﻿Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman, based on morphology and molecular phylogeny

﻿Abstract Hymenagaricus has small to medium-sized mushrooms and the cap surface with squamulose pellicles, consisting of hymeniform or pseudoparenchymatous cells and yellowish-brown basidiospores. The species of Hymenagaricus are very similar to those of Xanthagaricus and it is extremely difficult to differentiate the species of both genera in the field. However, phylogenetically, both the genera are clearly distinct. In this study, we describe two new species of Hymenagaricus, i.e. H.wadijarzeezicus and H.parvulus from the southern part of Oman. Species descriptions are based on a combination of morphological characteristics of basidiomata and phylogenetic analyses of three gene regions: internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), the large subunit of nuclear ribosomal DNA (28S) and translation elongation factor one alpha (EF-1α). Full descriptions, micrographs and illustration of anatomical features, basidiomata photos and phylogenetic analyses results of the new taxa are provided. Morphological comparisons of new taxa with similar species and a key to species included in the phylogenetic analyses are also provided.


Introduction
Three species previously in Agaricus subgenus Conioagaricus, Agaricus hymenopileus, A. alphitochrous and A. nigrovinosus, were placed in a new and separate genus called Hymenagaricus Heinem.by Heinemann in 1981.This taxonomic change was likely due to the distinct features observed in the cap of these species during different stages of their development.At the young stage, the pilei are entirely covered with a pellicle, but as they mature, the pellicle is disrupted, leaving a single large squamulose pellicle at the centre of the pileus.The squamules are composed of hymeniform or pseudoparenchymatous cells, which set these species apart from others within the genus Agaricus (Heinemann 1981).The genus Hymenagaricus was typified by H. hymenopileus (Heinem.),belonging to the family Agaricaceae Chevall.(Heinemann 1981).
Species of Hymenagaricus are saprotrophic in nature and are mostly distributed in the Palaeotropical Regions.Members of this genus are recognised by the squamulose pellicle on the pileus surface that mostly consists of hymeniform cells or pseudoparenchymatous tissues, yellow to yellowish-brown basidiospores and the absence of both pleurocystidia and clamp connections (Heinemann and Little Flower 1984;Reid and Eicker 1995;Little Flower et al. 1997;Hosen et al. 2017;Al-Kharousi et al. 2022a).The number of known species in the genus is 17 (Hussain et al. 2018;Kumla et al. 2021Kumla et al. , 2023;;Syed et al. 2023).
Phylogenetically, species of Hymenagaricus are intermixed with the monotypic genus Heinemannomyces Watling (Hosen et al. 2017;Hussain et al. 2018).This intermixing may be due to limited molecular data available for the previously-described species of Hymenagaricus.However, morphologically, both genera can be differentiated.Heinemannomyces with single species H. splendidissimus Watling, distributed in southeast Asia, has medium-sized basidiomata, with woolly fibrillose cap surface, composed of pseudoparenchymatous cells and the spore print is leaden-grey to dark blue (Watling 1998).
During the years 2022-23, macrofungal exploration missions were conducted in the Dhofar Region, in which we collected ten (10) collections of Hymenagaricus.Morphological characterisation and multigene (ITS, 28S, EF-1α) phylogenetic analyses revealed that the 10 collections represent two new species, which are described in this study.

Study sites and field sampling
The specimens were collected in the Dhofar Region, located in the south of the Sultanate of Oman.The region experiences a monsoon-influenced climate with a distinct wet season known as the Khareef, which occurs from June to early September (Bookhagen et al. 2005).During this time period, the moist and cool air from the Indian Ocean is drawn in by the southwest monsoon, bringing significant rainfall into the region, which is extremely rare in the rest of the Arabian Peninsula, including Oman.This seasonal variation supports a diverse ecosystem and separates Dhofar from the arid desert conditions that prevail in the Arabian Peninsula (El-Sheikh 2013).The Khareef season triggers the growth of various plants and trees, including frankincense trees, creating a lush and vibrant landscape where a number of saprotrophic mushrooms can flourish (Al-Kharousi et al. 2022a).
In the current study, mushroom specimens were collected from three localities (Wadi Naheez, Wadi Jahaneen, Wadi Jarzeez) of the Dhofar Region, in the months of August-September 2022 to 2023.The specimens were photographed in the field and field characteristics such as the shape, colour, size and smell of basidiomata were noted.The samples were dried using a fruit dehydrator with temperature adjusted at 45 °C (Hu et al. 2022).After drying, the specimens were kept in zip lock plastic bags and stored at -80 °C for two weeks to kill all the insects/eggs/larvae.After the cold temperature treatment, the samples were characterised morpho-anatomically and phylogenetically.All the samples are deposited in Oman Animal and Plant Genetic Resources Center (Mawarid), AlKhoud, Muscat, Sultanate of Oman.

Morphological investigation
For microscopic study, handmade sections were made from lamellae, cap and stipe surfaces and annulus.Thin small sections were initially mounted in 5% aqueous potassium hydroxide (KOH) (w/v) and then re-hydrated in 1% aqueous Cong red (w/v) for a more obvious appearance.Microscopic features such as the size, shape and colour of basidiospores, basidia, cheilocystidia, pellicle structure, veil and annulus morphology were studied under a compound microscope (ECLIPSE Ni-U, Nikon Co., Ltd., Japan).For size measurements of these structures, Piximetre (http://ach.log.free.fr/Piximetre/)was used.For the morphological terminology, Vellinga and Noordeloos (2001) was followed.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from dried specimens using X-AMP DNA reagent kit (Dubuque, Iowa, USA), following the manufacturer's protocol.A volume of 200 µl X-AMP DNA reagent was taken in an Eppendorf tube containing the sample (approximately 5-15 mg of gills) and incubated for 15 minutes at 70 °C.After cooling, 2 µl solution from the sample was used as a DNA template directly for polymerase chain reaction (PCR) without any further treatment.We amplified three gene regions, including the internal transcribed spacer (ITS), the large subunit of nuc rDNA (28S) and the translation elongation factor 1 alpha (EF-1α) gene.The primer combinations were: ITS1F and ITS4 for ITS (White et al. 1990;Gardes and Bruns 1993), LR0R and LR5 for 28S (Vilgalys and Hester 1990;White et al. 1990), EF1-983 and EF1-1567R for EF-1α (Rehner and Buckley 2005).PuReTaqTM Ready-To-Go PCR beads (GE Healthcare UK Limited, Buckinghamshire, UK) were used for PCR amplification.We added 1.0 µl of each primer (10 µmol/l), 2 µl DNA template and 22 µl Nuclease free water to each bead.For ITS amplification, the PCR conditions were optimised as: initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 45 s and extension at 72 °C for 1 min.For the 28S and EF-1α regions, only the annealing temperature was optimised, 52 °C for 28S and 60 °C EF-1α, respectively (Hussain et al. 2022).The PCR products were purified and then sequenced from Macrogen Inc. © (Seoul, Republic of Korea) bidirectionally using the same primers.

Sequence alignment and phylogenetic analyses
Consensus sequences were created from the forward and reverse primer reads of the newly-generated ITS, 28S and EF-1α sequences using BioEdit v.7.0.9.0 (Hall 1999).We performed BLAST searches for the newly-generated sequences; only ITS and 28S regions showed maximum similarity with Hymenagaricus species.In the case of EF-1α sequences, the BLAST search re-vealed Heinemannomyces sp.(ZRL185) is the most similar species because, in GenBank, no EF-1α sequences of Hymenagaricus are available.This is the reason that we used only ITS and 28S sequences in the phylogenetic analyses.A combined ITS-28S dataset was constructed from the sequences used in the recent studies of Hymenagaricus (Mwanga and Tibuhwa 2014;Hosen et al. 2017;Kumla et al. 2021Kumla et al. , 2023;;Syed et al. 2023).The final ITS-28S dataset was comprised of 27 specimens, including 26 ITS and 20 28S sequences (Table 1).Agaricus campestris L. (LAPAG370) was used as the outgroup taxon.Sequences were aligned using MAFFT v.7 (Katoh et al. 2019) and visually inspected using BioEdit v.7.0.9.0 (Hall 1999).Maximum Likelihood (ML) and Bayesian Inference (BI) methods were used for the phylogenetic analyses.Maximum Likelihood (ML) phylogeny was performed with RAxML-HPC Black-Box, implemented on CIPRES Science Gateway (Miller et al. 2010;Stamatakis

Phylogenetic analyses
In this study, 19 new sequences (7 ITS, 6 28S and 6 EF-1α) were generated from our collections of Hymenagaricus.There were no EF-1α sequences of the genus available in GenBank; therefore, only combined ITS-28S sequences were used in the final data matrix.Diagnosis.The new species Hymenagaricus wadijarzeezicus can be differentiated from other species of the genus by its unique whitish woolly veil, covering both the cap and the stipe surfaces.
Etymology.The specific epithet 'wadijarzeezicus' refers to the valley Jarzeez in the south of Oman, where the holotype was found.
Description.Basidiomata small to medium-sized.Pileus 30-80 mm in diam., at the young stage, broadly ovoid to parabolic, covered completely by a smooth, pale brownish pellicle; at mature stage, pulvinate to convex, pellicle disrupting except at the centre where it is retained as one large, smooth, brownish squamule, surface is woolly, covered with whitish, strigose to vil-
Habit, habitat, and distribution.Occurring in July to early September, as saprotrophic, solitary or scattered in small groups, on or near the termite mounds, under the trees of Anogeissus dhofarica.Currently only known from southern Oman.Notes.The new species Hymenagaricus wadijarzeezicus with medium-sized basidiomata, can be distinguished from the known species of the genus by its remarkable woolly cap and stipe surfaces.In Hymenagaricus, there are four species with a cap diameter of 50 mm or above, which are: Hymenagaricus cf.kivuensis, H. mlimaniensis Mwanga & Tibuhwa, H. ardosiaecolor and H. alphitochrous (Berk & Broome) Heinem.Hymenagaricus wadijarzeezicus is the 5 th species with a cap diameter above 50 mm.None of these species has a woolly basidiomata surface, except Hymenagaricus wadijarzeezicus.
Etymology.The specific epithet 'parvulus' refers to the small-sized basidiomata of the new species.
Habit, habitat and distribution.Fruiting body formation occurs in early August to early September, saprotrophic, scattered in small groups, found on termite mounds.Currently only known from southern Oman.

Discussion
Species of Hymenagaricus and Xanthagaricus are morphologically very similar and it is extremely difficult to differentiate the species of these genera in the field.However, in most species of Xanthagaricus, the cap surface is covered with small, brownish to purplish scales.These scales are concentrated at the pileus centre, while a large central, undisrupted scale at the cap centre has been observed in the most species of Hymenagaricus.Phylogenetically, both the genera are clearly distinct.
Phylogenetically, the species of Hymenagaricus are closely related to the monotypic genus Heinemannomyces.Morphologically, both these genera are clearly distinct.Species of Hymenagaricus have a squamulose cap surface and these squamules consist of hymeniform or pseudoparenchymatous cells and yellowish-brown basidiospores.The monotypic genus with single species Heinemannomyces splendidissimus has a brownish to greyish-red pileus, covered with a finely woolly veil and greyish to dark bluish basidiospores (Watling 1998;Hosen et al. 2017).
In our phylogenetic analyses, the specimens representing Heinemannomyces formed a basal group.Species of Hymenagaricus were recovered in three groups.One group consisted of Hymenagaricus wadijarzeezicus, the new species, H. saisamornae and H. cf.kivuensis.In this group, Hymenagaricus saisamornae with small-sized basidiomata intermix with H. cf.kivuensis and H. wadijarzeezicus both with medium-sized basidiomata.Similarly, another group consisting of Hymenagaricus pakistanicus, H. parvulus the new species and unnamed species H. sp.(LAH35329).All these taxa, including the unnamed species, have a small fruiting body.The third group consists of Hymenagaricus ardosiaecolor (medium-sized basidiomata) and H. siamensis, the small-sized species.
Both basidiomata size and pellicle structure are species delimitation characters in the genus Hymenagaricus.Based on our analyses, we can predict that these characters could be used in the future for infrageneric classification of the genus.
The two new species, Hymenagaricus wadijarzeezicus and H. parvulus, were collected in the Dhofar Region, located in the southern part of Oman.Hymenagaricus wadijarzeezicus is medium-sized and H. parvulus is a small-sized species.Both are widespread in the Region, under the trees of Anogeissus dhofarica.It is interesting to note that both collections of H. parvulus (JRZ-22-002, JRZ2-22-004) and several collections of H. wadijarzeezicus  were found on termite mounds.However, we did not find any study reporting the association of Hymenagaricus with termites.However, secotioid fungal ge-nus Podaxis Desv. in the family Agaricaceae has an apparent relationship with termites (Conlon et al. 2016).It will be interesting to study the relationships of these mushrooms with termites.
Several species of Agaricaceae were recently reported from the Dhofar Region (Al-Kharousi et al. 2022a, 2022b;Hussain et al. 2022).It is evident that the area is rich in the diversity of Agaricaceae, including the genus Hymenagaricus.More new species of dark-spored agarics are likely occurring in the area.

Taxonomic key to the species of Hymenagaricus
A taxonomic key to the species of Hymenagaricus included in our phylogenetic analyses is presented below.This key is based on cap diameter (small-sized with cap less than 40 mm in diam.and medium-sized with cap ranging from 50-100 mm in diam.) and pellicle structure either hymeniform or pseudoparenchymatous cells.

Figure 1 .
Figure 1.Maximum Likelihood phylogeny of Hymenagaricus and Heinemannomyces, based on combined ITS-28S sequence data, with Agaricus campestris as the outgroup taxon.Values above the node represent ML bootstrap percentages and BI posterior probabilities; the new species are represented in bold fonts.0.02

Table 1 .
Taxa included in the molecular phylogenetic analyses.