DOI: 10.5061/dryad.v9s4mw704
title: A new species of Agaricus sect. Agaricus (Agaricaceae) from India

description: 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.
description: Morphological study <em>Agaricus </em>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 <em>Agaricus </em>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 <em>A. bisporus </em>(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
 (&lt;www.ebi.ac.uk/Tools/msa/mafft/&gt;) with default settings.
 The aligned data matrix of nrITS sequences from 60 taxa,
 including <em>A. bisporus</em> 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).