Genomic analyses of globally distributed Rhizopus microsporus populations indicate clinical isolates derived from environmental diversity reservoirs

Mucormycosis is a group of diseases that is increasing in frequency. A common opportunistic human fungal pathogen in this group is Rhizopus microsporus, which is a globally distributed species present in soil-associated environments. A subset of isolates in this species host endobacteria that are hypothesized to influence fungal pathogenicity in both clinical and environmental settings. We have limited understanding of how clinically and environmentally derived isolates are related or how physiological attributes, including thermotolerance and endosymbiosis, are correlated with population structure. Traditional molecular barcodes used to assess intraspecific relationships, such as ribosomal DNA internal transcribed spacer (ITS-rDNA)-based markers, do not provide species-level resolution, necessitating analyses of whole genome data. In this study, we generated novel whole genome sequencing data for six R. microsporus isolates and combined these data with publicly available whole genome sequences of 46 R. microsporus isolates. We evaluated these sequences to understand the evolutionary relationships among clinical and environmental isolates using phylogenomic and single nucleotide polymorphism (SNP)-based population genomics methods. We further studied their relationships by quantifying and comparing potential physiological differences and endosymbiont presence in a subset of 16 isolates with live cultures. We found that clinical isolates that originate from environmental settings contain higher molecular diversity than subpopulations isolated from clinical settings. We observed that environmental isolates grow faster than clinical isolates at temperatures between 22 and 37 C and that 7 of 16 (44%) contain endobacteria in the genus Mycetohabitans (Burkholderiales). Lastly, we observed that genome assembly size in R. microsporus is variable and that long-read sequencing technologies greatly enhance our ability to investigate the underlying genomic features. Our study provides a valuable backdrop for probing the basic biology and applied biomedical importance of Rhizopus and related fungi that cause mucormycosis.