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 consistent with ongoing genetic exchange. 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 associated with centromere inactivation following interchromosomal rearrangements. 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.

Department of Agricultural Food and Environmental Sciences University of Perugia Perugia Italy

Department of Biochemistry Genetics and Microbiology Forestry and Agricultural Biotechnology Institute University of Pretoria Pretoria South Africa

Department of Infectious Disease Epidemiology Imperial College London London United Kingdom

Department of Molecular Genetics and Microbiology Duke University Medical Center Durham North Carolina USA

G K Skryabin Institute of Biochemistry and Physiology of Microorganisms PSCBR RAS Pushchino Russia

Laboratory of Fungal Genetics and Metabolism Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague Czechia

Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures Braunschweig Germany

M 5 Lomonosov Moscow State University Moscow Russia

UCIBIO i4HB Departamento de Ciências da Vida Faculdade de Ciências e Tecnologia Universidade Nova de Lisboa Caparica Portugal

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