Two new species of Perenniporia (Polyporales, Basidiomycota)

Abstract Two new species of Perenniporia, P. pseudotephroporasp. nov. and P. subcorticolasp. nov., are introduced respectively from Brazil and China based on morphological characteristics and molecular data. Perenniporia pseudotephropora is characterised by perennial, pileate basidiocarps with distinctly stratified tubes, grey pores, tissues becoming dark in KOH, a dimitic hyphal system with slightly dextrinoid arboriform skeletal hyphae and broadly ellipsoid to subglobose, truncate, weakly dextrinoid, cyanophilous basidiospores, measuring 4.9–5.2 × 4–4.8 μm. Perenniporia subcorticola is characterised by resupinate basidiocarps, yellow pores with thick dissepiments, tissues becoming dark in KOH, flexuous skeletal hyphae, ellipsoid, truncate and slightly dextrinoid basidiospores, measuring 4.2–5 × 3.5–4.2 µm. The morphologically-similar species and phylogenetically closely-related species to the two new species are discussed.


Introduction
Perenniporia Murrill (Polyporales, Basidiomycetes) is typified by Polyporus unitus Pers. (Decock and Stalpers 2006). Species in the genus are important, not only for the wood-decaying, but also for their potential application in both biomedical engineering and biodegradation (Younes et al. 2007;Dai et al. 2009;Si et al. 2016). Perenniporia is characterised by mostly perennial, resupinate to pileate ba-sidiocarps, a dimitic to trimitic hyphal system with generative hyphae bearing clamp connections, cyanophilous and variably dextrinoid skeletal hyphae or skeletal-binding hyphae in most species and ellipsoid, to subglobose, truncate or not, thick-walled, variably dextrinoid and cyanophilous basidiospores. All Perenniporia species cause a white rot (Ryvarden and Gilbertson 1994;Decock and Ryvarden 1999;Cui et al. 2019).
According to the phylogenetic analysis, based on ITS and nuclear ribosomal partial LSU DNA sequences, Robledo et al. (2009) demonstrated the fundamental phylogeny of Perenniporia s.l., combined with such characteristics as a diversity of the vegetative hyphae and basidiospores morphology. In their study, Perenniporia s.s. and Perenniporia s.l. were scattered into distinct clades, which is also supported by different morphological traits.  divided Perenniporia s.l. into seven clades, based on ITS and nLSU DNA phylogenetic inferences, each of these seven clades being distinguished by a specific combination of morphological characteristics that supported recognition at the genus level. Some genera, having similar morphological characteristics to Perenniporia, such as Amylosporia B.K. Cui et al., Murinicarpus B.K. Cui & Y.C. Dai, Vanderbylia D.A. Reid, Truncospora Pilát and Hornodermoporus Teixeira, were also proved to form distinct lineages in DNA-based phylogenetic analyses . Besides, several new species were proved to belong to Perenniporia, based on morphological characteristics and phylogenetic evidence, which improved the understanding of the phylogenetic structure of Perenniporia (Jang et al. 2015;Huang et al. 2017;Ji et al. 2017;Liu et al. 2017;Zhao and Ma 2019).
During a study of wood-inhabiting polypore from Brazil and China, two unknown species of Perenniporia were distinguished by both morphology and molecular data. In this study, the two species are described and illustrated.

Morphological studies
The studied specimens are deposited in the herbaria of the Institute of Microbiology, Beijing Forestry University (BJFC) and Universidade Federal de Pernambuco (URM). Morphological descriptions are based on field notes and herbarium specimens. Microscopic analyses follow . In the description: KOH = 5% potassium hydroxide, IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, CB = Cotton Blue, CB+ = cyanophilous in Cotton Blue, CB-= acyanophilous, L = arithmetic average of all spore length, W = arithmetic average of all spore width, Q = L/W ratios, n = number of spores/measured from given number of specimens. Colour terms are cited from Anonymous (1969) and Petersen (1996).

Molecular studies and phylogenetic analysis
A CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain PCR products from dried specimens, according to the manufacturer's instructions with some modifications (Shen et al. 2019;Sun et al. 2020). Two DNA gene fragments, ITS and nrLSU were amplified using the primer pairs ITS5/ ITS4 (White et al. 1990) and LR0R/LR7 (http://www.biology.duke.edu/fungi/mycolab/primers.htm). The PCR procedures for ITS and nLSU followed  in the phylogenetic analyses. DNA sequencing was performed at Beijing Genomics Institute and the newly-generated sequences were deposited in the GenBank database. Sequences generated for this study were aligned with additional sequences downloaded from GenBank, using BioEdit (Hall 1999) and ClustalX (Thompson et al. 1997).
In the study, nuclear ribosomal RNA genes were used to determine the phylogenetic position of the new species. Sequence alignment was deposited at TreeBase (submission ID 26254). Sequences of Donkioporia expansa (Desm.) Kotl. and Pouzar and Pyrofomes demidoffii (Lév.) Kotl. and Pouzar, obtained from GenBank, were used as outgroups .
Phylogenetic analyses, used in this study, followed the approach of Han et al. (2016) and Zhu et al. (2019). Maximum parsimony (MP) and Maximum Likelihood (ML) analyses were conducted for the datasets of ITS and nLSU sequences. The bestfit evolutionary model was selected by hierarchical likelihood ratio tests (hLRT) and Akaike Information Criterion (AIC) in MrModeltest 2.2 (Nylander 2004) after scoring 24 models of evolution by PAUP* version 4.0b10 (Swofford 2002).
The MP topology and bootstrap values (MP-BS) obtained from 1000 replicates were performed using PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5,000, 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. Sequences were also analysed using Maximum Likelihood (ML) with RAxML-HPC2 through the CIPRES Science Gateway (www.phylo.org; Miller et al. 2009). Branch support (BT) for ML analysis was determined by 1000 bootstrap replicates.
Bayesian phylogenetic inference and Bayesian posterior probabilities (BPP) were performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Four Markov chains were run for 4,650,000 generations until the split deviation frequency value was less than 0.01 and trees were sampled every 100 generations. The first 25% of the sampled trees were discarded as burn-in and the remaining ones were used to reconstruct a majority rule consensus and calculate Bayesian posterior probabilities (BPP) of the clades.

Phylogeny results
The combined ITS and nLSU dataset contained 101 sequences from 101 specimens referring to 59 taxa in this study. They were downloaded from GenBank and the sequences about Perenniporia corticola, P. pseudotephropora and P. subcorticola are new ( Table 1). The dataset had an aligned length of 2089 characters in the dataset, of which, 1400 characters are constant, 181 are variable and parsimony-uninformative and 508 are parsimony informative. Maximum Parsimony analysis yielded one equally-parsimonious tree (TL = 2627, CI = 0.389, RI = 0.711, RC = 0.277, HI = 0.611) and a strict consensus tree of these trees is shown in Fig. 1. Best model applied in the Bayesian analysis: GTR+I+G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1, 1, 1, 1). Bayesian analysis resulted in a same topology with an average standard deviation of split frequencies = 0.009950.
From the phylogenetic tree ( Fig. 1), P. pseudotephropora and P. subcorticola were absorbed in the genus Perenniporia. Moreover, P. subcorticola formed a direct lineage with a high approval rating (98/99/1.00) and P. pseudotephropora produced an independent lineage.   Basidiocarps. Perennial, resupinate or effused-reflexed to pileate, without odour or taste when fresh, becoming hard corky when dry. Pilei applanate, semicircular to fan-shaped, projecting up to 1 cm, 3.5 cm wide and about 1 cm thick at base. Pile- al surface pinkish-buff, grey to greyish-brown, smooth. Pore surface greyish to pale brown; pores tiny, round, 8-9 per mm; dissepiments thick, thicker than pore diameter, entire. Context thin, fawn to brown, corky, up to 0.5 mm thick. Tubes buff to brown, darker than pore surface, distinctly stratified, hard corky, up to 9.5 mm long.
Macromorphologically, Perenniporia subcorticola is similar to P. corticola by its yellow pores and almost the same size of basidiospores and that is the reason why the specimens of P. subcorticola were previously treated as P. cf. subcorticola (Dai et al. 2002). However, P. corticola has arboriform branched skeletal hyphae and dendrohyphidia at dissepiments and it is a tropical species usually growing on the wood of Dipterocarpaceae (Decock 2001a); while P. subcorticola lacks arboriform branched skeletal hyphae and dendrohyphidia and it seems to be a warm temperate species growing on both gymnosperm and angiosperm wood.