Some Asian fungi are morphologically very similar to European species but belong to different species. A fungus that resembles Pyrenopeziza petiolaris, which commonly occurs on the petioles of Acer pseudoplatanus in Europe, was found on the petioles of Acer spp. and other tree leaves in Japan. The apothecia of this fungus were smaller than those of P. petiolaris, suggesting that it is a different species. To examine this possibility, specimens of this fungus were collected from various hosts in Japan. A detailed morphological examination elucidated that this fungus differed from P. petiolaris in smaller apothecia, marginal cells of the ectal excipulum, and conidia. The ITS sequence difference between this fungus and P. petiolaris was 3.3-4.3%, and they formed distinct clades in the phylogenetic analysis, supporting that they are different species. Consequently, a new species, P. orientalipetiolaris is described. Since an undescribed phialophora-state was observed in the cultures of P. petiolaris for the first time, the morphology under culture is also reported in detail.

More than 300 species epithets of Pyrenopeziza Fuckel (Ploettnerulaceae, Helotiales) are listed in the Index Fungorum (http://www.indexfungorum.org/names/names.asp; visited on May 14, 2022); however, only three species have been reported in Japan (Otani & Tubaki, 1976; Itagaki, Nakamura, & Hosoya, 2019). Considering the species diversity of Pyrenopeziza in temperate Europe, it is expected that more species are potentially distributed in Japan because a large part of Japan is in the temperate region.

Pyrenopeziza petiolaris (Alb. & Schwein.) Nannf., which occurs on the fallen petioles of Acer pseudoplatanus L. in spring, is one of the most common fungal species in Europe (Nannfeldt, 1932). Pyrenopeziza petiolaris also occurs on several other trees and herbaceous plants, showing a wide host range (Hütter, 1958). Pyrenopeziza petiolaris is relatively easy to recognize because of its specific occurrence on petioles and the blackish apothecia that are erumpent through the host epidermis.

In Japan, a P. petiolaris-like fungus is commonly found on petioles of Acer rufinerve Siebold & Zucc., endemic to Japan, and has been identified as P. petiolaris in the local pictorial book (Iguchi & Kutsuna, 2021). However, the apothecia of this fungus seem to be smaller than those of P. petiolaris; therefore, we suspected that the fungus belonged to a different species. In Asia, several fungi that are morphologically very similar to European species but belong to different entities are known, and geographic isolation may be one of the factors contributing to its speciation. Therefore, a detailed morphological and genetic comparison between this fungus from Japan and P. petiolaris from Europe is necessary to clarify the conspecificity.

Fortunately, the second author had the opportunity to obtain specimens and cultures of P. petiolaris from Switzerland. In this study, P. petiolaris-like specimens were collected from various sites in Japan, and their morphologies were compared in detail with those of Swiss specimens. To assess genetic differences in the DNA barcoding region, the sequences of the internal transcribed spacer (ITS1 and ITS2) and 5.8S ribosomal regions (ITS region) were analyzed from extracted DNA obtained from Japanese and Swiss isolates. In addition, the ITS sequences of the Japanese specimens were compared with those of European specimens through phylogenetic analysis.

The materials were collected from various hosts over a wide area of Japan (Table 1). Ascosporous isolates were obtained from fresh apothecia, following the procedure described by Itagaki et al. (2019). Germinated ascospores were transferred to potato dextrose agar (PDA, Nissui, Tokyo, Japan) slants and incubated in the dark at 20 ºC. The voucher specimens were dried at 60 ºC overnight and deposited in the mycological herbarium of the National Museum of Nature and Science (TNS, numbered with TNS-F-). Some isolates, including holotype cultures, were deposited at the Biological Resource Center, National Institute of Technology and Evaluation (NBRC, Chiba, Japan). The additionally examined isolates were deposited in the fungal culture collection in TNS (numbered with FC-).

To observe the colony morphology, mycelia cut from the developed colony on PDA slants were inoculated on 9 cm Petri dishes containing PDA. The inoculated plates were sealed with Parafilm (Bemis, Neenah, USA) and incubated for 2-3 mo at 20 ºC. As distinctive chlamydospores are known for P. petiolaris under culture (Hütter, 1958), we also attempted to induce hyphal structure formation on PDA using cultures derived from Japanese and Swiss specimens by prolonged incubation under black-light (FL15BLB, peak wavelength 352 nm, Toshiba, Tokyo, Japan). The overall appearance of colonies was photographed using a digital camera (D40, Nikon, Tokyo, Japan). Mycelia were picked up from the colonies using a sterilized needle, mounted in water or phloxine in water on slide glass, and gently squashed with a cover glass to observe the hyphal structure.

The overall appearance of fresh or dried apothecia was observed under a stereomicroscope (SZ61, Olympus, Tokyo, Japan) and photographed with a digital camera (DS-L4, Nikon). To observe the pigment dissolution and discoloration of apothecia in potassium hydroxide (KOH) solution, apothecia cut from fresh or dried specimens were immersed in 3% KOH (w/v) droplets and observed under a stereomicroscope.

To prepare the cross-section of the apothecia, a dried apothecium was rehydrated in water, embedded in mucilage (Tissue Tek II, Miles Laboratories, Inc., Naperville, USA), and sliced at 20-30 µm with a microtome (FX-801, Yamato Kouki, Miyazaki, Japan) equipped with an electric freezer (MC-802A, Yamato Kouki). The sliced pieces were mounted in lactic acid (LA), Melzer's solution with or without 3% KOH pretreatment, or cotton blue dissolved in LA (CB/LA). The cross sections of the apothecia and microstructures were observed in detail using an Olympus BX51-equipped with a Nomarski phase interference microscope (Olympus) and photographed with a digital camera (DS-L3, Nikon). The images were synthesized by depth synthetic processing using the CombineZP software (https://combinezp.software.informer.com/) when necessary.

Microstructures were measured in CB/LA preparations using an ocular micrometer. The mean ± standard deviation of each measured value with outliers in brackets is shown. Illustrations were prepared using line-drawing attachments (U-DA, Olympus). The colors of the apothecia and colonies were described by citing the codes in the CMYK systems using a color chart (DIC Corp., Tokyo, Japan).

DNA was extracted from 2-4 wk old mycelium incubated in 2% malt extract broth, following a previously described protocol (Itagaki et al., 2019). Polymerase chain reaction (PCR) and sequencing were conducted using the primer pair ITS1F and ITS4 (White, Bruns, Lee, & Taylor, 1990) to amplify the ITS regions (ITS-5.8S rDNA). The PCR master mix contained the following reagents: 1 µL of extracted DNA, 3.5 µL of DNA-free water, 5 µL of EmeraldAmp PCR Master Mix (Takara Bio, Gunma, Japan), and 0.25 µL each of forward/reverse primers. The ITS region was amplified using the following PCR protocol: initial denaturation at 94 ºC for 3 min, 35 cycles at 94 ºC for 35 s, 51 ºC for 30 s and 72 ºC for 60 s, and a final extension at 72 ºC for 10 min. The amplified PCR products were purified using ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol. Sequencing reactions were carried out using an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems Inc., Norwalk, CT, USA). The obtained sequences were assembled and trimmed using the software ATGC version 7.0.3 (Genetyx, Tokyo, Japan), aligned by Clustal W using the default parameters (Thompson, Higgins, & Gibson, 1994), and manually edited when necessary, using BioEdit ver. 7.0.5.2 (Hall, 1999). Representative sequence data were deposited in DDBJ (accession numbered with LC). To find existing sequence matches for the obtained sequences of P. petiolaris-like specimens, a basic local alignment search tool (BLAST) search in the GenBank database was performed.

Rhynchobrunnera orthospora (Caldwell) B.A. McDonald, U. Braun & Crous was selected as the outgroup because this genus was suggested to be phylogenetically related to Pyrenopeziza (Carmody et al., 2020). ITS sequences of P. petiolaris (CBS 335.58) and R. orthospora were obtained from GenBank. Phylogenetic analysis inferred from ITS sequence was performed using maximum-likelihood estimation (ML) and neighbor-joining estimation (NJ). The ML and NJ phylogenetic trees were constructed with MEGA 7 software (Kumar, Stecher, & Tamura, 2016), and bootstrap values (Felsenstein, 1985) were obtained using 1,000 pseudo-replications. The sequence alignments are available upon request.

We describe the Japanese fungus as a new species based on morphological and genetic comparisons (see text below). We also observed the morphology of P. petiolaris under culture, and discovered an asexual state, described in detail here.

Pyrenopeziza orientalipetiolaris Itagaki & Hosoya sp. nov. Figs. 1, 3, S1.

MycoBank no.: MB 843010.

Fig. 1 - Morphology of Pyrenopeziza orientalipetiolaris. C-E are mounted in LA. F-I are mounted in CB/LA. J is mounted in Melzer's solution after 3% KOH pretreatment. M-O are mounted in water, Q-S are mounted in phloxine. A: Fresh apothecia on a fallen petiole of Aesculus turbinata (TNS-F-86182). B: Dried apothecia on a fallen petiole of Acer pictum (TNS-F-86027). C: Vertical section of the apothecium (TNS-F-86017) shows subepidermal receptacle and margin. D: Outermost layer of ectal excipulum consisting of textura angularis. Note that thick-walled marginal cells cemented. E: Basal part of apothecium with thick-walled hyphae tightly packed in epidermal cells. F: Ascospores. G: Ascus arising from a crozier. H: Asci with ascospores. I: Paraphyses. J: Blue-stained apical pore of ascus. K: Colony incubated on PDA for 3 mo at 20 ºC (NBRC 115386). L: Aerial hyphae with abundant dark chlamydospores (FC-8528, TNS-F-86023). M: Partially thick-walled hyphal cells. N, O: Chlamydospores. P: Colony surface of the edge. Arrows indicate aggregated conidia as slimy heads (FC-8528). Q: Conidia. R, S: Conidiophores. Bars: A, B 1 mm; L 0.5 mm; P 0.25 mm; C 50 µm; D, E 20 µm; F-J, M-O, Q-S 10 µm.

Type: JAPAN, Nagano Pref., Ueda City, Sugadaira Montane Research Center, 6 Jun 2018, on fallen petioles of Aesculus turbinata Blume (holotype, TNS-F-86017; ex-type culture, NBRC 115386).

Gene sequence ex-holotype (ITS): LC683123.

Etymology: Orientalis in Latin means eastern, and petiolaris refers to the European species, P. petiolaris.

Japanese name: Haiiro-hira-saratake (Iguchi & Kustuna, 2021).

Apothecia scattered, erumpent and appearing superficial when fresh, subepidermal when dried, sessile, urceolate to saucer shape, flat to slightly convex when fresh, 0.1-0.2 mm in height, connecting at the bottom with thick-walled hyphae that penetrate and tightly packed in epidermal cells; disc 0.2-0.5 mm diam, dark grey (K80) or greyish brown (C60M60Y60) when fresh, shrunk to 0.1-0.3 mm diam, turned darker when dried; margin entire, smooth, paler color. Ectal excipulum textura prismatica at the base and middle of the receptacle, becoming textura porrecta incrassata at upper receptacle connecting with margin, 25-30 µm thick at the basal receptacle, 20-25 µm thick at the upper receptacle, without crystals or exudates, composed of thick-walled cells; outermost cells rounded rectangle, 5-8.5 × 3.5-4.5 µm at the base, (2.5-)3.5-5 × 2-3 µm at the mid; marginal cells long clavate with wider apex, 6-8 µm in length, coalescing with thick-walls, hyaline. Medullary excipulum textura intricata to prismatica, 45-56 µm thick, hyaline. Asci (45-)48-58(-63) × 3.8-5 µm, cylindrical clavate, eight-spored, arising from croziers, with apical pore blue-stained by Melzer's solution with or without 3% KOH pretreatment. Ascospores (7.5-)8-11 × 1.8-2.5 µm, ellipsoid to fusiform with rounded or pointed apices, sometimes contain 2-3 small guttles, thin-walled, aseptate, hyaline. Paraphyses (48-)50-58(-62.5) × 1-2.5 µm, filiform, straight to waving, with swelled apex up to 3.7-5 µm wide, occasionally adhering exudates or grains at the apex, frequently branching at the base, 2-3-septate below, thin-walled, hyaline, not exceed the tip of asci.

Colony of NBRC 115386 on PDA entire, flat to slightly convex at the center with aerial hyphae, dense, floccose to cottony, greyish brown (C30M30Y30) from the center, becoming beige (M10Y20-30K10) at the edge, darker from the reverse, with indistinct sectors; mycelium hyaline to pale brown, smooth, branching, septate, consisting of 1.5-5 µm wide hyphae, containing oil globes, thin- to thick-walled; soluble pigment and crystals not produced. Conidiophores macronematous, superficial, constricted, arising vertically or laterally from hyphae, hyaline, smooth, thin- or thick-walled, branched; conidiogenous cells phialidic, ampulliform, up to 10 µm long, 3-4 µm wide at the base, discrete to integrated, arranged terminally to intercalary, hyaline, with cylindrical collarettes 2.5-4 µm long and 3 µm wide; conidia, aseptate, ellipsoid, containing 1-2 guttles, 3-4.5 × 1-2 µm, hyaline, thin-walled, aggregated in slimy heads.

Notes. Pyrenopeziza orientalipetiolaris occurs on fallen petioles and midveins of deciduous broad-leaved trees from spring to early summer (May to Jul).

Pyrenopeziza orientalipetiolaris has a wide host range, being found on 26 species in 19 genera and 14 families, including several Japanese endemic species such as Aesculus turbinata, Alnus matsumurae Callier, Fagus crenata Blume, Fraxinus japonica Blume ex K.Koch, Magnolia obovata Thunb., and Wisteria brachybotrys Siebold & Zucc. Most hosts are woody plants, but an annual herb, Fallopia japonica (Houtt.) Ronse Decr. is included.

After 2 mo incubation on PDA, chlamydospore-like cells were observed at the tip and in the middle of the aerial hyphae (Fig. 1 L). Chlamydospore-like cells are pale to dark brown, lobe-shaped with thick wall, solitary or in 2-4 series of cells, constricted at the septa, smooth, and containing several oil globules in the cytoplasm (Fig. 1 N, O). A similar structure has been reported in cultures of P. petiolaris and P. protrusa (Berk. & M.A. Curtis) Sacc. (Hütter, 1958; Itagaki et al., 2019).

Additional specimens examined: See Table 1.

For comparison, we provide a detailed description of P. petiolaris (TNS-F-61891).

Pyrenopeziza petiolaris (Alb. & Schwein.) Nannf., 1932 Figs. 2, 3.

Fig. 2 - Morphology of Pyrenopeziza petiolaris. C-E are mounted in LA. G-J, L are mounted in CB/LA. K is mounted in Melzer's solution after 3% KOH pretreatment. N-Q are mounted in phloxine. A: Dried apothecia on a fallen petiole of Acer pseudoplatanus (TNS-F-61891). B: Dried apothecia on a fallen petiole of A. gultinosa (TNS-F-61861). C: Vertical section of the apothecium (TNS-F-61861). D: Outermost layer of ectal excipulum consisting of textura angularis. E: Thick-walled brown hyphae tightly packed in epidermal cells. F: Colony incubated on PDA for 3 mo at 20 ºC (FC-5740, TNS-F-61891). G: Ascospores. H: Asci. I, J: Paraphyses. Arrows denote adhesive materials attached to the top of the paraphysis. K: Blue-stained apical pore of ascus. L: Croziers at the base of ascus. M: Colony surface of the edge. Arrows indicate collapsed conidiophores with slimy conidial drops (FC-5740, TNS-F-61891). N: Conidia. O-Q: Conidiophores. Bars: A, B 1 mm; M 0.5 mm; C, D 50 µm; E 20 µm; G-L, N-Q 10 µm.

Fig. 3 - Line-drawing of hyphal structures. A: Conidiophore and conidia of Pyrenopeziza orientalipetiolaris (FC-8528). B: Conidiophore and conidia of P. petiolaris (FC-5740). Note that the conidia of P. petiolaris are slightly larger than those of P. orientalipetiolaris. C: Chlamydospores of P. orientalipetiolaris (FC-8528).

Basionym: Hysterium petiolare Alb. & Schwein.

Apothecia scattered, erumpent with a central longitudinal slit, entirely embedded in the dark epidermis when dried, sessile, deeply concave when rehydrated, 0.1-0.2 mm in height, connecting at the bottom with thick-walled brown hyphae that penetrate and tightly packed in epidermal cells; disc fusiform to ellipsoid, 0.5-1 × 0.2-0.5 mm when dried, wholly black. Ectal excipulum textura prismatica to angularis at the base, textura prismatica at the middle, becoming textura porrecta at upper receptacle connecting with margin, 30-35 µm thick at the basal receptacle, 20-25 µm thick at the upper receptacle, without crystals or exudates, composed of thick-walled cells, composed of 3-5 cell layers of thick-walled cells, darker toward the outermost layer; outermost cells globose to rounded rectangular, 10-18 × 8-12 µm at the base, elongating tangential direction to (10-)12-22 × 5-10 µm at the middle; marginal cells long clavate with blunt head to 3-5 µm wide, 15-25 µm in length, coalescing together, hyaline. Medullary excipulum textura intricata to prismatica, 30-45 µm thick, hyaline. Asci (40-)45-52(-57.5) × 3.8-5 µm, cylindrical clavate, eight-spored, arising from croziers, with apical pore blue-stained by Melzer's solution with or without 3% KOH pretreatment. Ascospores (6.2-)7.5-10 × 1.8-2.5 µm, ellipsoid to fusiform with rounded or pointed apices, sometimes contain 2-3 small guttles, thin-walled, aseptate, hyaline. Paraphyses (45-)50-58(-60) × 1-2.5 µm, filiform, straight to waving, with swelled apex up to approximately 3.5 µm wide, frequently adhering membranous exudates at the apex, sometimes branching at the base, 2-3-septate below, thin-walled, hyaline, projecting up to 5 µm beyond the asci.

Colony of FC-5740 on PDA entire, flat to slightly convex at the center with aerial hyphae, dense, pulveraceus to cottony, beige (C40M40Y40K10) from the center, paler toward to the edge, darker from the reverse, with indistinct sectors; soluble pigment and crystals not produced. Conidiophores macronematous, superficial, constricted, arising vertically or laterally from fascicular or coiled hyphae, smooth, thin- or thick-walled, branched; conidiogenous cells phialidic, ampulliform, up to 10 µm long, 3-4 µm wide at the base, discrete to integrated, arranged terminally to intercalary, hyaline, with cylindrical collarettes of 2.5-3 µm long and 2-3.5 µm wide; conidia aseptate, ellipsoid to oblong, containing small guttles, 4-6 × 2.5-3 µm, hyaline, thin-walled, aggregated in slimy heads.

Notes: Pyrenopeziza petiolaris is distributed in Austria, Czechia, France, Germany, and Switzerland. Apothecia occur in spring (Apr to Jun), and Hütter (1958) listed the following hosts; Acer spp., Aesculus hippocastanum L., Ailanthus glandulosa Desf. (Syn. Ai. altissima (Mill.) Swingle), Betula spp., Fagus sylvatica L., Ficus carica L., Geranium robertianum L., Mercurialis perennis L., Populus tremula L., Sambucus ebulus L., Sorbus aria (L.) Crantz and Tilia spp. Pyrenopeziza petiolaris shares some host genera with P. orientalipetiolaris (e.g., Acer, Aesculus, Fagus, Populus and Sorbus), but no common host species were found.

Additional specimens examined: TNS-F-61841, 61861, 61872, 65646, 65656 (for details, see Table 1).

Pyrenopeziza orientalipetiolaris overlaps with P. petiolaris in the dimension of ascospores, asci, and paraphyses but differs in its smaller apothecia (0.1-0.3 mm diam vs. 0.5-1 × 0.2-0.5 mm when dried), short marginal cells (6-8 µm vs. 15-25 µm in length) and small conidia (3-4.5 × 1-2 µm vs. 4-6 × 2.5-3 µm). In particular, the apothecia of P. petiolaris having fusiform to ellipsoid disc with longitudinal slit and being entirely embedded in the host epidermis (Fig. 2A and 2B), are morphologically distinct from those of P. orientalipetiolaris.

Hütter (1958) noted that the characteristic morphologies of P. petiolaris are ectal excipulum consisting of textura prismatica, thick upper receptacle, and brown hyphae that penetrate and tightly packed in epidermal cells (“klypeusartige Durchwucherung der Epidermiszellen mit Hyphen”). Since P. orientalipetiolaris has similar excipulum structures (Fig. 1D and 2D) and hyphal structures within the epidermal cells (Fig. 1E and 2E), these two features do not help distinguish the two species.

Hütter (1958) pointed out that P. petiolaris occurs abundantly on A. pseudoplatanus but relatively less on other hosts and that the size of the apothecia varies with the host substrate. He suggested that Trochila petiolicola f. fagi Rehm on the petioles of Tilia sp. and Pyrenopeziza aceris Nannf. on Acer negundo L. are synonymous with P. petiolaris, including their wide host range and variable apothecial sizes. In the present study, little morphological variation was observed in P. orientalipetiolaris in relation to its hosts (Supplementary Fig. S1). However, the size and shape of apothecia of Japanese specimens were different from those of any European specimens. When comparing the apothecial morphology of the two species, there were distinct differences, even within the same host genus, Acer spp.

The chlamydospores observed by Hütter (1958) in cultures of P. petiolaris were not found in Swiss cultures in the present study. In addition, the formation of the phialophora-state of P. petiolaris under Swiss cultures was observed in this study for the first time (Fig. 2M-Q). Similar asexual morphs have been observed in cultures of P. ebuli (Fr.) Sacc., P. eryngii Fuckel, P. lonicerae Nannf. and P. rubi (Fr.) Rehm but not in P. petiolaris (Hütter, 1958). However, the reference sequence for the ITS region derived from a culture isolated by Hütter (CBS 335.58) and sequences derived from specimens collected in Switzerland were identical (Fig. 4). Thus, these specimens were confirmed to be conspecifics. The production of anamorphs in Swiss cultures may have been induced by prolonged incubation and stimulation of black-light irradiation.

Fig. 4 – Phylogenetic tree of Pyrenopeziza orientalipetiolaris and P. petiolaris inferred from the ITS region sequence based on the neighbor-joining method (NJ) method. NJ and maximum-likelihood (ML) bootstrap values greater than 90% are shown above the nodes (NJ/ML). Holotype of P. orientalipetiolaris TNS-F-86017 is in boldface.

Pyrenopeziza orientalipetiolaris and P. petiolaris each formed phylogenetically strongly supported clades (Fig. 4). Both species showed no tendency for speciation in relation to the hosts, and the sequences were diverse, even for the same host species. In the phylogenetic tree, the branches of P. orientalipetiolaris were slightly shorter than those of P. petiolaris, suggesting that genetic divergence within the species might have occurred rapidly.

The sequence differences among the specimens were less than 1.5% and 0.7% in P. orientalipetiolaris and P. petiolaris, respectively, while the difference between the two species was 3.3-4.3%. This is more than the level suggested for the same species (≥1.5%) (Nilsson et al., 2012; Schoch et al., 2012), indicating that the two species are genetically distinct.

Some Asian species resemble European species but differ in the barcode region. For example, Hymenoscyphus albidus (Gillet) W. Phillips in Europe vs. H. pseudoalbidus Queloz, Grünig, Berndt, T. Kowalski, T.N. Sieber & Holdenr. in Asia (the interspecific difference is 2.2-2.4%) (Queloz et al., 2011; Barral & Bemmann, 2014), Sarcoscypha coccinea (Jacq.) Lambotte in Europe vs. S. hosoyae F.A. Harr. in Japan (10.5-12.2%) (Harrington, 1997; Harrington & Potter, 1997) and Poculum sydowianum (Rehm) Dumont in Europe vs. P. pseudosydowianum Yan J. Zhao & Hosoya in Japan (8-10%) (Hosoya, Zhao, & Degawa, 2014). The geographical distance between Asia and Europe may influence the speciation of these species, and it is speculated that P. orientalipetiolaris in Japan and P. petiolaris in Europe are morphologically and genetically differentiated by geographical isolation.

As a result of BLAST search, the ITS sequences of P. orientalipetiolaris matched some existing records with ≥1,000 max score, e-value = 0.0, ≥95% query coverage and ≥98.5% sequence identity, including isolates from symptomless Fraxinus petiole in Poland (MW446991), from dead Fraxinus petiole in Poland (MZ493124), from sea weeds in Italy (MK626719, KT699138), from permafrost in Russia (MF120200), as well as environmental sequence from fine roots of Triticum aestivum L. in Germany (KY430513), from beech litter in Austria (JF449547, JF449498), and from Myrica gale L. leaf in Japan (LC704392). The isolates of MW446991 and MK626719 were identified as Rhexocercosporidium carotae (Årsvoll) U. Braun, the soil-borne pathogen causing black spots on stored carrots (Braun, 1994), and the others are unidentified “Helotiales sp.” or “fungus”. These sequences were not included in the phylogenetic analysis due to the lack of appropriate voucher specimens with morphological features or their identification is doubtful. However, these samples may reflect that P. orientalipetiolaris infects the living leaves and roots of various plants and is distributed worldwide.

The authors declare that they have no conflicts of interest. This study was conducted in accordance with the current laws of Japan.

We thank Dr. Yousuke Degawa (Faculty of Life and Environmental Sciences, University of Tsukuba) for providing the sampling sites at the Sugadaira Research Station, Mountain Science Center, Nagano Pref. Appreciation is also due to Ms. Kyong-Ok Nam, Ms. Miyoko Uehara, and Ms. Nozomi Tsujino for their help with the molecular analysis and preparation of specimens and cultures. We also express our gratitude to Drs. Andrin Gross and Ottmar Holdenrieder, ETH Zurich for their help and guidance in collecting samples in Switzerland. We would like to thank Editage (www.editage.com) for English language editing. This study was financially supported by JSPS KAKENHI Grant Number JP21J11957, 28th Fujiwara Natural History Foundation, 2019-2020 and The University of Tokyo Edge Capital Partners Co., Ltd. Scholarship 2021.

• Barral, H. O., & Bemmann, M. (2014). Hymenoscyphus fraxineus vs. Hymenoscyphus albidus - A comparative light microscopic study on the causal agent of European ash dieback and related foliicolous, stroma-forming species. Mycology, 5, 228-290. https://doi.org/10.1080/21501203.2014.963720
• Braun, U. (1994). Miscellaneous notes on phytopathogenic hyphomycetes. Mycotaxon, 51, 37-68.
• Carmody, S. M., King, K. M., Ocamb, C. M., Fraaije, B. A., West, J. S., & du Toit, L. J. (2020). A phylogenetically distinct lineage of Pyrenopeziza brassicae associated with chlorotic leaf spot of Brassicaceae in North America. Plant Pathology, 69, 518-537. https://doi.org/10.1111/ppa.13137
• Felsenstein, J. (1985). Phylogenies and the comparative method. The American Naturalist, 125, 1-15. https://doi.org/10.1086/284325
• Hall, A. T. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98.
• Harrington, F. A. (1997). New species of Sarcoscypha (Sarcoscyphaceae, Pezizales). Harvard Papers in Botany, 10, 53-64.
• Harrington, F. A., & Potter, D. (1997). Phylogenetic relationships within Sarcoscypha based upon nucleotide sequences of the internal transcribed spacer of nuclear ribosomal DNA. Mycologia, 89, 258-267. http://dx.doi.org/10.2307/3761080
• Hosoya, T., Zhao, Y. J., & Degawa, Y. (2014). Poculum pseudosydowianum, sp. nov. (Rutstroemiaceae, Ascomycota) from Japan and its endophytic occurrence. Phytotaxa, 175, 216-224. https://doi.org/10.11646/phytotaxa.175.4.3
• Hütter, R. (1958). Untersuchungen über die Gattung Pyrenopeziza Fuck. Phytopathologische Zeitzschrift, 33, 2-53.
• Iguchi, K., & Kutsuna, M. (2021). Guidebook for identification of morel and cup fungi [In Japanese]. Bun-ichi Co., Ltd.
• Itagaki, H., Nakamura, Y., & Hosoya, T. (2019). Two new records of ascomycetes from Japan, Pyrenopeziza protrusa and P. nervicola (Helotiales, Dermateaceae sensu lato). Mycoscience, 60, 189-196. https://doi.org/10.1016/j.myc.2019.02.008
• Kumar, S., Stecher, G., & Tamura, K., (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0. for bigger datasets. Molecular Biology and Evolution, 33, 1870-1874. https://doi.org/10.1093/molbev/msw054
• Nannfeldt, J. A. (1932). Studien über die Morphologie und Systematik der nicht-lichenisierten inoperculaten Discomyceten. Nova Acta Reg. Soc. Sci. Upsal., Ser. IV, 8, 81-173.
• Nilsson, R. H., Tedersoo, L., Abarenkov, K., Ryberg, M., Kristiansson, E., Hartmann, Unterseher, M., Porter, T. M., Bengtsson-Palme, J., Walker D. M., de Sousa, F., Gamper, H. A., Larsson, E., Larsson, K. H., Kõljalg, U., Edger, R. C., & Abarenkov, K. (2012). Five simple guidelines for establishing basic authenticity and reliability of newly generated fungal ITS sequences. MycoKeys, 4, 37-63. https://doi.org/10.3897/mycokeys.4.3606
• Otani, Y., & Tubaki, K. (1976). Notes on the cup fungi and imperfect fungi in Yakushima [In Japanese]. Memoires of the National Science Museum, 9, 77-84.
• Queloz, V., Grünig, C. R., Berndt, R., Kowalski, T., Sieber, T. N., & Holdenrieder, O. (2011). Cryptic speciation in Hymenoscyphus albidus. Forest Pathology, 41, 133-142. https://doi.org/10.1111/j.1439-0329.2010.00645.x
• Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., Chen, W., & Fungal Barcoding Consortium (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America, 109, 6241-6246. https://doi.org/10.1073/pnas.1117018109
• Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673-4680. https://doi.org/10.1093/nar/22.22.4673
• White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols: A guide to methods and applications (pp. 315-322). Academic Press.