Understanding the distribution of agaric fungi through molecular phylogeny is critical for biodiversity assessment. This comprehensive investigation documented the presence of Panaeolus subbalteatus, a previously unreported agaric fungus, addressing a significant gap in the country's knowledge of fungal diversity. This psilocybin-containing mushroom species, known for its neurotoxic properties, was collected from humus-rich soil in the Gujranwala district of Punjab province. The study employed integrated morpho-anatomical and molecular approaches, including detailed microscopic examination of basidiospores, cystidia, and hyphal structures, coupled with DNA extraction and PCR amplification of the Internal Transcribed Spacer (ITS) region. Phylogenetic analysis using the Maximum Likelihood method with 23 nucleotide sequences of related members confirmed the specimen's identity as P. subbalteatus, with strong clustering with reference sequences from China, the Netherlands, and GenBank. The species exhibited characteristic features, including a creamy white to light brown pileus with a broad umbo, greyish-black lamellae, an absent annulus, and basidiospores measuring 10.8-14.2 × 5.7-8.5 µm with truncate germ pores. This first molecular record of P. subbalteatus from Pakistan expands the known geographical distribution of this psychoactive fungal species. It adds to the six previously documented Panaeolus species in the country. The findings underscore the importance of integrating morphological and molecular techniques for accurate fungal identification and provide a foundation for future investigations into the diversity, ecology, and toxicological significance of Panaeolus species in South Asian mycobiota, with implications for public health awareness regarding hallucinogenic mushrooms in the region.
