Based on morphological and molecular evidence, two new species, namely Hymenagaricus indicus and Xanthagaricus nigrosquamosus, are described from tropical region of Maharashtra, India. Hymenagaricus indicus is morphologically circumscribed by its small basidiomata, covered with brown plate-like squamules at the centre along with small, numerous squamules scattered towards margin, pileal squamules composed of globose to subglobose elements, and yellowish brown, ellipsoid to ovoid-ellipsoid, smooth basidiospores. Xanthagaricus nigrosquamosus is characterised by its yellowish pileus covered with black squamules, epithelial pileipellis, and broadly ellipsoid to ovoid-ellipsoid, rugulose-rough, basidiospores. Molecular phylogenetic analyses using rDNA ITS1-5.8S-ITS2, and partial 28S rDNA sequences also confirmed that they are distinct from their closest taxa.

The paleotropical genus Hymenagaricus Heinem. was erected by Heinemann in the family Agaricaceae and designated by H. hymenopileus Heinem. as the type species (Heinemann, 1981). Heinemann and Little Flower (1984) divided the genus Hymenagaricus into two subgenera, i.e., Hymenagaricus and Xanthagaricus (Heinem.) Little Flower, Hosag, & T.K. Abraham. Later on, Little Flower et al., (1997) elevated the subgenus Xanthagaricus as an independent genus typified by X. flavidorufus (Berk. & Broome) Little Flower, Hosag. & T.K. Abraham. Phylogenetic studies also supported their independent generic status (Al-Kharousi et al., 2022; Hosen et al., 2017, 2018; Hussain et al., 2018). Small to medium-sized basidiomata, squamulose pileus surface, pileipellis made up of mainly hymeniform cells at centre, becoming sub-hymeniform, epitheloid to trichoid towards the margin. Basidiospores usually smooth, ellipsoid, brownish tinged, and the absence of pleurocystidia and clamp connections are the defining features of the genus Hymenagaricus (Heinemann & Little Flower, 1984; Little Flower et al., 1997; Reid & Eicker, 1995), whereas the genus Xanthagaricus is characterised by its small to rarely medium-sized basidiomata, squamulose pileus surface, pileipellis composed of subhymeniform or globose to subglobose cells, yellowish brown or bluish tinged, smooth or mostly ornamented basidiospores, and the absence of pleurocystidia and clamp connections (Heinemann & Little flower, 1984; Hosen et al., 2017; Little Flower et al., 1997).

At present, there are twenty one species of Hymenagaricus (Crous et al., 2023; Heinemann, 1981, 1985; Heinemann & Little Flower, 1984; Hussain et al., 2024; Kumla et al., 2021, 2023; Mwanga & Tibuhwa, 2014; Pegler, 1977, 1986; Reid & Eicker, 1995, 1998, 1999; Syed et al., 2023; Yang et al., 2024) and thirty nine species of Xanthagaricus are described from the Paleotropical regions of the world (Al-Kharousi et al., 2022; Fatima & Khalid, 2023; Ge et al., 2008; Haqnawaz et al., 2023; Heinemann & Little Flower, 1984; Hosen et al., 2017, 2018; Hussain et al., 2018; Javeed et al., 2024; Kumla et al., 2018; Little Flower et al., 1997; Liu et al., 2020; Pegler, 1986; Reid & Eicker, 1998; Wang et al., 2018; Yang et al., 2024). So far, three species of Hymenagaricus (Heinemann & Little Flower, 1984) and fourteen species of Xanthagaricus (Heinemann & Little Flower, 1984; Little Flower et al., 1997) are described from the Kerala state of India. All the reports of taxa are based exclusively on morphological characters and further changes should be expected after the incorporation of molecular data.

During the exploration of macrofungi from 2017 to 2023 in the different locations of Matheran Hills, Maharashtra, India, amidst the monsoon seasons, we came across the interesting collections of Hymenagaricus and Xanthagaricus. After detailed morphological and molecular analyses based on nrITS and nrLSU, these collections represent two new species in the above genera.

Basidiomata were obtained after photographed fresh in the habitat using Canon EOS 700D Camera (Canon Inc., Tokyo), and macroscopic characters were recorded from fresh specimens. Colour terminology for fresh mushrooms were followed from the procedure of Kornerup and Wanscher (1978). The examined mushrooms were dried in an electric dryer for approximately 10-12 h at 40-45 °C temperature (Hu et al., 2022). The holotypes were deposited in the Ajrekar Mycological Herbarium (AMH), Pune, India, while the paratypes were maintained in the Botany department of Smt. Chandibai Himathmal Mansukhani College, Thane, India. The micromorphological characters were accomplished from thin hand-cut sections of dried samples, then mounted in 5% (w/v) KOH, stained with 1% (w/v) phloxin and 0.5% (w/v) Congo red in distilled water. Melzer’s reagent was used to test the amyloidity of basidiospores. The notation [50/3/3] indicates that the 50 basidiospores were measured from 3 basidiocarps of 3 collections. Sizes of basidia, cheilocystidia and pileal elements were measured from at least 20 elements of each character. The following abbreviations were given for dimensions of basidiospores: X_m for arithmetic mean of length by width of basidiospores (± standard deviation), Q for quotient of length divided by width of individual basidiospores and Q_m stands for the mean of Q values (± standard deviation). The abbreviation “H.” is used for Hymenagaricus, and “X.” for Xanthagaricus.

The CTAB (Doyle & Doyle, 1987) method was used to extract the total genomic DNA. Moreover, based on the results of previous studies, the DNA markers nrITS and nrLSU were opted for the molecular phylogenetic analyses (Al-Kharousi et al., 2022; Hosen et al., 2017, 2018; Hussain et al., 2018). For the selected markers, the PCR amplification and purification was done at Genematrix LLP (Pune, India). The nrITS was amplified using ITS1/ITS4 primers (White et al., 1990), while the nrLSU region was amplified using LROR/LR5 primers (Moncalvo et al., 2000; Vilgalys & Hester, 1990). After the markers were amplified successfully, the purified PCR products were dispatched to Apical Scientific Sdn Bhd (Seri Kembangan, Selangor, Malaysia) for Sanger sequencing. The obtained sequences were quality-checked using Chromas (Technelisium Pvt. Ltd, Australia) software, and then further curated using BioEdit v 7.2.5 (Hall, 1999). Consensus sequences were accomplished using both forward and reverse sequences, and subsequently submitted to GenBank (Table 1). In accordance with previous research, the ingroups and outgroups (Chlorophyllum molybdites (G. Mey.) Massee and C. rachodes (Vittad.) Vellinga as outgroups) were recovered from NCBI database to construct the phylogenetic trees (Al-Kharousi et al., 2022; Crous et al., 2023; Fatima & Khalid, 2023; Haqnawaz et al., 2023; Hosen et al., 2017, 2018; Hussain et al., 2018; Kumla et al., 2018, 2021, 2023; Liu et al., 2020; Syed et al., 2023; Vellinga et al., 2011; Wang et al., 2018; Yang et al., 2024). The newly produced sequences were aligned separately along with NCBI data using MAFFT v 7.0 with default settings (Katoh et al., 2019). At the outset, the nrITS and nrLSU trees were constructed individually so as to verify their topology. The topology of both the trees was precisely the same. Furthermore, to build the combined tree, both markers were perused for their combining efficacy by performing Partition Homogeneity Test (ILD) in PAUP v-4.0b10 (Swofford & Sullivan, 2009). Pertained to ILD test, the matrix of both markers was concatenated using TaxonDNA v-1.7.8 (Vaidya et al., 2010). The combined dataset consists of 145 nrITS and 85 nrLSU sequences (including eight newly generated sequences during present study). The phylogenetic trees were constructed using Maximum Likelihood (ML) and Bayesian inference (BI) criteria. For ML analysis, IQTree v1.6.8 (Nguyen et al., 2014) was used, and the best fit evolutionary model (TIM3+F+I+G4) was chosen according to BIC by ModelFinder (an inbuilt tool of IQTree, Kalyaanamoorthy et al., 2017). Bayesian analysis was executed using Metropolis Coupled MCMC method in MrBayes v-3.2.6 (Ronquist et al., 2012). Two parallel chains were run for four million generations, the standard deviation of split frequency was obtained less than 0.01, and the effective sample size (ESS) for each parameter was ensured exceeding 200. The nucleotide substitution model (GTR+I+G) was selected using jModeltest (Darriba et al., 2012). Moreover, FigTree v-1.4.2 displayed the consensus trees (Rambaut, 2014). The phylogram's statistical supports were calculated using posterior probabilities (PP) and bootstrap values (BS). The sequence alignments and phylogenetic trees were submitted to TreeBASE (accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S31602?x-access-code=3e881310d5998a542e04a42b76e40df0&format=html).

Hymenagaricus indicus P.B. Patil, S.A. Vaidya, N.P. Patil & S.K. Maurya, sp. nov. Figs. 1, 2.

MycoBank no.: MB 853990.

Fig. 1. - Hymenagaricus indicus (AMH 10699, holotype). A-D: Basidiomata in the natural habitat. E: Basidiomata with scale bar. F: Lamellae. Bars: A-D 1 cm.

Fig. 2. - Hymenagaricus indicus (AMH 10699, holotype). A: Basidiospores. B: Scanning electron photographs of basidiospores. C, D: Basidia. E-G: Cheilocystidia. H: Pileipellis. Bars: A, B 5 μm; C-H 10 μm.

Diagnosis: Differs from H. pakistanicus by its small basidiomata (up to 15 mm), and ellipsoid to ovoid-ellipsoid, smaller basidiospores and, differs from H. parvulus by its plate-like brown squamules, narrower and longer stipe and smaller basidiospores.

Type: INDIA, Maharashtra, Raigad District, Matheran Hills (18°58'48.00"N, 73°16'12.00"E, 800 m a.s.l.), 20 Aug 2022, P. B. Patil. (AMH 10699, Holotype).

DNA sequence ex-Holotype: PP217302 (nrITS).

Etymology: The species epithet “indicus” refers to the name of the country ‘India’ where the holotype was collected.

Basidiomata small. Pileus 5-15 mm diam, initially hemispherical to conico-campanulate, then convex to nearly applanate with age, dry, white (8A1) to pinkish white (8A2), central disk covered with smooth, plate-like, snuff brown (5F6), violet brown (10E7-8) to brown (6E8) squamules; small, numerous, squamules scattered towards margin, margin incurved, white (8A1) to pinkish white (8A2), striate, with appendiculate thin velar remnants; Lamellae free, depressed around the stipe, pinkish white (8A2) in juvenile, becoming greyish brown (11E3) to violet brown (11E5-6) at maturity, with entire edge, broadly ventricose, distant, lamellulae of 3-4 different lengths. Stipe 30-50 mm × 1-2 mm, central, cylindrical, fistulose, mostly curved, slightly attenuated towards base, white (8A1) to greyish brown (11E3), often covered with white, minute squamules. Annulus very thin, apical, concolorous with the pileus margin, mostly lost with age or due to handling.

Basidiospores [50/3/3] 4-5.5(-6) × 2.5-3.5(-3.7) µm, Q = 1.35-1.88, [X_m = 4.5 ± 0.4 × 3.0 ± 0.3 µm, Q_m = 1.52 ± 0.13], ellipsoid to ovoid-ellipsoid, smooth under light microscope and SEM, inamyloid, no germ pore, thick-walled (0.8 µm), guttulate, apiculate, yellowish brown to dark brown when observed in H₂O and 5% KOH. Basidia 15-20 × 5-7 µm, clavate, thin walled, hyaline, 2-4 spored, sterigmata up to 3.5 µm long. Lamellar trama regular to subregular, consist of thin-walled, cylindrical hyphae, 3-7 µm broad. Cheilocystidia 13.5-28 × 7.5-12 µm, numerous, clavate to broadly clavate, sometimes obovate to slightly fusoid, hyaline, thin-walled. Pleurocystidia absent. Pileipellis (pileal squamules) an epithelium, composed of globose to subglobose, thin-walled, hyaline cells measuring 6.5-20.5 × 7.5-18 µm. Stipitipellis consisting of parallel, hyaline hyphae, 7-11.5 µm wide. Stipe squamules intricate trichoid, composed of globose to subglobose, cylindrical to clavate cells measured 4-16 × 2.5-4.5 µm. Clamp connections absent in all observed tissues.

Habitat and distribution: Scattered to clustered on the soil, in semi-evergreen forest dominated by tree species like Memecylon umbellatum Burm.F., Garcinia talbotii Raizada ex Santapau., Olea dioica Roxb., Xantolis tomentosa (Roxb). Raf. So far known only from Matheran Hills, Maharashtra, India.

Additional specimens examined: INDIA, Maharashtra, Raigad District, Matheran Hills (18°58'48.00"N, 73°16'12.00"E), 22 Jul 2023 (MMH 1511, PP229193 for nrITS and PP217303 for nrLSU), 13 Aug 2023 (MMH 1512), Prashant B. Patil.

Xanthagaricus nigrosquamosus P.B. Patil, S.A. Vaidya, N.P. Patil & S.K. Maurya, sp. nov. Figs. 3, 4.

MycoBank no.: MB 853994.

Fig. 3. - Xanthagaricus nigrosquamosus (AMH 10700, holotype). A-D: Basidiomata in the natural habitat. E: Pileal surface. F: Lamellae with scale bar. Bars: A-E 1 cm.

Fig. 4. - Xanthagaricus nigrosquamosus (AMH 10700, holotype). A: Basidiospores. B: Scanning electron photographs of basidiospores. C, D: Basidia. E-H: Cheilocystidia. I: Pileipellis. Bars: A 5 μm; B 2 μm; C-I 10 μm.

Diagnosis: Differing from X. punjabensis and X. retisporus by its black squamules on pileus surface and smaller basidiospores.

Type: INDIA, Maharashtra, Raigad District, Matheran Hills (18°58'48.00"N, 73°16'12.00"E, 800 m a.s.l.), 12 Aug 2018, P. B. Patil. (AMH 10700, Holotype).

DNA sequence ex-Holotype: MK447559 (nrITS).

Etymology: The species epithet “nigrosquamosus” refers to the black squamules on the pileus surface.

Basidiomata small-sized. Pileus 10-30 mm diam, obtusely conical or convex when young, then broadly umbonate to plane with age, dry, light yellow (4A4-5), vivid yellow (2A8) to maize yellow (4A6), surface concentrically covered with greenish black (27H8) to tar black (H1) squamules, one or more large, darker squamules at the umbonate centre, scattered elsewhere; margin incurved, with prominent light yellow (4A4-5), appendiculate, triangular velar remnants; context up to 1 mm thick at centre, no change in colour upon cut. Lamellae free, depressed around the centre, pinkish white (10A2) in juvenile, becoming pale brown (5D4) to reddish brown (9D5) at maturity, with crenulate edge, broadly ventricose, moderately crowded, lamellulae of 4-5 different lengths. Stipe 20-30 mm × 1-2 mm, central, cylindrical, fistulose, often curved, equal, yellowish white (4A2), light brown (4A4) to pale brown (5D4), often covered with yellow (4A6), minute squamules. Annulus not prominent, thin, apical, concolorous with the pileus margin, mostly lost with age or due to handling.

Basidiospores [50/3/3] (3-)3.5-4.5(-4.7) × (2.2-)2.4-3 µm, Q = (1.2-)1.35-1.7(-1.9), [X_m = 4 ± 0.32 × 2.6 ± 0.22 µm, Q_m = 1.54 ± 0.12], broadly ellipsoid to ellipsoid to ovoid-ellipsoid, smooth under light microscope but rugulose-rough under SEM, no germ pore, slightly thick-walled (0.5 µm), guttulate, inamyloid, apiculate, yellowish brown when observed in H₂O and 5% KOH. Basidia 10-14 × 4-6 µm, clavate, thin walled, hyaline, 2-4 spored, sterigmata up to 2 µm long. Lamellar trama regular to subregular, consisting of thin-walled, cylindrical hyphae, 4-7 µm broad, Cheilocystidia 16-30 × 6-12 µm, numerous, clavate to broadly clavate, sometimes broadly fusoid, hyaline, thin-walled. Pleurocystidia absent. Pileipellis an epithelium, composed of globose to subglobose cells, terminal cells 7.5-21 × 6.5-12.5 µm. Stipitipellis consisting of parallel hyphae, 5.5-13 µm wide. Stipe squamules intricate trichoid, composed of cylindrical to clavate cells measured 4.5-14 × 2.5-4 µm. Clamp connections absent in all observed tissues.

Habitat and distribution: Scattered to clustered, usually strongly caespitose on the soil, in semi-evergreen forest dominated by tree species like Memecylon umbellatum Burm.F., Garcinia talbotii Raizada ex Santapau., Olea dioica Roxb., Xantolis tomentosa (Roxb). Raf. So far known only from Matheran Hills, Maharashtra, India.

Additional specimens (paratypes) examined: INDIA, Maharashtra, Raigad District, Matheran Hills (18°58'48.00"N, 73°16'12.00"E), 11 Aug 2019 (MMH 1411, PP263034 for nrITS and PP217230 for nrLSU), 13 Aug 2023 (MMH 1412, PP217227 for nrITS and PP217231 for nrLSU), Prashant B. Patil.

Hymenagaricus indicus is delineated by its small-sized basidiomata, squamulose pileus made up of pseudoparenchymatous tissues composed of globose to subglobose elements, ellipsoid to ovoid-ellipsoid, smooth, thick walled, yellowish brown basidiospores measuring 4-5.5 × 2.5-3.5 µm. The characters like globose to subglobose cells of pileal squamules are common with H. nigroviolaceus Heinem., H. pakistanicus M.F. Syed & M. Saba, H. siamensis J. Kumla, W. Phonrob, N. Suwannar & S. Lumyong. However, H. nigroviolaceus has smaller size of the pseudoparenchymatous cells (4-12 µm wide) and larger basidiospores (5.4-7.4 × 3.5-4.5 µm) (Heinemann, 1985). Hymenagaricus pakistanicus also has a striate margin but markedly differs from H. indicus by its smaller pileal squamulose cells (5.2-10.4 × 2-4 µm) and subglobose to broadly ellipsoid, larger basidiospores (4.9-6.2 × 3.3-4.9 µm) (Syed et al., 2023). Furthermore, H. siamensis differs from all above species by having remarkably largest basidiospores in the genus (6.5-8 × 4-5 µm) (Kumla et al., 2023). Morphologically, H. saisamornae J. Kumla & N. Suwannarach is also close to H. indicus by having white to pinkish white pileus, covered with violet brown plate-like squamules but distinguishable from H. indicus by its larger basidiospores (5.5-7 × 4-4.5 µm) and hymeniform pileal squamules cells (Kumla et al., 2021). Another recently described species from Pakistan, H. brunneodiscus M. Asif, Saba & M. Raza differs from H. indicus by its larger pileus size (28-43 mm diam) and larger basidiospores (5.1-6.2 × 3.3-3.9 µm) (Crous et al., 2023). Phylogenetically, H. parvulus Al‐Kharousi, Al‐Sadi, Al-Yahya’ei, & S. Hussain formed sister clade with H. indicus but differs by having pinkish to creamy, appressed pellicle squamules on pileus, shorter and broader stipe (25-35 × 2-5 mm), and larger basidiospores (5.0-6.5 × 4.0-4.5 µm) (Hussain et al., 2024). Comparison of morphological characters of H. indicus with Indian taxa of Hymenagaricus are depicted in Table 2.

Xanthagaricus nigrosquamosus is characterised by its black pileal squamules, broadly ellipsoid to ovoid-ellipsoid basidiospores with a rugulose-rough surface under SEM, and a pseudoparenchymatous epithelial pileipellis consist of globose to subglobose elements. Based on the overall appearance of the basidiomata, Xanthagaricus nigrosquamosus is macromorphologically close to X. epipastus (Berk. & Broome) Hussain, X. flavosquamosus T.H. Li, Iqbal Hosen & Z.P. Song, X. montgomeryensis N. Fatima & Khalid, X. necopinatus Iqbal Hosen, T.H. Li & G.M. Gates, X. pakistanicus Hussain, Afshan & Ahmad, and X. taiwanensis (Zhu L. Yang, Z.W. Ge & C.M. Chen) Hussain. However, X. epipastus has a yellow to yellowish brown squamules on the pileus surface, and larger basidiospores (4.2-4.9 × 2.8-3.4 µm) (Heinemann & Little Flower, 1984). Xanthagaricus flavosquamosus is distinguishable from X. nigrosquamosus by its larger and wider basidiospores (5-5.5 × 3-3.5 µm) which are verrucose or warty under SEM and smaller cheilocystidia (7-15 × 6-9 µm) (Hosen et al., 2017). Xanthagaricus montgomeryensis has a pileus covered by dark brown squamules, slightly larger and narrower basidiospores with higher Q value (3.99-4.87 × 2.12-2.85 µm; Qm = 1.75) (Fatima & Khalid, 2023). Xanthagaricus necopinatus is also shared common characters with X. nigrosquamosus such as broadly ellipsoid to ovoid-ellipsoid basidiospores with a rugulose-rough surface under SEM and similar Q value but differs from X. nigrosquamosus by having yellow to yellowish brown squamules on the pileus surface, slightly larger basidiospores (4-5 × 2.7-3.2 µm), and smaller and narrower cheilocystidia (15-20 × 4-6 µm) (Hosen et al., 2017). Xanthagaricus pakistanicus differs from all above species by its globose to subglobose basidiospores (Hussain et al., 2018). Xathagaricus taiwanensis also has a black squamules on the pileus surface but markedly differs from X. nigrosquamosus in having larger basidia (15-22 × 7-8 µm), and larger basidiospores (5-5.5 × 3-4 µm) (Ge et al., 2008). Another species, X. punjabensis Haqnawaz, Niazi & Khalid, and X. retisporus Kun L. Yang, Jia Y. Lin & Zhu L. Yang are phylogenetically close to X. nigrosquamosus. However, X. punjabensis having globose to subglobose, larger basidiospores (5.1-5.9 × 4.1-5.5 µm) (Haqnawaz et al., 2023), and X. retisporus has pudding orange, cherrywood brown to coffee-bean brown pileal squamules, distinctly larger basidiospores (5-6 × 3-4 µm), and smaller cheilocystidia (11-13 × 8-9 µm) (Yang et al., 2024). Comparison of morphological characters of X. nigrosquamosus with Indian taxa of Xanthagaricus are depicted in Table 2.

The combined dataset alignment contained 1323 characters, which includes the aligned sequence dataset composed of 668 bp from nrITS, and 655 bp from nrLSU for the analyses. The exhaustive ILD test analysis with 1000 bootstrap showed congruence with the p value 0.85 at the significance level of 0.05. So, the dataset was combined for the further analysis. Based on combined analysis using ML and Bayesian methods we obtained similar tree topologies. In phylogenetic analyses of Hosen et al. (2017), the genus Hymenagaricus appears to be polyphyletic and the species of Hymenagaricus are intermixed with the monotypic genus, Heinemannomyces Watling and showed its close relationship with Heinemannomyces. (Hosen et al., 2017). So, the Heinemannomyces would be grouped into the single genus Hymenagaricus or separate them into sub-genus/section level as suggested by Hosen et al. (2017). However, morphologically, Heinemannomyces differs from Hymenagaricus by its medium-sized to large basidiomata, pileus surface covered with wooly arachnoid velar remnants, a reddening of context when cut, bluish gray to leaden gray lamellae, and hyphae forming an irregular trichoderm in pileal squamules (Watling, 1998). Further investigation of the taxa, Heinemannomyces, Hymenagaricus and Xanthagaricus on the basis of multi-gene (nrITS, nrLSU, rpb2, tef-1α) phylogenetic analyses and detailed morphological studies by Yang et al. (2024) confirmed that the generic status of Heinemannomyces was no longer exist and hence Heinemannomyces was considered as a synonym under Hymenagaricus. The morphological interrogation of Hymenagaricus and Xanthagaricus taxa by Yang et al. (2024) showed that epitheloid or hymeniform cells of pileus squamules may be terminated with intricate trichoid elements, and both genera have a wider range of basidiospore colours and, also insinuated that the size of cells in pileus squamules and the structure of stipe squamules are usually deceptive for identification of taxa. Further, Yang et al. (2024) propounded the importance of morphological features for the identification of Xanthagaricus species such as basidiomata strongly caespitose or not, basidiospore ornamentation, shape and size of cheilocystidia, and length of the stipe.

The newly described species H. indicus placed in a supported clade (100/1) together with H. pakistanicus, and H. parvulus which appeared sister to H. indicus with strong bootstrap and posterior probability supports (92/1) (Fig. 5). In phylogenetic tree, the genus Xanthagaricus recovers as a monophyletic clade and appeared sister to Pseudolepiota Z.W. Ge, a monotypic genus recently reported from China, with strong bootstrap and posterior probability supports (91/1). Moreover, this study revealed monophyletic origin of X. nigrosquamosus and nested in a supported group (96/1) with X. montgomeryensis, X. punjabensis, X. retisporus, X. taiwanensis, X. thailandensis, and undescribed taxa, with two sequences from China (ITS: GenBank MN088737, MN088738) and one from Pakistan (ITS: GenBank MH778555) (Fig. 5).

Fig. 5. - Maximum likelihood phylogram generated from the combined dataset of two nuclear genes (nrITS and nrLSU). Bootstrap values (BS) ≥ 70 and Bayesian posterior probability (PP) ≥ 0.7 are given at the internodes. Chlorophyllum molybdites and Chlorophyllum rachodes are selected as outgroups. The new species, Hymenagaricus indicus and Xanthagaricus nigrosquamosus, are highlighted in bold on the phylogram. Voucher specimen numbers are provided after the species name. The scale bar represents a phylogenetic distance of 0.04 nucleotide substitutions per site.

The authors declare no conflicts of interest.

We greatly acknowledge the Principal, Smt. C.H.M. College, Ulhasnagar, Maharashtra, India for providing the laboratory facilities.

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