Phylogeny and Diversity of the Genus Pseudohydnum (Auriculariales, Basidiomycota)

The toothed jelly fungus Pseudohydnum gelatinosum was originally described from Europe. The name has a broad sense and the species has been widely reported almost all over the world. We have studied samples of so-called P. gelatinosum from Asia and Oceania. Based on morphology, hosts, geography, and phylogenetic analysis using the internal transcribed spacer regions (ITSs) and the large subunit of nuclear ribosomal RNA gene (nLSU), four new species, P. himalayanum, P. orbiculare, P. sinogelatinosum, and P. tasmanicum, from China, New Zealand, and Australia are described and illustrated, and a new combination, Pseudohydnum totarae, is proposed. The five new taxa can be differentiated by the shape of their basidiomata, pileal surface color when fresh, spine size, basidiospore dimensions, shape of hyphidia, hosts, and biogeography. Phylogenetically, most of these taxa are distantly related, and different base pairs among these taxa mostly account for >2% nucleotides in the ITS regions.

The 'toothed jelly' fungus Pseudohydnum gelatinosum is the type species of the genus and was originally described from Croatia in Europe as Hydnum gelatinosum Scop. [17]. The name is applied broadly and the species has been widely reported from Europe [3,18,19], North America [20][21][22], Central and South America [2,11,13,23], Asia [24][25][26], and The studied specimens are deposited in the herbaria of the Institute of Microbiology, Beijing Forestry University (BJFC) and Landcare Research (PDD), New Zealand. Morphological descriptions are based on field notes and voucher specimens. Color terms are from Anonymous [34], Petersen [35], and Kornerup and Wanscher [36]. Microscopic structures refer to Pippola and Kotiranta [37], Malysheva et al. [38], and Fan et al. [39]. Handmade sections of voucher basidiomata were examined using a Nikon Eclipse 80i microscope (magnification ×1000) after being mounted in 5% KOH for five minutes and treated with 1% Phloxine B (C 20 H 4 Br 4 Cl 2 K 2 O 5 ). Microscopic structures were photographed using a Nikon Digital Sight DS-L3 or Leica ICC50 HD camera. At least 20 basidia and basidiospores from each specimen were measured. Spore measurements are quoted as means and standard deviations (±1.5 σ) or with 5% of measurements excluded from each end of the range given in parentheses. Stalks were excluded for basidia measurements, and the hilar appendages were excluded for basidiospore measurements. The following abbreviations are used in the descriptions: IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, CB = Cotton Blue, CB-= acyanophilous in Cotton Blue, L = arithmetic average of all spore lengths, W = arithmetic average of all spore widths, Q = L/W ratios, n (a/b) = number of spores (a) measured from a given number (b) of specimens, and σ = standard deviation from the mean.

DNA Extraction, Amplification, and Sequencing
A CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) or DNA easy Plant Mini Kit (Aidlab Biotechnologies, Beijing, China) was used to obtain DNA from dried specimens and perform a polymerase chain reaction (PCR) according to the manufacturer's instructions, with some modifications [40,41]. Two DNA gene fragments, i.e., the internal transcribed spacer (ITS) and large subunit nuclear ribosomal RNA gene (nLSU), were amplified using the primer pairs ITS5/ITS4 [42] and LR0R/LR7 (https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/, accessed on 22 April 2022) [43]. The PCR procedure for ITS was as follows: initial denaturation at 95 • C for 3 min, followed by 35 cycles at 94 • C for 40 s, 54 • C for 45 s, and 72 • C for 1 min and a final extension of 72 • C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94 • C for 1 min, followed by 35 cycles at 94 • C for 30 s, 50 • C for 1 min, and 72 • C for 1.5 min and a final extension of 72 • C for 10 min. DNA sequencing was performed at the Beijing Genomics Institute (BGI) and Manaaki Whenua, Landcare Research, Auckland. All newly generated sequences have been submitted to GenBank and are listed in Table 1.

Molecular Phylogenetics Analysis
The concatenated ITS + nLSU dataset included 34 ITS sequences and 22 nLSU sequences from 34 samples representing twelve taxa. The dataset had an aligned length of 1942 characters, of which 1521 were constant, 107 were variable and parsimony-uninformative, and 294 were parsimony-informative. MP analysis yield a tree (TL = 690, CI = 0.799, RI = 0.915, RC = 0.721, HI = 0.201). The best model for the concatenated ITS + nLSU dataset estimated and applied in the BI analysis was 'GTR + F + I + G4', lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). The sequences generated for this study were aligned with additional sequences downloaded from GenBank. Both ITS and nLSU sequences were aligned using MAFFT v. 7 online (https://mafft.cbrc.jp/alignment/server/, accessed on 22 April 2022), adjusting the direction of nucleotide sequences according to the first sequence (accurate enough for most cases) and selecting the G-INS-i iterative refinement method [44]. Alignments were manually adjusted to maximum alignment and minimize gaps by BioEdit v. 7.0.9 [45]. A dataset composed of concatenated ITS + nLSU sequences was used to determine the phylogenetic position of the new species. Protomerulius subreflexus (Lloyd) O. Miettinen & Ryvarden and Protomerulius substuppeus (Berk. & Cooke) Ryvarden were selected as the outgroups, following Chen et al. [8].
Maximum Parsimony (MP) analysis was used for the dataset in PAUP* v. 4.0b10 [46]. All characters were equally weighted, and gaps were treated as missing data. A heuristic search option was used to infer the trees with TBR branch swapping and 1000 random sequence additions. All parsimonious trees were saved. The max-trees were set to 5000. Branches were collapsed if the minimum branch length was zero. Clade robustness was assessed using bootstrap analysis with 1000 replicates [47]. 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.
The CIPRES Science Gateway was used for Maximum Likelihood (ML) analysis based on the dataset in [48]. All parameters in the ML analysis used default settings, and statistical support values were obtained using nonparametric bootstrapping with 1000 replicates.
Bayesian Inference (BI) analysis based on the dataset was carried out using MrBayes v3.2.6 [49]. The best substitution model for the datasets was selected by ModelFinder [50], and trees were sampled every 100 generations. Four Markov chains were run from random starting trees for 2 million generations. Trees were sampled every 1000th generation. The first 25% of sampled trees were discarded as burn-in, whereas other trees were used to construct a 50% majority consensus tree and for calculating Bayesian posterior probabilities (BPPs).

Molecular Phylogeny
BI analysis yielded a similar topology to MP and ML analysis, with an average standard deviation of split frequencies = 0.004227. Only the MP tree is provided here ( Figure 1). Branches that received bootstrap support for MP (MP-BS), ML (ML-BS), and BI (BPP) greater than or equal to 70% (MP-BS and ML-BS) and 0.90 (BPP) were considered as significantly supported, respectively. The current phylogeny placed all samples of Pseudohydnum in a fully supported clade ( Figure 1). Two previously accepted species, P. brunneiceps and P. gelatinosum, received strong support. The samples from North America are treated as "Pseudohydnum gelatinosum-1", "Pseudohydnum gelatinosum-2", and "Pseudohydnum gelatinosum-3". Nine Chinese specimens formed two distinct lineages, and seven Oceanian samples formed three distinct linages nested in Pseudohydnum.  The current phylogeny placed all samples of Pseudohydnum in a fully supported clade ( Figure 1). Two previously accepted species, P. brunneiceps and P. gelatinosum, received strong support. The samples from North America are treated as "Pseudohydnum gelatinosum-1", "Pseudohydnum gelatinosum-2", and "Pseudohydnum gelatinosum-3". Nine Chinese specimens formed two distinct lineages, and seven Oceanian samples formed three distinct linages nested in Pseudohydnum.   Diagnosis-Differs from other Pseudohydnum species by stipitate and confluent basidiomata, clay-pink to cinnamon pileal surface when fresh, dense spines 5-6 per mm at base, growth on Abies in high altitude (>3000 m), and occurrence in Southwest China.

Taxonomy
Basidiomata-Annual, gelatinous when fresh, brittle when dry, with a lateral stipe, occasionally confluent sharing a stipe base. Pilei flabelliform, projecting up to 2.5 cm, 3.8 cm wide and 0.3 mm thick when dry. Pileal surface clay-pink (6D4) to cinnamon (6D6) when fresh, becoming purplish/date-brown when dry. Spines white and conical when fresh, become grayish brown when dry, 5-6 per mm at base, up to 0.2 mm long. Context translucent when fresh. Stipe concolorous with pileal surface, translucent when fresh, up to 2.5 cm long and 3 mm in diam. when dry.
Habitat-The species is rather common on rotten wood of Abies at altitudes higher than 3000 m. Etymology-Orbiculare (Lat.): refers to the species having orbicular basidiomata. Diagnosis-Differs from other Pseudohydnum species by pileate basidiomata without a well-formed pseudostipe, orbicular pilei, dark-gray to black pileal surface when fresh, hymenophore with sparse spines (0.5-1 per mm at base), growth mostly on angiosperm, and distribution so far restricted to New Zealand.
Habitat-The species is commonly associated with angiosperm hosts, including southern beech (Nothofagaceae), but several collections were obtained from rotten trunks of Pinus radiata.
Distribution-Distributed throughout New Zealand but perhaps more common in the south.  Diagnosis-Differs from other Pseudohydnum species by lateral stipitate basidiomata, pinkish buff to cinnamon-buff pileal surface when fresh, hymenophore with spines 3-4 per mm at base, strongly flexuous and branched hyphidia, growth on gymnosperm wood at high altitudes (>3000 m), and occurrence in Southwest China. Diagnosis-Differs from other Pseudohydnum species by lateral stipitate basidiomata, pinkish buff to cinnamon-buff pileal surface when fresh, hymenophore with spines 3-4 per mm at base, strongly flexuous and branched hyphidia, growth on gymnosperm wood at high altitudes (>3000 m), and occurrence in Southwest China.
Basidiomata-Annual, gelatinous when fresh, brittle when dry, usually solitary, with a lateral stipe. Pilei applanate, occasionally lobed with an undulating margin, projecting up to 4.7 cm, 2.9 cm wide, 1.2 mm thick when dry. Pileal surface pinkish buff (5A3) to cinnamon-buff (4/5B4) when fresh, becoming milk-coffee to cigar-brown when dry. Spines white and conical when fresh, cream when dry, 3-4 per mm at base, up to 1 mm long when fresh. Context translucent when fresh. Stipe concolorous with pileal surface, shrinking to the base, translucent when fresh, up to 3.2 cm long and 4 mm in diam. when fresh.
Basidiomata-Annual, pileate, with a rudimentary stipe base, a few imbricate, gelatinous when fresh, brittle when dry. Pilei dimidiate, shell-shaped, projecting up to 1.1 cm, 1.8 cm wide and 1.2 mm thick when dry. Pileal surface light vinaceous gray (13B2/3) to smoke-gray (F34) when fresh, become mouse-gray when dry. Spines white and conical when fresh, become buff when dry, 2-3 per mm at base, up to 1 mm long when dry. Context translucent, vinaceous buff when dry.
Habitat-The species was found on a fallen angiosperm trunk and dead standing angiosperm trees, e.g., Eucalyptus and Nothofagus.
Diagnosis-Differs from other Pseudohydnum species by stipitate and confluent basidiomata, spathulate pilei, grayish brown to reddish brown upper surface when fresh, hymenophore with sparse spines (0.8-1.2 per mm at base), growth on gymnosperm wood, and known distribution restricted to New Zealand.
Diagnosis-Differs from other Pseudohydnum species by stipitate and confluent basidiomata, spathulate pilei, grayish brown to reddish brown upper surface when fresh, hymenophore with sparse spines (0.8-1.2 per mm at base), growth on gymnosperm wood, and known distribution restricted to New Zealand.
Habitat-Originally described on rotten wood of Totara (Podocarpus totara). The species appears to be commonly associated with decaying gymnosperm wood; other recorded substrates include Kauri (Agathis australis) and Rimu (Dacrydium cupressinum). Notes-The species was described as Auricula totarae by C.G. Lloyd (USA) in 1920 from material sent to him by C.G. Cunningham in New Zealand. The original description is sparse but refers to the protuberances on the upper surface of the fruiting bodies and the presence of microscopic globose bodies that Lloyd assumed to be basidia. The isotype consists of several small fruiting bodies and fragments. The material appears to be immature, but some fragments do bear the characteristic hymenophore spines of Pseudohydnum as well as the upper surface protuberances mentioned by Lloyd. The contemporary writings of Lloyd indicate that he was familiar with Pseudohydnum gelatinosum and we must assume that his placement of the species in Auricula was due to his having failed to observe the hymenophore spines. No mature basidia or spores were found in the isotype (PDD 225), although the immature globose basidia mentioned by Lloyd were abundant. Several of the fruiting bodies show a well-developed stipe. There is no doubt that this taxon is the same as the one re-described here based on modern taxonomy.

Discussion
There is unrecognized cryptic diversity within the genus Pseudohydnum, including three taxa detected from North America in our phylogenetic analysis (Figure 1). The different base pairs among these taxa account for >2% nucleotides in the ITS regions, and these taxa are treated as "Pseudohydnum gelatinosum-1", "Pseudohydnum gelatinosum-2", and "Pseudohydnum gelatinosum-3" in our study because we did not study the representative collections.
Pseudohydnum himalayanum, P. sinogelatinosum, P. orbiculare, and P. tasmanicum have similar basidiospore dimensions. The former two species have dense spines (3-6 per mm at base) and are boreal species found at altitudes higher than 3000 m in Southwest China. The latter two species have sparse spines (0.5-3 per mm at base) and are temperate species found in Oceania.
Pseudohydnum himalayanum and P. sinogelatinosum have an overlapping distribution in Southwest China, but the former has 5-6 spines per mm at base and unbranched hyphidia, while the latter has 3-4 spines per mm at base and branched hyphidia.
Pseudohydnum tasmanicum and P. orbiculare are closely related phylogenetically ( Figure 1) and share pileate basidiomata with circular pilei and no stipe, but P. tasmanicum has a distribution in Tasmania, an island in the far south of Australia, while the distribution of P. orbiculare is restricted to New Zealand. In addition, P. tasmanicum has a vinaceous gray to smoke-gray pileal surface, a hymenophore with 2-3 spines per mm at base, while P. orbiculare has a dark-gray, brown to black pileal surface when fresh and a hymenophore with sparse spines (0.5-1 per mm at base).
The two New Zealand species of Pseudohydnum have a similar size and identical range of coloration, from translucent white to gray or brown to reddish brown. They are distinguished by different basidiomata morphology; being spathulate with a pseudostipe in P. totarae, and orbicular to fan-shaped and without a pseudostipe in P. orbiculare. In addition, the former species prefers gymnosperm hosts, especially podocarp wood, and the latter is more common on angiosperm hosts, although the distinction is not absolute because of confirmed records of P. orbiculare on rotten wood of Pinus radiata, which is an exotic species in New Zealand. Pseudohydnum orbiculare is closely related to P. tasmanicum from Australia. ITS sequences show a consistent difference of 1.8%. This pair of species exhibit few or no morphological or ecological differences, which is probably a consequence of a relatively recent dispersal event with subsequent genetic isolation and divergence or maybe when Gondwana split Tasmania off from New Zealand. They are nevertheless good independent phylogenetic species.
Pseudohydnum brunneiceps differs from our new taxa by its vinaceous brown to fuscous pileal surface when fresh, distinctly confluent basidiomata, and growth on Cryptomeria in a subtropical area of China [8].
Pseudohydnum translucens has pure white basidiomata when soaked and dry and subglobose basidiospores measuring 4-5 µm [7]. Lloyd thought that P. translucens and P. gelatinosum belonged to different genera since P. gelatinosum has a homogeneous texture both in spine and context, while P. translucens has a heterogeneous texture and the hymenial layer is different from the context, although both are white [7]. Anyhow, Pseudohydnum translucens is easily distinguished from our new taxa by its shorter basidiospores (4-5 µm vs. 5.5-9 µm long in our new taxa).

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