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Research Article
Crassisporus gen. nov. (Polyporaceae, Basidiomycota) evidenced by morphological characters and phylogenetic analyses with descriptions of four new species
expand article infoXing Ji, Dong-Mei Wu§, Shun Liu, Jing Si, Bao-Kai Cui
‡ Beijing Forestry University, Beijing, China
§ Biotechnology Research Institute, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi, China
Open Access

Abstract

A new poroid wood-inhabiting fungal genus, Crassisporus gen. nov., is proposed on the basis of morphological characters and molecular evidence. The genus is characterized by an annual growth habit, effused-reflexed to pileate basidiocarps with pale yellowish brown to yellowish brown, concentrically zonate or sulcate, and velutinate pileal surface, a trimitic hyphal system with clamped generative hyphae, tissues turning to dark in KOH, oblong to broadly ellipsoid, hyaline, smooth, and slightly thick-walled basidiospores. Phylogenetic analysis based on ITS+nLSU sequences indicate that Crassisporus belongs to the core polyporoid clade. The combined ITS+nLSU+mtSSU+EF1-α+RPB2 sequences dataset of representative taxa in the Polyporaceae demonstrate that Crassisporus is grouped with Haploporus but forms a monophyletic lineage. In addition, four new species of Crassisporus, C. imbricatus, C. leucoporus, C. macroporus, and C. microsporus are described.

Keywords

core polyporoid clade, molecular phylogeny, polypore, taxonomy, wood-decaying fungi

Introduction

Polyporales is one of the most diverse orders of Basidiomycota including more than 1800 described species in 216 genera and 13 families (Kirk et al. 2008). In the last 10 years, many new genera in Polyporales have been established, such as Datroniella B.K. Cui, Hai J. Li & Y.C. Dai (Li et al. 2014a), Fragifomes B.K. Cui, M.L. Han & Y.C. Dai (Han et al. 2016), Megasporia B.K. Cui, Y.C. Dai & Hai J. Li (Li and Cui 2013), Pseudofibroporia Yuan Y. Chen & B.K. Cui (Chen et al. 2017), and Pseudomegasporoporia X.H.Ji & F.Wu (Ji and Wu 2017).

During the investigations of species diversity and phylogeny of Polyporales, four new species were found that did not belong to any known genus, for which reason, a new genus is established to accommodate them. Morphologically, these four taxa do not fit any of the known polypore taxa. To confirm the position of the new genus, phylogenetic analyses of the new genus and related taxa within Polyporales were carried out based on the internal transcribed spacer (ITS) regions, the large subunit nuclear ribosomal RNA gene (nLSU), the small subunit mitochondrial rRNA gene sequences (mtSSU), the translation elongation factor 1-α gene (EF1-α), and the second largest subunit of RNA polymerase II (RPB2).

Materials and methods

Morphological studies

The studied specimens were deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Macro-morphological descriptions are based on the field notes and measurements of herbarium specimens. Color terms follow Petersen (1996). Micro-morphological data were obtained from the dried specimens and observed under a light microscope following Shen et al. (2019). Sections were studied at magnifications up to 1000× using a Nikon E80i microscope and phase contrast illumination (Nikon, Tokyo, Japan). 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. In presenting the variation of spore size, 5% of measurements were excluded from each end of the range, and were given in parentheses. The following abbreviations were used: KOH, 5% potassium hydroxide; IKI, Melzer’s reagent; IKI-, neither amyloid nor dextrinoid; CB, cotton blue; CB+, cyanophilous; CB-, acyanophilous; 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). 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 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). RPB2 was amplified with primer pairs fRPB2-5F and fRPB2-7cR or bRPB2-6F and bRPB2-7R (Liu et al. 1999; Matheny 2005). The PCR procedure for ITS, mtSSU and EF1-α was as follows: initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, 54 °C for ITS and mtSSU, 54–56 °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 was as follows: initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 50 °C for 1 min, 72 °C for 1.5 min, and a final extension of 72 °C for 10 min. The PCR procedure for RPB2 was as follows: 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, 55–57 °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 (Table 1).

Table 1.

Species, specimens and GenBank accession numbers of sequences used in this study.

Species Sample no. Locality GenBank accessions
ITS nLSU mtSSU EF1-α RPB2
Abortiporus biennis EL 65-03 Sweden JN649325 JN649325
Abundisporus fuscopurpureus Cui 10950 China KC456254 KC456256 KF051025 KF181154
A. pubertatis Dai 11310 China KC787568 KC787575 KF051031 KF181125
Dai 11927 China KC787569 KC787576 KF051034 KF181128
A. sclerosetosus MUCL 41438 Singapore FJ411101 FJ393868
A. violaceus Ryvarden 32807 Finland KF018127 KF018135 KF051038 KF181132
Antrodia albida CBS 308.82 USA DQ491414 AY515348
A. macra MUAF 887 Czech Republic EU340898
Bjerkandera adusta NBRC 4983 Unknown AB733156 AB733333
Cinereomyces lindbladii KHL 12078 Norway FN907906 FN907906
Climacocystis borealis KHL 13318 Estonia JQ031126 JQ031126
Coriolopsis brunneoleuca Cui 13911 China MK116480a MK116489a MK116498a MK124544a
C. brunneoleuca Dai 12180 China KC867414 KC867432 KF274655
C. polyzona BKW004 Ghana JN164978 JN164790 JN164881 JN164856
C. retropicta Cui 13849 China MK116481a MK116490a MK116499a MK122979a MK124545a
Cui 14030 China MK116482a MK116491a MK116500a MK122980a MK122987a
Crassisporus imbricatus Cui 6556 China KC867351 KC867426
C. imbricatus Dai 10788 China KC867350 KC867425 KX838374
C. leucoporus Cui 16801 Australia MK116488a MK116497a MK116507a MK122986a MK122993a
C. macroporus Cui 14465 China MK116485a MK116494a MK116504a MK122983a MK122990a
Cui 14468 China MK116486a MK116495a MK116505a MK122984a MK122991a
C. microsporus Cui 16221 China MK116487a MK116496a MK116506a MK122985a MK122992a
Daedaleopsis confragosa Cui 6892 China KU892428 KU892448 KX838381 KX838418 KU892507
D. hainanensis Cui 5178 China KU892435 KU892462 KX838413 KX838441 KU892495
Datronia mollis RLG6304sp USA JN165002 JN164791 JN164901 JN164872
Earliella scabrosa PR1209 Puerto Rico JN165009 JN164793 JN164866
Fomes fomentarius ES 2008-3 Sweden JX109860 JX109860
Fomitella supina JV0610 Guatemala KF274645 KF274646
F. supina Ryvarden 39027 Puerto Rico KF274643
Fomitopsis betulina Dai 11449 China KR605798 KR605737 KR605998 KR610726 KR610816
F. pinicola Cui 10405 China KC844852 KC844857 KR605961 KR610690 KR610781
Fragiliporia fragilis Dai 13080 China KJ734260 KJ734264
F. fragilis Dai 13559 China KJ734261 KJ734265
Yuan 5516 China KJ734263 KJ734267
Funalia gallica Dai 10977 China KC867378 KC867452 KU182651
F. trogii RLG4286Sp USA JN164993 JN164808 JN164898 JN164867
Gelatoporia subvermispora BRNU 592909 Czech Republic FJ496694 FJ496706
Grammethelopsis subtropica Cui 9035 China JQ845094 JQ845097 KF051030 KF181124
Cui 9041 China JQ845096 JQ845099 KF051039 KF181133
Haploporus latisporus Dai 11873 China KU941847 KU941871 KU941896 KU941934 KU941918
H. latisporus Dai 10562 China KU941848 KU941872 KU941897 KU941935 KU941919
H. odorus Yuan 2365 China KU941846 KU941870 KU941895 KU941933 KU941917
Dai 11296 China KU941845 KU941869 KU941894 KU941932 KU941916
H. subtrameteus Dai 4222 China KU941849 KU941873 KU941898 KU941936 KU941920
Cui 10656 China KU941850 KU941874 KU941899 KU941937 KU941921
Heterobasidion annosum PFC 5327 Greece KC492915 KC571655
Hexagonia apiaria Cui 6447 China KC867362 KC867481 MG847228 MG867697 KF274660
H. apiaria Dai 10784 China KX900635 KX900682 KX900732 KX900822 MG867677
H. hirta Dai 5081 China KC867486
Cui 4051 China KC867359 KC867471
Hornodermoporus latissimus Cui 6625 China HQ876604 JF706340 KF051040 KF181134
Dai 12054 China KX900639 KX900686 KF218297 KF286303
H. martius MUCL 41677 Argentina FJ411092 FJ393859
MUCL 41678 Argentina FJ411093 FJ393860
Hydnopolyporus fimbriatus LR 40855 Puerto Rico JN649347 JN649347
Hypochnicium lyndoniae NL 041031 UK JX124704 JX124704
Laetiporus montanus Cui 10011 China KF951274 KF951315 KX354570 KX354617 KT894790
L. sulphureus Cui 12388 China KR187105 KX354486 KX354560 KX354607 KX354652
Lenzites betulina HHB9942Sp USA JN164983 JN164794 JN164895 JN164860
Megasporia ellipsoidea Cui 13854 China MK116483a MK116492a MK116501a MK122981a MK122988a
M. major Cui 10253 China JQ314366 JQ780437 MK116502a
Megasporoporiella rhododendri Cui 10745 China MK116484a MK116493a MK116503a MK12298222982a MK122989a
M. subcavernulosa Cui 14247 China MG847213 MG847222 MG847234 MG867705 MG867685
Microporus affinis Cui 7714 China JX569739 JX569746 KX880696 KF274661
M. vernicipes Dai 9283 China KX880618 KX880658 KX880701 KX880926
M. xanthopus Cui 8284 China JX290074 JX290071 KX880703 KX880878 JX559313
Neodatronia sinensis Dai 11921 China JX559272 JX559283 JX559320
Neofomitella fumosipora Cui 8816 China JX569734 JX569741 KX900766
Cui 13581a China KX900664 KX900714 KX900767 KX900848 KX900815
N. rhodophaea TFRI 414 Unknown EU232216 EU232300
Obba rivulosa KCTC 6892 Canada FJ496693 FJ496710
Perenniporia hainaniana Cui 6364 China JQ861743 JQ861759 KF051044 KF181138
P. hainaniana Cui 6365 China JQ861744 JQ861760 KF051045 KF181139
P. medulla-panis MUCL 49581 Poland FJ411088 FJ393876
Cui 14515 China MG847214 MG847223 MG867707 MG867687
P. substraminea Cui 10177 China JQ001852 JQ001844 KF051046 KF181140
Cui 10191 China JQ001853 JQ001845 KF051047 KF181141
Perenniporiella chaquenia MUCL 47648 Argentina FJ411084 FJ393856 HM467610
P. micropora MUCL 43581 Cuba FJ411086 FJ393858 HM467608
P. neofulva MUCL 45091 Cuba FJ411080 FJ393852 HM467599
P. pendula MUCL 46034 Cuba FJ411081 FJ393853 HM467601
Phanerochaete chrysosporium BKM-F-1767 USSR HQ188436 GQ470643
Phlebia unica KHL 11786 Sweden EU118657 EU118657
Pycnoporus cinnabarinus Dai 14386 China KX880629 KX880667 KX880712 KX880885 KX880854
Skeletocutis amorpha Miettinen 11038 Finland FN907913 FN907913
Stereum hirsutum NBRC 6520 Unknown AB733150 AB733325
Trametes conchifer FP106793Sp USA JN164924 JN164797 JN164887 JN164849
T. pubescens FP101414Sp USA JN164963 JN164811 JN164889 JN164851
T. tephroleuca Cui 7987 China KC848293 KC848378 KX880755 KX880934 KX880869
T. versicolor FP135156Sp USA JN164919 JN164809 JN164878 JN164850
Truncospora detrita MUCL 42649 French Guyana FJ411099 FJ393866
T. macrospora Cui 8106 China JX941573 JX941596 KX880763 KX880920 KX880871
Yuan 3777 China JX941574 JX941597
T. ochroleuca MUCL 39726 China Taiwan FJ411098 FJ393865
Cui 5671 China JX941584 JX941602 KF218309 KF286315
T. ohiensis MUCL 41036 USA FJ411096 FJ393863
Tyromyces chioneus Cui 10225 China KF698756 KF698745
T. kmetii Penttilä 13474 China KF705040 KF705041
Vanderbylia fraxinea DP 83 Italy AM269789 AM269853
MUCL 39326 France FJ411094 FJ393861
V. robiniophila Cui 5644 China HQ876609 JF706342 KF051051 KF181145 MG867691
Cui 7144 China HQ876608 JF706341 KF051052 KF181146
V. vicina MUCL 44779 Ethiopia FJ411095 FJ393862
Whitfordia scopulosa Dai 10739 China KC867364 KC867482 KX880766 KX880922 MG867692

Phylogenetic analyses

Sequences used for phylogenetic analyses in this study are listed in Table 1. Sequences of ITS, nLSU, mtSSU, EF1-α, and RPB2 were aligned initially in MAFFT 7 (Katoh and Standley 2013; http://mafft.cbrc.jp/alignment/server/) and then manually adjusted in BioEdit (Hall 1999). Finally, these gene fragments were concatenated with Mesquite 3.2 (Maddison and Maddison 2017) for further phylogenetic analyses. Phylogenies were inferred from the combined 2-gene dataset (ITS+nLSU) and 5-gene dataset (ITS+nLSU+mtSSU+EF1-α+RPB2). Heterobasidion annosum (Fr.) Bref. and Stereum hirsutum (Willd.) Pers. obtained from GenBank were used as outgroups to root trees in the 2-gene based analysis. Laetiporus montanus Černý ex Tomšovský & Jankovský and L. sulphureus (Bull.) Murrill were selected as outgroups to root trees in the 5-gene based analysis. The final concatenated sequence alignments were deposited in TreeBase (https://treebase.org/treebase-web/home.html; submission ID 23521).

Phylogenetic analyses used in this study followed the approach of Zhu et al. (2019) and Song and Cui (2017). Maximum parsimony (MP) analysis was performed in PAUP* v. 4.0b10 (Swofford 2002). 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. 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 generated.

RAxML v. 7.2.6 (Stamatakis 2006) was used to perform maximum likelihood (ML) analysis 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 assess the reliability of the nodes.

MrModeltest v. 2.3 (Posada and Crandall 1998; Nylander 2004) was used to determine the best fit evolution model for the combined multi-gene dataset for Bayesian inference (BI). Bayesian inference was calculated with MrBayes v. 3.1.2 with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist and Huelsenbeck 2003). Four Markov chains were run for two runs from random starting trees for 2 million generations (ITS+nLSU), for 5 million generations (ITS+nLSU+mtSSU+EF1-α+RPB2) until the split deviation frequency value <0.01, and trees were sampled every 100 generation. The first quarter generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated.

Phylogenetic trees were viewed using FigTree v. 1.4.2 (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 2-gene dataset included sequences from 68 fungal samples representing 59 taxa. The dataset had an aligned length of 2111 characters, of which 1249 characters were constant, 196 were variable and parsimony-uninformative, and 666 were parsimony-informative. MP analysis yielded 37 equally parsimonious trees (TL = 4143, CI = 0.345, RI = 0.617, RC = 0.213, HI = 0.655). Best model for the combined 2-gene dataset estimated and applied in the BI was GTR+I+G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). MP, ML and BI analyses yielded similar tree topologies with an average standard deviation of split frequencies = 0.006293 (BI), and the ML topology is shown in Figure 1. The phylogeny (Fig. 1) inferred from the combined ITS+nLSU sequences demonstrated seven major clades for 59 species of the Polyporales. The new genus Crassisporus embed in the core polyporoid clade and grouped with Haploporus Bondartsev & Singer.

Figure 1. 

Phylogeny of Crassisporus and related genera in Polyporales based on combined ITS and nLSU sequences. Topology is from ML analysis with parsimony bootstrap support values (≥50 %), maximum likelihood bootstrap support values (≥50 %) and Bayesian posterior probability values (≥0.95).

The combined 5-gene (ITS, nLSU, mtSSU, EF1-α, RPB2) dataset included sequences of 82 fungal samples representing 57 taxa. The dataset had an aligned length of 4306 characters, of which 2521 characters were constant, 258 were variable and parsimony-uninformative, and 1527 were parsimony-informative. MP analysis yielded 1 equally parsimonious tree (TL = 8989, CI = 0.339, RI = 0.620, RC = 0.210, HI = 0.661). Bayesian and ML analyses resulted in a similar topology as the MP analysis, with an average standard deviation of split frequencies = 0.006328 (BI); and the ML topology is shown in Figure 2. A further phylogeny (Fig. 2) inferred from the combined 5-gene dataset was obtained for more representative taxa in the Polyporaceae and showed that the new genus grouped with Haploporus clade but distinctly formed a monophyletic lineage.

Figure 2. 

Phylogeny of Crassisporus and related species obtained for more representative taxa in the Polyporaceae based on combined sequences dataset of ITS+nLSU+mtSSU+EF1-α+RPB2. Topology is from ML analysis with parsimony bootstrap support values (≥50 %), maximum likelihood bootstrap support values (≥50 %), and Bayesian posterior probability values (≥0.95).

Taxonomy

Crassisporus B.K. Cui & Xing Ji, gen. nov.

MycoBank No: 828486

Notes

Differs from other genera by the combination of effused-reflexed to pileate basidiocarps, pale yellowish brown to yellowish brown, concentrically zonate or sulcate, velutinate pileal surface, a trimitic hyphal system with clamped generative hyphae, tissues darkening in KOH, and oblong to broadly ellipsoid, hyaline, smooth and slightly thick-walled basidiospores.

Etymology

Crassisporus (Lat.): referring to thick-walled basidiospores.

Type species

Crassisporus macroporus B.K. Cui & Xing Ji.

Basidiocarps annual, effused-reflexed to pileate. Pileal surface pale yellowish brown, yellowish brown to umber-brown when dry, concentrically zonate or sulcate, velutinate. Pore surface usually white, cream buff to cinnamon-buff when fresh, buff, pale yellowish brown to yellowish brown when dry. Context pale yellowish brown to yellowish brown, leathery to corky when dry. Tubes concolorous with the context, corky when dry. Hyphal system trimitic with clamped generative hyphae, skeletal hyphae hyaline to pale yellowish brown, binding hyphae hyaline to pale yellowish brown, negative in Melzer’s reagent, tissues turning to black in KOH. Cystidia absent, thin-walled cystidioles usually present. Basidiospores oblong to broadly ellipsoid, hyaline, smooth, slightly thick-walled, IKI-, CB-. Causing a white rot.

Crassisporus imbricatus B.K. Cui & Xing Ji, sp. nov.

MycoBank No: 828487
Figs 3, 4

Notes

Crassisporus imbricatus is characterized by imbricate basidiocarps, pale greyish-brown pore surface when dry, round to angular pores (3–5 per mm), and oblong ellipsoid basidiospores (10–14 × 4.5–6.2 μm).

Holotype

CHINA. Hainan Province, Changjiang County, Bawangling Nature Reserve, on dead angiosperm tree, 9 May 2009, Dai 10788 (BJFC).

Etymology

Imbricatus (Lat.): referring to the imbricate basidiocarps.

Description

Fruitbody: Basidiocarps annual, effused-reflexed to pileate, imbricate, soft corky, without odor or taste when fresh, leathery to corky upon drying. Pilei semicircular or elongated, projecting up to 1.5 cm, 3.5 cm wide, and 2.5 mm thick at base. Pileal surface yellowish brown, velutinate, concentrically zonate. Pore surface buff when fresh, becoming pale greyish brown when dry; sterile margin indistinct, pores round to angular, 3–5 per mm; dissepiments slightly thick, entire to slightly lacerate. Context yellowish brown, leathery, up to 2.5 mm thick. Tubes concolorous with context, corky, up to 1.5 mm long.

Hyphal structure: Hyphal system trimitic; generative hyphae bearing clamp connections; skeletal and binding hyphae IKI-, CB-; tissues turning to black in KOH.

Context: Generative hyphae infrequent, hyaline, thin-walled, unbranched, 2–3.5 μm in diam.; skeletal hyphae dominant, hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, rarely branched, straight, interwoven, occasionally simple-septate, 2.5–5.5 μm in diam.; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 1.2–2.5 μm in diam.

Tubes: Generative hyphae infrequent, hyaline, thin-walled, occasionally branched, 1.5–3 μm in diam.; skeletal hyphae dominant, hyaline to pale yellowish brown, thick-walled, occasionally branched, strongly interwoven, rarely simple-septate, 1.5–3.5 μm in diam.; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 1–2 μm in diam. Cystidia and cystidioles absent. Basidia clavate, bearing four sterigmata and a basal clamp connection, 19–32 × 9–12 μm; basidioles dominant, in shape similar to basidia, but distinctly smaller.

Spores: Basidiospores oblong ellipsoid, hyaline, slightly thick-walled, smooth, IKI-, CB-, 10–14(–15) × 4.5–6.2(–6.6) μm, L = 12.33 μm, W = 5.34 μm, Q = 2.27–2.36 (n = 60/2).

Type of rot

White rot.

Additional specimen (paratype) examined

CHINA. Hainan Province, Changjiang County, Bawangling Nature Reserve, on fallen branch of Pinus latteri, 10 May 2009, Cui 6556 (BJFC).

Figure 3. 

Basidiocarps of Crassisporus imbricatus. Scale bar: 2 cm.

Figure 4. 

Microscopic structures of Crassisporus imbricatus (drawn from the holotype) A basidiospores B basidia and basidioles C hyphae from trama D hyphae from context.

Crassisporus leucoporus B.K. Cui & Xing Ji, sp. nov.

MycoBank No: 828488
Figs 5, 6

Notes

Crassisporus leucoporus is characterized by a white pore surface when fresh, round to angular pores (3–4 per mm) and oblong ellipsoid basidiospores (8.4–11.2 × 4.2–5.4 μm).

Holotype

AUSTRALIA. Queensland, Cairns, Roadside of Mount Whitfield Park, on fallen angiosperm branch, 18 May 2018, Cui 16801 (BJFC).

Etymology

Leucoporus (Lat.): referring to the white pore surface when fresh.

Description

Fruitbody: Basidiocarps annual, effused-reflexed to pileate, corky, without odor or taste when fresh, soft leathery to corky upon drying. Pilei semicircular or elongated, projecting up to 1.5 cm, 3 cm wide, and 6 mm thick at base. Pileal surface yellowish brown to umber-brown, finely velutinate, concentrically sulcate. Pore surface white when fresh, becoming cream, clay buff to pale yellowish brown when dry; sterile margin distinct, cream to pale yellowish brown, up to 1.5 mm wide; pores round to angular, 3–4 per mm; dissepiments slightly thick, entire. Context pale yellowish brown to fulvous, leathery, up to 3 mm thick. Tubes pale yellowish brown, corky, up to 2.5 mm long.

Hyphal structure: Hyphal system trimitic; generative hyphae bearing clamp connections; skeletal and binding hyphae IKI-, CB-; tissues turning to black in KOH.

Context: Generative hyphae infrequent, hyaline, thin-walled, unbranched, 1.1–2.6 μm in diam.; skeletal hyphae in context dominant, pale yellowish brown, thick-walled with a narrow to wide lumen, unbranched, straight, interwoven, occasionally simple-septate, 1.8–3.9 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.

Tubes: Generative hyphae infrequent, hyaline, thin-walled, occasionally branched, 1–2.8 μm in diam.; skeletal hyphae dominant, hyaline to pale yellowish brown, thick-walled with a narrow to wide lumen, occasionally branched, more or less straight, strongly interwoven, 0.9–3.3 μm in diam.; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 0.8–2.1 μm in diam. Cystidia absent, cystidioles fusoid, sometimes septate at the tips, hyaline, thin-walled, 16.7–28.1 × 5.1–6.3 μm. Basidia clavate, bearing four sterigmata and a basal clamp connection, 18.1–29.2 × 6.4–9.8 μm; basidioles dominant, in shape similar to basidia, but smaller.

Spores: Basidiospores oblong ellipsoid, hyaline, smooth, slightly thick-walled, IKI-, CB-, (7.9–)8.4–11.2(–11.5) × (4–)4.2–5.4(–5.7) μm, L = 9.49 μm, W = 4.79 μm, Q = 1.99 (n = 60/1).

Type of rot

White rot.

Figure 5. 

Basidiocarps of Crassisporus leucoporus. Scale bars: 1 cm (A); 2 cm (B).

Figure 6. 

Microscopic structures of Crassisporus leucoporus (drawn from the holotype) A basidiospores B basidia and basidioles C cystidioles D hyphae from trama E hyphae from context.

Crassisporus macroporus B.K. Cui & Xing Ji, sp. nov.

MycoBank No: 828489
Figs 7, 8

Notes

Crassisporus macroporus is characterized by cream-buff to cinnamon-buff colored pore surface with distinct sterile margin when fresh, large pores (2–3 per mm) with thin dissepiments, a trimitic hyphal system with cyanophilous skeletal hyphae, the presence of fusoid cystidioles, and oblong ellipsoid basidiospores (9.5–13.2 × 4–6.2 μm).

Holotype

CHINA. Guangxi Autonomous Region, Huanjiang County, Mulun Nature Reserve, on fallen angiosperm branch, 10 July 2017, Cui 14468 (BJFC).

Etymology

Macroporus (Lat.): referring to the large pores.

Description

Fruitbody: Basidiocarps annual, effused-reflexed to pileate, corky to leathery, without odor or taste when fresh, soft leathery upon drying. Pilei flabelliform, semicircular or elongated, projecting up to 1.5 cm, 4 cm wide and 5 mm thick at base; resupinate part up to 7 cm long, 4 cm wide, and 5 mm thick at center. Pileal surface buff to yellowish brown when fresh, becoming yellowish brown upon drying, finely velutinate, concentrically sulcate. Pore surface cream, buff to cinnamon-buff when fresh, becoming buff, pale yellowish brown to yellowish brown when dry; sterile margin distinct, buff to pale yellowish brown, up to 2 mm wide; pores round to angular, 2–3 per mm; dissepiments thin, entire to lacerate. Context yellowish brown to pale yellowish brown, leathery, up to 1.5 mm thick. Tubes pale yellowish brown, corky, up to 2 mm long.

Hyphal structure: Hyphal system trimitic; generative hyphae bearing clamp connections; skeletal and binding hyphae IKI-, CB+; tissues turning to black in KOH.

Context: Generative hyphae infrequent, hyaline, thin-walled, unbranched, 1.5–3.5 μm in diam.; skeletal hyphae dominant, pale yellowish brown, thick-walled with a narrow lumen to subsolid, unbranched, more or less straight, interwoven, occasionally simple-septate, 2–5.5 μm in diam.; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 1–3 μm in diam.

Tubes: Generative hyphae infrequent, hyaline, thin-walled, occasionally branched, 1–2 μm in diam.; skeletal hyphae dominant, hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, occasionally branched, more or less straight, strongly interwoven, 1.5–3 μm in diam.; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 0.8–2 μm in diam. Cystidia absent, cystidioles fusoid, hyaline, thin-walled, 13–20 × 4.5–6 μm. Basidia clavate, bearing four sterigmata and a basal clamp connection, 17–28 × 7–8 μm; basidioles dominant, in shape similar to basidia, but smaller.

Spores: Basidiospores oblong ellipsoid, hyaline, smooth, slightly thick-walled, IKI-, CB-, 9.5–13.2(–14) × 4–6.2(–6.5) μm, L = 11.24 μm, W = 4.96 μm, Q = 2.26–2.31 (n = 60/2).

Type of rot

White rot.

Additional specimen (paratype) examined

CHINA. Guangxi Autonomous Region, Huanjiang County, Mulun Nature Reserve, on dead angiosperm tree, 10 July 2017, Cui 14465 (BJFC).

Figure 7. 

Basidiocarps of Crassisporus macroporus. Scale bars: 2 cm.

Figure 8. 

Microscopic structures of Crassisporus macroporus (drawn from the holotype) A basidiospores B basidia and basidioles C cystidioles D hyphae from trama e hyphae from context.

Crassisporus microsporus B.K. Cui & Xing Ji, sp. nov.

MycoBank No: 828514
Figs 9, 10

Notes

Crassisporus microsporus is characterized by pileate basidiocarps, small pores (5–7 per mm), and small, broadly ellipsoid basidiospores (4–5 × 3–3.7 μm).

Holotype

CHINA. Yunnan Province, Ruili, Mori Tropical Rainforest Park, on living angiosperm tree, 17 September 2017, Cui 16221 (BJFC).

Etymology

Microsporus (Lat.): referring to the small basidiospores.

Description

Fruitbody: Basidiocarps annual, pileate, sessile, corky, without odor or taste when fresh, soft leathery to corky upon drying. Pilei semicircular, projecting up to 2 cm, 4 cm wide, and 4.5 mm thick at base. Pileal surface pale yellowish brown to yellowish brown, finely velutinate, concentrically sulcate. Pore surface cream, buff to cinnamon-buff when fresh, buff, pale yellowish brown to yellowish brown when dry; sterile margin distinct, buff, up to 1 mm wide; pores round to angular, 5–7 per mm; dissepiments slightly thick, entire. Context pale yellowish brown to yellowish brown, leathery to corky when dry, up to 1.5 mm thick. Tubes concolorous with context, soft corky to corky, up to 3 mm long.

Hyphal structure: Hyphal system trimitic; generative hyphae bearing clamp connections; skeletal and binding hyphae IKI-, CB-; tissues turning to deep brown in KOH.

Context: Generative hyphae infrequent, hyaline, thin-walled, occasionally branched, 1.2–3.5 μm in diam.; skeletal hyphae dominant, hyaline to pale yellowish brown, thick-walled with a narrow lumen, rarely branched, straight, interwoven, occasionally simple-septate, 2.5–6 μm in diam.; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 0.8–2.5 μm in diam.

Tubes: Generative hyphae infrequent, hyaline, thin-walled, rarely branched, 1.2–3 μm in diam.; skeletal hyphae dominant, hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, moderately branched, more or less straight, strongly interwoven, 1.5–3 μm in diam.; binding hyphae hyaline to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 0.8–2.5 μm in diam. Cystidia absent, cystidioles fusoid, hyaline, thin-walled, 12.5–18 × 4–5.5 μm. Basidia clavate, bearing four sterigmata and a basal clamp connection, 14–21 × 4.5–6 μm; basidioles in shape similar to basidia, but distinctly smaller.

Spores: Basidiospores broadly ellipsoid, hyaline, smooth, slightly thick-walled, IKI-, CB-, 4–5(−5.2) × (−2.8)3–3.7(−3.9) μm, L = 4.5 μm, W =3.23 μm, Q = 1.4 (n = 60/1).

Type of rot

White rot.

Figure 9. 

Basidiocarps of Crassisporus microsporus. Scale bars: 1 cm.

Figure 10. 

Microscopic structures of Crassisporus microsporus (drawn from the holotype) A basidiospores B basidia and basidioles C cystidioles D hyphae from trama E hyphae from context.

Discussion

In the present study, Crassisporus is proposed based on morphological characters and phylogenetic analyses. In the ITS+nLSU analysis, Crassisporus was nested in the core polyporoid clade with strong support (100% MP, 100% ML, 1.00 BPP; Fig. 1). A further study based on combined ITS+nLSU+mtSSU+EF1-α+RPB2 sequences data indicated that Crassisporus grouped with Haploporus with low support, but formed a monophyletic lineage with a strong support (100% MP, 100% ML, 1.00 BPP; Fig. 2). Morphologically, Crassisporus is characterized by the combination of an annual growth habit, effused-reflexed to pileate basidiocarps, pale yellowish brown to yellowish brown, concentrically zonate or sulcate pilei, velutinate pileal surface, a trimitic hyphal system with clamped generative hyphae, tissues turning to dark in KOH, oblong to broadly ellipsoid, hyaline, smooth and slightly thick-walled basidiospores.

Morphologically, the four Crassisporus species can be easily distinguished from each other. Crassisporus microsporus differs from other species by its small pores (5–7 per mm), and small broadly ellipsoid basidiospores (4–5 × 3–3.7 μm). Except for C. imbricatus, C. leucoporus, C. macroporus, and C. microsporus, all have fusoid cystidioles in the hymenium; moreover, C. imbricatus produces imbricate basidiocarps. Previously, the type specimen of C. imbricatus was identified as Coriolopsis byrsina (Mont.) Ryvarden based on morphological characters (Li and Cui 2010). After careful examination of the basidiospores along with DNA sequences analyses, the specimen was found to represent an unknown taxon. Here, we describe it as a new species of Crassisporus based on morphological characters and phylogenetic analysis. Crassisporus macroporus may be confused with C. leucoporus due to their similarity in effused-reflexed to pileate basidiocarps and oblong ellipsoid basidiospores, but C. leucoporus is distinguished from C. macroporus by its smaller pores (3–4 per mm), white pore surface when fresh, and acyanophilous skeletal and binding hyphae.

Phylogenetically, Haploporus groups together with Crassisporus (Figs 1, 2), but the former differs by its annual to perennial growth habit, dimitic to trimitic hyphal system, and ornamented, cyanophilous basidiospores (Shen et al. 2016; Cui et al. 2019).

Crassisporus is similar to Hexagonia Fr. and Neofomitella Y.C. Dai, Hai J. Li & Vlasák, because these genera share pileate brown basidiocarps, a trimitic hyphal system with clamped generative hyphae, and tissues becoming dark in KOH. However, Hexagonia is distinguished from Crassisporus by its larger hexagonal pores and thin-walled basidiospores (Núñez and Ryvarden 2001). Neofomitella differs from Crassisporus in having crusted basidiocarps with the cuticle developing from base to margin, and smaller, thin-walled basidiospores (Li et al. 2014b).

Both Perenniporia Murrill and Crassisporus have hyaline and thick-walled basidiospores, but species of Perenniporia have cyanophilous, and variable dextrinoid skeletal hyphae. In addition, Perenniporia usually has truncate basidiospores (Gilbertson and Ryvarden 1987; Núñez and Ryvarden 2001; Zhao et al. 2013; Cui et al. 2019).

Truncospora Pilát is similar to Crassisporus in having pileate basidiocarps and variable presence of cystidioles. However, Truncospora is distinguished from Crassisporus by variable dextrinoid and cyanophilous skeletal hyphae and truncate, strongly dextrinoid basidiospores (Zhao and Cui 2013; Cui et al. 2019).

Abundisporus Ryvarden and Crassisporus share effused-reflexed or pileate basidiocarps, but Abundisporus differs by its pale-umber to deep-purplish-brown or greyish- to umber-brown context, dimitic hyphal system, and pale-yellowish basidiospores (Ryvarden 1998; Zhao et al. 2015; Cui et al. 2019).

Perenniporiella Decock & Ryvarden also has annual, pileate basidiocarps, and hyaline, thick-walled basidiospores, but it differs from Crassisporus in having a dimitic hyphal system (Decock and Ryvarden 2003).

Grammothelopsis Jülich is similar to Crassisporus in having thick-walled basidiospores; however, it differs from Crassisporus in its resupinate to effused basidiocarps with shallow irregular pores, and variable dextrinoid skeletal hyphae (Robledo and Ryvarden 2007; Zhao and Cui 2012).

Key to species of Crassisporus

1 Cystidioles absent C. imbricatus
Cystidioles present 2
2 Basidiospores broadly ellipsoid C. microsporus
Basidiospores oblong ellipsoid 3
3 Pore surface cream, buff to cinnamon-buff when fresh, pores 2–3 per mm C. macroporus
Pore surface white when fresh, pores 3–4 per mm C. leucoporus

Acknowledgments

We express our gratitude to Drs Tom May (Royal Botanic Gardens Victoria, Australia) and Yu-Cheng Dai (Beijing Forestry University) for arrangement of and assistance during field collections. The research was financed by the National Natural Science Foundation of China (Project Nos. 31670016, 31870008) and Beijing Forestry University Outstanding Young Talent Cultivation Project (No. 2019JQ03016).

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