Taxonomic and phylogenetic contributions to Fuscoporia (Hymenochaetales, Basidiomycota): two new species from Hawaii with a key to North American species

Fuscoporia is a cosmopolitan, poroid, wood-decaying genus, belonging to the Hymenochaetales. During a study of wood-inhabiting fungi in the USA, four unknown specimens were collected from Hawaii. Both morphological criteria and molecular genetic analyses based on the ITS+nLSU+EF1-α datasets and the nLSU dataset confirmed that these four specimens represent two new species of Fuscoporia, and they are described as F. hawaiiana and F. minutissima. Fuscoporia hawaiiana is characterized by pileate basidiocarps, the absence of cystidioles, hooked hymenial setae, broadly ellipsoid to subglobose basidiospores measuring 4−6 × 3.5−4.5 μm. Fuscoporia minutissima is distinguished by small pores (10−13 per mm) and basidiospores (3.4−4 × 2.4−3 μm). The taxonomic status of the two new species is briefly discussed. A key to the North American species of Fuscoporia is provided.


Introduction
Most wood-rotting fungi, belonging to Basidiomycetes, can use different types of wood as their nutritional source and are indispensable participants and important biological regeneration resources in natural ecosystems. The genus Fuscoporia Murrill (Hymenochaetales, Basidiomycota), erected by Murrill (1907) with F. ferruginosa (Schrad.) as generic type, is a cosmopolitan fungal group that encompasses also important species with medicinal effects, such as F. gilva (Schwein.) T. Wagner & M. Fisch. and F. torulosa (Pers.) T. Wagner & M. Fisch. (Wu et al., 2019). Fuscoporia is widely distributed in Asia, Europe, Oceania, and America. The genus has been considered a synonym of Poria Adans. or Phellinus Queĺ. for a long time (Overholts, 1953;Lowe, 1966;Ryvarden and Johansen, 1980;Gilbertson and Ryvarden, 1987;Larsen and Cobb-Poulle, 1990). However, Fiasson and Niemela (1984) recognized the genus as monophyletic morphologically, characterized by annual to perennial and resupinate to pileate basidiomata, a dimitic hyphal system with crystal encrustations on generative hyphae, the presence of hymenial setae, and hyaline, thin-walled and smooth basidiospores. Later, Wagner and Fischer, 2001;Wagner and Fischer, 2002) by means of nLSU sequence data and morphological studies of European Hymenochaetales reconfirmed the taxonomic status of the genus of Fuscoporia, with six species of Phellinus being classified into it.
Large number of Fuscoporia new species and new combinations have been discovered in recent years, and at present, more than 90 species are accepted in the genus Tchoumi et al., 2020;Vlasaḱ et al, 2020;Yuan et al., 2020;Dai et al., 2021;Chen et al., 2022;Hussain et al., 2022;Wu et al., 2022a, b). Fuscoporia is considered to be a complex genus with still unsettled taxonomy.
Striking diversity in the Hawaiian Islands with multiple cooccurring species leads to many species unreported in the literature. During a study of wood-inhabiting fungi, four unknown specimens were collected from Hawaii, with the features of Fuscoporia. To explore their relationships, phylogenetic analyses based on the ITS +nLSU+EF1-a datasets and the nLSU dataset were carried out. Both morphological criteria and molecular genetic analyses confirmed that these four specimens represent two new species of Fuscoporia. So, we describe them as F. hawaiiana and F. minutissima in the present paper. A key to the North American species of Fuscoporia is also provided.

Morphological studies
Some studied specimen materials are deposited in the fungoria of the Institute of Microbiology, Beijing Forestry University (BJFC). The other ones are deposited in private fungoria of Josef Vlasak (JV) and then will be transferred into Prague Museum Herbarium (PRM). Morphological descriptions are based on field notes and herbarium specimens. The macroscopic color terms cited from Anonymous (1969) and Petersen (1996). Sections were studied at a magnification up to × 1,000 using a Nikon Eclipse 80i microscope with phase contrast illumination (Nikon, Tokyo, Japan). The microscopic analyses were performed accroding to Liu et al. (2022) and Si et al. (2023). Microscopic features, measurements, and drawings were prepared from slides stained with Cotton Blue. The following abbreviations are used: KOH = 5% potassium hydroxide, CB− = acyanophilous in Cotton Blue, IKI− = neither amyloid nor dextrinoid in Melzer's reagent, L = arithmetic average of all spore length, W = arithmetic average of all spore width, Q = variation in the L/W ratios, and (n = x/y) = number of measured spores (x) measured from a given number of specimens (y).

DNA extraction, PCR, and sequencing
A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to extract total genomic DNA from dried specimens following the manufacturer's instructions with some modifications Zhang et al., 2023;Zhou et al., 2023). For generation of PCR amplicons, the following primer pairs were used: ITS5 (GGA AGT AAA AGT CGT AAC AAG G) and ITS4 (TCC TCC GCT TAT TGATAT GC) for internal transcribed spacer (ITS, White et al., 1990); LR0R (ACC CGC TGA ACT TAA GC) and LR7 (TAC TAC CAC CAA GAT CT) for nuclear large subunit rDNA (nLSU, Vilgalys and Hester, 1990); EF1-983F (GCY CCY GGH CAY CGT GAY TTY AT) and EF1-1567R (ACH GTR CCR ATA CCA CCR ATC TT) for translation elongation factor-1 alpha (EF1-a, Rehner and Buckley, 2005). The PCR procedures for ITS and EF1-a were as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 94°C for 40 s, annealing at 54°C for 45 s and extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94°C for 1 min, followed by 35 cycles of denaturation at 94°C for 1 min, 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 with the same primers and the sequences are deposited in GenBank. All newly generated sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov ) and are listed in Table 1.

Phylogenetic analysis
The following softwares were used for data processing and phylogenetic analyses: Mesquite, MAFFT 7.110, BioEdit 7.0.1 (Hall, 1999), ClustalX 2.0P10 (Thompson et al., 1997), MrModeltest 2.3 (Posada and Crandall, 1998;Nylander, 2004), PAUP* 4.0b10 (MP, Swofford, 2002), raxmlGUI 1.2 (ML, Stamatakis, 2006;Silvestro and Michalak, 2012), TreeView 1.5.0, and PowerPoint. Maximum parsimony (MP) and maximum likelihood (ML) methods were adopted to perform phylogenetic analyses of the two aligned datasets. The two phylogenetic methods resulted in similar topology for each data set. Thus, only the topology of the MP analysis appears along and branches that received bootstrap supports greater than or equal to 75% (MP and ML) were considered as significantly supported at the nodes.
The ITS+nLSU+EF1-a dataset (Figure 2) included 98 ITS, 92 nLSU, and 62 EF1-a sequences from 100 fungal specimens representing 52 pecies of Fuscoporia. The dataset had an aligned length of 2705 characters, of which 1721 were constant, 99 variable Fruiting body. -Basidiocarps perennial, pileate, laterally fused to imbricate, without odor or taste when fresh, hard corky when dry. Pilei mostly semicircular, projecting up to 11 cm long, 5 cm wide, and 5 cm thick at the base, more or less convex towards margin. Pileal surface reddish brown, concentrically sulcate with zones, glabrous, sometimes covered with mosses; margin obtuse, yellowish brown, up to 5 mm wide. Pore surface honey-yellow to deep olive, slight glancing; margin narrow, olivaceous buff, up to 1 mm wide; pores circular, 5−7 per mm; dissepiments thin to fairly thick, entire, abundant hymenial setae in tube cavities (under anatomical lens). Context clay-buff, hard corky, about 8 mm thick. Tubes olivaceous buff, hard corky, up to 2 cm long.
Spores. -Basidiospores broadly ellipsoid, hyaline, thin-walled, smooth, IKI−, CB−, some of them bearing a guttule, 4−6 × (3.4−)3.5 Phylogeny of Fuscoporia and related species generated inferred from nLSU sequences. Statistical values (MP/ML) are indicated for each node. Names of new species are in bold.  Etymology. -Minutissima (Lat.): refers to the very small size of the pores exhibiting by these species.
Fruiting body. -Basidiocarps perennial, pileate, imbricate, without odor or taste when fresh, hard corky when dry. Pilei mostly imbricate, projecting up to 5 cm long, 3 cm wide, and 1 cm thick at the base. Pileal surface reddish brown, concentrically sulcate with zones, nodulose; margin obtuse to slightly acute, honey-yellow, up to 1 mm wide. Pore surface greyish brown to deep olive, glancing; margin narrow, honey-yellow, up to 1 mm wide; pores circular, 10−13 per mm; dissepiments fairly thick, entire and matted, abundant hymenial setae in tube cavities (under anatomical lens). Context honey-yellow, hard corky, up to 5 mm thick. Tubes olivaceous buff, paler than pores, hard corky, up to 4 mm long.

Discussion
The islands far from the mainland have attracted scientific researchers for a long time, due to the close combination of ecological environment and evolution process here that deepen our understanding of the formation process of biological diversity (Cotoras et al., 2018). Hawaii is a biological hotspot with a variety of climates and habitats. 160 genera and 400 species of wood-rotting basidiomycetes are reported from 110 native and exotic substrate species on the Hawaiian Islands (Gilbertson et al., 2002;Ashiglar et al., 2015). There are few natural forests on the Hawaii islands (Chambers et al., 2007). Most forests are filled with introduced trees, and our new species may be introduced as well. The two new wood-rotting fungal species Fuscoporia hawaiiana and F. minutissima were collected from the northernmost and oldest Kauai Island, and the largest island Big Island in the Hawaiian Islands, both with a tropical sea climate.
Fuscoporia hawaiiana is characterized by perennial and pileate basidiocarps, circular and medium pores (5−7 per mm), the absence of cystidioles, hooked hymenial setae, broadly ellipsoid to subglobose basidiospores measuring 4−6 × 3.5−4.5 mm. Fuscoporia hawaiiana may also be distributed in Yunnan Province, China, considered to be F. torulosa derived from CLZhao 10146 (OM959398) on GenBank. The ITS of CLZhao 10146 shows that only 2 base differences from our new samples, but unfortunately, it lacks nLSU or EF1-a sequence. Furthermore, F. torulosa can be easily distinguished from F. hawaiiana by its subungulate basidiocarps and straight hymenial setae. Morphologically, seven species, F. semihispida   (Figures 1, 2) and morphology. F. atlantica differs from F. hawaiiana by its annual basidiocarps (Pires et al., 2015); F. australasica is described from Southern China and Viet Nam and differs from F. hawaiiana by its glabrous basidiocarps and the presence of cystidioles ; F. wahlbergii is described from Europe, East-African, Australia, and USA, and distinguishes from F. hawaiiana by its smaller pores (7−9 per mm) and the presence of cystidioles ; F. eucalypti is distributed Australia, and differs from F. hawaiiana by its spores effusedreflexed to pileate and wider (4.3−5.5 × 4−4.5 mm) ; F. marquesiana is a South American species, and can be easily distinguished from F. hawaiiana by its smaller pores (8−9 per mm) (Wu et al., 2022b). Even without molecular data, F. semihispida and F. rufa can be easily distinguished from F. hawaiiana by narrower or wider spores (2−2.5 mm in F. semihispida; 4−4.5 mm in F. rufa) (Wu et al., 2022a).
Fuscoporia minutissima stands out in the genus by its perennial and pileate basidiocarps, and very small pores (10−13 per mm) and spores (3.4−4 × 2.4−3 mm). Fuscoporia minutissima is usually sterile after collecting, and such specimens are very similar to F. gilva by pileate basidiocarps, glabrous to rugose sometimes with nodulose pileal surface and broadly ellipsoid to subglobose spores, but the later differs by having bigger pores (6−8 per mm). Fuscoporia plumeriae Q. Chen, F. Wu & Y.C. Dai and F. chinensis Q. Chen, F. Wu & Y.C. Dai clustered together with F. minutissima into a group with a strong support (100/100, Figures 1, 2). Morphologically, these species share the effused-reflexed to pileate basidiocarps with lacerate dissepiments and ellipsoid basidiospores; but F. plumeriae and F. chinensis have annual basidiocarps and  (Wu et al., 2022b). Fuscoporia minutissima may be confused with F. rhabarbarina in field, but the pileal surface of F. rhabarbarina is glabrous and dark brown crust at the base. Moreover, the two species are not closely related in the phylogenetic tree ( Figure 1).
Fuscoporia hawaiiana and F. minutissima are described in this study. Traditionally, they were most probably treated as F. wahlbergii and F. gilva, respectively. The phylogenetic analyses provide molecular evidence to support these two new species derive from the F. wahlbergii complex and the F. gilva complex (Figures 1, 2). The similar results are demonstrated in many polypores complex, for instances, Megasporoporia setulosa (Henn.) Rajchenb. (Wang et al., 2021;Wang et al., 2022), Heterobasidion annosum (Fr.) Bref. , (Mao et al., 2023), Sidera vulgaris (Fr.) Miettinen (Liu et al., 2021), and Phaeolus schweinitzii (Fr.) Pat. . It seems that the diversity of wood-inhabiting fungi is extremely rich in tropics, some traditional definition on tropical species should be re-evaluated, and the concepts of taxa in tropics should be modified after molecular phylogeny.
A key to North American species of Fuscoporia.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Author contributions QC, LL, JS, and JV designed the research and contributed to data analysis and interpretation. JV prepared the samples. QC and LL conducted the molecular experiments and analyzed the data. QC prepared the samples and drafted the manuscript. JV and JS discussed the results and edited the manuscript. All authors contributed to the article and approved the submitted version.