Biology subjects

Rokas, A.

Publications and source records attributed to Rokas, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

Heterogeneity in the transcriptional response of the human pathogen Aspergillus fumigatus to the antifungal agent caspofungin

Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin is the second line therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, caspofungin induces a tolerance phenotype with partial reestablishment of fungal growth called caspofungin paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high caspofungin concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in caspofungin tolerance, and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the {Delta}crzACEA17 mutant, the {Delta}crzAAf293 mutant fails to activate cell wall remodeling genes upon caspofungin exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This work describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

microbiology↗

bioRxiv2021

A gene coevolution network provides insight into eukaryotic cellular and genomic structure and function

Orthologous gene coevolution--which refers to gene pairs whose evolutionary rates covary across speciation events--is often observed among functionally related genes. We present a comprehensive gene coevolution network inferred from the examination of nearly three million orthologous gene pairs from 332 budding yeast species spanning [~]400 million years of eukaryotic evolution. Modules within the network provide insight into cellular and genomic structure and function, such as genes functioning in distinct cellular compartments and DNA replication. Examination of the phenotypic impact of network perturbation across 14 environmental conditions using deletion mutant data from the bakers yeast Saccharomyces cerevisiae suggests that fitness in diverse environments is impacted by orthologous gene neighborhood and connectivity. By mapping the network onto the chromosomes of S. cerevisiae and the opportunistic human pathogen Candida albicans, which diverged [~]235 million years ago, we discovered that coevolving orthologous genes are not clustered in either species; rather, they are most often located on different chromosomes or far apart on the same chromosome. The budding yeast coevolution network captures the hierarchy of eukaryotic cellular structure and function, provides a roadmap for genotype-to-phenotype discovery, and portrays the genome as an extensively linked ensemble of genes.

evolutionary biology↗

bioRxiv2021

Aspergillus fumigatus acetate utilisation impacts virulence traits and pathogenicity

Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilisation of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolised via different pathways in A. fumigatus and that acetate utilisation is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilisation is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, to anti-fungal drugs and to human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilisation in A. fumigatus and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of anti-fungal strategies. ImportanceAspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonisation and invasion. This work shows that A. fumigatus metabolises acetate via different pathways, a process that is dependent on the transcription factor FacB. Furthermore, the type and concentration of the extracellular available carbon source were determined to shape A. fumigatus virulence determinants such as secondary metabolite secretion and cell wall composition. Subsequently, interactions with immune cells are altered in a carbon source-specific manner. FacB is required for A. fumigatus in vivo virulence in both insect and mammalian models of invasive aspergillosis. This is the first report that characterises acetate utilisation in A. fumigatus and highlights the importance of available host-specific carbon sources in shaping virulence traits and potentially subsequent disease outcome.

microbiology↗

bioRxiv2021

Examination of gene loss in the DNA mismatch repair pathway and its mutational consequences in a fungal phylum

The DNA mismatch repair (MMR) pathway corrects mismatched bases produced during DNA replication and is highly conserved across the tree of life, reflecting its fundamental importance for genome integrity. Loss of function in one or a few MMR genes can lead to increased mutation rates and microsatellite instability, as seen in some human cancers. While loss of MMR genes has been documented in the context of human disease and in hypermutant strains of pathogens, examples of entire species and species lineages that have experienced substantial MMR gene loss are lacking. We examined the genomes of 1,107 species in the fungal phylum Ascomycota for the presence of 52 genes known to be involved in the MMR pathway of fungi. We found that the median ascomycete genome contained 49 / 52 MMR genes. In contrast, four closely related species of obligate plant parasites from the powdery mildew genera Erysiphe and Blumeria, have lost between 5 and 21 MMR genes, including MLH3, EXO1, and DPB11. The lost genes span MMR functions, include genes that are conserved in all other ascomycetes, and loss of function of any of these genes alone has been previously linked to increased mutation rate. Consistent with the hypothesis that loss of these genes impairs MMR pathway function, we found that powdery mildew genomes with higher levels of MMR gene loss exhibit increased numbers of mononucleotide runs, longer microsatellites, accelerated sequence evolution, elevated mutational bias in the A|T direction, and decreased GC content. These results identify a striking example of macroevolutionary loss of multiple MMR pathway genes in a eukaryotic lineage, even though the mutational outcomes of these losses appear to resemble those associated with detrimental MMR dysfunction in other organisms. Significance StatementThe DNA mismatch repair pathway corrects nucleotide base errors that occur during the replication of DNA; loss of these genes leads to cancer. We examined the conservation of the DNA mismatch repair pathways genes across more than 1,000 species in a fungal phylum and found a lineage of powdery mildews, a group of fungi that infect the leaves of plants, which have experienced extensive loss of multiple, otherwise highly conserved, genes. The genomes of these powdery mildews show elevated rates of diverse types of mutation, raising the hypothesis that these organisms have diversified while lacking genes thought to be essential for the accurate replication of DNA.

evolutionary biology↗

bioRxiv2021

An evolutionary genomic approach reveals both conserved and species-specific genetic elements related to human disease in closely related Aspergillus fungi

Aspergillosis is an important opportunistic human disease caused by filamentous fungi in the genus Aspergillus. Roughly 70% of infections are caused by Aspergillus fumigatus, with the rest stemming from approximately a dozen other Aspergillus species. Several of these pathogens are closely related to A. fumigatus and belong in the same taxonomic section, section Fumigati. Pathogenic species are frequently most closely related to non-pathogenic ones, suggesting Aspergillus pathogenicity evolved multiple times independently. To understand the repeated evolution of Aspergillus pathogenicity, we performed comparative genomic analyses on 18 strains from 13 species, including 8 species in section Fumigati, which aimed to identify genes, both ones previously connected to virulence as well as ones never before implicated, whose evolution differs between pathogens and non-pathogens. We found that most genes were present in all species, including approximately half of those previously connected to virulence, but a few genes were section- or species-specific. Evolutionary rate analyses identified hundreds of genes in pathogens that were faster-evolving than their orthologs in non-pathogens. For example, over 25% of all single-copy genes examined in A. fumigatus were faster-evolving. Functional testing of deletion mutants of 17 transcription factor-encoding genes whose evolution differed between pathogens and non-pathogens identified eight genes that affect either fungal survival in a model of phagocytic killing, host survival in an animal model of fungal disease, or both. These results suggest that the evolution of pathogenicity in Aspergillus involved both conserved and species-specific genetic elements, illustrating how an evolutionary genomic approach informs the study of fungal disease.

evolutionary biology↗

bioRxiv2020

Genomic and phenotypic analysis of COVID-19-associated pulmonary aspergillosis isolates of Aspergillus fumigatus

The ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) first described from Wuhan, China. A subset of COVID-19 patients has been reported to have acquired secondary infections by microbial pathogens, such as fungal opportunistic pathogens from the genus Aspergillus. To gain insight into COVID-19 associated pulmonary aspergillosis (CAPA), we analyzed the genomes and characterized the phenotypic profiles of four CAPA isolates of Aspergillus fumigatus obtained from patients treated in the area of North Rhine-Westphalia, Germany. By examining the mutational spectrum of single nucleotide polymorphisms, insertion-deletion polymorphisms, and copy number variants among 206 genes known to modulate A. fumigatus virulence, we found that CAPA isolate genomes do not exhibit major differences from the genome of the Af293 reference strain. By examining virulence in an invertebrate moth model, growth in the presence of osmotic, cell wall, and oxidative stressors, and the minimum inhibitory concentration of antifungal drugs, we found that CAPA isolates were generally, but not always, similar to A. fumigatus reference strains Af293 and CEA17. Notably, CAPA isolate D had more putative loss of function mutations in genes known to increase virulence when deleted (e.g., in the FLEA gene, which encodes a lectin recognized by macrophages). Moreover, CAPA isolate D was significantly more virulent than the other three CAPA isolates and the A. fumigatus reference strains tested. These findings expand our understanding of the genomic and phenotypic characteristics of isolates that cause CAPA.

microbiology↗

bioRxiv2020

Rooting the animal tree of life

There has been considerable debate about the placement of the root in the animal tree of life, which has emerged as one of the most challenging problems in animal phylogenetics. This debate has major implications for our understanding of the earliest events in animal evolution, including the origin of the nervous system. Some phylogenetic analyses support a root that places the first split in the phylogeny of living animals between sponges and all other animals (the Porifera-sister hypothesis), and others find support for a split between comb jellies and all other animals (Ctenophora-sister). These analyses differ in many respects, including in the genes considered, species considered, molecular evolution models, and software. Here we systematically explore the rooting of the animal tree of life under consistent conditions by synthesizing data and results from 15 previous phylogenomic studies and performing a comprehensive set of new standardized analyses. It has previously been suggested that site-heterogeneous models favor Porifera-sister, but we find that this is not the case. Rather, Porifera-sister is only obtained under a narrow set of conditions when the number of site-heterogeneous categories is unconstrained and range into the hundreds. Site-heterogenous models with a fixed number of dozens of categories support Ctenophora-sister, and cross-validation indicates that such models fit the data just as well as the unconstrained models. Our analyses shed light on an important source of variation between phylogenomic studies of the animal root. The datasets and analyses consolidated here will also be a useful test-platform for the development of phylogenomic methods for this and other difficult problems.

evolutionary biology↗

bioRxiv2020

PhyKIT: a UNIX shell toolkit for processing and analyzing phylogenomic data

Diverse disciplines in biology process and analyze multiple sequence alignments (MSAs) and phylogenetic trees to evaluate their information content, infer evolutionary events and processes, and predict gene function. However, automated processing of MSAs and trees remains a challenge due to the lack of a unified toolkit. To fill this gap, we introduce PhyKIT, a toolkit for the UNIX shell environment with 30 functions that process MSAs and trees, including but not limited to estimation of mutation rate, evaluation of sequence composition biases, calculation of the degree of violation of a molecular clock, and collapsing bipartitions (internal branches) with low support. To demonstrate the utility of PhyKIT, we detail three use cases: (1) summarizing information content in MSAs and phylogenetic trees for diagnosing potential biases in sequence or tree data; (2) evaluating gene-gene covariation of evolutionary rates to identify functional relationships, including novel ones, among genes; and (3) identify lack of resolution events or polytomies in phylogenetic trees, which are suggestive of rapid radiation events or lack of data. We anticipate PhyKIT will be useful for processing, examining, and deriving biological meaning from increasingly large phylogenomic datasets. PhyKIT is freely available on GitHub (https://github.com/JLSteenwyk/PhyKIT) and documentation including user tutorials are available online (https://jlsteenwyk.com/PhyKIT).

evolutionary biology↗

bioRxiv2020

Transcriptomic, protein-DNA interaction, and metabolomic studies of VosA, VelB, and WetA in Aspergillus nidulans asexual spores

In filamentous fungi, asexual development involves morphological differentiation and metabolic changes leading to the formation of asexual spores. The process of asexual spore formation in Aspergillus is precisely regulated by multiple transcription factors (TFs), including VosA, VelB, and WetA, and these three TFs are key regulators of the formation and maturation of asexual spores (conidia) in Aspergillus including the model fungus Aspergillus nidulans. To gain a mechanistic insight on the complex regulatory roles of these TFs in asexual spores, we conducted genome-wide studies on the expression, protein-DNA interactions, and primary and secondary metabolism employing A. nidulans conidia. RNA sequencing and chromatin immunoprecipitation-sequencing data have revealed that the three TFs directly or indirectly regulate the expression of genes associated with spore-wall formation/integrity, asexual development, and secondary metabolism. In addition, metabolomics analyses of wild-type and mutant conidia indicate that these three TFs regulate a diverse array of primary and secondary metabolism. In summary, WetA, VosA, and VelB play inter-dependent and distinct roles governing morphological development and primary/secondary metabolic remodeling in Aspergillus conidia. ImportanceFilamentous fungi produce a vast number of asexual spores that act as reproductive and propagator cells. These spores affect humans, due to the infectious or allergenic nature of the propagule. Aspergillus species produce asexual spores called conidia and their formation involves morphological development and metabolic changes, and the associated regulatory systems are coordinated by spore-specific transcription factors. To understand the underlying global regulatory programs and cellular outcomes associated with conidia formation, functional genomic and metabolomic analyses were performed in the model fungus Aspergillus nidulans. Our results show that the fungus specific WetA/VosA/VelB transcription factors govern the coordination of morphological and chemical developments during sporogenesis. The results of this study provide insights into the genetic regulatory networks about how morphological developments and metabolic changes are coordinated in fungi. The findings are relevant for other Aspergillus species such as the major human pathogen Aspergillus fumigatus and the aflatoxin-producer Aspergillus flavus.

microbiology↗

bioRxiv2020

A genome-scale phylogeny of Fungi; insights into early evolution, radiations, and the relationship between taxonomy and phylogeny

Phylogenomic studies based on genome-scale amounts of data have greatly improved understanding of the tree of life. Despite their diversity, ecological significance, and biomedical and industrial importance, large-scale phylogenomic studies of Fungi are lacking. Furthermore, several evolutionary relationships among major fungal lineages remain controversial, especially those at the base of the fungal phylogeny. To begin filling these gaps and assess progress toward a genome-scale phylogeny of the entire fungal kingdom, we compiled a phylogenomic data matrix of 290 genes from the genomes of 1,644 fungal species that includes representatives from most major fungal lineages; we also compiled 11 additional data matrices by subsampling genes or taxa based on filtering criteria previously shown to improve phylogenomic inference. Analyses of these 12 data matrices using concatenation- and coalescent-based approaches yielded a robust phylogeny of the kingdom in which [~]85% of internal branches were congruent across data matrices and approaches used. We found support for several relationships that have been historically contentious (e.g., for the placement of Wallemiomycotina (Basidiomycota), as sister to Agaricomycotina), as well as evidence for polytomies likely stemming from episodes of ancient diversification (e.g., at the base of Basidiomycota). By examining the relative evolutionary divergence of taxonomic groups of equivalent rank, we found that fungal taxonomy is broadly aligned with genome sequence divergence, but also identified lineages, such as the subphylum Saccharomycotina, where current taxonomic circumscription does not fully account for their high levels of evolutionary divergence. Our results provide a robust phylogenomic framework to explore the tempo and mode of fungal evolution and directions for future fungal phylogenetic and taxonomic studies.

evolutionary biology↗

bioRxiv2020

Aspergillus fumigatus G-protein coupled receptors GprM and GprJ are important for the regulation of the cell wall integrity pathway, secondary metabolite production, and virulence

G-protein coupled receptors (GPCRs) are extracellular signalling receptors that sense environmental cues to coordinate a biological response. Fungi sense their environment primarily through GPCR-mediated signalling pathways, which in turn regulate fungal development, metabolism, virulence and mycotoxin biosynthesis. A. fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate when compared to the wild-type (WT) strain, and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the {Delta}gprM and {Delta}gprJ strains and decreased in the overexpression mutants when compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. RNA-sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the {Delta}gprM and {Delta}gprJ strains, and that this regulation occurs, at least partially, through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signalling. Finally, we show that the {Delta}gprM and {Delta}gprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. This work further contributes to unravelling functions of A. fumigatus GPCRs and shows that GprM and GprJ are essential for CWI, secondary metabolite production and virulence. Author summaryA. fumigatus is the main ethiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immuno-compromised humans. Withstanding the host environment is essential for A. fumigatus virulence and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which in turn regulate fungal development, metabolism, virulence and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

microbiology↗

bioRxiv2020

Signatures of optimal codon usage predict metabolic ecology in budding yeasts

Reverse ecology is the inference of ecological information from patterns of genomic variation. One rich, heretofore underutilized, source of ecologically-relevant genomic information is codon optimality or adaptation. Bias toward codons that match the tRNA pool is robustly associated with high gene expression in diverse organisms, suggesting that codon optimization could be used in a reverse ecology framework to identify highly expressed, ecologically relevant genes. To test this hypothesis, we examined the relationship between optimal codon usage in the classic galactose metabolism (GAL) pathway and known ecological niches for 329 species of budding yeasts, a diverse subphylum of fungi. We find that optimal codon usage in the GAL pathway is positively correlated with quantitative growth on galactose, suggesting that GAL codon optimization reflects increased capacity to grow on galactose. Optimal codon usage in the GAL pathway is also positively correlated with human-associated ecological niches in yeasts of the CUG-Ser1 clade and with dairy-associated ecological niches in the family Saccharomycetaceae. For example, optimal codon usage of GAL genes is greater than 85% of all genes in the major human pathogen Candida albicans (CUG-Ser1 clade) and greater than 75% of genes in the dairy yeast Kluyveromyces lactis (family Saccharomycetaceae). We further find a correlation between optimization in the thiamine biosynthesis and GAL pathways. As a result, optimal codon usage in thiamine biosynthesis genes is also associated with dairy ecological niches in Saccharomycetaceae, which may reflect competition with co-occurring microbes for extracellular thiamine. This work highlights the potential of codon optimization as a tool for gaining insights into the metabolic ecology of microbial eukaryotes. Doing so may be especially illuminating for studying fungal dark matter--species that have yet to be cultured in the lab or have only been identified by genomic material.

evolutionary biology↗

bioRxiv2020

Repeated horizontal gene transfer of GALactose metabolism genes violates Dollo's law of irreversible loss

Dollos law posits that evolutionary losses are irreversible, thereby narrowing the potential paths of evolutionary change. While phenotypic reversals to ancestral states have been observed, little is known about their underlying genetic causes. The genomes of budding yeasts have been shaped by extensive reductive evolution, such as reduced genome sizes and the losses of metabolic capabilities. However, the extent and mechanisms of trait reacquisition after gene loss in yeasts have not been thoroughly studied. Here, through phylogenomic analyses, we reconstructed the evolutionary history of the yeast galactose utilization pathway and observed widespread and repeated losses of the ability to utilize galactose, which occurred concurrently with the losses of GALactose (GAL) utilization genes. Unexpectedly, we detected three galactose-utilizing lineages that were deeply embedded within clades that underwent ancient losses of galactose utilization. We show that at least two, and possibly three, lineages reacquired the GAL pathway via yeast-to-yeast horizontal gene transfer. Our results show how trait reacquisition can occur tens of millions of years after an initial loss via horizontal gene transfer from distant relatives. These findings demonstrate that the losses of complex traits and even whole pathways are not always evolutionary dead-ends, highlighting how reversals to ancestral states can occur.

genetics↗

bioRxiv2020

Feature Frequency Profile-based phylogenies are inaccurate

Choi and Kim (PNAS, 117: 3678-3686; first published February 4, 2020; https://doi.org/10.1073/pnas.1915766117) used the alignment-free Feature Frequency Profile (FFP) method to reconstruct a broad sketch of the tree of life based on proteome data from 4,023 taxa. The FFP-based reconstruction reports many relationships that strongly contradict the current consensus view of the tree of life and its accuracy has not been tested. Comparison of FFP with current standard approaches, such as concatenation and coalescence, using simulation analyses shows that FFP performs poorly. We conclude that the phylogeny of the tree of life reconstructed by Choi and Kim is suspect based on methodology as well as prior phylogenetic evidence.

evolutionary biology↗

bioRxiv2020

ClipKIT: a multiple sequence alignment-trimming algorithm for accurate phylogenomic inference

Highly divergent sites in multiple sequence alignments, which stem from erroneous inference of homology and saturation of substitutions, are thought to negatively impact phylogenetic inference. Trimming methods aim to remove these sites before phylogenetic inference, but recent analysis suggests that doing so can worsen inference. We introduce ClipKIT, a trimming method that instead aims to retain phylogenetically-informative sites; phylogenetic inference using ClipKIT-trimmed alignments is accurate, robust, and time-saving.

evolutionary biology↗

bioRxiv2020

Genome-scale phylogeny and contrasting modes of genome evolution in the fungal phylum Ascomycota

Ascomycota, the largest and best-studied phylum of fungi, contains three subphyla: Saccharomycotina (budding yeasts), Pezizomycotina (filamentous fungi), and Taphrinomycotina (fission yeasts); organisms from all three subphyla have been invaluable as models in diverse fields (e.g., biotechnology, cell biology, genetics, and medicine). Despite its importance, we still lack a comprehensive genome-scale phylogeny or understanding of the similarities and differences in the mode of genome evolution within this phylum. To address these gaps, we examined 1,107 genomes from Saccharomycotina (332), Pezizomycotina (761), and Taphrinomycotina (14) species to infer the Ascomycota phylogeny, estimate its timetree, and examine the evolution of key genomic properties. We inferred a robust genome-wide phylogeny that resolves several contentious relationships and estimated that the Ascomycota last common ancestor likely originated in the Ediacaran (~563 {+/-} 68 million years ago). Comparisons of genomic properties revealed that Saccharomycotina and Pezizomycotina, the two taxon-rich subphyla, differed greatly in their genome properties. Saccharomycotina typically have smaller genomes, lower GC contents, lower numbers of genes, and higher rates of molecular sequence evolution compared to Pezizomycotina. Ancestral state reconstruction showed that the genome properties of the Saccharomycotina and Pezizomycotina last common ancestors were very similar, enabling inference of the direction of evolutionary change. For example, we found that a lineage-specific acceleration led to a 1.6-fold higher evolutionary rate in Saccharomycotina, whereas the 10% difference in GC content between Saccharomycotina and Pezizomycotina genomes stems from a trend toward AT bases within budding yeasts and toward GC bases within filamentous fungi. These results provide a robust evolutionary framework for understanding the diversification of the largest fungal phylum.

evolutionary biology↗

bioRxiv2020

Disentangling biological and analytical factors that give rise to outlier genes in phylogenomic matrices

The genomic data revolution has enabled biologists to develop innovative ways to infer key episodes in the history of life. Whether genome-scale data will eventually resolve all branches of the Tree of Life remains uncertain. However, through novel means of interrogating data, some explanations for why evolutionary relationships remain recalcitrant are emerging. Here, we provide four biological and analytical factors that explain why certain genes may exhibit "outlier" behavior, namely, rate of molecular evolution, alignment length, misidentified orthology, and errors in modeling. Using empirical and simulated data we show how excluding genes based on their likelihood or inferring processes from the topology they support in a supermatrix can mislead biological inference of conflict. We next show alignment length accounts for the high influence of two genes reported in empirical datasets. Finally, we also reiterate the impact misidentified orthology and short alignments have on likelihoods in large scale phylogenetics. We suggest that researchers should systematically investigate and describe the source of influential genes, as opposed to discarding them as outliers. Disentangling whether analytical or biological factors are the source of outliers will help uncover new patterns and processes that are shaping the Tree of Life.

evolutionary biology↗

bioRxiv2020

Beyond the biosynthetic gene cluster paradigm: Genome-wide co-expression networks connect clustered and unclustered transcription factors to secondary metabolic pathways

Fungal secondary metabolites are widely used as therapeutics and are vital components of drug discovery programs. A major challenge hindering discovery of novel secondary metabolites is that the underlying pathways involved in their biosynthesis are transcriptionally silent in typical laboratory growth conditions, making it difficult to identify the transcriptional networks that they are embedded in. Furthermore, while the genes participating in secondary metabolic pathways are typically found in contiguous clusters on the genome, known as biosynthetic gene clusters (BGCs), this is not always the case, especially for global and pathway-specific regulators of pathways activities. To address these challenges, we used 283 genome-wide gene expression datasets of the ascomycete cell factory Aspergillus niger generated during growth under 155 different conditions to construct two gene co-expression networks based on Spearmans correlation coefficients (SCC) and on mutual rank-transformed Pearsons correlation coefficients (MR-PCC). By mining these networks, we predicted six transcription factors named MjkA - MjkF to concomitantly regulate secondary metabolism in A. niger. Over-expression of each transcription factor using the Tet-on cassette modulated production of multiple secondary metabolites. We found that the SCC and MR-PCC approaches complemented each other, enabling the delineation of global (SCC) and pathway-specific (MR-PCC) transcription factors, respectively. These results highlight the great potential of co-expression network approaches to identify and activate fungal secondary metabolic pathways and their products. More broadly, we argue that novel drug discovery programs in fungi should move beyond the BGC paradigm and focus on understanding the global regulatory networks in which secondary metabolic pathways are embedded. ImportanceThere is an urgent need for novel bioactive molecules in both agriculture and medicine. The genomes of fungi are thought to contain vast numbers of metabolic pathways involved in the biosynthesis of secondary metabolites with diverse bioactivities. Because these metabolites are biosynthesized only under specific conditions, the vast majority of fungal pharmacopeia awaits discovery. To discover the genetic networks that regulate the activity of secondary metabolites, we examined the genome-wide profiles of gene activity of the cell factory Aspergillus niger across hundreds of conditions. By constructing global networks that link genes with similar activities across conditions, we identified six global and pathway-specific regulators of secondary metabolite biosynthesis. Our study shows that elucidating the behavior of the genetic networks of fungi under diverse conditions harbors enormous promise for understanding fungal secondary metabolism, which ultimately may lead to novel drug candidates.

microbiology↗