Introduction

Neofomitella Y.C. Dai, Hai J. Li & Vlasák (Polyporaceae, Polyporales) was recently derived from Fomitella Murrill and typified by N. rhodophaea (Lév.) Y.C. Dai, Hai J. Li & Vlasák, it differs from Fomitella by its distinctly crusted basidiomata with the cuticle developing from base to margin (Li et al. 2014). Species in Neofomitella have annual or perennial growth habit; pileate or effused-reflexed basidiomata; concentrically zonate or sulcate and glabrous to velutinate pileal surface; corky to hard corky context with a dark agglutinated crust developing from base to margin; and a trimitic hyphal system with clamped generative hyphae, tissues turning to black in KOH, oblong ellipsoid to cylindrical, hyaline, smooth and thin-walled basidiospores (Li et al. 2014). Phylogenetic analysis showed that Neofomitella clustered with Microporus P. Beauv., with no close relationships to Fomitella (Li et al. 2014). Currently, three species are accepted in Neofomitella: N. rhodophaea; N. fumosipora (Corner) Y.C. Dai, Hai J. Li & Vlasák; and N. polyzonata Y.C. Dai, Hai J. Li & Vlasák.

During the investigations on species diversity and taxonomy of polypores, two undescribed taxa matching the definitions of Neofomitella were found. In order to confirm the affinity of these taxa with the Neofomitella species, phylogenetic analysis was carried out based on a combined 7-gene dataset.

Materials and methods

Morphological studies

The examined specimens were deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC), and some duplicates were deposited at the herbarium of Royal Botanic Gardens Victoria, Australia (MEL). Macro-morphological descriptions were based on the field notes and measurements of herbarium specimens. Color terms followed Petersen (1996). Micro-morphological data were obtained from the dried specimens, and observed under a light microscope following Han et al. (2016) and Shen et al. (2019). Sections were studied at a magnification of up to × 1000 using a Nikon E 80i microscope and phase contrast illumination. Drawings were made with the aid of a drawing tube. Microscopic features, measurements, and drawings were made from slide preparations stained with Cotton Blue and Melzer’s reagent. Spores were measured from sections cut from the tubes. To present the variation of spore size, 5% of measurements excluded from each end of the range were given in parentheses. The following abbreviations were used: IKI = Melzer’s reagent, IKI– = neither amyloid nor dextrinoid, CB = cotton blue, CB– = acyanophilous, KOH = 5% potassium hydroxide, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of 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.

DNA extraction and sequencing

A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co. Ltd., Beijing) was used to extract total genomic DNA from dried specimens, and performed the polymerase chain reaction (PCR) according to the manufacturer’s instructions with some modifications (Chen et al. 2015; Zhou et al. 2016). The ITS region was amplified with primer pairs ITS5 and ITS4 (White et al. 1990). The nLSU region was amplified with primer pairs LR0R and LR7 (http://www.biology.duke.edu/fungi/mycolab/primers.htm). The nSSU region was amplified with primer pairs PNS1 and NS41 (Hibbett 1996). The mtSSU region was amplified with primer pairs MS1 and MS2 (White et al. 1990). Part of EF1-α was amplified with primer pairs EF1-983F and EF1-1567R (Rehner and Buckley 2005). RPB1 was amplified with primer pairs RPB1-Af and RPB1-Cr (Matheny et al. 2002). RPB2 was amplified with primer pairs fRPB2-5F and fRPB2-7cR (Liu et al. 1999). The PCR procedure for ITS, mtSSU, and EF1-α included an initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, 54 °C for ITS and mtSSU, 56 °C for EF1-α for 45 s, 72 °C for 1 min, and a final extension of 72 °C for 10 min. The PCR procedure for nLSU and nSSU included an initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 50 °C for nLSU and 53 °C for nSSU for 1 min, 72 °C for 1.5 min, and a final extension of 72 °C for 10 min. The PCR procedure for RPB1 and RPB2 included an initial denaturation at 94 °C for 2 min, followed by 10 cycles at 94 °C for 40 s, 60 °C for 40 s and 72 °C for 2 min, then followed by 37 cycles at 94 °C for 45 s, 53 °C–58 °C for 1.5 min and 72 °C for 2 min, and a final extension of 72 °C for 10 min. The PCR products were purified and sequenced at Beijing Genomics Institute, China, with the same primers. All newly generated sequences were submitted to GenBank and listed in Table 1.

Table 1 A list of species, specimens and GenBank accession numbers of sequences used in this study
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Phylogenetic analyses

Additional sequences were selected from GenBank (Table 1). All sequences were aligned in MAFFT 7 (Katoh and Standley 2013; http://mafft.cbrc.jp/alignment/server/) and manually adjusted in BioEdit (Hall 1999). These gene fragments were spliced with Mesquite 3.2 (Maddison and Maddison 2017) for further phylogenetic analyses. The final concatenated sequence alignment was deposited in TreeBase (https://treebase.org/treebase-web/home.html; submission ID 23613). Sequences of Laetiporus montanus Černý ex Tomšovský & Jankovský and L. sulphureus (Bull.) Murrill were used as outgroups to root trees.

Maximum parsimony (MP) analysis was applied to the combined multiple genes datasets, and the tree construction procedure was performed in PAUP* version 4.0b10 (Swofford 2002). Settings for phylogenetic analyses followed Song et al. (2016) and Song and Cui (2017). 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. Maxtrees were set to 5000; branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates (Felsenstein 1985). 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 (MPT) generated.

RAxML v7.2.6 (Stamatakis 2006) was used to construct a maximum likelihood (ML) tree involved 200 ML searches under the GTR+GAMMA model and only the best tree from all searches was kept. In addition, 200 rapid bootstrap replicates were run with the GTR+CAT model to obtain the ML bootstrap values.

MrModeltest 2.3 (Posada and Crandall 1998; Nylander 2004) was used to determine the best-fit evolution model for the combined dataset for Bayesian inference (BI). Bayesian inference (BI) was calculated with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003) with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites. Four Markov chains were run for two runs from random starting trees for 2 million generations and sampled every 100 generations. The first quarter generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated.

Phylogenetic trees were visualized using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). Branches that received bootstrap support for maximum parsimony (MP), maximum likelihood (ML) and Bayesian posterior probabilities (BPP) greater than or equal to 75% (MP and ML) and 0.95 (BPP) were considered as significantly supported, respectively.

Results

Molecular phylogeny

The combined dataset (ITS+nLSU+mtSSU+nSSU+EF1-α+RPB1+RPB2) included sequences of 63 fungal specimens representing 37 taxa. The dataset had an aligned length of 6528 characters, of which 4464 characters are constant, 235 are variable and parsimony-uninformative, and 1829 are parsimony-informative. MP analysis yielded 40 equally parsimonious trees (TL = 7826, CI = 0.440, RI = 0.735, RC = 0.323, HI = 0.560). The best model for the combined dataset estimated and applied in the BI was GTR + I + G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). Bayesian and ML analyses resulted in a similar topology as MP analysis, with an average standard deviation of split frequencies = 0.003063 (BI); and the ML topology was shown in Fig. 1.

Fig. 1
figure 1

Strict consensus tree illustrating the phylogeny of Neofomitella generated by ML analysis based on ITS+nLSU+mtSSU+nSSU+EF1-α+RPB1+RPB2 sequences. Branches are labeled with parsimony bootstrap proportions higher than 50%, maximum likelihood bootstrap higher than 50% and Bayesian posterior probabilities more than 0.95

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Samples of Neofomitella clustered together, then grouped with the samples of Microporus P. Beauv. Sampled specimens of the two new species formed distinctly well-supported lineages (Fig. 1).

Taxonomy

Neofomitella australiensisB.K. Cui & Xing Ji, sp. nov. (Figs. 2a–b, 3)

Fig. 2
figure 2

Basidiomata of Neofomitella species. a, bN. australiensis; c, dN. guangxiensis (scale bars: a–b = 3 cm; c–d = 2 cm)

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Fig. 3
figure 3

Microscopic structures of Neofomitella australiensis (drawn from the holotype). a Basidiospores; b Basidia and basidioles; c Cystidioles; d Hyphae from trama; e Hyphae from context. Bars: a = 5 μm; a–e = 10 μm

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MycoBank: MB 828729

Diagnosis. Differs from other Neofomitella species by its distinctly pileate basidiomata, white pore surface when fresh, dextrinoid skeletal and binding hyphae, and presence of orange-red colored context close to the pileal surface when fresh.

Holotype. Australia. Victoria, Yarra Ranges National Park, on dead tree of Nothofagus, 10 May 2018, Cui 16,571 (BJFC, isotype in MEL).

Etymology. Australiensis (Lat.): referring to the locality (Australia) of the type specimens.

Basidiomata. Annual to perennial, pileate, sessile, hard corky, without odor or taste when fresh, woody hard upon drying. Pilei flabelliform or semicircular, sometimes ungulate, projecting up to 10 cm, 20 cm wide, and 8 cm thick at base. Pileal surface buff, grayish-brown, yellowish-brown to umber, glabrous, concentrically sulcate with different colored zones, margin obtuse. Pore surface white when fresh, becoming buff to cinnamon-buff when dry; sterile margin distinct, buff, up to 2 mm wide; pores round to angular, 6–7 per mm; dissepiments moderate thick to thick, entire. Context cream when fresh, buff upon drying, presence of an orange red line near pileal surface when fresh, which becoming cinnamon upon drying, woody hard, up to 6 cm thick. Tubes concolorous with context, hard corky, up to 2 cm long.

Hyphal structure. Hyphal system trimitic; generative hyphae bearing clamp connections; skeletal and binding hyphae dextrinoid, CB–; tissues unchanged in KOH.

Context. Generative hyphae in infrequent, hyaline, thin-walled, unbranched, 1.6–2.8 μm in diam; skeletal hyphae dominant, pale yellowish brown, thick-walled with a narrow lumen to subsolid, unbranched, more or less regularly arranged, 2.8–5.2 μm in diam; binding hyphae pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 1.2–2.4 μm in diam.

Tubes. Generative hyphae infrequent, hyaline, thin-walled, occasionally branched, 1.4–2.3 μm in diam; skeletal hyphae dominant, pale yellowish brown, thick-walled with a narrow lumen to subsolid, unbranched, interwoven, 2.2–4.3 μm in diam; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 0.7–2.2 μm in diam. Cystidia absent, fusoid cystidioles present, hyaline, thin-walled, 9.8–14.7 × 2.8–4.2 μm. Basidia clavate, bearing four sterigmata and a basal clamp connection, 10.2–15 × 4.4–5.3 μm; basidioles in shape similar to basidia, but slightly smaller.

Spores. Basidiospores cylindrical, hyaline, thin-walled, smooth, IKI–, CB–, (3.7–)3.8–4.9(−5.2) × (1.7–)1.8–2.3(−2.5) μm, L = 4.2 μm, W = 2.02 μm, Q = 2.04–2.12 (n = 90/3).

Type of rot. White rot.

Additional specimens (paratypes) examined. Australia. Victoria, Yarra Ranges National Park, on fallen trunk of Eucalyptus, 10 May 2018, Cui 16542 & Cui 16543 (BJFC, duplicates in MEL). Victoria, Yarra Ranges National Park, on living tree of Nothofagus, 10 May 2018, Cui 16558, Cui16561 & Cui 16570 (BJFC, duplicates in MEL). Tasmania, on living tree of Eucalyptus, 10 May 2018, Cui 16642 & Cui 16679 (BJFC).

Neofomitella guangxiensisB.K. Cui & Xing Ji, sp. nov. (Figs. 2c–d, 4).

Fig. 4
figure 4

Microscopic structures of Neofomitella guangxiensis (drawn from the holotype). a Basidiospores; b Basidia and basidioles; c Cystidioles; d Hyphae from trama; e Hyphae from context. Bars: a = 5 μm; a–e = 10 μm

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MycoBank: MB 828730

Diagnosis. Differs from other Neofomitella species by its effused-reflexed to pileate basidiomata, round to angular pores (5–6 per mm), weakly dextrinoid skeletal and binding hyphae, and large basidiospores (5.5–7.5 × 1.8–2.2 μm).

Holotype. China. Guangxi Auto. Reg., Shangsi County, Shiwandashan National Forest Park, on fallen angiosperm branch, 6 July 2016, Cui 14029 (BJFC).

Etymology. Guangxiensis (Lat.): referring to the locality (Guangxi Auto. Reg.) of the type specimens.

Basidiomata. Annual, effused-reflexed to pileate, corky, without odor or taste when fresh, corky to hard corky upon drying. Pilei semicircular, projecting up to 1 cm, 3 cm wide, and 3 mm thick at base; up to 12.5 cm long, 3.5 cm wide, and 3 mm thick in the resupinate parts. Pileal surface straw-yellow, glabrous, concentrically sulcate, margin acute. Pore surface cream to clay-pink when fresh, becoming straw-yellow to clay-buff when dry; sterile margin indistinct, cream, up to 1 mm wide; pores round to angular, 5–6 per mm; dissepiments thin, entire. Context straw-yellow, corky, up to 1 mm thick. Tubes concolorous with context, corky, up to 2 mm long.

Hyphal structure. Hyphal system trimitic; generative hyphae bearing clamp connections; skeletal and binding hyphae weakly dextrinoid, CB–; tissues turning to brown in KOH.

Context. Generative hyphae infrequent, hyaline, thin-walled, rarely branched, 2–3.5 μm in diam; skeletal hyphae dominant, pale yellowish brown, thick-walled with a wide to narrow lumen, rarely branched, strongly interwoven, 2–5 μm in diam; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 1–2.5 μm in diam.

Tubes. Generative hyphae infrequent, hyaline, thin-walled, unbranched, 1.5–3 μm in diam; skeletal hyphae dominant, pale yellowish brown, thick-walled with a narrow lumen to subsolid, rarely branched, strongly interwoven, rarely simple-septate, 2–3 μm in diam; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 1–2 μm in diam. Cystidia absent, fusoid cystidioles present, hyaline, thin-walled, 10.5–14.5 × 3–4 μm. Basidia clavate, bearing four sterigmata and a basal clamp connection, 13–16.5 × 4–5 μm; basidioles in shape similar to basidia, but slightly smaller.

Spores. Basidiospores cylindrical, hyaline, smooth, thin-walled, IKI–, CB–, 5.5–7.5(−8) × (1.7–)1.8–2.2(−2.4) μm, L = 6.38 μm, W = 1.99 μm, Q = 3.12–3.31 (n = 90/3).

Type of rot. White rot.

Additional specimens (paratypes) examined. China. Guangxi Auto. Reg., Longzhou County, Nonggang Nature Reserve, on fallen angiosperm branch, 4 July 2016, Cui 13968 (BJFC); Shangsi County, Shiwandashan National Forest Park, on fallen angiosperm branch, 6 July 2016, Cui 13984 & Cui 14005 (BJFC).

Discussion

In the present study, Neofomitella australiensis from southern Australia and N. guangxiensis from southern China are supported in Neofomitella by a combined multi-gene dataset and can be distinguished from other Neofomitella species by morphological characters and phylogenetic evidence.

Neofomitella australiensis is phylogenetically related to N. fumosipora and N. rhodophaea (Fig. 1), but morphologically N. fumosipora has grayish-red context, smaller pores (7–9 per mm; Hattori 2005) and smaller basidiospores (3–4 × 1.7–2.2 μm; Li et al. 2014). Neofomitella rhodophaea differs from N. australiensis in its oblong-ellipsoid basidiospores and absence of cystidioles (Li et al. 2014). Both N. polyzonata and N. australiensis have cystidioles and cylindrical basidiospores, but N. polyzonata has velutinate pileal surface, larger pores (3–4 per mm), non-dextrinoid skeletal and binding hyphae (Li et al. 2014).

Neofomitella guangxiensis and N. fumosipora share glabrous pileal surface, and presence of cystidioles, but N. fumosipora has distinct smaller pores (7–9 per mm; Hattori 2005) and basidiospores (3–4 × 1.7–2.2 μm; Li et al. 2014). Neofomitella polyzonata is similar to N. guangxiensis in producing annual basidiomata and presence of cystidioles, but N. polyzonata has distinctly pileate basidiomata, velutinate pileal surface, larger pores (3–4 per mm) and smaller basidiospores (3.9–5 × 1.9–2.1 μm; Li et al. 2014). Both N. guangxiensis and N. rhodophaea have glabrous pileal surface, but N. rhodophaea has oblong-ellipsoid basidiospores (3.5–4.5 × 2.5–3 μm) and smaller pores (7–8 per mm), and lacks cystidioles (Li et al. 2014).

Neofomitella is phylogenetically close to Microporus. Morphologically, they both have a trimitic hyphal system, and hyaline, thin-walled, smooth, non-dextrinoid and non-amyloid basidiospores, but Microporus has centrally to laterally stipitate basidiomata with white to cream context (Gilbertson and Ryvarden 1986; Núñez and Ryvarden 2001; Li et al. 2014). Neofomitella was derived from Fomitella, they both have annual to perennial and effused-reflexed to pileate basidiomata, a trimitic hyphal system, and hyaline and thin-walled basidiospores, but Neofomitella has distinctly crusted basidiomata with the cuticle developing from base to margin, while Fomitella has the cuticle that develops from the base but does not usually extend to the very margin (Hattori 2005; Li et al. 2014). Coriolopsis strumosa (Fr.) Ryvarden is similar to Neofomitella in having encrusted basidiomata with glabrous pileal surface and a trimitic hyphal system, but C. strumosa has olivaceous-brown context and distinctly larger basidiospores (9–12 × 3–3.7 μm; Núñez and Ryvarden 2001; Li et al. 2014).

Until now, 5 species are accepted in Neofomitella. An identification key to the species of Neofomitella is provided.

Key to species of Neofomitella

  • 1 Pileal surface velutinate, pores 3–4 per mm...................................................................................... N. polyzonata

  • 1 Pileal surface glabrous, pores 5–9 per mm................. 2

  • 2 Cystidioles absent, basidiospores oblong-ellipsoid........................................................... N. rhodophaea

  • 2 Cystidioles present, basidiospores cylindrical to oblong-ellipsoid.................................................................................. 3

  • 3 Pore surface dark brown when dry, pores 7–9 per mm; basidiospores cylindrical to oblong-ellipsoid........................................................................................ N. fumosipora

  • 3 Pore surface buff, cinnamon-buff to straw-yellow when dry, pores 5–7 per mm; basidiospores cylindrical.......... 4

  • 4 Basidiomata annual, effused-reflexed to pileate, margin obtuse; basidiospores 5.5–7.5 × 1.8–2.2 μm...................................................................................... N. guangxiensis

  • 4 Basidiomata annual to perennial, pileate, margin acute; basidiospores 3.8–4.9 × 1.8–2.3 μm........ N. australiensis