﻿Introduction of two novel species of Hymenopellis (Agaricales, Physalacriaceae) from Thailand

﻿Abstract Hymenopellis is the most diverse genus in the group of oudemansielloid/xeruloid taxa (Physalacriaceae). This genus has a worldwide distribution with records mostly from Europe and America. Asian taxa are least represented. In this paper on Hymenopellis from Thailand, two novel species are introduced, and a Hymenopellis collection affine to H.orientalis is described. Macro and micromorphological characters are described. Maximum likelihood and Bayesian phylogenetic analyses were performed on combined ITS and nrLSU regions to confirm taxonomical placement and infer the phylogenetic affinities of the studied species. Hymenopellisstraminea sp. nov. is straw-yellow, with medium-sized basidiomata, abundant and diverse in form cheilocystidia, few, narrowly lageniform to fusiform pleurocystidia, and clamp connections at the lower part of the stipe. Hymenopellisutriformis sp. nov. has mostly utriform pleurocystidia and 2-spored basidia. In the inferred phylogenies, the new species from this study formed distinct clades well supported by bootstrap proportions and posterior probabilities. The studied specimen affine to H.orientalis produced 2-spored basidia whereas published descriptions of other specimens mention 4-spored basidia. Moreover, the genetic distance between ITS sequences of this specimen and that of a Hymenopellisorientalis specimen from GenBank was 1.30–2.57%. Therefore, the conspecificity of our specimen with H.orientalis is uncertain, and additional specimens are needed to fully confirm its identity.

Introduction of two novel species of Hymenopellis (Agaricales, Physalacriaceae) from Thailand Introduction Hymenopellis R.H. Petersen, one of the genera in the Physalacriaceae Corner, was circumscribed by Petersen and Hughes (2010) as a new genus, covering those species with moist to glutinous pileus. It is the largest genus in the oudemansielloid/xeruloid complex and has a worldwide distribution. Hymenopellis species were previously classified in the section Radicatae of Oudemansiella (Clémençon 1979;Pegler and Young 1986;Yang et al. 2009). The presence of a pseudorrhiza separated O. sect. Radicatae from sect. Hygrophoroides (Clémençon 1979). The type species, H. radicata was first described by a British botanist, Richard Relhan, in 1780, under the name Agaricus radicatus, which is also synonymous with Oudemansiella pseudoradicata M.M. Moser, Oudemansiella radicata (Relhan: Fr.) Singer and Xerula radicata (Relhan: Fr.) Dörfelt. There are around 50 species of Hymenopellis (He et al. 2019) of which 13 were first described from Asia (Petersen and Hughes 2010).
Hymenopellis is widely distributed in tropical and temperate regions (He et al. 2019). The majority of the literature on this genus has focused on Europe and the United States, where its taxonomy and distribution have been extensively researched. The most thorough study on Hymenopellis was done by Petersen and Hughes (2010), in which descriptions of all known species were provided. Out of the 50 described species, only 19 have sequences available in GenBank. The majority of sequences found in GenBank are from specimens collected in the eastern United States. Asian taxa are least represented (Petersen and Hughes 2010) with limited studies in this genus from Asian countries. Thirteen species of Hymenopellis have been recorded from Asia, of which 12 were first described from Asian countries. Six species were first described from temperate regions in China and Japan while another six species were described from tropical countries, namely H. altissima (Massee) R.H. Petersen (Petersen and Hughes 2010). In Thailand, only two species have been recorded, namely H. raphanipes (Petersen and Nagasawa 2006;Yang et al. 2009), and H. radicata (as X. radicata) (Chandrasrikul et al. 2011). However, the H. radicata recorded in Thailand has no associated sequence available. Thailand has a forested area of around 16.3 million ha (FAO 2020), a thriving habitat for diverse macrofungal species (Hyde et al. 2018). Many macrofungal species have been discovered in this country and many more remain to be introduced to science. Additional collections and further studies are necessary to improve our knowledge of Asian Hymenopellis taxonomy.
In this study, two new tropical species of Hymenopellis are introduced and a Hymenopellis specimen affine to H. orientalis is described from Thailand, adding to the limited number of Asian taxa.

Sample collection and morphological observations
The specimens were collected from Chiang Rai and Chiang Mai provinces, Thailand during rainy season in June and August 2019. Photographs of the fresh samples were taken on the field, and information about habitat, habit, and other important features (e.g., color of the basidiomata, gills and stipe) of the specimen were noted. The basidiomata were carefully collected and kept in aluminum foil, labeled, and brought to the laboratory. Once in the laboratory, each specimen was photographed, measured, and described. Spore prints were collected on both black and white paper. Specimens were dried using a hot air dryer set to 45-50 °C for 24 hours. They were carefully labelled and stored in zip-lock bags to be used for further analyses. All samples were deposited in the Mae Fah Luang University fungarium (MFLU).
Macromorphological characters of the specimens (i.e., pileus, lamellae, and stipe) were described based on the fresh basidiomata. Naming of original colors was based on Methuen Handbook of Color, 3 rd ed. (Kornerup and Wanscher 1978). Preparation of macrofungal samples to describe micromorphological characters was based on the laboratory techniques by Clémençon (2009). Important features were examined using Motic SMZ-171 dissecting microscope and specific features were noted based on the terminology of Vellinga and Noordeloos (2001). Microscopic characters were observed using Nikon Eclipse Nἰ, DS-Ri2 compound microscope with dried samples rehydrated and mounted in water or in 3-5% KOH to retain original color. The prepared slides were stained with ammoniacal Congo Red to bring out hyaline structures. Specific features, i.e., basidiospores, basidia, cystidia and pellis, were drawn by free hand using standard microscopic techniques and described following the glossary of Vellinga and Noordeloos (2001). Dimension of at least 30 basidiospores per collection were measured in side view. The notation [A, B, C] preceding measurements of basidiospores, basidia and cystidia indicates the number (A) of those cells measured from the number (B) of basidiomata in the number (C) of collections. Measurements are presented as (a)b-c-d(e), where 'a' and 'e' are the extreme values, 'b-d' are the 5 th and 95 th percentiles, and 'c' is the average. Q represents the length/width ratio and Q*, the average value.

DNA extraction, PCR and sequencing
DNA was isolated from samples taken from the dried specimens, using the Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology, Hangzhou, China), following the manual's procedure. The DNA loci amplified by PCR were the ITS region (including ITS1, 5.8S, ITS2) with the primers ITS1-F and ITS4 (White et al. 1990;Gardes and Bruns 1993), and nrLSU, with the primers LR0R and LR5 (Vilgalys and Hester 1990;White et al. 1990). PCR products were purified and sequenced in both directions, using the PCR primers, by Sangon Biological Engineering Technology and Services (Shanghai, China). The quality of each generated sequence read was checked using Bioedit Sequence Alignment Editor version 7.0.9.0 (Hall 1999) and sequence reads were assembled using SEQMan Pro software (DNA Star, Madison, USA).

Phylogenetic analyses
Ten new sequences were generated in this study and were deposited in GenBank (Table 1). Each sequence was compared with sequences in GenBank (National Center for Biotechnology Information, NCBI) with the Basic Local Alignment Search Tool (BLAST). Forty-nine related accessions retrieved from GenBank, including three outgroup taxa, Paraxerula americana (Dörfelt) R.H. Petersen, Strobilurus conigenoides (Ellis) Singer and X. pudens (Pers.) Singer, were used to infer phylogenetic relationships with the newly generated sequences (Table 1). Outgroup taxa were chosen based on the ITS+nrLSU phylogeny in Hao et al. (2016). ITS and nrLSU were the only gene regions used to infer phylogenetic relationships with the newly generated sequences in this study. Other Table 1. List of sequences used in the phylogenetic analysis from GenBank with geographic origin and accession numbers of gene regions. The sequences newly generated for this study are in bold.  (Huelsenbeck and Ronquist 2001). Two runs of five simultaneous MCMC chains were run for 5,000,000 generations with trees and parameters sampled every 1,000 th generation, for a total of 10,000 samples. The first 25% of samples were discarded as burn-in phase. The remaining samples were used to calculate the majority rule consensus tree and associated posterior probabilities (PP). The trees were viewed using FigTree v1.4.2 (Rambaut 2012). Genetic distances between closely related sequences were measured from MAFFT aligned sequences. The genetic distances between ITS sequences were computed based on the combined ITS1 and ITS2 regions, excluding the 5.8S gene. For LSU, the full sequence between the primers LR0R and LR5 was used.

DNA sequence analyses
The BLAST search results from the sequences of both loci (ITS and nrLSU) all matched with Hymenopellis taxa, thus indicating that all sequences generated from this study belong to this genus. In the combined ITS and nrLSU phylogeny, the new species H. straminea, represented by the specimens MFLU22-0138 (holotype) and MFLU22-0139, was monophyletic with 99% bootstrap support and 1.00 probability (Fig. 1). The ITS and nrLSU genetic distances between the two accessions were 0.52% (3/573) and 0.44% (4/905), respectively, and therefore are supported as conspecific.
"Hymenopellis raphanipes" TENN 59800 and "H. furfuracea" JM98-155 were separated from their respective species clades (Fig. 1). Therefore, it is likely that they were not identified correctly. The ITS genetic distance between H. raphanipes TENN 59800 and H. raphanipes TENN 59791 from Thailand was 8.68% (48/553). The ITS genetic distance between H. furfuracea JM98-155 and H. furfuracea HKAS 93109, both specimens from China, was 11.20% (57/509). The ITS genetic distances between specimens are much higher than the highest threshold value (3.0%) of species hypotheses in the Unite database , or the weighted average of the intraspecific ITS variability of Basidiomycota is 3.33% (Nilsson et al. 2008). The distances we observed therefore support separate species. BCC56836, on the other hand, was misidentified as "Xerula sp." since it is clearly closely related to Hymenopellis species. However, BCC56836 is a culture collection only published for its bioactivity, without a corresponding herbarium specimen. Therefore, its morphology cannot be checked. The ITS genetic distances between "Xerula sp." BCC56836 and H. raphanipes TENN 59800 and H. furfuracea JM98-155 were 3.91% (22/562) and 3.8% (20/527), respectively, while the ITS genetic distance between H. raphanipes TENN 59800 and H. furfuracea JM98-155 was 2.67% (14/527). It is possible that H. raphanipes TENN 59800, H. furfuracea JM98-155 and "Xerula sp." BCC56836 are conspecific but further taxonomic studies, especially morphological comparisons among specimens belonging to this clade, are needed to confirm this assumption. Also, a detailed study of the holotypes of H. raphanipes and H. furfuracea is needed to confirm which of the sequenced specimens identified as those two species, if any, actually belong to them.
The Hymenopellis sp. MFLU22-0142 fell into the clade of H. orientalis with 64% bootstrap support and 0.92 posterior probability. The ITS genetic distances between Hymenopellis sp. MFLU22-0142 from this study and H. orientalis TMI-2IX2002c1 and HKAS70323 are 2.57% (14/545) and 1.30% (7/539), respectively. ITS1 and ITS2 are fast-evolving loci and are very useful in species delimitation in Hymenopellis. The often advocated 3% threshold to separate interspecific and intraspecific ITS genetic distances worked well for the two new species Figure 1. Phylogenetic tree generated from ML analysis of combined ITS and nrLSU data set for Hymenopellis with three outgroup species. Bootstrap support values (≥70%) and posterior probabilities (≥0.9) (BS/PP) are given above the branches. All termini are with species name and voucher ID, with the newly generated sequences from this study in bold.
here described, with interspecific distances from their closest relatives being well above this value. However, for the specimen related to Hymenopellis orientalis, while the ITS genetic distance between our specimen and the Japanese specimen was lower than 3%, morphological differences were observed. More specimens related to H. orientalis must be studied to determine if they belong to one or more than one species. The 3% threshold should not be considered as universal. Some Basidiomycota genera have indeed been reported to exhibit lower intraspecific ITS variability such as Amanita muscaria (0.9%) and Boletus edulis (0.3%) (Nilsson et al. 2008 Etymology. The name refers to the straw-yellow color of the pileus. Diagnosis. Differentiated from similar Hymenopellis species by the small (< 5 cm), straw-yellow pileus and lamellae without decurrent tooth.
Habitat and distribution. Solitary, in tropical hill forest of Chiang Rai Province, Thailand.
Hymenopellis straminea is also quite similar to some specimens of H. furfuracea (Peck) R.H. Petersen in having a broadly convex to nearly flat pileus with bald and moderately wrinkled surface. Hymenopellis furfuracea basidiomata are more diverse in color (dark brown to gray brown or yellow brown) and size (very small to large). Lamellae also have slight decurrent tooth (Yang et al. 2009;Petersen and Hughes 2010). Hymenopellis straminea on the other hand is consistently small in pileus size (35-45 mm), evenly straw-yellow. Finally, Hymenopellis raphanipes is different from the new species by having mostly dark colored basidiomata but they can sometimes be "buckthorn brown" (5D6), and also vary in size from small to large (Petersen and Hughes 2010). Strains of H. raphanipes also have 2-and 4-spored basidia. H. straminea basidia, however, are always 4-spored. When compared with H. raphanipes TENN 59800, the herbarium specimen with which H. straminea formed a clade, the morphology is quite different. The most obvious difference is the much bigger basidiospores of H. raphanipes TENN 59800 [(13.7) 14-15.8-17 (18) × (11) 12.5-13.3-14 (15) µm]. The terminal elements of the pileipellis of Etymology. The name refers to the most common utriform or narrowly utriform pleurocystidia of the type specimen.
Habitat and distribution. Solitary to clustered, in soil covered with degrading leaves and other organic matters, in deciduous forest of Chiang Mai Province, Thailand. Notes. Hymenopellis utriformis is similar to H. rubrobrunnescens (Redhead, Ginns & Shoemaker) R.H. Petersen, having small to large but gracile basidiomata. The color is "tawny olive" (5C5) with rugose to rugulose surface.
Habitat and distribution. Solitary, on the soil covered with litter, in tropical hill forest of Chiang Mai Province, Thailand.
Specimen examined. Thailand. Chiang Mai Province: Mae Taeng District, Ban Pa Daeng, elev. 1,110 m, tropical evergreen hill forest, 08 August, 2019, A.G. Niego, MFLU22-0142; GenBank OP265166-ITS, OP265161-nrLSU. Notes. The specimen described in this study is morphologically quite similar to H. orientalis, which was first described from Japan (Petersen and Nagasawa 2006). However, it has a smaller pileus (15 mm diam.). It also produces 2-spored basidia whereas those of the holotype are 4-spored. The ITS genetic distances from the two most closely related H. orientalis TMI-2IX2002c1 and HKAS70323 were 2.57% and 1.30%, respectively. Such distances may be compatible with conspecificity. However, some morphological differences were noted, but based only on the single specimen we collected. Therefore, we use the name H. aff. orientalis until additional collections are available from tropical and temperate Asia to ascertain its taxonomic identity and properly describe it if it is confirmed to be a new species different from H. orientalis.