The Morphological Characteristics and Phylogenetic Analyses Revealed an Additional Taxon in Heteroradulum (Auriculariales)

: Auriculariales is diverse, embracing a number of corticioid, poroid, and hydnoid genera. The present study covers a new wood-inhabiting fungal species of Heteroradulum niveum sp. nov that is proposed on the basis of a combination of morphological features and molecular evidence. The species is characterized by the resupinate basidiomata, a monomitic hyphal system with generative hyphae with clamp connections; tubular cystidia; two-to four-celled basidia; and allantoid, colorless, thin-walled, smooth, IKI–, CB–, basidiospores (6.5–13.5 (cid:2) 2.7–5.5 (cid:22) m). Sequences of ITS and nLSU rRNA gene regions of the specimens were generated, and phylogenetic analyses were carried out with methods of maximum parsimony, maximum likelihood, and Bayesian inference. These phylogenetic analyses inferred from ITS+nLSU indicated that H. niveum is nested in Heteroradulum within Auriculariales. Further study within Heteroradulum on the basis of ITS+nLSU dataset revealed that it formed a monophyletic lineage with a strong support (100% BS, 100% BP, 1.00 BPP) and then grouped with H. yunnanensis .


Introduction
The Auriculariales is a characteristic order of Agaricomycetes (Basidiomycota), in which it has been introduced to embrace a number of corticioid, poroid, and hydnoid genera on the basis of a result of morphological, phylogenetic, and cytological studies [1][2][3].The Auriculariales are wood decomposers inhabiting various hosts, from the tropics to the subarctic zone, in which some of them are able to survive under extreme climatic conditions [4].Currently, the delimitation of Auriculariales has been principally based on the characteristics observed under optical and electron microscopy and supports by molecular analyses [5].
The molecular systematics involving Heteroradulum based on the internal transcribed spacer (ITS) regions and the large subunit nuclear ribosomal RNA gene (nLSU) sequences revealed that the taxonomy and phylogeny of the Auriculariales with stereoid basidiocarps were employed, in which H. adnatum Spirin & Malysheva and H. semis Spirin & Malysheva were described as new taxa [4].The genus Protohydnum A. Möller [12] was revised, which showed that Heteroradulum grouped with Exidiopsis (Bref.)Möller [13] and Tremellochaete Raitv [14].Phylogenetic analyses based on ITS+nLSU DNA sequence data indicated that Heteroradulum belonged to Auriculariaceae, which showed that Heteroradulum was to be related to Grammatus H.S. Yuan & C. Decock [15].Sequences of ITS and LSU nrRNA gene regions of the studied samples were generated, which showed that H. yunnanensis C.L. Zhao formed a monophyletic lineage with a strong support and then grouped with H. adnatum [11].
In the present study, we collected the material supposedly belonging to an undescribed species of corticioid fungi from Yunnan Province, P.R. China.We present morphological and molecular phylogenetic evidence that supports the recognition of a new species within the Heteroradulum, on the basis of the internal transcribed spacer (ITS) region nLSU sequences.

Morphological Studies
The studied specimens are deposited at the herbarium of Southwest Forestry University (SWFC), Yunnan Province, China.Macromorphological descriptions are based on photos captured in the field and lab and field notes.Color terminology follow Petersen [16].Micromorphological data were obtained from the dried specimens and observed under a light microscope following Dai [17].The following abbreviations were used: KOH = 5% potassium hydroxide water solution, CB-= acyanophilous, CB = Cotton Blue, IKI-= both inamyloid and indextrinoid, IKI = Melzer's reagent, L = mean spore length (arithmetic average for all spores), W = mean spore width (arithmetic average for all spores), Q = variation in the L/W ratios between the specimens studied, n = a/b (number of spores (a) measured from given number (b) of specimens).
Maximum parsimony analysis was applied to two combined ITS+nLSU datasets.Its approaches followed Zhao and Wu [18], and the tree construction procedure was performed in PAUP* version 4.0b10 [27].All characters were equally weighted, and gaps were treated as missing data.Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions.Max-trees were set to 5000, branches of zero length were collapsed, and all parsimonious trees were saved.Clade robustness was assessed using bootstrap (BT) analysis with 1000 replicates [28].Descriptive tree statistics: tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each maximum parsimonious tree generated on the basis of PAUP* version 4.0b10 [27].Datamatrix was also analyzed using maximum likelihood (ML) approach with RAxML-HPC2 through the Cipres Science Gateway (www.phylo.org,accessed on 5 January 2022) [29].Branch support (BS) for ML analysis was determined by 1000 bootstrap replicates.
MrModeltest 2.3 [30] was used to determine the best-fit evolution model for each dataset for Bayesian inference (BI).The soft of MrBayes 3.1.2was calculated for BI [31].Four Markov chains were run for 2 runs from random starting trees for 1.1 million generations for ITS+nLSU (Figure 1), or for 0.25 million generations for ITS+nLSU (Figure 2).The first quarter of all generations was discarded as burn-in.The majority rule consensus tree of all remaining trees was calculated.brinkmannii (Bres.)J. Erikss.and Bjerkandera adusta (Willd.)P. Karst.were selected as an outgroup for phylogenetic analyses of ITS+nLSU dataset following a previous study [26] (Figure 1); Exidiopsis effusa (Bref.ex Sacc.)Möller and Tremellochaete japonica (Lloyd) Raitv [14] obtained from GenBank were used as an outgroup to root trees following Guan et al [11] in the ITS+nLSU analysis (Figure 2).are labeled with maximum likelihood bootstrap higher than 70%, parsimony bootstrap prop tions higher than 50%, and Bayesian posterior probabilities more than 0.95.
Maximum parsimony analysis was applied to two combined ITS+nLSU datasets.approaches followed Zhao and Wu [18], and the tree construction procedure was p formed in PAUP* version 4.0b10 [27].All characters were equally weighted, and ga were treated as missing data.Trees were inferred using the heuristic search option w Figure 2. Maximum parsimony strict consensus tree illustrating the phylogeny of Heteroradulum niveum and related species in genus Heteroradulum on the basis of ITS+nLSU sequences.Branches are labeled with maximum likelihood bootstrap higher than 70%, parsimony bootstrap proportions higher than 50%, and Bayesian posterior probabilities more than 0.95.

Molecular Phylogeny
The dataset of ITS+nLSU included sequences from 24 fungal specimens representing 22 species (Figure 1).The dataset includes 1922 characters an aligned for the length, of which 1350 characters are constant, 410 are parsimony-informative, and 309 are variable and parsimony-uninformative. Maximum parsimony analysis yielded one equally parsimonious tree (TL = 1922, CI = 0.5723, RI = 0.4207, HI = 0.4277, RC = 0.2408).The best model for the dataset of ITS+nLSU estimated and applied in the Bayesian analysis was GTR+I+G (lset nst = 6, prset statefreqpr = dirichlet (1,1,1,1), rates = invgamma).ML analysis and Bayesian analysis resulted in a similar topology to MP analysis, and Bayesian analysis has an average standard deviation of split frequencies = 0.008747 (BI), and the effective sample size (ESS) across the two runs was the double of the average ESS (avg ESS) = 1108.The phylogram based on ITS+nLSU sequences (Figure 1) demonstrated that the new taxon clustered into genus Heteroradulum and formed a well-supported lineage within Auriculariales.
As shown in Figure 2, the ITS+nLSU dataset included 22 fungal specimens representing 7 species.The dataset had an aligned length of 1967 characters, of which 1724 characters are constant, 159 are parsimony-informative, and 84 are variable and parsimony-uninformative. Maximum parsimony analysis yielded 42 equally parsimonious trees (TL = 412, HI = 0.2694, RI = 0.8490, CI = 0.7306, RC = 0.6202).The best model for the ITS+nLSU dataset estimated and applied in the Bayesian analysis was GTR+I+G (lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1)).ML analysis and Bayesian analysis resulted in a similar topology to MP analysis, Bayesian analysis had an average standard deviation of split frequencies = 0.009184 (BI), and the effective sample size (ESS) across the two runs was double the average ESS (avg ESS) = 247.The phylogenetic tree (Figure 2) inferred from ITS+nLSU sequences revealed that Heteroradulum niveum sp.nov.formed a monophyletic lineage with high supports of 100% BS, 100% BP, and 1.00 BPP, and then grouped with H. yunnanensis.
Fruiting body-Basidiomata annual, resupinate, leathery, without odor or taste when fresh, becoming membranaceous upon drying, up to 15 cm long, 4 cm wide, 100-200 µm thick.Hymenial surface smooth, white when fresh, becoming white to slightly cream upon drying, older basidiomata with more or less pronounced black stains.Sterile margin white.

Discussion
In the present study, a new species, Heteroradulum niveum, is described on the basis of phylogenetic analyses and morphological characters.
Heteroradulum niveum is reminiscent of two species of Heteroradulum, H. deglubens and H. kmetii, on the basis of the character by the smooth hymenophore, but H. deglubens differs from H. niveum by having a greyish pink hymenial surface with reddish tints and larger basidiospores (13.1-19.8× 6.1-8.1 µm) [4], whereas H. kmetii differs in its perennial,

Discussion
In the present study, a new species, Heteroradulum niveum, is described on the basis of phylogenetic analyses and morphological characters.
On the basis of biogeography of Heteroradulum (Figure 5), the species is mainly distributed in Europe, such as in Austria, Russia, France, Germany, Poland, the United Kingdom, the Netherlands, Portugal, Sweden, Italy, Denmark, Norway, Finland, and Spain [32].On the other hand, H. adnatum has been found in Mexico, H. brasiliense has been found in Brazil, H. spinolosum has been found in North America, and H. yunnanensis was discovered as a new species by Guan et al. in China [4,11].
Wood-rotting fungi are an extensively studied group of Basidiomycota [17,[32][33][34], but Chinese wood-rotting fungal diversity is still not well known, especially in the subtropics and tropics.Many recently described taxa of fungi are from subtropical and tropical areas [17,[35][36][37][38].The new species presented in the current study is also from the subtropics.It is possible that new taxa will be found after further investigations and molecular analyses.

Figure 1 .
Figure1.Maximum parsimony strict consensus tree illustrating the phylogeny of Heteroradulum niveum and related species in Auriculariaceae based on ITS+nLSU sequences.Branches are labeled with maximum likelihood bootstrap higher than 70%, parsimony bootstrap proportions higher than 50%, and Bayesian posterior probabilities more than 0.95.

Table 1 .
List of species, samples, and GenBank numbers of sequences employed in present study.