Morphological and Phylogenetic Reevaluation of the Genera Mycobonia and Pseudofavolus (Polyporaceae) Melissa Palacio (  melissapalacio@gmail.com ) Universidade Federal do Rio Grande do Sul https://orcid.org/0000-0003-2890-5189 Mauro Westphalen Universidade Federal do Rio Grande do Sul Yuan Yuan Beijing Forestry University Yingda Wu China Fire and Rescue Institute Rosa Mara Borges Da Silveira Universidade Federal do Rio Grande do Sul Research Article Keywords: polypores, core polyporoid clade, phylogeny, hymenophore con guration Posted Date: September 30th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-925842/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/25 Abstract Mycobonia and Pseudofavolus (Polyporales, Basidiomycota) are polyporoid genera with tropical and subtropical distribution. Both genera are morphologically similar in presenting abelliform to conchate subsessile basidiomata, with a dimitic hyphal system, consisting of clamped generative hyphae and skeleto-binding hyphae that produce large basidiospores with smooth, thin walls. However, while Pseudofavolus species present a poroid hymenophore, in Mycobonia it is stereoid with hyphal pegs that resemble thin teeth. Mycobonia and Pseudofavolus have a controversial taxonomy, and the phylogenetic relationships between their species have yet to be assessed. For this reason, we performed molecular phylogenetic analyses on specimens of Mycobonia and Pseudofavolus from both the Neotropics and Asia, using internal transcribed spacers (ITS), the large subunit of nuclear rDNA (nc LSU rDNA), and also the genes encoding the second largest subunit of RNA polymerase II (RPB2). Furthermore, in order to develop an evolutionary analysis of the hymenophore con guration, we performed stochastic character mapping of ancestral states for the hymenophore type presented in Polyporus s.l. Our study revealed that Pseudofavolus is an arti cial group and its species actually nest in a clade within Mycobonia. Therefore, in order to establish a monophyletic group, based upon priority of publication, we re-circunscribed Mycobonia to encompass both stereoid and poroid hymenophore species. Two new combinations are presented from the Neotropics: Mycobonia cucullata and M. miquelii. A new species from tropical Asia, M. yuchengii, is also described. We presente a summary of stochastic mapping of ancestral states estimates of hymenophore type in Polyporus s.l. The ancestral state for Mycobonia clade is estimated to have angular pores. Introduction Mycobonia Pat. and Pseudofavolus Pat. are genera of wood-decaying Basidiomycota belonging to the family Polyporaceae with tropical to subtropical distribution. Both genera are morphologically similar in presenting abelliform to conchate subsessile basidiomata, a dimitic hyphal system with clamped generative hyphae, skeleto-binding hyphae and large, smooth basidiospores with thin and smooth walls (Corner 1984). Macroscopically, the shape, color and consistency of the basidiomata are very similar in both genera; however, while Pseudofavolus includes species with poroid hymenophore, Mycobonia presents stereoid hymenophore with hyphal pegs (Corner 1984; Julich 1976). Currently, two species are recognized in Mycobonia, M. brunneoleuca and M. ava (type species), and are distributed throughout the Americas in tropical and subtropical areas, though they differ in their basidiospores differ in shape, size and altitudinal occurrence (Julich 1976; Gerlach and Loguercio-Leite 2011). Currently nine species are accepted in the Pseudofavolus genus, with tropical and subtropical worldwide distribution (Ryvarden and Johansen 1981; Ryvarden 2016). Speci cally, four of them were originally described from the Neotropics: P. cucullatus (Mont.) Pat., P. miquelii (Mont.) Pat. (type species), P. nigrus Ryvarden, and P. orinocensis (Pat. and Gaillard) Ryvarden. Additionally, P. cucullatus, which was Page 2/25 originally described from Cuba, has subsequently been recorded from several areas of subtropical and tropical Asia (Dai 2007, 2012; Dai et al. 2011; Cui et al. 2019; Wu et al. 2020). The taxonomy of Mycobonia and Pseudofavolus is controversial. Due to the stereoid hymenophore, Mycobonia has been included in several families, such as Corticiaceae, Mycoboniaceae, Stereaceae and Thelephoraceae (Corner 1984; Krueger 2002). However, in several studies, the a nities of Mycobonia and Pseudofavolus with Polyporus s.l. were described and discussed based on morphological characteristics (Singer 1951, 1986; Corner 1984). These similarities were subsequently corroborated by phylogenetic studies (Kruguer 2002; Krueger and Gargas 2004, Motato-Vásquez et al. 2018). Mycobonia and Pseudofavolus have also been treated as synonyms of Polyporus (Silveira and Wright 2005; Nakasone 2015). Nevertheless, phylogenetic studies have placed Mycobonia next to Pseudofavolus, and in distant relation to Polyporus s.s. (Krüger and Gargas 2004; Motato-Vasquez et al. 2018), thus representing different genera in the core polyporoid clade. In the present study, we aim to investigate the relationships between Mycobonia and Pseudofavlus based on morphological and molecular evidence. In addition, we compare the Asian specimens of P. cucullatus to neotropical samples in order to determine if they are in fact conspeci c. Finally, considering the traditional taxonomic value of hymenophore differentiation, we reconstruct the ancestral state for the hymenophore con guration of Polyporus s.l. Material And Methods Specimens and morphological studies The studied specimens are deposited in ICN, BJFC, and SP herbaria. Herbarium acronyms follow Thiers (continuously updated, http://sweetgum.nybg.org/science/ih/). Freehand cross sections of dried materials were mounted on slides and observed under the microscope in Melzer’s reagent, 5% KOH and/or 1% phloxine, cresyl blue and/or cotton blue (CB). To observe hyphal systems, small pieces of basidiomata were kept in 3% NaOH solution at 45 ºC for about 2 h; then, pieces were dissected under a stereomicroscope (Decock et al. 2013). Basidiospores were measured (n = 40) in Melzer’s reagent. The following abbreviations were also used: IKI– = inamyloid and non-dextrinoid, CB+/– = cyanophilous/acyanophilous, m = arithmetic mean and Q = the ratio of length/width of basidiospores. We followed Stalpers (1996) and the Stalpers database (http://www.cbs.knaw. nl/russulales/) for basidiospore shape terminology. DNA extraction and sequencing Following the manufacturer's protocols, DNA was extracted from dried specimens using CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) and FH plant DNA kit II (Demeter Biotech Co., Ltd., Beijing, China). Primer pairs ITS4/ITS5 (White et al. 1990), LR0R/LR7 (Vilgalys and Hester 1990) and fRPB2-5F/bRPB2-7.1R (Matheny 2005; Frøslev et al. 2005) were used to amplify the ITS, nc LSU rDNA and RPB2 regions, respectively, by a qualitative simplex Polymerase Chain Reaction (PCR). The parameters of the PCR for each region were followed as described by Dentinger et al. (2010), Page 3/25 Vilgalys and Hester (1990), Frøslev et al. (2005) and Matheny (2005), respectively. The PCR products were puri ed and sequenced with the same primers at Beijing Genomics Institute in China. Phylogenetic analyses Chromatograms were assembled and manually edited using Geneious v. 7.1.9 (http://www.geneious.com). The newly generated ITS, nc LSU rDNA and RPB2 sequences were deposited in GenBank and were later combined with additional sequences retrieved from GenBank to compose the entire dataset (Table 1). The ITS, nc LSU rDNA and RPB2 matrices were individually aligned using MAFFT v.7 (Katoh and Standley 2013) under the Auto strategy, then inspected and edited using Aliview (Larsson 2014). We used PartitionFinder v.2 (Lanfear et al. 2017) to estimate the best- t partitioning strategy and the best- t model of nucleotide evolution with the following settings: branch lengths = linked, models = mr bayes, model selection = AICc and search = greedy. Two distinct analyses were performed: Bayesian Inference (BI) and Maximum Likelihood (ML). Bayesian Inference analysis was conducted using MrBayes 3.2 (Ronquist et al., 2012) with two independent runs, each one with four chains and starting from random trees. The runs performed 20 million generations, and trees were sampled every 1,000th generation. Of the sampled trees, 25% were discarded as burn-in, while the remainder were used for calculating a consensus tree and Bayesian Posterior Probabilities (BPP). Page 4/25 Table 1 List of species, collections, geographic origin and GenBank accession numbers for the ITS, nc LSU rDNA, mtSSU and RPB2 sequences used in the phylogenetic analyses in this study. New sequences generated from this study are marked in bold. Taxon Voucher Country GenBank accession number ITS nc LSU rDNA RPB2 Atroporus diabolicus DS1266 Brazil KY631757 KY631768 — Atroporus rufoatratus DS816 Brazil KY631759 KY631770 KY744947 Bresadolia craterella TENN59383 Ecuador AJ487944 — Bresadolia paradoxa MV23 Brazil KY777230 KY777235 — Bresadolia paradoxa Robledo1958 Argentina KY777233 KY777237 — Cerioporus squamosus AFTOLID704 DQ267123 AY629320 DQ408120 Datronia mollis RLG6304sp USA JN165002 JN164791 JN164872 Datronia stereoides Holonen Finland KC415179 KC415196 KC415202 Datroniella mellanocarpa Cui10646 China KC415186 KC415194 KC415201 Datroniella scutellata RLG9584T USA JN165004 JN164792 JN164873 Dichomitus campestris O103769 Norway — AJ487512 — Dichomitus sp. IFP14643 China KX832053 KX832062 — Echinochaete brachypora TFMF24996 Japan AB462321 AB462309 — Echinochaete russiceps TFMF15716 Japan AB462310 AB368065 AB368123 Echinochaete sp. MN272 Ecuador AF518754 Favolus brasiliensis Kellermann s.n. Brazil MN648682 MN648708 — Favolus emerici WD2379 Japan AB587628 AB587619 AB368147 Favolus roseus PEN33 Malaysia AB735975 AB368099 AB368156 Favolus rugulosus MP191 Brazil MN648684 MN648712 Favolus yanomami ACM1295 Brazil MN648686 MN648714 — Lentinus bertieri TENN59773 Dominican Republic GU207303 AY615984 — Page 5/25 Taxon Voucher Country GenBank accession number ITS nc LSU rDNA RPB2 Lentinus crinitus DSH9243C Costa Rica KP283495 KP283523 — Lentinus tigrinus MUCL22821 Belgium AB478881 AB368072 AB368130 Megasporoporia cavernulosa Wu9508328 China — AY333800 — Mycobonia brunneoleuca GAS625 Brazil MZ997324 MZ996883 OK032602 Mycobonia brunneoleuca TENN57579 Costa Rica AY513570 AJ487934 — Mycobonia cucullata MP204 Brazil MZ997329 MZ996888 Mycobonia cucullata TENN58910 Argentina AF516600 AJ488124 — Mycobonia ava MP207 Brazil MZ997326 MZ996885 — Mycobonia ava MP213 Brazil MZ997325 MZ996884 — Mycobonia ava TENN59088 Argentina AY513571 AJ487933 — Mycobonia miquelii GAS1122 Brazil MZ997327 MZ996886 — Mycobonia miquelii VOG213 Brazil MZ997328 MZ996887 OK032603 Mycobonia orientalis Cui8707 China KX880623 KX880662 — Mycobonia orientalis Dai13584 China KX900071 KX900185 — Mycobonia orientalis WD2157 Japan AB587637 AB368114 AB368170 Neodatronia gaoligongensis Cui8055 China JX559269 JX559286 JX559317 Neodatronia sinensis Dai11921 China JX559272 JX559283 JX559320 Neodictyopus atlanticae DS1286 Brazil KY631763 KY631774 KY744950 Neodictyopus dictyopus GAS272 Brazil KY631766 KY631777 KY744952 Neofavolus alveolaris WD2340 Japan AB735970 AB368077 AB368135 Neofavolus cremealbidus TUMH 50009 Japan AB735980 AB735957 — Neofavolus mikawae TFMF-27417 Japan AB735963 AB735943 — Page 6/25 Taxon Voucher Country GenBank accession number ITS nc LSU rDNA RPB2 Neofavolus suavissimus ADD7 USA KP283501 KP283527 — Neofavolus subpurpurascens CG6242 Brazil MH544277 MH544275 — Picipes austroandinus MR10701 Argentina AF516569 — — Picipes badius WD2341 Japan AB587625 AB368083 AB368140 Picipes tubaerformis WD1839 Japan AB587634 AB368101 AB368158 Polyporellus arcularius RGT830522 Canada AF516523 AB368081 AB368138 Polyporellus brumalis TENN61760 USA FJ596883 AB368084 AB368141 Polyporellus ciliatus TENN57698 Denmark AB070882 AJ487943 — Polyporus tricholoma TENN56503 Puerto Rico AF516555 AB368100 AB368157 Polyporus tuberaster DAOM7997B USA AY218420 AF261544 — Polyporus tuberaster WD2382 Japan AB474086 AB368104b AB368161 Polyporus varius WD619 Japan AB587635 AB368110 AB368167 Trametes hirsuta RLG5133T USA JN164941 JN164801 JN164854 Trametes versicolor FP135156 USA JN164919 JN164809 JN164850 ML analysis was performed on the CIPRES SCIENCE GATEWAY 3.1 (Miller et al. 2010) using RAxML 8.1.4 (Stamatakis 2014). The analysis rst involved 100 ML searches, each starting from one randomized stepwise-addition parsimony tree, under a GTRGAMMA model, with all other parameters estimated by the software. We provided a partition le with the de ned partitions to force RAxML to search for a separate evolution model for each partition. Bootstrap support values (BS) were obtained under the same models and partitioning schemes allowing the program to automatically halt bootstrapping by using the autoMRE option. A node was considered to be strongly supported if it showed BPP ≥ 0.95 and/or BS ≥ 80%. Trametes hirsuta (Wulfen) Pilát and T. versicolor (L.) Lloyd were used as the outgroup based on Motato-Vásquez et al. (2018). Ancestral character state reconstruction of hymenophore con guration In order to reconstruct hymenophore evolution of the phylogeny of Polyporus s.l. we evaluated two models of character state evolution: Equal Rates (ER) and All Rates Different (ARD) using the tDiscrete function of the R package Geiger 2.0 (Harmon et al., 2008). We selected the best model based on the Page 7/25 corrected Akaike Information Criterion (AIC) and used this for the reconstruction. The evolutionary analysis of the hymenophore was performed by stochastic character mapping (Huelsenbeck et al. 2003; Bollback 2006) implemented in the R package Phytools (Revell 2012), using the make.simmap and describe.simmap functions (Revell 2012). A total of 1000 stochastic maps were generated. Results were subsequently summarized and plotted on the phylogeny using the R package Ape 5.3 (Paradis et al. 2004). Hymenophore types were assigned as: circular pores = 0, angular pores = 1, subporoid lamellae = 2, lamellate = 3, stereoid = 4 (Supp Table). Results Phylogenetic analyses Phylogenetic analyses included 57 terminals representing 48 putative species. The nal alignment consisted of 2481 characters, of which 1389 were conserved and 908 were parsimony-informative. Best partitioning scheme was six subsets: ITS1-ITS2, 5.8S, nc LSU rDNA, RPB2 1st codon position, 2nd codon position and 3rd codon position. The evolutionary model selected were GTR + I + G (ITS1 and ITS2), SYM + I + G (5.8S), GTR + I + G (nc LSU rDNA), GTR + G (RPB2 1st codon position), HKY + I + G (RPB2 2nd codon position) and GTR + I + G (RPB2 3rd codon position). The nal alignment was deposited at TreeBASE (28731). Our phylogenetic analyses showed that Pseudofavolus and Mycobonia represent arti cial groups according to current circumscriptions, as Pseudofavolus species actually nest in a clade together with Mycobonia species, which are phylogenetically distant from Polyporus s.s. (Fig. 1). Therefore, in order to accommodate these species in a natural group based upon priority of publication, we propose to transfer Pseudofavolus species into the genus Mycobonia (see Taxonomy section). Results from phylogenetic analyses recovered ve strongly supported linages within the Mycobonia clade, representing ve species that can also be recognized by morphological and ecological differences: M. brunneoleuca (PP = 1.0, BS = 99), M. cucullata (PP = 1.0, BS = 96), M. ava (PP = 1.0, BS = 91), M. miquelii (PP = 1.0, BS = 96) and the new species M. yuchengii (PP = 1.0, BS = 100). Mycobonia yuchengii clade is composed by Asian specimens previously identi ed as “P. cucullatus”, which will be treated below in the Taxonomy section. Phylogenetic relationships between Mycobonia species are not well resolved and ITS, nc LSU rDNA and RPB2 sequences are highly conserved in Mycobonia clade species, with only 100 variable sites of the 2481 in nal alignment. Additionally, the following were recovered as monophyletic: Atroporus (PP = 1.0, BS = 100), Bresadolia (PP = 1.0, BS = 100), Datronia (PP = 1.0, BS = 97), Datroniella (PP = 1.0, BS = 100), Dichomitus (PP = 1.0, BS = 100), Echinochaete (PP = 1.0, BS = 100), Favolus (PP = 1.0, BS = 100), Lentinus/Polyporellus clade (PP = 1.0, BS = 100), Neodictyopus (PP = 1.0, BS = 100), Neofavolus (PP = 1.0, BS = 82), and Picipes (PP = 1.0, BS = 100). Ancestral character state reconstruction of hymenophore con guration Page 8/25 The best tting model for hymenophore con guration evolution was estimated to be the ER (AIC = 93.25), compared with ARD (AIC = 107.55). In the supplemental gures, we present estimates of the hymenophore type in Polyporus s.l. based on stochastic mapping of ancestral states. Probabilities are also showed in (Supplementary Fig. 1). The stochastic mapping of this hymenophore transition presented high probabilities of internal nodes, but low probabilities of back-bone nodes. Furthermore, the mapping indicated that the ancestral state for Mycobonia clade is estimated to have had angular pores (0.92), as well as for Neofavolus (1), as previously shown by Seelan et al. (2015). For the Lentinus/Polyporellus clade we found that the probability for circular pores was low, only 0.22, though in contrast Seelan et al. (2015) did estimate them to be circular. Polyporus s. l. likely had angular pores (0.72). The number of transitions from circular to angular pores was estimated to be four, while the number from angular to circular was three, and there was a single transition from angular to stereoid which was placed in the Mycobonia clade. Taxonomy Mycobonia Pat., Bull. Soc. mycol. Fr. 10(2): 76 (1894), emend. Palacio & Westphalen. = Pseudofavolus Pat., Essai Tax. Hyménomyc. (Lons-le-Saunier): 80 (1900) Type species: Mycobonia ava (Sw.) Pat. Basidiomata annual, solitary to imbricate, pileate, abelliform, cucullate, conchate- abelliform, subsessile with a very short lateral stipe to sessile; exible to hard when fresh, hard and boney consistency when dry; pilear surface glabrous, smooth to tessellate (when poroid), pale ochraceous to brownish ochraceous; context thin up to 3 mm, homogeneous, beige. Hymenophore surface poroid or stereoid with hyphal pegs; when poroid pores large angular to circular, 0.2–3 mm wide, dissepiments entire to lacerated; tubes short up to 4 mm long; when stereoid with sterile prominent hyphal pegs up to 180 µm long. Stipe reduced to subdiscoid, attached to substrate by a basal disc, concolorous with the pilear surface, to absent (Fig. 2). Hyphal system dimitic; generative hyphae 2–5 µm wide, with clamp connections, hyaline, thin-walled, scanty, CB−, IKI−; skeletal-binding hyphae 2–6 µm wide, densely interwoven, thick-walled, exuous, densely branched and interwoven dominating throughout the basidiomata, CB − to CB+, IKI − to slightly dextrinoid in mass. Pileipellis not well differentiated. Cystidia not seen. Dendrohypidia present in the dissepiments. Basidia clavate, with four sterigmata. Basidiospores large, up to 25 × 11 µm, ellipsoid to cylindrical, thin-walled, smooth, hyaline, IKI−, CB− (Fig. 3). Ecology and distribution: growing on fallen branches of unidenti ed angiosperms, causing white-rot; known from tropical to subtropical areas. Comments: Mycobonia is characterized by the pileate, cucullate and boney consistency of the basidiomata with the hymenophore being stereoid with hyphal pegs or poroid with shallow tubes pores (Fig. 2), and large basidiospores (Fig. 3). Page 9/25 Mycobonia cucullata (Mont.) Palacio & R.M. Silveira, comb. nov. MycoBank: MB841083 ≡ Favolus cucullatus Mont., Annls Sci. Nat., Bot., sér. 2 17: 125 (1842) ≡ Pseudofavolus cucullatus (Mont.) Pat., Essai Tax. Hyménomyc. (Lons-le-Saunier): 81 (1900) ≡ Hexagonia cucullata (Mont.) Murrill, Bull. Torrey bot. Club 31(6): 332 (1904) ≡ Polyporus miquelii var. cucullatus (Mont.) Corner, Beih. Nova Hedwigia 78: 90 (1984) = Favolus curtipes Berk. & M.A. Curtis, Hooker's J. Bot. Kew Gard. Misc. 1: 234 (1849) = Polyporus curtipes (Berk. & M.A. Curtis) Ryvarden, Syn. Fung. (Oslo) 5: 213 (1991) Description: Basidiomata annual, solitary to imbricate, laterally short stipitate, dimidiate to subsessile. Pileus 15–40 mm from the base to margin of the pileus, 30–60 mm wide, and 2 mm thick; dimidiate to abelliform, conchate- abelliform when dry, exible when fresh, very tough when dry; surface glabrous, smooth or slightly tessellate, not zoned, beige to pale ochraceous, slightly reddish brown towards the margin when dry; margin entire to mbriate, involute when dry, pale ochraceous; context of the pileus up to 2 mm wide, homogeneous, beige. Hymenophore surface poroid, light brown, pores 2–3 per mm, angular to circular; dissepiments entire to lacerated; tubes up to 1 mm long. Stipe reduce subdiscoid up to 2 mm long, concolorous with pilear surface, or practically non-existent and attached to the substrate by a basal disc up to 10 mm in diam. Hyphal system dimitic; generative hyphae 2–4 µm wide, with clamp connections, hyaline, thin-walled, scanty, CB−, IKI−; skeletal-binding hyphae 2–6 µm wide, densely interwoven, thick-walled, exuous, branched 4–6 times, branches tapering to liform tips 0.5 µm wide, dominating throughout the basidiomata, CB−, IKI − to slightly dextrinoid in mass in the dissepiments. Hyphal pegs absent. Pileipellis not well differentiated. Cystidia not seen; hymenium with some nal tips of skeletal-binding hyphae among the basidia. Basidia 25–36 × 7–14 µm, mostly clavate, with four sterigmata. Basidiospores 14– 20 × 5–7 µm (m = 17.5 × 5.7 µm) Q = 2.3–4 (m = 3; n = 100/5), narrowly cylindrical to sub-cylindrical, thinwalled, smooth, hyaline, IKI−, CB−. Ecology and distribution: growing on fallen branches of unidenti ed angiosperms; known from Argentina (Robledo and Rajchenberg 2007 as Polyporus curtipes), Brazil (Baltazar and Gibertoni 2009 as Pseudofavolus cucullatus), Costa Rica (Velázquez and Ruíz-Boyer 2005 as Pseudofavolus cucullatus), Cuba (type locality), and Mexico (Nava-Mora and Valenzuela (1997) as Polyporus curtipes). Specimens examined: Brazil, Paraná, Foz do Iguaçu, Parque Nacional do Iguaçu, 25°37'21.1"S, 54°28'11.0"W, 22 Jan 2017, leg. M. Palacio 204, (ICN197323). — Cuba, leg. Ramón de la Sagra & C. Wright (PC) (holotype of Pseudofavolus cucullatus). Page 10/25 Comments: Mycobonia cucullata can be distinguished by the hexagonal pores 2–3 per mm, short tubes up to 0.7 mm long, thinner context up to 0.2 mm wide, and by the narrowly cylindrical basidiospores. This species has been recorded in South America under the name Favolus curtipes Berk. & M.A. Curtis (Silveira and Wright 2005). However, according to the protologue of F. curtipes, this species can be differentiated from M. cucullata by the smaller and less rigid pores, eshier context and darker pilear surface (Hooker 1849). Mycobonia miquelii (Mont.) Palacio & Westphalen, comb. nov. MycoBank: MB841084 ≡ Polyporus miquelii Mont., Annls Sci. Nat., Bot., sér. 3 4: 357 (1845) ≡ Hexagonia miquelii (Mont.) Sacc., Syll. fung. (Abellini) 6: 361 (1888) ≡ Scenidium miquelii (Mont.) Kuntze, Revis. gen. pl. (Leipzig) 3(3): 516 (1898) ≡ Pseudofavolus miquelii (Mont.) Pat., Essai Tax. Hyménomyc. (Lons-le-Saunier): 81 (1900) = Favolus induratus Berk., Ann. Mag. nat. Hist., Ser. 2 9: 197 (1852) = Hexagonia indurata (Berk.) Murrill, Bull. Torrey bot. Club 31(6): 332 (1904) = Favolus daedaleoides Speg., Revista Argent. Hist. Nat. 1(2): 108 (1891) Description: Basidiomata annual, solitary, laterally short stipitate to subsessile. Pileus 20–40 mm from the base to margin of the pileus, 20–60 mm wide, and 2–4 mm thick; exible when fresh, very tough and convex when dry, abelliform, conchate- abelliform; surface glabrous, tessellate bullate-reticulate, not zoned, pale ochraceous to brownish ochraceous with reddish-brown spots mainly towards the margin; margin entire to lacerate, wavy, incurved when dry, reddish-brown; context of the pileus 2–3 mm wide, homogeneous, beige. Hymenophore surface poroid, pale ochraceous to brownish ochraceous, pores 1– 1.5 per mm, circular to angular; dissepiments entire to lacerated; tubes up to 4 mm long. Stipe reduce subdiscoid up to 2 mm long, concolorous with pilear surface, or practically non-existant and attached to substrate by a basal disc up to 7 mm in diam. Hyphal system dimitic; generative hyphae 2–5 µm wide, with clamp connections, hyaline, thin-walled, scanty, CB−, IKI−; skeletal-binding hyphae 2–6 µm wide, densely interwoven, thick-walled, exuous, branched 4–7 times, branches tapering to liform tips − 1 µm wide, dominating throughout the basidiomata, CB−, IKI−. Hyphal pegs absent. Pileipellis not well differentiated. Cystidia not seen; hymenium with some nal tips of skeleto-binding hyphae between the basidia. Basidia 20–35 × 9–15 µm, mostly clavate, with four sterigmata. Basidiospores 11–17 × 5.5–8.5 µm (m = 14 × 6.5 µm) Q = 2.3– 2.8 (m = 2.4; n = 100/5), sub-cylindrical, thin-walled, smooth, hyaline, IKI−, CB−. Page 11/25 Ecology and distribution: growing on fallen branches of unidenti ed angiosperms; common species originally described from Surinam and also registered from Argentina, Brazil (Capelari and Maziero 1988), Costa Rica (Velázquez and Ruíz-Boyer 2005), and the Dominican Republic. Specimens examined: Brazil, Espírito Santo, Santa Tereza, Rebio Augusto Ruschi, Trilha da Cachoeira, 13 Dez 2016, leg. A. Magnago 1305 (ICN197324); Rio Grande do Sul, Viamão, Parque Estadual de Itapuã, 21 Apr 2017, leg. G. Alves-Silva 1120 (ICN197320); ibid, leg. G. Alves-Silva 1121 (ICN197321); ibid, leg. G. Alves-Silva 1122, (ICN197322); Guaiba, Fazenda Maximiniano, 11 Nov 2017, leg. V. Oliveira-Garcia 213 (ICN197423); Porto Alegre, Morro Santana, 14 Dez 2007, leg. M. Westphalen 85/07 (ICN134137); Santa Catarina, Florianópolis, Parque Ecológico Córrego Grande, 27°35'55''S, 48°30'36''W, 10 May 2018, leg. M. Monteiro 160 (FLOR); São Paulo, Cananéia, Parque Estadual da Ilha do Cardoso, Trilha do Poço da Anta, 7 Jan 2019, leg. M.P. Drewinski 463, (SP499085); ibid., Parque Estadual das fontes do Ipiranga, 23°38'36.1"S, 46°37'21.6"W, 28 Mar 2019, leg. M.P. Drewinski 459 (SP499083). Comments: Large and angular pores, 1–1.5 per mm, and a tessellate pilear surface with a reddish-brown margin, which differentiates M. miquelii from other Mycobonia. Mycobonia yuchengii Yuan Yuan & Palacio, sp. nov. Figure 4. MycoBank: MB841082 Type. — China, Hainan Province, Lingshui County, Diaoluoshan Forest Park, on fallen angiosperm trunk, 13 Jun 2014, leg. Dai 13584A (BJFC017323). Etymology: — Yuchengii (Lat.): in honor of the Chinese mycologist, Prof. Yu Cheng Dai. Description: Basidiomata annual, pileate, attached to substrate with a short laterally stipe-like base, leathery when fresh, becoming hard or rigid upon drying. Pileus dimidiate to abelliform, projecting up to 3.5 cm, 4 cm wide, 1.5 mm thick at the center. Pilear surface cream to pale yellow-brown, glabrous, azonate, sometimes radiate-striate; margin acute, wavy when dry. Pore surface yellow-brown to umber; pores irregular, sometimes hexagonal, 1–3 per mm; dissepiments thin, entire to slightly lacerate. Context pale yellow-brown, hard corky, thin, less than 0.5 mm thick. Tube layer concolorous with the poroid surface, up to 2 mm long. Stipe short, concolorous with the pileal surface, up to 0.5 cm long, often forming a small disc at the base. Hyphal system dimitic; generative hyphae bearing clamp connections; skeletal-binding hyphae dominant, thick-walled, frequently branched, slightly dextrinoid, especially at dissepimental edge, CB+, tissue unchanged in KOH. Contextual generative hyphae infrequent, hyaline, thin-walled, rarely branched, 2.5– 3.5 µm in diam; contextual skeletal-binding hyphae dominant, hyaline, thick-walled with a wide to narrow lumen, some of them sub-solid, exuous, frequently branched, tightly interwoven, 2.5–4 µm in diam. Tramal generative hyphae scanty, hyaline, thin-walled, rarely branched, 2–3.5 µm in diam,; tramal skeletalbinding hyphae thick-walled to almost solid, frequently branched, tightly interwoven, 2–4.5 µm in diam. Cystidia and cystidioles absent. Dendrohyphidia present along the hymenium, especially abundant in the Page 12/25 dissepiments. Basidia broadly clavate, with four sterigmata and a basal clamp connection, 20–35 × 12– 15 µm; basidioles in shape similar to basidia, but slightly smaller. Basidiospores ellipsoid to mangoshaped, hyaline, thin-walled, smooth, sometimes with one or two guttule, IKI–, CB–, (11)13–17(18) × (5.5)5.8–8.5(8.8) µm, L = 14.8 µm, W = 7.1 µm, Q = 2.08–2.25 (n = 60/2). Ecology and distribution: When Wu et al. (2020) made a systematic study on the polypores from subtropical and tropical China, M. yuchengii was found as a common species in South China, usually grows on angiosperm trunks or branches and occurring in open areas of the forest. Specimens examined: China, Hainan Province, Wuzhishan County, Wuzhishan Forest Park, on fallen angiosperm trunk, 10 Jun 2016 Dai 16493 (BJFC022610); Hunan Province, Wulingyuan District, on fallen angiosperm trunk, 17 Ago 2010, leg. Dai 11682 (BJFC008806); Guangdong Province, Shixing County, Chebaling Nature Reserve, on fallen angiosperm branch, 23 Jun 2010, Cui 8707 (BJFC007647); ibid., 23 Jun 2010, leg. Cui 8747 (BJFC007687); Yunnan Province, Mengla County, Xishuangbanna Botanical Garden, on fallen angiosperm branch, 23 Jul 2014, leg. Dai 13893 (BJFC017623); ibid., leg. Dai 13894 (BJFC017624); Green Stone Forest Park, on fallen angiosperm branch, 4 Ago 2005, leg. Dai 6678 (IFP015327); Puer County, Laiyanghe Forest Park, on fallen angiosperm trunk, 6 Jun 2011, leg. Dai 12207 (BJFC010490). Comments: Mycobonia yuchengii was previously identi ed as “P. cucullatus” (Dai 2007). Mycobonia yuchengii basidiomata have shallow pores, so macroscopically it resembles species of Grammothele Berk. & M.A. Curtis. In addition, species in both genera have dendrohyphidia along the hymenia, and their skeletal hyphae are dextrinoid (Zhou and Dai 2012). However, Grammothele lacks both stipe and binding hyphae. Mycobonia brunneoleuca (Berk. & M.A. Curtis) Pat., Enum. Champ. Guadeloupe (Lons-le-Saunier): 23 (1903). ≡ Hydnum brunneoleucum Berk. & M.A. Curtis, Trans. Linn. Soc. London 22: 129. 1857, non Polyporus brunneoleucus Berk. 1846. ≡ Polyporus polyacanthophorus Nakasone, Mycotaxon 130(2): 383. 2015. Description: see Nakasone (2015). Ecology and distribution: growing on fallen branches of unidenti ed angiosperms, in cloud forest at 700– 2700 m.a.s.l. in Brazil (Gerlach and Loguercio-Leite 2011), Colombia, Costa Rica, Honduras (Nakasone 2015), Martinique (Burt 1919), Panamá (Martin 1939), Paraguay, Puerto Rico (Nakasone 2015), and Venezuela (type locality). Page 13/25 Specimens examined: Brazil, Rio Grande do Sul, São Francisco de Paula, Floresta Nacional de São Francisco de Paula, 25 May 2019, leg. M. Palacio 438 (ICN202959); Santa Catarina, Joaçaba, Parque Ecológico Municipal Rio do Peixe, leg. G. Alves-Silva 625, (FLOR60335). Comments: Mycobonia brunneoleuca is principally characterized by the large basidiospores 15–24(25) × (6)7–11 µm, narrowly ellipsoid, thin walled and hyaline. Macroscopically is characterized by the concave and shell-like basidiomata with stereoid hymenophore projecting sterile hyphal pegs, which is almost identical to M. ava. Mycobonia brunneoleuca and M. ava; can be differentiated by the basidiospores, which are sub-cylindrical and smaller (up to 5–7 µm wide) in M. ava, and also by the ecological niches that these two species occupy (Gerlach and Loguercio-Leite 2011). Mycobonia brunneoleuca occurs in cloud forests of the Caribbean, and throughout Central and South America between 700 and 2,700 m asl., while M. ava occurs at elevations up to 700 m asl. from North America to Argentina. In addition to these morphological and ecological differences, our phylogenetic analysis showed that M. brunneoleuca and M. ava represent two different linages (Fig. 1). Mycobonia ava (Sw.) Pat., Bull. Soc. mycol. Fr. 10(2): 77. 1894. ≡ Peziza ava Sw.: Fr., Prod.: 150. 1788. ≡ Hydnum avum (Sw.: Fr.) Berk., Ann. Mag. Nat. Hist., 10: 380. 1843 [“1842”]. ≡ Bonia ava (Sw.: Fr.) Henn., Hedwigia 36: 192. 1897. ≡ Auricularia ava (Sw.: Fr.) Farl., Bibl. Index N. Amer. Fung.: 307. 1905. ≡ Grandinioides ava (Sw.: Fr.) Banker, Mem. Torrey Bot. Club 12: 179. 1906. ≡ Polyporus curtipes subsp. avus (Sw.: Fr.) D. Krüger, Cryptog. Mycol. 31: 399. 2010. ≡ Polyporus epitheloides Nakasone, Mycotaxon 130(2): 383. 2015. Description: see Julich (1976). Ecology and distribution: growing on fallen branches of unidenti ed angiosperms, in Argentina, Colombia (Jülich 1975), Cuba (Burt 1919), Jamaica (Swartz 1788) (type locality), North America (Burt 1919), Brazil (Jülich 1975; Baltazar and Gibertoni 2009) and Venezuela (Corner 1984). Specimens examined: Brazil, Amazonas, Novo Aripuanã, BR-230, 23 Apr 1985, leg. K.F. Rodrigues 325 (INPA136956); Paraná, Foz do Iguaçu, Parque Nacional do Iguaçu, 25°37'21.1"S, 54°28'11.0"W, 23 Jan 2017, leg. M. Palacio 207 (ICN197318); ibid., leg. M. Palacio 213, (ICN197319); Rio Do Janeiro, Rio de Janeiro, Parque Nacional da Tijuca, 8 Feb 2017, leg. F.T.F. Linhares 228 (ICN197346); São Paulo, São Paulo, Parque Estadual da Cantareira, Trilha da cachoeira, 25 Mar 2019, leg. M.P. Drewinski 449, (SP499082). Page 14/25 Comments: Microscopically M. ava is very similar to M. miquelii, especially by the sub-cylindrical basidiospores, however M. ava can be differentiated by the hymenophore being stereoid with hyphal pegs, while in M. miquelii it is poroid. Other species possibly included in the genus Mycobonia: Favolus curtipes Berk. & M.A. Curtis, Hooker's J. Bot. Kew Gard. Misc. 1: 234 (1849) Favolus tenuis Fr., Syst. orb. veg. (Lundae) 1: 76 (1825) Hexagonia bipindiensis Henn., Bot. Jb. 38(1): 122 (1905) [1907] Hexagonia pulchella Lév., Annls Sci. Nat., Bot., sér. 3 2: 200 (1844) Polyporus orinocensis Pat. & Gaillard, Bull. Soc. mycol. Fr. 4(2): 31 (1888) Polyporus polygrammus Mont., Annls Sci. Nat., Bot., sér. 2 8: 365 (1837) Pseudofavolus nigrus Ryvarden, Mycotaxon 28(2): 537 (1987) KEY TO MYCOBONIA NEOTROPICAL SPECIES 1 Hymenophoral surface poroid 2 1* Hymenophoral surface stereoid with hyphal pegs 5 2 Pores 1–1.5 per mm; pilear surface tessellate, bullate-reticulate, pale ochraceous to brownish ochraceous with reddish-brown spots mainly towards the margin ...........................M. miquelii 2* Pores 2–3 per mm; pilear surface smooth to slightly tessellate, or radially wrinkled, beige to pale ochraceous, to chesnut to black 3 3 Pores 0.2–0.5 per mm; pilear surface smooth or slightly tessellate, beige to pale ochraceous, slightly reddish brown towards the margin when dry; known from Mexico to Argentina M. cucullata 3* Pores 4–5 per mm; pilear surface smooth darker, only known from Venezuela. 4 4 Pilear surface pale chestnut to reddish-brown M. yuchenguii 4* Pilear surface black P. nigrus 5 Basidiospores narrowly ellipsoid 15–24(25) × (6)7–11 µm; growing in cloud forests of the Caribbean and Central and South America between 700 and 2,700 m asl. M. brunneoleuca Page 15/25 5* Basidiospores sub-cylindrical 12–18 × 5–7 µm; growing at elevations up to 700 m asl. from North America to Argentina M. ava Discussion Mycobonia and Pseudofavolus in their traditional circumscriptions are paraphyletic groups. The differences between ITS, nc LSU rDNA and RPB2 sequences within Mycobonia species are signi cantly small, with just 100 variable sites from 2481 characters, thus showing that the phylogenetic separation between both genera is arti cial. Following the guidelines to de ne fungi genera formulated by Vellinga et al. (2015), we consider Pseudofavolus a synonym of Mycobonia (older name with priority). Therefore, we emended Mycobonia to encompass stereoid and also poroid species. We combined M. cucullata and M. miquelii since we could assess phyllogenetic data of those species through molecular analysis. Regarding other Pseudofavolus species, such as P. nigrus and P. orinocensis, we preferred to avoid nomenclatural changes until sequences of those species become available and their phylogenetic position can be con rmed, especially in a group with several genera with similar morphology. Phylogenetic relationships within Mycobonia clade are still not well resolved, especially due to the similarity in the sequences among the different species. More molecular data including other DNA regions could help clarify that in the future. However, Mycobonia species can be differentiated and recognized by ecologically through distribution patterns and elevation, while morphologically through the basidiospore shape and size, as well as the hymenophore type. Hymenophoral transitions within genera have often been documented in other groups of Polyporales, as Antrodia (Runnel et al. 2019), Steccherinum (Westphalen et al. 2018; Miettinen et al. 2012), Metuloidea (Miettinen and Ryvarden 2016), and also within the core polyporoid clade in Neofavolus, Lentinus/Polyporellus (Seelan et al. 2015). Additionally, Seelan et al. (2015) estimated the ancestral hymenophoral con guration for Neofavolus and Lentinus/Polyporellus. However, ancestral character state reconstruction including both stereoid and poroid hymenophore had not been estimated previously in the core polyporoid clade. Our results suggest a transition from angular pores to stereoid hymenophore in Mycobonia clade, and that the plesiomorphic condition for Polyporus s.l. is to have angular pores. Other groups in the core polyporoid clade can also present stereoid hymenophore with hyphal pegs, as Dichomitus, Ephitele, and Gramothele (Nakasone 2015). However, these groups present resupinate basidiomata, while the basidiomata in Mycobonia are pileate. Speci cally, hyphal pegs are also present in other clades of Polyporaceae, as Favolus, Lentinus, and Neofavolus, which are different in presenting basidiomata with lamellate hymenophore. The presence of hyphal pegs could represent an anatomical advantage, considering it has been suggested that there exists a possible relation with moisture retention and also protection from insect invasion (Pegler 1983). However, the function of the hyphal pegs is still unknown (Pegler and Young 1983), and it is not a homologous character (Hibbett and Vilgalys 1993). Mycobonia shares with Polyporus s.l. a dimitic hyphal system with skeletal-binding hyphae, hyaline, and thin-walled basidiospores, and caising white-rot, which appear to be a conserved characteristic across all Page 16/25 Polyporus s.l. species. However, relationships between genera, like Mycobonia in Polyporus s.l. are yet to be solved. Declarations ACKNOWLEDGEMENTS The authors would like to thank the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) for giving permission to sample the collections and also the curators of the herbaria mentioned above for the specimen loans. We would like to thank Felipe Bittencourt and Genivaldo Alves Silva for sharing photos and Hunter Daniel for for the English revision of the manuscript. We also thank to Professor YuCheng Dai and Xiao-Hong Ji for their assistance and support in the molecular procedures performed at the Institute of Microbiology (Beijing Forestry University, China). Rosa Mara Borges da Silveira is supported by Conselho Nacional de Desenvolvimento Cientí co e Tecnológico (process no. 308122/2019-4). Melissa Palacio is grateful for Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), which provided a PhD scholarship, as well as the International Association for Plant Taxonomy for an IAPT research grant. Funding: Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) provided a PhD scholarship to Melissa Palacio, and the International Association for Plant Taxonomy awarded her an IAPT Research Grant. Con icts of interest/Competing interests: Not applicable Availability of data and material: the manuscript data are deposited in world-reference repositories (GenBank, TreeBase) and the fungal collections are deposited in herbaria recognized by the Index Herberiorum. Code availability: Not applicable References 1. Baltazar JM, Gibertoni TB (2009) A checklist of the aphyllophoroid fungi (Basidiomycota) recorded from the Brazilian Atlantic Forest. Mycotaxon 109:439–442 2. Bollback JP (2006) SIMMAP: stochastic character mapping of discrete traits on phylogenies. BMC Bioinformatics 7:88. https://doi.org/10.1186/1471-2105-7-88 3. Burt EA (1919) The Thelephoraceae of North America. XI. Tulasnella, Veluticeps, Mycobonia, Epithele, and Lachnocladium. 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Westphalen MC, Rajchenberg M, Tomšovský M, Gugliotta AM (2018) A re-evaluation of Neotropical Junghuhnia s. lat. (Polyporales, Basidiomycota) based on morphological and multigene analyses. Persoonia: Molecular Phylogeny Evolution of Fungi 41:130–141 4 . Wu F, Yuan HS, Zhou LW, Yuan Y, Cui BK, Dai YC (2020) Polypore diversity in South China. Mycosystema 39:653–681 47. Zhou LW, Dai YC (2012) Wood-inhabiting fungi in southern China 5. New species of Theleporus and Grammothele (Polyporales, Basidiomycota). Mycologia 104:915–924 Figures Page 20/25 Figure 1 Phylogenetic relationships in Polyporus s.l. from ITS, nc LSU rDNA and RPB2 sequences. Topology from ML analysis. Bayesian posterior probability above 0.95 and bootstrap values above 80% are shown. Pies at nodes represent probability of each state from the summary of stochastic mapping of ancestral state estimates of hymenophore con guration across Polyporus s.l. (blue = circular pores, green = angular pores, orange = subporoid lamellae, pink = lamellae, grey = stereoid). Page 21/25 Figure 2 Basidiomata and hymenophore detail of Mycobonia species. A–D. Mycobonia brunneoleuca (GAS625). E. Mycobonia ava (MP207). F–H. M. miquelii (Monteiro 160; GAS 1122). I–J. M. cucullata. K. M. yuchengii (type Dai 13584A). Photo A. by G. Alves-Silva and F. by Felipe Bittencourt. Page 22/25 Figure 3 Microscopic features of Mycobonia species. A. Hymenium, tramal hyphae and sterile hyphal pegs in M. ava (MP207). B–E. Basidiospores. B. M. ava. C. M. brunneoleuca. D. M. cucullata. E. M. miquelii. Page 23/25 Figure 4 Microscopic features of Mycobonia yuchengii from type Dai 13584A (BJFC017323). A. Basidiospores. B. Basidia. C. Dendrohypidia. D. Hyphae from the trama. E. Hyphae from the context. Supplementary Files Page 24/25 This is a list of supplementary les associated with this preprint. Click to download. SuppFig1.png Page 25/25