Introduction

Neofavolus Sotome & T. Hatt., which was previously treated as member of Polyporus P. Micheli ex Adans. (Núñez and Ryvarden 1995), was founded based on phylogenetical and morphological analyses (Sotome et al. 2013). It is characterized by a glabrous pileus with or without scales, a hyaline to brown cutis composed of hyaline to brown agglutinated generative hyphae. The segregation of Neofavolus from Polyporus was accepted in recent studies (Dai et al. 2014; Seelan et al. 2015; Sotome et al. 2016; Zhou et al. 2016; Zmitrovich and Kovalenko 2016; Zhou and Cui 2017; Cui et al. 2019; Luo et al. 2019; Palacio et al. 2019). Currently, six species are accepted in the genus Neofavolus: N. alveolaris (DC.) Sotome & T. Hatt. (known from North America, Europe, and East Asia, Sotome et al. 2013; Zhou and Cui 2017), N. cremeoalbidus Sotome & T. Hatt. (known from East Asia, Sotome et al. 2013), N. mikawai (Lloyd) Sotome & T. Hatt. (known from China and Japan, Palacio et al. 2019), N. suavissimus (Fr.) Seelan, Justo & Hibbett (known from the USA, Europe, and Japan, Seelan et al. 2015), N. subpurpurascens (Murrill) Palacio & Robledo (known from the neotropics, Palacio et al. 2019) and N. yunnanensis C.L. Zhao (known from China, Luo et al. 2019).

During the taxonomic and phylogenetic studies of polyporoid fungi, two new Neofavolus species were discovered and confirmed after morphological and molecular analyses. Their phylogenetic relationships were analyzed based on ITS+nLSU dataset and ITS+nLSU+nSSU+mtSSU+TEF + TUB + RPB1 + RPB2 dataset.

Materials and methods

Morphological studies

The examined specimens were mainly deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC, Beijing, China); duplicates will be sent to Center for Forest Mycology Research, Northern Research Station, US Forest Service (CFMR, Madison, USA). Macro-morphological characters were described based on field notes and dried specimens. Color descriptions follow Petersen (1996). Microscopic features were observed from free-hand sections mounted in 5% KOH solution after staining in 1% Congo red. Cotton Blue (CB) and Melzer’s reagents were used to test the cyanophilous and amyloid reactions, respectively. Nikon Digital Sight DS-Fi1 microscope (Nikon Corporation, Tokyo, Japan) was used to observe and photograph the microscopic elements at magnifications of up to ×1000. Then, all the microscopic elements were measured using the Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, USA). The following abbreviations are used in this article: IKI– = neither amyloid nor dextrinoid, KOH = 5% potassium hydroxide, CB– = acyanophilous, L = mean spore length (arithmetic average of all basidiospores), W = mean spore width (arithmetic average of all basidiospores), Q = variation in the L/W ratios between the specimens studied, Qm = mean Q, n = number of basidiospores measured here.

Molecular phylogeny

A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co. Ltd., Beijing) was used to extract the total genomic DNA from dried specimens and performed the polymerase chain reaction (PCR) according to the manufacturer’s instructions with some modifications (Han et al. 2016; Shen et al. 2019). Primer pairs used in this study were listed in Table 1.

Table 1 The primers for each DNA fragment used in this study
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Final polymerase chain reaction (PCR) reaction volumes were 50 μl, included 1.5 μl for each primer (10 pM), 2 μl DNA extract, 20 μl ddH2O, and 25 μl 2 × EasyTaq PCR Supermix (TransGen Biotech Co., Ltd., Beijing, China). All DNA fragments were amplified in the S1000TM Thermal Cycler (Bio-Rad Laboratories, California, USA) and sequenced by the BGI (Beijing Genomics Institute, China) using the same primers. The PCR procedures for different DNA sequences followed those used by Zhou et al. (2016).

Besides the sequences generated in this study, other reference sequences for our phylogenetic analysis were selected from GenBank. The information of all the specimens used in this study are shown in Table 2. Sequences of Trametes conchifer (Schwein.) Pilát and T. polyzona (Pers.) Justo obtained from GenBank were selected as outgroups. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) phylogenetic analyses were performed as reported in Zhou et al. (2016) and Song and Cui (2017). RAxML v.7.2.8 was used to construct a maximum likelihood (ML) tree. Maximum parsimony (MP) analysis was applied to the combined multiple genes datasets, and the tree construction procedure was performed in PAUP* version 4.0b10. 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. 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. Bayesian inference (BI) was calculated with MrBayes 3.1.2. PhyloSuite v1.1.16 was used to determine the best-fit evolution model for the combined multi-gene dataset for Bayesian inference (BI).

Table 2 Species, specimens, and GenBank accession number of sequences used in this study
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Trees were viewed and derived by using of the FigTree 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/). The topology of the ML analysis was used, with ML bootstrap support > 50%, MP bootstrap support > 50%, and BI posterior probabilities > 0.95 shown on branches (Fig. 1 and 2).

Results

The combined ITS+nLSU dataset had an aligned length of 2017 characters, of which 1516 characters are constant, 125 are variable and parsimony-uninformative, and 376 are parsimony-informative. Maximum parsimony analysis yielded 12 equally parsimonious trees (TL = 1357, CI = 0.541, RI = 0.664, RC = 0.359, HI = 0.459). Best model for the combined ITS+nLSU dataset estimated and applied in the Bayesian analysis was GTR + F + I + G4, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). Bayesian analysis and ML analysis resulted in a similar topology as MP analysis, with an average standard deviation of split frequencies = 0.007016 (BI). The ML topology was shown in Fig. 1.

Fig. 1
figure 1

Strict consensus tree illustrating the phylogeny of Neofavolus and related taxa generated by ML analysis based on ITS+nLSU sequences. Branches are labeled with maximum likelihood bootstrap higher than 50%, parsimony bootstrap proportions higher than 50%, and Bayesian posterior probabilities more than 0.95. New species are indicated in bold

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The combined dataset (ITS+nLSU+nSSU+mtSSU+TEF + TUB + RPB1 + RPB2) had an aligned length of 7286 characters, of which 5098 characters are constant, 529 are variable and parsimony-uninformative, and 1659 are parsimony-informative. Maximum parsimony analysis yielded 2 equally parsimonious trees (TL = 6104, CI = 0.559, RI = 0.618, RC = 0.345, HI = 0.441). Best model for the combined 8-gene dataset estimated and applied in the Bayesian analysis was SYM + I + G4, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). Bayesian analysis and ML analysis resulted in a similar topology as MP analysis, with an average standard deviation of split frequencies = 0.002240 (BI). The ML topology was shown in Fig. 2.

Fig. 2
figure 2

Strict consensus tree illustrating the phylogeny of Neofavolus and related taxa generated by ML analysis based on ITS+nLSU+nSSU+mtSSU+TEF + TUB + RPB1 + RPB2 sequences. Branches are labeled with maximum likelihood bootstrap higher than 50%, parsimony bootstrap proportions higher than 50%, and Bayesian posterior probabilities more than 0.95. New species are indicated in bold

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In the two phylogenetic results, both Favolus and Neofavolus are well supported as individual monophyletic groups, and the materials examined are highly supported as two new species in the Neofavolus clade.

Taxonomy

Neofavolus americanus J.H. Xing, J.L. Zhou & B.K. Cui, sp. nov. (Figs. 3 and 4)

Fig. 3
figure 3

Basidiomata of Neofavolus americanus (Dai 12761). Scale bar = 1 cm

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Fig. 4
figure 4

Microscopic structures of Neofavolus americanus. a Basidiospores; b Basidia and basidioles; c Hyphae from context; d Hyphae from trama; e Hyphae from stipe. Scale bars = 10 μm

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MycoBank no.: MB 834716

Etymology. americanus (Lat.) referring to the geographic distribution in America(USA).

Fruiting body: Basidiomata annual, laterally stipitate, leathery when fresh, and hard when dry. Pilei semicircular, depressed toward the stipe, 1.8–2.3 cm long from base to margin, 2.8–3.5 cm wide, and up to 3 mm thick. Pileal surface glabrous, white to cream when fresh, buff to buff-yellow and with slightly radially aligned stripes on drying; margin plane when fresh and straight to slightly incurved upon drying. Pore surface white to cream when fresh, cream to saffron yellow when dry; pores angular to elongate, 1–3 mm long and 0.5–1 mm wide; dissepiments thin, entire to lacerate. Context white when fresh and ivory to cream when dry, woody hard upon drying, up to 1 mm thick. Tubes concolorous with pore surface, decurrent on one side of the stipe, up to 2 mm thick. Stipe short, glabrous, concolorous with pileal surface or slightly lighter when dry, up to 8 mm long and 7 mm in diameter.

Hyphal structure: Hyphal system dimitic; generative hyphae bearing clamp connections; skeleto-binding hyphae IKI–, CB+; tissues unchanged in KOH.

Context: Generative hyphae frequent, hyaline, thin-walled, frequently branched, and 2–6.4 μm in diameter; skeleto-binding hyphae dominant, hyaline, thick-walled with a wide to narrow lumen, frequently branched, interwoven, and 1.8–9.7 μm in diameter.

Tubes: Generative hyphae frequent, hyaline, thin-walled, frequently branched, and 2–3.8 μm in diameter; skeleto-binding hyphae dominant, hyaline, thick-walled with a wide to narrow lumen, frequently branched, interwoven, and 2–4.5 μm in diameter. Cystidia and cystidioles absent. Basidia clavate, with a basal clamp and four sterigmata, 17.7–28 × 7–8.7 μm; basidioles in shape similar to basidia, but smaller.

Stipe: Generative hyphae frequent, hyaline, thin-walled, frequently branched, and 2–7 μm in diameter; skeleto-binding hyphae dominant, thick-walled with a narrow to wide lumen, frequently branched, interwoven, and 2–9 μm in diameter.

Spores: Basidiospores cylindrical, hyaline, thin-walled, smooth, frequently bearing small guttules, IKI–, CB–, (9.5–)10.4–12(−12.7) × (3.7–)3.8–4.5 μm, L = 11.18 ± 0.68 μm, W = 4.07 ± 0.18 μm, Q = 2.4–3.1, and Qm = 2.75 ± 0.17 (n = 53/1).

Rot type: A white rot.

Specimens examined: USA, CT, New Haven, Sleeping Giant State Park, on fallen branch of Quercus, 21 July 2012, Dai 12761 (holotype, BJFC).

Neofavolus squamatus J.H. Xing, J.L. Zhou & B.K. Cui, sp. nov. (Figs. 5 and 6)

Fig. 5
figure 5

Basidiomata of Neofavolus squamatus (Cui 12175). Scale bar = 1 cm

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Fig. 6
figure 6

Microscopic structures of Neofavolus squamatus. a Basidiospores; b Basidia and basidioles; c Hyphae from context; d Hyphae from pileal squamae; e Hyphae from trama; f Hyphae from stipe. Scale bars = 10 μm

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MycoBank no.: MB 834717

Etymology: squamatus (Lat.) referring to the squamae on the pileal surface.

Fruiting body: Basidiomata annual, solitary, laterally stipitate, and soft-leathery when fresh and corky when dry. Pilei suborbicular, depressed toward the stipe, 1.1–2.7 cm long from base to margin, 3–3.8 cm wide, and up to 3 mm thick. Pileal surface white when fresh and buff to festucine when dry, covered by yellowish orange squamae when fresh, discoloring to apricot orange when dry, and more or less radially wrinkling on drying; margin incurved upon drying. Pore surface white to cream when fresh and buff when dry; pores angular, 0.7–3 mm long and 0.5–1.5 mm wide; dissepiments thin, entire. Context white, corky upon drying, up to 2.5 mm thick. Tubes white when fresh and light ivory to cream when dry, up to 1 mm thick, and decurrent on one side of the stipe. Stipe short, glabrous, white when fresh and buff yellow after drying, up to 6 mm long, and 5.5 mm in diameter.

Hyphal structure: Hyphal system dimitic; generative hyphae bearing clamp connections, hyaline, and thin-walled; skeleto-binding hyphae hyaline, thick-walled with a wide to narrow lumen, occasionally branched and with tapering ends, IKI–, and CB+; tissue unchanged in KOH.

Context: Generative hyphae frequent, hyaline, thin-walled, frequently branched, 2.8–9 μm in diameter, and occasionally inflated up to 27.7 μm in diameter at branched area; skeleto-binding hyphae dominant, hyaline, thick-walled with a wide lumen, occasionally branched, interwoven, 2.6–9.5 μm in diameter, and occasionally inflated up to 13.5 μm in diameter. Hyphae in squamae with buff inclusion inside, thin-walled hyphae bearing clamp connections, thick-walled hyphae simple-septate with a wide lumen, and 3.5–8.4 μm in diameter.

Tubes: Generative hyphae frequent, hyaline, thin-walled, frequently branched, and 2.2–4.2 μm in diam; skeleto-binding hyphae dominant, hyaline, thick-walled with a wide lumen, occasionally branched, interwoven, and 1.9–6.4 μm in diameter. Cystidia and cystidioles absent. Basidia clavate, with a basal clamp and four sterigmata, 27.7–51.7 × 6.5–9.8 μm; basidioles in shape similar to basidia, but smaller.

Stipe: Generative hyphae infrequent, hyaline, thin-walled, frequently branched, 2.2–6.3 μm in diameter, and occasionally inflated up to 16 μm in diameter at clamping area; skeleto-binding hyphae dominant, hyaline, thick-walled with a wide to narrow lumen, occasionally branched, interwoven, and 2.6–8.6 μm in diameter.

Spores: Basidiospores cylindrical to navicular, hyaline, thin-walled, smooth, occasionally bearing one or two guttules, IKI–, CB–, (7.8–)8.9–12(−14.5) × 3.1–4.1(−4.3) μm, L = 10.34 ± 1.09 μm, W = 3.63 ± 0.29 μm, Q = 2.17–3.72, and Qm = 2.86 ± 0.31 (n = 70/1).

Rot type: A white rot.

Specimens examined: CHINA, Xizang, Linzhi County, Lulang, Sejila Mountain, on fallen angiosperm branch, 18 September 2014, Cui 12175 (holotype, BJFC).

Discussion

In the present study, N. americanus, from USA, and N. squamatus, from China, are described in Neofavolus based on morphological evidence and multi-gene phylogenetic inferences.

Neofavolus americanus is a species known so far from the temperate USA. It has glabrous basidiomata, large and elongate pores, and large basidiospores. Phylogenetically, N. americanus clustered with N. alveolaris (Figs. Fig. 1 and 2). However, the basidiospores of N. americanus are much larger than those of N. alveolaris (7–10 × 2.5–4 μm; Sotome et al. 2013). Neofavolus cremeoalbidus is similar to N. americanus; both share the light colored pileus and pore surface and short and lateral stipe, but based on our specimens collected from China, the former has much smaller pores (2–4 per mm) and basidiospores (8–10.7 × 3–3.8 μm). Moreover, N. cremeoalbidus is only known from East Asia, in China and Japan, while N. americanus is limited to northern–eastern USA. Neofavolus mikawai shares the cream pore surface and the short lateral stipe with N. americanus, but it differs in its smaller pores (3–5 per mm; Sotome et al. 2013) and basidiospores (6–9.5 × 2.3–3.6 μm; Sotome et al. 2013).

Neofavolus squamatus is a special species which in known from the Tibetan Plateau. It is characterized by its bright squamae on the pileus. It was initially thought to be a member of Polyporus squamosus (Huds.) Fr. for its squamae and large pores. However, phylogenetic analyses (Figs. Fig. 1 and 2) showed that it nested within the Neofavolus lineage. Morphologically, N. squamatus differs from other species in the genus in having smaller basidiospores, thinner basidiomata, and a white stipe. Neofavolus yunnanensis is another Neofavolus species with scaled pileal surface and large angular pores, but it has light-colored pileus and much smaller basidiospores (5.5–7.5 × 2–3 μm, L = 6.4 μm, W = 2.65 μm; Luo et al. 2019) compared with N. squamatus. Although N. alveolaris occasionally has scales on the pileus, it has smaller basidiospores (7–10 × 2.5–4 μm, L = 8.29 μm, W = 3.01 μm; Sotome et al. 2013) and basidia (17.5–26 × 4–7 μm; Sotome et al. 2013) compared to those of N. squamatus. Favolus roseus Lloyd also has orangish basidiomata, large pores, and cylindrical basidiospores (7–12 × 2.4–4.2 μm), which are similar to N. squamatus, but its glabrous pileal surface, yellowish orange to brownish orange pore surface, and tropical distribution (Sotome et al. 2013) are different from N. squamatus.

Sotome et al. (2013) concluded that species of Neofavolus occur in temperate regions and are unknown from the tropics. We also previously suggested that temperature may be a critical variable affecting the distributions of Neofavolus species (Zhou and Cui 2017). However, several tropical specimens of N. mikawai and N. alveolaris collected from China (Zhou and Cui 2017; Luo et al. 2019) and the neotropical species (N. subpurpurascens) reported from Jamaica, Brazil (Coelho and Silveira 2014), and Bolivia (Palacio et al. 2019) may overthrow the inference, and it will be more convincing if sequences of N. subpurpurascens could be available from the type locality of Jamaica.