Introduction

The genus Fulvifomes Murrill, typified by F. robiniae (Murrill) Murrill (Murrill 1914; Pyropolyporus robiniae Murrill, basionym), consists of bracket polypores with perennial basidiomes, rough to rimose pileal surfaces, and coloured basidiospores (Murrill 1903, 1904). The taxonomic status of this genus has long been debated. One argument supported Fulvifomes as a genus (Kotlaba and Pouzar 1978; Fiasson and Niemelä 1984), whilst the other considered it a synonym (Gilbertson 1980; Ryvarden and Johansen 1980; Ryvarden 1991; Ryvarden 1994) or a subgenus of Phellinus Quél. (Dai 1999). This debate was resolved by phylogenetic analyses of the nuclear large ribosomal subunit (nLSU) rDNA, which strongly supported Fulvifomes as an independent genus (Wagner and Fischer 2002).

The generic morphological diagnosis of Fulvifomes has been redefined based on the phylogenetic analysis of internal transcribed spacer (ITS) and nLSU sequences, which separated Fulvifomes species from the others. This revision encompassed characteristics such as annual to perennial growth, sessile, effused-reflexed to substipitate basidiomes with solitary to imbricate pilei, homogeneous or duplex context, a hyphal system ranging from monomitic to dimitic, the absence of hyphoid and hymenial setae, and subglobose to ellipsoid, flattened ventral side, yellowish to brown and slightly thin to thick-walled basidiospores (Zhou 2014; Salvador-Montoya et al. 2018).

Most Fulvifomes species are distributed in the tropical and subtropical regions of Asia (Zhou and Zhang 2012; Hattori et al. 2014; Zhou 2014, 2015; Ji et al. 2017), America (Ji et al. 2017; Salvador-Montoya et al. 2018; Zhou et al. 2023), and Africa (Olou et al. 2019). The Federated States of Micronesia (FS Micronesia) is a biodiversity hotspot in the tropical region of the Pacific Ocean. To date, four Fulvifomes species have been reported from the FS Micronesia, namely Aurificaria luteoumbrina (Romell) D.A. Reid (≡ Fulvifomes luteoumbrinus (Romell) Y.C. Dai & Vlasák), Phellinus fastuosus (Lév.) S. Ahmad (≡ Fulvifomes fastuosus (Lév.) Bondartseva & S. Herrera), P. mangrovicus (Imazeki) Imazeki (≡ Fulvifomes mangrovicus (Imazeki) T. Hattori), and P. merrillii (Murrill) Ryvarden (≡ Fulvifomes merrilli (Murrill) Baltazar & Gibertoni) (Zhou 2015; Gilbert et al. 2008). However, as various Fulvifomes species have been reported in tropical regions, the diversity of Fulvifomes species in FS Micronesia is expected to be higher.

In this study, we aimed to examine Phellinus-like specimens collected from the FS Micronesia based on ITS and nLSU phylogenetic analyses, and on morphological characteristics. We also generated sequence data for RNA polymerase II (RPB2) and translation elongation factor-1 alpha (TEF1) protein-coding genes. Three species of Fulvifomes were determined, two of which are proposed as new to science. Detailed morphological descriptions are provided for the new taxa. Our findings contribute to the diversity of Fulvifomes species in the FS Micronesia.

Material and methods

Morphological studies

Six fruiting bodies were collected from three islands in the FS Micronesia; Chuuk Weno, Kosrae Lelu and Kosrae Tafunsak, in 2016 and 2017 (Fig. 1). Sampling and transport processes were performed with the permission of the FS Micronesia Government. Dried fungal specimens were deposited at the Seoul National University Fungus Collection (SFC). Photographs of the fruiting bodies were captured in the field.

Fig. 1
figure 1

A Geographical map of Fulvifomes distribution around the world. Distribution of phylogenetically confirmed Fulvifomes species from Table 1 is shown. Red dots indicate the tropical region, green indicates the sub-tropical region, and blue indicates the temperate region. B Sampling locations of Fulvifomes species in FS Micronesia. Orange dots indicate the locations where specimens were found

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Table 1 List of Fulvifomes specimens and GenBank accessions
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Microscopic examination of the dried specimens was performed by sectioning the tissue into 1 cm × 1 cm pieces. Tissue sections were mounted in 5% KOH on a glass slide and then observed using a Nikon Eclipse 80i microscope (Nikon, Japan). Approximately 20 basidia and basidiospores were measured per specimen using ImageJ software (Collins 2007). The Q values of the basidiospores indicate their length-to-width ratios. Colours were described based on ‘Methuen Handbook of Colour’ (Kornerup and Wanscher 1967). The pore surface and pore size were measured using a Nikon SMZ1500 stereomicroscope (Nikon, Japan). The following abbreviations were used: IKI = Melzer’s reagent, IKI −  = neither amyloid nor dextrinoid, CB = Cotton Blue, CB −  = acyanophilous, L = mean spore length, W = mean spore width, Q = ratios of L/W of specimens studied, and n = number of spores measured from the given number of specimens.

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from dried specimens (1 cm × 1 cm of tissue) using the CTAB (Rogers and Bendich 1994) protocol and the AccuPrep® Genomic DNA Extraction Kit (Bioneer, Daejeon, Republic of Korea). Polymerase Chain Reaction (PCR) was performed for four genetic regions. The ITS region was amplified using ITS1F/ITS4 (White et al. 1990) or ITS1/ITS4B primers (Gardes and Bruns 1993). LR0R/LR7 (Vilgalys and Hester 1990) or LR0R/LB-y (Tedersoo et al. 2008) primers were used for nLSU. PCR for the ITS region and nLSU was performed under the following conditions: 5 min initial denaturation at 95 °C, followed by 35 cycles of 40 s at 95 °C, 40 s at 54 °C and 90 s at 72 °C, with a final extension step for 5 min at 72 °C. For the protein-coding genes, RPB2 region was amplified with primers bRPB2-6F/bRPB2-7.1R (Matheny 2005), under the following conditions: initial denaturation at 94 °C for 2 min, 36 cycles of 94 °C for 45 s, 53 °C for 90 s, 72 °C for 90 s, and a final extension at 72 °C for 10 min. The TEF1 region was amplified with primers EF595F/EF1160R (Kauserud and Schumacher 2001) under the following conditions: initial denaturation at 95 °C for 4 min, 35 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 7 min.

The amplification of the PCR products was verified by gel electrophoresis using 1% agarose gel with EcoDye DNA staining solution (SolGent Co., Daejeon, Republic of Korea). The products were purified using an ExpinTM PCR Purification Kit (GeneAll Biotechnology, Seoul, Republic of Korea). Sequencing was performed by Macrogen (Seoul, Republic of Korea) using an ABI 3730xL DNA analyser (Life Technologies, Gaithersburg, MD, USA). All sequences were proofread and edited using MEGA version 11 (Kumar et al. 2016) and FinchTV v.1.4. All the newly generated sequences were deposited at GenBank (Table 1).

Multiple peaks were found in the ITS region for three specimens (SFC20170118-26, SFC20170120-06 and SFC20170120-09), which were separated into their respective ITS type through cloning. ITS amplicons were cloned into the pTOP TA V2 blunt end vector (Enzynomics, Korea) and transformed into competent cells (Escherichia coli, DH10β). Transformed clones were re-amplified as described by Hattori et al. (2022). Sequencing of each ITS type was performed as above described.

Phylogenetic analysis

Phylogenetic trees were constructed using ITS and nLSU sequences. Newly generated sequences and published reference sequences of Fulvifomes from GenBank were collected with GenMine (Seo et al. 2022), and used for phylogenetic analyses. Inocutis dryophilus and Fomitiporella americana were used as outgroups due to their close phylogenetic relationships with Fulvifomes (Guglielmo et al. 2008a, b; Pildain et al. 2018). Sequence data collection, outgroup selection, multiple sequence alignment, trimming, concatenation, model selection and maximum likelihood (ML) tree construction were performed with FunID ver 0.3.4 with ‘accurate’ preset (https://github.com/Changwanseo/FunID). The final sequence alignments from FunID are in Online Resource 1. Phylogenetic trees were visualised by a modified ‘tree_interpretation.py’ function of FunID. All processed data are shown in Table 1.

Results

Molecular phylogeny

The final combined dataset (ITS1 + 5.8S + ITS2 + nLSU) included 64 specimens from 40 species. The concatenated sequences comprised 1604 nucleotides, including gaps. The ITS1, 5.8S, ITS2 and nLSU regions comprised 365, 142, 216 and 881 bases, respectively. Protein-coding gene sequences (RPB2 and TEF1) were not included in the phylogenetic analyses because of the lack of reference sequences.

The six newly analysed specimens formed three well-supported clades in the ML analyses (Fig. 2). One specimen (SFC20170120-02) grouped with Fulvifomes halophilus with 100% bootstrap value. The other two clades were distinct from any described Fulvifomes species (bootstrap = 100). After comparing the morphological descriptions of these specimens with those of Fulvifomes species without molecular data (Lowe 1957; Ryvarden and Johansen 1980; Hattori 1999; Dai 2010; Hattori et al. 2014), we proposed them as two new species: Fulvifomes labyrinthus sp. nov. and Fulvifomes rhizophorus sp. nov. The two new species of Fulvifomes were further validated as novel based on their distinguishable morphological characteristics from other known Fulvifomes species.

Fig. 2
figure 2

Maximum likelihood tree of Fulvifomes constructed based on ITS and nLSU regions. The symbols (+/+) indicate the availability of ITS/nLSU sequences or the absence (‒/‒). Taxa in bold correspond to the type specimens. Purple boxes indicate new species from the FS Micronesia

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Three ITS types were identified in Fulvifomes rhizophorus sp. nov. (SFC20170120-06, SFC20170120-09 and SFC20170118-26). Each specimen had two different types of ITS sequences: Type1 ITS was shared across all three specimens, Type2 ITS was found in SFC20170120-06, and Type3 ITS was found in SFC20170120-09 and SFC20170118-26. Two indels and 13 substitutions were detected (Fig. 3).

Fig. 3
figure 3

ITS sequence types of Fulvifomes rhizophorus. Three ITS types were identified in Fulvifomes rhizophorus and two different types of ITS were present in each specimen. In the ITS1 region, there were five substitutions and one indel. In the ITS2 region, there were eight substitutions and one indel

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Taxonomy

Fulvifomes labyrinthus H. Suh, Y. Cho, D. Kim & Y.W. Lim, sp. nov. (Fig. 4)

Fig. 4
figure 4

Morphological characters of Fulvifomes labyrinthus (SFC20160126-30, holotype). A Basidiome. B Pore surface. C Drawings of microscopic features: ‘sp’ basidiospores, ‘b1’ basidia, ‘b2’ basidioles, ‘h1’ generative hypha and ‘h2’ skeletal hypha in trama

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MycoBank Number: MB849263

Diagnosis: Fulvifomes labyrinthus grows on the trunk of the mangrove tree, Xylocarpus granatum, and is characterised by irregular pores.

Holotype: Federated States of Micronesia, Chuuk State, Weno Island; on a rotting trunk of X. granatum, 26 January 2016. SFC20160126-30 (dried specimen). GenBank accession numbers: OR168711 (ITS) and OR168721 (nLSU).

Etymology: The epithet ‘labyrinthus’ referring to its partly irregular pores.

Description: Basidiome perennial, pileate, sessile, broadly attached, solitary, woody hard, without odour or taste. Pileus dimidiate, triquetrous to subungulate, projecting up to 10 cm, 14 cm wide, 7 cm thick at the base. Pileal surface reddish-brown (8E5) to dark-brown (8F5), velutinate, slightly rugose, concentrically sulcate with shallow grooves, irregularly cracked, sometimes covered with moss at the centre. Pore surface light brown (7D5) to reddish-gold (6C7); sterile margin distinct, up to 4 mm wide. Pores somewhat circular and regular near the sterile margin, 7‒8 pores per mm, but irregularly elongated to sinuous at the centre; dissepiments entire, thick. Tubes brown (6E7), stratified, woody hard, up to 3.5 cm thick in total, each annual layer up to 7 mm thick without layer of contextual tissue in between. Context darker than tubes, woody hard, up to 1.5 cm thick.

Hyphal system dimitic; generative hyphae hyaline to pale yellowish (3A3), thin- to slightly thick-walled, frequently septate, occasionally branched, present mostly in subhymenium and trama, rare in context, 1.8–3.3 μm wide; skeletal hyphae yellow (3B8) to dark brown (7F7), thick-walled, aseptate, unbranched, dominant in both trama and context, 2–3 μm wide in trama, 1.8–3.2 μm wide in context, IKI−, CB−.

Basidia rarely observed in the type material, barrel-shaped to subutriform, 4-sterigmate, simple septate, 11.8 × 4.8 μm. Basidiospores subglobose to broadly ellipsoid, yellowish orange (4A8) to brownish-yellow (5C8), thick-walled, smooth, IKI−, CB−, (3.7–)4.1–4.8(–5) × (2.9–)3.2–3.9(–4.1) μm, L = 4.44 μm, W = 3.57 μm, Q = 1.25 (n = 60/2). Cystidioles absent. Setae absent.

Additional specimen examined: SFC20160126-34; Federated States of Micronesia, Chuuk States, Weno Island; on a rotten trunk of Xylocarpus granatum, 26 January 2016. GenBank accession numbers: OR168712 (ITS) and OR168722 (nLSU).

Notes: Fulvifomes labyrinthus is phylogenetically closely related and morphologically similar to Fulvifomes xylocarpicola T. Hatt., Sakayaroj & E.B.G. Jones, they share the same host, Xylocarpus granatum (Hattori et al. 2014). However, F. labyrinthus can be distinguished from F. xylocarpicola by a reddish-brown pileus and partly irregular pores.

Fulvifomes rhizophorus H. Suh, Y. Cho, D. Kim & Y.W. Lim, sp. nov. (Fig. 5)

Fig. 5
figure 5

Morphological characters of Fulvifomes rhizophorus (SFC20170118-26, holotype). A Basidiome. B Pore surface. C Drawings of microscopic features: ‘sp’ basidiospores, ‘b1’ basidia, ‘b2’ basidioles, ‘h1’ generative hypha, ‘h2’ skeletal hypha in trama, and ‘h3’ skeletal hypha in context

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MycoBank Number: MB849264

Diagnosis: Fulvifomes rhizophorus grows on the trunk of the mangrove tree, Rhizophora apiculata, and is characterised by an ungulate basidiome.

Holotype: Federated States of Micronesia, Kosrae Lelu, 18 January 2017. SFC20170118-26 (dried specimen). GenBank accession numbers: OR168715 (ITS) and OR168725 (nLSU).

Etymology: The epithet ‘rhizophorus’ referring to the genus of its host tree.

Description: Basidiome perennial, pileate, sessile, broadly attached, solitary, woody hard and without odour or taste. Pileus dimidiate, applanate, projecting 6.5 cm up, 11.5 cm wide and 3 cm thick at the base. Pileal surface yellowish (5E8) to reddish-brown (8F8), furrowed, concentrically sulcate with deep grooves, radially cracked. Pore surface golden-brown (5D7) to linoleum-brown (5E7); sterile margin distinct, up to 4 mm wide. Pores primarily round in shape, featuring closed slits, typically not connected to adjacent pores, 6‒7 pores per mm; dissepiments entire, thick. Tubes light brown (6D8) to dark brown (6F8), stratified, woody hard, up to 1.4 cm thick in total, each annual layer up to 1 cm thick without layer of contextual tissue in between. Context darker than tubes, woody hard, duplex, the lower layer dark blond (5D4), up to 8 mm thick, the upper layer dark yellow (4C8), up to 9 mm thick.

Hyphal system dimitic; generative hyphae hyaline to pale yellowish (3A3), thin- to slightly thick-walled, frequently septate, occasionally branched, present mostly in subhymenium and trama, rare in context, 2.5–3 μm wide in trama, 3.3–5.2 wide in context; skeletal hyphae aseptae, unbranched, dominant in both trama and context, brown (6E8), in trama thick-walled with narrow lumen, 3.7–7.5 μm wide, dark brown (7F7), thick-walled with wide lumen, 6.2–8 μm in context, IKI−, CB−.

Basidia rarely observed in the type material, clavate to subutriform, 4-sterigmate, simple septate, 10.8 × 4.7 μm. Basidiospores subglobose to broadly ellipsoid, yellowish-orange (4A8) to brownish-yellow (5C8), thick-walled, smooth, IKI−, CB−, (5–)5.3–6(–6.8) × (4.3–)4.4–5.2(–6) μm, L = 5.6 μm, W = 5 μm, Q = 1.14 (n = 81/3). Cystidioles absent. Setae absent.

Additional specimens examined: SFC20170120-06; Federated States of Micronesia, Kosrae Tafunsak, on a trunk of R. apiculata, 20 January 2017. GenBank accession numbers: ITS OR168713 and nLSU OR168723. SFC20170120-09; Federated States of Micronesia, Kosrae Tafunsak, on a trunk of R. apiculata tree on 20 January 2017. GenBank accession numbers: OR168714 (ITS) and OR168724 (nLSU).

Notes: Fulvifomes rhizophorus is found on trunks of Rhizophora apiculata, which is submerged in the shallow waters of the mangrove forest. Amongst the Fulvifomes species in the FS Micronesia, Fulvifomes rhizophorus can be distinguished by larger basidiospores compared to those of Fulvifomes labyrinthus (4.1–4.8 × 3.2–3.9 μm) and Fulvifomes halophilus (4–5.5 × 3.5–5 μm) (Hattori et al. 2022).

Discussion

During the exploration of FS Micronesia in 2016 and 2017, six fruiting bodies of Fulvifomes were found. These were identified as three species, including two newly proposed species, based on morphological examination and phylogenetic analyses. Two specimens are described here as Fulvifomes labyrinthus sp. nov. and three are described as Fulvifomes rhizophorus sp. nov. The remaining specimen was identified as Fulvifomes halophilus, which is being reported for the first time in the FS Micronesia. Fulvifomes rhizophorus has three different types of ITS sequences, whereas other Fulvifomes species in FS Micronesia have only one ITS type. Various ITS types within a single species have been observed across Basidiomycota (Anderson and Kohn 2007; Lim et al. 2008). With various environmental changes occurring in mangrove forests, this phenomenon may indicate the initiation of sympatric speciation, where divergence occurs under non-disruptive selection (Giraud et al. 2008; Druzhinina et al. 2010). Therefore, our results suggest that Fulvifomes rhizophorus was introduced early in the FS Micronesia with some geographical and host restrictions, resulting in a diverse population (Burnett 1983; Griffith and Hedger 1994).

Fulvifomes species have relatively simple basidiome features, which make them difficult to distinguish amongst species. Species differentiation based on microscopic features is challenging because setae and cystidioles are absent. Furthermore, basidia are difficult to find, where some studies have noted that basidia are not observed (Zhou 2014; Olou et al. 2019; Salvador-Montoya et al. 2022). Similarly, basidia of these three species have been extensively searched. We observed basidia from the pore layer closer to the context of our perennial specimens by thin-sectioning in the vertical direction. Basidia may not be the key morphological character in distinguishing Fulvifomes species, however, providing the overall morphological profile of a species when designating a new species may ensure greater confidence in species delimitation.

According to Gilbert et al. (2008), four species were found in the FS Micronesia, namely Aurificaria luteoumbrina, Phellinus fastuosus, P. mangrovicus and P. merrillii, which have been transferred to Fulvifomes (Bondarceva et al. 1992; Hattori et al. 2014; Zhou 2015; Wu et al. 2022). As phylogenetic analyses had not been thoroughly conducted at the time, these species were classified and identified based only on morphological characteristics. Besides Fulvifomes species, other polypores were also transferred to other genera later on (Zmitrovich and Malysheva 2013). It is essential to re-identify the FS Micronesian specimens collected in the past using DNA sequencing and morphological characterisation to fully understand the Fulvifomes and other fungal distribution in this area.

Geographical and ecological features help to distinguish Fulvifomes from other genera in Hymenochaetaceae. Amongst the species that have validated sequences, three species were found in the temperate region: Fulvifomes robiniae from USA in the early 1900s (Murrill 1914), F. rimosus of which the lectotype was found in Tasmania, and F. imazekii from Kochi, Japan (Hattori et al. 2022). Other Fulvifomes species have been detected mainly in tropical and subtropical regions worldwide (Fig. 1). Fulvifomes halophilus, an unrecorded species in the FS Micronesia, also followed the distribution pattern of the majority of Fulvifomes; this species was also found in Thailand and Borneo, in the tropical and subtropical regions of Southeast Asia (Sakayaroj et al. 2012; Wu et al. 2022).

Limited protein-coding gene sequences have been found for Fulvifomes in open databases, such as for RPB2 and TEF1, which are commonly analysed in phylogenetic studies of Hymenochaetaceae. In this study, we provide sequences of protein-coding genes (RPB2 and TEF1) for the three Fulvifomes species, hoping to increase the resolution of phylogenetic analyses in future studies. Until now, a total of 40 Fulvifomes species have been discovered around the world. Two new species (Fulvifomes labyrinthus and Fulvifomes rhizophorus) and one previously unrecorded species (Fulvifomes halophilus) were identified in this study. Including species previously reported in the FS Micronesia, a total of seven Fulvifomes species are detected in the country. Considering the unique environments of the Pacific Islands in tropical and subtropical areas (Mueller-Dombois et al. 1998; Rehman et al. 2013), a more intensive study in these regions may reveal more Fulvifomes species.