*Article* **New Species and New Records of** *Otidea* **from China Based on Molecular and Morphological Data**

**Yu-Yan Xu † , Ning Mao † , Jia-Jia Yang and Li Fan \***

> College of Life Science, Capital Normal University, Xisanhuanbeilu 105, Haidian, Beijing 100048, China; 2190801004@cnu.edu.cn (Y.-Y.X.); 2210801013@cnu.edu.cn (N.M.); 2210802122@cnu.edu.cn (J.-J.Y.)

**\*** Correspondence: fanli@mail.cnu.edu.cn

† These authors contributed equally to this work.

**Abstract:** Species of genus *Otidea* previously reported in China are mainly distributed in the northeast, northwest and southwest regions of China, but the species diversity of *Otidea* in north China is not very clear. In this study, newly collected *Otidea* specimens from northern China and some herbarium specimens deposited in three important Chinese fungus herbaria (HMAS, HKAS, HMJAU) were studied using morphological and phylogenetic methods. The internal transcribed spacers of the nrDNA (ITS), the nrRNA 28S subunit (nrLSU), the translation elongation factor 1-alpha (*tef1-α*), and the second largest subunit of RNA polymerase II (*rpb2*), were employed to elucidate the phylogenetic relationships between *Otidea* species. Results identified 16 species of *Otidea*, of which seven new species are described, namely *O. aspera*, *O. cupulata*, *O. filiformis*, *O. khakicolorata*, *O. parvula*, *O. plicara* and *O. purpureobrunnea*. *Otidea bicolor* and *O. pruinosa* are synonymized as *O. subpurpurea*. Two species, *O. mirabilis* and *O. nannfeldtii*, are being reported for the first time in China. The occurrence of *O. bufonia*, *O. leporina* and *O. onotica* are confirmed by molecular data in China.

**Keywords:** Ascomycota; Pyronemataceae; phylogeny; seven new taxa; taxonomy

## **1. Introduction**

The genus *Otidea* (Pers.) Bonord. (Pyronemataceae, Pezizales), with *O. onotica* (Pers.) Fuckel as the type species, was established in the mid-19th century [1,2]. The genus is characterized by the following criteria: epigeous, cup- to ear-like apothecia, split to the base on one side (less often entirely), and being stipitate or not; or as in a single species, hypogeous with enclosed ascomata, subcylindrical, nonamyloid, operculate asci, ellipsoid to fusoid, biguttulate ascospores, and filiform paraphyses [3–5]. *Otidea* species are considered to form ectomycorrhizae with both broad leaf and conifer trees [3,4], and are widely distributed across the temperate regions of Europe, North America and Asia in the northern hemisphere [4–10]. The taxonomy of *Otidea* species are mainly based on the morphological characteristics studied in work before 2006 [5,6,11–17]. Since then, taxonomists began to introduce molecular methods into the taxonomy and identification of *Otidea* species. Molecular techniques have revolutionized phylogenetics and species delimitation of *Otidea* [3,4,10,18–25]. In 2009, a whole new set of morphological and histochemical features was also introduced in *Otidea* by Harmaja [15] and further by Hansen and Olariaga [3] and Olariaga et al. [4]. These features have made it possible to recognize the species morphologically to a large extent and have since been employed. *Otidea* species in Europe (c. 32 accepted species) and North America (c. 14 accepted species) have been comprehensively and systematically studied in recent years [3,4,10,21,22].

China has a huge temperate area in the northern hemisphere and likely has diverse *Otidea* species. However, only a few taxonomic works focus on this genus, and about a quarter of Chinese *Otidea* species are not supported by molecular evidence [6,8,16–18,23–29]. Currently, a total of 24 *Otidea* species are reported in China, including 17 native species and seven known species originally described from Europe and/or North America.

**Citation:** Xu, Y.-Y.; Mao, N.; Yang, J.-J.; Fan, L. New Species and New Records of *Otidea* from China Based on Molecular and Morphological Data. *J. Fungi* **2022**, *8*, 272. https:// doi.org/10.3390/jof8030272

Academic Editors: Cheng Gao and Lei Cai

Received: 31 January 2022 Accepted: 6 March 2022 Published: 8 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

During our investigation of fungal resources in northern China since 2017, many apothecia of the genus *Otidea* were collected. On the bases of these new collections and some of herbarium specimens deposited in three important Chinese fungus herbaria (HMAS, HKAS, HMJAU), we recognized seven species and two records new to China based on both morphological examination and molecular analysis. Also, both *O. bicolor* W.Y. Zhuang & Zhu L. Yang, and *O. pruinosa* Ekanayaka, Q. Zhao & K.D. Hyde were conspecific with *O. subpurpurea* W.Y. Zhuang. Our aim in this paper is to describe and illustrate these new species and new records and to synonymize *O. bicolor* and *O. pruinosa* as *O. subpurpurea*. Molecular data for some known species existing in China are additionally provided.

## **2. Materials and Methods**

## *2.1. Morphological Studies*

Fresh specimens were collected and photographed in the field from the Shanxi and Hebi provinces, as well as Beijing, China. The specimens were dried and deposited in the BJTC (Herbarium, Biology Department, Capital Normal University) and the HSA (Herbarium Institute of Edible Fungi, Shanxi Academy of Agricultural Science, Taiyuan, China). Other specimens were studied from the HMAS (Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences), HKAS (Herbarium of Cryptogams at the Kunming Institute of Botany, Chinese Academy of Sciences) and HMJAU (Herbarium of Mycology, Jilin Agricultural University). Macroscopic characteristics were recorded from fresh specimens. Standardised color values matching the described colour were taken from ColorHexa (http://www.colorhexa.com/, accessed on 30 January 2022). Microscopic characteristics were observed in thin sections of dry specimens mounted in 5% KOH and Melzer's reagent [30]. The dimensions for ascospores are given using notation of the form (a-)b-c(-d). The range b-c contains a minimum of 90% of the measured values. The extreme values, i.e., a and d, are given in the parentheses. L<sup>m</sup> and W<sup>m</sup> indicate the average ascospore length and width for the measured ascospores, respectively. Q is used to represent length/width ratio of a ascospore in side view and Q<sup>m</sup> represents average Q of all specimens. The number of populations that the statistics are based on is indicated by n.

## *2.2. DNA Extraction, PCR Amplification, Sequencing*

Herbarium specimens were crushed by shaking for 30 s at 30 Hz 2–4 times (Mixer Mill MM 301, Retsch, Haan, Germany) in a 1.5 mL tube together with one 3 mm diameter tungsten carbide ball, and total genomic DNA was extracted using the modified CTAB method [31]. The following primers were used for PCR amplification and sequencing: ITS1f/ITS4 [31,32] were used for the internal transcribed spacers of the nrDNA (ITS1-5.8S-ITS2 = ITS), LR0R/LR5 [33] for the nrDNA 28S subunit (nrLSU), EF1-983F/EF1-2218R [34] for the translation elongation factor 1-alpha (*tef1-α*), and RPB2-Otidea6F/RPB2-Otidea7R and fRPB2-7cF/fRPB2-11aR [3] for the RNA polymerase II second largest subunit (*rpb2*), respectively. PCRs were performed in 50 µL reactions containing 4 µL DNA template, 2 µL of per primer (10 µM), 25 µL 2× Master Mix (Tiangen Biotech Co., Beijing, China), and 17 µL ddH2O. PCR reactions were performed as follows: for the ITS gene: initial denaturation at 94 ◦C for 3 min, followed by 35 cycles at 94 ◦C for 30 s, 56 ◦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 94 ◦C for 4 min, followed by 35 cycles at 94 ◦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 *tef1-α* gene: initial denaturation at 94 ◦C for 3 min, followed by 35 cycles at 94 ◦C for 30 s, 60 ◦C for 45s, 72 ◦C for 1min, and a final extension at 72 ◦C for 10 min; for the *rpb2* gene: initial denaturation at 94 ◦C for 3 min, followed by 10 cycles (including denaturation) at 94 ◦C for 30 s, annealing temperature started at 62 ◦C (decreased by 1 ◦C per cycle, until to 52 ◦C) for 45 s and extension at 72 ◦C for 1 min, then followed by 30 cycles at 94 ◦C for 35 s, 55 ◦C for 45 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, sequencing, and editing. The newly generated sequences were assembled and edited using SeqMan (DNA STAR package, DNAStar Inc., Madison, WI, USA) with generic-level identities for sequences confirmed via BLAST queries of GenBank.

## *2.3. Sequence Alignment and Phylogenetic Analyses*

A total of 730 sequences from 283 collections of *Otidea* were used in the molecular phylogenetic analyses. The detail information about them is provided in Supplementary Table S1, including the geographic origin and accession numbers. Sequences of all DNA regions generated in this study were deposited in GenBank. The sequences obtained from GenBank are based on published literature or selected by using BLASTn search to find similar matches with taxa in *Otidea*. Two datasets were assembled for this study. Dataset I (ITS/nrLSU) and datasets II (ITS/nrLSU/*tef1-α*/*rpb2*) contained the backbone species and all phyloclades of *Otidea*, which were used to infer the phylogenetic status of Chinese *Otidea* species of the genus *Otidea*. The taxa *Monascella botryosa* Guarro & Arx and *Warcupia terrestris* Paden & J.V. Cameron were selected as outgroups. The ITS, nrLSU, *tef1-α* and *rpb2* sequences were respectively aligned using the MAFFT v.7.110 online program under the default parameters [35], and manually adjusted to allow for maximum sequence similarity in Se-Al version.2.03a [36]. Ambiguously aligned regions of each sequence were detected and excluded using Gblocks 0.91b [37] before the phylogenetic analyses. Unsampled gene regions were coded as missing data and all introns of *tef1*-*α* and *rpb2* were excluded because of the alignment difficulty. To examine the conflict among topologies with maximum likelihood (ML), separate single-gene analyses were conducted. Alignments were concatenated using SequenceMatrix v1.7.8 [38] and are provided in Supplementary Files S2 and S3. We conducted maximum likelihood (ML) and Bayesian inference (BI) analyses on the two datasets.

Maximum likelihood (ML) analyses of the two datasets were carried out using RAxML 8.0.14 [39] with all parameters kept to the default settings using a GTRGAMMAI model. The ML bootstrap replicates (1000) were computed in RAxML using a rapid bootstrap analysis searching for the best scoring ML tree. Bayesian inference (BI) analyses were performed with MrBayes v3.1.2 [40] based on the best substitution models for each gene region as determined by MrModeltest 2.3 [41]. The GTR + I+G model was the best model for ITS, nrLSU and *rpb2*, whereas the best model for *tef1-α* was the SYM + I+G model. Two independent executions of four chains were conducted: 3,485,000 for ITS/nrLSU and 765,000 for the ITS/nrLSU/*tef1-α*/*rpb2* datasets. Markov chain Monte Carlo generations were conducted using the default settings and sampled every 100 generations. The temperature value was lowered to 0.20, burn-in was set to 0.25, and the program was automatically stopped as soon as the average standard deviation of split frequencies reached below 0.01. A 50% majority-rule consensus tree was constructed. Clades with a bootstrap support (BS) ≥ 70% and a Bayesian posterior probability (PP) ≥ 0.95 were considered as significantly supported [42,43]. All phylogenetic trees were viewed with TreeView32 [44].

## **3. Results**

## *3.1. Phylogenetic Analyses*

No topological incongruence was detected when the four genes were analyzed individually. Dataset I (ITS/nrLSU) contained 528 sequences from 51 species, including 93 novel sequences the two genes from Chinese collections, and four from the outgroups (*Monascella botryosa* and *Warcupia terrestris*). The dataset had an aligned length of 1363 characters (551 bp from ITS and 812 bp from nrLSU), of which 647 were constant, 716 were variable, and 609 of these variable sites were informative. ML and BI analyses yielded similar tree topologies. Only the tree inferred from the ML analyses is shown (Figure 1). The species of *Otidea* formed a monophyletic clade with high support values (BS = 100%, PP = 1.00). A total of 10 clades were recognized in the two-gene phylogram, which is consistent with Olariaga et al. [4] and Hansen and Olariaga [3]. Newly obtained Chinese *Otidea* specimens were nested in six clades: *O. bufonia-onotica*, *O. formicarum*, *O. leporina*, *O. cantharella*, *O. alutacea*, and *O. platyspora* clade (Figure 1). A total of 16 species were recognized.

In the *O. bufonia-onotica* clade, 24 Chinese specimens were clearly placed in eight well-supported clades, represented by five known species and three new species. The known species are *O. bufonia* (Pers.) Boud., *O. subpurpurea*, *O. mirabilis* Bolognini & Jamoni, *O. onotica* and *O. brevispora* (W.Y. Zhuang) Olariaga & K. Hansen. The three new species are respectively described as *O. filiformis*, *O. cupulata* and *O. purpureobrunnea* in this study. It is interesting that the sequences from the type specimens of *O. bicolor* and *O. pruinosa* fall into the clade of *O. subpurpurea* and shared 98.87–99.84% similarity in the ITS region, which implied they may be conspecific, although they have some difference in apothecial color. Fortunately, we borrowed the type specimens of these three species for observation and research. We thought that they should be conspecific and formally synonymized *O. bicolor* and *O. pruinosa* in this study.

**Figure 1.** *Cont*.

**Figure 1.** *Cont*.

**Figure 1.** Phylogenetic tree generated from maximum likelihood analysis based on ITS and nrLSU sequences, showing the phylogenetic relationships of *Otidea*. *Monascella botryosa* and *Warcupia terrestris* are the outgroups. Maximum likelihood bootstrap support values (≥50%) are indicated above the nodes as BS. Thick black branches received Bayesian posterior probabilities (BPP) ≥ 0.95. Novel sequences are printed in bold red. Mycorrhizal or environmental sequences are printed in blue. The new species are in bold font and highlighted by blue boxes.

In the *O. formicarum* clade, seven Chinese specimens were clearly placed in two well supported clades. One of clades corresponds to *O. nannfeldtii* Harmaja, marking the first time it is discovered in China. The other one is a new species described as *O. khakicolorata* in this study. In the *O. leporina* clade and *O. cantharella* clade, the Chinese specimens were identified as the previously known species, *O. leporina* (Batsch) Fuckel and *O. propinquata* (P. Karst.) Harmaja, respectively. In the *O. alutacea* clade, seven Chinese collections were clearly placed into three well supported clades, represented by the known species *O. alutacea* (Pers.) Massee and two new species described as *O. parvula* and *O. aspera* in this paper. In the *O. platyspora* clade, the two Chinese collections formed a distinct clade with high evidential support, which was described as *O. plicara* in this study.

In order to further verify the phylogenetic positions of the seven new species and whether *O. bicolor* and *O. pruinosa* are conspecific with *O. subpurpurea*, we performed a further phylogenetic analysis based on the four-gene dataset II. This dataset II does not include sequences from seven confirmed known species, namely *O*. *bufonia*, *O*. *mirabilis*, *O*. *onotica*, *O*. *nannfeldtii*, *O*. *leporina*, *O*. *propinquata* and *O*. *alutacea*. Dataset II (ITS/nrLSU/*tef1-α*/*rpb2*)

contained 501 sequences from 49 species, including 73 novel sequences these four genes from Chinese collections, and 8 from the outgroups (*M. botryosa* and *W. terrestris*). The dataset had an aligned length of 4400 characters (551 bp from ITS, 812 bp from nrLSU, 1383bp from *tef1-α* and 1654 bp from *rpb2)*, of which 2456 were constant, 1854 were variable, and 1753 of these variable sites were informative. ML and BI analyses yielded similar tree topologies. Only the tree inferred from the ML analysis is shown (Figure 2).

**Figure 2.** *Cont*.

**Figure 2.** Phylogenetic tree generated from a maximum likelihood analysis based on ITS, nrLSU, *tef1-α* and *rpb2* sequences, showing the phylogenetic relationships of *Otidea*. *Monascella botryosa* and *Warcupia terrestris* are the outgroups. Maximum likelihood bootstrap support values (≥50%) are indicated above the nodes as BS. Thick black branches received Bayesian posterior probabilities (BPP) ≥ 0.95. The new species are in bold font and highlighted by blue boxes.

The species of *Otidea* formed a monophyletic clade with high support values (BS = 100%, PP = 1.00). A total of 10 clades were recognized in the four-gene phylogram, which is consistent with Hansen and Olariaga [3]. Similar to the two-gene phylogram results, the specimens from China formed seven apparently independent clades with high support values, representing seven new species. The type sequences of *O. bicolor*, *O. pruinosa* and *O. subpurpurea* clustered into a clade with high support values (BS = 100%, PP = 1.00), indicating that they are indeed the same species, and *O. bicolor* and *O. pruinosa* are placed in synonymy as *O. subpurpurea* in the taxonomy section below. Compared with the two-gene tree phylogram (Figure 1), the 10 clades identified in the four-gene tree are consistent, but inside the *O. bufonia-onotica* clade, the *O. concinna* clade, and the *O. alutacea* clade, the topological positions of some species are slightly different (e.g., *O.* *purpureobrunnea* and *O. cupulata* in the *O. bufonia-onotica* clade), which may be due to the fact that these species have a lower support value between them.

## *3.2. Taxonomy*

Based on our phylogenies and morphological data, a total of seven new species, a known species, and two new records of *Otidea* from China were described and illustrated here.

*Otidea aspera* L. Fan & Y.Y. Xu, sp. nov. (Figure 3).

**Figure 3.** *Otidea aspera* (HSA 278) (**A**) apothecia, (**B**) anatomy of apothecium, (**C**) ascospores, (**D**) asci and paraphyses, (**E**) ectal excipulum in water, and (**F**) basal mycelium. Scale bars: (**A**) = 1 cm, (**B**) = 100 µm, (**C**) = 10 µm; (**D**) = 20 µm; (**E**) = 50 µm; (**F**) = 10 µm.

MycoBank: MB843176.

Etymology: *aspera*, referring to the rough receptacle surface.

Holotype: China. Shanxi Province, Jiaocheng County, Pangquangou National Nature Reserve, HaoJiagou, alt. 1800m, in the mixed forest dominated by *Pinus tabuliformis* Carrière and *Quercus mongolica* Fisch. ex Ledeb., 29 August 2018, J.Z. Cao, LH278 (HSA 278).

Saprobic on soil. Apothecia solitary or caespitose in nature, 30–45 mm high, 25–60 mm wide, initially ear-shaped, then expanding and sometimes becoming irregularly ear-shaped or shallowly to deeply cup-shaped, sometimes elongated on one side or obconical, split, stipitate. Hymenium surface greyish yellow (#fbe8c5) to light brown (#baab98) when fresh, ochre brown (#a87832) when dry, subsmooth. Receptacle surface pale yellow (#fafad2) to yellowish brown (#b5a27f) when fresh, slightly hygrophanous, pale whitish ochre (#c8b99f) when dry, finely warty. Stipe 5–12 × 4–8 mm. Basal tomentum and mycelium white. Apothecial section 600–900 µm thick. Ectal excipulum of *textura angularis*, 100–150 µm

thick, cells thin walled, hyaline to pale brown, 11–28 × 8–20 µm. Medullary excipulum of *textura intricata*, 300–500 µm thick, hyphae 3–10 µm wide, sometimes slightly swollen, thin to thick walled, septate, hyaline to light brown. Subhymenium c. 50–120 µm thick, visible as a brown zone of densely arranged cylindrical to swollen cells. Paraphyses septate, straight to slightly curved, of uniform width or slightly enlarged at the apices to 3–4.7 µm wide, without or rarely with 1–2 low notches. Asci 150–200 × 9–13 µm, 8-spored, unitunicate, cylindrical, hyaline, long pedicellate, arising from croziers, non-amyloid, ascospores released from an eccentric split at the apical apex. Ascospores ellipsoid to slightly subfusoid, inequilateral, with two large guttules, sometimes only with one big guttule, smooth, hyaline, (12–) 12.8–15 (–15.5) × (5.8–) 6.5–7.5 (–8) µm (L<sup>m</sup> × W<sup>m</sup> = 14 × 7 µm, Q = 1.8–2.2, Q<sup>m</sup> = 2, n = 50). Receptacle surface with warts, 50–80 µm high, formed by short, fasciculate, hyphoid hairs, of 5–7 subglobose to elongated cells, constricted at septa, 6–13 µm wide. Resinous exudates absent to scarce. Basal mycelium of 2–5 µm wide, septate, hyaline to pale brown hyphae, smooth, unchanged in KOH, smooth, turning yellow in MLZ.

Other materials examined: China. Inner Mongolia Autonomous Region, Daqinggou National Nature Reserve, on the broad-leaved woodland, 24 August 2005, Tolgor Bau (HMJAU 4166).

Notes: *Otidea aspera* is diagnosed by the combination of the stipitate, broadly ear shaped to cup-shaped, greyish yellow to light brown hymenium, pale-yellow to yellowishbrown receptacle surface, ellipsoid to slightly subfusoid ascospores and straight to slightly curved paraphyses. *Otidea aspera* and *O. parvispora* have comparable apothecia color, however *O. parvispora* differs from *O. aspera* by the smaller ascospores ((11.0–) 11.5–13.0 × 5.0–6.5 µm) and shorter asci. DNA analysis showed that *O. aspera* shared less than 93.39% ITS sequence similarity with other *Otidea* species. Phylogenetic analyses revealed that the sequences of *O. aspera* were grouped into an independent clade with a strong support value (Figures 1 and 2). These supported the erection of the new species.

*Otidea cupulata* L. Fan & Y.Y. Xu, sp. nov. (Figure 4).

**Figure 4.** *Otidea cupulata* (HSA 218) (**A**) apothecia, (**B**) ascospores, (**C**) asci and paraphyses, (**D**) paraphyses, (**E**) basal mycelium, and (**F**) ectal and medullary excipulum in water, (**G**) ectal excipulum in water, (**H**) ectal excipulum in MLZ. Scale bars: (**A**) = 1 cm, (**B**) = 10 µm, (**C**) = 50 µm, (**D**) = 10 µm, (**E**) = 25 µm, (**F**) = 50 µm, (**G**,**H**) = 30 µm.

MycoBank: MB843177.

Etymology: *cupulata*, referring to the apothecia shape of the fungus.

Holotype: China. Shanxi Province, Jiaocheng County, Pangquangou Township, Badaogou valley, alt. 2200m, on soil in mixed forest of *Larix* sp. and *Betula* sp., 28 August 2018, L.J. Guo, LH218 (HSA 218).

Saprobic on soil. Apothecia gregarious or caespitose in nature, 25–40 mm high, 15–50 mm wide, initially ear-shaped, soon expanding, becoming broadly ear-shaped or deeply cup-shaped, split, often broader above, margin sometimes lobate, stipitate. Hymenium surface dark orange brown (#734a12) to dark purple brown (#4d282d) when fresh, and usually with bluish-lilaceous shades, gray ochraceous brown (#37290e) when dry, subsmooth. Receptacle surface yellowish brown (#a1805f) to brown (#8a6660) when fresh, slightly hygrophanous, surface with shallow wrinkles, dark brown (#261600) when dry, furfuraceous. Stipe 5–10 × 5–7 mm. Basal tomentum and mycelium whitish to grayish yellow (#c6cbac). Apothecial section 900–1200 µm thick. Ectal excipulum of *textura angularis*, 80–110 µm thick, cells thin walled, brownish, 10–25 × 8–22 µm. Medullary excipulum of *textura intricata*, 500–700 µm thick, hyphae 3.5–10 µm wide, sometimes slightly swollen, thin to slightly thick walled, septate, hyaline to light brown. Subhymenium c. 60–100 µm thick, visible as a brown zone, of densely arranged cylindrical to swollen cells, with scattered brown resinous exudate at septa. Paraphyses septate, curved to hooked of uniform width or slightly enlarged at the apices to 2.6–4.2 µm wide, without or rarely with 1–2 low notches, sometimes forked near the apex. Asci 150–200 × 8–11 µm, 8-spored, unitunicate, cylindrical, hyaline, long pedicellate, arising from croziers, non-amyloid, ascospores release from an eccentric split at the apical apex. Ascospores ellipsoid to slightly subfusoid, inequilateral, with two large guttules, sometimes with only one big guttule, smooth, hyaline, (12–) 12.5–15 (–15.5) × (6–) 6.5–7.4 (–7.9) µm (L<sup>m</sup> × Wm= 13.9 × 7 µm, Q = 1.9–2.1, Q<sup>m</sup> = 2, n = 50). Receptacle surface with low warts, 25–45 µm high, formed by short, fasciculate, hyphoid hairs, of 3–4 subglobose to elongated cells, constricted at septa, 6–11 µm wide, sometimes with a gelatinous sheath. Resinous exudates abundant on the outer surface, dark brown, partly dissolving and converting into small particles in MLZ, entirely dissolving and turning bright yellow in KOH. Basal mycelium of 3–6.5 µm wide, septate, hyaline to pale brown hyphae, bright yellow in KOH, with abundant, very small, irregularly, brown, resinous exudates on the surface, dissolving and turning bright yellow in KOH, unchanged in MLZ.

Other materials examined: China. Shanxi Province, Jiaocheng County, Pangquangou Township, Badaogou Valley, alt. 1800m, on soil under *Larix* sp., 6 September 2018, L.J. Guo, LH 406 (HSA 406).

Notes: *Otidea cupulata* is recognized by the stipitate, broadly cup-shaped, dark-orangebrown to dark-purple-brown hymenium, yellowish-brown to brown receptacle surface, ellipsoid to subfusoid ascospores, forked or notched paraphyses, and furfuraceous receptacle surface. The forked paraphyses is rarely found in other species in the O. bufonia-onotica clade. Several species in the O. bufonia-onotica clade are similar to *O. cupulata* in apothecial shape and color, including *O. bufonia*, *O. filiformis*, *O. mirabilis,* and *O. olivaceobrunnea* Harmaja, but *O. bufonia* can be distinguished by its distinctly narrowly fusoid ascospores, the presence of hyphae with striate resinous exudates in the medullary excipulum, and resinous exudates of the ectal excipulum that does not turn bright yellow in KOH. *Otidea filiformis* differs in its apothecia with pinkish shades, distinctly narrowly fusoid ascospores, unenlarged and curved to hooked paraphyses, and higher warts (40–75 µm) on the receptacle surface. *Otidea mirabilis* differs by having purple to lilaceous-bluish shades on the receptacle surface and narrowly fusoid ascospores (Q<sup>m</sup> = 2.1–2.3). *Otidea olivaceobrunnea* can be separated by its olive-brown hymenium and wider ascospores (14–17 × 8–8.5 µm). Four species (*O. purpureogrisea* Pfister, F. Xu & Z.W. Ge, *O. purpureobrunnea*, *O. simithii* Kanouse and *O. subpurpurea*) have more or less bluish or lilaceous shades on apothecia that resembles that of *O. cupulata*. However, *O. purpureogrisea* is distinguished by its ear-shaped apothecia, dark-purple-brown to purple-gray receptacle surface, and the resinous exudate

in the ectal excipulum turning amber and brown in KOH. *Otidea purpureobrunnea* is distinguished by its grayish-purple-brown to dark-purple-brown receptacle surface and mostly smooth basal mycelium. *Otidea simithii* differs in having typically narrower, ear-shaped apothecia, and resinous exudates of the ectal excipulum that does not turn bright yellow in KOH. *Otidea subpurpurea* in having smaller ascospores (9–12 × 4.5–6 µm) and lilac to purplish receptacle surface.

In the two-gene phylogenetic tree (Figure 1), two specimens (K(M)41595 and K(M)156077) from England fall into the clade of *O*. *cupulata*, which are noted to belong to an unnamed taxon related to *O. bufonia* and *O. subpurpurea* by Parslow et al. [10]. Regrettably, they did not further describe the morphology of these two specimens. According to the ITS sequence similarity analysis (ITS: 98%) and phylogenetic analyses (Figure 1), they may be conspecific with *O. cupulata*. Final confirmation requires morphological observation of these two specimens.

*Otidea filiformis* C.L. Hou, Y.Y. Xu & H. Zhou, sp. nov. (Figure 5).

**Figure 5.** *Otidea filiformis* (BJTC C505) (**A**) apothecia, (**B**) ascospores, (**C**) asci and paraphyses, (**D**) basal mycelium, (**E**) ectal excipulum in water, and (**F**) ectal excipulum in KOH. Scale bars: (**A**) = 1 cm, (**B**) = 10 µm, (**C**) = 30 µm, (**D**) = 20 µm, (**E**,**F**) = 50 µm.

MycoBank: MB843178.

Etymology: *filiformis*, referring to the filiform paraphyses in the hymenium.

Holotype: China. Beijing City, Huairou District, Sunshanzi Village, alt. 770m, on soil in mixed forest of *Populus* sp. and *Larix* sp., 28 August 2020, G.Q. Chen C505 (BJTC C505).

Saprobic on soil. Apothecia gregarious or caespitose in nature, 15–55 mm high, 15–55 mm wide, initially ear-shaped, soon expanding, becoming shallowly or deeply cupshaped, split, margin sometimes lobate, sessile or shortly stipitate, regular or sometimes undulate in the margin. Hymenium surface yellowish brown (#b19461) to ochre yellow (#cd9575) with pinkish shades, sometimes with brown spots or stains when fresh, margin dark brown when bruised, gray brown (#321f15) when dry, subsmooth. Receptacle surface yellowish brown (#b19461) to orange brown (#967059) when fresh, slightly hygrophanous, dark brown (#321f15) when dry, furfuraceous to finely warty. Stipe not well developed. Basal tomentum and mycelium whitish to pale brown (#dccdbf). Apothecial section 800–1000 µm thick. Ectal excipulum of *textura angularis*, 75–110 µm thick, cells thin walled, brown, 10–35 × 7–26 µm. Medullary excipulum of *textura intricata*, 400–500 µm thick, hyphae 4–10 µm wide, sometimes slightly swollen, thin to thick walled, septate, hyaline to light brown, without resinous exudates. Subhymenium ca. 75–100 µm thick, visible as a brown zone of densely arranged cylindrical to swollen cells, with scattered brown resinous exudate at septa. Paraphyses septate, curved to hooked of uniform width at the apices to 2–3 µm wide, without or with a low notch. Asci 140–175 × 10–14 µm, 8-spored, unitunicate, cylindrical, hyaline, long pedicellate, arising from croziers, non-amyloid, ascospores released from an eccentric split at the apical apex. Ascospores narrowly fusoid, narrowed at both ends, inequilateral, with two large guttules, sometimes only with one big guttule, smooth, hyaline, (12.5–) 13–14.5 (–15) × (6–) 6.5–7 (–7.3) µm (L<sup>m</sup> × W<sup>m</sup> = 13.5 × 6.8 µm, Q = 1.9–2.1, Q<sup>m</sup> = 2, n = 50). Receptacle surface with broad conical warts, 40–75 µm high, formed by short, fasciculate, hyphoid hairs, of 6–7 subglobose to elongated cells, constricted at septa, 6–10 µm wide. Resinous exudates abundant on the outer surface, dark yellow brown, partly dissolving and converting into small particles in MLZ, partially dissolving and turning yellowish brown in KOH. Basal mycelium of interwoven, 3–6 µm wide, septate, hyaline to pale brown hyphae, turning yellow in KOH, with abundant small, regularly arranged, spheroid, pale brown, resinous exudates, partly dissolving in KOH, unchanged in MLZ.

Other materials examined: China. Hebei Province, Chicheng County, Dahaituo nature reserve, alt. 1640m, on soil under *Betula platyphylla* Suk., 22 August 2019, J.Q. Li L482 (BJTC L482); China. Inner Mongolia Autonomous Region, Balinyou Banner, Saihanwula Nature Reserve, on soil in mixed forest of *Larix gmelinii* (Rupr.) Kuzen. and *Betula platyphylla* Suk., 2 September 2008, T.Z. Liu, H.M. Zhou & C. Sun 3858 (HMAS 188468).

Notes: *Otidea filiformis* diagnosed by the combination of yellowish-brown to ochreyellow apothecia, sometimes with brown spots or stains, narrowly fusoid ascospores, uniform width, narrow paraphyses (≤3 µm) and basal mycelium with abundant spheroid, pale brown, resinous exudates. *Otidea bufonia* and *O*. *mirabilis* are similar to *O. filiformis* in apothecia color and ascospore shape. *Otidea bufonia* differs from *O. filiformis* in having the longer ascospores (12–) 13–16.5 (–18) × 6–7.5 (–8) µm, and brown striate exudates on some hyphae of the medullary excipulum. *O*. *mirabilis* differs in dark-brown apothecia, purple to lilaceous-bluish shades on the receptacle surface and biflabellate crystal-like exudates in the medullary excipulum. The apothecia of *Otidea korfii* Pfister, F. Xu & Z.W. Ge, *O. olivaceobrunnea* and *O. saliceticola* Cartabia, M. Carbone & P. Alvarado also have brown tones. *Otidea korfii* is distinguished from *O. filiformis* by its ear-shaped apothecia, olivaceous brown receptacle surface, ellipsoid to broadly ellipsoid bigger ascospores (14.5–17 × 6.5–9 µm), resinous exudate of the ectal excipulum dissolving in MLZ and smooth basal mycelium. *Otidea olivaceobrunnea* differs from *O. filiformis* in olive-brown hymenium, ellipsoid ascospores. *Otidea saliceticola* differs in pale alutaceous-greyish hymenium surface, dark brown receptacle surface, wider ascospores (14–15 × 7.5–8 µm) with low Q value of 1.75–1.87.

Phylogenetic analyses revealed that the sequences of *O. filiformis* were grouped into an independent clade with a strong support value (Figures 1 and 2). DNA analysis showed that *O. filiformis* shared less than 95.88% ITS sequence similarity with other *Otidea* species. These supported the erection of the new species. One Finnish collection (MCVE 29372) identified as *O. bufonia* by Carbone et al. [22] is clustered into the *O. filiformis* clade with strong support values in our phylogenetic trees (Figures 1 and 2), and it shared more than 98% ITS similarity with our *O. filiformis* and less than 94.6% with *O. bufonia*. This evidence showed MCVE 29372 is more closely related to *O. filiformis,* and whether it is conspecific with *O. filiformis* need further morphological identification.

*Otidea khakicolorata* L. Fan & Y.Y. Xu, sp. nov. (Figure 6).

**Figure 6.** *Otidea khakicolorata* (BJTC FM107) (**A**) apothecia, (**B**) ascospores, (**C**) asci and paraphyses, (**D**) ectal excipulum in KOH, (**E**) ectal excipulum in water, (**F**) basal mycelium, and (**G**) amber drops on the outermost ectal excipulum cells in Melzer's reagent. Scale bars: (**A**) = 0.5 cm, (**B**) = 10 µm, (**C**) = 20 µm, (**D**,**E**) = 25 µm, (**F**,**G**) = 20 µm.

MycoBank: MB843179.

Etymology: *khakicolorata*, referring to the khaki color of apothecia.

Holotype: China. Shanxi Province, Ningwu County, Guancen Mountain, Dashidong Forest Farm, alt. 2200m, among moss under coniferous forest dominated by *Picea wilsonii* Mast. and *Larix principis-rupprechtii* Mayr, 24 August 2017, X.Y. Yan YXY170824 (BJTC FM107).

Saprobic on soil. Apothecia solitary or gregarious in nature, 15–25 mm high, 5–8 mm wide, narrowly long ear-shaped, margin rounded, split, sessile or sub-stipitate. Hymenium surface khaki (#ca9a67) to pale ochre (#c08649) when fresh, yellowish ochre(#e3c57f) when dry, subsmooth. Receptacle surface concolorous with hymenium when fresh, slightly hygrophanous, dark reddish brown (#8c5738) when dry, furfuraceous. Warts absent or very low. Stipe, if present, very short. Basal tomentum and mycelium whitish to grayish whitish (#e7e2d9). Apothecial section 500–800 µm thick. Ectal excipulum of *textura angularis*, 70–120 µm thick, cells thin walled, brown, 10–25 × 6–20 µm. Medullary excipulum of *textura intricata*, 250–500 µm thick, hyphae 3.5–8 µm wide, sometimes slightly swollen, thin to slightly thick walled, septate, hyaline to light brown. Subhymenium ca. 60–90 µm thick, visible as a yellowish-brown zone, of densely arranged cylindrical to swollen cells. Paraphyses septate, curved to hooked, of uniform width at the apices, 2.5–3.7 µm wide, without notch. Asci 150–180 × 8.5–11 µm, 8-spored, unitunicate, cylindrical, hyaline, long pedicellate, arising from croziers, non-amyloid, ascospores released from an eccentric split at the apical apex. Ascospores ellipsoid, sometimes slightly inequilateral, with two large guttules, smooth, hyaline, (8.5–) 9–10 (–10.5) × (4.5–) 5–6 (–6.5) µm (L<sup>m</sup> × Wm= 9.5 × 5.5 µm, Q= 1.6–1.9, Qm= 1.75, n = 50). Receptacle surface with broadly conical warts, 30–40 µm high, formed by hyphoid hairs, of 2–5 subglobose to elongated cells, constricted at septa, 6–10 µm wide, sometimes with a gelatinous sheath. Resinous exudates abundant on the outer surface, yellow brown to dark brown, partly dissolving into amber drops in MLZ, turning reddish brown to dark reddish brown in KOH. Basal mycelium of 3–5.5 µm wide, septate, hyaline to pale-brown hyphae, unchanged in KOH, smooth or with little resinous exudates on the surface, partly dissolving in MLZ, and partly dissolving and more slowly in KOH.

Notes: *Otidea khakicolorata* is characterized by khaki to pale-ochre, long, narrowly earshaped apothecia, small ascospores and resinous exudates on the ectal excipulum turning reddish brown in KOH. *Otidea nannfeldtii* and *O. khakicolorata* share similar apothecia shape and the reaction of the resinous exudate in the ectal excipulum and basal mycelium in MLZ and KOH, but *O. nannfeldtii* can be distinguished by an ochre to orangish-ochre hymenium surface with pink tones, higher warts (45–85 µm) on the apothecial outer surface, medullary excipulum of textura intricata differentiated into two parts, and relatively bigger ascospores ((9–) 9.5–10.5 (–11.5) × 5.5–6.5 (–7) µm). *Otidea khakicolorata* is phylogenetically close to *O. nannfeldtii*; however, they are separated by a low support value (Figures 1 and 2). DNA analyses showed that *O. khakicolorata* shared less than 91% similarity in ITS sequence with *O. nannfeldtii*.

*Otidea parvula* L. Fan & Y.Y. Xu, sp. nov. (Figure 7).

**Figure 7.** *Otidea parvula* (BJTC FM210-A) (**A**,**B**) apothecia, (**C**) ascospores, (**D**) paraphyses and asci, (**E**) anatomy of apothecium, and (**F**) basal mycelium. Scale bars: (**A**,**B**) = 1 cm, (**C**,**D**) = 10 µm, (**E**) = 100 µm, (**F**) = 10 µm.

MycoBank: MB843180.

Etymology: *parvula*, referring to the small apothecia of the fungus.

Holotype: China. Shanxi Province, Jiaocheng County, Guandi Mountain, Pangquangou National Nature Reserve, alt. 2000m, on soil in the mixed forest dominated by *Picea wilsonii* Mast., 7 September 2017, J.Z. Cao, Cao170803 (BJTC FM210-A).

Saprobic on soil. Apothecia gregarious to caespitose in nature, 8–15 mm high, 5–13 mm wide, initially narrowly to broadly ear-shaped, margin rounded, then expanding and sometimes becoming irregularly ear-shaped or cup-shaped, split, stipitate, or sessile. Hymenium surface pale whitish ochre (#ffffed) to ochre yellow (#b39a7f) when fresh, pale ochre brown (#c2a461) when dry, subsmooth. Receptacle surface pale ochre (#d7c498) to orangish ochre (#dba35e) when fresh, hygrophanous, light yellowish brown (#d7c498) when dry, furfuraceous. Stipe 3–7 × 3–5 mm. Basal tomentum and mycelium white. Apothecial section 500–700 µm thick. Ectal excipulum of *textura angularis*, 70–120 µm thick, cells thin walled, pale brown, 9–24 × 7–20 µm. Medullary excipulum of *textura intricata*, 80–160 µm

thick, hyphae 3–7 µm wide, sometimes slightly swollen, thin to thick walled, septate, hyaline to light brown. Subhymenium c. 50–80 µm thick, visible as a brown zone, of densely arranged cylindrical to swollen cells, with scattered brown resinous exudate at septa. Paraphyses septate, bent to curved, sometimes straight, of uniform width or slightly enlarged at the apices, 2.5–4.5 µm wide, sometimes with 1–2 notches near the apex. Asci 150–200 × 10.5–13.5 µm, 8-spored, unitunicate, cylindrical, hyaline, long pedicellate, arising from croziers, non-amyloid, ascospores released from an eccentric split at the apical apex. Ascospores ellipsoid, slightly inequilateral, with two large guttules, sometimes with only one big guttule, smooth, hyaline, (12.5–) 13–15.5 (–16) × (6.5–) 6.8–8 (–8.6) µm (L<sup>m</sup> × W<sup>m</sup> = 14.3 × 7.5 µm, Q = 1.7–2.1, Q<sup>m</sup> = 1.9, n = 50). Receptacle surface with low warts, 30–50 µm high, formed by short, fasciculate, hyphoid hairs, of 5–6 subglobose to elongated cells, constricted at septa, 5–10 µm wide. Resinous exudates absent. Basal mycelium of 2.5–5 µm wide, septate, hyaline to pale brown hyphae, smooth, unchanged in KOH, turning yellow in MLZ.

Other materials examined: China. Shanxi Province, Jiaocheng County, Guandi Mountain, Pangquangou National Nature Reserve, alt. 2000m, on soil in the mixed forest dominated by *Picea wilsonii*, 7 September 2017, J.Z. Cao, Cao170803 (BJTC FM210-B).

Notes: *Otidea parvula* is easily recognized by the stipitate, irregularly ear-shaped or cup-shaped, small, pale-whitish-ochre, ochre-yellow to orangish-ochre apothecia, straight to bent paraphyses and furfuraceous receptacle surface. *Otidea parvula* and *O. adorniae* Agnello, M. Carbone & P. Alvarado are somewhat similar in the color of the apothecia, but *O. adorniae* differs in its larger apothecia and smaller ascospores (11.8 × 6.4 µm). *Otidea parvula* and *O. parvispora* (Parslow & Spooner) M. Carbone, Agnello, Kautmanová, Z.W. Ge & P. Alvarado have the highest ITS sequence similarity of 96%, but upon examination of the phylogenetic tree (Figures 1 and 2), they don't seem to be closely related, and *O. parvispora* is easily distinguished by its pale hymenium and smaller ascospores ((11.0–) 11.5–13.0 × 5.0–6.5 µm).

*Otidea plicara* L. Fan & Y.Y. Xu, sp. nov. (Figure 8).

**Figure 8.** *Otidea plicara* (BJTC FM262-A) (**A**,**B**) apothecia, (**C**) ascospores, (**D**) paraphyses and asci, (**E**) ectal excipulum in water, and (**F**) basal mycelium. Scale bars: (**A**,**B**) = 1 cm, (**C**) = 10 µm, (**D**,**E**) = 30 µm, (**F**) = 20 µm.

MycoBank: MB843181.

Etymology: *plicara*, referring to the small fold on the apothecia.

Holotype: China. Shanxi Province, Jiaocheng County, Guandi Mountain, Badaogou valley, alt. 2000m, on soil in the mixed forest dominated by *Picea wilsonii*, 7 September 2017, J.Z. Cao, Cao170855 (BJTC FM262-A).

Saprobic on soil. Apothecia solitary or gregarious in nature, 15–28 mm high, 12–42 mm wide, initially spoon-shaped or broadly ear-shaped, soon expending, in the end almost deeply cup-shaped, often broader above, margin entire, with a small fold on the apothecia, seemingly spilt yet not split, stipitate. Hymenium surface pale greyish brown (#6f6a61) to dark brown (#665856), margin yellow ochre (#837050) when fresh, when dry becoming slightly lighter but dull, light brown (#ab9876), subsmooth. Receptacle surface dark reddish brown (#7c6052) when fresh, slightly hygrophanous, pale ochre brown (#a48e6a) when dry, finely furfuraceous, wrinkle veined at the base. Stipe 8–15 × 5–8 mm. Basal tomentum and mycelium abundant, white to pale cream (#f1f9ed). Apothecial section 650–1000 µm thick. Ectal excipulum of *textura angularis*, 60–100 µm thick, cells thin walled, brown, 11–34 × 9–28 µm. Medullary excipulum of *textura intricata*, 300–500 µm thick, hyphae 3–7.5 µm wide, sometimes slightly swollen, thin walled, septate, hyaline to light brown. Subhymenium ca. 80–130 µm thick, visible as a yellowish-brown zone. Paraphyses septate, curved to hooked, usually enlarged at the apices, 3.5–5.5 µm wide at apex, 2–3 µm below. Asci 160–220 × 10–14 µm, 8-spored, unitunicate, cylindrical, hyaline, long pedicellate, arising from croziers, non-amyloid, ascospores released from an eccentric split at the apical apex. Ascospores ellipsoid, sometimes slightly inequilateral, with one to two large guttules, smooth, hyaline, (12.5–) 13.5–16 (–17) × (6–) 6.5–8 (–8.5) µm (L<sup>m</sup> × W<sup>m</sup> = 14.7 × 7.4 µm, Q = 1.8–2.2, Q<sup>m</sup> = 2, n = 50). Receptacle surface with hyphoid hairs, 50–80 µm long, of 3–6 ovoid or subglobose to elongated cells, constricted at septa, 4–9 µm wide. Resinous exudates absent. Basal mycelium of interwoven, 2.5–6 µm wide, septate, hyaline to pale brown hyphae, unchanged in KOH, smooth, turning yellow in MLZ.

Other materials examined: China. Shanxi Province, Jiaocheng County, Guandi Mountain, Badaogou Scenic Area, alt. 2000m, on soil in the mixed forest dominated by *Picea wilsonii* Mast., 7 September 2017, J.Z. Cao, Cao170855 (BJTC FM262-B).

Notes: *Otidea plicara* is characterized by greyish-brown to dark-brown, stipitate, rarely split, deeply cup-shaped apothecia, small ascospores, enlarged paraphyses and the lack of resinous exudates on the ectal excipulum and basal mycelium. Macroscopically, *Otidea apophysata* (Cooke & W. Phillips) Sacc. and *O. platyspora* Nannf. have similar apothecial shape and color to *O. plicara*, but *O. apophysata* can be distinguished by the larger ascospores (20–24.5 × 9–11 µm) and frequently branched paraphyses. *Otidea platyspora* can be distinguished by split apothecia and larger ascospores (18–22 × (9.5–)10.5–12 µm). DNA analyses showed that *O*. *plicara* shared less than 92% similarity in ITS sequence with other species of *Otidea*. Phylogenetic analyses revealed that the sequences of *O. plicara* were grouped into an independent clade with a strong support value (Figures 1 and 2). These supported the erection of the new species.

*Otidea purpureobrunnea* L. Fan & Y.Y. Xu, sp. nov. (Figure 9).

MycoBank: MB843182.

Etymology: *purpureobrunnea*, referring to the purple-brown tone of apothecia.

Holotype: China. Shanxi Province, Qinshui County, Tuwo Township, Shangwoquan Village, alt. 1200m, on soil under *Quercus* sp., 25 August 2020, H. Liu 1065 (BJTC FM1048).

**Figure 9.** *Otidea purpureobrunnea* (BJTC FM1048) (**A**) apothecia, (**B**) ascospores, (**C**) anatomy of apothecium, (**D**) paraphyses, (**E**) asci, (**F**) ectal and medullary excipulum in water, and (**G**) basal mycelium. Scale bars: (**A**) = 1 cm, (**B**) = 10 µm, (**C**) = 100 µm, (**D**,**E**) = 20 µm, (**F**) = 50 µm, (**G**) = 20 µm.

Saprobic on soil. Apothecia gregarious to caespitose in nature, 25–55 mm high, 50–80 mm wide, initially ear shaped, soon expanding, becoming broadly ear shaped or deeply cup shaped, often elongated on one side, split, margin sometimes lobate, stipitate or sessile. Hymenium surface ochraceous brown (#804618), grayish purple (#5e4f5f) to purple brown (#39242f) when fresh, gray brown to dark brown (#3e2c1c) when dry, subsmooth. Receptacle surface grayish purple brown (#816e71) to dark purple brown (#483131) when fresh, sometimes partly dark yellow brown, slightly hygrophanous, some apothecia with shallowly wrinkled, dark brown (#492615) when dry, furfuraceous to finely warty. Stipe 5–10 × 4–8 mm. Basal tomentum and mycelium whitish to pale brown (#dccdbf). Apothecial section 900–1300 µm thick. Ectal excipulum of *textura angularis*, 80–120 µm thick, cells thin walled, brownish, 13–33 × 7–26 µm. Medullary excipulum of *textura intricata*, 500–900 µm thick, formed of loosely woven cylindrical to slightly swollen thin-walled hyphae, 4.5–11 µm wide, septate, hyaline to light brown, with brown resinous exudates at septa. Subhymenium c. 100–150 µm thick, visible as a brown zone, of densely arranged cylindrical to swollen cells, with scattered brown resinous exudate at septa. Paraphyses septate, curved to hooked, a few curved, sometimes forming a coil or helix, of the same width or often enlarged at the apices, 3.5–5 µm wide, 2–3.3 µm below, sometimes with 1–2 notches, or with an obvious bulge near the apex. Asci 140–190 × 8.5–15 µm, 8-spored, unitunicate, cylindrical, hyaline, long pedicellate, arising from croziers, non-amyloid, ascospores released from an eccentric split at the apical apex. Ascospores ellipsoid to slightly subfusoid, inequilateral, with two large guttules, sometimes with only one big guttule, smooth, hyaline, (12.5–) 13–15 (–15.5) × (6–) 6.5–7 (–7.5) µm (L<sup>m</sup> × Wm= 14 × 6.5 µm, Q= 1.9–2.3, Qm= 2.1, n = 50). Receptacle surface with broad conical warts, 35–60 µm high, formed by short, fasciculate, hyphoid hairs, of 2–5 subglobose to elongated cells, constricted at septa, 6–13 µm wide. Resinous exudates abundant on the outer surface, yellow brown to dark brown, partly dissolving into particles in MLZ, entirely dissolving and turning yellow

in KOH. Basal mycelium of 3.5–6 µm wide, septate, hyaline to pale brown hyphae, turning yellow in KOH, mostly smooth, a few with very small, spheroid, pale-brown, resinous exudates, dissolving in KOH, partially dissolving in MLZ.

Other materials examined: China. Shanxi Province, Qinshui County, Tuwo Township, Shangwoquan Village, alt. 1200m, on soil under *Quercus* sp., 25 August 2020, H. Liu 1079 (BJTC FM1061).

Notes: *Otidea purpureobrunnea* is characterized by the stipitate, broadly ear-shaped to cup-shaped, grayish-purple to purple-brown apothecia, ellipsoid to slightly subfusoid ascospores, paraphyses enlarged at the apices, with 1–2 notches or an obvious bulge and smooth basal mycelium. Similar to *O. purpureobrunnea*, the apothecia of *O. bufonia, O. cupulata, O. mirabilis*, *O. purpurea*, *O. purpureogrisea*, *O. smithii*, and *O. subpurpurea* all have some purple tones, but *Otidea bufonia* differs in its fusoid ascospores, the presence of hyphae with striate resinous exudates in the medullary excipulum, resinous exudates of the ectal excipulum not turning bright yellow in KOH, and in having abundant resinous exudates on the basal mycelium. *Otidea mirabilis* differs by having fusoid ascospores, resinous exudates of the ectal excipulum that do not turn bright yellow in KOH and when present, biflabellate, crystal-like exudates in the medullary excipulum. *Otidea purpurea* and *O. subpurpurea* are easily distinguished by the obviously smaller spores (*O. subpurpurea*: 9–12 × 4.5–6 µm; *O. purpurea*: 8–10 × 4.5–6 µm). *Otidea purpureogrisea* is distinguished by the purple-gray tone of the receptacle surface near the base and resinous exudates of the ectal excipulum turning amber in MLZ and turning brown in KOH. *Otidea smithii* is distinguished by typically narrower, ear-shaped apothecia, relatively shorter ascospores (12–14 × 6–7.5 µm) with a lower Q<sup>m</sup> value (1.9–2), and resinous exudates of the ectal excipulum not turning bright yellow in KOH. For a comparison with *O. cupulata* see under that species below.

Phylogenetic analyses revealed that *O. purpureobrunnea* and *O. filiformis* are grouped together with a low support value (Figure 2), but *O. filiformis* is easy to distinguish from *O. purpureobrunnea* by its apothecia without purple tones, fusoid ascospores, same width and narrow paraphyses (≤3 µm), as well as its basal mycelium with abundant spheroid, pale brown, resinous exudates. DNA analysis showed that *O. purpureobrunnea* shared less than 94.53% similarity in its ITS sequence with *O. filiformis*. These indicate that they are two different species.

*Otidea subpurpurea* W.Y. Zhuang, Mycologia Montenegrina 10: 238 (2007).

Holotype: China, Yunnan Province, Kunming City, Kunming Institute of Botany, alt. 1980m, 8 October 2005, Z.L. Yang 4602, (HKAS 49443); Isotype (HMAS 97530).

= *Otidea bicolor* W.Y. Zhuang & Zhu L. Yang, Mycotaxon 112: 35 (2010).

Holotype: China, Yunnan Province, Kunming City, Heilongtan Park, 16 August 2008, Z.L. Yang 5156, (HKAS 54453); Isotype (HMAS 188415).

= *Otidea pruinosa* Ekanayaka, Q. Zhao & K.D. Hyde, Fungal Diversity 87: 130 (2017).

Holotype: China, Yunnan Province, Kunming City, Xishan Scenic Area, 15 September 2012, T. Guo 617, (HKAS 81819).

Materials examined: China, Yunnan Province, Kunming City, Kunming Institute of Botany, alt. 1980m, 9 October 2005, Z.L. Yang 4602, (HKAS 49443); Isotype (HMAS 97530). China, Yunnan Province, Kunming City, Heilongtan Park, 16 August 2008, Z.L. Yang 5156, (HKAS 54453); Isotype (HMAS 188415). ibid., (HKAS 54449). China, Yunnan Province, Kunming City, Xishan Scenic Area, 15 September 2012, T. Guo 617, (HKAS 81819).

Notes: *Otidea bicolor*, *O. pruinosa* and *O. subpurpurea* are highly similar species. In fact, previous scholars have also noticed the phenomenon that the type sequences of the three species are clustered together [22,25]; however, due to the unavailability of specimens, this issue has not been formally addressed. In this study, we examined the type specimens and obtained multiple loci sequences from them. DNA analyses revealed that *O. pruinosa*, *O. bicolor*, and *O. subpurpurea* share high sequence similarity (ITS: >98.87%; nrLSU: >99.53%; *tef1-α*: >99.72%; *rpb2*: >99.45%). We performed morphological observation on these type specimens and found that there was no obvious difference in microscopic features. The reaction of the resinous exudate in the ectal excipulum and basal mycelium in MLZ and

KOH are also the same. Although the receptacle surface of *O. bicolor* and *O. subpurpurea* is purplish in tint when fresh, the receptacle surface of *O. pruinosa* is without a purplish tint [23,28,29], but that may be influenced by its habitat. *Otidea pruinosa* is proposed as a new species because of receptacle surface with pruinose, but we found a similar granulate on the surface of dry specimens of *O. bicolor* and *O. subpurpurea*. In addition, phylogenetic analyses based on the two-gene and four-gene datasets also confirmed that they represent the same species, so here we formally treat *O. bicolor* and *O. pruinosa* as synonyms of *O. subpurpurea*. The sequence from ZMU124 (label as *O. bufonia*) from Guizhou province of China grouped with *O. subpurpurea* with a high support value (Figure 1), indicating that *O. subpurpurea* seems widely distributed in southwest China. Similarly, the sequence from JS150904-08 from Korea named *O. bufonia* [45] was also grouped into this clade (Figure 1). We checked the original morphological description by Jin et al. [45] and found that its ascospores size does not conform to *O. bufonia*, but instead to *O. subpurpurea*. This indicates that *O. subpurpurea* also occurs in Korea.

*Otidea mirabilis* Bolognini & Jamoni in Jamoni, Funghi e Ambiente 85–86: 56 (2001). (Figure 10).

**Figure 10.** Two new record species from China. (**A**) *Otidea mirabilis* (HSA 234), (**B**) *Otidea nannfeldtii* (BJTC FM236). Scale bars: (**A**,**B**) = 1 cm.

Habitat: on soil under mixed forest of *Larix principis-rupprechtii* and *Betula* sp.

Distribution: Known from the northeast, northern, northwest and southwest regions of China.

Materials examined: China, Yunnan Province, Jingdong County, Ailao Mountain, Xujiaba Village, alt. 2500m, 24 August 1994, M. Zang, 12389 (HKAS 28129). China, Gansu Province, Wudu County, Liangshui Town, Gongba River Beach, alt. 2600m, 11 July 1996, M.S. Yuan, 2213 (HKAS 30708). China, Sichuan Province, Hongyuan County, Kangle Town, alt. 3400m, 19 August 1998, M.S. Yuan, 3433 (HKAS 33633). China, Jilin Province, Fusong County, Songjiang River, 19 August 2000, M.S. Yuan, 4725 (HKAS 37272). China, Xinjiang Autonomous Region, Jimusa'er, alt. 1700m, 1 August 2003, W.Y. Zhuang & Y. Nong, 4657 (HMAS 83568). China, Inner Mongolia Autonomous Region, Chifeng City, Baiyin Aobao National Nature Reserve, 2 August 2013, Tolgor Bau (HMJAU 26926). China, Shanxi Province, Jiaocheng County, Guandi Mountain, Shanshui Village, alt. 1800m, 8 September 2017, J.Z. Cao, CAO170863 (BJTC FM292). China, Shanxi Province, Jiaocheng County, Pangquangou Nature Reserve, alt. 2100m, 28 August 2018, H. Liu, LH234 (HSA 234).

Notes: The occurrence of *O. mirabilis* is confirmed in China based on morphological and DNA evidence in this study. Olariaga et al. [4] showed already that *O. mirabilis* occur in China using nrLSU sequences from two Chinese collections in GenBank (identified as *O. leporina* by Zhuang [17] and Liu and Zhuang [18], but by morphology it has not been previously confirmed, as Olariaga et al. did not study those two collections morphologically. It is interesting that two distinct clades were revealed, one comprising Chinese specimens, and another comprising specimens from Europe. The Chinese specimens shared 98.46–99.84% ITS sequence similarity and the European ones had 99.54–99.85% similarity, while the similarities between the two proveniences were 97–98.5%. However, we found no significant morphological differences between the Chinese and European specimens, which probably resulted from the geographic distance.

*Otidea nannfeldtii* Harmaja, Karstenia 15: 31 (1976). (Figure 10).

Habitat: on soil under mixed forest of *Larix principis-rupprechtii*.

Distribution: Known in northern China and northwest China.

Materials examined: China, Shanxi Province, Ningwu County, Guancen Mountain, Qiuqiangou Village, alt. 2100m, on soil under *L. principis-rupprechtii* Mayr, 25 August 2017, X.Y. Yan, YXY170836 (BJTC FM168); ibid., X.Y. Yan, YXY170837 (BJTC FM169); ibid., X.Y. Yan, YXY170838 (BJTC FM170). China, Shanxi Province, Jiaocheng County, Guandi Mountain, Pangquangou Nature Reserve, alt. 2000m, 6 September 2017, J.Z. Cao, CAO170829 (BJTC FM236); ibid., J.Z. Cao, CAO170836 (BJTC FM243). China, Xinjiang Autonomous Region, Jimusa'er, alt. 1700m, 1 August 2003, W.Y. Zhuang & Y. Nong, 4655 (HMAS 83573).

Notes: The occurrence of *O. nannfeldtii* in China is first confirmed based on molecular and morphological evidence. *Otidea nannfeldtii* is originally described in Europe, and also reported from North America [4]. Before this study, there are no DNA data that support the existence of this species in China.

## **4. Discussion**

Temperate China is surely rich in *Otidea* species. Nine species are added to this genus by this study. A total of 31 species is thus recorded in this huge country currently. Of these species, 27 species are supported by morphological and molecular data, but four species (*O. cochleata* (L.) Fuckel, *O. purpurea* (M. Zang) Korf & W.Y. Zhuang, *O. smithii*, *O. tianshuiensis* J.Z. Cao, L. Fan & B. Liu) still lack DNA evidence. Compared to the records from the continents of Europe (c. 32 accepted species) and North America (c. 14 accepted species), more studies of this large and widely distributed temperate fungal group in China are needed. From the present point of view, the *Otidea* species is widely distributed in the southwest and northern regions of China. Four species, *O. alutacea*, *O. bufonia*, *O. mirablilis*, and *O. onotica*, are widely distributed and are often encountered in the wild. So far, almost no *Otidea* species have been reported from south-central China and east China, which also have abundant forest resources, so it is necessary to investigate fungal resources in these regions in the future.


//www.mdpi.com/article/10.3390/jof8030272/s1, Table S1: Information on sequences used in molecular phylogenetic analyses for Otidea; File S1: Aligned, concatenated dataset of Otidea, consisting of two partitions (ITS, nrLSU).; File S2: Aligned, concatenated dataset of Otidea, consisting of four partitions (ITS, nrLSU, tef-α, rpb2).

**Author Contributions:** L.F. conceived and designed the study; Y.-Y.X. and N.M. wrote the manuscript; Y.-Y.X. conducted phylogenetic analyses and morphological observations; N.M. and J.-J.Y. conducted the experiments. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Natural Science Foundation of China (No. 31750001) and the Beijing Natural Science Foundation (No. 5172003).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The sequencing data were submitted to GenBank.

**Acknowledgments:** J.Z. Cao is appreciated for collecting specimens and providing valuable suggestions. Many thanks to the curator Lei Cai and staff Zhuo Du (HMAS), to the curator Tao Deng and staff Wen-Zhang Ma (HKAS), and to the curator Tolgor Bau (HMJAU) for loan of specimens used in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


**Ning Mao † , Yu-Yan Xu † , Tao-Yu Zhao, Jing-Chong Lv and Li Fan \***

> College of Life Science, Capital Normal University, Xisanhuanbeilu 105, Haidian, Beijing 100048, China; 2210801013@cnu.edu.cn (N.M.); 2190801004@cnu.edu.cn (Y.-Y.X.); 2200802110@cnu.edu.cn (T.-Y.Z.); 2200802057@cnu.edu.cn (J.-C.L.)

**\*** Correspondence: fanli@mail.cnu.edu.cn

† These authors contributed equally to this work.

**Abstract:** Within the family Inocybaceae, many species of *Mallocybe* and *Pseudosperma* have been reported, but there are only a few reports on these two genera from north China. In this study, six collections of *Mallocybe* and 11 collections of *Pseudosperma* were studied by morphological and phylogenetic methods. Phylogenetic analyses based on sequence data from three or two different loci (ITS, LSU, and *rpb2* for *Mallocybe*; ITS and LSU for *Pseudosperma*) are performed to infer species relationships within genera *Mallocybe* and *Pseudosperma*, respectively. Results indicate that eight species of *Mallocybe* and *Pseudosperma* are found in Shanxi province, north China; two new species of *Mallocybe*, *M*. *depressa* and *M*. *picea*, are described. Overall, six species belong to *Pseudosperma*, of which three are new: *P*. *gilvum*, *P*. *laricis* and *P*. *pseudoniveivelatum*.

**Keywords:** inocybaceae; multigene; phylogeny; taxonomy

## **1. Introduction**

Inocybaceae Jülich (Basidiomycota, Agaricales) is an ecologically important fungal family, and is estimated to contain 1050 species [1]. These ectomycorrhizal fungi of Inocybaceae form a mutually symbiotic association with as many as 23 families of vascular plants [2]. Inocybaceae was initially considered by many researchers to include only one or two genera [3–9]. Recently, Matheny et al. [2] revised Inocybaceae to include seven genera based on a six-locus phylogeny, namely *Auritella* Matheny & Bougher, *Inocybe* (Fr.) Fr., *Inosperma* (Kühner) Matheny & Esteve-Rav., *Mallocybe* (Kuyper) Matheny, Vizzini & Esteve-Rav., *Nothocybe* Matheny & K.P.D. Latha, *Pseudosperma* Matheny & Esteve-Rav., and *Tubariomyces* Esteve-Rav. & Matheny.

The genus *Mallocybe* was originally described as a subgenus of *Inocybe*. It is elevated to the genus level by Matheny et al. [2], with *Mallocybe terrigena* (Fr.) Matheny, Vizzini & Esteve-Rav. As the type species. The species of this genus are very widely distributed, reported in Africa, Asia, Australia, Europe, New Zealand, and North America [2]. Approximately 56 species are recorded in Index Fungorum [www.indexfungorum.org/Names/Names.asp (accessed on 5 February 2022)]. This genus is mainly characterized by a coarsely fibrillose or woolly-squamulose and often flattened pileus, which becomes noticeably dark upon the application of 5% potassium hydroxide; adnate lamellae; a short stipe; necropigmented basidia; short cheilocystidia (<50 µm long); and the absence pleurocystidia [2,7,10–14]. The genus *Pseudosperma* was originally included in *Inocybe* section *Rimosae* sensu stricto (= clade *Pseudosperma*) [15–17], and traditionally placed in the subgenus *Inosperma*. Now, it is one of the seven genera in Inocybaceae [2]. There are 93 records listed in Index Fungorum, and approximately 70 species are accepted according to Matheny et al. [2]. The species of this genus are characterized by fibrillose or rarely squamulose, often rimose pileus; furfuraceous to furfuraceous–fibrillose stipe, distinctly pruinose stipe apex; adnexed to sinuate lamellae; hyaline or not necropigmented basidia; cylindrical to clavate cheilocystidia; absent pleurocystidia; and spermatic odor [2,17–19].

**Citation:** Mao, N.; Xu, Y.-Y.; Zhao, T.-Y.; Lv, J.-C.; Fan, L. New Species of *Mallocybe* and *Pseudosperma* from North China. *J. Fungi* **2022**, *8*, 256. https://doi.org/10.3390/jof8030256

Academic Editors: Lei Cai and Cheng Gao

Received: 6 February 2022 Accepted: 1 March 2022 Published: 2 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

During an investigation of Inocybaceae fungi in Shanxi (north China), some fruitbodies of *Mallocybe* and *Pseudosperma* were collected. Subsequent morphological examination and molecular analyses showed they represented eight species, including five undescribed species. The aim of this study is to improve the knowledge of the genus *Pseudosperma* and *Mallocybe* by adding descriptions of five new species, and provide the DNA data of three previously described *Pseudosperma* species from China.

## **2. Materials and Methods**

## *2.1. Morphological Studies*

Collections were obtained and photographed in the field from Shanxi and Hebei province in China, dried in a fruit drier at 40–50 ◦C, and deposited in the herbarium of Capital Normal University, Beijing, China (BJTC) and the Herbarium Institute of Edible Fungi, Shanxi Academy of Agricultural Science, Taiyuan, China (HSA). Standardized color values were obtained from ColorHexa [http://www.colorhexa.com.asp (accessed on 5 February 2022)]. Microscopic characteristics were observed in sections obtained from dry specimens mounted in 3% KOH, Congo Red, or Melzer's reagent [20]. The term '[n/m/p]' means n. basidiospores from m. basidiomata of p collections. Dimensions of basidiospores are given using the following format '(a–)b–c(–d)', where the range 'b–c' represents at least 90% of the measured values, and 'a' and 'd' are the most extreme values. L<sup>m</sup> and W<sup>m</sup> indicate the average basidiospore length and width (± standard deviation) for the measured basidiospore, respectively. 'Q' refers to the length/width ratio of basidiospores in side-view; 'Qav' refers to the average Q of all basidiospores ± standard deviation.

## *2.2. DNA Extraction, PCR Amplification, Sequencing*

A small amount of basidiomata material (20−30 mg) was crushed by shaking for 45 s at 30 Hz 2−4 times (Mixer Mill MM301, Retsch, Haan, Germany) in a 1.5 mL tube, together with a 3 mm diam tungsten carbide ball. Total genomic DNA was extracted from the powdered basidiomata using NuClean Plant Genomic DNA Kit (CWBIO, Beijing, China), following the manufacturer's instructions. Primers ITS1F and ITS4 were employed for the ITS [21,22], while LR0R and LR5 for LSU [23], and bRPB2-6F and bRPB2-7R2 for the *rpb2* were used [16,24]. Polymerase chain reactions (PCR) for the ITS region, LSU region, and *rpb2* gene were performed in 25 µL reaction containing 2 µL DNA template (concentration: 12−20 ng/µL), 1 µL primer (10 µM) each, 12.5 µL of 2 × Master Mix [Tiangen Biotech (Beijing) Co., Beijing, China], 8.5 µL ddH2O.

PCR reactions were implemented as follows: an initial denaturation at 94 ◦C for 5 min, then to 35 cycles of the following denaturation at 94 ◦C for 30 s, annealing at 52 ◦C for 45 s (ITS), 60 s (LSU and *rpb2*), 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. The newly generated sequences were assembled and edited using SeqMan (DNA STAR package; DNAStar Inc., Madison, WI, USA) with generic-level identities for sequences confirmed via BLAST queries of GenBank. These sequences of *Mallocybe* and *Pseudosperma* were mainly selected from those used by previous studies [2,12,14–19,25–32]. The accession numbers of all sequences employed are provided in Supplementary Tables S1 and S2.

## *2.3. Sequence Alignment and Phylogenetic Analyses*

For this study, two datasets were assembled. Dataset I (ITS/LSU/*rpb2*) was used to investigate the phylogenetic placement of the *Mallocybe* species. *Pseudosperma triaciculare* Saba & Khalid and *P*. *breviterincarnatum* (D.E. Stuntz ex Kropp, Matheny & L.J. Hutchison) Matheny & Esteve-Rav. were selected as outgroup taxon. Dataset II (ITS/LSU) was used to investigate the phylogenetic placement of the *Pseudosperma* species. *Mallocybe velutina* Saba & Khalid and *M*. *africana* Aïgnon, Yorou & Ryberg were selected as outgroup taxon. The sequences of each marker were independently aligned in MAFFT v.7.110 [33] under

default parameters, and edited by BioEdit 1.8.1. Maximum Likelihood (ML) and Bayesian Inference (BI) analyses were conducted on the resulting concatenated dataset.

Maximum Likelihood (ML) was performed using RAxML 8.0.14 [34] by running 1000 bootstrap replicates under the GTRGAMMAI model (for all partitions). Bayesian Inference (BI) analyses was performed with MrBayes v3.1.2 [35] based on the best substitution models (GTR + I + G for ITS and LSU; GTR + G for *rpb2*) determined by MrModeltest 2.3 [36]. A total of two independent runs with four Markov chains were conducted for 10 M generations under the default settings. Average standard deviations of split frequency (ASDSF) values were far lower than 0.01 at the end of the runs. Trees were sampled every 100 generations after burn-in (25% of trees were discarded as the burn-in phase of the analyses, set up well after convergence), and a 70% majority-rule consensus tree was constructed.

Trees were visualized with TreeView32 [37]. Bootstrap values (BS) ≥ 70% and Bayesian Posterior Probability values (BPP) ≥ 0.99 were considered significant [38,39].

## **3. Results**

## *3.1. Phylogenetic Analyses*

In this study, 36 sequences of ITS, LSU and *rpb2* were newly generated from our collections. Dataset I (ITS/LSU/*rpb2*) contained 122 sequences from 39 species, including 15 novel sequences of all three genes from our collections. *P*. *triaciculare* and *P*. *breviterincarnatum* were selected as the outgroup. The length of the aligned dataset was 2175 bp after exclusion of poorly aligned sites, with 620 bp for ITS, 884 bp for LSU, and 671 bp for *rpb2*. The topologies of ML and BI phylogenetic trees obtained in this dataset were practically the same, therefore only the tree inferred from the ML analyses is shown (Figure 1). The *Mallocybe* species formed a monophyletic lineage with strong support (MLB = 93%, BPP = 1.00). The sequences of our six collections formed two independent clades, which were respectively recognized and described as two new species: *Mallocybe depressa* and *Mallocybe picea*. *M*. *depressa* was sister to *M*. *velutina* Saba & Khalid with high supports, implying that they are closely related to each other. Another species *M*. *picea* was sister to *M*. *arthrocystis* (Kühner) Matheny & Esteve-Rav., with strong support (MLB = 96%, BPP = 1.00), and then grouped with *M*. *multispora* (Murrill) Matheny & Esteve-Rav. and *M*. *unicolor* (Peck) Matheny & Esteve-Rav. without supported data.

Dataset II (ITS/LSU) contained 1409 total characters (539 from ITS, 870 from LSU, gaps included) and included of 123 samples of 65 taxa. Since the topologies of ML phylogenetic trees is similar to that of the BI phylogenetic tree, only the tree inferred from the ML analyses is shown (Figure 2). A total of 21 sequences newly generated from our collections were resolved as six strong support clades, which indicated that they were six distinct species. Of them, the sequences of five collections clustered well with the authentic sequence of *Pseudosperma bulbosissimum* (Kühner) Matheny & Esteve-Rav., *P*. *rimosum* (Bull.) Matheny & Esteve-Rav., and *P*. *solare* Bandini, B. Oertel & U. Eberh., showing their identities with these three species, respectively. The remaining sequences of our collections formed three independent clades, which was recognized and described as three new species *Pseudosperma gilvum*, *Pseudosperma laricis,* and *Pseudosperma pseudoniveivelatum*. *P*. *gilvum* was sister to *P*. *citrinostipes* Y.G. Fan & W.J. Yu. *P*. *laricis* was closely grouped with *P*. *huginii* Bandini & U. Eberh and *P*. *arenicola* (R. Heim) Matheny & Esteve-Rav. with lower supports. *P*. *pseudoniveivelatum* was sister to *P*. *notodryinum* (Singer, I.J.A. Aguiar & Ivory) Matheny & Esteve-Rav.

**Figure 1.** Phylogeny derived from Maximum Likelihood analysis of the combined (ITS/LSU/*rpb2*) dataset of *Mallocybe* and related genera in the family Inocybaceae. *Pseudosperma triaciculare* and *P*. *breviterincarnatum* were employed to root the tree as an outgroup. Numbers representing likelihood bootstrap support (BS ≥ 70%, left) and significant Bayesian posterior probability (BPP ≥ 0.99, right) are indicated above the nodes. New sequences are highlighted in black bold.

**Figure 2.** *Cont*.

**Figure 2.** Phylogeny derived from Maximum Likelihood analysis of the ITS/LSU sequences from *Pseudosperma* and related genera in the family Inocybaceae. *Mallocybe velutina* and *M*. *africana* were employed to root the tree as an outgroup. Numbers representing likelihood bootstrap support (BS ≥ 70%, left) and significant Bayesian posterior probability (BPP ≥ 0.99, right) are indicated above the nodes. New sequences are highlighted in black bold. The red symbol represents the type species.

## *3.2. Taxonomy*

*Mallocybe depressa* L. Fan, H. Zhou & N. Mao, sp. Nov. (Figures 3C and 4)

**Figure 3.** Basidiomata *of Mallocybe* and *Pseudosperma*. (**A**,**B**). *Mallocybe picea* (BJTC FM555, holotype), (**C**) *Mallocybe depressa* (BJTC C643), (**D**) *Pseudosperma laricis* (BJTC FM887, holotype), (**E**–**G**) *Pseudosperma pseudoniveivelatum* (BJTC FM1660, holotype), (**H**–**J**). *Pseudosperma gilvum* (BJTC FM1941, holotype). Scale bars: (**A**–**J**) = 10 mm.

**Figure 4.** *Mallocybe depressa* (BJTC FM1695). (**A**) Basidiospores, (**B**,**C**) Cheilocystidia, (**D**) Basidia, (**E**) Caulocystidia, (**F**) Pileipellis. Scale bars: (**A**–**F**) = 10 µm.

## MycoBank: MB843127

Diagnosis: *Mallocybe depressa* is characterized by its golden yellow to yellowish-brown pileus, central depression of pileus when old, pileus margin splitting when mature, amygdaloid, and subamygdaloid to subcylindrical basidiospores, clavate to broadly clavate, and septate cheilocystidia, and usually grow in coniferous forest dominated by *Pinus* sp. It is most similar to *M*. *velutina,* but differs by its narrower basidiospores and broadly clavate cheilocystidia.

Etymology: *depressa*, refers to the depression in the center of the pileus with age.

Holotype: China. Shanxi Province, Taiyuan City, Xishan Forest Park, 37◦82.390 N, 112◦46.990 E, alt. 1100 m, 22 July 2021, on the ground in coniferous forest dominated by *Pinus* sp., J.Z. Cao CF1014 (BJTC FM1695).

Description—Pileus 10–35 mm wide, convex to plano-convex at young age, then applanates to uplifted, with a shallow depression at the center; margin initially decurved, becoming flattened and splitting with age; surface dry, strongly fibrous towards the margin, squamulose at the center, dark brown (#4e3000) around the disc, golden yellow (#ff9f00) to yellowish-brown (#cd7f00) elsewhere. Lamellae regular, adnate, brown (#915b25) to dark brown (#68421b) when mature, 1–2 tiers of lamellulae and concolorous with lamellae. Stipe 20–32 × 2.5–5 mm, central, equal with a slightly swollen apex and base, longitudinally fibrillose downwards the stipe, yellowish-brown (#cd7f00) to orange-brown (#9a4d00). Context pale yellow brown. Odor unrecorded.

Basidiospores [60/2/2] (7–)7.5–9(–11) × 4–5 µm, L<sup>m</sup> × W<sup>m</sup> = 8.33 (± 0.72) × 4.65 (± 0.34), Q = (1.4–)1.6–1.9(–2.2) (Qav = 1.79 ± 0.16), smooth, amygdaloid, subamygdaloid to subcylindrical, sometimes ellipsoid, thick-walled, yellowish-brown. Basidia with yellowish necropigment, 20–30 × 6–8 µm, clavate, four-spored, occasionally two-spored. Cheilocystidia 15–36 × 9–12(–16) µm, often in clusters, septate, clavate to broadly clavate, occasionally balloon-shaped, apices rounded to obtuse, hyaline, thin-walled. Pleurocystidia absent. Caulocystidia only near the apex, 20–58 × 6–15 µm, clavate to elongate clavate, hyaline or pale yellow. Pileipellis a cutis, composed of parallel arranged of yellowish-brown to brown, cylindrical hyphae, often septate, 3–14 µm wide, thin-walled. Stipitipellis a cutis, composed of parallel, compactly arranged, hyaline, cylindrical hyphae, 4–12 µm wide, thin-walled. Clamp connections abundant in all tissues.

Habitat: In groups on the ground in coniferous forest dominated by *Pinus* sp., Hebei province and Shanxi province, China.

Additional specimens examined: China. Shanxi Province, Taiyuan City, Jinci Park, 38◦57.18' N, 113◦30.52' E, alt. 1370 m, 20 August 2020, on the ground in coniferous forest dominated by *Pinus* sp., H. Liu LH1266A (BJTC FM1300). Hebei Province, Chicheng county, Yanshan Mountains, 38◦57.18' N, 113◦30.52' E, alt. 947 m, 26 August 2020, on the ground in coniferous forest dominated by *Pinus* sp., H. Zhou 130732MFBPC643 (BJTC C643).

Notes: *Mallocybe velutina* is sister to *M*. *depressa* in our phylogenetic analyses (Figure 1). Morphologically, *M*. *velutina* differs from *M*. *depressa* by its pileus center fulvous, margin light yellow, larger, and broader basidiospores (9.0 × 5.4 µm on average) [13]. Molecular analyses reveal that *M*. *velutina* shares less than 96.04% similarity in ITS sequence with *M*. *depressa*, supporting their separation. *Inocybe caesariata* (Fr.) P. Karst. is recorded in Shanxi province, China [40]. It is similar to the new species by its pileus color and size. However, it can be differentiated from *M*. *depressa* by its pileus not splitting, white lamellae edge, and ellipsoid basidiospores. Another collected species *M*. *picea* is distinguished from *M*. *depressa* based on its larger basidiospores (10.44 × 5.69 µm on average) and broadly clavate to balloon-shaped cheilocystidia.

*Mallocybe picea* L. Fan & N. Mao, sp. nov. (Figure 3A,B and Figure 5)

MycoBank: MB843129

Diagnosis: *Mallocybe picea* is characterized by its flattened and splitting pileus margin when mature, broadly clavate to balloon-shaped cheilocystidia, and usually grows in coniferous forest dominated by *Picea asperata*. It is most similar to *M*. *arthrocystis* but differs by its slightly broader basidiospores and often splitting pileus margin.

Etymology: *picea*, refers to the habitat of the species amongst forest of Picea.

Holotype: China. Shanxi Province, Wutai County, Wutai Mountain, 38◦57.130 N, 113◦29.580 E, alt. 2038 m, 25 July 2019, on the ground in coniferous forest dominated by *Picea asperata* Mast., L.J. Guo GLJM004 (BJTC FM555).

**Figure 5.** *Mallocybe picea* (BJTC FM555) (**A**) Basidiospores, (**B**) Basidia, (**C**) Caulocystidia, (**D**) Cheilocystidia, (**E**) Pileipellis. Scale bars: (**A**–**E**) = 10 µm.

Description—Pileus 20–55 mm wide, hemispherical to broadly convex at young age, becoming plano-convex to applanate with age, with distinctly umbo; margin turned down when young, then flattened and splitting; surface dry, uniformly grayish velutinous when young, fibrillose to tomentose, earthy yellow (#e1a95f), yellowish-brown (#cd8526), sometimes dark brown (#764d16) towards the margin when mature. Lamellae regular, adnate, subdistant, yellowish-brown (#cc8526) to dark brown (#764d16), 2–3 tiers of lamellulae and concolorous with lamellae. Stipe 21–45 × 4–8 mm, hollow, central, equal, or sometimes slight widening at base, longitudinally fibrillose downwards the stipe, with white tomentose hyphae at the base, earthy yellow (#e1a95f) to yellowish-brown (#cd8526), paler white (#f5f5f5) at base. Context yellowish white. Odor unrecorded.

Basidiospores [100/2/3] (9–)9.5–11.5(–12) × (5–)5.2–6(–6.5) µm, L<sup>m</sup> × W<sup>m</sup> = 10.44 (±0.69) × 5.69 (±0.35), Q = 1.5–2.2 (Qav = 1.84 ± 0.16), smooth, subamygdaloid to subcylindrical, cylindrical, thick-walled, yellowish-brown. Basidia with yellowish necropigment, (24–)28–39 × 8–10 µm, cylindrical to clavate, four-spored, rarely two-spored; sterigmata 2–5 µm long. Cheilocystidia 18–35 × 9–16 µm, often in clusters, septate, broadly clavate to balloon-shaped, apices rounded to subcapitate, hyaline, thin-walled. Pleurocystidia absent. Caulocystidia only near the apex, 22–50 × 7–10 µm, clavate to cylindric, hyaline or pale yellow. Pileipellis a cutis, composed of dense layers of repent hyphae; hyphae cylindrical, often septate, 6–15 µm wide and with yellowish-brown to brown intracellular or parietal pigment, thin-walled. Stipitipellis a cutis, made up of parallel, compactly arranged, thin-walled, cylindrical hyphae, 3.5–12 µm wide, hyaline or pale brown in KOH. Clamp connections abundant in all tissues.

Habitat: In groups on the ground in coniferous forest dominated by *Picea asperata*, Shanxi province, China.

Additional specimens examined: China. Shanxi Province, Wutai County, Wutai Mountain, 38◦57.520 N, 113◦31.90 E, alt. 1910 m, 25 July 2019, on the ground in coniferous forest dominated by *Picea asperata*, H. Liu LH636 (BJTC FM569). Ibid, 38◦57.180 N, 113◦30.520 E, alt. 2013 m, 27 August 2019, on the ground in coniferous forest dominated by *P*. *asperata*, Y. Shen SYM078 (BJTC FM896).

Notes: *Mallocybe picea* and *M*. *arthrocystis* are not only closely related phylogenetically, but also morphologically very similar. *Mallocybe arthrocystis* is originally reported from France and distinguished from *M*. *picea* by its pileus margin not splitting, slightly narrower

basidiospores of 9.5–1.2 × 4.5–5.5 µm [12]. Molecular analyses also revealed that *M*. *arthrocystis* shares less than 90.23% similarity in ITS sequence with *M. picea*, supporting their separation. The species *Inocybe dulcamara* (Pers.) P. Kumm. is easily confused with *M*. *picea* in morphology, which is reported from China, but classified into *Mallocybe* by Fan and Tolgor [41]. *Inocybe dulcamara* differs from *M*. *picea* by its longer basidia 30–60 × 8–12 µm and white context in cap [12].

*Pseudosperma gilvum* L. Fan & N. Mao, sp. nov. (Figure 3H,J and Figure 6)

**Figure 6.** *Pseudosperma gilvum* (BJTC FM1941) (**A**) Basidiospores, (**B**) Basidia, (**C**) Cheilocystidia, (**D**,**E**). Caulocystidia, (**F**) Pileipellis. Scale bars: (**A**–**F**) = 10 µm.

## MycoBank: MB843130

Diagnosis: *Pseudosperma gilvum* is characterized by convex to broadly convex pileus with subacute or obtuse umbo, mostly subphaseoliform, subcylindrical to cylindrical basidiospores, cylindrical, clavate to broadly clavate cheilocystidia. It is most similar to *P*. *triaciculare* but differs by its narrower basidiospores and paler pileus color.

Etymology: *gilvum*, Latin indicating light yellow, refers to the color of the pileus and stipe.

Holotype: China. Shanxi Province, Wenshui County, Lvliang Mountains, 37◦28.280 N, 111◦34.220 E, alt. 1750 m, 30 July 2021, on the ground in coniferous and broad-leaved mixed forest dominated by *Pinus* sp., L. FAN CF1115 (BJTC FM1941).

Description—Pileus 30–45 mm wide, convex to broadly convex with subacute or obtuse umbo; margin decurved or straight, not splitting; surface dry, fibrillose-rimulose, presence of a pale velipellis coating over the disc, light yellow (#ffffd4), becoming yellowishbrown (#ffc000) in some places with age, background pallid to cream white. Lamellae regular, adnate to sinuate, pale white (#f2f2f2) to grayish white (#e6e6e6) when young, becoming yellowish-brown (#ffb31a) with age, 1–2 tiers of lamellulae and concolorous with lamellae. Stipe 47–104 × 4–6 mm, solid, central, nearly terete, base slightly swollen, covered with whitish tomentum at young age, longitudinally fibrillose, pale yellow (#ffffd4) to yellowish brown (#ffab00), pale white at apex and base. Context white. Odor unrecorded.

Basidiospores [70/2/2] 10.5–12.5(–14) × (5.5–)6–7 µm, L<sup>m</sup> × W<sup>m</sup> = 11.40 (± 0.80) × 6.34 (±0.39), Q = 1.7–2.0 (Qav = 1.80 ± 0.12), smooth, mostly subphaseoliform, subcylindrical to cylindrical, occasionally ellipsoid, slightly thick-walled, yellowish-brown to reddish-brown. Basidia 30–40 × 9–11 µm, clavate to broadly clavate, occasionally rounded-swollen at apex, primarily with four spored, rarely two spored, often with oily inclusions, hyaline in KOH. Cheilocystidia 25–75 × 9–14 µm, often in clusters, septate, cylindrical, clavate to broadly clavate, sometimes ovoid or subfusiform, often catenate with much shorter elements below the terminal element, hyaline to pale brown, thin-walled. Pleurocystidia absent. Caulocystidia only near the apex, 20–85 × 8–12 µm, clavate to cylindric, similar to cheilocystidia, hyaline or pale yellow. Pileipellis a cutis, composed of parallel, compactly arranged, thin-walled, hyaline or yellowish-brown, cylindrical hyphae, 4–12.5 µm wide. Stipitipellis a cutis, composed of compactly hyphae, 4–10 µm wide, hyaline or pale brown in KOH. Clamp connections abundant in all tissues.

Habitat: Scattered or in groups on the ground in mixed coniferous and broad-leaved forest dominated by *Pinus* sp., Shanxi Province, China.

Additional specimens examined: China. Shanxi Province, Pu County, Wulu Mountain, 36◦33.340 N, 113◦30.520 E, alt. 1910 m, 28 July 2021, on the ground in coniferous and broad-leaved mixed forest dominated by *Pinus* sp., N. Mao MNM275 (BJTC FM1875).

Notes: *Pseudosperma gilvum* is clustered with *P*. *citrinostipes* and *P*. *triaciculare* Saba & Khalid, and forms a distinct monophyletic group. This indicates that the three species are phylogenetically closely related to each other. However, there are clear differences in morphology among them. *Pseudosperma citrinostipes* has brownish yellow or straw yellow to golden yellow pileus, mostly ellipsoid spores, subfusiform, utriform to lageniform cheilocystidia and a different phylogenetic position (Figure 2) [19], that separates it well from our new species. *Pseudosperma triaciculare* can be distinguished by its darker pileus (brownish-orange to fulvous) with radially rimose margin, broader basidiospores (9.0 × 5.4 µm on average) and slightly smaller cheilocystidia (23–54 × 9–16 µm) [17]. Molecular analyses also reveal that *P. gilvum* shares less than 89.20% similarity in ITS sequence with *P*. *triaciculare*, supporting their separation. Both *P*. *brunneoumbonatum* Saba & Khalid and *P. gilvum* are presumed to be associated with *Pinus,* and have similar pileus shape. However, *P*. *brunneoumbonatum* differs from *P. gilvum* by its strongly rimose pileus margin and lager basidiospores (12.5 × 7.5 µm on average) [17]. We also collected some specimens of *P*. *bulbosissimum* (Kühner) Matheny & Esteve-Rav from Shanxi province, China. Its pileus is pale yellow, then ochraceous to reddish brown, and covered with fibrillose-rimose, similar to that of *P. gilvum*. The basidiospores of *P*. *bulbosissimum*, however, are remarkably larger (12–15 × 6–8 µm) [15].

*Pseudosperma laricis* L. Fan & N. Mao, sp. nov. (Figures 3D and 7)

**Figure 7.** *Pseudosperma laricis* (BJTC FM887) (**A**) Basidiospores, (**B**) Basidia, (**C**) Caulocystidia, (**D**) Cheilocystidia, (**E**) Pileipellis. Scale bars: (**A**–**E**) = 10 µm.

## MycoBank: MB843131

Diagnosis: *Pseudosperma laricis* is characterized by the pileus surface with fibrillose and strongly rimose, subcylindrical to cylindrical basidiospores and an ecological association with *Larix principis-rupprechtii*. It is most similar to *P*. *rimosum,* but differs by its narrower basidiospores and orange brown or brown pileus.

Etymology: *laricis*, refers to the habitat of the species amongst forest of *Larix*.

Holotype: China. Shanxi Province, Wutai County, Wutai Mountains, 38◦57.70 N, 113◦30.160 E, alt. 2075 m, 27 August 2019, on the ground in coniferous forest dominated by *Larix principis-rupprechtii* Mayr, Y. Shen SYM069 (BJTC FM887).

Description—Pileus 20–35 mm wide, convex, broadly convex or plane with subacute or obtuse umbo; margin at first incurved, then straight to somewhat wavy, not splitting; surface dry, smooth at the umbo, fibrillose and strongly rimose cracked towards center, yellowish-orange (#e6a800) to orange brown (#cc8400) or brown (#d18e4a), background pale white. Lamellae regular, adnate, grayish white (#e6e6e6) when young, becoming yellowishbrown (#ffb31a) to brown (#d18e4a) with age, 1–3 tiers of lamellulae and concolorous with lamellae. Stipe 51–65 × 4–6 mm, hollow, central, equal, longitudinally fibrillose, yellowish brown (#ffdf80) in different intensity, grayish white (#e6e6e6) at apex of the stipe. Context white. Odor unrecorded.

Basidiospores [85/2/2] (10–)11–14(–17) × (5–)5.5–7(–7.5) µm, L<sup>m</sup> × W<sup>m</sup> = 12.77 (± 1.38) × 6.16 (± 0.55), Q = 1.8–2.4 (Qav = 2.07 ± 0.12), smooth, subcylindrical to cylindrical, slightly thick-walled, yellowish-brown to reddish-brown. Basidia 30–40 × 10–12 µm, clavate to broadly clavate, rounded-swollen at apex, generally with four spored, rarely two spored, often with oily inclusions, hyaline in KOH. Cheilocystidia 25–57 × 9–20 µm, often in clusters, mostly cylindrical, clavate to broadly clavate, rarely ovoid or fusiform, hyaline to pale brown, thin-walled. Pleurocystidia absent. Caulocystidia only near the apex, 17–47 × 8–11 µm, clavate or cylindrical, similar to cheilocystidia, often catenate with much shorter elements below the terminal element, hyaline or pale yellow. Pileipellis a cutis, composed of parallel, compactly arranged, thin-walled, yellowish-brown, cylindrical hyphae, with 4–13 µm wide. Stipitipellis a cutis, composed of parallel, compactly hyphae, 3–9 µm wide, hyaline or pale brown in KOH. Clamp connections abundant in all tissues.

Habitat: Scattered or in groups on the ground in coniferous forest dominated by *L*. *principis-rupprechtii*, Shanxi province, China.

Additional specimens examined: China. Shanxi Province, Wutai County, Wutai Mountains, 38◦58.250 N, 113◦31.130 E, alt. 1900 m, 27 August 2019, on the ground in coniferous forest dominated by *L*. *principis-rupprechtii*, H. Liu LH842 (BJTC FM924).

Notes: *Pseudosperma laricis* is clustered with *P*. *arenicola* (R. Heim) Matheny & Esteve-Rav. and *P*. *mimicum* (Massee) Matheny & Esteve-Rav. However, *P*. *arenicola* is distinguished by its stipe solid, often deeply buried in sand, and a broad host range, including species in *Salicaceae* and *Pinaceae*, and *P*. *mimicum* by its larger pileus (>65 mm) and ellipsoid basidiospores [15]. *Pseudosperma rimosum* (Bull.) Matheny & Esteve-Rav. is similar to *P*. *laricis,* and we also collected the fruit-bodies of *P. rimosum* from Shanxi Province, north China. It differs from the new species by its ellipsoid basidiospores (9.5–12.5 × 6–7 µm) [15]. Another species, *P*. *gilvum*, is easily distinguished from *P. laricis* by its paler color pileus and smaller basidiospores (11.4 × 6.34 µm on average).

*Pseudosperma pseudoniveivelatum* L. Fan & N. Mao, sp. nov. (Figure 3E–G and Figure 8)

MycoBank: MB843132

Diagnosis: *Pseudosperma pseudoniveivelatum* is characterized by pileus surface with a distinct pale grayish to pale white velipellis, pileus margin splitting with age, ellipsoid basidiospores, broadly clavate to papillate or utriform cheilocystidia. It is most similar to *P*. *niveivelatum,* but differs by its smaller basidiospores and darker (yellowish-brown to brown) pileus.

Etymology: *pseudoniveivelatum*, refers to this species is similar to *P*. *niveivelatum*.

Holotype: China. Shanxi Province, Qinshui County, Lishan Mountains, 35◦29.480 N, 112◦4.120 E, alt. 1690 m, 7 July 2021, on the ground in coniferous and broad-leaved mixed forest dominated by *Quercus* sp., N. Mao MNM232 (BJTC FM1660).

**Figure 8.** *Pseudosperma pseudoniveivelatum* (BJTC FM1660) (**A**) Basidiospores, (**B**) Cheilocystidia, (**C**) Basidia, (**D**) Caulocystidia, (**E**) Pileipellis. Scale bars: (**A**–**E**) = 10 µm.

Description—Pileus 20–45 mm wide, conical to conical-convex at first, then broadly convex to plane-convex with obtuse umbo; margin decurved or straight, becoming splitting with age; surface dry, with a distinct pale greyish to pale white velipellis, indistinctly fibrillose-rimulose, yellowish-brown (#cd9900) to brown (#8b4513), sometimes dark brown (#5e2f0d) at the center, background cream white. Lamellae regular, crowded, adnate, pale white (#ffffff) or yellowish white (#ffffe7) when young, later yellowish-brown (#ffbf00) to ochraceous (#a5682a), 1–2 tiers of lamellulae and concolorous with lamellae. Stipe 33–75 × 4–9 mm, solid, central, cylindrical, equal, or base slightly swollen, covered with whitish tomentum for a long time, later longitudinally fibrillose, pale orange (#ffae1a) to pale brownish (#997654). Context white. Odor unrecorded.

Basidiospores [70/2/2] (8.5–)9.5–11(–12) × (5–)5.5–7(–7.5) µm, L<sup>m</sup> × W<sup>m</sup> = 10.19 (± 0.77) × 6.22 (± 0.55), Q = 1.4–1.8 (Qav = 1.63 ± 0.10), smooth, mostly ellipsoid, occasionally broadly ellipsoid or subcylindrical, slightly thick-walled, yellowish-brown to reddishbrown. Basidia 23–33 × 9–12.5 µm, clavate to broadly clavate, often rounded-swollen at apex, primarily four-spored, occasionally two-spored, usually with oily inclusions, hyaline in KOH. Cheilocystidia 30–65 × 7–16 µm, often in clusters, mostly clavate, broadly clavate to papillate or utriform, sometimes cylindrical, hyaline, thin-walled. Pleurocystidia absent. Caulocystidia only near the apex, 32–95 × 9–25 µm, in clusters, broadly clavate or utriform, at times with apices tapered or papillate, similar to cheilocystidia but larger, hyaline or pale yellow. Pileipellis a cutis, composed of parallel, compactly arranged, thin-walled, hyaline or yellowish-brown, cylindrical hyphae, 4–15 µm wide, with some encrustations, septate. Stipitipellis a cutis, composed of compactly hyphae, 5–14 µm wide, hyaline or pale brown in KOH. Clamp connections abundant in all tissues.

Habitat: Scattered or in groups on the ground in mixed coniferous and broad-leaved forests dominated by *Quercus* sp., north China, south China, and Europe.

Additional specimens examined: China. Shanxi Province, Qinshui County, Lishan Mountains, 35◦29.140 N, 112◦1.200 E, alt. 1660 m, 7 July 2021, on the ground in coniferous and broad-leaved mixed forest dominated by *Quercus* sp., N. Mao MNM224 (BJTC FM1656).

Notes: *Pseudosperma niveivelatum* is easily confused with *P*. *pseudoniveivelatum* in morphology, due to the presence of a white, abundant velipellis in *P*. *niveivelatum* that covers the pileus. However, this species has pale brown or yellow hues pileus, larger basidiospores (13.9 × 6.4 µm on average) and a different phylogenetic position (Figure 2) that separates it well from our new species [29]. *Pseudosperma notodryinum* is sister to *P*. *pseudoniveivelatum* in our phylogenetic analyses (Figure 2), implying that they have a close relationship. However, there are clear differences in the morphology. *P*. *notodryinum* can be distinguished by its darker pileus (yellow-ocher to rich yellowish-fuscous) and narrower basidiospores (9–12 × 5–6 µm) [42]. Molecular analyses revealed that *Pseudosperma notodryinum* shares less than 91.88% similarity in ITS sequence with *P. pseudoniveivelatum*, supporting their separation. A total of three species reported in China in previous studies, *P*. *obsoletum* (Quadr.) Valade, *P*. *perlatum* (Cooke) Matheny & Esteve-Rav. and *P*. *yunnanense* (T. Bau & Y.G. Fan) Matheny & Esteve-Rav., are all easily confused with *P. pseudoniveivelatum* in morphology [41,43]. However, *P*. *obsoletum* differs from *P. pseudoniveivelatum* by its gray brown to pinkish gray pileus, the absence of velipellis and narrower basidiospores (9–13 × 5–6 µm) [29]; it differs from *P*. *perlatum* by its larger basidiomata (pileus 35–100 mm, stipe 80–120 × 8–13 mm), and pileus color without yellow tinges [15]; and *P*. *yunnanens* by its fibrillose with densely squamules stipe and slightly smaller and narrower basidiospores (9–10.5 × 5–6 µm) [43]. Moreover, eleven ITS sequences respectively labelled '*P*. *obsoletum*', '*Inocybe obsoleta*' and '*P*. aff. *perlatum*' are conspecific to the new species *P. pseudoniveivelatum* since they clustered together with *P*. *pseudoniveivelatum* in ITS tree (not shown), and have more than 98.66% similarity in ITS region. Of them, two (MT072905, MG367271) are respectively from Inner Mongolia in northern China, Hainan Province in southern China, and nine from Europe (UDB035861, UDB015340, MW355002, MG367270, HG937630, JF908256, MZ410669, JX625280, JQ994477). These show that the new species *P. pseudoniveivelatum* is distributed in both northern and southern China and in Europe.

## **4. Discussion**

Shanxi Province is located in north China, where the climate ranges from subtropical to cold temperate. Our analyses revealed two species of *Mallocybe* and six species of *Pseudosperma* in this region, i.e., *M*. *depressa*, *M*. *picea*, *P. bulbosissimum*, *P*. *gilvum*, *P*. *laricis*, *P*. *pseudoniveivelatum*, *P*. *rimosum* and *P. solare*. They all are associated with coniferous forests. *Pseudosperma bulbosissimum* is the most commonly encountered species, which is distributed in the central and northern regions in Shanxi Province. *Pseudosperma gilvum* is found in both central and southern regions. The remaining species probably have distribution limitations: *P*. *pseudoniveivelatum*, *P*. *rimosum,* and *P*. *solare* are distributed in the southern region, *M*. *depressa* are distributed in the central region, and *M*. *picea* and *P*. *laricis* are distributed in the northern region.

In China, the species diversity of the two genera of *Mallocybe* and *Pseudosperma* are scarce. A total of five species are reported in *Mallocybe* and 13 species in *Pseudosperma* [19,40–46]. With the exception of *P*. *citrinostipes*, *P*. *neoumbrinellum* (T. Bau & Y.G. Fan) Matheny & Esteve-Rav. and *P*. *yunnanense*, and the new species described in this study, the remaining seven species all need to be reexamined and verified with molecular data. They are *M*. *heimii* (Bon) Matheny & Esteve-Rav. [= *Inocybe heimii* Bon], *M*. *leucoloma* (Kühner) Matheny & Esteve-Rav. [= *Inocybe leucoloma* Kühner], *M*. *terrigena* (Fr.) Matheny, Vizzini & Esteve-Rav. [= *Inocybe terrigena* (Fr.) Kühner], *P*. *avellaneum* (Kobayasi) Matheny & Esteve-Rav. [= *I*. *avellanea* Kobayasi], *P*. *obsoletum* [= *I*. *obsoleta* Romagn.], *P*. *perlatum* [= *I*. *perlata* (Cooke) Sacc.], and *P*. *sororium* (Kauffman) Matheny & Esteve-Rav. [= *I*. *sororia* Kauffman] [19,40–46].


**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8030256/s1, Table S1: Information on sequences used in molecular phylogenetic analyses for *Mallocybe*; Table S2: Information on sequences used in molecular phylogenetic analyses for *Pseudosperma*.

**Author Contributions:** L.F. conceived and designed the study; N.M. and Y.-Y.X. wrote the manuscript; N.M. conducted phylogenetic analyses and morphological observations; Y.-Y.X., T.-Y.Z. and J.-C.L. conducted the experiments. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Natural Science Foundation of China (No. 31750001) and the Beijing Natural Science Foundation (No. 5172003).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.

**Acknowledgments:** J.Z. Cao was appreciated for collecting specimens and providing valuable suggestions.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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