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The genus Agaricus includes more than 500 species mostly containing the edible and cultivated species worldwide. As part of the ongoing studies on the biodiversity of genus Agaricus in Pakistan, our objective was to focus on A. sect. Minores which is the largest section of the genus. In the first phylogenetic analyses based on the ITS region of the nuclear ribosomal DNA, our sample included specimens of 97 named species, 27 unnamed species, and 31 specimens (29 newly generated sequences in this study) from subtropical climate zones of Pakistan that likely belong to this section based on their morphology. The 31 specimens grouped into five distinct, well-supported clades corresponding to five species: A. glabriusculus already known from Pakistan and India, A. robustulus first recorded from Pakistan and briefly described here but already known from Bénin, Malaysia, China, and Thailand, and three possibly endemic new species described in detail A. badiosquamulosus sp. nov., A. dunensis sp. nov., and A. violaceopunctatus sp. nov. The sixth species currently known in Pakistan, including A. latiumbonatus also found in Thailand, were included in a multigene tree based on ITS, LSU, and Tef-1α sequence data. They all belong to a large pantropical paraphyletic group while most temperate species belong to a distinct clade, which includes about half of the species of the section. The current study aims to propose three novel species of genus Agaricus based on comprehensive morphological as well as molecular phylogenetic evidences from Pakistan., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bashir et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Agaricus is a genus with more than 500 species. Most of the new species reported since 2000 are tropical or subtropical. The study area, the Malakand region, located in the north of Pakistan, has a subtropical climate. In this study, nine species, including three new species, of Agaricus subgenus Pseudochitonia , are reported from this region. Description of the new species are based on morphological characteristics and phylogenetic analyses using three DNA regions: nuc ribosomal DNA internal transcribed spacers (ITS), fragments of the large subunit of nuc ribosomal DNA (28S), and the translation elongation factor 1-alpha gene ( TEF1 ). One new species, Agaricus lanosus , with wooly squamules on its cap, forms a lineage within Agaricus sect. Bivelares and cannot be classified with certainty in one of the two subsections ( Cupressorum and Hortenses ) of this section. Agaricus rhizoideus with rhizoid-like structure at the base of the stipe forms a basal clade in Agaricus sect. Hondenses . Specimens of the third new species, Agaricus malakandensis , form a species-level clade within Agaricus sect. Catenulati and exhibits the morphological characteristics of this section. Due to their similar ITS sequences, two previously unnamed specimens from Thailand ( A . sp. LD2012162 and CA799) are considered conspecific with A. malakandensis .

In a recent revision of the genus Agaricus, A. section Xanthodermatei was split into two sections A. sect. Hondenses and A. sect. Xanthodermatei. Our objectives were to investigate the species diversity of both sections in Pakistan and to give an overview of the major clades. Phylogenetic analyses based on the combined nucLSU, ITS and TEF1 dataset from 35 specimens of both sections revealed three major clades. Analyses based on ITS dataset and 106 specimens, including 33 from Pakistan, reveal eight new species and one new record species. These nine species are described in detail. It is noteworthy that intraspecific variability as well as interspecific variability between closely related species were very low in ITS sequences in many cases. In the case of the two new species A. xanthochromaticus and A. griseovariegatus, TEF1 sequence data were much more efficient than ITS to distinguish these species from each other. The other new species are A. atroumbonatus, A. fumidicolor, A. macropeplus, A. parviniveus, A. swaticus and A. bambusetorum. The latter is the only new species of A. sect. Hondenses in which it is morphologically atypical and also the unique (sub)tropical species. Agaricus gregariomyces is recorded for the first time in Pakistan. In addition, brief descriptions are provided not only for A. bisporiticus, A. endoxanthus and A. punjabensis, which are reported again in Pakistan, but also for A. californicus, which is reported for the first time in Spain and outside North America. In total 12 species of both sections were reported in Pakistan and half of them were from subtropical climatic areas, underlining the contribution of the climatic diversity to the high species richness in this country.