Exidia qinghaiensis, a new species from China

Abstract

A novel, wood-inhabiting jelly fungus from China is described as a new species, Exidia qinghaiensis (Basidiomycota: Auriculariaceae). 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-α (Tef1) regions. Sequences of the new taxon formed a sister group to Exidia thuretiana, 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 μm; 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 Auriculariaceae will require more extensive taxon sampling.

   Exidia Fr. is a genus 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 Auricularia Bull. (Weiss & Oberwinkler, 2001). As in Auricularia, basidiocarps (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 described 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 on phylogenetic analyses of the nuclear ribosomal DNA internal transcribed spacer region (nrITS) and large subunit (nrLSU), RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-a (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 spores and hypobasidia were carried out using oil immersion at 400× and 1000× magnification with differential interference microscopy on a Zeiss AxioImager Z1. All spore 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 Q_avg. Color codes (e.g., 8B5) follow Kornerup and Wanscher (1978)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, & 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-terminator 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). 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 Exidia glandulosa (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, this 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.

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	AF291277	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	—	—

^aSequence derived from Y C Dai 13648, China

^bSequence 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.

   Phylogenetic 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 [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.

Fig. 1 - Phylogeny of selected members of Exidia based on a maximum likelihood (ML) analysis of nrITS, nrLSU, RPB2 and Tef1 generegions, with ML bootstrap support (bss) values above 50% shown above the nodesand posterior probabilities from Bayesian analysis below the nodes. 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.

Taxonomy

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

MycoBank no.: MB 838343.

Fig. 2 - Rehydrated Exidia qinghaiensis (HMAS 156328, holotype), on dead stem of Betula sp. A: Glossy hymenium. B: Slightly granular, areolate abhymenium. Bars: A 5 mm; B 1 mm.

Fig. 3 - Microscopy of Exidia qinghaiensis (HMAS 156328, holotype). A–E: Basidiospores; F, G: Immaturehypobasidia with arrows indicating basal clamp connection. Bars: 5 μm.

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 μm, with Q_avg = 3.7, and the host is Betula. Basidiospores of Exidia saccharina Fr., on conifers, are slightly wider (10–14 × 3.5–4.0 μm), and those of E. thuretiana, also on angiosperms, are both longer and broader (13–19 × 4.5–6.0 μm), 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).

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

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

   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) μm, on average 12.7 × 3.4 μm, Q = (2.7–)3.1–4.3(–4.5), Q_avg = 3.7; probasidia amygdaloid-limoniform; hypobasidia pyriform to subglobose, cruciately septate at maturity, astipitate or substipitate, with basal clamp connection, (10.5–)11.3–15.5(–17.2) × (7.5–)7.6–9.4(–10.0) μm, on average 12.8 × 8.5 μm; Q_avg = 1.5, with longitudinal septa, developing 4 long, finger-like epibasidia (sterigmata), 8–18 × 1.5–2.2 μm. 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 μm diam at their base, some with brownish contents, sparingly and irregularly branched, the branches narrowing to 0.4 μm diam; gloeocystidia not seen. A thin, apparently acellular, brownish epithecium that is present in some mounts (but poorly-preserved and with abundant rod-shaped 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.

   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 μm; 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 μm; 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 μm; 12 × 3μm on average; Aoki & Tubaki, 1986).Wojewoda (1977) reported E. repanda from Betula, Alnus, Fraxinus, 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 narrow 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 collections that we studied of E. qinghaiensis were recorded from Betula, the same host genus as reported for E. repanda in Japan (Aoki et al., 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 Collaborative 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. SW also thank K. Nygard at the Biotron, University of Western Ontario for guidance in the use of the microscope.

References

• Aoki, T. (1991). Heterobasidiomycetes in and around Sugadaira, Central Japan. I. Exidia. Bulletin of the Sugadaira Montane Research Center, 12, 13–26.
• 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, A. 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). Agaricomycetes. 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 and jelly fungi. The Korean Journal of Mycology, 21, 51–63.
• 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 (Auriculariales, 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 and 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 (vol. 3) (pp. 97–104). Copenhagen: Nordsvamp.
• 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 genetic 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 Nauk Instytut Botaniki.
• 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