Abstract

Fungal secondary metabolite core genes including nonribosomal peptide synthetases, polyketide synthases, terpenes, and alkaloids are among the most rapidly evolving of fungal gene families. These core secondary metabolite genes are often co-localized in the genome with accessory genes involved in modifying the backbone product, forming secondary metabolite biosynthetic clusters (SMBC). The rapid evolution of both core secondary metabolite gene families and SMBCs is thought to be in response to selective pressures in the environment and may enable fungi to adapt to new environments or result in rapid host-shifts. We investigate the population genomic variation of secondary metabolite core genes and clusters in two insect pathogenic fungi, the beetle pathogen Tolypocladium inflatum and the wide host-range insect pathogen and endophyte Beauveria bassiana. Using Pac Bio single molecule real time sequencing, we sequenced six geographically diverse strains of T. inflatum to investigate the role of genome rearrangement in generating diversity in secondary metabolism. For the previously sequenced NRRL8044 strain we also used a Hi-C chromosome mapping approach to improve the chromosomal level assembly and provide support for rearrangement events. The nearly complete chromosomal assemblies produced using these methods have allowed investigation of fine-scale evolutionary genetic mechanisms contributing to the rapid evolution of secondary metabolite genes and clusters, including transposition, duplication/deletion, cluster rearrangement, and horizontal transfer in cluster evolution. Similarly, using Illumina technology, we have sequenced ten strains of B. bassiana and several outgroup Beauveria species (B. brongniartii, B. asiatica, and B. australis) to analyze the evolution of secondary metabolite clusters and other genes involved in host-interactions (e.g. G-protein coupled receptors, small cysteine-rich secreted proteins). Specifically, we investigate population level variation in secondary metabolism with potential roles in virulence. The results of our analysis shed light on the role of secondary metabolites in shaping the interaction of these fungi with distinct hosts and conversely on host-interactions in shaping the evolution of secondary metabolism.

Abstract

The yeast Saccharomyces cerevisiae has been an essential component of human civilization and used worldwide for baking, brewing, distilling and winemaking for thousands of years. However, the diversity, origin and evolutionary history of both the wild and domestic populations of the yeast remain elusive. Here we employed a total of 106 wild S. cerevisiae isolates from diversified natural sources including primeval forests and 160 domestic isolates from various fermentation processes including traditional fermented foods from countryside in China. We performed phenotypic profiling, sporulation and flow cytometry analyses, and high coverage genome re-sequencing in the natural ploidy of the isolates. Based on phylogenomic analysis, we identified 10 wild and 12 domestic lineages including the oldest lineage of the species. The genetic diversities of both the wild and domestic populations of the species in China are much higher than those revealed in other regions of the world, supporting the Far East Asian origin hypothesis of the species. The domestic lineages which are all heterozygous share a single ancestor which was probably formed by outcrossing between diverse wild lineages which are all homozygous. The domestic isolates evolved into two major groups adapting to solid- and liquid-state fermentation, respectively, and share elevated maltose utilization ability, no matter maltose is present or not in their living environments. The data imply that the domestic population of S. cerevisiae might originate from an ancestor initially adapting to maltose-rich niches rather than fruit. The sporulation efficiency and spore viability of domestic isolates were much lower than those of wild isolates, explaining the maintenance of heterozygosity of the domestic population. We found consistent expansion and contraction of genes in domestic lineages, acquisition of new traits through lineage specific introgression and horizontal gene transfer, and metabolic remodeling for adapting to specific fermentation environments. Our integrated phenotypic and genomic analyses based on a set of S. cerevisiae isolates representing the largest genetic diversity of the species documented so far show a nearly panoramic view of the evolutionary history of S. cerevisiae and provide new insights into the origin and domestication of the species.

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen of enormous clinical importance. To characterize the population diversity, subdivisions, and the level of genetic exchange of C. neoformans var grubii, we sequenced the genomes of over 400 diverse isolates. While our data supports the three previously identified lineages (VNI, VNII, and VNB), phylogenetic analysis highlighted a deep, non-recombining split in VNB (VNBI and VNBII) and that VNI was further subdivided into three distinct clades, two of which were globally distributed and one of which was restricted to sub-Saharan Africa. Despite the higher prevalence of MATa isolates in VNB compared to VNI, we find similar levels of linkage disequilibrium in VNI, VNBI and VNBII. While we did not detect recombination between lineages based on genome wide comparisons, we identified introgressions (5 to 260 kb) in all pairwise combinations of VNI, VNBI, and VNBII as recipient and donor. Notably, some haploid isolates show more widespread hybrid ancestry of multiple lineages, including isolates that appear to have originated from recent interbreeding. Excluding hybrid isolates and recombinant regions of other isolates impacts estimates of the timing of VNI global dispersal and of VNBI and VNBII diversification. By assembling and annotating multiple isolates of VNI, VNII, and VNB, we find that gene content is highly conserved, with few examples of lineage-specific genes. Rapidly evolving genes between the lineages include transcription factors and transferases, many of which have been implicated in virulence or oxidative stress resistance.

To evaluate how selective pressures in the environment coincidentally adapted C. neoformans for human virulence, we focused on a set of clinical and environmental VNB isolates from sub-saharan Africa. We found that the VNBII group was enriched for clinical samples relative to VNBI, while phenotypic profiling of sequenced isolates demonstrated that VNBI isolates were significantly more resistant to oxidative stress and more heavily melanized than VNBII isolates. Lack of melanization in both lineages was associated with loss-of-function mutations in the BZP4 transcription factor. A genome-wide association study across all VNB isolates revealed sequence differences between clinical and environmental isolates in virulence factors and stress response genes. Inositol transporters and catabolism genes, which process sugars present in plants and the human nervous system, were identified as targets of selection in all three lineages. These data highlight the complex evolutionary interplay between adaptation to natural environments and opportunistic infections, and that selection on specific pathways may predispose isolates to human virulence.

Desjardins CA et al. Population genomics and the evolution of virulence in the fungal pathogen Cryptococcus neoformans. Genome Res. 2017 Jul;27(7):1207-1219.

Rhodes J et al. Tracing Genetic Exchange and Biogeography of Cryptococcus neoformans var. grubii at the Global Population Level. Genetics. 2017 Jul 5. pii: genetics.117.203836.

Abstract

Epichloë species (Clavicipitaceae, Hypocreales) are systemic symbionts of cool season grasses (Poaceae subfamily Poöideae), some of which are strictly seed-borne (vertical transmission), whereas others can produce stromata that disrupt host seed production (choke disease) and, following heterothallic mating, give rise to airborne ascospores for horizontal transmission. Epichloë typhina is a choke pathogen of Brachypodium pinnatum, Dactylis glomerata, Holcus lanatus, Lolium perenne, Poa nemoralis, Poa trivialis, Puccinellia distans and other grasses, but of these it is apparently capable of vertical transmission only in Poa nemoralis and P. distans. The species is also phylogenetically indistinguishable from Epichloë sylvatica, strains of which can be either horizontally transmitted choke pathogens or nonpathogenic, vertically transmitted symbionts of Brachypodium sylvaticum. A feature of Epichloë species is their production of bioprotective alkaloids that deter insect and, in some cases, mammalian herbivores. Four classes of Epichloë alkaloids are known: aminopyrrolizidines (e.g., lolines), ergot alkaloids (e.g., ergovaline), indole-diterpenes (e.g., lolitrems) and pyrrolopyrazines (e.g., peramine). Genes for each class are typically arranged in a cluster, and the genes or entire clusters typically exhibit presence-absence polymorphism, giving a wide diversity of alkaloid profiles. Thus, in the context of the E. typhina pan-genome, the alkaloid biosynthesis genes are accessory genes, and part of the accessory (a.k.a., “flexible”) genome, in contrast to the core genome of genes present in all genomes of the species (e.g., housekeeping genes). In published pan-genome analyses of bacteria, indications are that the accessory genome may have many times more genes than the core genome. We have begun a similar investigation of the E. typhina/ E. sylvatica pan-genome. A total of 21 genomes from hosts listed above were sequenced and annotated with FGENESH and MAKER, and arranged into putative orthologous groups (OGs) with OrthoMCL followed by COCO-CL. In the process, filters for minimum length and maximum A-T content were used to minimize the inclusion of spurious gene calls, although it is likely that some actual genes were also removed by those filters. With a 55% AT maximum and aligned gene lengths of at least 50 amino acid positions, we identified 10,901 OGs with genes shared between at least two genomes. Of these, the core contained 6377 OGs that were shared between all genomes, leaving 4524 accessory OGs. In addition, there were 7818 singletons, identified in no more than one sequenced genome, to give a total of 18,719 genes. Although spot checks suggest that the filters were reasonably effective, we will further assess these and other filtering approaches. Then, we will apply rarefaction, capture-recapture and binomial mixture models to estimate the pan-genome size. Further we will assess how the number of genomes sequenced affects the estimation of the total pan-genome size.

Abstract

Fungi have different life-history traits, which may result in highly variable population genetic structure across species. Polypore fungi producing conspicuous fruiting bodies with measurable species traits may serve as good candidates to elucidate fundamental life-history processes such as dispersal. The aim of this study was to investigate the manner and degree in which life-history traits are affecting both spatial genetic structure and in the long-term demographic history of fungi. We compared population genetic statistics among eleven polypore species, using large SNP datasets generated from RAD sequencing. The eleven species with different life-history traits are phylogenetically, geographically and demographically comparable. Traits, related mainly to reproduction and dispersal have a significant influence on the degree of inbreeding among various species. Species producing perennial fruit bodies exhibited higher levels of inbreeding compared to species with ephemeral fruit bodies. Hence, producing short-lived fruit bodies, possibly coupled to quicker life cycles and faster population turnover, seems associated with lower inbreeding. In correspondence with these observations, species with perennial fruit bodies showed signs of local-scale spatial genetic structuring, implying limited dispersal capacity. Spatial population genetics analyses inferred from recent haplotype coancestry, revealed isolation by distance within the population, with variable response among species. Most of the species have undergone a similar demographic history, showing sign of expansion in the population sizes, likely coupled with the co-migration of their host substrate Picea abies after the last glaciation. One rare species, Amylocystis lapponica, showed a divergent demographic history, with signatures of a population contraction. Further ecological and evolutionary genomics features of the 11 non-model fungal species will be presented.