New Species of Tomentella (Thelephorales, Basidiomycota) from Temperate Continental Mountain Climate of China (Xinjiang Region)

: Species in the genus Tomentella are distributed throughout the temperate and tropical regions worldwide, but few studies associated with the taxonomy and phylogeny of this genus had been reported from Northwest China. In this paper, molecular phylogenetic analyses of the nuclear ribosomal ITS (internal transcribed spacer: ITS1-5.8S-ITS2) and LSU (large subunit: 28S) sequences combined with morphological characteristics identiﬁed three new species from Xinjiang Autonomous Region in Northwest China, which were named T. aurantispora , T. kanasensis , and T. schrenkiana . Similar macromorphological and anatomical characteristics are shared by these new species: arachnoid basidiocarps; byssoid sterile margins; utriform basidia with a clamp connection at the base; the absence of rhizomorphs and cystidia; and slightly thick-walled, subglobose to globose basidiospores. Among these new species, the color of the hymenophoral surface, the size of the basidiospores, and some other features can be used for species delimitation. The new species and closely related species in the phylogenetic tree were discussed, and a key to the identiﬁed species of Tomentella from China was provided.


Introduction
Studies on the genus Tomentella Pers.have undergone a long and convoluted history in several regions worldwide.The name Tomentella was used first by Persoon in 1799 [1] and was published as a genus by Patouillard in 1887 [2].The initial studies on taxonomy of this genus were mainly from North America and Eurasia [3,4], and some species were also reported from West India, Australia, and New Zealand [5][6][7].In recent years, more species were subsequently reported from many regions worldwide, including Asia, Africa, South America, and so on [8][9][10][11].
Tomentella is closely related to Odontia Pers.and Thelephora Ehrh.ex Willd.They belong to Thelephoraceae Chevall., Thelephorales Corner ex Oberw. of Basidiomycota R.T. Moore [3][4][5].Odontia is morphologically similar to Tomentella by the resupinate basidiocarps with gray to brown colors and the generative hyphae with simple septa or clamps [3,12].The basidiocarps of Odontia and Tomentella are often found on fallen branches, leaves, twigs, and decayed wood debris in coniferous and deciduous forests [3,9,13].The similar morphological features and habitats make it difficult to distinguish these two genera, and species of Odontia were originally recognized as Tomentella.Species of Tomentella had been found to form ectomycorrhizae with jack pine since the 1980s [14].However, Odontia was declared a non-mycorrhizal genus, verified by stable isotope and phylogenetic analyses, and treated as a separate Tomentella [12,15].Thelephora was categorized as an ectomycorrhizal genus slightly earlier than Tomentella [16,17], and species in this group often form paraphyletic clades in the phylogenetic trees [9,18].Thelephora and Tomentella possess obviously different types of basidiocarps, which are epigeous and resupinate, respectively [3,19].The tomentella-thelephora lineage has been found to be one of the most species-rich ectomycorrhizal (ECM) lineages in forest ecosystems [18,20,21].
ECM fungi have attracted much attention over the years, because they can play an essential role in nutrient cycling [22], seedling establishment [23], tree growth rate and survival [24,25], and biodiversity maintenance [26] in natural ecosystems.ECM fungi can produce conspicuous or inconspicuous basidiocarps, and about 70% of basidiomycete species with inconspicuous basidiocarps are comprised of resupinate tomentelloid fungi [27].In China, a few studies on ECM fungal diversity and the community structure of Fagaceae, Pinaceae, and Salicaceae revealed that Tomentella was one of the most species-rich genera in some forests [28][29][30][31].Studies on the ECM fungal communities of Quercus fabri Hance., Q. liaotungensis Koidz. in the family Fagaceae have identified 6 and 33 taxonomic units or species of Tomentella, respectively [29,32,33].In addition, tomentella-thelephora lineages associated with Fagaceae species from 30 forest sites in China account for 26.4% of the total sequences in 92.8% of the samples [31].Studies have also reported that the tomentella-thelephora lineage can account for 38.2% of the total ECM fungal reads from the mycorrhizal root tips of Betulaceae, Fagaceae, Pinaceae, and Tiliaceae [34].These studies revealed that there are a large number of Tomentella species in China, but studies based on basidiocarps of Tomentella species are scarce in Xinjiang Autonomous Region of Northwest China.At present, 48 species distributed in Liaoning, Heilongjiang, and Jilin Provinces of northeastern China have been reported based on the collected specimens [10,11].
In this study, basidiocarps were collected from three regions in Xinjiang Autonomous Region, including the Kanas National Nature Reserve, Tianshan Tianchi National Park, and Tianshan Grand Canyon National Forest Park.The regions belong to the typical temperate continental mountain climate with the obvious temperature fluctuations between day and night.Their average annual temperatures are -0.2 • C, 2.6 • C, and 5 • C, respectively [35,36], with average annual precipitation of 1065, 610, and 600-800 mm [35][36][37].Kanas National Nature Reserve is the only area characterized by European Siberian flora in China [35], with the domination of Abies sibirica Ledeb., Betula pendula Roth., Larix sibirica Ledeb., Picea obovata Ledeb., Pinus sibirica (Loud.)Mayr, and Populus tremula Linn [36].Tianshan Tianchi National Park is dominated by the Schrenk spruce, P. schrenkiana Fisch.et Mey., mixed with B. tianschanica Ruprecht, B. pendula, and L. sibirica [38].P. schrenkiana accounts for more than 91% of the trees in the Tianshan Grand Canyon National Forest Park, with a mixture of Populus davidiana Dode., Betula spp.and some other coniferous and deciduous species [37].During this study, three undescribed species of Tomentella were detected using morphological and phylogenetic analyses of DNA sequences.The aim of this work is to update the species diversity of Tomentella in China and assess its taxonomic and phylogenetic status.

Morphological Studies
The specimens were collected from the Kanas National Nature Reserve (KNNR), Tianshan Tianchi National Park (TTNP), and Tianshan Grand Canyon National Forest Park (TGCNFP) in Xinjiang Autonomous Region of Northwest China and were deposited at the herbarium of the Institute of Applied Ecology, Chinese Academy of Sciences (IFP).
The macromorphological characteristics of the basidiocarps were observed using a stereomicroscope (Nikon SMZ 1000: Tokyo, Japan) at 4× magnification, and the special color terms followed Kornerup [39].Observations of the microscopic characteristics followed Lu et al. [10], and these were examined on dried specimens rehydrated in 2.5% KOH and stained in cotton blue and Melzer's reagent.The following abbreviations were used in the text: IKI = Melzer's reagent; IKI-= neither amyloid nor dextrinoid; KOH = 2.5% potassium hydroxide; CB = cotton blue; CB+ = cyanophilous; CB-= acyanophilous; L = mean spore length (arithmetic average of all spores in lateral face excluding ornamentation); W = mean spore width (arithmetic average of all spores in lateral face excluding ornamentation); Q = variation in the L/W ratios; and n = number of spores measured from every specimen.Micromorphological characteristics were studied at magnifications up to 1000× using a Nikon Eclipse E600 microscope (Tokyo, Japan) with phase contrast illumination.Drawings were made with a drawing tube.The surface morphologies of the basidiospores were investigated by scanning electron microscopy (SEM) (Phenom ProX, Phenom-World, Eindhoven, The Netherlands) at 10 kV accelerating voltage and in a secondary electron detector (SED) imaging mode.A thin layer of gold was sputter coated on the samples to avoid electrostatic charging.

Molecular Procedures and Phylogenetic Analyses
DNA was extracted from dried basidiocarps using a Thermo Scientific Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA).ITS and LSU regions were amplified with SSU1318-Tom/LSU-Tom4 [40] and LROR/LR7 primer pairs [41], respectively.Protocols for the polymerase chain reaction (PCR) amplifications of ITS and LSU regions were as follows: initial denaturation at 95 • C for 5 min, followed by 39 cycles at 95 • C for 30 s, × • C for 30 s (the annealing temperatures for SSU1318-Tom/LSU-Tom4 and LROR/LR7 were 62 • C and 47.2 • C, respectively) [40,41], 72 • C for 20 s, and a final extension at 72 • C for 1 min.PCR reaction products were confirmed on 1% agarose electrophoresis gels stained with ethidium bromide [42] and sequenced at the Beijing Genomics Institute (BGI) with the same primers.The sequencing data were assembled and manually modified with the software DNAMAN 8.0 (Lynnon Biosoft, San Ramon, CA, USA).All newly generated sequences have been verified [43] and were deposited at GenBank (Table S1).Two hundred and thirteen sequences published in previous studies were downloaded from NCBI [44] and UNITE [45], and six newly generated sequences were used in this study.These sequences were aligned with MAFFT, which was available at http://mafft.cbrc.jp/alignment/server/large.html(accessed on 22 February 2021) [46] with manual adjustment.Output files in the FASTA format were directly loadable in the MAFFT online service and converted into NEXUS and PHYLIP files by ClustalX and EasyCodeMLv1.0,respectively [47].Maximum likelihood (ML) analysis was conducted in RAxMLGUI1.5b2with PHYLIP files under the GTRGAMMA model for 100 rapid bootstrap replicates under ML + rapid bootstrap mode [48].Bayesian analysis was carried out in MrBayes 3.2.6 with NEXUS files [49] based on the Markov chain Monte Carlo (MCMC) technique, and the optimal models (K80 + G for ITS1 + 5.8S + ITS2 and GTR + I + G for LSU) for the ITS1, 5.8S, ITS2 and LSU regions were predicted using jModelTest 2.1.10according to the corrected Akaike information criterion (AICc) [50].As the model K80 + G was not available in MrBayes, and HKY + G as the most similar model was run instead of K80 + G. Four Markov chains were executed, starting from random trees and keeping one tree every 1000 generations until the average standard deviation of split frequencies was below 0.01.The initial 25% of trees were removed as burn-in.

Phylogenetic Analysis
The combined dataset of the 217 sequences had an aligned length of 1675 sites with 669 bp ITS and 1006 bp LSU, which included 1077 constant characters, 87 parsimonyuninformative variable characters, and 511 parsimony-informative positions.A similar topology was obtained using ML and Bayesian analyses with two specimens of Odontia ferruginea as the outgroup [15], and only the ML tree is shown in Figure 1 with the ML bootstrap values and Bayesian posterior probabilities.The Bayesian analysis ran 10 million generations with an average standard deviation of split frequencies = 0.005051.Members of the genus Tomentella from regions in the world have been divided into 18 major clades with moderate support or high support (52 in ML/0.99 BPP for clade 1, 80 in ML/1.00 BPP for clade 2, 62 in ML/1.00 BPP for clade 3, 100 in ML/1.00 BPP for clade 4, 53 in ML for clade 5, 98 in ML/1.00 BPP for clade 6, 81 in ML/1.00 BPP for clade 7, 75 in ML/0.95BPP for clade 8, 0.96 BPP for clade 9, 84 in ML/1.00 BPP for clade 10, 95 in ML/1.00 BPP for clade 11, 60 in ML/1.00 BPP for clade 12, 95 in ML/1.00 BPP for clade 13, 89 in ML/1.00 BPP for clade 14, 96 in ML/1.00 BPP for clade 15, 99 in ML/1.00 BPP for clade 16, 82 in ML/0.97 BPP for clade 17, and 60 in ML) based on the phylogenetic analyses of combined ITS and LSU sequence data.The six sampled specimens formed three strongly supported lineages (T.aurantispora, T. kanasensis, and T. schrenkiana: 100 in ML/1.00 BPP for T. aurantispora and T. schrenkiana and 99 in ML/1.00 BPP for T. kanasensis), which clustered together with other species from The six sampled specimens formed three strongly supported lineages (T.aurantispora, T. kanasensis, and T. schrenkiana: 100 in ML/1.00 BPP for T. aurantispora and T. schrenkiana and 99 in ML/1.00 BPP for T. kanasensis), which clustered together with other species from Northeast China, Estonia, Finland, and Seychelles in clade 1 with moderate support (52 in ML/0.99 BPP).The confirmed distribution range of a new species, T. kanasensis, spans Xinjiang and Jilin Provinces located in the northwest and northeast of China, respectively.

Discussion
Three new Tomentella species distributed in Xinjiang of northwest China were id tified by phylogenetic analysis based on a combined ITS and LSU sequence dataset a morphological characteristics.Phylogenetic analyses and morphological features w able to distinguish the three new species from other Tomentella species.The deeper nod of the tree are weakly supported, which is consistent with previous studies [9].In phylogenetic tree, most species from global regions have scattered in the 18 main clad

Discussion
Three new Tomentella species distributed in Xinjiang of northwest China were identified by phylogenetic analysis based on a combined ITS and LSU sequence dataset and morphological characteristics.Phylogenetic analyses and morphological features were able to distinguish the three new species from other Tomentella species.The deeper nodes of the tree are weakly supported, which is consistent with previous studies [9].In the phylogenetic tree, most species from global regions have scattered in the 18 main clades.Species in the clade with moderate support or high support present unique features.Species in clade 1 have basidiocarps with similar color; species in clade These species share some similar characteristics: clamped hyphal system, globose to subglobose basidiospores, and the absence of rhizomorphs and cystidia [3,11] except for the ellipsoid, triangular, or lobed basidiospores of T. lammiensis and T. stipitata [11,51].T. aurantispora also presents some other similar morphological characteristics with T. changbaiensis and T. megaspora: byssoid sterile margin, short and inflated subhymenial hyphal cell, and utriform basidia.But T. changbaiensis differs from T. aurantispora by the separable basidiocarps and larger basidiospores with longer echinuli (L = 8.45 µm, W = 7.83 µm, echinuli up to 1.5 µm) [11].T. megaspora can be distinguished from T. aurantispora based on the larger basidiospores with longer echinuli (L = 9.88 µm, W = 9.08 µm, echinuli up to 2.5 µm) [11].Morphologically, T. aurantispora and T. asiae-orientalis also share some other common characteristics: arachnoid basidiocarps adherent to the substrate and byssoid sterile margin.Moreover, T. asiae-orientalis can be delimited from T. aurantispora by the more or less uniform subhymenial hyphal cells, clavate basidia, and basidiospores with longer echinuli (echinuli up to 2.5 µm) [11].
Macroscopically, T. kanasensis and T. aurantispora possess the same arachnoid basidiocarps with similar color (yellowish brown to brown and light brown to dark brown) and byssoid sterile margin.But T. aurantispora differs by the length of sterigmata (3-5 µm long) and the hyphal system without simple septa in comparison with T. kanasensis.Some features of T. changbaiensis can be also observed in T. kanasensis, such as the byssoid sterile margin and the equally sized basidiospores.However, T. changbaiensis differs markedly from T. kanasensis by the duplex basidiocarps, pale brown sterile margin, and hyphal system without simple septa [11].T. bryophila also forms short and inflated subhymenial hyphal cells and utriform basidia similar to those of T. kanasensis.However, the encrusted hyphae of T. bryophila are different from T. kanasensis [3].
As Picea spp.constituted the dominant tree distributed in the investigated regions of Xinjiang Autonomous Region, we speculate that T. aurantispora and T. schrenkiana may form ectomycorrhizae with P. schrenkiana.Specimens of T. kanasensis have also been observed in Jilin Province, and the climate of this region is apparently different from Xinjiang.Picea-dominated forests in Xinjiang have quite different tree species in comparison to the mixed broadleaf-conifer forests in Jilin Province dominated by Pinus koraiensis Siebbold & Zucc., Acer pictum subsp.mono (Maxim.)Ohashi, A. mandshuricum Maxim., Tilia amurensis Rupr., and Carpinus cordata Blume mixed with shrub species [53].The host tree species of T. kanasensis from Xinjiang and Jilin Provinces may be P. obovata and P. koraiensis, respectively.At present, 51 species of Tomentella have been identified based on morphological and molecular characteristics of the Tomentella collections from Northeast China (Liaoning, Heilongjiang, and Jilin Provinces) and Northwest China (Xinjiang Autonomous Region) [10,11].While several Tomentella spp. of ECM associated with plant roots in Pinaceae and Fagaceae were also reported in previous studies from China, they were not available for identification [29,30,34], and the cryptic or atypical basidiocarps of Tomentella may be easily overlooked in diverse studies.Therefore, we speculate that an increasing number of species in this genus will be found at more sites in China.

The Types of Rhizomorphs Used in the Key
Agerer recognized six categories of rhizomorph structure (types A-F) [54], and the dimitic hypal system of rhizomorphs named type G was reported in 2020 [11].

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10.3390/f12111531/s1,Table S1: GenBank/UNITE accession numbers, voucher numbers, substrates, localities and references for the specimens included in this study.The newly generated sequences are in bold.

Figure 1 .
Figure 1.Maximum likelihood tree illustrating the phylogeny of Tomentella aurantispora, T. kanasensis, T. schrenkiana, and related taxa based on ITS + LSU nuclear rDNA sequences dataset.Branches are labeled with maximum likelihood bootstrap higher than 50% and Bayesian posterior probabilities higher than 0.95.The terminals indicate the regions where they were distributed.New species are in bold (black).
7 and clade 8 have rhizomorphs; species in clade 10 have a simple-septate hyphal system and basidiospores with the same size; species in clade 14 have separable basidiocarps with similar color; species in clade 15 have similar anatomical structure; species in clade 16 have cystidia.In addition, T. brunneocystidiata Yorou & Agerer, T. capitata Yorou & Agerer, T. capitatocystidiata Yuan et al., T. cystidiata Yuan et al., T. gloeocystidiata Yuan et al., and T. pilosa (Burt) Bourdot & Galzin in clade 4 also share common features: the capitate cystidia.T. aureomarginata Yuan et al., T. brunneoflava Yuan et al., and T. separata Yuan et al. in clade 6 also have common features: light brown to brown basidiocarps and the presence of rhizomorphs.In the phylogenetic tree, T. aurantispora and T. kanasensis are closely related to T. bryophila (Pers.)M.J.Larsen, T. changbaiensis Yuan et al., T. interrupta Yuan et al., T. longiaculeifera Yuan et al., T. megaspora Yuan et al., and T. pallidobrunnea Yuan et al. with strong support (85 in ML and 1.00 BPP), and they formed a highly supported (0.97 BPP) relationship with T. asiae-orientalis Yuan et al., T. lammiensis Lu et al., and T. stipitata Yuan et al.

Author Contributions:
Conceptualization, X.L. and H.-S.Y.; methodology, X.L. and H.-S.Y.; software, X.L.; validation, formal analysis, and investigation, X.L. and H.-S.Y.; resources, H.-S.Y.; data curation, X.L. and H.-S.Y.; writing-original draft preparation, X.L.; writing-review and editing, X.L. and H.-S.Y.; visualization, X.L. and H.-S.Y.; supervision, project administration, and funding acquisition, H.-S.Y.All authors have read and agreed to the published version of the manuscript.Funding: This research was financed by the National Natural Science Foundation of China (Project Nos.31970017, 31770028 & 31470148) and the Special Funds for the Young Scholars of Taxonomy of the Chinese Academy of Sciences (Project No. ZSBR-015).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.