Expanded phylogeny of extremely halophilic archaea shows multiple independent adaptations to hypersaline environments.

Extremely halophilic archaea (Haloarchaea, Nanohaloarchaeota, Methanonatronarchaeia and Halarchaeoplasmatales) thrive in saturating salt concentrations where they must maintain osmotic equilibrium with their environment. The evolutionary history of adaptations enabling salt tolerance remains poorly understood, in particular because the phylogeny of several lineages is conflicting. Here we present a resolved phylogeny of extremely halophilic archaea obtained using improved taxon sampling and state-of-the-art phylogenetic approaches designed to cope with the strong compositional biases of their proteomes. We describe two uncultured lineages, Afararchaeaceae and Asbonarchaeaceae, which break the long branches at the base of Haloarchaea and Nanohaloarchaeota, respectively. We obtained 13 metagenome-assembled genomes (MAGs) of these archaea from metagenomes of hypersaline aquatic systems of the Danakil Depression (Ethiopia). Our phylogenomic analyses including these taxa show that at least four independent adaptations to extreme halophily occurred during archaeal evolution. Gene-tree/species-tree reconciliation suggests that gene duplication and horizontal gene transfer played an important role in this process, for example, by spreading key genes (such as those encoding potassium transporters) across extremely halophilic lineages.

Improving Orthologous Signal and Model Fit in Datasets Addressing the Root of the Animal Phylogeny.

There is conflicting evidence as to whether Porifera (sponges) or Ctenophora (comb jellies) comprise the root of the animal phylogeny. Support for either a Porifera-sister or Ctenophore-sister tree has been extensively examined in the context of model selection, taxon sampling, and outgroup selection. The influence of dataset construction is comparatively understudied. We re-examine five animal phylogeny datasets that have supported either root hypothesis using an approach designed to enrich orthologous signal in phylogenomic datasets. We find that many component orthogroups in animal datasets fail to recover major lineages as monophyletic with the exception of Ctenophora, regardless of the supported root. Enriching these datasets to retain orthogroups recovering ≥3 major lineages reduces dataset size by up to 50% while retaining underlying phylogenetic information and taxon sampling. Site-heterogeneous phylogenomic analysis of these enriched datasets recovers both Porifera-sister and Ctenophora-sister positions, even with additional constraints on outgroup sampling. Two datasets which previously supported Ctenophora-sister support Porifera-sister upon enrichment. All enriched datasets display improved model fitness under posterior predictive analysis. While not conclusively rooting animals at either Porifera or Ctenophora, we do see an increase in signal for Porifera-sister and a decrease in signal for Ctenophore-sister when data are filtered for orthologous signal. Our results indicate that dataset size and construction as well as model fit influence animal root inference.

Filtering artifactual signal increases support for Xenacoelomorpha and Ambulacraria sister relationship in the animal tree of life.

Conflicting studies place a group of bilaterian invertebrates containing xenoturbellids and acoelomorphs, the Xenacoelomorpha, as either the primary emerging bilaterian phylum1,2,3,4,5,6 or within Deuterostomia, sister to Ambulacraria.7,8,9,10,11 Although their placement as sister to the rest of Bilateria supports relatively simple morphology in the ancestral bilaterian, their alternative placement within Deuterostomia suggests a morphologically complex ancestral bilaterian along with extensive loss of major phenotypic traits in the Xenacoelomorpha. Recent studies have questioned whether Deuterostomia should be considered monophyletic at all.10,12,13 Hidden paralogy and poor phylogenetic signal present a major challenge for reconstructing species phylogenies.14,15,16,17,18 Here, we assess whether these issues have contributed to the conflict over the placement of Xenacoelomorpha. We reanalyzed published datasets, enriching for orthogroups whose gene trees support well-resolved clans elsewhere in the animal tree.16 We find that most genes in previously published datasets violate incontestable clans, suggesting that hidden paralogy and low phylogenetic signal affect the ability to reconstruct branching patterns at deep nodes in the animal tree. We demonstrate that removing orthogroups that cannot recapitulate incontestable relationships alters the final topology that is inferred, while simultaneously improving the fit of the model to the data. We discover increased, but ultimately not conclusive, support for the existence of Xenambulacraria in our set of filtered orthogroups. At a time when we are progressing toward sequencing all life on the planet, we argue that long-standing contentious issues in the tree of life will be resolved using smaller amounts of better quality data that can be modeled adequately.19.

Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching.

Ribosomes have long been thought of as homogeneous macromolecular machines, but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. Here, we have characterised ribosomal protein heterogeneity across 4 tissues of Drosophila melanogaster. We find that testes and ovaries contain the most heterogeneous ribosome populations, which occurs through a combination of paralog-enrichment and paralog-switching. We have solved structures of ribosomes purified from in vivo tissues by cryo-EM, revealing differences in precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-switching could alter the ribosome surface, enabling different proteins to regulate translation. One testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of ribosome heterogeneity. Overall, this work allows us to propose that mRNA translation might be regulated in the gonads through ribosome heterogeneity, providing a potential means of ribosome specialisation.

Whole Genome Sequence of the Commercially Relevant Mushroom Strain Agaricus bisporus var. bisporus ARP23.

Agaricus bisporus is an extensively cultivated edible mushroom. Demand for cultivation is continuously growing and difficulties associated with breeding programs now means strains are effectively considered monoculture. While commercial growing practices are highly efficient and tightly controlled, the over-use of a single strain has led to a variety of disease outbreaks from a range of pathogens including bacteria, fungi and viruses. To address this, the Agaricus Resource Program (ARP) was set up to collect wild isolates from diverse geographical locations through a bounty-driven scheme to create a repository of wild Agaricus germplasm. One of the strains collected, Agaricus bisporus var. bisporus ARP23, has been crossed extensively with white commercial varieties leading to the generation of a novel hybrid with a dark brown pileus commonly referred to as 'Heirloom'. Heirloom has been successfully implemented into commercial mushroom cultivation. In this study the whole genome of Agaricus bisporus var. bisporus ARP23 was sequenced and assembled with Illumina and PacBio sequencing technology. The final genome was found to be 33.49 Mb in length and have significant levels of synteny to other sequenced Agaricus bisporus strains. Overall, 13,030 putative protein coding genes were located and annotated. Relative to the other A. bisporus genomes that are currently available, Agaricus bisporus var. bisporus ARP23 is the largest A. bisporus strain in terms of gene number and genetic content sequenced to date. Comparative genomic analysis shows that the A. bisporus mating loci in unifactorial and unsurprisingly highly conserved between strains. The lignocellulolytic gene content of all A. bisporus strains compared is also very similar. Our results show that the pangenome structure of A. bisporus is quite diverse with between 60-70% of the total protein coding genes per strain considered as being orthologous and syntenically conserved. These analyses and the genome sequence described herein are the starting point for more detailed molecular analyses into the growth and phenotypical responses of Agaricus bisporus var. bisporus ARP23 when challenged with economically important mycoviruses.

Pangloss: A Tool for Pan-Genome Analysis of Microbial Eukaryotes.

Although the pan-genome concept originated in prokaryote genomics, an increasing number of eukaryote species pan-genomes have also been analysed. However, there is a relative lack of software intended for eukaryote pan-genome analysis compared to that available for prokaryotes. In a previous study, we analysed the pan-genomes of four model fungi with a computational pipeline that constructed pan-genomes using the synteny-dependent Pan-genome Ortholog Clustering Tool (PanOCT) approach. Here, we present a modified and improved version of that pipeline which we have called Pangloss. Pangloss can perform gene prediction for a set of genomes from a given species that the user provides, constructs and optionally refines a species pan-genome from that set using PanOCT, and can perform various functional characterisation and visualisation analyses of species pan-genome data. To demonstrate Pangloss's capabilities, we constructed and analysed a species pan-genome for the oleaginous yeast Yarrowialipolytica and also reconstructed a previously-published species pan-genome for the opportunistic respiratory pathogen Aspergillus fumigatus. Pangloss is implemented in Python, Perl and R and is freely available under an open source GPLv3 licence via GitHub.

Antibiotic resistomes of healthy pig faecal metagenomes.

Antibiotic resistance reservoirs within food-producing animals are thought to be a risk to animal and human health. This study describes the minimum natural resistome of pig faeces as the bacteria are under no direct antibiotic selective pressure. The faecal resistome of 257 different genes comprised 56 core and 201 accessory resistance genes. The genes present at the highest relative abundances across all samples were tetW, tetQ, tet44, tet37, tet40, mefA, aadE, ant(9)-1, ermB and cfxA2. This study characterized the baseline resistome, the microbiome composition and the metabolic components described by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in healthy pig faeces, without antibiotic selective pressures. The microbiome hierarchical analysis resulted in a cluster tree with a highly similar pattern to that of the accessory resistome cluster tree. Functional capacity profiling identified genes associated with horizontal gene transfer. We identified a statistically significant positive correlation between the total antibiotic resistome and suggested indicator genes, which agree with using these genes as indicators of the total resistomes. The correlation between total resistome and total microbiome in this study was positive and statistically significant. Therefore, the microbiome composition influenced the resistome composition. This study identified a core and accessory resistome present in a cohort of healthy pigs, in the same conditions without antibiotics. It highlights the presence of antibiotic resistance in the absence of antibiotic selective pressure and the variability between animals even under the same housing, food and living conditions. Antibiotic resistance will remain in the healthy pig gut even when antibiotics are not used. Therefore, the risk of antibiotic resistance transfer from animal faeces to human pathogens or the environment will remain in the absence of antibiotics.

Pan-genome analyses of model fungal species.

The concept of the species 'pan-genome', the union of 'core' conserved genes and all 'accessory' non-conserved genes across all strains of a species, was first proposed in prokaryotes to account for intraspecific variability. Species pan-genomes have been extensively studied in prokaryotes, but evidence of species pan-genomes has also been demonstrated in eukaryotes such as plants and fungi. Using a previously published methodology based on sequence homology and conserved microsynteny, in addition to bespoke pipelines, we have investigated the pan-genomes of four model fungal species: Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans var. grubii and Aspergillus fumigatus. Between 80 and 90 % of gene models per strain in each of these species are core genes that are highly conserved across all strains of that species, many of which are involved in housekeeping and conserved survival processes. In many of these species, the remaining 'accessory' gene models are clustered within subterminal regions and may be involved in pathogenesis and antimicrobial resistance. Analysis of the ancestry of species core and accessory genomes suggests that fungal pan-genomes evolve by strain-level innovations such as gene duplication as opposed to wide-scale horizontal gene transfer. Our findings lend further supporting evidence to the existence of species pan-genomes in eukaryote taxa.

Evolutionary, structural and functional analysis of the caleosin/peroxygenase gene family in the Fungi.

BACKGROUND: Caleosin/peroxygenases, CLO/PXG, (designated PF05042 in Pfam) are a group of genes/proteins with anomalous distributions in eukaryotic taxa. We have previously characterised CLO/PXGs in the Viridiplantae. The aim of this study was to investigate the evolution and functions of the CLO/PXGs in the Fungi and other non-plant clades and to elucidate the overall origin of this gene family. RESULTS: CLO/PXG-like genes are distributed across the full range of fungal groups from the basal clades, Cryptomycota and Microsporidia, to the largest and most complex Dikarya species. However, the genes were only present in 243 out of 844 analysed fungal genomes. CLO/PXG-like genes have been retained in many pathogenic or parasitic fungi that have undergone considerable genomic and structural simplification, indicating that they have important functions in these species. Structural and functional analyses demonstrate that CLO/PXGs are multifunctional proteins closely related to similar proteins found in all major taxa of the Chlorophyte Division of the Viridiplantae. Transcriptome and physiological data show that fungal CLO/PXG-like genes have complex patterns of developmental and tissue-specific expression and are upregulated in response to a range of biotic and abiotic stresses as well as participating in key metabolic and developmental processes such as lipid metabolism, signalling, reproduction and pathogenesis. Biochemical data also reveal that the Aspergillus flavus CLO/PXG has specific functions in sporulation and aflatoxin production as well as playing roles in lipid droplet function. CONCLUSIONS: In contrast to plants, CLO/PXGs only occur in about 30% of sequenced fungal genomes but are present in all major taxa. Fungal CLO/PXGs have similar but not identical roles to those in plants, including stress-related oxylipin signalling, lipid metabolism, reproduction and pathogenesis. While the presence of CLO/PXG orthologs in all plant genomes sequenced to date would suggest that they have core housekeeping functions in plants, the selective loss of CLO/PXGs in many fungal genomes suggests more restricted functions in fungi as accessory genes useful in particular environments or niches. We suggest an ancient origin of CLO/PXG-like genes in the 'last eukaryotic common ancestor' (LECA) and their subsequent loss in ancestors of the Metazoa, after the latter had diverged from the ancestral fungal lineage.

Genome analysis of the yeast Diutina catenulata, a member of the Debaryomycetaceae/Metschnikowiaceae (CTG-Ser) clade.

Diutina catenulata (Candida catenulata) is an ascomycetous yeast that has been isolated from humans, animals and environmental sources. The species is a contaminant of dairy products, and has been linked to superficial and invasive infections in both humans and animals. Previous phylogenetic analyses have assigned the species to the Saccharomycetales, but failed to identify its specific clade. Here, we report the genome sequence of an environmental isolate of D. catenulata. Examination of the tRNA repertoire and coding potential of this species shows that it translates the CUG codon as serine and not leucine. In addition, two phylogenetic analyses using 204 ubiquitous gene family alignments and 3,826 single-copy genes both confirm the placement of the species in the Debaryomycetaceae/Metschnikowiaceae, or CTG-Ser clade. The sequenced isolate contains an MTLα idiomorph. However, unlike most MTL loci in related species, poly (A) polymerase (PAP) is not adjacent to MTLα1.

Multiple Approaches to Phylogenomic Reconstruction of the Fungal Kingdom.

Fungi are possibly the most diverse eukaryotic kingdom, with over a million member species and an evolutionary history dating back a billion years. Fungi have been at the forefront of eukaryotic genomics, and owing to initiatives like the 1000 Fungal Genomes Project the amount of fungal genomic data has increased considerably over the last 5 years, enabling large-scale comparative genomics of species across the kingdom. In this chapter, we first review fungal evolution and the history of fungal genomics. We then review in detail seven phylogenomic methods and reconstruct the phylogeny of 84 fungal species from 8 phyla using each method. Six methods have seen extensive use in previous fungal studies, while a Bayesian supertree method is novel to fungal phylogenomics. We find that both established and novel phylogenomic methods can accurately reconstruct the fungal kingdom. Finally, we discuss the accuracy and suitability of each phylogenomic method utilized.

Phylogenomic Reconstruction of the Oomycete Phylogeny Derived from 37 Genomes.

The oomycetes are a class of microscopic, filamentous eukaryotes within the Stramenopiles-Alveolata-Rhizaria (SAR) supergroup which includes ecologically significant animal and plant pathogens, most infamously the causative agent of potato blight Phytophthora infestans. Single-gene and concatenated phylogenetic studies both of individual oomycete genera and of members of the larger class have resulted in conflicting conclusions concerning species phylogenies within the oomycetes, particularly for the large Phytophthora genus. Genome-scale phylogenetic studies have successfully resolved many eukaryotic relationships by using supertree methods, which combine large numbers of potentially disparate trees to determine evolutionary relationships that cannot be inferred from individual phylogenies alone. With a sufficient amount of genomic data now available, we have undertaken the first whole-genome phylogenetic analysis of the oomycetes using data from 37 oomycete species and 6 SAR species. In our analysis, we used established supertree methods to generate phylogenies from 8,355 homologous oomycete and SAR gene families and have complemented those analyses with both phylogenomic network and concatenated supermatrix analyses. Our results show that a genome-scale approach to oomycete phylogeny resolves oomycete classes and individual clades within the problematic Phytophthora genus. Support for the resolution of the inferred relationships between individual Phytophthora clades varies depending on the methodology used. Our analysis represents an important first step in large-scale phylogenomic analysis of the oomycetes. IMPORTANCE The oomycetes are a class of eukaryotes and include ecologically significant animal and plant pathogens. Single-gene and multigene phylogenetic studies of individual oomycete genera and of members of the larger classes have resulted in conflicting conclusions concerning interspecies relationships among these species, particularly for the Phytophthora genus. The onset of next-generation sequencing techniques now means that a wealth of oomycete genomic data is available. For the first time, we have used genome-scale phylogenetic methods to resolve oomycete phylogenetic relationships. We used supertree methods to generate single-gene and multigene species phylogenies. Overall, our supertree analyses utilized phylogenetic data from 8,355 oomycete gene families. We have also complemented our analyses with superalignment phylogenies derived from 131 single-copy ubiquitous gene families. Our results show that a genome-scale approach to oomycete phylogeny resolves oomycete classes and clades. Our analysis represents an important first step in large-scale phylogenomic analysis of the oomycetes.

Systematic Search for Evidence of Interdomain Horizontal Gene Transfer from Prokaryotes to Oomycete Lineages.

While most commonly associated with prokaryotes, horizontal gene transfer (HGT) can also have a significant influence on the evolution of microscopic eukaryotes. Systematic analysis of HGT in the genomes of the oomycetes, filamentous eukaryotic microorganisms in the Stramenopiles-Alveolates-Rhizaria (SAR) supergroup, has to date focused mainly on intradomain transfer events between oomycetes and fungi. Using systematic whole-genome analysis followed by phylogenetic reconstruction, we have investigated the extent of interdomain HGT between bacteria and plant-pathogenic oomycetes. We report five putative instances of HGT from bacteria into the oomycetes. Two transfers were found in Phytophthora species, including one unique to the cucurbit pathogen Phytophthora capsici. Two were found in Pythium species only, and the final transfer event was present in Phytopythium and Pythium species, the first reported bacterium-inherited genes in these genera. Our putative transfers included one protein that appears to be a member of the Pythium secretome, metabolic proteins, and enzymes that could potentially break down xenobiotics within the cell. Our findings complement both previous reports of bacterial genes in oomycete and SAR genomes and the growing body of evidence suggesting that interdomain transfer from prokaryotes into eukaryotes occurs more frequently than previously thought. IMPORTANCE Horizontal gene transfer (HGT) is the nonvertical inheritance of genetic material by transfer between different species. HGT is an important evolutionary mechanism for prokaryotes and in some cases is responsible for the spread of antibiotic resistance from resistant to benign species. Genome analysis has shown that examples of HGT are not as frequent in eukaryotes, but when they do occur they may have important evolutionary consequences. For example, the acquisition of fungal genes by an ancestral Phytophthora (plant destroyer) species is responsible for the large repertoire of enzymes in the plant-degrading arsenal of modern-day Phytophthora species. In this analysis, we set out to systematically search oomycete genomes for evidence of interdomain HGT (transfer of bacterial genes into oomycete species). Our results show that interdomain HGT is rare in oomycetes but has occurred. We located five well-supported examples, including one that could potentially break down xenobiotics within the cell.