New Species of Mallocybe and Pseudosperma from North China

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.

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.

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 m and W m 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; 'Q av ' refers to the average Q of all basidiospores ± standard deviation.

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. 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.

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 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       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.
Habitat: In groups on the ground in coniferous forest dominated by Picea asperata, Shanxi province, China.
Additional 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]. 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.
Habitat: Scattered or in groups on the ground in mixed coniferous and broad-leaved forest dominated by Pinus sp., Shanxi Province, China.
Additional 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]. 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.
Habitat: Scattered or in groups on the ground in coniferous forest dominated by L. principis-rupprechtii, Shanxi province, China.
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.
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.