Taxonomic Study of the Genus Abundisporus in Korea

Abstract

The polypore genus Abundisporus Ryvarden is characterized by resupinate to pileate fruitbodies with a purplish brown hymenophore, slightly thick-walled, pale yellowish and non-dextrinoid basidiospores, and causing white rot. A purple color hymenophore, an easily observable and striking character, was considered the main distinctive feature at the generic level within polypores. However, due to highly similar basidiocarp features, species identification within these purple polypores is particularly difficult. Three species of purple colored polypores have been reported in Korea (Abundisporus fuscopurpureus, A. pubertatis, and Fomitopsis rosea). Based on morphological re-examination, ecological information, and sequence analysis of the internal transcribed spacer, we showed that previous classification was incorrect and there is only one species (A. pubertatis) in Korea. We provide a detailed description of A. pubertatis in Korea, as well as a taxonomic key to distinguish wood rot fungi with a purple hymenophore.

The polypore genus Abundisporus [1] is characterized by resupinate to pileate fruibodies with pale umber to deep purplish brown or greyish to amber brown context; ellipsoidal, slightly thick-walled, pale yellowish and non-dextrinoid basidiospores; dimitic hyphal systems with skeletal hyphae; and causing white rot [2]. This genus is mostly distributed in tropical and subtropical areas [1], but was recently found in temperate areas [3,4,5]. Eight Abundisporus species have been reported worldwide: A. fuscopurpureus (Pers.) Ryvarden, A. mollissimus Zhao, A. pubertatis (Lloyd) Parmasto, A. quercicola Y. C. Dai, A. sclerosetosus C. Decock and O. Laurence, A. subflexibilis (Berk. & Curtis) Parmasto (synonym of A. roseoalbus), A. roseoalbus (Jungh.) Ryvarden, and A. violaceus (Wakef.) Ryvarden [5]. Six of these species (A. fuscopurpureus, A. mollissimus, A. pubertatis, A. quercicola, A. roseoalbus, and A. sclerosetosus) are recorded in Asia [3,4,5,6,7,8,9].

Phylogenetic analysis based on the internal transcribed spacer (ITS) and the 28S nuclear ribosomal large subunit (LSU) showed that Abundisporus formed a monophyletic group [10,11,12,13]. This result was supported by a multigene phylogenetic analysis that included ITS, LSU, the small subunit mitochondrial rRNA gene (mtSSU), and the translation elongation factor 1-α gene (tef1) [5].

Before 2006, there were no records of Abundisporus in Korea. After detailed morphological and molecular analyses, two Abundisporus species were identified: A. fuscopurpureus [4] and A. pubertatis [8]. Both of these species were previously misidentified as Fomitopsis rosea [14], as they are superficially similar (e.g., have a purple hymenophore). In fact, these three species can be distinguished using microscopic features [1,9,15], DNA [5], or ecology [9,16,17]. In order to clarify the status of Abundisporus species in Korea, we investigated Abundisporus and F. rosea specimens collected across South Korea using microscopic characters, DNA sequence analyses of the ITS region, and ecology (habitat). Based on our findings, we address the status of Abundisporus in Korea and provide a taxonomic key to distinguish these species.

MATERIALS AND METHODS

Samples and morphological analysis

Specimens originally identified as A. fuscopurpureus, A. pubertatis, Abundisporus sp., or F. rosea at the Seoul National University Fungus Collection (SFC) were used in this study. When available, information on host species was noted. First, we sorted specimens using macro- and micro-morphological observations [5,6,15]. Initially, three morphological features were observed for 45 specimens: the number of pores per mm, basidiospore shape, and basidiospore size. For observations of micro-morphological features for the representative specimens of A. pubertatis, slide preparations were made from dried tissue mounted in 3% KOH and viewed using a light microscope (Nikon 80i; Nikon, Tokyo, Japan). We specifically noted the shape and wall thickness of the basidiospores, as these are features that distinguish Abundisporus from Fomitopsis-Abundisporus basidiospores are ellipsoid in shape and have thick walls, while Fomitopsis basidiospores are cylindrical in shape and have thin walls [1,15]. All specimens were identified and used in latter parts of this study (Table 1).

Table 1. Abundisporus pubertatis used in this study.

^aRepresentative specimens for sequencing.

DNA extraction, amplification, and sequencing

Fifteen specimens were chosen for DNA sequencing (Table 1). Genomic DNA was extracted using a modified CTAB extraction protocol [16]. The ITS region was amplified using primers ITS1F and ITS4b [18,19]. PCR reactions were performed on a thermal cycler (C1000TM; Bio-Rad, Richmond, CA, USA) using AccuPower PCR premix (Bioneer Co., Daejeon, Korea) following Park et al. [20]. PCR products were electrophoresed through a 0.8% agarose gel stained with EcoDye (SolGent Co., Daejeon, Korea) and purified using the Expin PCR Purification Kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer's instructions. DNA sequencing was performed at Macrogen (Seoul, Korea) using an ABI3700 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA).

Phylogenetic analysis

Sequences were assembled and proofread using MEGA 6 [21] and aligned using the default settings of MAFFT v7 [22]. Alignments were checked by eye and ambiguous positions adjusted manually. We included available GenBank sequences of Abundisporus and Fomitopsis. Bjerkandera adusta (KJ704813) and Phanerochaete chrysosporium (KP689211) were used as outgroups following Binder et al. [10]. A maximum likelihood (ML) phylogenetic analysis was conducted with RAxML 8.0.2 [23] using the GTRGAMMA model of evolution and 1,000 bootstrap replicates.

RESULTS AND DISCUSSION

In Korea, all wood rot fungi with purple hymenophores were originally identified as F. rosea [14]. After detailed morphological and molecular studies, some specimens were identified as A. fuscopurpureus [4], A. pubertatis [8], or a new species F. incarnatus [24]. Although superficially similar, these species can be differentiated based on micromorphology, DNA, and ecology. In this study, we used these three data types to evaluate the status of Abundisporus in Korea.

The first step was to distinguish Abundisporus from Fomitopsis. These two genera can be differentiated based on rot type (Abundisporus, white rot; Fomitopsis, brown rot) [1,17], but these data are seldom determined during field collection. For microscopic features differentiating Abundisporus and Fomitopsis, hyphal system, along with pore, basidia, and basidiospore sizes are slightly different, but measurements overlap in their ranges [6,15]. Two clear microscopic characters that can be used to differentiate these two genera are basidiaspore shape and wall thickness; Abundisporus has ellipsoid basidiospores with thick walls, while Fomitopsis has cylindrical basidiospores with thin walls [5,15,24,25]. Based on these two characters, we identified 45 specimens in SFC that are Abundisporus (Table 1).

The next step was to verify which Abundisporus species are present in Korea. All the specimens used in this study had similar morphology (Fig. 1); The hyphal systems were all dimitic, generative hyphae with clamp connections and skeletal hyphae. Depending on their growth condition, basidiocarps were either sessile or effused-reflexed. Hymenophores of old speciemens were dark brown or ocher, while fresh samples were purple or pinkish. Based on microscopic characters, A. pubertatis can be distinguished from A. fuscopurpureus by its larger pores (6~7 per mm vs. 7~9 per mm) and larger basidiospores (3.5~5.0 × 2.5~3.5 µm vs. 2.0~3.0 × 1.0~1.5 µm) [9]. For all 45 specimens, there was an average of 5 to 7 pores per mm and basidiospores were 4.5~5.0 × 2.4~3.0 µm. These features were more similar to A. pubertatis, despite some specimens originally being identified as A. fuscopurpureus or F. rosea.

Fig. 1. Abundisporus pubertatis. A, Basidiocarp (SFC20030921-11); B, Basidiocarp (SFC20110921-31); C, Microscopic features (scale bars: A, B = 1 cm, C = 10 µm). a, basidiospores; b, basidia; c, generative hyphae; d, skeletal hyphae.

ITS was sequenced for 15 specimens, selecting representatives specimens originally identified as A. pubertatis, A. fuscopurpureus, Abundisporus sp., or F. rosea. All sequences were approximately 600 bp in length. Sequences of SFC specimens were highly similar (similarity 99.6~100%). All the samples in Korea formed a monophyletic clade with A. pubertatis (ML bootstrap = 100%) (Fig. 2). Based on sequence similarity and ML analysis, all sequenced specimens were identified as A. pubertatis. Sequences were deposited in GenBank (accession numbers in Fig. 2).

Fig. 2. Phylogenetic tree for Abundisporus and related species based on a maximum likelihood analysis of the internal transcribed spacer. Bootstrap scores of > 50 are presented at the nodes. The scale bar indicates the number of nucleotide substitutions per site.

Ecological data should not be the only data used for identification, but it may help. It is noted in the key to differentiating between the two Abundisporus species in East Asia [9] that A. fuscopurpureus is found in tropical to subtropical climates, while A. pubertatis is found in temperate climates. The climate in Korea is temperate, implying the existence of only A. pubertatis in Korea.

Data from micro-morphology, DNA, and ecology all support the existence of a single Abundisporus species in Korea-A. pubertatis. To check our results, we reexamined one of the specimens in the original record of A. fuscopurpureus in Korea (SFC20030927-10) [4]. Phylogenetic analysis of the ITS sequence clearly identified this species to be A. pubertatis (Fig. 2), and re-examination of the micro-morphology measurments also matched this specimen with A. pubertatis. Below we provide a detailed description of A. pubertatis in Korea, as well as a key to distinguish wood rot fungi with purple hymenophores.

Taxonomy

Abundisporus pubertatis (Lloyd) Parmasto, Karstenia 40: 133 (2000).

Basidiocarps partly resupinate, perennial, vinaceous brown to fuscous, forming a thick pileus along upper edges; pileal surface narrow, smooth, often faintly sulcate, developing grey to dark brown crust; margin distinct, entire, obtuse, usually purple and pinkish when fresh; pore 5~7 per mm, angulate ellipsoid or round, pore surface pale purple to lilac grey; tubes 1~3 mm long; context vinaceous brown, soft corky, becoming hard.

Hyphal system dimitic, generative hyphae septate with clamps, 2~3 µm wide; skeletal hyphae straight to moderately branched, 3~5 µm wide; basidia broadly clavate, 18.3~20.0 × 6.3~7.9 µm, 4 sterigmata; basidiospores pale brown, broadly ellipsoid, 4.5~5.0 × 2.4~3.0 µm.

Specimen examined

On a fallen trunk of Quercus serrata, Mt. Deogyu (SFC20030927-10).

Additional specimens examined: SFC20061013-24, SFC20120703-08, SFC20111029-20, SFC20111227-18, SFC20140626-27, SFC20021108-12, SFC20110921-31, SFC20050817-85, SFC20140411-03, SFC20041105-11, SFC20040205-02, SFC20030927-10, SFC20030921-11, SFC20011114-35, SFC20021011-22, SFC20150320-02.

Remarks

Abundisporus pubertatis is similar to A. fuscopurpureus and F. rosea, having a purple colored hymenophore. It can be distinguished from F. rosea by rot type and basidiospores with thick walls and ellipsoid shape. Size of pore, basidia, and basidiospore can be used to distinguish A. pubertatis from A. fuscopurpureus. Although smaller basidia size (10.0~13.0 × 3.0~4.5 µm) of A. pubertatis (KUC20080726-14) was proposed [8], we correct the basidia size as 18.3~20.0 × 6.3~7.9 µm through the re-examination of the originally recorded specimen (KUC20080726-14).

Taxonomic key to distinguish wood rot fungi with purple colored hymenophores (based on [5,9,15]).

1. Basidiospore thin wall and cylindrical shape, brown rot ... 2

1. Basidiospore thick wall and ellipsoid shape, white rot ... 4

2. Pores 6~8 per mm ... F. incarnatus

2. Pores less than 5 per mm ... 3

3. Basidiospore curved cylindrical ... F. cajanderi

3. Basidiospore non-curved cylindrical ... F. rosea

4. Pores 7~9 per mm, basidiospores 2.5~3.3 × 1.7~2.1 ... A. fuscopurpureus

4. Pores 5~7 per mm, basidiospores 4.5~5.0 × 2.4~3.0 ... A. pubertatis