The Genus Xanthagaricus: An Updated Global Species Distribution and Phylogeny with the Description of Two New Species from Oman

Xanthagaricus is a saprotrophic mushroom genus with small-sized basidiomata in the family Agaricaceae (Agaricales). Prior to this study, 26 species belonging to this genus have been described and published. In this study, we reported Xanthagaricus for the first time from Oman with the description of two new species. Basidiomata of the new species Xanthagaricus appendiculatus and X. omanicus were collected during the monsoon rains of summer in 2018 in the southern coastal region of Oman. Species descriptions are based on morphological and molecular characterization. Phylogenetic analyses based on internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS) of the nuclear ribosomal DNA clustered the new species in the Xanthagaricus clade with strong statistical support. The new species Xanthagaricus appendiculatus can be distinguished from other species by its purplish pileus with umbonate disc and X. omanicus with the largest pileus (70–90 mm diameter) among the known species of the genus. A detailed description, photographs, line drawings, and a phylogenetic tree showing the position of both new species are provided. A dichotomous key to the known taxa of Xanthagaricus is proposed. Morphological comparisons of new species with known Xanthagaricus taxa are provided. Our observations highlight the diversity of Xanthagaricus and other lepiotaceous mushrooms in southern Oman and further document the need for additional systematic focus on the region’s fungi.

Oman has a subtropical dry and hot climate. The climatic conditions are varied according to geography. From October to April, the climate in Oman is mostly warm and sunny; daytime temperature rises up to 37 • C and is at night at about 17 • C [20]. From May to September, it is dry and hot in most parts of the country, and the temperature reaches up to 50 • C or above. The annual rainfall is about 100 mm [21]. Most regions of interior Oman are desert areas, known as the Rub'al-Khali [22]. The coastline has greater plant biodiversity [23]. The southern coastline of Oman, consisting of the Dhofar Governorate, is the most fertile area and receives plenty of monsoon rain from May to September [24,25]. The monsoon climate can influence the emergence of various macrofungi on different substrata, e.g., soil, log, stump, wood, leaf-litter, and dung.
The Sultanate of Oman has about 1390 species of vascular plants; most of these plants are endemic to the region [26]. Based on the most conservative approach of the 6:1 (six fungal species associated with one plant species) ratio used by Hawksworth [27], it is estimated that nearly 7000-8000 fungal species may occur in Oman. A checklist of the known fungi from Oman has been compiled, comprised of 318 fungal species belonging to 173 genera, mostly of Ascomycota [17]. It seems that only 4-5% of fungi of Oman are discovered, and the vast majority are unexplored. A similar case has been observed in northern Thailand, where fungal explorative expeditions showed that up to 96% of species in most agaric genera are new to science [28]. The reported fungi from Oman are mostly those causing diseases in different crops [29][30][31], and few of these belong to the macrofungi. It seems that the coastal region of Oman, which receives an ample amount of precipitation in summer, would be the hotspot of macrofungi. The fungal diversity studies in these areas will be particularly highly productive.
During the macrofungal exploration of Dhofar Governorate of Oman in 2018, some interesting specimens of lepiotaceous mushrooms were collected. According to morpho-logical and molecular phylogenetic analyses, the specimens represent two new species of Xanthagaricus which are subsequently described in detail.

Sampling and Morphological Observations
Specimens were collected from the Gogob area, around Salalah, Dhofar Governorate, Oman (Figure 2), during the monsoon rainy season in the month of September in the year 2018. The sampling area is arid with hot desert (BWh) climate using the latest Köppen climate map and classification [32,33]. However, the mountains of Dhofar are located in the monsoon belt. The area receives southwest monsoon from mid-June to mid-September every year, as part of the Inter Tropical Convergence Zone [34]. With such high precipitation in the monsoon, the vegetation of the area is mainly covered by deciduous trees [35]. Photographs of basidiomata were captured and labeled, and field notes were made. Munsell's soil color charts were used for color determination [36]. Specimens examined in this study were submitted to SQUH herbarium, Department of Plant Sciences, CAMS, Sultan Qaboos University, Muscat, Oman.
For microscopic observations, slides were prepared in 5% aqueous KOH (w/v), followed by 1% aqueous Congo red (w/v). Microscopic characters, including the size and shape of basidiospores, basidia, cheilocystidia, caulocystidia, and pileipellis were studied under a light microscope (ECLIPSE Ni-U, Nikon Co., Ltd., Tokyo, Japan), with at least 20 structures measured in each instance. For basidiospores, measurements were done for 60 spores using 1000× magnification with a calibrated Nikon DS-Ri2 microscopic camera. Measurements of basidiospores are given as (a)b-c(d), where b-c includes a minimum of 90% of the measured values. Extreme values (a and d) are given in parentheses. The Q was calculated as the length/width ratio of spores, and av. Q is the mean length/width ratio of all basidiospores.

Molecular Identification
Extraction of the genomic DNA was done from dried specimens following a modified CTAB protocol [37]. The internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS) of the nuclear ribosomal DNA was amplified using ITS1F/ITS4-B primer pair [37]. Polymerase chain reaction (PCR) was performed using PuReTaq TM Ready-To-Go PCR beads, with 1.0 µL of each primer (10 µM/µL), 22 µL H 2 O, and 1 µL template DNA. PCR amplification was performed with 4 min initial denaturation at 95 • C, followed by 34 cycles of 50 s at 94 • C, 40 s at 54 • C, 50 s at 72 • C, and a final extension of 7 min at 72 • C followed the last cycle, following Al-Sadi et al. [38]. Purification of PCR products and sequencing with the same primers were carried out at ©Macrogen Inc. (238, Teheran-ro, Gangnam-gu, Seoul, Korea).
Maximum Likelihood (ML) and Bayesian Inference (BI) methods were used for phylogeny. The BI phylogeny was estimated using BEAST version 1.8.4 [42] with an uncorrelated lognormal relaxed clock. A Birth-Death Incomplete Sampling speciation model tree was selected [43]. After that, selection of the best-fit model (TIM2 + I + G) was done using jModelTest2 [44]. The analyses were run from the BEAST on XSEDE tool on the Cipres Science Gateway [45]. The obtained log files were entered in Tracer [46]. Then, log files and trees files were combined in LogCombiner 1.8.2 [47]. TreeAnnotator 1.8.2 [47] was used to produce the Maximum Clade Credibility tree.
Maximum Likelihood analyses were run in RAxML-VI-HPC on XSEDE tool [45]. The best-fit model (TIM2 + F + I + G4) was selected using ModelFinder [48]. Node support was obtained with 1000 pseudoreplicates under the GTRCAT model. Branch support was calculated by 1000 bootstrap replicates.
For phylogenetic tree visualization, FigTree 1.4.2 [49] was used, and the tree was annotated using Adobe Illustrator CC2018.
Habitat and distribution: Scattered in small groups, saprotrophic, on humus-rich soil with dead leaves and wood under trees of Anogeissus dhofarica. So far only known from southern Oman.
In our phylogenetic analyses, X. appendiculatus is shown to be related to X. pakistanicus and X. necopinatus. Xanthagaricus pakistanicus is recently described from Pakistan [6] and X. necopinatus from Bangladesh [5]. Our newly described species shares the pileus squamules characters with these species. Xanthagaricus appendiculatus can be separated from these taxa by the characteristic purplish pileal squamules, while the squamules are dark brown in X. pakistanicus and yellowish-brown in X. necopinatus.  Diagnosis: The diagnostic characteristics of the new species Xanthagaricus omanicus are: the largest pileus (70-90 mm diam) among the known species of the genus, which may be convex to applanate, with a slightly depressed center and covered with yellowish to yellowish-green squamules; broadly ellipsoid to ellipsoid, yellowish-brown basidiospores, measuring 7.0-8.5 × 5.0-5.5 µm.
Microscopically, the basidiospores of the new species are broadly ellipsoid to ellipsoid, yellowish-brown, measuring 7.0-8.5 × 5.0-5.5 µm, and the cheilocystidia are variable in shape, from narrowly clavate to broadly clavate or ventricose to napiform.
Our new species Xanthagaricus omanicus and X. siamensis Yuan S. Liu and S. Lumyong, recently described from Thailand [10], are morphologically similar by their pileus surfaces which are more or less fibrillose. Macroscopically as well as microscopically, both species can be differentiated. Pileus is comparatively smaller (31-54 mm diam) and covered with grayish-orange to violet-brown fibrils in X. siamensis. Basidiospores in X. siamensis are substantially smaller (4.0-5.5 × 2.5-3.0 µm) and ellipsoid to oblong. Moreover, on the basis of ML phylogeny, both the species are phylogenetically well differentiated (Figure 3).
In our molecular analyses, X. omanicus is shown to be related to X. necopinatus and allies. The new species shares basidiospores morphology with X. necopinatus, X. appendiculatus, X. pakistanicus, and X. purpureosquamulosus. However, X. omanicus, with its large pileus, can be differentiated from these species due to their smaller pilei. Similarly, X. omanicus is also related to X. appendiculatus. Xanthagaricus appendiculatus and X. omanicus share similar habitat, and both species occur in the same area around Salalah, Oman. Macroscopically, in X. omanicus, the pileus is substantially larger (70-90 mm diam) and covered with appressed, yellowish squamules. Microscopically, the new species can be differentiated on the basis of cheilocystidia. Cheilocystidia in X. omanicus are comparatively smaller (20.5-29.5 × 9.0-13.0 µm), variable in shape, from narrowly clavate to broadly clavate, ventricose to napiform. The cystidia in X. appendiculatus are larger (26.0-33.0 × 7.0-8.5 µm) and uniformly shaped, i.e., ventricose. The pileus is covered by an irregular epithelium in X. appendiculatus, while it is an irregular trichoderm to intricate trichoderm in X. omanicus. A detailed comparison of morpho-anatomical features of X. appendiculatus and X. omanicus with those of other species of the genus is provided in Table 1.

Discussion
The genus Xanthagaricus was introduced by Little Flower et al. [2] with the following characteristics: basidiomata smaller to rarely medium-sized, pileus with distinctive woolly squamules and appendiculate margin; lamellae free, brown, in some cases ink-blue at maturity; stipe cylindrical, almost equal, slightly broader at the apex, annulus rudimentary or absent; spores brown, sometimes yellowish, subglobose to ellipsoid, smooth or slightly ornamented, thick-walled; cheilocystidia present, clavate, subclavate or ventricose; pleurocystidia absent; pileal surface a disrupted epicutis of radial hyphae with plenty of spherical or subspherical cells at the scales; clamp connections absent.
In the Agaricus clade of Agaricaceae, Xanthagaricus is recovered as a monophyletic group, forming a subclade with Pseudolepiota, a monotypic genus recently reported from China [50]. Pseudolepiota is differentiated by the white color of the lamellae, hyaline basidiospores, and an ixocutis layer of pileipellis made up of slightly interwoven cylindrical hyphae [50]. However, the synapomorphic characters of the two genera are their pileus squamules, the absence of both pleurocystidia, and clamp connections.
During this study, we collected and described two new species of Xanthagaricus from southern Oman. Both the new species Xanthagaricus appendiculatus and X. omanicus were collected from the Gogob region around Salalah of Dhofar Governorate, Oman. We have examined the distribution of our collection sites following the updated Köppen climate map and classification [32,33]. All the specimens were found in an area with an arid hot desert climate. However, the mountains of Dhofar come under the influence of monsoon, where it usually rains from mid-June to mid-September [34]. This results in dense vegetation and subsequently high diversity of fungal species.
The present study indicates that the mountain and scrublands of southern Oman are rich in mushrooms, and fungal surveys are much needed in the future. This is the first report describing new species of mushrooms from Oman, based on morphology and phylogeny. This study provides a baseline for future scientific studies of these fascinating organisms, which contribute a lot to humanity in terms of food, medicine, pharmacy, etc.
Key to the Known Taxa of Xanthagaricus