Umbellaceae fam. nov. (Hymenochaetales, Basidiomycota) for Umbellus sinensis gen. et sp. nov. and Three New Combinations

Hymenochaetales, belonging to Agaricomycetes, Basidiomycota, comprises most polypores and corticioid fungi and, also, a few agarics. The latest taxonomic framework accepts 14 families in this order. When further exploring species diversity of Hymenochaetales, two corticioid specimens collected from China producing cystidia with large umbrella-shaped crystalline heads attracted our attention. This kind of cystidia was reported only in three unsequenced species, viz. Tubulicrinis corneri, T. hamatus and T. umbraculus, which were accepted in Tubulicrinaceae, Hymenochaetales. The current multilocus-based phylogeny supports that the two Chinese specimens formed an independent lineage from Tubulicrinaceae as well as the additional 13 families and all sampled genera in Hymenochaetales. Therefore, a monotypic family, Umbellaceae, is newly described with the new genus Umbellus as the type genus to represent this lineage. The two Chinese specimens are newly described as U. sinensis, which differs from T. corneri, T. hamatus, and T. umbraculus in a combination of a smooth to grandinioid hymenophoral surface, not flattened, broadly ellipsoid basidiospores with a tiny apiculus, and growth on angiosperm wood. Due to the presence of the unique cystidia, the three species of Tubulicrinis, even though they lack available molecular sequences, are transferred to Umbellus as U. corneri, U. hamatus, and U. umbraculus. Hereafter, all known species with large umbrella-shaped crystalline-headed cystidia are in a single genus. In summary, the current study provides a supplement to the latest taxonomic framework of Hymenochaetales and will help to further explore species diversity and the evolution of this fungal order.


Introduction
Hymenochaetales was described as a monotypic order to accommodate Hymenochaetaceae by Frey et al. [1].This fungal order, belonging to Agaricomycetes, Basidiomycota [2], is globally distributed in the forest ecosystem, and for now comprises 14 families and 83 genera, of which 19 genera have no certain position at the family level [3].Most of the species in Hymenochaetales are polypores and corticioid fungi, whereas certain species, like those in the genera Blasiphalia, Contumyces, and Rickenella, are agarics.In addition to the morphological diversity, various trophic modes, including saprotrophs, parasites, and symbiotes (with both tree and moss), also exist in Hymenochaetales.More importantly, some polypores of Hymenochaetales, like those in the genera Sanghuangporus and Phylloporia among others, are highly valuable medicinal fungi [4,5].Therefore, species in Hymenochaetales can be important in the forest ecosystem and for economic development as strategic biological resources [6].
While the species diversity has been well explored all over the world [7][8][9][10][11][12][13][14][15][16][17], the systematics of Hymenochaetales at the family level were poorly established.The families recorded in several papers were even contradictory.This phenomenon was mainly due to the samplings in phylogenetic analyses with a biased emphasis on target fungal groups [18,19] and was also caused by the unreliable phylogenetic analyses inferred only from one or two ribosomal loci [18,20].This was the case until, recently, Wang et al. [3] systematically summarized the taxonomic background and updated the taxonomic framework of Hymenochaetales via multilocus phylogenetic analyses on the basis of the most comprehensive samplings.This update provides a crucial basis for further exploring species diversity and the taxonomic positions of species in Hymenochaetales.
The cystidium is a sterile structure but possesses unique importance in fungal taxonomy, especially for corticioid fungi that normally have simple morphological traits.Among various kinds of cystidia, large umbrella-shaped crystalline-headed cystidia are rarely present and are known only in three species, viz.Tubulicrinis corneri, T. hamatus, and T. umbraculus [21][22][23].Tubulicrinis, typified by T. glebulosus, was placed in Tubulicrinaceae, Hymenochaetales for the first time by Larsson [20].This opinion is accepted by Wang et al. [3], treating Tubulicrinaceae as a monotypic family.Unfortunately, the molecular sequences are unavailable from T. corneri, T. hamatus, and T. umbraculus.Therefore, the phylogenetic relationships among these three species and other species in Tubulicrinis are unknown.
When examining two corticioid specimens collected in China, umbrella-shaped crystallineheaded cystidia were observed.To identify these two specimens at a species level and determine their taxonomic position at higher ranks, careful morphological examinations and phylogenetic analyses were performed.In addition to the unique cystidia, other key taxonomic morphological characters of these two specimens were different from T. corneri, T. hamatus, and T. umbraculus.Moreover, these two specimens occupied an independent lineage from Tubulicrinaceae as well as the additional 13 families and all sampled genera in Hymenochaetales.Therefore, these two specimens are described as a new species belonging to a new genus in a new monotypic family.In addition, T. corneri, T. hamatus, and T. umbraculus are transferred to the new genus.

Morphological Examination
The two studied specimens were deposited at the Fungarium, Institute of Microbiology, Chinese Academy of Sciences (HMAS), Beijing, China.
Macromorphological characters were examined with the aid of a Leica M125 stereomicroscope (Wetzlar, Germany) at magnifications of up to 100×.Special color terms followed Petersen [24].Micromorphological characters were examined with an Olympus BX43 light microscope (Tokyo, Japan) at magnifications of up to 1000×, following Wang et al. [25].Specimen sections were separately mounted in Cotton Blue, Melzer's reagent, and 5% potassium hydroxide.All measurements were made from the sections mounted in Cotton Blue.When presenting the variation in basidiospore sizes, 5% of the measurements were excluded from each end of the range and are given in parentheses.Drawings were made with the aid of a drawing tube.The following abbreviations are used in the descriptions: L = mean basidiospore length (arithmetic average of all measured basidiospores), W = mean basidiospore width (arithmetic average of all measured basidiospores), Q = variation in the L/W ratios between the studied specimens, and (n = a/b) = number of basidiospores (a) measured from given number of specimens (b).
The detailed structure of cystidia was examined with a Hitachi SU8000 scanning election microscope (Tokyo, Japan).The sections of basidiomes were sprayed with gold and platinum using Leica EM ACE600 (Wetzlar, Germany).

Molecular Sequencing
Crude DNA was extracted from basidiomes of dry specimens as templates for subsequent PCR amplifications using FH Plant DNA Kit (Beijing Demeter Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions.The nrSSU, ITS, nrLSU, mtSSU, and RNA polymerase II second largest subunit (RPB2) regions were amplified using the selected primer pairs PNS1/NS41 [26], ITS1F/ITS4 [27], LR0R/LR7 [28], MS1/MS2 [29], and fRPB2-5F/fRPB2-7cR [30] and bRPB2-6F/bRPB2-7.1R[31], respectively.The PCR procedures for nrSSU and mtSSU regions were as follows: initial denaturation at 94 • C for 3 min, followed by 34 cycles at 94 • C for 40 s, 55 • C for 45 s, and 72 • C for 1 min and a final extension at 72 • C for 10 min.For ITS region, they were as follows: initial denaturation at 95 • C for 3 min, followed by 34 cycles at 94 • C for 40 s, 57.2 • C for 45 s, and 72 • C for 1 min and a final extension at 72 • C for 10 min.For nrLSU region they were as follows: initial denaturation at 94 • C for 1 min, followed by 34 cycles at 94 • C for 30 s, 47.2 • C for 1 min, and 72 • C for 1.5 min and a final extension at 72 • C for 10 min.And, for RPB2 region, they were as follows: initial denaturation at 94 • C for 2 min, followed by 9 cycles at 94  1).

Phylogenetic Analyses
In addition to the newly generated sequences for this study, additional related sequences, mainly following Wang et al. [3], were also integrated in phylogenetic analyses (Table 1).
The dataset with a combination of nrSSU, ITS, nrLSU, mtSSU, and RPB2 regions was used to explore the phylogenetic position of the newly sequenced specimens in Hymenochaetales.Within Hymenochaetales, all sequenced species with uncertain taxonomic positions at the family level and selected representatives of all 14 previously accepted families were included.Meanwhile, two species from Polyporales, viz.Fomitopsis pinicola and Grifola frondosa, were also included, and two species from Thelephorales, viz.Boletopsis leucomelaena and Thelephora ganbajun, were selected as outgroup taxa [3].
Each of the five regions was separately aligned using MAFFT v.7.110 [32] under the "G-INS-i" option [33].Due to the crucial role of gaps for delimiting taxa at the higher taxonomic level [34], they were reserved as the fifth character for all five regions.Then, the alignments of the five regions were concatenated as a single alignment (File S1).The best-fit evolutionary models of the concatenated alignment and each single-region alignment were estimated using jModelTest v.2.1.10 [35,36] under Akaike information criterion.Maximum Likelihood (ML) and Bayesian Inference (BI) algorithms were utilized for phylogenetic analyses of the concatenated alignment, and ML algorithm was utilized for phylogenetic analyses of each single-region alignment.The ML algorithm was conducted using raxml-GUI v.8.2.12 [37] and the bootstrap (BS) replicates were calculated under the auto FC option [38].The BI algorithm was conducted using MrBayes v.3.2.7 [39].Two independent runs, with each run including four chains and starting from random trees, were employed.Trees were sampled every 1000th generation.Of the sampled trees, the first 25% were removed while the other 75% were retained for constructing a 50% majority consensus tree and calculating Bayesian posterior probabilities (BPPs).Chain convergence was judged using Tracer v.1.7.1 [40] after discarding 25% of samples.The final phylogenetic tree was edited and visualized using tvBOT (https://www.chiplot.online/tvbot.html;accessed on 24 June 2023) [41].The newly generated sequences are in boldface.

Molecular Phylogeny
In this study, nine sequences for the five regions used in phylogenetic analyses were newly generated from the two studied specimens, viz.LWZ 20190615-27 and LWZ 20190615-39, with the absence of the mtSSU sequence from the specimen LWZ 20190615-39 (Table 1).
The phylogenies generated from the five single-region alignments under the best-fit evolutionary model of GTR + I + G generally share rather similar topologies in their main lineages (Figures S1-S5).However, in each phylogeny, several species are not located in their supposed positions and the BS values are not high enough.This phenomenon indicates that a single region cannot well delimit the taxonomic relationship of Hymenochaetales.Therefore, multilocus-based phylogenetic analyses are necessary.
The combined dataset of nrSSU, ITS, nrLSU, mtSSU, and RPB2 regions from 96 collections generated a concatenated alignment of 5190 characters with GTR + I + G as the best-fit evolutionary model.In the ML algorithm, the BS search stopped after 150 replicates.In the BI algorithm, after 25 million generations with an average standard deviation of split frequencies of 0.008948, all chains converged, which was indicated by the effective sample sizes of all parameters being above 6600 and all potential scale reduction factors being equal to 1.000.ML and BI algorithms generated similar topologies in main lineages, and thus, the topology generated by the ML algorithm is presented along with BS values and BPPs above 50% and 0.8, respectively at the nodes (Figure 1).In this phylogeny, the monophyly of Hymenochaetales receives full statistical support, and within Hymenochaetales, the two newly sequenced specimens collected from Guangdong, China, group together as an independent lineage (BS = 100%, BPP = 1) from all sampled families and genera.Taking the unique characters of the two specimens into consideration together, we describe them as members of a new species of a new genus in a new family.Description: Basidiomes annual, adnate and resupinate.Hymenophore smooth to grandinioid or odontioid to hydnoid, white to cream; margin thinning out, arachnoid, concolorous or paler than subiculum.Hyphal system monomitic; generative hyphae with clamp connections.Cystidia dimorphic: (1) arising from subhymenium and more or less enclosed in the hymenium or strongly projecting for the greater part of their length, cylindrical, unevenly thick-walled with a narrow or wide lumen, rooted at the base, gradually tapering, broadly rounded at the apex and covered by a large umbrella-shaped crystalline head; (2) originating laterally on subicular hyphae, with the same morphology as those arising from subhymenium but smaller in size and stalk slightly thick-walled.Basidia subclavate to clavate-cylindrical, barrel-shaped or suburniform, with a basal clamp connection and four sterigmata.Basidiospores oblong-ellipsoid or broadly ellipsoid, hyaline, smooth, thin-walled, indextrinoid, inamyloid, acyanophilous.
Hymenochaetales, the two newly sequenced specimens collected from Guangdong, China, group together as an independent lineage (BS = 100%, BPP = 1) from all sampled families and genera.Taking the unique characters of the two specimens into consideration together, we describe them as members of a new species of a new genus in a new family.Diagnosis: Distinguished by capitate cystidia with a large umbrella-shaped crystalline head.
Type: Umbellus sinensis Xue W. Wang & L.W. Zhou.Description: Basidiomes annual, adnate and resupinate.Hymenophore smooth to grandinioid or odontioid to hydnoid, white to cream; margin thinning out, arachnoid, concolorous or paler than subiculum.Hyphal system monomitic; generative hyphae with clamp connections.Cystidia dimorphic: (1) arising from subhymenium and more or less enclosed in the hymenium or strongly projecting for the greater part of their length, cylindrical, unevenly thick-walled with a narrow or wide lumen, rooted at the base, gradually tapering, broadly rounded at the apex and covered by a large umbrella-shaped crystalline head; (2) originating laterally on subicular hyphae with the same morphology as those arising from subhymenium but smaller in size and stalk slightly thick-walled.Basidia subclavate to clavate-cylindrical, barrel-shaped or suburniform, with a basal clamp connection and four sterigmata.Basidiospores oblong-ellipsoid or broadly ellipsoid, hyaline, smooth, thin-walled, indextrinoid, inamyloid, acyanophilous.
Notes: The two studied specimens, described as Umbellus sinensis below, are distinguished by the capitate cystidia with umbrella-shaped crystalline heads.Previously, three species of Tubulicrinis, viz.T. corneri, T. hamatus, and T. umbraculus, were reported to have this kind of cystidium [21][22][23].In the current phylogeny, the lineage formed by the two studied specimens is separated from Tubulicrinis (Figure 1).Therefore, they cannot be placed in Tubulicrinis.In addition, while T. corneri was originally described in Tubulicrinis [21], the basionyms of T. hamatus and T. umbraculus belong to Peniophora [22,23].Peniophora is a genus accepted in Russulales and thus cannot accommodate the two studied specimens.Therefore, a new genus, Umbellus, is introduced to accommodate species with the unique cystidia.Previously, the three species with the large umbrella-shaped crystalline-headed cystidia were all placed in the same genus, Tubulicrinis.For now, the fourth species with this kind of cystidium has been phylogenetically proven in a new genus, Umbellus.Therefore, although the molecular sequences of T. corneri, T. hamatus, and T. umbraculus are unavailable for phylogenetic analyses, these three species are transferred to Umbellus on the basis of their unique cystidia that hereafter are only known in this genus.

Discussion
In this paper, the latest taxonomic framework of Hymenochaetales, proposed by Wang et al. [3], is supplemented by describing a new family, Umbellaceae.Although Umbellaceae is a monotypic family with the new genus Umbellus as the type genus, it occupies an independent phylogenetic position from all sampled families and genera in Hymenochaetales (Figure 1).Similarly, Chaetoporellaceae was also a monotypic family in Hymenochaetales when being reinstated; however, later study soon added one more genus to this family [3].Therefore, it is reasonable to describe monotypic families to provide certain taxonomic positions at the family level for as many genera as possible, as if the phylogenetic evidence is solid.More importantly, the large umbrella-shaped crystalline-headed cystidia in Umbellaceae are unique in all fungal groups to our knowledge.In addition to the presence of unique cystidia, Umbellaceae also differs from Tubulicrinaceae in its lack of cylindrical, conical, multi-radicate cystidia with a capitate or subulate apex [3].Therefore, the description of Umbellaceae is supported from both phylogenetic and morphological perspectives.
In the molecular era of fungal taxonomy, the generic position of a species can be easily determined using accurate molecular phylogenetic analyses [42].Therefore, the transfer of a fungal species to another genus normally needs molecular evidence.However, in the current case, Umbellus corneri, U. hamatus, and U. umbraculus are rather old species, and we cannot sequence them now and in the foreseeable future.Moreover, the large umbrellashaped crystalline heads of cystidia are an extremely unique morphological character in taxonomy, and could be tentatively considered to be synapomorphy.In addition to sharing the unique cystidia, Umbellus corneri, U. hamatus, and U. umbraculus also resemble U. sinensis in annual, adnate, resupinate basidiomes and a monomitic hyphal system with clamp-connected generative hyphae.Therefore, we transfer these species to Umbellus based on the morphological perspective, even though their molecular sequences are unavailable.Then, all known species with the unique cystidia are in a single genus.
After the description of Umbellaceae and Umbellus, a total of 15 families accommodating 65 genera are accepted in Hymenochaetales while an additional 19 genera in Hymenochaetales have no certain taxonomic positions at the family level [3].The species diversity in most of these 19 genera has rarely been systematically explored with the aid of molecular evidence [43,44], and their morphological and phylogenetic relationships with the 15 known families have still not been resolved [3].Therefore, it is too mature to assign them to any known or new families.Given above, the taxonomic framework of Hymenochaetales still needs to be further updated.

Conclusions
In summary, two Chinese corticioid specimens are newly described as Umbellus sinensis, and a new monotypic family Umbellaceae, typified by a new genus, Umbellus, is described to accommodate the new species in Hymenochaetales.Moreover, three combinations, viz.Umbellus corneri, U. hamatus, and U. umbraculus, are proposed for the species previously belonging to Tubulicrinis.The updated taxonomic framework of Hymenochaetales will help further explore species diversity and the evolution of this fungal order, which are the main aims of fungal taxonomy [45].

Figure 1 .
Figure 1.Phylogenetic position of Umbellaceae (marked with a red star) within Hymenochaetales, inferred from the combined dataset of nrSSU, ITS, nrLSU, mtSSU, and RPB2 regions.The topology has been generated using the maximum likelihood algorithm.The maximum likelihood bootstrap values and the Bayesian posterior probability values above 50% and 0.8, respectively are shown at the nodes.Boletopsis leucomelaena and Thelephora ganbajun from Thelephorales have been selected as outgroup taxa.

Figure 1 .
Figure 1.Phylogenetic position of Umbellaceae (marked with a red star) within Hymenochaetales, inferred from the combined dataset of nrSSU, ITS, nrLSU, mtSSU, and RPB2 regions.The topology has been generated using the maximum likelihood algorithm.The maximum likelihood bootstrap values and the Bayesian posterior probability values above 50% and 0.8, respectively are shown at the nodes.Boletopsis leucomelaena and Thelephora ganbajun from Thelephorales have been selected as outgroup taxa.
• C for 40 s, 60 • C for 40 s, and 72 • C for 2 min and 36 cycles at 94 • C for 45 s, 55 • C for 1.5 min, and 72 • C for 2 min, and a final extension at 72 • C for 10 min.With the same primers used in PCR amplifications, the PCR products were sequenced at the Beijing Genomics Institute, Beijing, China, and the resulting new sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/;accessed on 7 July 2023; Table

Table 1 .
Information on taxa in Agaricomycetes used in phylogenetic analyses.