Poriella subacida Gen. & Comb Nov. for Perenniporia subacida (Peck) Donk

: Poriella subacida gen. & comb. nov., previously known as Perenniporia subacida , which causes white rot, has been documented in temperate and tropical forests. Specimens from Asia, North America, and Europe were examined, including the type specimen of Polylorus subacidus . Sequences of the ITS1-5.8S-ITS2 region, the 28S rDNA, the mitochondrial rDNA small subunit (mtSSU), and the gene encoding the translation elongation factor 1- α (EF1) were generated. In multigene phylogenies (maximum parsimony, maximum likelihood, Bayesian inferences), “ Perenniporia subacida ” formed a well-supported lineage, distinct from the core “ Perenniporia ” clade (type species: “ P . medulla - panis ”), and sister to the “ Yuchengia narymica ” lineage. We therefore conclude that “ P . subacida ” should be placed in the new genus “ Poriella ”gen. nov. Morphologically, “ Poriella ” is characterized by a di- to trimitic hyphal system, non-truncate basidiospores, and strongly dextrinoid, cyanophilic skeletal hyphae.

Molecular studies involving Polyporaceae, mainly based on ITS and/or nLSU sequences, have been carried out [21][22][23][24][25][26][27]. Further studies employing a six-gene dataset have helped to clarify the generic relationships of polyporoid fungi for 373 taxa. The latter study showed that Perenniporia subacida clustered in the core polyporoid clade in which it was related to P. medulla-panis (Jacq.) Donk [23]. Further studies of Perenniporia inferred from nuclear ribosomal 28S and ITS sequence data revealed that P. subacida formed a monophyletic lineage distant from the Perenniporia s.s. [15,16,28].
To resolve the placement of this species, phylogenetic research was carried out employing the ITS, 28S, TEF1 and mtSSU and a comparative morphological study of the type specimen was conducted. We conclude that Perenniporia subacida should be treated as a distinct, new genus as described below.

Morphological Studies
Specimens studied are deposited at the Farlow Herbarium of Harvard University (FH), MA, USA, Beijing Forestry University (BJFC), Beijing, China and United States National Fungus Collections (BPI), New York, NY, USA. Macromorphological descriptions were on the basis of field notes and study of specimens. Color terms followed previous studies [29]. Microscopic measurements were made from slide preparations of dried specimens stained with Cotton Blue and Melzer's reagent by light microscopy [30,31]. Sections were studied using an Olympus BX40 compound microscope (Tokyo, Japan). In presenting spore size variation, 5% of measurements were excluded from each end of the range. The following abbreviations are used: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous; IKI = Melzer's reagent, IKI-= both non-amyloid and non-dextrinoid, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = mean each spore length/width ratio, n (a/b) = number of spores (a) measured from given number of specimens (b).

Molecular Techniques and Phylogenetic Analyses
CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to extract DNA from dried specimens from China and other regions, and 2xTaq PCR Mix (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to perform PCR products [30]. The primer pair ITS5 and ITS4 were amplified for the ITS region [32]. The primer pair LR0R and LR7 were for the nuclear r28S region [33]. The primer pair MS1 and MS2 were for the mitochondrial SSU region [32]. The primer pair EF1-983F and EF1-2218R were for Tef1 [34]. The PCR cycling program for ITS, mtSSU, 28S and TEF1 followed previous studies [27]. The Beijing Genomics Institute (Beijing, China) was in charge of purifying and direct sequencing of the PCR products. All sequences used in the phylogeny were downloaded from GenBank (Table 1) with references. The sequence alignment was deposited in TreeBase (submission ID 23826). A previous study [30] was followed for maximum parsimony analysis. The combined multiple genes dataset was analyzed under heuristic search and 1000 homogeneity replicates, giving a P value of 1.000, which is much greater than the 0.01 used in PAUP* version 4.0b10, which means there is no discrepancy among the four loci in reconstructing phylogenetic trees. Trees were constructed in PAUP* version 4.0b10 [44]. 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 bootstraps. 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 [45]. DNA sequence data was also analyzed using Maximum Likelihood (ML) with RAxML-HPC2 on Abe through the Cipres Science Gateway [46], with default settings except that branch support was obtained with 1000 rapid bootstrap replicates.
Mr Modeltest 2.3 was used to estimate the best-fit evolution model for each data set for Bayesian inference (BI). The best fit models were general time reversible (GTR)+G for ITS, and general time reversible (GTR)+I+G for nr28S, mtSSU, the exons of Tef1, and the combined dataset. The partitioned mixed model, which allows for model parameters to be estimated separately for each genetic marker, was used in the Bayesian analysis. BI Agronomy 2021, 11, 1308 5 of 13 was performed using MrBayes 3.1.2 [47][48][49]. Two runs of four Markov chains were run from random starting trees for two datasets: (ITS+28S) dataset for 5 million generations and (ITS+28S+mtSSU+Tef1) dataset for 10 million generations. Trees were sampled every 100 generations. The first quarter of the generations was by default discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for maximum likelihood (ML-BS), maximum parsimony (MP-BT), and Bayesian posterior probabilities (BPP) greater than or equal to 75% (ML-BS and MP-BS) and 0.95 (BPP) were considered significantly supported.

Molecular Phylogeny
The ITS+28S dataset included sequences from 63 fungal specimens representing 56 taxa.  1,1,1). Bayesian analysis and ML analyses resulted in a topology similar to the MP analysis, with an average standard deviation of split frequencies = 0.003420.
A further phylogeny ( Figure 2) inferred from the combined ITS+28S+mtSSU+Tef1 sequences was obtained for ten genera in Perenniporia s.l. and demonstrated that this taxon formed a clade together with Y. narymica, with strong support (100% BS, 100% BP, 1.00 BPP). The clade is distinct from P. medulla-panis (Jacq.) We, along with Donk, conclude that it belongs to a distinct, new genus, hereafter called Poriella.
Type species: Poriella subacida (Peck) C.L. Zhao. Etymology: Poriella (Lat.): referring to its similar appearance to the genus Perenniporia. Fruiting body: Perennial, resupinate to effused-reflex, becomes corky when dried, about 22 cm or more at the longest dimension, 15 cm or more at the widest dimension, and up to 17 mm thick at the center. Pore surface pale yellowish to dingy-yellowish when fresh, dingy-yellowish to pale tan to ochraceous when dry; pores round to angular, 4-6 per mm; dissepiments thin, entire. Subiculum thin, cream to buff, up to 1 mm thick. Tubes concolorous with pore surface, up to 16 mm thick.
Fruiting body: Perennial, resupinate to effused-reflex, becomes corky when dried, about 22 cm or more at the longest dimension, 15 cm or more at the widest dimension, and up to 17 mm thick at the center. Pore surface pale yellowish to dingy-yellowish when fresh, dingy-yellowish to pale tan to ochraceous when dry; pores round to angular, 4-6 per mm; dissepiments thin, entire. Subiculum thin, cream to buff, up to 1 mm thick. Tubes concolorous with pore surface, up to 16 mm thick. Hyphal structure: Hyphal system di-trimitic; generative hyphae with clamp connections; skeletal hyphae strongly dextrinoid, CB+; dissolving in KOH.
Tubes: Generative hyphae infrequent, hyaline, thin-walled, 2.5-3.5 µ m in diameter; skeletal hyphae dominant, thick-walled with a wide lumen, unbranched, subparallel, 3-  Associated wood-rot: White. Substrates and distribution: Mainly on conifers, but also on hardwood, causes white rot in Abies Mill and Tsuga Carr. and common on dead fallen trees in many areas. A boreal eastern species in Europe, widely distributed in forest regions of Asia and North America [8,10,11].

Discussion
The taxonomic position of this taxon has been long debated [16][17][18][19][20]. It was transferred to Perenniporia by Donk [20], and this placement has been generally accepted [8][9][10][11][12]. Due to a combination of characteristics (completely unbranched skeletal hyphae and ellipsoid and non-truncate basidiospores), Decock and Staplers [13] mentioned that the species did not belong to Perenniporia. Based on phylogenetic and morphological grounds, Robledo et al. confirmed that this taxon should be separated from Perenniporia, and could be recognized as a distinct genus morphologically as well [28]. Previous studies also confirmed that this species formed a clade distinct from the P. medulla-panis clade [5,6,28]. The present study confirms these results from previous studies and formally describes the new genus, Poriella with P. subacida as type species.
Other species of Perenniporia and Poriella subacida share the morphological features of unbranched skeletal hyphae and non-truncate basidiospores, such as Perenniporia africana (Ipulet and Ryvarden) in Uganda [50,51], P. contraria (Berk. and M.A. Curtis) Ryvarden in Cuba, and P. ellipsospora (Ryvarden and Gilb.) in North America [10]. A comparison of Poriella and related genera is presented in Table 2. Three species have been mentioned as possible synonyms of this highly variable species. Poria colorea Overh. & Englerth, described from Western Tsuga Carr. and has generally been considered to be conspecific with Polyporus subacidus. Poria fuscomarginata Berk. ex Cooke was commented on by Murrill [52] who found the type material to be badly preserved and scanty. He concluded that "it was suggested little." Poria subaurantia Berk. ex Cooke was considered a synonym of Polyporus subacidus by Murrill [53]. Judging by their descriptions, these fungi show a range of variation that is acceptable for P. subacidus.
Poriella subacida is primarily a boreal taxon and is widely distributed in forest regions of northern Asia, North America and Europe [8,10,11]. It mainly grows on conifers, especially Picea Dietr., but also on Larix and Pinus Linn. In Europe, it has also been found occasionally on hardwoods like Populus L. and Prunus L. [8], and other hardwoods in North America and Asia [10,11]. The species is also present in tropical areas (e.g., in Africa) [57], but these records should be treated with caution. Ipulet and Ryvarden [50] recently described Perenniporia africana Ipulet and Ryvarden, from Uganda, with seemingly the same combination of characteristicss, i.e., unbranched skeletal hyphae and non-truncate basidiospores [57,58]. A list of characteristics of Poriella subacida comb. nov. from different regions is presented in Table 3.  Poriella subacida causes white rot of conifers and hardwoods and also butt and root rots of living conifers. Due to the cream to golden yellow mycelia felts that develop in the decayed wood, this rot is commonly called "feather rot" [10].  Data Availability Statement: Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/; https://www.mycobank.org/page/Simple%20 names%20search; http://purl.org/phylo/treebase, submission ID 23826; accessed on 25 June 2021.