Agaricus albovariabilis is presented as a new species from peninsular India on the basis of morphological features and ITS sequence analysis. It is charcterized by small to medium sized white basidiomata, becoming brownish at maturity, unchanging context, mild anise odor, negative Schäffer’s and KOH reactions, and clavate to vesiculose clavate cheilocystidia. It is presented with detailed descriptions, photographs, phylogenetic reconstruction and a comparison with phenetically similar species.

Morphological study Agaricus specimens were gathered during the monsoon seasons of 2016 and 2021 from the grasslands of the JNTBGRI campus. Photographs of fresh specimens were taken in the field along with information about habit, habitat, odor and color changes. Macroscopic characters were drawn from fresh specimens with color codes following Kornerup and Wanscher (1978). Schäffer’s and KOH reactions were tested on fresh basidiomata surfaces. Basidiomata were dried overnight in a hot air oven at 50°C and sealed in zip lock covers. The holotype was deposited in the Central National Herbarium (CAL) in Calcutta, India, with CAL accession numbers. All additional specimens (paratypes) investigated are preserved at the Jawaharlal Nehru Tropical Botanic Garden and Research Institute’s Mycological Herbarium Thiruvananthapuram (TBGT). For microscopic examination, sections of the dried basidiomata were made and mounted in 3% aqueous KOH and stained with 1% Congo red. Features of basidiospores, basidia, cheilocystidia, pileipellis and annulus were measured and illustrated based on at least 30 measurements using an Olympus CX43 microscope. Basidiospore measurements include the range of all spores, together with the range of spore quotient ((Q), length/width ratio) and its mean value (Qm). The hilar appendix was excluded in the basidiospore measurements.DNA extraction, PCR and sequencingGenomic DNA was extracted from fresh specimens of the new Agaricus species following Izumitsu et al. (2012). The ITS1-5.8S-ITS4 region of rDNA were amplified with the primer pair ITS1 and ITS4 (White et al. 1990). The protocols for PCR amplification and sequencing followed Kumar et al. (2018a). The PCR products were sequenced at the commercial facility AgriGenome, Kochi. All the sequences obtained in this work have been deposited in GenBank (ON555779, ON555770).Sequence alignment and phylogenetic analysisThe dataset comprised two sequences from the specimens collected in the present study, and 58 reference sequences representing 40 species that were downloaded from GenBank, including A. bisporus (J.E.Lange) Imbatch which was used as the outgroup following Liu et al. (2020). The GenBank accession numbers of retrieved and newly generated sequences are given Table 1. Sequences were aligned by the MAFFT web tool (<www.ebi.ac.uk/Tools/msa/mafft/>) with default settings. The aligned data matrix of nrITS sequences from 60 taxa, including A. bisporus as outgroup, was then manually adjusted in BioEdit v7.2.6.1 (Hall 1999). Maximum likelihood (ML) analysis was performed in RAxMLGUI 2.0 (Edler et al. 2021) under a GTR+GAMMA model with 1000 rapid bootstrap replicates. The best substitution model was selected as per the BIC score by running model test in the same software. The phylogram inferred from the ML analysis was visualised with MEGA X (Kumar et al. 2018b).