<8) MycoKeys MycoKeys 105: 1-19 (2024) DOI: 10.3897/mycokeys.105.113591 Research Article Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman, based on morphology and molecular phylogeny Shah Hussain'’”®, Moza Al-Kharousi', Dua’a Al-Magbali', Arwa A. Al-Owaisi', Rethinasamy Velazhahan®, Mohamed N. Al-Yahya’ei™®, Abdullah M. Al-Sadi2® 1 Oman Animal and Plant Genetic Resources Center (Mawarid), Ministry of Higher Education, Research and Innovation, P.O. Box 515, PC. 123, Muscat, Oman 2 Department of Plant Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, PO Box 34, AlKhoud 123, Oman Corresponding authors: Mohamed N. Al-Yahya‘ei (mohamed. alyahyaei@moheri.gov.om); Abdullah M. Al-Sadi (alsadi@squ.edu.om) OPEN Qaceess Academic editor: Alfredo Vizzini Received: 2 October 2023 Accepted: 28 March 2024 Published: 17 April 2024 Citation: Hussain S, Al-Kharousi M, Al- Maqpbali D, Al-Owaisi AA, Velazhahan R, Al-Yahya’ei MN, Al-Sadi AM (2024) Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman, based on morphology and molecular phylogeny. MycoKeys 105: 1-19. https://doi.org/10.3897/ mycokeys.105.113591 Copyright: © Shah Hussain et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Hymenagaricus has small to medium-sized mushrooms and the cap surface with squa- mulose pellicles, consisting of hymeniform or pseudoparenchymatous cells and yellow- ish-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-1a). Full descrip- tions, micrographs and illustration of anatomical features, basidiomata photos and phy- logenetic 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. Key words: Dhofar, diversity, taxonomy, termite mounds, two new taxa Introduction Three species previously in Agaricus subgenus Conioagaricus, Agaricus hy- menopileus, 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 (Heine- mann 1981). The genus Hymenagaricus was typified by H. hymenopileus (Hei- nem.), belonging to the family Agaricaceae Chevall. (Heinemann 1981). Species of Hymenagaricus are saprotrophic in nature and are mostly dis- tributed in the Palaeotropical Regions. Members of this genus are recognised Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman by the squamulose pellicle on the pileus surface that mostly consists of hy- meniform 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 spe- cies in the genus is 17 (Hussain et al. 2018; Kumla et al. 2021, 2023; Syed et al. 2023). Phylogenetically, species of Hymenagaricus are intermixed with the mono- typic 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, morphological- ly, 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 pseudoparenchy- matous cells and the spore print is leaden-grey to dark blue (Watling 1998). Four species of Agaricaceae, namely Agaricus arabiensis S. Hussain & Al- Sadi, Micropsalliota ventricocystidiata Al-Sadi & S. Hussain, Xanthagaricus appendiculatus Al-Sadi & S. Hussain and X. omanicus Al-Kharousi, Al-Sadi & S. Hussain have recently been described from Dhofar Region, Oman (Al-Kharousi et al. 2022a, 2022b; Hussain et al. 2022). However, no Hymenagaricus species has been reported from the country. During the years 2022-23, macrofungal exploration missions were conduct- ed in the Dhofar Region, in which we collected ten (10) collections of Hyme- nagaricus. Morphological characterisation and multigene (ITS, 28S, EF-1a) phy- logenetic analyses revealed that the 10 collections represent two new species, which are described in this study. Materials and methods 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 lo- calities (Wadi Naheez, Wadi Jahaneen, Wadi Jarzeez) of the Dhofar Region, in the months of August-September 2022 to 2023. The specimens were pho- tographed 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, MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 9 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman 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 Cen- ter (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% aque- ous 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 micro- scope (ECLIPSE Ni-U, Nikon Co., Ltd., Japan). For size measurements of these structures, Piximetre (http://ach.log.free.fr/Piximetre/) was used. For the mor- phological 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, lowa, USA), following the manufacturer's protocol. A volume of 200 ul X-AMP DNA reagent was taken in an Eppendorf tube containing the sam- ple (approximately 5-15 mg of gills) and incubated for 15 minutes at 70 °C. After cooling, 2 pl solution from the sample was used as a DNA template di- rectly 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-1a) gene. The primer combinations were: ITS1F and ITS4 for ITS (White et al. 1990; Gardes and Bruns 1993), LROR and LR5 for 28S (Vilgalys and Hester 1990; White et al. 1990), EF1-983 and EF1-1567R for EF-1a (Rehner and Buck- ley 2005). PuReTaqTM Ready-To-Go PCR beads (GE Healthcare UK Limited, Buckinghamshire, UK) were used for PCR amplification. We added 1.0 ul of each primer (10 umol/I), 2 ul DNA template and 22 ul 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-1a regions, only the annealing temperature was optimised, 52 °C for 28S and 60 °C EF-1a, 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-1a 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 Hy- menagaricus species. In the case of EF-1a sequences, the BLAST search re- MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 3 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman vealed Heinemannomyces sp. (ZRL185) is the most similar species because, in GenBank, no EF-1a sequences of Hymenagaricus are available. This is the reason that we used only ITS and 28S sequences in the phylogenetic analy- ses. 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. 2021, 2023; Syed et al. 2023). The final ITS-28S data- set was comprised of 27 specimens, including 26 ITS and 20 28S sequences (Table 1). Agaricus campestris L. (LAPAG370) was used as the outgroup tax- on. 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 Table 1. Taxa included in the molecular phylogenetic analyses. Species GenBank accession Agaricus campestris Heinemannomyces splendidissimus Hei. splendidissimus Hei. splendidissimus Hei. splendidissimus Hei. splendidissimus Hei. sp. Hymenagaricus ardosiaecolor H. ardosiaecolor H. cf. kivuensis wadijarzeezicus wadijarzeezicus wadijarzeezicus wadijarzeezicus wadijarzeezicus pakistanicus pakistanicus parvulus parvulus siamensis siamensis saisamornae saisamornae saisamornae saisamornae saisamornae sp. sp. a a Origin Voucher number 7 58 Reference Spain LAPAG370 KM657927 KP739803 Parra et al. (2016) China Q. Zhao 2591 KY039571 KY039576 Yang and Ge (2017) China Z.W. Ge2540 KY039570 KY039575 Yang and Ge (2017) China GDGM46633 MF621038 MF621039 Hosen et al. (2017) China GDGM46634 = MF621040 Hosen et al. (2017) Thailand ecv3586 HM488760 HM488769 Vellinga et al. (2011) Thailand ZRL185 KT951346 KT951527 Zhao et al. (2016) Togo LAPAF9 JF727840 = Zhao et al. (2011) Tanzania ZA KM360160 = Mwanga and Tibuhwa (2014) Burundi BR6089 KM982454 = Mwanga and Tibuhwa (2014) Oman JRZ2-22-015 OR613000 0R613018 This study Oman JRZ2-22-013 OR612999 OR613019 This study Oman NHZ-22-019 OR612997 OR613020 This study Oman JHN-22-019 OR612998 OR613021 This study Oman JRZ-22-005 OR612996 OR613022 This study Pakistan FAK196 OP082405 - Syed et al. (2023) Pakistan FAK195 OP082404 = Syed et al. (2023) Oman JRZ-22-004 OR612994 OR613017 This study Oman JRZ2-22-002 OR612995 = This study Thailand SDBR-CMUWP038 OP837533 OP836600 Kumla et al. (2023) Thailand SDBR-CMUNK1508 OP836301 OP836385 Kumla et al. (2023) Thailand ZRL3103 KM982450 KM982452 Kumla et al. (2021) Thailand SDBRCMUNKNW0474 MW349605 MW349603 Kumla et al. (2021) Thailand SDBR-CMUNK0369 MW345912 MW345917 Kumla et al. (2021) Thailand SDBR-CMUNK0567 MW349602 MW349604 Kumla et al. (2021) Thailand LD2012186 KM982451 KM982453 Kumla et al. (2021) Pakistan LAH35329 0Q998344 = Usman (2018) Thailand CA833 JF727858 > Zhao et al. (2011) MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman 2014). The best model (GTR+F+I+G4) was chosen following jModelTest2 (Dar- riba et al. 2012). Branch support for the ML phylogeny was executed with 1000 bootstrap replicates. For BI analyses, we used BEAST v.1.8.2 (Drummond et al. 2012). The combined ITS-28S alignment was converted to XML datafile using BEAUti v.1.8.2 (Bayesian Evolutionary Analysis Utility; Drummond et al. (2012)). A Birth-Death Incomplete Sampling speciation model (Stadler 2009) was selected. Four independent runs were performed with BEAST on XSEDE tool on the CIPRES Science Gateway (Miller et al. 2010). Resulting log files were checked in Tracer (Rambaut et al. 2014) for effective sample size (ESS) values. All ESS values were well over 200. Tree files were combined in LogCombiner v.1.8.2 (Drummond and Rambaut 2007). A maximum clade credibility (MCC) tree was obtained using the TreeAnnotator v.1.8.2 (Drummond and Rambaut 2007). The ML bootstrap (BT) percentage = 70 and BI posterior probabilities (PPs) = 0.80, respectively, were considered significant. For phylogenetic tree visualisation, FigTree v.1.4.2 (Rambaut 2012) was used and the tree was anno- tated using Adobe Illustrator CC2019. The alignment file is submitted to Tree- Base (http://purl.org/phylo/treebase/phylows/study/TB2:S30802). Results Phylogenetic analyses In this study, 19 new sequences (7 ITS, 6 28S and 6 EF-1a) were generated from our collections of Hymenagaricus. There were no EF-1a sequences of the genus available in GenBank; therefore, only combined ITS-28S sequences were used in the final data matrix. The final ITS-28S dataset was comprised of 1516 characters including 1272 constant sites, 141 informative sites and 103 unin- formative sites. The topology of trees revealed similar patterns in both ML and BI methods; therefore, the phylogeny inferred from ML analysis is presented here with values from both BT and PPs in Fig. 1. In both ML and BI analyses, six specimens of Heinemannomyces formed a basal group, sharing a clade with Hymenagaricus species. This clade is weakly supported in ML analyses and well supported with BI (BT 59%, PPs 0.94). However, the subclade repre- senting Heinemannomyces specimens is strongly supported in both analyses (BT 100%, PPs 1). Species of Hymenagaricus are distributed in three clades. Clade-I with good statistical support (BT 93%, PPs 1) consisted of three spe- cies, the new species Hymenagaricus wadijarzeezicus, H. saisamornae J. Kum- la & N. Suwannarach and H. cf. kivuensis Heinem. Each of these three spe- cies has its unique position, confirming their unique identity. Similarly, clade-ll was strongly supported (BT 100%, PPs 1), with three taxa, H. pakistanicus M.F. Syed & M. Saba, the new species H. parvulus and an unnamed species H. sp. (LAH35329). The third is a subclade in the clade consisting of Hymenagaricus and Heinemannomyces taxa. This subclade consisted of H. siamensis J. Kum- la, W. Phonrob, N. Suwannar & S., Lumyong, H. ardosiaecolor Heinem. and an unnamed species H. sp. (CA833). However, the two specimens (LAPAF9, Z4) representing H. ardosiaecolor, were recovered with different branch lengths. This variation in branch length could be the result of using only ITS sequences of H. ardosiaecolor in the phylogenetic analyses. MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 5 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman Hymenagaricus wadijarzeezicus JRZ2-22-015 Hymenagaricus wadijarzeezicus NHZ-22-019 1/100 | Hymenagaricus wadijarzeezicus JRZ2-22-013 -I71 1/93 -/50 Hymenagaricus wadijarzeezicus JHN-22-019 Hymenagaricus wadijarzeezicus JRZ-22-005 Hymenagaricus saisamornae SDBR-CMUNKNW0474 I Hymenagaricus saisamornae SDBR-CMUNK0369 Hymenagaricus saisamornae SDBR-CMUNK0562 1/100 Hymenagaricus saisamornae ZRL3103 Hymenagaricus saisamornae LD2012186 Hymenagaricus cf. kivuensis BR6089 - — — — — G enus Hymenagaricus | 1/100, Hymenagaricus parvulus JRZ-22-004 Hymenagaricus parvulus JRZ2-22-002 90/87 1/100); Hymenagaricus pakistanicus FAK195 1/100 1/100 1/98 -/60 0.86/56 0.94/59 Hymenagaricus pakistanicus FAK196 Hymenagaricus sp. LAH35329 - — — — - Hymenagaricus siamensis SDBR-CMUWP038 Hymenagaricus siamensis SDBR-CMUNK1508 Hymenagaricus ardosiaecolor ZNM4 Il Hymenagaricus ardosiaecolor LAPAF9 Hymenagaricus sp. CA833 - — — — — — Heinemannomyces splendidissimus GDGM-46633 Heinemannomyces sp. ZRL185 Heinemannomyces splendidissimus GDGM46634 A einemannomyces splendidissimus Heinemannomyces splendidissimus Q. Zhao 2591 Heinemannomyces splendidissimus Z.W. Ge 2540 Agaricus campestris LAPAG370 Outgroup 0.02 Figure 1. Maximum Likelihood phylogeny of Hymenagaricus and Heinemannomyces, based on combined ITS-28S se- quence data, with Agaricus campestris as the outgroup taxon. Values above the node represent ML bootstrap percentag- es and BI posterior probabilities; the new species are represented in bold fonts. Taxonomy Hymenagaricus wadijarzeezicus Al-Sadi, Al-Yahya’ei, A. Al-Owaisi & S. Hussain, sp. nov. MycoBank No: 850249 Figs 2-4 Diagnosis. The new species Hymenagaricus wadijarzeezicus can be differenti- ated from other species of the genus by its unique whitish woolly veil, covering both the cap and the stipe surfaces. Holotype. SULTANATE OF OMAN: Dhofar, Salalah, Wadi Jarzeez, on termite mounds, under the trees of Anogeissus dhofarica, 11 August 2022, S. Hussain, A. Al-Owaisi & Al-Yahya’ei, JRZ2-22-013 (holotype Mawarid-JRZ2-22-013), Gen- Bank accession: ITS = OR612999, 28S = 0R613019, EF-1a = OR729599. 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, pelli- cle disrupting except at the centre where it is retained as one large, smooth, brownish squamule, surface is woolly, covered with whitish, strigose to vil- MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 6 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman lose or floccose veil towards the margin; margin appendiculate with long, whitish, fibrils of veil. Context dark pinkish on cutting, 3-5 mm thick at the pileus centre. Lamellae free, pale pinkish at young stage, at mature stage greyish-pink to brownish, ventricose, up to 3 mm wide, densely crowded, with 1-3 series of lamellulae. Stipe 30-60 x 5-10 mm, equal, with a slightly bul- bous base, with root-like rhizoid structure at the base, annulus floccose, con- ie "% We? @ f Figure 2. Basidiomata of Hymenagaricus wadijarzeezicus A-C holotype collection (JRZ2-22-013) D, E NHZ-22-019 F young fruiting bodies where the cap is entirely covered by pellicle represented by arrows (JRZ2-22-015) G context changed into pinkish on cutting, the arrow represents the root-like rhizoid (JRZ2-22-015). Scale bars: 20 mm. MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 - Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman = iid , { Figure 3. Light microscopy of anatomical features of Hymenagaricus wadijarzeezicus (based on holotype collection JRZ2-22-013) A basidiospores B, C basidia D cheilocystidia E annulus F veil elements G pellicle structure. Scale bars: 10 um (A); 15 um (B-D); 15 um (E-G). = colorous to veil; stem covered with floccose veil below the annulus, smooth above the annulus, context pinkish on cutting, fistulose. Smell pleasant. Taste not recovered. Basidiospores (6.5)7.0-8.0(8.5) x (4.0)4.5-5.5(6.0) um, average size 7.5 x 5.0 um, Q = 1.4-1.6, av. Q = 1.5; ellipsoid to broadly ellipsoid, yellowish to dark brown, smooth, thick-walled, apiculus visible, germ-pore not observed. Basidia 20-25 x 7-9 um, on average 22.5 x 8.0 um, clavate to cylindrical, smooth, hya- line in KOH, mostly tetrasporic, rarely bisporic. Cheilocystidia 16-23 x 7-9 um, on average 19.5 x 8.0 um, ellipsoid to subclavate, smooth, thin-walled, hyaline in KOH. Pleurocystidia absent. Lamellar trama regular, with 4—6.6 um diam., cylindrical to inflated, thin-walled, hyaline hyphae. Subhymenium consisted of subglobse to irregular cells, measuring 12-18 um diam. Pellicle is a hymeni- form, consisting of chains of two or three elements, measuring 13-17 x 10- 16 um each element, globose to subglobose or ovoid, hyaline, or pale yellowish, smooth, thin-walled, these chains of elements attached to inflated hyphae with encrusted walls. Pileus veil is a cutis to ixocutis, consisting of elongated or cy- lindrical elements, easily detached, hyaline, thin-walled, each element measur- ing 13-45 x 6-9 um. Annulus is an intricate trichoderm, composed of hyaline hyphae, 6-8 um diam., cylindrical, constituted by short elements, constricted at septa, easily disarticulated. Stipe veil similar to pileus veil. Clamp connections absent in all tissues. 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. MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 9 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman Figure 4. Line drawings of anatomical features of Hymenagaricus wadijarzeezicus (based on holotype collection JRZ2- 22-013) A basidiospores B basidia C cheilocystidia D pellicle structure E annulus F veil elements. Scale bars: 10 um (A); 15 um (B-D); 15 um (E, F). MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 9 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman Additional specimens examined. SULTANATE OF OMAN: Dhofar, Salalah, Wadi Naheez, on termite mounds, under the trees of Anogeissus dhofar- ica, 07 August 2022, S. Hussain, A. Al-Owaisi, Al-Yahya’ei & Al-Sadi, NHZ- 22-019 (Mawarid-NHZ-22-019), GenBank accession: ITS = OR612997, 28S = OR613020, EF-la = OR729602; Wadi Jarzeez, under the trees of Anogeissus dhofarica, 08 August 2022, S. Hussain, A. Al-Owaisi, Al-Ya- hya’ei & Al-Sadi, JRZ-22-005 (Mawarid-JRZ-22-005), GenBank accession: ITS = OR612996, 28S = OR613022, EF-1a = OR/729603; Wadi Jaheen, under the trees of Anogeissus dhofarica, 10 August 2022, S. Hussain, A. Al-Owaisi, Al-Yahya’ei & Al-Sadi, JHN-22-019 (Mawarid-JHN-22-019), GenBank acces- sion: ITS = O0R612998, 28S = OR613021, EF-1a = OR/29600; Wadi Jarzeez, on termite mounds, under the trees of Anogeissus dhofarica, 11 August 2022, S. Hussain, A. Al-Owaisi & Al-Yahya’ei, JRZ2-22-015 (Mawarid-JRZ2-22-015), GenBank accession: ITS = OR613000, 28S = OR613018, EF-1a = OR/29601; Wadi Gogob, on termite mounds, under the trees of Anogeissus dhofarica, 22 August 2023, S. Hussain & Al-Yahya’ei, GOB-23-008 (Mawarid-GOB-23-008); Sahalanawt, on termite mounds, 27 August 2023, S. Hussain & Muhammad Salim, Sahalanawt-23-001 (Mawarid-Sahalanawt-23-001); Tetam, on termite mounds, 30 August 2023, S. Hussain & Amer Qattan, Tetam-23-001 (Mawar- id-Tetam-23-001). 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. alphi- tochrous (Berk & Broome) Heinem. Hymenagaricus wadijarzeezicus is the 5‘ species with a cap diameter above 50 mm. None of these species has a woolly basidiomata surface, except Hymenagaricus wadijarzeezicus. In ML phylogeny, the most similar species to the new species H. wadijar- zeezicus is H. saisamornae. Hymenagaricus saisamornae is a recently de- scribed species from Thailand, with substantially smaller basidiomata (10- 25 mm cap diam.), pileus surface covered with minute brownish squamules, stipe smooth to finely whitish squamulose and smaller basidiospores (5.5- 7.0 x 4.0-4.5 um; Kumla et al. (2021)). Similarly, Hymenagaricus cf. kivuen- sis and H. mlimaniensis, both African species, shared medium-sized pileus with H. wadijarzeezicus. Hymenagaricus cf. kivuensis has pileus of 50-100 diam., with smaller basidiospores (4.0-6.5 x 3.0-4.5 um), narrower basid- ia (16-20 x 4.5-6) and broader hymeniform cells (Pegler 1977; Heinemann 1984). Hymenagaricus mlimaniensis has a broadly umbonate, reddish-brown disc, with sparsely squamulose surface and smaller basidiospores (4.0—7.0 x 3.5-4.5 um; Mwanga and Tibuhwa (2014)) than H. wadijarzeezicus (7.0-8.0 x 4.5-5.5 um). Hymenagaricus siamensis differs from H. wadijarzeezicus by its smaller basidiomata with brownish cap, measuring 22-32 mm diam., squa- mules consisting of pseudoparenchymatous cells (Kumla et al. 2023). Anoth- er small-sized species Hymenagaricus pakistanicus with pileus 24-30 mm diam., covered with dark brownish squamules at the cap centre, smaller ba- sidiospores (5.0-6.0 x 3.5-5.0 um) and a pseudoparenchymatous pellicle (Syed et al. 2023). MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 10 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman Hymenagaricus parvulus Al-Kharousi, Al-Sadi, Al-Yahya’ei, & S. Hussain, sp. nov. MycoBank No: 850248 Figs 5-7 Diagnosis. The new species Hymenagaricus parvulus can be differentiated from other species of the genus by its small-sized, creamy basidiomata, um- bonate pileus covered with appressed pellicle. Holotype. SULTANATE OF OMAN: Dhofar, Salalah, Wadi Jarzeez, on termite mounds, under the trees of Anogeissus dhofarica, 8 August 2022, S. Hussain, A. Al-Owaisi, Al-Yahya’ei & Al-Sadi, JRZ-22-004 (holotype Mawarid-JRZ-22-004), GenBank accession: ITS = OR612994, 28S = OR613017, EF-1a = OR735176. Etymology. The specific epithet ‘parvulus’ refers to the small-sized basidi- omata of the new species. Description. Basidiomata small-sized. Pileus 15-25 mm in diam., at young stage globose to parabolic, surface floccose squamulose, squamules light pinkish to creamy, with appressed pellicle at the centre, margin appendiculate; at mature stage cap convex to hemispherical with the broadly umbonate disc, with appressed, pale brownish pellicle at the disc, surface finely floccose squamulose, squamules pale creamy to light greyish, margins striate, just exceeding the lamellae; context membranous, pinkish on cutting. Lamellae free, pale pinkish to brownish, ventri- cose, sparsely crowded, with 1-2 series of lamellulae. Stipe 25-35 x 2-5 mm, equal, annulus cortinate, concolorous to squamules; stem surface creamy, Cov- ered with finely floccose squamules below the annulus, smooth above the annu- lus, context pinkish on cutting, fistulose. Smell pleasant. Taste not recorded. Basidiospores 5.0-6.5 x 4.0-4.5 um, average size 6.0 x 4.2 um, Q = 1.3- 1.5, av. Q = 1.4; ellipsoid to broadly ellipsoid, yellowish to dark brown, smooth, thick-walled, apiculus visible, germ-pore not observed. Basidia 16.5-22.5 x 6.5-8.5 um, on average 19.0 x 7.5 um, clavate to cylindrical, smooth, hyaline in KOH, tetrasporic. Cheilocystidia 19-25 x 9-11 um, on average 22 x 10 um, clavate to broadly clavate, often turning to one side, with multiseptate base, smooth, thin-walled, hyaline in KOH. Pleurocystidia absent. Subhymenium con- sisting of cylindrical to elongated cells, measuring 6-9 um diam. Pellicle is a hymeniform, consisting of chains of several elements, each element measuring 14-22 x 12-17 um, globose to subglobose or ovoid, hyaline or pale yellowish, smooth, thin-walled; these chains of elements attached to inflated hyphae with encrusted walls. Veil is a cutis to ixocutis, consisting of elongated or cylindrical elements, not easily detached, hyaline, thin-walled, with terminal element fusi- form with papillate end, each element measuring 15-18 x 5-7 um. Annulus is an intricate trichoderm, composed of hyaline hyphae, 4-7 um diam., cylindrical, constituted by short elements, constricted at septa and easily disarticulated. Clamp connections absent in all tissues. Habit, habitat and distribution. Fruiting body formation occurs in early Au- gust to early September, saprotrophic, scattered in small groups, found on ter- mite mounds. Currently only known from southern Oman. Additional specimens examined. SULTANATE OF OMAN: Dhofar, Salalah, Wadi Jarzeez, on termite mounds, under the trees of Anogeissus dhofarica, 11 Au- gust 2022, S. Hussain, A. Al-Owaisi, Al-Yahya’ei & Al-Sadi, JRZ2-22-002 (Mawar- id-NHZ-22-002), GenBank accession: ITS = 0R612995. MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 i Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman _=>- » * =. P .—" a 4 * * > * : \ ' y = Figure 5. Basidiomata of Hymenagaricus parvulus (based on holotype collection JRZ-22-004) A mature and young basid- iomata B mature basidimata. Notes. Hymenagaricus parvulus is a small, cream-coloured species, differ- entiated from other species of the genus by its whitish to pale pinkish floc- cose squamules on pileus and stipe surfaces with a broadly umbonate centre. Hymenagaricus parvulus shares basidiomata size and basidiospores morphol- ogy with H. pakistanicus. However, H. pakistanicus can be differentiated from the new species by its caesptiose fruiting habit, pileus with pinkish to brown- MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 12 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman Figure 6. Light microscopy of anatomical features of Hymenagaricus parvulus (based on holotype collection JRZ-22-004) A basidiospores B basidia C cheilocystidia D annulus E pellicle structure F veil elements. Scale bars: 5 um (A); 10 um (B, C); 15 um (D-F). ish squamulose pellicle, consisting of pseudoparenchymatous cells (Syed et al. 2023). Hymenagaricus saisamornae differs from the new species by its smaller pileus (up to 15 mm diam. Vs. 15-25 mm of H. parvulus), covered with brownish pellicles and larger basidiospores (5.5—7.0 x 4—4.5 um; Kumla et al. (2021)). Hy- menagaricus siamensis, another small-sized species is distinguished from the Mycokeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 13 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman Figure 7. Line drawings of anatomical features of Hymenagaricus parvulus (based on holotype collection JRZ-22-004) A basidiospores B basidia C annulus elements D cheilocystidia E pellicle structure F veil elements. Scale bars: 5 um (A); 10 um (B, D); 15 um (C, E, F). new species by its pinkish-brown cap, pellicle comprised of pseudoparenchyma- tous cells and larger basidiospores (6.5—8.0 x 4.0-5.0 ym; Kumla et al. (2023)). Similarly, Hymenagaricus canoruber (Berk. & Br.) Heinem. & Little Flower, known from India and Sri Lanka, is characterised by a small-sized pileus (15-25 mm diam.), with greyish-brown squamules, hymeniform pellicle and smaller basidio- MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 14 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman spores (4.6-5.7 3.5-4.3 um; Heinemann and Little Flower (1984)). Hymenagar- icus pallidodiscus D.A. Reid & Eicker, the smallest mushroom in the genus with pileus diam. up to 11 mm, covered with brownish squamules and smaller basid- iospores (4.2—5.4 x 3.1-3.8 um; Reid and Eicker (1999)). Hymenagaricus cylin- drocystis Heinem. & Little Flower another small-sized species, has been report- ed in Singapore and India, with a brownish cap, larger basidiospores (6.4—8.4 x 4.5-5.6 um) and a pseudoparenchymatous pellicle (Heinemann 1956; Heine- mann and Little Flower 1984). Hymenagaricus cf. kivuensis and H. wadijarzeez- icus with their medium-sized pilei can be easily differentiated from H. parvulus. 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, cov- ered 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. wadijarzeezi- cus 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 (medi- um-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. Hyme- nagaricus 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 (NHZ-22-019, JRZ2-22-013, JRZ2-22-015, GOB-23-008, Sahalanawt-23-001, Tetam-23-001) were found on termite mounds. However, we did not find any study reporting the association of Hymenagaricus with termites. However, secotioid fungal ge- MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 15 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman 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 Re- gion (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 pseudoparen- chymatous cells. 1 Basidiomata small-sized, pileus diam. below 40 MM...............cccceeeseeee 2 - Basidiomata medium-sized, pileus diam. up to 100 mM. ............. eee 5 2 « hruning Bodies-selitary OROreQanlOUS,. cht .00 cues henre eased ental as eee adele 3 - Fruiting bodies appear in cluster (casepitose), pileus 20-30 mm diam., pellicle consisted of pseudoparenchymatous cells ....... Hymenagaricus pakistanicus 3. Pileus whitish or pinkish-brown, pellicle consisted of hymeniform cells....... 4 - Pileus brownish, pellicle consisted of pseudoparenchymatous cells ......... bate epit desi ubett: tate Nee Pan Uester shih Sate vale RODEN ONAN RS tree td Or we ete a H. siamensis 4 Pileus 15-25 mm diam., whitish to creamy, pellicle smooth, finely ap- POS SOG cc dans syvweten sp vv ensenewadedervanutre dasusnearecs any ecteinsenebn wshente dana nay atrae H. parvulus - Pileus 10-15 mm diam., pinkish to brownish, squamulose pellicle............ devsonsal Wate cmueeck encnatvi ects ac Nccaitin cles Seta ooh thee 0 Basle, eR RA H. saisamornae 5 _ Pileus covered with brownish Squamulls .................cccsscccessssecsesteeesesseeesens 6 - Pileus and stipe surfaces covered with whitish woolly fibrils ...................... sae ete aarearee east or cng amiatred sea Ta aha eoaae et eee ager rh eereanA eager H. wadijarzeezicus 6 Pileus diam. 40-60 mm, basidiospores 5.8-6.5 x 3.9-4.5 UM... nage Fe GRO ic ers te a ene ag I caer eae etna een a eRe te H. ardosiaecolor - Pileus diam. up to 100 mm, hymeniform squamules ......... H. cf. kivuensis Acknowledgements We would like to thank Bader Al Quyudhi, Shamsa Al Balushi and Maryam Al Hinai for their invaluable help in the sampling trip to the Dhofar Region, which was es- sential to the success of our research. We would also like to thank Amer Qattan and Mohammed Al Jahwari for their guidance and support throughout the project. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. MycoKeys 105: 1-19 (2024), DOI: 10.3897/mycokeys.105.113591 16 Shah Hussain et al.: Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman Funding This research was funded by the Agriculture and Fisheries Development Fund, Sultanate of Oman (grant number 104/1/1). Author contributions Shah Hussain and Moza Al-Kharousi: conceptualisation, writing - original draft and review and editing, data curation, formal analysis, investigation, methodology and vi- sualisation. Dua’a Al-Magqbali and Arwa A. Al-Owaisi: sampling, data curation and in- vestigation. Mohamed N. Al-Yahyaei and Abdullah M. Al-Sadi: project administration, resources, supervision , writing - review and editing. Rethinasamy Velazhahan: writing - review and editing, formal analysis. Author ORCIDs Shah Hussain © https://orcid.org/0000-0002-5772-7206 Rethinasamy Velazhahan © https://orcid.org/0000-0002-9263-4371 Mohamed N. Al-Yahya’ei ® https://orcid.org/0000-0002-951 6-5339 Abdullah M. Al-Sadi © https://orcid.org/0000-0002-3419-8268 Data availability All of the data that support the findings of this study are available in the main text. References Al-Kharousi M, Hussain S, Al-Muharabi MA, Al-Shabibi Z, Al-Magbali D, Al-Balushi AH, Al-Yahya’ei MN, Al-Saady N, Velazhahan R, Al-Sadi AM (2022a) The genus Xanthagar- icus: An updated global species distribution and phylogeny with the description of two new species from Oman. 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