Taxonomy and multigene phylogeny of Pseudohydnum (Auriculariales, Basidiomycota)

Taxonomy of Pseudohydnum gelatinosum and its sister taxa is revised via morphological data and a four-gene dataset (ITS, nc LSU rDNA, TEF1, RPB1). Identity of P. gelatinosum and Tremellodon pusillus is re-established based on newly collected and sequenced material from their type localities. Pseudohydnum alienum from Europe; P. umbrosum from temperate East Asia; P. cystidiatum, P. meridianum, and P. placibile from Vietnam; and P. omnipavum from North America are described as new to science; P. translucens and P. brunneiceps from East Asia are redescribed. Most Pseudohydnum collections from North America belong to P. gelatinosum ssp. pusillum. A significant divergence of TEF1 sequences in P. gelatinosum is discussed.


Introduction
Pseudohydnum gelatinosum (Scop.) P. Karst. is a jelly fungus recognizable because of its substantial, pale-coloured, stipitate basidiocarps with a spiny hymenophore. The species was considered as having a worldwide distribution, with records from Europe, North America, and South America, as well as Asia, Australia, and New Zealand (Wojewoda 1981). A highly peculiar habit of the basidiocarps and rather uniform anatomical structure of specimens collected in different continents maintained this viewpoint although Bourdot and Galzin (1927) pointed at morphologically deviating collections of P. gelatinosum from Europe. Holtermann (1898), Hennings (in Warburg 1899), Martin (1944), Lowy (1959Lowy ( , 1971, and Courtecuisse and Lowy (1990) described aberrant specimens of P. gelatinosum from the tropics. Recently, Chen et al. (2020) introduced a new species from the southeastern part of China, P. brunneiceps Y.L. Chen, M.S. Su & L.P. Zhang, albeit identity of P. gelatinosum elsewhere remained unquestioned. Thereafter, Zhou et al. (2022Zhou et al. ( , 2023 introduced eight more Pseudohydnum species from China, Australia, and New Zealand. A high divergence of P. gelatinosum DNA sequences in public repositories induced us to study the identity of this species and related taxa more closely. Because of the lack of recent material from Slovenia where P. gelatinosum was originally described from, we conducted extensive collecting there in 2019-2020 and supplemented it with sampling in Asia (Siberia, Russian Far East, and Vietnam), other parts of Europe, and North America. Here, we present results of this study. Blue for a few hours. All measurements were made with the use of phase contrast and oil immersion lens (Leitz Diaplan microscope, × 1250 magnification). At least 20 basidia, 20 hyphae from the context, 20 subhymenial hyphae, and 30 basidiospores were measured per each specimen studied. The following abbreviations are used in taxonomic section: L -mean basidiospore length, W -mean basidiospore width, Q′ -L/W ratio, Q -mean L/W ratio, and n -number of measurements per specimens measured.
A matrix with 1440 values was constructed, where each species was represented by at least 30 measurements of the basidiospore length and width. Statistical analysis and scatter plot of these data was performed using the programming language R 3.5.1 (R Core Team 2018) in the software environment RStudio 2022.07.2 (RStudio Team 2022).

DNA study
In total, 54 specimens of Pseudohydnum spp. were selected for molecular sampling ( Table 1). The procedure of DNA extraction completely corresponded to the manufacturer's protocol of the Phytosorb Kit (ZAO Syntol, Russia). The following primers were used for both amplification and sequencing: the primers ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990) for the ITS1-5.8S-ITS2 region, primers EF1-983F and EF1-1567R (Rehner & Buckley 2005) for a part of the TEF1 region, primers RPB1-Af and RPB1-C2f (Matheny et al. 2002) for RNA polymerase II subunit 1 (RPB1), and primers JS1 (Landvik 1996) and LR5 (Vilgalys & Hester 1990) for D1-D3 domains of nc LSU rDNA region. PCR products were purified applying the GeneJET Gel Extraction and DNA Cleanup Micro Kit (Thermo Scientific, USA). Sequencing was performed with an ABI model 3500 Genetic Analyser (Applied Biosystems, USA). Raw data were edited and assembled in MEGA X (Kumar et al. 2018). Molecular studies were carried out at the centre for collective use of scientific equipment "Cellular and molecular technology of studying plants and fungi" (BIN RAS) and Finnish Museum of Natural History, University of Helsinki (Finland).
We compiled six datasets for phylogenetic analyses: (1) ITS + nc LSU rDNA dataset for main lineages in the Auriculariales. The final alignment contained 966 characters of which 211 bp were parsimony informative. Substitution models: SYM + I + G (ITS) and GTR + I + G (LSU), (2) ITS + nc LSU rDNA dataset for Pseudohydnum spp.
Phylogenetic reconstructions were performed with maximum likelihood (ML) and Bayesian inference (BI) analyses. Before the analyses, a best-fit substitution model for each marker in each of the alignments was estimated based on the Akaike information criterion (AIC) using ModelTest-NG v0.2.0. A partition homogeneity test (PHT) between different datasets was performed with PAUP 4.0b10* (Swofford 2002). The PHT resulted in a p value of 0.01 for combined ITS + TEF1 dataset, indicating that gene sequences are incongruent and these datasets should be analysed separately.
Maximum likelihood analysis was run on RAxML-NG 1.1.0 (Kozlov et al. 2019) with one thousand bootstrap replicates. Bayesian analyses were performed with the MrBayes 3.2.6 software (Ronquist et al. 2012) by implementing three independent runs each with eight chains and 4 million generations (except 8 million for TEF1 dataset) sampling every 2000 generations, temp = 0.1. In all cases, average standard deviation of split distances reached < 0.01, indicating convergence of the runs. All phylogenetic analyses were conducted in CSC-IT Center for Science (Espoo, Finland) multi-core computing environment.

Results
In ITS-nc LSU rDNA-based phylogeny of the Auriculariales, eight described Pseudohydnum spp. and a number of undescribed species recover in the well-supported clade (pp = 1, bs = 83%) (Fig. 1). In ITS-nc LSU rDNA-based phylogeny of Pseudohydnum, the species included in the analysis are divided into seven lineages and sixteen species (Fig. 2). The ITS-nc LSU rDNA-RPB1 phylogeny corroborates this division as far as the two overlap, while providing more information on how these lineages are related (Fig. 3). The lineages (subclades) are as follows (see Fig. 2): (1) P. totarae (Lloyd) J.A. Cooper known only from New Zealand (Zhou et al. 2022).
(  (Fig. 3), but in the wider ITS-LSU dataset, it is weakly supported in the Bayesian analysis only (pp = 0.92) (Fig. 2). Taxonomic interpretation of this group is difficult. ITS sequences of P. gelatinosum from Eurasia are different in seven positions from the North American ones and up to sixteen positions versus P. abietinum and P. sinogelatinosum. These differences exceed those ones between P. alienum and P. translucens, two sister species from the fifth subclade (see below). As opposed to these two species, European and North American specimens of P. gelatinosum show no differences in the LSU region, and morphology does not allow to separate them either. In the ITS phylogeny of P. gelatinosum complex, European and North American sequences of P. gelatinosum recover in one clade (pp = 1, bs = 85%) (Fig. 4).
(4) Two Vietnamese newly introduced species, P. Both ITS-LSU and ITS-LSU-RPB1 phylogenies (Figs. 2 and 6) support separation of closely related P. alienum and P. translucens, but this is not the case with the ITS, as shown by the phylogeny constructed for the P. translucens subclade (data not shown). Reason behind this is that ITS differs only a little between P. alienum and P. translucens (pairwise distance < 1%, 3 bp), while the differences are more robust in LSU (pairwise distance 0.7-1%, 5 bp), RPB1 (pairwise distance < 1%, 5 bp), and TEF1 (pairwise distance 4.8-5%, 21 bp). Morphological traits separating the two species are discussed in taxonomic section (see under P. translucens). The ITS dataset of P. translucens complex indicates that there seems to be yet more species in this complex. Two highly similar sequences (GenBank KC152166, KT875091) originated from Mexico; however, it is impossible to decide from ITS region only if they belong to one or two species. Four sequences from the northwestern part of North America (UDB034836, UDB0778273, GenBank MF954690, HM488590) have 4-5 unique positions versus P. alienum and P. translucens and show 1-3 bp difference from each other. As in the case of the Mexican sequences, more genetic markers and proper morphological study are necessary to understand how many species they could represent.
(6) This single-species lineage subclade is represented by the newly described P. umbrosum from temperate East Asia. The three-marker phylogeny places this species as a sister to the fifth subclade around P. translucens (Fig. 3). Morphologically, P. umbrosum is distinguishable from other species All sequences generated for this study are indicated in bold. Gen-Bank accession numbers are given for all additional sequences. Support values (pp/bs) are given above the branches. Scale bar shows expected changes per site due to dwarf-sized, dark-coloured basidiocarps with a rudimentary stipe and long, ellipsoid basidiospores.
(7) The last subclade encompasses P. brunneiceps Y.L. Chen, M.S. Su & L.P. Zhang recently described from the southern part of China and a new species, P. meridianum from Vietnam. They both have substantial, dark-coloured basidiocarps with a pronounced, long stipe differentiating them from other members of the genus.
The TEF1 phylogeny (Fig. 5) makes the whole picture more complicated. TEF1 sequences of P. gelatinosum are divided among three clades, and each of them reveals strong inner variation. All but two TEF1 sequences of P. gelatinosum (all originated from Eurasian specimens) belong to two related clades; however, genetic distance between them is comparable to other sister species within the genus. The larger clade covers Eurasia from the northern part of Slovenia up to Kuril Islands, while the smaller clade embraces an epitype of P. gelatinosum from the western part of Slovenia (Spirin 13369) and another specimen from the southern part of the country, as well as three other collections from very distant areas (i.e., Finland, South Ural, and Central Siberia) (Fig. 5). This certainly points at the lack of a geographic pattern reflecting these two genetic groups. No morphological or ecological data differentiate them either. The single TEF1 sequence from the North American specimen of P. gelatinosum (Miettinen 19671) clearly deviates from the Eurasian ones although it forms a strongly supported lineage with TEF1 sequence of P. gelatinosum from Kunashir, Kuril Islands (LE 313565). ITS sequence of the latter specimen shows no differences versus other P. gelatinosum sequences from Eurasia. Therefore, we are unwilling to interpret the high divergence among TEF1 sequences of P. gelatinosum s. lato as an argument for splitting it into several species. Nevertheless, we redescribe the North American specimens of P. gelatinosum as belonging to ssp. pusillum, in anticipation of better solution in the future. In the combined ITS-LSU-RPB1 phylogeny, P. gelatinosum from Eurasia and ssp. pusillum from North America form one highly supported clade (pp = 1, bs = 98%) (Fig. 3). The taxonomic status of P. abietinum and P. sinogelatinosum as separate species deserves further clarification with the use of RPB1 and TEF1 sequences. To conclude, we can only recognize one species in this lineage, P. gelatinosum, with high certainty, while one to two further species (including P. sinogelatinosum) might also be recognized pending better genetic data.
Remarks. Pseudohydnum alienum is one of two representatives of the genus distributed in Europe. From another European species, P. gelatinosum, it differs in having smaller basidiocarps, distinctly narrower tramal and subhymenial hyphae, as well as slenderer hyphidia. In addition, subhymenial hyphae of P. alienum are more regularly packed, predominantly ascending, and basidia are normally arranged in Fig. 2 Combined phylogenetic ITS-nc LSU rDNA topology from Bayesian analysis for Pseudohydnum spp. All sequences generated for this study are indicated in bold. GenBank accession numbers are given for all additional sequences. Support values (pp/bs) are given on the branches. Scale bar shows expected changes per site ◂ a palisade manner. On the contrary, the subhymenial structure of P. gelatinosum looks much more disorderly, with hyphae mostly oriented in a random way. Differences of P. alienum from the phylogenetically close P. omnipavum and P. translucens are discussed under these species.
Pseudohydnum alienum is a poorly sampled species known from two collections from the Caucasus and two specimens from the southwestern Finland. One of the latter specimens was collected from remains of a cultivated  Basidiocarps up to 3 cm in widest dimension and 2-3 mm thick, pileate, laterally stipitate, gelatinous. Pileal surface strigose, reddish-brownish to brown. Pileal edge sharp, fertile. Spines sharp-tipped, white to cream-coloured, 0.5-2 mm long, 4-5 per mm. Stipe up to 3 cm long and 5 mm in diam., equally thickened along the whole length, greyish-or reddishbrown, strigose or partly covered by spines.
Distribution and ecology. Southeast Asia (Vietnam); decayed wood in lowland evergreen mixed forest.
Remarks. The presence of hymenial cystidia differentiate P. cystidiatum from other species described in the present paper. From two other Vietnamese species (P. meridianum and P. placibile) described here, P. cystidiatum differs in having small basidiocarps with a rudimentary stipe and larger, broadly ellipsoid basidiospores. The species is so far known only from two localities in Vietnam.
Distribution and ecology. Europe, Asia (Ural, Siberia, Russian Far East); various wood remnants of conifers.
Remarks. Hydnum gelatinosum was described by Scopoli (1772) from the present-day Slovenia, and the name was subsequently sanctioned by Fries (1821: 407). No authentic material of H. gelatinosum survives except Scopoli's oil painting stored in the National Museum of Natural History, Paris (A. Piltvater, pers. comm. 13.XII.2020). However, we see no strong reasons to designate this illustration as a lectotype of H. gelatinosum. First, there is no reference to the oil painting (or any other illustrative material) in the protologue. Second, Scopoli's oil paintings have a formal status of copyright material, and therefore, they are currently not available for a broader audience. The current version of the Code allows us to select a lectotype from the sanctioning treatment, i.e., Fries' Systema Mycologicum (1821). Therein, Fries referred to a colour figure of H. gelatinosum by Jacquin (1778); this book is available online and can be easily accessed by researchers. We designate the latter illustration as a lectotype (iconotype) of H. gelatinosum. Additionally, a recent sequenced specimen of P. gelatinosum from Idrija (Slovenia), the place where Scopoli lived and collected many of his species, is selected as epitype (see above).
Fries (1821) (Müller 1777; Batsch 1783), and we use them for lectotypification of these taxa. In both cases, distinct brownish coloration of the pileal surface was depicted: this feature certainly refers to P. gelatinosum as redefined here and rules out another European species, P. alienum (see description above). Donk (1966) treated H. auriculatum Fr. as one more synonym of P. gelatinosum. The protologue (Fries 1838) seemingly refers to P. gelatinosum. The single specimen of H. auriculatum in Fries' herbarium (UPS F-117034) consists of two pieces with different labels (Å. Kruys, pers. comm. 19.IV.2023). Unfortunately, it is impossible to decide whether they were collected before 1838. Buxbaum's illustration cited by Fries in the protologue of H. auriculatum makes the idea of this species even more obscure. This illustration (Buxbaum 1728: Table 56; Fig. 2) is almost incomprehensible, and the accompanying description (as "Agaricus gelatinosus, parte prona erinaceus," p. 36) refers to a hydnoid fungus with bluish or light-violet fructifications growing on wood of Alnus ("dilute coeruleus aut purpurascens, antiquaram Alnorum truncis adnascitur," ibid.). These indications are certainly at odds with Fries's own description of H. auriculatum ("pileis … murinis, aculeis teretibus brevibus albis… In truncis Pini" -Fries, op. cit., p. 513). At the moment, we leave this problem unresolved. Bourdot and Galzin (1927) introduced Tremellodon crystallinum var. exidiodon as a taxon deviating from P. gelatinosum (treated by them as T. crystallinum (O.F. Müll.) Quél.) due to its globose basidiocarps and a peculiar host (Populus). This variety was even suggested to represent a separate species (Pilát 1957) although never formally raised to the species rank. We studied the single authentic collection of T. crystallinum var. exidiodon (Aveyron, Crouzette, 15.XII.1913 Galzin 14918 (herb. Bourdot 12384, PC)) and concluded it shows no morphological differences from typical specimens of P. gelatinosum. However, we are unaware of other collections of this species from deciduous trees. Newly collected material from the southern part of France is highly desirable for settling taxonomic status of this variety.
Pseudohydnum gelatinosum is the most common representative of the genus in temperate-boreal forests of Eurasia. Morphologically, it is a highly diverse species, and its recognition versus sister taxa demands a meticulous microscopic study. The variation range of basidiospores in P. gelatinosum is huge, and it covers the variation range of all other temperate-boreal species except P. umbrosum (Fig. 11). Therefore, P. gelatinosum can be confidently distinguished from P. alienum in Europe, P. translucens in Asia, and P. omnipavum in North America due to distinctly wider tramal hyphae and hyphidia ( Table 2). The North-American collections of P. gelatinosum are described below as P. gelatinosum ssp. pusillum. Fig. 6 Basidiocarps of Pseudohydnum spp.: a P. alienum (holotype); b P. meridianum (LE F-347447); c P. cystidiatum (holotype); d P. cystidiatum (LE 313657); e, f P. placibile (holotype). Scale bar = 1 cm ◂ Basidiocarps up to 5 cm in widest dimension and 1-3 mm thick, pileate, laterally stipitate, gelatinous. Pileal surface first strigose, watery-greyish or greyish-brownish, then almost smooth, brown, pale ochraceous or greyishbrownish in dry condition. Pileal edge sharp to rather blunt, fertile. Spines sharp-tipped, white to greyish, 1-3 mm long, 6-8 per mm. Stipe up to 0.5 cm long, watery-greyish, usually covered by spines up to the very base, sometimes rudimentary.
Distribution and ecology. North America (USA -New York, North Carolina, Tennessee, Washington); fallen logs of conifers.
Remarks. Tremellodon pusillus was described from the Olympic Peninsula, the northwestern part of the USA (Ellis & Everhart 1894). The species quickly became forgotten, most likely because of the lack of the surviving type material. The single specimen (a presumable type) was cited in the protologue. We could not trace this collection in the contemporary public herbaria and thus consider it lost. Therefore, the only remaining source for understanding of T. pusillus is the original description. Ellis and Everhart described their species as having smoky-brown upper surface with sparse hairs and producing a rather long (up to 1 cm) stipe. These features preclude P. omnipavum (see description below) distributed in the same geographic region but fit well to the North-American subspecies of P. gelatinosum (henceforth treated as P. gelatinosum ssp. pusillum). A neotype for T. pusillus from the Olympic Peninsula is selected here to support our viewpoint.
Pseudohydnum gelatinosum spp. pusillum normally produces somewhat larger and darker basidiocarps than P. gelatinosum s. str. from Eurasia. Its basidiospores are not so variable as in the latter taxon; nevertheless, their dimensions totally fall within the range limits of P. gelatinosum s. str. No reliable anatomical traits were detected by us to distinguish these two taxa, and P. gelatinosum ssp. pusillum is treated here as a separate entity mainly because of its small genetic differences from P. gelatinosum s. str. Further studies with the use of additional markers may provide better interpretation of P. gelatinosum ssp. pusillum.  Basidiocarps up to 4 cm in widest dimension and 1-2 mm thick, pileate, laterally stipitate, gelatinous. Pileal surface papillate or smooth, greyish-brown when fresh, dark brown to almost black in dry condition. Pileal edge sharp, somewhat undulating, fertile. Spines sharp-tipped, white to cream-coloured, 0.5-1 mm long, 3-4 per mm. Stipe up to 2 cm long and 5 mm in diam., gradually tapering to the base, concolorous with pileal surface, finely tomentose to almost smooth.
Distribution and ecology. Southeast Asia (Vietnam); decayed wood in lowland evergreen mixed forest.
Remarks. Dark-coloured basidiocarps with a pronounced stipe and nearly smooth pileal surface, as well as small, predominantly subglobose basidiospores differentiate P. meridianum from other species distributed in the Southeast Asia. The species is so far known from a few localities in Vietnam.
Distribution and ecology. East Asia (Siberia, Russian Far East); decayed wood of conifers.
Remarks. Of the temperate-boreal species treated here, P. umbrosum is morphologically most distinctive due to a dark-coloured, fuscous-brown upper surface of pilei covered by short hairs. In addition, it possesses the largest spores in the genus; they are more or less regularly ellipsoid and have highest Q values comparing to other Pseudohydnum species.

Discussion
In the present study, we re-established identity of P. gelatinosum and described six new Pseudohydnum species from Eurasia and North America, in addition to eight species previously introduced by Chen et al. (2020) and Zhou et al. (2022Zhou et al. ( , 2023. However, the species diversity in the genus seems to be much higher, as we could judge from the already existing data. Descriptions of P. gelatinosum s. lato from the neotropics provided by Möller (1895) and Lowy (1971) do not fit to any of the species treated here, likely referring to species yet to be described. Future taxonomic studies of the genus outside Europe and boreal North America should carefully reconsider already existing older names associated with P. gelatinosum, e.g. Hydnum hirneoloides Berk. & M.A. Curtis from Cuba.
The species concepts advocated above have been based on combined morphological and DNA evidence. The main obstacle for morphological definition of Pseudohydnum spp. is the large variability of basidiospores in the most common species, P. gelatinosum. Therefore, we propose to pay attention to other morphological traits for introducing new species. Among macroscopic characters, the pileal surface, basidiocarp colour(s), and a presence of stipe are the most important. Species-specific microscopic traits are the width of contextual and tramal hyphae, shape, and width of hyphidia and dimensions of basidia. Combined, these features provide a comprehensive background for the species delimitation. However, we cannot preclude an existence of morphologically indistinguishable Pseudohydnum species which could be identified via DNA tools only.
Of four generic markers used in this study, ITS, nc LSU rDNA, and to a lesser extent, RPB1 sequences allow of species recognition. The large divergence of TEF1 detected in P. gelatinosum deserves further investigations. Intraspecific variation of TEF1 is higher in this complex than interspecific variation of some other species, and phylogenetic signal is also mixed, placing some TEF1 copies far outside of the core P. gelatinosum clade (Fig. 5). Our previous studies of the Auriculariales (Spirin et al. 2018(Spirin et al. , 2019(Spirin et al. , 2020(Spirin et al. , 2021 revealed no conflicts between ITS-LSU-and TEF1-based phylogenies. Therefore, Pseudohydnum is the first documented case of obvious discordance between these markers. Among the Auriculariales, an unnamed Auricularia species was shown to have multiple copies of TEF1 region (Matheny et al. 2007). Further studies are needed to understand whether Pseudohydnum represents the same case.
Data on geographic distribution of most Pseudohydnum spp. remain rather fragmentary. Four species, P. brunneiceps, P. cystidiatum, P. meridianum, and P. placibile, seem to have a subtropical-tropical distribution in East and Southeast Asia; however, they all are known from a few records only. The rest of species are distributed in temperate-boreal forests of the northern hemisphere. Of them, P. gelatinosum is the most common Eurasian species, occurring on various wood remains of (almost exclusively) coniferous trees. Pseudohydnum translucens is seemingly restricted to East Asia, predominantly inhabiting remnants of Abies spp. Its closest relative, P. alienum, was so far found in Finland and Caucasus although it surely has a wider distribution. Therefore, much denser sampling of jelly fungi even in Europe is highly desirable.
The existence of two Pseudohydnum species in Europe made it necessary to collect the epitype material in the locus classicus of P. gelatinosum (Idrija, Slovenia). Although fulfilling minimal requirements for solid epitypification, recollecting specimens in the type locality should consider changes of forest types in the targeted area. The Norway spruce (Picea abies) is currently the most common coniferous tree in the type locality of P. gelatinosum. In Scopoli's time, the dominating tree species was evidently Abies alba. Additional sampling in the intact fir-dominated forest in Slovenia and sequencing this material for four markers confirmed that it is identical with our specimen of P. gelatinosum from the type locality, thus validating designation of the latter collection as an epitype of this species.
Funding Open Access funding provided by University of Helsinki including Helsinki University Central Hospital. The research was supported by the projects no. 315927 (Academy of Finland) and "A survey of Auriculariales and Sebacinales in Sweden" (SLU Artdatabanken) (the author VS), by the project no. 122011900033-4 (Komarov Botanical Institute RAS) (field work) and the agreement no. 075-15-2021-1056 (molecular study) (the authors VM and VD), by the research project J4-3098 "The unrevealed information on soil biodiversity in leached waters" and the Research Program in Forest Biology, Ecology, and Technology (P4-0107) of the Slovenian Research Agency (the author TG).
Data availability DNA sequences used in the present study are available in GenBank. Alignments were deposited in PlutoF (https:// doi. plutof. ut. ee/ doi/ 10. 15156/ BIO/ 29121 06). Fungal specimens are stored in public herbaria (as indicated under Specimens examined).

Declarations
Ethics approval and consent to participate Not applicable.

Competing interests The authors declare no competing interests.
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