\ te. MycoKeys 106: 1-21 (2024) V ycokeys DOI: 10.3897/mycokeys.106.121840 Research Article Morphological and molecular data reveal Cerrena caulinicystidiata sp. nov. and Polyporus minutissimus sp. nov. in Polyporales from Asia Zi-Wei Zheng’®, Qiu-Yue Zhang’™®, Li-Rong Zhang?, Hai-Sheng Yuan®®, Fang Wu'® 1 State Key Laboratory of Efficient Production of Forest Resources, School of Ecology and Nature Conservation, Beijing Forestry University, Beijing 100083, China 2 Center for Biodiversity and Nature Reserve, Chinese Academy of Environmental Planning, Beijing 100043, China 3 Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, Liaoning, China Corresponding author: Fang Wu (fangwubjfu2014@bjfu. edu. cn) Abstract Two new species of Polyporales, Cerrena caulinicystidiata and Polyporus minutissimus, are illustrated and described on the basis of morphological studies and phylogenetic analyses from southern China and Vietnam. C. caulinicystidiata is characterized by an- nual, resupinate, sometimes effused-reflexed basidiocarps, greyish orange to brownish orange pore surface, irregular pores (3-8 per mm), a trimitic hyphal system, pyriform to ventricose cystidia, and subglobose basidiospores 3.2-4.5 x 2.8-3.5 um in size. P minutissimus is characterized by annual, solitary, fan-shaped with a depressed center or infundibuliform basidiocarps, obvious black stipe, cream to buff yellow pileal surface with glabrous, occasionally zonate and radially aligned stripes, angular pores (6-9 per mm), a dimitic hyphal system, and cylindrical basidiospores, 5—9.2 x 2.2—4 um. Detailed descriptions and illustrations of the two new species are provided. The differences between the two new species and their morphologically similar and phylogenetically related species are discussed. OPEN Qrceess Academic editor: Maria P. Martin Received: 28 February 2024 Accepted: 6 May 2024 Key words: Cerrenaceae, phylogeny, Polyporaceae, taxonomy, wood-decaying fungi Published: 13 June 2024 Introduction Citation: Zheng Z-W, Zhang Q-Y, The order Polyporales presents a great diversity of basidiocarp types and hy- Zhang L-R, Yuan H-S, Wu F (2024) menophore configurations (Binder et al. 2013). It is an important group of fungi, Morphological and molecular data as Polyporales species can cause wood-decay and thus play an essential role reveal Cerrena caulinicystidiata sp. nov. and Polyporus minutissimus Sp. nov. in Polyporales from Asia. in the carbon cycle. In addition, some species of Polyporales may have me- dicinal properties (Dai 1996, 1999, 2012; Dai et al. 2009). The order has long MycoKeys 106: 1-21. https://doi. been the subject of research on taxonomic diversity, distribution patterns, and org/10.3897/mycokeys.106.121840 ecological functions (Hibbett et al. 2014). As of early 2024, more than 1,800 species are recognized in the order (Martinez et al. 2004; Martinez et al. 2009; Copyright: © Zi-Wei Zheng et al. Kirk et al. 2008; Grigoriev et al. 2013; Zhao et al. 2015; Justo et al. 2017). Due to iiss 2 apen isles ace ls iibL ed Ande its great diversity, the order is intensively studied worldwide (Justo et al. 2017). terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). * These authors contributed equally to this paper. Zi-Wei Zheng et al.: Two new species of Cerrena and Polyporus (Polyporales) Cerrena Gray is the type genus of Cerrenaceae within the Polyporales (Ry- varden and Gilbertson 1993; Justo et al. 2017), and it is typified by C. unicol- or (Bull.) Murrill. It is widely distributed throughout the world. The genus is characterized by resupinate, effused-reflexed or pileate basidiocarps, irregular hymenophore, dimitic or trimitic hyphal systems, cylindric to ellipsoid basid- iospores, and white rot (Ryvarden and Gilbertson 1993; Ryvarden et al. 2022). The genus Cerrena published in 1821 has priority above Trametes Fr., pub- lished in 1838, and the two genera were considered as a single taxon in several studies. Thus, a huge number of new combinations in Cerrena. (Cunningham and Cunningham1 965; Gilbertson and Ryvarden 1987; Corner 1989; Ryvarden and Melo 2022). However, Ryvarden (1991) recommended to keep Cerrena as a separate genus based on their anatomical characters. According to Index Fungorum (http://www.indexfungorum.org) and Yuan (2014), Cerrena currently comprises around 10 species. Polyporus P. Micheli ex Adans., the type genus of the Polyporaceae, is a well-known polypore genus (Gilbertson and Ryvarden 1987). Given that Micheli (1729) did not originally select a type species for Polyporus, there is no consen- sus on the selection of the type. Since Donk (1933) selected P tuberaster (Jacq. ex Pers.) Fr. as the type species, this lectotype was accepted by most subse- quent mycologists (Cunningham and Cunningham 1965; Singer 1986; Niemela and Kotiranta 1991; Ryvarden 1991; Sotome et al. 2008; Ji et al. 2022). Morpho- logically, Polyporus is a heterogeneous genus including many species belong- ing to six morphological groups described by Nufiez and Ryvarden (1995), viz, the Polyporus group, the Favolus group (= Favolus Fr.), the Melanopus group (= Melanopus Pat.), the Polyporellus group (= Polyporellus Karst.), the Admirabi- lis group, and the Dendropolyporus group (= Dendropolyporus (Pouz.) Julich). Phylogenetically, Polyporus s. str. is known as a polyphyletic genus (Kriiger et al. 2006; Sotome et al. 2008, 2011; Ji et al. 2022). Phylogenetic analyses of Polyporus did not conform to the six morphological groups, for which further in-depth study of the group is needed (Sotome et al. 2008; Zhou et al. 2016). During investigations on wood-decaying polypores from South China and Vietnam, specimens that morphologically fit the definitions of Cerrena and Polyporus were collected. Phylogenetically, these samples formed two distinct lineages within Cerrena and Polyporus, respectively, and they are different from their morphologically similar and phylogenetically related species. Therefore, we describe and illustrate two new species, Cerrena caulinicystidiata sp. nov. and Polyporus minutissimus sp. nov. within the Polyporales on the basis of mor- phological studies and phylogenetic analyses. Materials and methods Morphological studies The studied specimens are deposited in the Fungarium of Beijing Forestry Uni- versity (BUFC) and the Institute of Applied Ecology of the Chinese Academy of Sciences (IFP). Macro-morphological descriptions were based on field notes and voucher herbarium specimens. Microscopic measurements and drawings were made from slides prepared from voucher tissues and stained with Cotton Blue and Melzer’s reagent. The following abbreviations were used: KOH = 5% MycoKeys 106: 1-21 (2024), DOI: 10.3897/mycokeys.106.121840 5 Zi-Wei Zheng et al.: Two new species of Cerrena and Polyporus (Polyporales) potassium hydroxide; CB = Cotton Blue; CB+ = cyanophilous in Cotton Blue; CB- = acyanophilous in Cotton Blue; IKI = Melzer’s; IKI- = neither amyloid nor dextrinoid in Melzer’s reagent; L = mean basidiospore length (arithmetic aver- age of basidiospores); W = mean basidiospore width (arithmetic average of ba- sidiospores); Q = variation in the L/W ratios between specimens studied; n (a/b) = number of basidiospores (a) measured from the given number of specimens (b). When we present basidiospore size variation, 5% of measurements were excluded from each end of the range. These excluded values are given in pa- rentheses. Special color terms follow Anonymous (1969) and Petersen (1996). DNA extraction and sequencing A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies, Co., Ltd., Bei- jing, China) was used to obtain DNA products from voucher specimens follow- ing the manufacturer's instructions with some modifications (Wu et al. 2020, 2022). The following primer pairs were used to amplify the DNA: ITS5 and ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and LROR and LR7 for the nuclear large subunit (nLSU) rDNA gene (Vilgalys and Hester 1990). The procedures for DNA extraction and polymerase chain reaction (PCR) used in this study were the same as described by Wu et al. (2022). The PCR products were purified and sequenced by Beijing Genomics Institute (BGI), Chi- na. All newly generated sequences in this study were deposited in GenBank (Sayers et al.2024; http://www.ncbi.nIm.nih.gov/genbank/) and listed in Table 1. Phylogenetic analysis Phylogenetic trees of Cerrena and Polyporus were constructed using the two concatenated ITS1-5.8S-ITS2-nLSU sequences dataset, respectively, and phy- logenetic analyses were performed with Maximum Likelihood (ML) and Bayes- ian Inference (BI) methods. New sequences generated in this study and ref- erence sequences retrieved from GenBank (Table 1) were partitioned to ITS1, 5.8S, ITS2, nLSU and then aligned separately using MAFFT v.74 (Katoh et al. 2019; http://mafft.cbre.jp/alignment/server/) with the G-INS-I iterative refine- ment algorithm and optimised manually in BioEdit v.7.0.5.3 (Hall 1999). The separate alignments were then concatenated using PhyloSuite v.1.2.2 (Zhang et al. 2020). Unreliably aligned sections were removed before the analyses, and efforts were made to manually inspect and improve the alignment. The data matrix was edited in Mesquite v3.70. Irpex latemarginatus (Durieu & Mont.) C.C. Chen & Sheng H. Wu was used as an outgroup in the phylogenetic analysis of Cerrena (Parmasto and Hallenberg 2000). Trametes conchifer (Schwein.) Pilat, T. elegans (Spreng.) Fr. and 7. polyzona (Pers.) Justo were selected as out- groups in the phylogenetic analysis of Polyporus (Ji et al. 2022). The final align- ments and the retrieved topologies were deposited in TreeBASE (http://www. treebase.org) under accessions 31102, 31103. RAXxML 7.2.8 was used to infer ML trees for both datasets with the GTR+I+G model of site substitution, including estimation of Gamma-distributed rate het- erogeneity and a proportion of invariant sites (Stamatakis 2006). The branch support was evaluated with a bootstrapping method of 1,000 replicates (Hillis and Bull 1993). MycoKeys 106: 1-21 (2024), DOI: 10.3897/mycokeys.106.121840 3 Zi-Wei Zheng et al.: Two new species of Cerrena and Polyporus (Polyporales) Table 1. Taxa information and GenBank accession numbers of the sequences used in this study. Species Cerrena albocinnamomea Cerrena albocinnamomea Cerrena albocinnamomea Cerrena albocinnamomea Cerrena caulinicystidiata Cerrena caulinicystidiata Cerrena caulinicystidiata (Cerrena sp. 1) Cerrena caulinicystidiata (Cerrena sp. 1) Cerrena caulinicystidiata (Cerrena sp. 1) Cerrena caulinicystidiata* Cerrena cystidiata Cerrena gilbertsonii Cerrena gilbertsonii Cerrena multipileata Cerrena multipileata Cerrena multipileata Cerrena sp. 2 Cerrena sp. 2 Cerrena sp. 2 Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena unicolor Cerrena zonata Cerrena zonata Cerrena zonata Cerrena zonata Cerrena zonata Cerrena zonata Specimen No. Miettinen 10511 NIBRFG0000102423 Dai 12892 KUC20121102-06 Yuan 12664 Yuan 12666 BJ2-11 G1669 Otu0185 Wu 661 548/17 JV 1609/29 Vandevender 94-144 JV 1407/63 Ryvarden 43881 Kout A36 F12 7-SU-3-B-77(M)-B NTOU5117 B2 D.T6.5_2 CBS 154.29 He6082 GSM-10 Han 849 CU2 H:Otto Miettinen 9443 MUT 10 um in length............... ee eeeeeeereees P. tuberaster TAA CYSTUCIOISS ADSEM Ue reenact ala tenccuabent leaned erst rhs s hee Reheat, 15 =) | "CYSTIGIGlESAINMFEQuUeNnt 2, «1! ase. .c.ctausactorccdees oaqutanenteesstenetnate P. austrosinensis NED Mp EMER ICI Fea et yg PEN TOL Uy,/o stn denna andere SOM dates cet hte Ae wnaihdnalee cog a maad Aathaaseh eeteds 16 SABA St eae EINE Tos 8 Fe a2, ese tes tale ceeaeme tee aa P. lamelliporus 16. Basidiospores. smialler6-8.3°* 22-3 UM scs2.cheeese seen P. arcularius —