Cryptic Diversity and Ecological Overlap in Sporothrix schenckii: Insights from Multilocus Phylogenetics of Clinical and Environmental Isolates

<i>Sporothrix schenckii</i> is a pathogenic fungus with both clinical and environmental origins that was traditionally described as a single species but is increasingly recognized as being genetically diverse. In this study, we analyzed multiple isolates recovered from human sporotrichosis cases and environmental sources across Latin America (Mexico, Guatemala, Colombia). We conducted a polyphasic analysis of 16 isolates, integrating morphological data with multilocus sequence analysis (MLSA) targeting the internal transcribed spacer (ITS), calmodulin (<i>CAL</i>), β-tubulin (<i>BT2</i>), and translation elongation factor 1-α (<i>TEF</i>) gene regions. Phylogenetic relationships were resolved via maximum likelihood, and genetic structure was corroborated via four independent clustering methods: minimum spanning tree, principal component analysis, multidimensional scaling, and self-organizing maps. MLSA reidentified six isolates as <i>S. globosa</i> and confirmed the absence of <i>S. brasiliensis</i> in the cohort. The remaining <i>S. schenckii s. str.</i> isolates were resolved into three clades (A, B, and C). Notably, clade B (EH748, EH194, and EH257) formed a genetically divergent cluster with the highest nucleotide diversity (π = 0.03556) and was consistently segregated by all clustering algorithms. Clinical and environmental isolates were phylogenetically intermingled, supporting an active environmental reservoir for human infections. Phenotypic data, including colony size and conidial and yeast dimensions, varied but did not clearly distinguish between clinical and environmental origins. Our study provides compelling molecular evidence for a previously unrecognized, highly divergent clade within <i>S. schenckii s. str.</i>, indicative of ongoing cryptic speciation. These findings refine the taxonomy of medically important <i>Sporothrix</i> species and reveal a distinct epidemiological profile for sporotrichosis in the studied regions, separate from the <i>S. brasiliensis</i>-driven epizootic. This highlights the critical role of molecular surveillance for accurate diagnosis, treatment, and public health strategies.