogen Malassezia globosa, but these enzymes are present in the sequenced plant pathogens in smut fungi. Interestingly, these genes are also absent in cultivable oomycete animal pathogens, while nitrate reductase has been lost in cultivable oomycete plant pathogens. Similar patterns were also observed for obligate biotrophic and hemi-biotrophic fungal and oomycete pathogens. Furthermore, it was found that both fungal and oomycete animal pathogen genomes are lacking cutinases and pectinesterases. Overall, these findings highlight the parallel evolution of certain genomic traits, revealing potential common evolutionary trajectories among fungal and oomycete pathogens, shaping the pathogen genomes according to their lifestyle.
Background: Xanthophyllomyces dendrorhous is a basal agaricomycete with uncertain taxonomic placement, known for its unique ability to produce astaxanthin, a carotenoid with antioxidant properties. It was the aim of this study to elucidate the organization of its CoA-derived pathways and to use the genomic information of X. dendrorhous for a phylogenomic investigation of the Basidiomycota.
Results: The genome assembly of a haploid strain of Xanthophyllomyces dendrorhous revealed a genome of 19.50 Megabases with 6385 protein coding genes. Phylogenetic analyses were conducted including 48 fungal genomes. These revealed Ustilaginomycotina and Agaricomycotina as sister groups. In the latter a well-supported sister-group relationship of two major orders, Polyporales and Russulales, was inferred. Wallemia occupies a basal position within the Agaricomycotina and X. dendrorhous represents the basal lineage of the Tremellomycetes, highlighting that the typical tremelloid parenthesomes have either convergently evolved in Wallemia and the Tremellomycetes, or were lost in the Cystofilobasidiales lineage. A detailed characterization of the CoA-related pathways was done and all genes for fatty acid, sterol and carotenoid synthesis have been assigned.
Conclusions: The current study ascertains that Wallemia with tremelloid parenthesomes is the most basal agaricomycotinous lineage and that Cystofilobasidiales without tremelloid parenthesomes are deeply rooted within Tremellomycetes, suggesting that parenthesomes at septal pores might be the core synapomorphy for the Agaricomycotina. Apart from evolutionary insights the genome sequence of X. dendrorhous will facilitate genetic pathway engineering for optimized astaxanthin or oxidative alcohol production.
Measurement of electrons from heavy-flavour hadron decays in p–Pb collisions at √sNN = 5.02 TeV
(2015)
The production of electrons from heavy-flavour hadron decays was measured as a function of transverse momentum (pT) in minimum-bias p–Pb collisions at √sNN = 5.02 TeV using the ALICE detector at the LHC. The measurement covers the pT interval 0.5 < pT < 12 GeV/c and the rapidity range −1.065 < ycms < 0.135 in the centre-of-mass reference frame. The contribution of electrons from background sources was subtracted using an invariant mass approach. The nuclear modification factor RpPb was calculated by comparing the pT-differential invariant cross section in p–Pb collisions to a pp reference at the same centre-of-mass energy, which was obtained by interpolating measurements at √s = 2.76 TeV and √s = 7 TeV. The RpPb is consistent with unity within uncertainties of about 25%, which become larger for pT below 1 GeV/c. The measurement shows that heavy-flavour production is consistent with binary scaling, so that a suppression in the high-pT yield in Pb–Pb collisions has to be attributed to effects induced by the hot medium produced in the final state. The data in p–Pb collisions are described by recent model calculations that include cold nuclear matter effects.
We present measurements of the azimuthal dependence of charged jet production in central and semi-central √sNN=2.76 TeV Pb–Pb collisions with respect to the second harmonic event plane, quantified as v2ch jet. Jet finding is performed employing the anti-kT algorithm with a resolution parameter R=0.2 using charged tracks from the ALICE tracking system. The contribution of the azimuthal anisotropy of the underlying event is taken into account event-by-event. The remaining (statistical) region-to-region fluctuations are removed on an ensemble basis by unfolding the jet spectra for different event plane orientations independently. Significant non-zero v2ch jet is observed in semi-central collisions (30–50% centrality) for 20<pTch jet<90 GeV/c. The azimuthal dependence of the charged jet production is similar to the dependence observed for jets comprising both charged and neutral fragments, and compatible with measurements of the v2 of single charged particles at high pT. Good agreement between the data and predictions from JEWEL, an event generator simulating parton shower evolution in the presence of a dense QCD medium, is found in semi-central collisions.
The pseudorapidity (η) and transverse-momentum (pT) distributions of charged particles produced in proton–proton collisions are measured at the centre-of-mass energy √s=13 TeV. The pseudorapidity distribution in |η|<1.8 is reported for inelastic events and for events with at least one charged particle in |η|<1. The pseudorapidity density of charged particles produced in the pseudorapidity region |η|<0.5 is 5.31±0.18 and 6.46±0.19 for the two event classes, respectively. The transverse-momentum distribution of charged particles is measured in the range 0.15<pT<20 GeV/c and |η|<0.8 for events with at least one charged particle in |η|<1. The evolution of the transverse momentum spectra of charged particles is also investigated as a function of event multiplicity. The results are compared with calculations from PYTHIA and EPOS Monte Carlo generators.
Background: Bacteria within the genus Photorhabdus maintain mutualistic symbioses with nematodes in complicated lifecycles that also involves insect pathogenic phases. Intriguingly, these bacteria are rich in biosynthetic gene clusters that produce compounds with diverse biological activities. As a basis to better understand the life cycles of Photorhabdus we sequenced the genomes of two recently discovered representative species and performed detailed genomic comparisons with five publically available genomes.
Results: Here we report the genomic details of two new reference Photorhabdus species. By then conducting genomic comparisons across the genus, we show that there are several highly conserved biosynthetic gene clusters. These clusters produce a range of bioactive small molecules that support the pathogenic phase of the integral relationship that Photorhabdus maintain with nematodes.
Conclusions: Photorhabdus contain several genetic loci that allow them to become specialist insect pathogens by efficiently evading insect immune responses and killing the insect host.
The centrality dependence of the charged-particle pseudorapidity density measured with ALICE in Pb–Pb collisions at √sNN=2.76 TeV over a broad pseudorapidity range is presented. This Letter extends the previous results reported by ALICE to more peripheral collisions. No strong change of the overall shape of charged-particle pseudorapidity density distributions with centrality is observed, and when normalised to the number of participating nucleons in the collisions, the evolution over pseudorapidity with centrality is likewise small. The broad pseudorapidity range (−3.5<η<5) allows precise estimates of the total number of produced charged particles which we find to range from 162±22(syst.) to 17170±770(syst.) in 80–90% and 0–5% central collisions, respectively. The total charged-particle multiplicity is seen to approximately scale with the number of participating nucleons in the collision. This suggests that hard contributions to the charged-particle multiplicity are limited. The results are compared to models which describe dNch/dη at mid-rapidity in the most central Pb–Pb collisions and it is found that these models do not capture all features of the distributions.
The production of K∗(892)0 and ϕ(1020) mesons has been measured in p–Pb collisions at √sNN = 5.02 TeV. K∗0 and ϕ are reconstructed via their decay into charged hadrons with the ALICE detector in the rapidity range - 0.5 < y < 0. The transverse momentum spectra, measured as a function of the multiplicity, have a pT range from 0 to 15 GeV/c for K∗0 and from 0.3 to 21 GeV/c for ϕ. Integrated yields, mean transverse momenta and particle ratios are reported and compared with results in pp collisions at √s = 7 TeV and Pb–Pb collisions at √sNN = 2.76 TeV. In Pb–Pb and p–Pb collisions, K∗0 and ϕ probe the hadronic phase of the system and contribute to the study of particle formation mechanisms by comparison with other identified hadrons. For this purpose, the mean transverse momenta and the differential proton-to-ϕ ratio are discussed as a function of the multiplicity of the event. The short-lived K∗0 is measured to investigate re-scattering effects, believed to be related to the size of the system and to the lifetime of the hadronic phase.