﻿Molecular and morphological data reveal two new species of Tropicoporus (Hymenochaetaceae, Basidiomycota) from Australia and tropical Asia

﻿Abstract Phylogenetic analyses and morphological examination confirmed two new species in the tropical polypore genus Tropicoporus, T.oceanianus and T.zuzaneae, from Australia and tropical Asia, respectively. A phylogenetic analysis based on the two DNA markers including the nuclear ribosomal internal transcribed spacer (ITS) region and the large subunit (nLSU) gene shows that these two new species form two independent lineages nested in the genus Tropicoporus. T.oceanianus is characterized by perennial and ungulate basidiomata, the occasional presence of hymenial setae, a trimitic hyphal structure in the context and a dimitic hyphal system in the trama, and broadly ellipsoid to subglobose basidiospores measuring 5.2–6 × 4–5 μm. T.zuzaneae is characterized by perennial and resupinate basidiomata with distinct receding margin, glancing pores, very thin to almost lacking subiculum, a dimitic hyphal structure, the absence of any setal elements, broadly ellipsoid to subglobose basidiospores measuring 3.8–4.9 × 3–4.2 µm. The differences among the new species and their phylogenetically related and morphologically similar species are discussed.


Introduction
Tropicoporus L.W. Zhou et al. (Hymenochaetaceae, Basidiomycota) is mainly a tropical polypore genus, and it is characterized by annual to perennial, resupinate to distinctly pileate basidiomata with yellow-brown to umber pore surface, a dimitic hyphal system at least in the trama, the presences of hymenial setae, and yellowish, slightly thick-walled, smooth, and usually collapsed basidiospores which become darker in a 5% KOH solution in a few species (Salvador-Montoya et al. 2018, 2020).Most species of the genus grow on angiosperm wood and cause a white rot (Zhou et al. 2016).As of early 2024, 49 species are accepted in the genus, 40 species exist in tropical region, and 25 species occur in tropical Asia and Australia (Tian et al. 2013;Xavier de Lima et al. 2022;Wu et al. 2022a, b;Gunaseelan et al. 2024;Liu et al. 2024).Tropicoporus excentrodendri L.W. Zhou & Y.C. Dai is the type species of the genus.
Tropical Pacific areas are rich for species of Hymenochaetales, and many new taxa have been described from these areas recently (Ji et al. 2017;Bian and Dai 2020;Chen et al. 2020;Du et al. 2020;Guo et al. 2022;Wu et al. 2022a;Zhao et al. 2022;Cui et al. 2023;Dong et al. 2023).However, there are still many unknown taxa in Hymenochaetales from certain regions of tropical Pacific areas.
A study on tropical polypores recovered four specimens from Australia and tropical Asia that morphologically fit the definition of Tropicoporus.Phylogenetic analyses assigned these specimens to two independent lineages nested in the Tropicoporus clade.Morphological comparison with all the taxa in Phellinus s.l. was carried out, and no existing taxa fit them.We thus describe two new species based on our studied samples and molecular data.

Morphological studies
The studied specimens are deposited in the Fungarium of the Institute of Microbiology, Beijing Forestry University (BJFC), the private herbarium of Josef Vlasák (JV), and the Royal Botanic Gardens Victoria (MEL).Morphological descriptions are based on field notes and voucher specimens.The microscopic analysis follows Dai (2010) and Wu et al. (2022a).Sections were studied at a magnification of up to 1 000× using a Nikon Eclipse 80i microscope and phase contrast illumination.Microscopic features and measurements were made from slide preparations stained with Cotton Blue and Melzer's reagent.Basidiospores were measured from sections cut from the tubes stained with Cotton Blue.To represent the variation in the size of spores, 5% of measurements were excluded from each end of the range and are given in parentheses.In the description: KOH = 5% potassium hydroxide, IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, CB = Cotton Blue, CB(+)= weakly cyanophilous in Cotton Blue, CB-= acyanophilous in Cotton Blue, L = arithmetic average of spore length, W = arithmetic average of spore width, Q = L/W ratios, and n = number of basidiospores/measured from given number of specimens.Color terms follow Anonymous (1969) and Petersen (1996).
The PCR procedure for ITS was as follows: initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, annealing at 54 °C for 45 s and extension 72 °C for 1 min, and a final extension of 72 °C for 10 min.The PCR procedure for nLSU was as follows: initial denaturation at 94 °C for 1 min, followed by 34 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 1 min and extension at 72 °C for 1.5 min, and a final extension at 72 °C for 10 min.The PCR products were purified and sequenced at the Beijing Genomics Institute (BGI), China, with the same primers.DNA sequencing was performed at the Beijing Genomics Institute and the newly generated sequences were deposited in GenBank.All sequences analysed in this study are listed in Table 1.Sequences generated from this study were aligned with additional sequences downloaded from GenBank using BioEdit (Hall 1999).The final ITS and nLSU datasets were subsequently aligned using MAFFT v.7 under the G-INS-i strategy with no cost for opening gaps and equal cost for transformations (command line: mafft -genafpair -maxiterate 1000) (Katoh and Standley 2013) and visualized in BioEdit (Hall 1999).

Phylogenetic analyses
The two genetic markers were concatenated into a single multiple sequence alignment for phylogenetic analysis ( Sequences were analysed using Maximum Likelihood (ML) with RAxML-HPC through the CIPRES Science Gateway (www.phylo.org;Miller et al. 2009).Branch support for ML analysis was determined by 1000 bootstrap replicates.Bayesian phylogenetic inference was done in MrBayes 3.2.7a(Ronquist et al. 2012).Four Markov chains were run for 2 million generations (2-gene dataset) until the split deviation frequency value was less than 0.01, and trees were sampled every 1000 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.All trees were viewed in FigTree v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).Branches that received ML bootstrap support of at least ≥75% and BPP of at least ≥ 0.90 BPP were considered as significantly supported.The significant ML bootstrap values and the BBP are presented on the topology from the ML analysis, respectively.

Molecular phylogeny
The concatenated two-marker dataset included sequences from 77 samples representing 41 taxa.The dataset had an aligned length of 2371 characters, of which 1664 (70%) were constant, 193 (8%) were variable and parsimony-uninformative, and 514 (22%) were parsimony informative.The phylogenetic reconstructions performed with Maximum Likelihood (ML) and Bayesian Inference (BI) analyses produced similar topologies and only minor differences in statistical support.The best model-fit applied in the Bayesian analysis was GTR+I+G.Bayesian analysis resulted in a nearly congruent topology with respect to the ML analysis, and thus only the ML tree is provided (Fig. 1).And the average standard deviation of split frequencies was 0.005467 (BI).Etymology.Oceanianus (Lat.):refers to the species being found in Oceania.Description.Basidiomata.Perennial, pileate, solitary, woody hard and without odor or taste when fresh, bone hard when dry; pilei ungulate to triquetrous, projecting up to 2 cm, 3 cm wide, and 2.5 cm thick at base; pileal surface vinaceous gray to black when fresh and dry, concentrically sulcate with narrow zones, velutinate to glabrous, encrusted with age, distinctly cracked; margin more or less acute, snuff brown.Pore surface fawn brown when fresh, becoming umber when dry, glancing; sterile margin fawn brown when fresh and dry, distinctly paler than pores, up to 2 mm wide; pores circular, 6-7 per mm; dissepiments thick, entire.Context homogeneous, fulvous, woody hard, up to 3 mm thick, a black crust present at pileal surface.Tubes concolorous with pore surface, bone hard to brittle, up to 22 mm long, annual layers indistinct.

Discussion
Tropicoporus oceanianus is characterized by perennial and ungulate basidiomata with glancing pores, hymenial setae occasionally context with a trimitic and tube trama with a dimitic hyphal system, and broadly ellipsoid to subglobose basidiospores measuring 5.2-6 × 4-5 μm.Although we studied a single specimen (Dai 18859), three samples (MEL 2382654, MEL 2382727 and MEL 238278) from Australia have available sequences in GenBank, and their sequences (KP013017, KP012908 and KP012961) are identical to those of Dai 18859.We thus treat MEL 2382654, MEL 2382727 and MEL 238278 as Tropicoporus oceanianus in the present paper.
Tropicoporus zuzaneae is characterized by perennial and resupinate basidiomata with receding margin, glancing pores as 6-8 per mm, very thin to almost lacking subiculum, a dimitic hyphal structure, the absence of any setal elements, broadly ellipsoid to subglobose basdiospores measuring 3.8-4.9× 3.1-4.2µm.We studied two Chinese specimens (Dai 18859, Dai 22168)  Wu by sharing perennial and resupinate basidiomata with pore 6-9 per mm, and broadly ellipsoid to subglobose basidiospores, but the latter four species are different from T. zuzaneae by the presence of hymenial setae (Wu et al. 2022a).
Two new members of Tropicoporus are described in the present paper.Tropicoporus oceanianus is unique in the genus by its trimitic hyphal structure in context, and T. zuzaneae is unique in the genus by its absence of any setal elements.We thus modify the definition of Tropicoporus to be annual to perennial, resupinate to distinctly pileate basidiomata with yellow-brown to umber pore surface, mostly a dimitic hyphal system at least in trama, a few with trimitic or monomitic hyphal sys-tem in context, hymenial setae present in most species, and yellowish, slightly thicksmooth, usually collapsed basidiospores which become darker in a 5% KOH solution in a few species, growing on angiosperm wood and causing a white rot.

Figure 1 .
Figure1.Phylogeny of Tropicoporus generated by ML analyses based on combined ITS+nLSU sequences.Branches are labelled with maximum likelihood bootstrap higher than 75% and Bayesian posterior probabilities higher than 0.90.New species are indicated in bold.0.03

Table 1 .
Taxa information and GenBank accession numbers of the sequences used in this study.New species are shown in bold.* Holotype.
T. cambodiensis (L.W. Zhou & W.M. Zhang) Y.C. Dai & F. Wu and T. inamoenus (Mont.)Y.C. Dai & F. Wu by sharing pileate and solitary basidiomata with concentrically sulcate and zonate at pileal surface, similar size of pores and basidiospores, but T. cambodiensis differs from T. oceanianus by a dimitic hyphal structure without skeleto-binding hyphae in context, and it has a distribution in Cambodia