Two new species of Haploporus (Polyporales, Basidiomycota) from China and Ecuador based on morphology and phylogeny

At present, 25 species are accepted in Haploporus and are distributed in Asia, Europe, North America, South America, Australia, and Africa. In this study, two new species, Haploporus ecuadorensis from Ecuador and H. monomitica from China, are described and illustrated based on morphological examination and phylogenetic analyses. H. ecuadorensis is characterized by annual, resupinate basidiomata with pinkish buff to honey yellow hymenophore when dry, round to angular pores of 2–4 per mm, a dimitic hyphal structure with generative hyphae bearing clamp connections, hyphae at dissepiment edge usually with one or two simple septa, the presence of dendrohyphidia and cystidioles, and oblong to ellipsoid basidiospores measuring 14.9–17.9 × 6.9–8.8 µm. Haploporus monomitica differs from other Haploporus species in that it has a monomitic hyphal system and strongly dextrinoid basidiospores. The differences between the new species and morphologically similar and phylogenetically related species are discussed. In addition, an updated key to 27 species of Haploporus is provided.

During a study on polypores from Ecuador and China, we collected specimens that morphologically fit the definition of Haploporus. After further examination and phylogenetic analysis, they formed two distinct lineages within Haploporus, and are morphologically different from the existing species in the genus. Thus, we describe them here as two new species.

Morphological studies
The studied Haploporus specimens are deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC), the private herbarium of Josef Vlasaḱ (JV), and the National Museum Prague of Czech Republic (PRM). For the morphological description, we followed the method from a previous study (Wu et al., 2022b). Color terms are from Anonymous (1969) and Petersen (1996).

DNA extraction, PCR, and sequencing
The DNA was extracted from the dried specimens using a rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing, China), following the manufacturer's protocol. The internal transcribed spacers (ITS), large subunit of nuclear ribosomal RNA gene (LSU), and small subunit mitochondrial rRNA gene (mtSSU) were amplified with primer pairs ITS 5 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′) and ITS 4 (5′-TCC TCC GCT TAT TGATAT GC-3′; White et al., 1990), LR0R (5′-ACC CGC TGA ACT TAA GC-3′) and LR7 (5′-TAC TAC CAC CAA GAT CT-3′; http://www. biology.duke.edu/fungi/mycolab/primers.htm ), and MS1 (5′-CAG CAG TCA AGA ATA TTA GTC AAT G-3′) and MS2 5′-GCG GAT TAT CGA ATT AAA TAA C-3′; White et al., 1990), respectively. The PCR procedures were as follows: for ITS and mtSSU regions, an initial denaturation at 95°C for 3 min, followed by 34 cycles at 94°C for 40 s, 54°C for ITS and 55°C for mtSSU for 45 s and 72°C for 1 min, and a final extension of 72°C for 10 min; for the LSU region, an initial denaturation at 94°C for 1 min, followed by 34 cycles at 94°C for 30 s, 50°C for 1 min and 72°C for 1.5 min, and a final extension of 72°C for 10 min Zhao et al., 2022c). The PCR products were sequenced using BGI Tech Solutions (Beijing Liuhe Co., Ltd., Beijing, China). Finally, all the new sequences were submitted to GenBank, and the accession numbers are shown in Table 1.

Phylogenetic analyses
The sequences generated were aligned with sequences downloaded from GenBank (Table 1) using MAFFT (version 7) and then manually adjusted (Katoh & Standley, 2013). A dataset of 34 specimens consisting of ITS, LSU, and mtSSU sequences was analyzed using Maximum Likelihood (ML), Maximum Parsimony (MP), and Bayesian Inference (BI) phylogenetic analyses using RAxML (version 8; Stamatakis, 2014), PAUP (version 4.0b10; Swofford, 2002), and MrBayes (version 3.2.7a; Ronquist et al., 2012), respectively, following Zhao et al, 2021;Zhao et al, 2022a;Zhao et al, 2022b). The ModelTest-NG (version 0.1.7; Darriba et al., 2020) determined the best models of ITS, LSU, and mtSSU sequences. The ML analysis was carried out with 1,000 bootstrap replications using the GTR + I + G substitution model. The MP analysis was conducted using 1,000 bootstrap replications with the heuristic search option. The BI analysis was performed for two million generations with random initial trees, using the GTR + I + G substitution model and the first 25% were set as burn-in.
The phylogenetic tree was visualized using FigTree version 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/ ). Branches that received bootstrap support for ML, BP, and Bayesian Posterior Probabilities (BPP) greater than or equal to 50% (ML/BP) and 0.95 (BPP) were considered as significantly supported, respectively.

Phylogeny
In this study, the combined ITS + LSU + mtSSU dataset included sequences from 37 specimens, representing 25 species of Haploporus and 2 species of Perenniporia Murrill as the outgroup (Table 1 and Figure 1). The aligned dataset had a length of 1,932 characters, of which 540 were constant characters, 122 were The sequences generated in this study are in bold. "−" represents sequences unavailable in GenBank.
parsimony-uninformative characters, and 221 were parsimonyinformative characters. The MP analysis yielded a tree with a length of 812, a consistency index of 0.5246, a homoplasy index of 0.4754, a retention index of 0.7551, and a rescaled consistency index of 0.3961. The best model for the ITS + LSU + mtSSU aligned dataset was GTR + I + G in the Bayesian analysis, and the average standard deviation of split frequencies is 0.00424. The phylograms of Bayesian analysis, MP analysis, and ML analysis are similar in topology, and the ML tree was chosen to represent the phylogenetic relationships ( Figure 1). The phylogenetic tree suggests that the specimen of H. ecuadorensis forms an independent lineage in the Haploporus clade, and specimens of H. monomitica are closely related to H. odorus with strong support. Basidiomata resupinate, annual, inseparable from the substrate, more or less corky when dry, up to 5 cm long, 1.5 cm wide, and 1.5 mm thick at the center. Hymenophore pinkish buff to honey yellow when dry, without distinct margin; pores angular to round, 2-4 per mm; dissepiments thick, entire. Subiculum paler than tubes, more or less corky, up to 0.5 mm thick. Tubes olivaceous buff, hard corky, up to 1.0 mm long.
Additional materials studied: China, Beijing, Mentougou, Xiaolongmen National Forest Park, on fallen trunk of Quercus sp., 30 August 2022, Yu-Cheng Dai, Dai 24429, Dai 24451. Distribution and ecology: Haploporus monomitica is distributed in temperate area of Beijing, China; it grows on fallen trunk of Quercus, and causes a white rot.