Integration and holistic analysis of multiple multidimensional soil data sets.

Complex matrices such as soil have a range of measurable characteristics, and thus data to describe them can be considered multidimensional. These characteristics can be strongly influenced by factors that introduce confounding effects that hinder analyses. Traditional statistical approaches lack the flexibility and granularity required to adequately evaluate such matrices, particularly those with large dataset of varying data types (i.e. quantitative non-compositional, quantitative compositional). We present a statistical workflow designed to effectively analyse complex, multidimensional systems, even in the presence of confounding variables. The developed methodology involves exploratory analysis to identify the presence of confounding variables, followed by data decomposition (including strategies for both compositional and non-compositional quantitative data) to minimise the influence of these confounding factors such as sampling site/location. These data processing methods then allow for common patterns to be highlighted in the data, including the identification of biomarkers and determination of non-trivial associations between variables. We demonstrate the utility of this statistical workflow by jointly analysing the chemical composition and fungal biodiversity of New Zealand vineyard soils that have been managed with either organic low-input or conventional input approaches. By applying this pipeline, we were able to identify biomarkers that distinguish viticultural soil from both approaches and also unearth links and associations between the chemical and metagenomic profiles. While soil is an example of a system that can require this type of statistical methodology, there are a range of biological and ecological systems that are challenging to analyse due to the complex interplay of global and local effects. Utilising our developed pipeline will greatly enhance the way that these systems can be studied and the quality and impact of insight gained from their analysis.

Novel endolithic bacteria of phylum Chloroflexota reveal a myriad of potential survival strategies in the Antarctic desert.

The ice-free McMurdo Dry Valleys of Antarctica are dominated by nutrient-poor mineral soil and rocky outcrops. The principal habitat for microorganisms is within rocks (endolithic). In this environment, microorganisms are provided with protection against sub-zero temperatures, rapid thermal fluctuations, extreme dryness, and ultraviolet and solar radiation. Endolithic communities include lichen, algae, fungi, and a diverse array of bacteria. Chloroflexota is among the most abundant bacterial phyla present in these communities. Among the Chloroflexota are four novel classes of bacteria, here named Candidatus Spiritibacteria class. nov. (=UBA5177), Candidatus Martimicrobia class. nov. (=UBA4733), Candidatus Tarhunnaeia class. nov. (=UBA6077), and Candidatus Uliximicrobia class. nov. (=UBA2235). We retrieved 17 high-quality metagenome-assembled genomes (MAGs) that represent these four classes. Based on genome predictions, all these bacteria are inferred to be aerobic heterotrophs that encode enzymes for the catabolism of diverse sugars. These and other organic substrates are likely derived from lichen, algae, and fungi, as metabolites (including photosynthate), cell wall components, and extracellular matrix components. The majority of MAGs encode the capacity for trace gas oxidation using high-affinity uptake hydrogenases, which could provide energy and metabolic water required for survival and persistence. Furthermore, some MAGs encode the capacity to couple the energy generated from H2 and CO oxidation to support carbon fixation (atmospheric chemosynthesis). All encode mechanisms for the detoxification and efflux of heavy metals. Certain MAGs encode features that indicate possible interactions with other organisms, such as Tc-type toxin complexes, hemolysins, and macroglobulins.IMPORTANCEThe ice-free McMurdo Dry Valleys of Antarctica are the coldest and most hyperarid desert on Earth. It is, therefore, the closest analog to the surface of the planet Mars. Bacteria and other microorganisms survive by inhabiting airspaces within rocks (endolithic). We identify four novel classes of phylum Chloroflexota, and, based on interrogation of 17 metagenome-assembled genomes, we predict specific metabolic and physiological adaptations that facilitate the survival of these bacteria in this harsh environment-including oxidation of trace gases and the utilization of nutrients (including sugars) derived from lichen, algae, and fungi. We propose that such adaptations allow these endolithic bacteria to eke out an existence in this cold and extremely dry habitat.

Metagenomics untangles potential adaptations of Antarctic endolithic bacteria at the fringe of habitability.

Survival and growth strategies of Antarctic endolithic microbes residing in Earth's driest and coldest desert remain virtually unknown. From 109 endolithic microbiomes, 4539 metagenome-assembled genomes were generated, 49.3 % of which were novel candidate bacterial species. We present evidence that trace gas oxidation and atmospheric chemosynthesis may be the prevalent strategies supporting metabolic activity and persistence of these ecosystems at the fringe of life and the limits of habitability.

A pile of pipelines: An overview of the bioinformatics software for metabarcoding data analyses.

Environmental DNA (eDNA) metabarcoding has gained growing attention as a strategy for monitoring biodiversity in ecology. However, taxa identifications produced through metabarcoding require sophisticated processing of high-throughput sequencing data from taxonomically informative DNA barcodes. Various sets of universal and taxon-specific primers have been developed, extending the usability of metabarcoding across archaea, bacteria and eukaryotes. Accordingly, a multitude of metabarcoding data analysis tools and pipelines have also been developed. Often, several developed workflows are designed to process the same amplicon sequencing data, making it somewhat puzzling to choose one among the plethora of existing pipelines. However, each pipeline has its own specific philosophy, strengths and limitations, which should be considered depending on the aims of any specific study, as well as the bioinformatics expertise of the user. In this review, we outline the input data requirements, supported operating systems and particular attributes of thirty-two amplicon processing pipelines with the goal of helping users to select a pipeline for their metabarcoding projects.

Highly diverse and unknown viruses may enhance Antarctic endoliths' adaptability.

BACKGROUND: Rock-dwelling microorganisms are key players in ecosystem functioning of Antarctic ice free-areas. Yet, little is known about their diversity and ecology, and further still, viruses in these communities have been largely unexplored despite important roles related to host metabolism and nutrient cycling. To begin to address this, we present a large-scale viral catalog from Antarctic rock microbial communities. RESULTS: We performed metagenomic analyses on rocks from across Antarctica representing a broad range of environmental and spatial conditions, and which resulted in a predicted viral catalog comprising > 75,000 viral operational taxonomic units (vOTUS). We found largely undescribed, highly diverse and spatially structured virus communities which had predicted auxiliary metabolic genes (AMGs) with functions indicating that they may be potentially influencing bacterial adaptation and biogeochemistry. CONCLUSION: This catalog lays the foundation for expanding knowledge of virosphere diversity, function, spatial ecology, and dynamics in extreme environments. This work serves as a step towards exploring adaptability of microbial communities in the face of a changing climate. Video Abstract.

Distinct and Temporally Stable Assembly Mechanisms Shape Bacterial and Fungal Communities in Vineyard Soils.

Microbial communities in agricultural soils are fundamental for plant growth and in vineyard ecosystems contribute to defining regional wine quality. Managing soil microbes towards beneficial outcomes requires knowledge of how community assembly processes vary across taxonomic groups, spatial scales, and through time. However, our understanding of microbial assembly remains limited. To quantify the contributions of stochastic and deterministic processes to bacterial and fungal assembly across spatial scales and through time, we used 16 s rRNA gene and ITS sequencing in the soil of an emblematic wine-growing region of Italy.Combining null- and neutral-modelling, we found that assembly processes were consistent through time, but bacteria and fungi were governed by different processes. At the within-vineyard scale, deterministic selection and homogenising dispersal dominated bacterial assembly, while neither selection nor dispersal had clear influence over fungal assembly. At the among-vineyard scale, the influence of dispersal limitation increased for both taxonomic groups, but its contribution was much larger for fungal communities. These null-model-based inferences were supported by neutral modelling, which estimated a dispersal rate almost two orders-of-magnitude lower for fungi than bacteria.This indicates that while stochastic processes are important for fungal assembly, bacteria were more influenced by deterministic selection imposed by the biotic and/or abiotic environment. Managing microbes in vineyard soils could thus benefit from strategies that account for dispersal limitation of fungi and the importance of environmental conditions for bacteria. Our results are consistent with theoretical expectations whereby larger individual size and smaller populations can lead to higher levels of stochasticity.

Genome sequencing provides new insights on the distribution of Erwinia amylovora lineages in northern Italy.

Erwinia amylovora is a Gram-negative bacterium that colonizes a wide variety of plant species causing recurrent local outbreaks of fire blight in crops of the Rosaceae family. Recent genomic surveys have documented the limited genomic diversity of this species, possibly related to a recent evolutionary bottleneck and a strong correlation between geography and phylogenetic structure of the species. Despite its economic importance, little is known about the genetic variability of co-circulating strains during local outbreaks. Here, we report the genome sequences of 82 isolates of E. amylovora, collected from different host plants in a period of 16 years in Trentino, a small region in the Northeastern Italian Alps that has been characterized by recurrent outbreaks of fire blight in apple orchards. While the genome isolated before 2018 are closely related to other strains already present in Europe, we found a novel subclade composed only by isolates that were sampled starting from 2018 and demonstrate that the endemic population of this pathogen can be composed by mixture of strains.

Rocks support a distinctive and consistent mycobiome across contrasting dry regions of Earth.

Rock-dwelling fungi play critical ecological roles in drylands, including soil formation and nutrient cycling; however, we know very little about the identity, function and environmental preferences of these important organisms, and the mere existence of a consistent rock mycobiome across diverse arid regions of the planet remains undetermined. To address this knowledge gap, we conducted a meta-analysis of rock fungi and spatially associated soil communities, surveyed across 28 unique sites spanning four major biogeographic regions (North America, Arctic, Maritime and Continental Antarctica) including contrasting climates, from cold and hot deserts to semiarid drylands. We show that rocks support a consistent and unique mycobiome that was different from that found in surrounding soils. Lichenized fungi from class Lecanoromycetes were consistently indicative of rocks across contrasting regions, together with ascomycetous representatives of black fungi in Arthoniomycetes, Dothideomycetes and Eurotiomycetes. In addition, compared with soil, rocks had a lower proportion of saprobes and plant symbiotic fungi. The main drivers structuring rock fungi distribution were spatial distance and, to a larger extent, climatic factors regulating moisture and temperature (i.e. mean annual temperature and mean annual precipitation), suggesting that these paramount and unique communities might be particularly sensitive to increases in temperature and desertification.

Power Play of Commensal Bacteria in the Buccal Cavity of Female Nile Tilapia.

Fish are widely exposed to higher microbial loads compared to land and air animals. It is known that the microbiome plays an essential role in the health and development of the host. The oral microbiome is vital in females of different organisms, including the maternal mouthbrooding species such as Nile tilapia (Oreochromis niloticus). The present study reports for the first time the microbial composition in the buccal cavity of female and male Nile tilapia reared in a recirculating aquaculture system. Mucus samples were collected from the buccal cavity of 58 adult fish (∼1 kg), and 16S rRNA gene amplicon sequencing was used to profile the microbial communities in females and males. The analysis revealed that opportunistic pathogens such as Streptococcus sp. were less abundant in the female buccal cavity. The power play of certain bacteria such as Acinetobacter, Acidobacteria (GP4 and GP6), and Saccharibacteria that have known metabolic advantages was evident in females compared to males. Association networks inferred from relative abundances showed few microbe-microbe interactions of opportunistic pathogens in female fish. The findings of opportunistic bacteria and their interactions with other microbes will be valuable for improving Nile tilapia rearing practices. The presence of bacteria with specific functions in the buccal cavity of female fish points to their ability to create a protective microbial ecosystem for the offspring.

Interactions between parasitic helminths and gut microbiota in wild tropical primates from intact and fragmented habitats.

The mammalian gastrointestinal tract harbours a highly complex ecosystem composed of a variety of micro- (bacteria, fungi, viruses, protozoans) and macro-organisms (helminths). Although most microbiota research focuses on the variation of single gut components, the crosstalk between components is still poorly characterized, especially in hosts living under natural conditions. We investigated the gut micro-biodiversity (bacteria, fungi and helminths) of 158 individuals of two wild non-human primates, the Udzungwa red colobus (Procolobus gordonorum) and the yellow baboon (Papio cynocephalus). These species have contrasting diets and lifestyles, but live sympatrically in both human-impacted and pristine forests in the Udzungwa Mountains of Tanzania. Using non-invasive faecal pellets, helminths were identified using standard microscopy while bacteria and fungi were characterized by sequencing the V1-V3 variable region of the 16S rRNA gene for bacteria and the ITS1-ITS2 fragment for fungi. Our results show that both diversity and composition of bacteria and fungi are associated with variation in helminth presence. Although interactions differed by habitat type, in both primates we found that Strongyloides was negatively associated and Trichuris was positively associated with bacterial and fungal richness. To our knowledge, this is one of the few studies demonstrating an interaction between helminth and gut microbiota communities in wild non-human primates.

Breeding Strategy Shapes the Composition of Bacterial Communities in Female Nile Tilapia Reared in a Recirculating Aquaculture System.

In industrial animal production, breeding strategies are essential to produce offspring of better quality and vitality. It is also known that host microbiome has a bearing on its health. Here, we report for the first time the influence of crossbreeding strategy, inbreeding or outbreeding, on the buccal and intestinal bacterial communities in female Nile tilapia (Oreochromis niloticus). Crossbreeding was performed within a family and between different fish families to obtain the inbred and outbred study groups, respectively. The genetic relationship and structure analysis revealed significant genetic differentiation between the inbred and outbred groups. We also employed a 16S rRNA gene sequencing technique to understand the significant differences between the diversities of the bacterial communities of the inbred and outbred groups. The core microbiota composition in the mouth and the intestine was not affected by the crossbreeding strategy but their abundance varied between the two groups. Furthermore, opportunistic bacteria were abundant in the buccal cavity and intestine of the outbred group, whereas beneficial bacteria were abundant in the intestine of the inbred group. The present study indicates that crossbreeding can influence the abundance of beneficial bacteria, core microbiome and the inter-individual variation in the microbiome.

Genome Recovery, Functional Profiling, and Taxonomic Classification from Metagenomes.

Recovering and annotating bacterial genomes from metagenomes involves a series of complex computational tools that are often difficult to use for researches without a specialistic bioinformatic background. In this chapter we review all the steps that lead from raw reads to a collection of quality-controlled, functionally annotated bacterial genomes and propose a working protocol using state-of-the-art, open source software tools.

Pre-Cambrian roots of novel Antarctic cryptoendolithic bacterial lineages.

BACKGROUND: Cryptoendolithic communities are microbial ecosystems dwelling inside porous rocks that are able to persist at the edge of the biological potential for life in the ice-free areas of the Antarctic desert. These regions include the McMurdo Dry Valleys, often accounted as the closest terrestrial counterpart of the Martian environment and thought to be devoid of life until the discovery of these cryptic life-forms. Despite their interest as a model for the early colonization by living organisms of terrestrial ecosystems and for adaptation to extreme conditions of stress, little is known about the evolution, diversity, and genetic makeup of bacterial species that reside in these environments. Using the Illumina Novaseq platform, we generated the first metagenomes from rocks collected in Continental Antarctica over a distance of about 350 km along an altitudinal transect from 834 up to 3100 m above sea level (a.s.l.). RESULTS: A total of 497 draft bacterial genome sequences were assembled and clustered into 269 candidate species that lack a representative genome in public databases. Actinobacteria represent the most abundant phylum, followed by Chloroflexi and Proteobacteria. The "Candidatus Jiangella antarctica" has been recorded across all samples, suggesting a high adaptation and specialization of this species to the harshest Antarctic desert environment. The majority of these new species belong to monophyletic bacterial clades that diverged from related taxa in a range from 1.2 billion to 410 Ma and are functionally distinct from known related taxa. CONCLUSIONS: Our findings significantly increase the repertoire of genomic data for several taxa and, to date, represent the first example of bacterial genomes recovered from endolithic communities. Their ancient origin seems to not be related to the geological history of the continent, rather they may represent evolutionary remnants of pristine clades that evolved across the Tonian glaciation. These unique genomic resources will underpin future studies on the structure, evolution, and function of these ecosystems at the edge of life. Video abstract.

Large-scale quality assessment of prokaryotic genomes with metashot/prok-quality.

Metagenomic sequencing allows large-scale identification and genomic characterization. Binning is the process of recovering genomes from complex mixtures of sequence fragments (metagenome contigs) of unknown bacteria and archaeal species. Assessing the quality of genomes recovered from metagenomes requires the use of complex pipelines involving many independent steps, often difficult to reproduce and maintain. A comprehensive, automated and easy-to-use computational workflow for the quality assessment of draft prokaryotic genomes, based on container technology, would greatly improve reproducibility and reusability of published results. We present metashot/prok-quality, a container-enabled Nextflow pipeline for quality assessment and genome dereplication. The metashot/prok-quality tool produces genome quality reports that are compliant with the Minimum Information about a Metagenome-Assembled Genome (MIMAG) standard, and can run out-of-the-box on any platform that supports Nextflow, Docker or Singularity, including computing clusters or batch infrastructures in the cloud. metashot/prok-quality is part of the metashot collection of analysis pipelines. Workflow and documentation are available under GPL3 licence on GitHub.

Large scale genome reconstructions illuminate Wolbachia evolution.

Wolbachia is an iconic example of a successful intracellular bacterium. Despite its importance as a manipulator of invertebrate biology, its evolutionary dynamics have been poorly studied from a genomic viewpoint. To expand the number of Wolbachia genomes, we screen over 30,000 publicly available shotgun DNA sequencing samples from 500 hosts. By assembling over 1000 Wolbachia genomes, we provide a substantial increase in host representation. Our phylogenies based on both core-genome and gene content provide a robust reference for future studies, support new strains in model organisms, and reveal recent horizontal transfers amongst distantly related hosts. We find various instances of gene function gains and losses in different super-groups and in cytoplasmic incompatibility inducing strains. Our Wolbachia-host co-phylogenies indicate that horizontal transmission is widespread at the host intraspecific level and that there is no support for a general Wolbachia-mitochondrial synchronous divergence.

Raw milk and fecal microbiota of commercial Alpine dairy cows varies with herd, fat content and diet.

The factors that influence the diversity and composition of raw milk and fecal microbiota in healthy commercial dairy herds are not fully understood, partially because the majority of metataxonomic studies involve experimental farms and/or single factors. We analyzed the raw milk and fecal microbiota of 100 healthy cows from 10 commercial alpine farms from the Province of Trento, Italy, using metataxonomics and applied statistical modelling to investigate which extrinsic and intrinsic parameters (e.g. herd, diet and milk characteristics) correlated with microbiota richness and composition in these relatively small traditional farms. We confirmed that Firmicutes, Ruminococcaceae and Lachnospiraceae families dominated the fecal and milk samples of these dairy cows, but in addition, we found an association between the number of observed OTUs and Shannon entropy on each farm that indicates higher microbiota richness is associated with increased microbiota stability. Modelling showed that herd was the most significant factor affecting the variation in both milk and fecal microbiota composition. Furthermore, the most important predictors explaining the variation of microbiota richness were milk characteristics (i.e. percentage fat) and diet for milk and fecal samples, respectively. We discuss how high intra-herd variation could affect the development of treatments based on microbiota manipulation.

The Gut Microbiota Communities of Wild Arboreal and Ground-Feeding Tropical Primates Are Affected Differently by Habitat Disturbance.

Human exploitation and destruction of tropical resources are currently threatening innumerable wild animal species, altering natural ecosystems and thus, food resources, with profound effects on gut microbiota. Given their conservation status and the importance to tropical ecosystems, wild nonhuman primates make excellent models to investigate the effect of human disturbance on the diversity of host-associated microbiota. Previous investigations have revealed a loss of fecal bacterial diversity in primates living in degraded compared to intact forests. However, these data are available for a limited number of species, and very limited information is available on the fungal taxa hosted by the gut. Here, we estimated the diversity and composition of gut bacterial and fungal communities in two primates living sympatrically in both human-modified and pristine forests in the Udzungwa Mountains of Tanzania. Noninvasively collected fecal samples of 12 groups of the Udzungwa red colobus (Procolobus gordonorum) (n = 89), a native and endangered primate (arboreal and predominantly leaf-eating), and five groups of the yellow baboon (Papio cynocephalus) (n = 69), a common species of least concern (ground-feeding and omnivorous), were analyzed by the V1-V3 region of the 16S rRNA gene (bacterial) and ITS1-ITS2 (fungal) sequencing. Gut bacterial diversities were associated with habitat in both species, most likely depending on their ecological niches and associated digestive physiology, dietary strategies, and locomotor behavior. In addition, fungal communities also show distinctive traits across hosts and habitat type, highlighting the importance of investigating this relatively unexplored gut component.IMPORTANCE Gut microbiota diversity has become the subject of extensive research in human and nonhuman animals, linking diversity and composition to gut function and host health. Because wild primates are good indicators of tropical ecosystem health, we developed the idea that they are a suitable model to observe the consequences of advancing global change (e.g., habitat degradation) on gut microbiota. So far, most of the studies focus mainly on gut bacteria; however, they are not the only component of the gut: fungi also serve essential functions in gut homeostasis. Here, for the first time, we explore and measure diversity and composition of both bacterial and fungal microbiota components of two tropical primate species living in highly different habitat types (intact versus degraded forests). Results on their microbiota diversity and composition are discussed in light of conservation issues and potential applications.

Tissue age, orchard location and disease management influence the composition of fungal and bacterial communities present on the bark of apple trees.

Plants host microbial communities that can be affected by environmental conditions and agronomic practices. Despite the role of bark as a reservoir of plant pathogens and beneficial microorganisms, no information is available on the effects of disease management on the taxonomic composition of the bark-associated communities of apple trees. We assessed the impact of disease management strategies on fungal and bacterial communities on the bark of a scab-resistant apple cultivar in two orchard locations and for two consecutive seasons. The amplicon sequencing revealed that bark age and orchard location strongly affected fungal and bacterial diversity. Microbiota dissimilarity between orchards evolved during the growing season and showed specific temporal series for fungal and bacterial populations in old and young bark. Disease management did not induce global changes in the microbial populations across locations and seasons, but specifically affected the abundance of some taxa according to bark age, orchard location and sampling time. Therefore, the disease management applied to scab-resistant cultivars, which is based on a limited use of fungicides, partially changed the taxonomic composition of bark-associated fungal and bacterial communities, suggesting the need for a more accurate risk assessment regarding possible pathogen outbreaks.

Metagenomes in the Borderline Ecosystems of the Antarctic Cryptoendolithic Communities.

Antarctic cryptoendolithic communities are microbial ecosystems dwelling inside rocks of the Antarctic desert. We present the first 18 shotgun metagenomes from these communities to further characterize their composition, biodiversity, functionality, and adaptation. Future studies will integrate taxonomic and functional annotations to examine the pathways necessary for life to evolve in the extremes.

Alpine headwaters emerging from glaciers and rock glaciers host different bacterial communities: Ecological implications for the future.

Mountain glacier shrinkage represents a major effect of the current global warming and 80-100% of the Alpine glaciers are predicted to vanish within the next few decades. As the thawing rate of mountain permafrost ice is much lower than for glacier ice, a shift from glacial to periglacial dynamics is predicted for Alpine landscapes during the 21st century. Despite the growing literature on the impacts of deglaciation on Alpine hydrology and ecosystems, chemical and biological features of waters emerging from Alpine rock glaciers (i.e. permafrost landforms composed by a mixture of ice and debris) have been poorly investigated so far, and knowledge on microbial biodiversity of headwaters is still sparse. A set of glacier-, rock glacier- and groundwater/precipitation-fed streams was investigated in the Italian Central Alps in late summer 2016, aiming at exploring bacterial community composition and diversity in epilithic and surface sediment biofilm and at verifying the hypothesis that rock glacier-fed headwaters represent peculiar ecosystems from both a chemical and biological point of view. Rock glacier-fed waters showed high values of electrical conductivity and trace elements related to their bedrock lithology, and their highly diverse bacterial assemblages significantly differed from those detected in glacier-fed streams. Bacterial taxonomic composition appeared to be mainly related to water and substrate type, as well as to water chemistry, the latter including concentrations of nutrients and trace metals. The results of this study confirm the chemical and biological peculiarity of rock glacier-fed waters compared to glacial waters, and suggest a potential driving role of thawing permafrost in modulating future ecological traits of Alpine headwaters within the context of progressing deglaciation.