Mycoscience: Advance Publication doi: 10.47371/mycosci.2021.03.002 Short Communication (Received December 30, 2020; Accepted March 11, 2021) J-STAGE Advance Published Date: March 27, 2021

Short communication

Exidia qinghaiensis, a new from China

Shurong Wanga, R. Greg Thornb, *

a College of Food Science and Engineering, Shanxi Agricultural University, Taigu, 030801,

China b Department of Biology, University of Western Ontario, London, N6A 5B7, Canada.

*Corresponding author. Department of Biology, University of Western Ontario, London, N6A

5B7, Canada.

E-mail address: [email protected] (G. Thorn).

Text: 12 pages; tables: 1; figures: 3

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ABSTRACT

A novel, wood-inhabiting jelly from China is described as a new species, qinghaiensis (: ). Phylogenetic analyses were based on sequences of the nuclear ribosomal DNA internal transcribed spacer (nrITS) and large subunit

(nrLSU), RNA polymerase II second largest subunit (RPB2), and translation elongation factor

1- (Tef1) regions. Sequences of the new taxon formed a sister group to , a species known from Europe and Asia, and distant to sequences of Exidia repanda from Europe.

Fruiting bodies are cushion-shaped to irregularly lobed and yellowish brown, basidiospores are hyaline, allantoid (averaging 12.7  3.4 m; average length/width is 3.7), and the host is Betula.

The new species also can be distinguished by nrITS, nrLSU, RPB2, and Tef1 sequences. Our multigene phylogeny supports an Exidia including Exidia japonica, type species of Tremellochaete, but defining generic limits in AuriculariaceaePublication will require more extensive taxon sampling.

Keywords: Auriculariaceae, Basidiomycota, one new species, phylogeny,

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Exidia Fr. is a of wood-inhabiting fungi (Basidiomycota: Auriculariaceae)

(Hibbett et al., 2014) growing on dead branches and logs, and best known from the temperate regions. Exidia forms a sister group to the much better known “wood ears” of the genus

Auricularia (Weiss & Oberwinkler, 2001). The basidia of Exidia are pear-shaped and have longitudinal septa, unlike the tubular and transversely septate basidia of Bull.

(Weiss & Oberwinkler, 2001). As in Auricularia, (fruiting bodies) are gelatinous and these are diverse in form, ranging from pustular to cup-shaped (Wojewoda, 1977; Moore,

1997); indeed, a few species of Exidia with rather ear-shaped fruiting bodies have regularly been misidentified as Auricularia (Barber, Thorn, & Voitk, 2011).

The study of fungal diversity plays an important role in its preservation, not only in

China but also on a worldwide scale. The mycota of China, including Exidia, still has not been well investigated because of its great geographical extent, and most Exidia species reported lack supporting molecular data. On the basis of morphological data, a total of 8 species of

Exidia, including synonyms and three recently describedPublication species, have been reported in recent years from China (Wu et al., 2020; Ye, Zhang, Wu, & Liu, 2020). Regionally, five species have been reported from Japan (Aoki & Tubaki, 1986; Imazeki, Otani, & Hongo, 1988; Aoki, 1991), three from Korea (Jung, 1993) and ten species from the Russian Far East (Govorova, 1998;

Malysheva, 2012; Malysheva & Spirin, 2017). To add to the knowledge of Exidia in China, the first author has undertaken field collection and morphological and molecular studies of Chinese

Exidia specimens in herbaria. During our work we detected one additional species-level clade based onAdvance phylogenetic analyses of the nuclear ribosomal DNA internal transcribed spacer region (nrITS) and large subunit (nrLSU), RNA polymerase II second largest subunit (RPB2),

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and translation elongation factor 1- (Tef1). Herein we describe this new species based on specimens from Qinghai Province, China.

For light microscopic observations, freehand sections of a rehydrated portion of specimens were mounted in 2% (w/v) KOH. All measurements of and hypobasidia were carried out using oil immersion at 400 and 1000 magnification with differential interference microscopy on a Zeiss AxioImager Z1. All dimensions exclude the hilar appendage and are reported as length, width and Q (length/width), given as the 80th percentile range with outliers in parentheses; the average value of Q is reported as Qavg. Color codes (e.g., 8B5) follow Kornerup and Wanscher (1978). The specimens we borrowed and examined are from

HMAS (Mycological Herbarium, Institute of Microbiology, Academia Sinica, Beijing, China).

Genomic DNA was extracted from dried materials using the E.Z.N.A. Forensic DNA extraction kit (Omega Bio-Tek, Norcross, Georgia, USA). PCR amplification was performed with primers ITS8F and ITS6R (Dentinger, Margaritescu,Publication & Moncalvo, 2010) or ITS8F/5.8S and 5.8SR/LS1R (Vilgalys & Hester, 1990; Hausner, Reid, & Klassen, 1993) for the ITS region, primers LS1 and LR3 (Vilgalys & Hester, 1990) for the 5'-LSU region, primers b-6F and b-7.1R (Matheny, 2005) or b-6F/f-7cR and b-6.9F/b-7.1R (Raja, Miller, Pearce, &

Oberlies, 2017) for the RPB2 region, and ef1-983-F and ef1-1567-R (Rehner & Buckley, 2005) for Tef1. Successfully amplified products were cleaned using an EZ-10 Spin Column PCR

Products Purification Kit (BioBasic Canada, Markham, Ontario) and sequenced using dye- terminatorAdvance sequencing at Robarts Research Institute (London, Canada) or Sangon Biotech Co., Ltd. (Shanghai, China). Following DNA sequencing, chromatograms of partial sequences were cleaned and assembled using SeqEd v1.0.3 (Applied Biosystems, Foster City, California, USA).

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Newly acquired sequences have been deposited in GenBank as MW353408–MW353409 and

MW358923–MW358926.

Few Exidia sequences available from GenBank contained all of the chosen gene regions but, after preliminary analyses based solely on the ITS region that found no matches to our putative new species (data not shown), we chose to focus on a small dataset that had coverage of as many of these regions as possible (Table 1). For (Bull.) Fr. (HHB

12029) and the outgroup Auricularia heimuer F. Wu, B.K. Cui & Y.C. Dai (Dai 13782), draft genome sequences are available and were queried using BLASTn to obtain sequences of RPB2 or Tef1. Sequences of the different gene regions were separately aligned using MAFFT v7 online (Katoh & Standley, 2013) with the G-INS-i strategy and “leave gappy regions” option invoked, then the rough ends of alignments trimmed using MEGA X (Kumar, Stecher, &

Tamura, 2016) before concatenating to yield a combined matrix of 2,572 aligned bases. Prior to concatenation, evolutionary models were compared using MEGA X, and since the GTR+G+I model received the best ln(L) score in all cases, Publicationthis model was used in maximum likelihood

(ML) analyses implemented in MEGA X for the combined dataset. Bayesian analyses were conducted in MrBayes 3.2.6 (MB; Ronquist et al., 2012) with 5 000 000 generations, 4 chains, and a burn-in of 25% (when the average standard deviation of split frequencies between chains had stabilized below 0.001). Node support was determined as posterior probabilities in

MrBayes, and as bootstrap support in ML analyses using 100 replicates. The alignments and trees have been deposited to TreeBase (http://www.treebase.org) as S27878.

AdvancePhylogenetic analyses of the combined nrITS, nrLSU, RPB2 and Tef1 data (Fig. 1) supports the segregation of Exidia qinghaiensis as a sister species to Exidia thuretiana (Lév.)

Fr. A sample representing the type species of Tremellochaete Raitv., Exidia japonica Yasuda

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[syn. Tremellochaete japonica (Yasuda) Raitv.], was placed with strong support in the genus

Exidia, as the sister to Exidia candida Lloyd. However, a more inclusive taxon sample will be required to define generic limits in the Auriculariaceae.

Taxonomy

Exidia qinghaiensis S.R. Wang & Thorn, sp. nov. Figs. 2, 3.

MycoBank no.: MB 838343.

Diagnosis: Fruiting bodies are cushion-shaped to irregularly lobed and adpressed, becoming confluent, yellowish brown (drying fuscous), with paler flesh, basidiospores are hyaline, allantoid, averaging 12.7  3.4 m, with Qavg = 3.7, and the host is Betula.

Basidiospores of Exidia saccharina Fr., on conifers, are slightly wider (10–14  3.5–4.0 m), and those of E. thuretiana, also on angiosperms,Publication are both longer and broader (13–19  4.5–6.0 m), but similar in shape to those of E. qinghaiensis. These species can be distinguished by their nrITS, nrLSU, RPB2, and Tef1sequences.

Holotype: CHINA, Qinghai Province, Menyuan County, Xianmi wood farm, approx.

37°17' N, 101°57' E, 2,850 m above sea level (a.s.l.), on a fallen branch of Betula, 19 Oct 2004, leg. Zhang Xiaoqing, HMAS 156328 (Mycological Herbarium, Institute of Microbiology,

Academia Sinica). AdvanceGene sequences ex-holotype: MW353409 (nrITS, nrLSU), MW358924 (RPB2) and MW358926 (Tef1).

Etymology: qinghaiensis (Latin), referring to Qinghai Province, where the holotype was collected.

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Basidiomata gelatinous, cushion-shaped to irregularly lobed, adpressed, orbicular, typically growing separately and adhering to the substrate, sometimes fusing together and becoming confluent, up to 3 cm in widest dimension and 0.5 cm thick; when dried uniformly greyish or fuscous brown (7F3) on both hymenial and abhymenial surfaces, rehydrating to yellowish brown or caramel (5DE7 to 6E7), with paler flesh; the hymenial, downward-facing surface smooth, glabrous and a little shining, becoming slightly eroded in age; abymenial surface slightly granular, usually without wrinkles and alveolate-venose. Flesh thin and pliant- gelatinous; odor and taste not noted.

Basidiospores (30 measured from 2 collections) narrowly allantoid, smooth, hyaline in

KOH, nonamyloid, (10.4–)11.2–13.5(–14.9)  (3.0–)3.1–3.8(–4.1) m, on average 12.7  3.4

m, Q = (2.7–)3.1–4.3(–4.5), Qavg = 3.7; probasidia amygdaloid-limoniform; hypobasidia pyriform to subglobose, cruciately septate at maturity, astipitate or substipitate, with basal , (10.5–)11.3–15.5(–17.2)  (7.5Publication–)7.6–9.4(–10.0) m, on average 12.8  8.5 m; Qavg = 1.5, with longitudinal septa, developing 4 long, finger-like epibasidia (sterigmata),

8–18  1.5–2.2 m. Tramal hyphae hyaline, thin-walled and 1.5–2.5 μm diam, embedded in gelatinous matrix, with simple clamp connections. Dikaryophyses tortuous-cylindrical and 2.0–

3.2 m diam at their base, some with brownish contents, sparingly and irregularly branched, the branches narrowing to 0.4 m diam; gloeocystidia not seen. A thin, apparently acellular, brownish epithecium that is present in some mounts (but poorly-preserved and with abundant rod-shapedAdvance and spherical bacteria present) appears responsible for the laccate, caramel color of the hymenial surface.

Habitat and distribution: Saprobic; growing on attached or fallen hardwood branches, the substrate identified as Betula. Collected in fall, distributed in Qinghai Province, China.

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Additional specimen examined (paratype): CHINA, Qinghai Province, Menyuan

County, Xianmi wood farm, approx. 37°17' N, 101°57' E, 2,850 m a.s.l., on dead branch of

Betula, 19 Oct 2004, Zhang Xiaoqing, HMAS 156376.

Comments: Exidia thuretiana, reported on hardwood branches in Europe and Asia

(Wojewoda, 1977; Moore, 1997; Spirin, Malysheva, & Larsson, 2018) has the greatest tendency to form extensive and cushion-shaped to irregularly lobed, adpressed fruiting bodies, but its spores are longer and broader (13–19  4.5–6.0 m; Malysheva, 2012) than those of this species. Exidia saccharina, reported in Europe (Wojewoda, 1977; Moore, 1997) and Asia

(Russia; Govorova, 1998), has fruiting bodies of similar colors and spores of similar size and shape (10–14  3.5–4.0 m; Malysheva, 2012) but occurs on conifers. Exidia repanda Fr., as reported from Japan, resembles E. qinghaiensis in both macromorphology and size of basidiospores (8.5–16  2–4.0 m; 12  3 m on average; Aoki & Tubaki, 1986). Wojewoda (1977) reported E. repanda from Betula, Alnus, PublicationFraxinus, Prunus, and Tilia and compared the species with E. recisa (Ditmar) Fr., which occurs primarily on Salix. Sequences of material from France on Prunus identified as E. repanda (Wu et al. 2020) are very distinct and were placed in the E. recisa complex by Wu et al. (2020) and in our analyses (Fig. 1). All of these species can be distinguished by their ITS sequences, so sequence data for Japanese and topotypical Swedish collections of E. repanda are highly desirable.

Within the genus Exidia, there seems to be a range in host specificity reported, from quite narrowAdvance to very broad, but only a few host records are supported by molecular data. Exidia thuretiana occurs on a broad range of hardwood species, particularly Fagus, Ribes and Ulmus

(Spirin et al., 2018). Exidia saccharina is reported on dead branches of Abies, Larix, Picea and

Pinus (Wojewoda, 1977; Govorova, 1998; Kirschner, 2010) in Europe and Asia. The two

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collections that we studied of E. qinghaiensis were recorded from Betula, the same host genus as reported for E. repanda in Japan (Aoki and Tubaki, 1986; Aoki, 1991). Further studies based on more extensive collecting, with culturing, mating studies, and DNA sequence data, may elucidate the possible presence of other members of Exidia in Asia, and the substrates used by

E. qinghaiensis.

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the countries where they were performed.

Acknowledgments

This study was supported in part by the Scientific Start-up Program of Shanxi

Agricultural University (No. 2014YJ18), the CollaborativePublication Innovation Center of The Loess

Plateau Edible Fungi (Shanxi Province) and the Faculty of Science and Department of Biology at the University of Western Ontario. We thank T.-Z. Wei, Y.-J. Yao and L. Cai (Curator,

HMAS) for the loan of specimens for our study. We also thank K. Nygard at the Biotron,

University of Western Ontario for guidance in the use of the microscope.

References

Aoki, T.Advance (1991). in and around Sugadaira, Central Japan. I. Exidia.

Bulletin of the Sugadaira Montane Research Center, 12, 13–26.

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Aoki, T., & Tubaki, K. (1986). Cultural study of Exidia repanda, previously unreported from

Japan. Mycologia, 78, 877–887.

Barber, M., Thorn, G., & Voitk, A. (2011). The mushroom basket: when is an ear not an ear?

Osprey, 42(3), 25.

Dentinger, B. M., Margaritescu, S., & Moncalvo, J. M. (2010). Rapid and reliable high-

throughput methods of DNA extraction for use in barcoding and molecular systematics

of mushrooms. Molecular Ecology, 10, 628–633. https://doi.org/10.1111/j.1755-

0998.2009.02825.x

Govorova, O. K. (1998). The genus Exidia (heterobasidiomycetes) from the Russian far east.

Mikologiya I Fitopatologiya, 32, 11–13

Hausner, G., Reid, J., & Klassen, G. R. (1993). On the subdivision of Ceratocystis s.l., based on

partial ribosomal DNA sequences. Canadian Journal of Botany, 71, 52–63.

https://doi.org/10.1139/b93-007

Hibbett, D. S., Bauer, R., Binder, M., Giachini, PublicationA. J., Hosaka, K., Justo, A., Larsson, E.,

Larsson, K. H., Lawrey, J. D., Miettinen, O., Nagy, L. G., Nilsson, R. H., Weiss, M. &

Thorn, R. G. (2014). . In: D. J. McLaughlin & J. W. Spatafora (Eds.),

The Mycota VIIA, part A. Systematics and Evolution (2nd ed., pp. 373–428). Berlin:

Springer-Verlag.

Imazeki, R., Otani, Y., & Hongo, T. (1988). Fungi of Japan. Tokyo: Yamakei.

Jung, H. S. (1993). Floral studies on Korean wood-rotting fungi (I): on the flora of ascomycetes

Advanceand jelly fungi. The Korean Journal of , 21, 51–63.

- 10 - Exidia qinghaiensis, a new species from China

Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7:

improvements in performance and usability. Molecular Biology and Evolution, 30, 772–

780. https://doi.org/10.1093/molbev/mst010.

Kirschner. R. (2010). The synnematous anamorph of Exidia saccharina (,

Basidiomycota): morphology, conidiogenesis and association with bark beetles. Polish

Botanical Journal, 55, 335–342.

Kornerup. A., & Wanscher. J. H. (1978). Methuen handbook of colour (3rd ed.). London: Eyre

Methuen.

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

Malysheva, V. F. (2012). A revision of the genus Exidia (Auriculariales, Basidiomycota) in

Russia. Mikologiya i Fitopatologiya, 46, 365–376.

Malysheva, V., & Spirin, V. (2017). Taxonomy Publicationand phylogeny of the Auriculariales

(Agaricomycetes, Basidiomycota) with stereoid basidiocarps. Fungal Biology, 121,

689–715. https://doi.org/10.1016/j.funbio.2017.05.001

Matheny, P. B. (2005). Improving phylogenetic inference of mushrooms with RPB1 and RPB2

nucleotide sequences (Inocybe, Agaricales). Molecular Phylogenetics and Evolution,

35, 1–20. https://doi.org/10.1016/j.ympev.2004.11.014

Moore, R. T. (1997). Exidiaceae. In: L. Hansen & H. Knudsen (Eds.), Nordic Macromycetes

Advance(vol. 3) (pp. 97–104). Copenhagen: Nordsvamp.

- 11 - Mycoscience: Advance Publication

Raja, H. A., Miller, A. N., Pearce, C. J., & Oberlies, N. H. (2017). Fungal identification using

molecular tools: a primer for the natural products research community. Journal of

Natural Products, 80, 756–770. https://doi.org/10.1021/acs.jnatprod.6b01085.s001

Rehner, S. A., & Buckley, E. (2005). A Beauveria phylogeny inferred from nuclear ITS and

EF1-a sequences: evidence for cryptic diversification and links to Cordyceps

teleomorphs. Mycologia, 97, 84–98. https://doi.org/10.3852/mycologia.97.1.84.

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B.,

Liu, L., Suchard, M. A., & Huelsenbeck, J. P. (2012). MrBayes 3.2: efficient Bayesian

phylogenetic inference and model choice across a large model space. Systematic

Biology, 61, 539–542. https://doi.org/10.1093/sysbio/sys029

Spirin, V., Malysheva, V., & Larsson, K. H. (2018). On some forgotten species of Exidia and

Myxarium (Auriculariales, Basidiomycota). Nordic Journal of Botany, 36, e01601.

https://doi.org/10.1111/njb.01601.

Vilgalys, R., & Hester, M. (1990). Rapid geneticPublication identification and mapping of enzymatically

amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology,

172, 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990

Weiss, M., & Oberwinkler, F. (2001). Phylogenetic relationship in Auriculariales and related

groups – hypotheses derived from nuclear ribosomal DNA sequences. Mycological

Research, 105, 403–415. https://doi.org/10.1017/s095375620100363x

Wojewoda, W. (1977). Grzyby (Mycota) (vol. VIII) (pp. 137–163). Warsaw: Polska Akademia

AdvanceNauk Instytut Botaniki.

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Wu, F., Zhao, Q., Yang, Z. L., Ye, S. Y., Rivoire, B., & Dai, Y. C. (2020). Exidia yadongensis,

a new edible species from East Asia. Mycosystema, 39(7), 1–12.

https://doi.org/10.13346/j.mycosystema.200205

Ye, S. Y., Zhang, Y. B., Wu, F., & Liu, H. X. (2020). Multi-locus phylogeny reveals two new

species of Exidia (Auriculariales, Basidiomycota) from China. Mycological Progress,

19, 859–868. https://doi.org/10.1007/s11557-020-01601-8

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Figure legends

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Fig. 1 – Phylogeny of selected members of Exidia based on a maximum likelihood (ML) analysis of nrITS, nrLSU, RPB2 and Tef1 gene regions, with ML bootstrap support (bss) values above 50% shown above the nodes and posterior probabilities from Bayesian analysis below the nodesAdvance. The new species E. qinghaiensis clustered with 100% bss as sister to E. thuretiana from Russia and Finland. All terminals are labelled by their collection number and name by which they were identified. Accession numbers are listed in Table 1. HT = holotype.

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Fig. 2 – Rehydrated Exidia qinghaiensis (HMAS 156328, holotype), on dead stem of Betula sp.

A: Glossy . B: Slightly granular, areolate abhymenium. Bars: A 5 mm; B 1 mm.

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Fig. 3 – Microscopy of Exidia qinghaiensis (HMAS 156328, holotype). A–E: Basidiospores; F,

G: Immature hypobasidia with arrows indicating basal clamp connection. Bars: 5 m.

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Table 1 Sequences of Auricularia and Exidia used in phylogenetic analyses. New sequences derived for this study are in bold font.

Name Voucher Country ITS LSU RPB2 Tef1 A. americana P. B. Matheny 2295 USA: WA DQ200918 AY634277 DQ366278 DQ408143 A. heimuer Y. C. Dai 13782 China MG925288 KM396842a MH020878 NEKD01000005 E. candida V. Spirin 3921 Russia: KHA KY801867 KY801892 — KY801918 E. candida V. Spirin 8588 USA KY801870 KY801895 — KY801920 E. candida var. cartilaginea V. Spirin 10105 Russia: LEN KY801873 KY801898 — KY801923 E. crenata F. Wu 26 Canada: ON MT663361 MT664780 MT679213 MW358922b E. glandulosa H. H. Burdsall 12029 USA: WI MW353407 MW353407 LOAW01000284 LOAW01000070 E. glandulosa M. Weiss 355 Germany AF291273 AF291319 — — E. glandulosa Y. C. Dai 17633 China MH213393 MH213425 MH213456 — E. japonica F. Wu 251 China MN850378 MN850367 MN819823 — E. qinghaiensis HMAS 156376 China MW353408 MW353408 MW358923 MW358925 E. qinghaiensis HT HMAS 156328 China MW353409 MW353409 MW358924 MW358926 E. recisa M. Weiss 315 Germany AF291276 AF291322 — — E. repanda LY BR 7046 France MT663367 MT664784 MT679214 — E. saccharina Roki 88 Germany AF291277Publication AF291323 — — E. thuretiana M. Weiss 373 Germany AF291278 AF291324 — — E. thuretiana V. Spirin 9999 Finland KY801878 KY801905 — KY801927 E. truncata M. Weiss 365 Germany AF291279 AF291325 — — E. uvapassa AFTOL-ID 461 Japan DQ241776 AY645056 — — a Sequence derived from Y. C. Dai 13648, China b Sequence derived from R. G. Thorn 200424/02 (University of Western Ontario herbarium), Canada: ON Abbreviations: AFTOL-ID, Assembling the Fungal Tree of Life identifier; HMAS, Mycological Herbarium, Institute of Microbiology, Academia Sinica, Beijing, China; HT, holotype; KHA, Khabarovsk Krai; LEN, Leningrad Oblast; LY BR, unknown (Wu et al., 2020); ON, Ontario; WA, Washington; WI, Wisconsin. Advance

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