Five New Species of the Genus Hymenogaster (Hymenogastraceae, Agaricales) from Northern China

In this study, five new species from China, Hymenogaster latisporus, H. minisporus, H. papilliformis, H. perisporius, and H. variabilis, are described and illustrated based on morphological and molecular evidence. Hymenogaster latisporus was distinguished from other species of the genus by the subglobose, broad ellipsoidal, ovoid basidiospores (average = 13.7 μm × 11.6 μm) with sparse verrucose and ridge-like ornamentation (1–1.2 μm high); H. minisporus by the ellipsoidal to broadly ellipsoidal and small basidiospores (average = 11.7 μm × 9.5 μm); H. papilliformis was characterized by the whitish to cream-colored basidiomes, and broadly fusiform to citriform basidiospores with a pronounced apex (2–3 μm, occasionally up to 4 μm high), papillary, distinct warts and ridges, and pronounced appendix (2–3 μm long); H. perisporius by the dirty white to pale yellow basidiomes, broad ellipsoidal to ellipsoidal, and yellow-brown to dark-brown basidiospores with warts and gelatinous perisporium; H. variabilis by the peridium with significant changes in thickness (167–351 μm), and broad ellipsoidal to subglobose basidiospores ornamented with sparse warts and ridges. An ITS/LSU-based phylogenetic analysis supported the erection of the five new species. A key for Hymenogaster species from northern China is provided.


Introduction
Hymenogaster (Hymenogastraceae, Agaricales), established by Carolo Vittadini based on eight species found in Europe [1], is one of the most species-rich genera of false truffles [2][3][4].Hymenogaster citrinus was designated as the type species [5].Species within this genus can form ectomycorrhiza with a wide range of tree species, mainly including Betulaceae, Ericaceae, Fagaceae, Myrtaceae, Pinaceae, Salicaceae, and Tiliaceae, and display no significant host specificity [6][7][8][9][10][11][12][13][14]; thus, they can assist host plants in nutrient uptake and play an important role in the conservation, restoration, or rebuilding of ecosystems.Since Vittadini's original description of this genus, the reliance on morphological features alone has led to persistent taxonomic errors and confusion [5,[15][16][17].The primary morphological characteristics for species delimitation are the color or discoloration of the basidiomes when fresh, and the features of basidiospores (including color, shape, and ornamentation).Molecular sequencing technology significantly altered our understanding of the species delineation within this genus.For instance, Stielow et al. [2] re-examined the genus using ITS analysis and described two new species; Smith et al. [18] employed multiple gene regions to demonstrate that H. mcmurphyi was in fact a sequestrate species of Xerocomellus (Boletineae, Boletales), rather than a Hymenogaster species as previously believed based solely on morphological data.However, molecular sequence-based studies of Chinese Hymenogaster remain scarce.
A total of 32 species and variants of Hymenogaster have been reported in China based on morphology [19].Clearly, it is necessary that the occurrence of these Hymenogaster species be re-examined and verified with molecular data.
In this study, we employed a combination of morphological and molecular methods to systematically investigate Hymenogaster species collected from Beijing and Shanxi Province in China.This approach led to the identification and description of five new species.Two known species reported in previous studies in China [19], H. arenarius and H. citrinus, were confirmed with morphological and molecular evidence (Figures 1 and 2).A total of 32 species and variants of Hymenogaster have been reported in China based on morphology [19].Clearly, it is necessary that the occurrence of these Hymenogaster species be re-examined and verified with molecular data.
In this study, we employed a combination of morphological and molecular methods to systematically investigate Hymenogaster species collected from Beijing and Shanxi Province in China.This approach led to the identification and description of five new species.Two known species reported in previous studies in China [19], H. arenarius and H. citrinus, were confirmed with morphological and molecular evidence (Figures 1 and  2).

Sample Collections
Samples were systematically collected over a period of six years in China and subsequently examined.These voucher specimens have been accessioned in the Herbarium of the Biology Department at Capital Normal University (BJTC).Macroscopic characteristics of these specimens were described from both fresh and dried materials.For microscopic analysis, thin sections were prepared from dried specimens by hand.These sections were then immersed in a 3% KOH (w/v) solution and Melzer's reagent [20] for detailed study.For the scanning electron microscopy (SEM) analysis, basidiospores were positioned onto double-sided adhesive tape affixed to the SEM stub.Subsequently, these samples were uniformly coated with a platinum-palladium film utilizing an ion sputter-coater (HITACHI E-1010).The coated samples were then examined and documented using a Hitachi S-4800 SEM (Hitachi, Tokyo, Japan).

Sample Collections
Samples were systematically collected over a period of six years in China and subsequently examined.These voucher specimens have been accessioned in the Herbarium of the Biology Department at Capital Normal University (BJTC).Macroscopic characteristics of these specimens were described from both fresh and dried materials.For microscopic analysis, thin sections were prepared from dried specimens by hand.These sections were then immersed in a 3% KOH (w/v) solution and Melzer's reagent [20] for detailed study.For the scanning electron microscopy (SEM) analysis, basidiospores were positioned onto double-sided adhesive tape affixed to the SEM stub.Subsequently, these samples were uniformly coated with a platinum-palladium film utilizing an ion sputter-coater (HITACHI E-1010).The coated samples were then examined and documented using a Hitachi S-4800 SEM (Hitachi, Tokyo, Japan).

Phylogenetic Analysis
The ITS-LSU combined dataset was assembled and aligned utilizing the MAFFT algorithm [24], adhering to default parameters.This alignment was further refined through manual adjustments in Se-Al v2.03a [25], ensuring optimal sequence similarity.Alignments of all datasets used in this study were submitted to TreeBASE (No. 31242).ML and BI analysis were used together to construct phylogenetic tree.Maximum likelihood (ML) analysis was performed with RAxML 8.0.14 [26] employing the GTRGAMMAI substitution model with parameters unlinked.ML bootstrap replicates (1000) were computed in RAxML using a rapid bootstrap analysis and search for the best-scoring ML tree.The ML trees were visualized with TreeView32 [27].Clades with bootstrap support (BS) ≥ 70% were considered significant [28].Bayesian inference (BI) was conducted using MrBayes v3.1.2[29] as an additional method of evaluating branch support.In BI analysis, after selecting the best substitution models (GTR + I + G for all positions) determined by MrModeltest v2.3 [30], two independent runs of four chains were conducted for 1,065,000 Markov chain Monte Carlo (MCMC) generations with the default settings.Average standard deviations of split frequency (ASDSF) values were far less than 0.01 at the end of the generations.Trees were sampled every 100 generations after burn-in (well after convergence), and a 50% majorityrule consensus tree was constructed and visualized with TreeView 32 [27].Clades with Bayesian posterior probability (PP) ≥ 0.95 were considered significantly supported [31].

Molecular Phylogenetics
The ITS-LSU combined dataset was compiled to elucidate the phylogenetic position of the new species in this study.This comprehensive dataset comprises 97 sequences from 23 different species, inclusive of 72 newly generated sequences derived from Chinese collections.The length of the aligned dataset was 1465 bp after the exclusion of poorly aligned sites, with 651 bp for ITS and 814 bp for nrLSU.Both Maximum Likelihood (ML) and Bayesian Inference (BI) analyses resulted in similar phylogenetic tree topologies.The tree, as deduced from the ML analysis, is presented here, which showed robust statistical bootstrap support from ML and posterior probability values from BI, confirming the reliability of the findings (Figure 1).Our collections were resolved in seven independent clades with strong statistical bootstrap support, indicating they represented seven distinct species.Two of them represented the known species H. arenarius and H. citrinus, respectively.The remaining five species are novel to science.

Taxonomy
Hymenogaster latisporus L. Fan and T. Li, sp.nov.(Figure 3)  Description-the basidiome is subglobose to irregular globose, 1.2-1.8cm in diameter, soft and elastic, earth yellow when fresh, yellow-brown to brown when dry, with a distinct depression at the sterile base.Its surface is smooth and glabrous.
The ITS-LSU-based phylogeny supported the description of this new species.DNA analysis showed that H. latisporus shared less than 97% identity in ITS sequence to other Hymenogaster species.Hymenogaster minisporus T. Li & L. Fan, sp.nov.(Figure 4) Description-the basidiome is subglobose to globose, 0.5-1.7 cm in diameter, soft and elastic, dirty white when fresh, stained pale brown, with a distinct depression at the MycoBank: MB852601 Etymology: minisporus, referring to small basidiospores.Holotype: China.Beijing, Miyun County, Bulaotun Town, alt.273 m. 27 July 2020, in soil under Castanea mollissima Blume, ZH571 (BJTC FAN1244).
Description-the basidiome is subglobose to globose, 0.5-1.7 cm in diameter, soft and elastic, dirty white when fresh, stained pale brown, with a distinct depression at the sterile base.The surface is smooth and glabrous.
Habit and habitat: hypogeous, gregarious, in the soil under Castanea mollissima.
Notes: Hymenogaster minisporus is characterized by the ellipsoidal to broadly ellipsoidal and small basidiospores.This new species was grouped into a clade with the 'cryptic species 1' of H. niveus provisionally proposed by Stielow et al. [2] but without statistical support (Figure 1).Morphologically, the latter has white basidiomes that change to red when touched or bruised [2].ITS-LSU-based phylogeny supports the erection of this new species.DNA analysis showed that H. minisporus shared less than 97% identity in the ITS sequence to other Hymenogaster species.
Description-the basidiome is subglobose to irregular globose, 0.8-2.5 cm in diameter, soft and elastic, whitish to cream-colored when fresh, with pale yellow to pale taupe spots, yellowish to yellow-brown when dry, with a distinct depression at the sterile base.The surface is smooth and glabrous.
'cryptic species 1′ of H. niveus provisionally proposed by Stielow et al. [2] but without statistical support (Figure 1).Morphologically, the latter has white basidiomes that change to red when touched or bruised [2].ITS-LSU-based phylogeny supports the erection of this new species.DNA analysis showed that H. minisporus shared less than 97% identity in the ITS sequence to other Hymenogaster species.
Hymenogaster papilliformis L. Fan & T. Li, sp.nov.(Figure 5) Description-the basidiome is subglobose to irregular globose, 0.8-2.5 cm in diameter, soft and elastic, whitish to cream-colored when fresh, with pale yellow to pale taupe spots, yellowish to yellow-brown when dry, with a distinct depression at the sterile base.The surface is smooth and glabrous.
The peridium is 140-300 μm thick, pseudoparenchymatous, composed of elliptic cells of 14-20 μm in diam, pale yellow to nearly hyaline.The gleba are reddish-brown to brown when fresh, deep brown when dry, and loculate; the locules are rectangle to ir-  Description-the basidiome is subglobose to irregular globose, 0.5-2 cm diameter, soft and elastic, dirty white to pale yellow when fresh, sometimes brownish, with a distinct depression at the white sterile base.Surface smooth, glabrous.
The peridium is 223-310 μm thick, pseudoparenchymatous, composed of elliptic cells of 8-12 μm in diameter and interwoven hyphae of 6.8-8.5 μm broad, light yellowish to nearly hyaline.The gleba are reddish-brown to brown when fresh, deep reddish-brown when dry, loculate, with locules irregular, empty, and filled with spores at  Description-the basidiome is subglobose to irregular globose, 1-1.8 cm diameter, soft and elastic, pale yellow to earth yellow when fresh, yellow-brown to brown when dry, with a distinct depression at the sterile base.The surface is smooth and glabrous.
Habit and habitat: hypogeous, gregarious, in the soil under Pinus tabulaeformis.Description-the basidiome is subglobose to irregular globose, 1-1.8 cm diameter, soft and elastic, pale yellow to earth yellow when fresh, yellow-brown to brown when dry, with a distinct depression at the sterile base.The surface is smooth and glabrous.
Habit and habitat: hypogeous, gregarious, in the soil under Pinus tabulaeformis.

Figure 1 .
Figure 1.Phylogeny derived from maximum likelihood analyses of the ITS/LSU sequences from Hymenogaster and related species.Two sequences of Anamika lactariolens were selected as the outgroup.Values on the left represent the likelihood bootstrap support values (≥70%).Values on the right represent significant Bayesian posterior probability values (≥0.95).Novel sequences are in bold.Super index "H" means "Holotype".The green background represents the five new species described in this study, the red background represents the two old species from China supported by molecular data.

Figure 1 .
Figure 1.Phylogeny derived from maximum likelihood analyses of the ITS/LSU sequences from Hymenogaster and related species.Two sequences of Anamika lactariolens were selected as the outgroup.Values on the left represent the likelihood bootstrap support values (≥70%).Values on the right represent significant Bayesian posterior probability values (≥0.95).Novel sequences are in bold.Super index "H" means "Holotype".The green background represents the five new species described in this study, the red background represents the two old species from China supported by molecular data.
• C for 30 s, 55 • C for 45 s, 72 • C for 1 min, and a final extension at 72 • C for 10 min; for the nrLSU gene, initial denaturation at 95 • C for 4 min, followed by 35 cycles at 95 • C for 30 s, 55 • C for 60 s, 72 • C for 1 min, and a final extension at 72 • C for 10 min.The PCR products were sent to Beijing Zhongkexilin Biotechnology Co., Ltd.(Beijing, China) for purification and sequencing.Validated sequences are stored in the NCBI database (http://www.ncbi.nlm.nih.gov/)(accessed on 30 April 2023) under the accession numbers provided (Table

Table 1 .
Sources of specimens and GenBank accession numbers for sequences used in this study.Newly generated sequences are in bold.