Márcia David-Palma

Malassezia pachydermatis is a zoophilic yeast found on the skin and in the outer ear canal of many mammals. It normally maintains a commensal lifestyle, but can cause dermatitis and otitis in predisposed hosts, particularly in atopic dogs. M. pachydermatis is genetically diverse, with strains clustering into at least three phylogroups based on molecular typing, a pattern we now confirm through whole-genome sequencing (WGS). Accurate species and strain-level identification is essential for understanding its epidemiology, pathogenic potential, and response to treatment. In this study, we established Fourier transform infrared (FTIR) spectroscopy as a rapid, cost-effective method for distinguishing M. pachydermatis from other Malassezia species, including M. globosa , M. furfur , M. restricta , and M. sympodialis . Within M. pachydermatis , FTIR spectroscopy resolved even closely related strains with high accuracy, producing clusters congruent with multilocus sequence typing (MLST)- and WGS-based phylogeny. The incorporation of an artificial neural network classifier further enhanced the discriminatory power, enabling robust and automated strain assignment. These findings demonstrate the potential of FTIR spectroscopy as a practical tool for large-scale epidemiological surveillance of M. pachydermatis and for clinical and veterinary applications where strain-level identification could inform treatment and management of Malassezia -associated diseases. IMPORTANCE: Malassezia pachydermatis is a yeast that commonly inhabits the skin and ear canals of mammals but can cause dermatitis and otitis in predisposed hosts, especially dogs with allergies or immunosuppression. This species displays substantial genetic diversity, with strains falling into distinct phylogroups that may differ in their biology and clinical significance. Determining these differences has typically required advanced molecular or genomic methods, which can be costly and time-consuming. In this study, we demonstrate that Fourier transform infrared spectroscopy can rapidly and accurately distinguish M. pachydermatis from other Malassezia species and resolve genetic groups within the species in a way that reflects whole-genome relationships. This capability offers a practical tool for investigating the epidemiology and inter-/intraspecies diversity of M. pachydermatis and for guiding targeted management of Malassezia -associated diseases in both veterinary and, potentially, human medicine.

1 Canine atopic dermatitis (CAD) is a chronic inflammatory skin condition sometimes associated with microbial dysbiosis, including alterations in colonization by the lipophilic yeast Malassezia pachydermatis . This study investigated the population diversity of M. pachydermatis in the ear canals of healthy and CAD-affected dogs using Fourier-transform infrared (FTIR) spectroscopy and whole genome sequencing (WGS). Among 60 dogs, M. pachydermatis prevalence was significantly higher in CAD cases than in healthy controls. FTIR spectroscopy revealed greater strain heterogeneity in CAD-affected dogs, often with distinct genotypes in each ear, while healthy dogs exhibited more homogeneous populations. Using a previously developed FTIR-based artificial neural network classifier, we assigned strains to three phylogroups. Strains from phylogroups I and III were significantly enriched in CAD-affected dogs, while phylogroup II was most prevalent overall and the dominant phylogroup in healthy controls. This suggests that CAD-associated inflammation may favor specific M. pachydermatis phylogroups and sub-clusters within phylogroups, shaping colonization dynamics. FTIR-based typing showed full concordance with WGS across 35 sequenced isolates, recapitulating relationships among phylogenetically related isolates and their similar phenotypic profiles. Overall, our findings reveal strain-level shifts in M. pachydermatis populations associated with CAD and establish FTIR spectroscopy as a rapid, cost-effective tool for large-scale epidemiological studies.

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.

1 Malassezia pachydermatis is a zoophilic yeast found on the skin and in the outer ear canal of many mammals. It normally maintains a commensal lifestyle but can cause dermatitis and otitis in predisposed hosts, particularly in atopic dogs. M. pachydermatis is genetically diverse, with strains clustering into at least three phylogroups based on molecular typing, a pattern we now confirm through whole-genome sequencing (WGS). Accurate species and strain-level identification is essential for understanding its epidemiology, pathogenic potential, and response to treatment. In this study, we established Fourier Transform Infrared (FTIR) spectroscopy as a rapid, cost-effective method for distinguishing M. pachydermatis from other Malassezia species, including M. globosa, M. furfur, M. restricta , and M. sympodialis . Within M. pachydermatis , FTIR spectroscopy resolved even closely related strains with high accuracy producing clusters congruent with WGS-based phylogeny. The incorporation of an Artificial Neural Network classifier further enhanced the discriminatory power, enabling robust and automated strain assignment. These findings demonstrate the potential of FTIR spectroscopy as a practical tool for large-scale epidemiological surveillance of M. pachydermatis and for clinical and veterinary applications where strain-level identification could inform treatment and management of Malassezia -associated diseases. Importance: Malassezia pachydermatis is a yeast that commonly inhabits the skin and ear canals of mammals but can cause dermatitis and otitis in predisposed hosts, especially dogs with allergies. This species displays substantial genetic diversity, with strains falling into distinct phylogroups that may differ in their biology and clinical significance. Determining these differences has typically required advanced molecular or genomic methods, which can be costly and time-consuming. In this study, we demonstrate that Fourier transform infrared spectroscopy can rapidly and accurately distinguish M. pachydermatis from other Malassezia species and resolve genetic groups within the species in a way that reflects whole-genome relationships. This capability offers a practical tool for investigating the epidemiology and inter-/intraspecies diversity of M. pachydermatis and for guiding targeted management of Malassezia -associated diseases in both veterinary and, potentially, human medicine.

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 Summary: Cryptococcus 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.

Sexual reproduction in basidiomycete fungi is governed by MAT loci ( P/R and HD ), which exhibit remarkable evolutionary plasticity, characterized by expansions, rearrangements, and gene losses often associated with mating system transitions. The sister genera Cryptococcus and Kwoniella provide a powerful framework for studying MAT loci evolution owing to their diverse reproductive strategies and distinct architectures, spanning bipolar and tetrapolar systems with either linked or unlinked MAT loci. Building on recent comparative genomic analyses, we generated additional chromosome-level assemblies, uncovering distinct trajectories shaping MAT loci organization. Contrasting with the small-scale expansions and gene acquisitions observed in Kwoniella , our analyses revealed independent expansions of the P/R locus in tetrapolar Cryptococcus , possibly driven by pheromone gene duplications. Notably, these expansions coincided with a pronounced GC-content reduction best explained by reduced GC-biased gene conversion following recombination suppression, rather than relaxed codon usage selection. Diverse modes of MAT locus linkage were also identified, including three previously unrecognized transitions: one resulting in a pseudobipolar arrangement and two leading to bipolarity. All three transitions involved translocations. In the pseudobipolar configuration, the P/R and HD loci remained on the same chromosome but genetically unlinked, whereas the bipolar transitions additionally featured rearrangements that fused the two loci into a nonrecombining region. Mating assays confirmed a sexual cycle in C. decagattii , demonstrating its ability to undergo mating and sporulation. Progeny analysis in K. mangrovensis revealed substantial ploidy variation and aneuploidy, likely stemming from haploid-diploid mating, yet evidence of recombination and loss of heterozygosity indicates that meiotic exchange occurs despite irregular chromosome segregation. Our findings underscore the importance of continued diversity sampling and provide further evidence for convergent evolution of fused MAT loci in basidiomycetes, offering new insights into the genetic and chromosomal changes driving reproductive transitions.

In exploring the evolutionary trajectories of both pathogenesis and karyotype dynamics in fungi, we conducted a large-scale comparative genomic analysis spanning the Cryptococcus genus, encompassing both global human fungal pathogens and nonpathogenic species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species, covering virtually all known diversity within these genera. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at preadaptive pathogenic potential, our analysis found evidence of gene gain (via horizontal gene transfer) and gene loss in pathogenic Cryptococcus species, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the 2 genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5, or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion–fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes showed reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Overall, our findings advance our understanding of genetic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

A large-scale comparative genomic analysis was conducted for the global human fungal pathogens within the Cryptococcus genus, compared to non-pathogenic Cryptococcus species, and related species from the sister genus Kwoniella . Chromosome-level genome assemblies were generated for multiple species of both genera, resulting in a dataset encompassing virtually all of their known diversity. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at pre-adaptive pathogenic potential, our analysis found evidence in pathogenic Cryptococcus species of specific examples of gene gain (via horizontal gene transfer) and gene loss, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the two genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5 or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus . Instead, Cryptococcus species with less than 14 chromosomes, underwent chromosome reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Taken together, our findings advance our understanding of genomic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.