Genomic and phenotypic insights into the expanding phylogenetic landscape of the Cryptococcus genus

The fungal genus Cryptococcus includes several life-threatening human pathogens as well as diverse saprobic species whose genome architecture, ecology, and evolutionary history remain less well characterized. Understanding how some lineages evolved into major pathogens remains a central challenge and may be advanced by comparisons with their nonpathogenic counterparts. Integrative approaches have become essential for delimiting species and reconstructing evolutionary relationships, particularly in lineages with cryptic diversity or extensive chromosomal rearrangements. Here, we formally characterize six Cryptococcus species representing distinct evolutionary lineages, comprising both newly discovered and previously recognized but unnamed taxa, through a combination of phylogenomic analyses, divergence metrics, chromosomal comparisons, mating assays, and phenotypic profiling. Among pathogenic taxa, we formally name Cryptococcus hyracis sp. nov., corresponding to the previously characterized VGV lineage within the C. gattii complex. In parallel, we describe five saprobic, nonpathogenic species isolated from fruit, soil, and bark beetle galleries, spanning four phylogenetic clades. We identify a strong ecological association with bark beetles for Cryptococcus porticicola sp. nov., the only newly described nonpathogenic species with multiple sequenced strains from diverse sites. In this species, we detect strain-level chromosomal variation and evidence of sexual reproduction, along with population-level signatures of recombination. Across the genus, chromosome-level comparisons reveal extensive structural variation, including species- and strain-specific rearrangements that may restrict gene flow. We also identify multiple instances of chromosome number reduction, often accompanied by genomic signatures consistent with centromere inactivation or loss of centromeric identity. Comparative metabolic profiling with Biolog phenotype microarrays reveals clade-level differentiation and distinct substrate preferences, which may reflect metabolic divergence and habitat-specific diversification. Notably, we confirm that thermotolerance is restricted to clinically relevant taxa. These findings refine the species-level taxonomy of Cryptococcus, broaden its known genomic and ecological diversity, and strengthen the framework for investigating speciation, adaptation, and the emergence of pathogenicity within the genus. Author SummaryCryptococcus is a genus of fungi that includes both pathogenic species capable of causing life-threatening infections in humans and many environmental species that inhabit soil, fruit, decaying wood, and insect-associated environments with no known link to disease. Here, we formally describe six previously unnamed species based on genome-wide comparisons, genetic divergence metrics, chromosome structure, reproductive behavior, and metabolic profiling. One of these species belongs to the pathogenic lineage, while the others are distinct environmental taxa, including several recovered from bark beetles or their galleries, indicating repeated occurrence in this ecological niche. For one of these insect-associated species, represented by multiple isolates, we detected signatures of sexual reproduction and genetic exchange in natural populations. This study expands the framework for investigating how certain Cryptococcus lineages adapt to new environments or evolve traits relevant to pathogenicity. It also emphasizes that continuous environmental sampling remains essential for capturing fungal diversity and illustrates the power of combining genomic and phenotypic data to define species boundaries and uncover lineages that may otherwise remain undetected.