A contribution to the taxonomy and phylogeny of the genus Pisolithus (Sclerodermataceae, Boletales) in Mexico

Background and Aims: The genus Pisolithus is characterized by its angiocarpic basidiomes, which have a pulverulent gleba composed of peridioles, apical maturation, and echinulated basidiospores. In Mexico, two closely related species have been cited based exclusively on morphology: Pisolithus tinctorius and P. arhizus, associated with Pinus, Quercus, Carya and Eucalyptus. However, it has been pointed out that none of those species form associations with eucalyptus, and P. arhizus is distributed just in Eurasia based on ITS sequences. This study aims to contribute to the taxonomy and phylogeny of the genus Pisolithus in Mexico by characterizing Pisolithus specimens from temperate and arid zones, based on morphology and ITS barcode sequences of fungarium specimens, as well as soil metabarcoding sequences. Methods: Thirty-two specimens from the MEXU herbarium were characterized macro- and microscopically by light and scanning electron microscopy. Specialized literature was used for morphological identification. DNA was extracted with a CTAB 3% protocol. The ITS region was amplified with the ITS5 and ITS4 primer pairs. Phylogenetic analyses included 21 newly generated sequences, metagenomic soil sequences and sequences deposited in NCBI, and were performed using maximum likelihood and Bayesian Inference methods. Key results: We confirmed the presence of P. tinctorius associated with Pinus, Quercus, and Carya illinoinensis. Pisolithus albus, a new record for Mexico, is distinguished by its association with Eucalyptus sp., a whitish exoperidium, ochraceous gleba, and echinulate basidiospores with short, isolated spines up to 1 µm high. Phylogenetic analysis supports the identity of the Mexican collections. Conclusions: Among the Mexican samples analyzed, two well-defined species of Pisolithus were taxonomically identified, P. albus and P. tinctorius. Based on the material considered in this work, we did not find any that corresponded to P. arhizus.