Two new Cortinarius species in subgenus Leprocybe from Southwest China

Morphological study

Specimens were collected from the Xizang Autonomous Region, Sichuan and Yunnan Provinces, China. The fresh basidiomata were photographed after collecting from the field and the macro-morphological characters were recorded in detail before drying in an oven at 45 °C. A 20% KOH solution was used on fresh pileus and stipe surface, lamella, and context for chemical reaction. Observation of basidiomata was performed under ultraviolet light at a wave length of 360 nm. The specimens were deposited in Fungarium, Institute of Microbiology, Chinese Academy of Sciences (also as Herbarium Mycologicum Academiae Sinicae, HMAS). Descriptions of the microscopical characters are from dried collections. Thin sections were prepared by hand with a razor blade. Sections were mounted in 5% KOH solution. Basidiospores, basidia, tramal hyphae, context, and pileipellis of pileus and stipe were measured using an ocular micrometer. At least 30 basidiospores and 20 basidia of each mature collection were measured.

DNA extraction, amplification and sequencing

Genomic DNA was extracted from dried specimens using standard protocol (Rogers & Bendich, 1994). The DNA extracts were used as templates for PCR. Amplification reactions were performed to obtain sequences of nuclear ribosomal internal transcribed spacer region (ITS) using primer pairs ITS5/ITS4 (White et al., 1990). The amplification was carried out under the following conditions: initial denaturation for 3 min at 95 °C, followed by 30 cycles, at 95 °C for 30 s, at 55 °C for 45 s, at 72 °C for 60 s, and a final elongation step at 72 °C for 10 min.

Phylogenetic analysis

The newly generated ITS sequences were submitted to GenBank. The ITS sequences for the phylogenetic analyses were selected based on results of BLASTn (>94% identity) in GenBank. Two species, C. veronicae (KC017355) and C. veronicoides (GQ890324), were chosen as outgroup. Seventy-three sequences (Table 1) were aligned and edited with BioEdit 7.2.2 (Hall, 1999). Bayesian inference (BI) and maximum likelihood (ML) methods were implemented to analyses in this study. MrModeltest 2.3 was used to calculate the best model (HKY+I+G) for BI analysis (Nylander, 2004). The BI analysis was performed with MrBayes 3.2.6 (Ronquist & Huelsenbeck, 2003), and the ML analysis was conducted in MEGA X (Kumar et al., 2018). The matrix contained 75 ITS sequences with 681 nucleotide sites is available from GenBank (see Data Availability section). Trees were viewed in FigTree 1.4.4 and processed in Adobe Photoshop 2019.

Nomenclature

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition, new names contained in this work have been submitted to Index Fungorum from where they will be made available to the Global Names Index (https://gni.globalnames.org/). The unique Index Fungorum number can be resolved and the associated information viewed through any standard web browser by appending the Index Fungorum number contained in this publication to the prefix “https://www.indexfungorum.org/Names/Names.asp”. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed, Central SCIE, and CLOCKSS.

Molecular phylogeny

The ITS matrix for phylogenetic analyses included 73 sequences, representing 15 species. The resulting alignments were deposited at TreeBASE (http://www.treebase.org; submission ID 30908; accessed on 1 November 2023). The BI and ML trees showed similar topologies, and the ML tree was selected as the representative phylogeny (Fig. 1). The independences of Cortinarius hengduanensis and C. yadingensis were well- supported by phylogenetic analyses. Cortinarius hengduanensis has a close relationship with C. flavifolius Peck, C. yadingensis nests in a clade of sect. Leprocybe and clusters together with C. contoneus, C. hughesiae Ammirati, Matheny, Liimat. & Niskanen, C. selinolens Bidaud & Bellanger and C. subcotoneus Bidaud. The BLASTn against GenBank and UNITE databases taked the holotype specimensas the examples. The percent identity of C. hengduanensis with C. flavifolius (TENN068695, epitype) and C. leproleptopus (R. Henry8409, holotype) are 95% and 94%, respectively. The ITS sequence of C. yadingensis (HMAS254819, holotype) has 97%, 97%, 96% identity with C. cotoneus (CFP1032, neotype), C. subcotoneus (PML2143, holotype) and C. hughesiae (JFA13086, holotype), respectively.

Taxonomy

Cortinarius hengduanensis P. Hong, Ke Wang, Z. Du, M.L. Xie, Di Liu & T.Z. Wei, sp. nov. Fig. 2.

Index Fungorum: IF901373.

Holotype:—CHINA, Yunnan Province, Shangri-La County, Haba Mountains Nature Reserve, alt. 3,000 m, in mixed forest with Pinus densata and Quercus aquifolioides, 16 Aug. 2008, T.-Z. Wei, X.-Q. Zhang & F.-Q. Yu 194, HMAS 250455 (GenBank ID: KX513581 ITS).

Etymology:—“hengduan”, Chinese, referring Hengduan Mountains of southwest China, the locality of the type collection.

Diagnosis:—Cortinarius hengduanensis has olive tinged basidiomata, squamulose pileus, and small subglobose to broadly ellipsoid basidiospores (5.5–7.5 × 5–6.5 μm). It differs from C. flavifolius for having greenish tint on pileus surface and differs from C. venetus by its obviously annulate stipe. ITS sequences of the new species (GenBank ID: KX513581–KX513583, OR538887 and OR538888) deviate from that of C. flavifolius by at least 28 substitutions and indel positions. In mixed forest with Pinus yunnanensis, P. densata and Quercus aquifolioides.

Description:—Pileus 4.5–12.5 cm diam., hemispherical at first, later becoming convex to applanate, with a shallow, obtuse umbo when mature, margin sometimes uplifted or undulate, surface uneven, tomentose, persistently covered with small olive-brown (RAL 8008) to dark olive-brown (RAL 8022) squamules, margin radially striate, olive-green (RAL 6003) with brownish (RAL 8024) tint at first, then brownish olive (RAL 8000), olive-brown (RAL 8008) to dull brown (RAL 8019) with paler margin, sometimes darker at the center. Context up to 8 mm thick at pileus center, whitish (RAL 9010) to pale when dry, olive-gray (RAL 7015) when moist, fleshy, soft; odor indistinct, taste mild. Lamellae adnate to emarginate, 3–8 mm wide, close to moderately crowded, olive to grayish green (RAL 6006) when young, brown with olive (RAL 8000) tint to rust-brown (RAL 8012) when mature, edges paler. Stipe 5.5–12 × 0.7–2.6 cm, cylindrical or base enlarged and up to 3 cm in diam., surface completely covered with universal veil at first, then longitudinally fibrillose above and with conspicuous annular girdles below; olivaceous yellow (RAL 1020), tinged rust-brown (RAL 8012) from basidiospores, at first solid, then soft to hollow. Partial veil arachnoid, greenish to olive (RAL 6003), then becoming rust brown (RAL 8012) from mature basidiospores. Universal veil distinct and persistent, fibrillose, covering whole basidiomata at first, then forming fibrils and squamules on pileus and fibrils and girdles on stipe surface, pale with olive tint at first, brownish with olive (RAL 8000) tint to olive-brown (RAL 8008) when mature. Basidiospore deposit rust brown (RAL 8012). Chemical reaction with 20% KOH dark reddish brown on pileus and lamella, brown on stipe and reddish brown on context. Fluorescence reaction under ultraviolet light distinctly bright yellow on lamellae, and weaker on surfaces of pileus and stipe.

Basidiospores (5.2–) 5.5–7 (–7.5) × (4.8–) 5–6 (–6.5) μm, Q = (1.08–) 1.1–1.2 (–1.23) (av. = 1.15), subglobose to broadly ellipsoid, yellowish brown, moderately to distinctly verrucose. Basidia 28–35 × 8–10 μm, clavate, thin-walled, mostly subhyaline, with four sterigmata. Lamella edges heterogeneous, with sterile cells, 20–30 × 6–9 μm, clavate, subhyaline, thin-walled. Pleurocystidia absent. Subhymenial layer up to 10 μm thick, of narrow and branched hyphae, hyaline, thin-walled, 2–4 μm diam. Hymenophoral trama regular, 80–100 μm wide, of hyaline and thin-walled hyphae, 5–20 μm diam. Pileipellis: epicutis well developed, hyphae 9–15 μm wide, subcylindrical, colourless, yellowish to brownish, thin-walled, smooth; Hypodermium present, hyphae 3–8 µm wide, irregular, with yellowish intracellular pigment in 5% KOH. Hyphae of the cortina 3–6 μm diam., subhyaline to yellowish, thin-walled. Clamp connections present.

Additional Specimens Examined: CHINA, Yunnan Province, Songming County, Aziying Town, alt. 2,000 m, in mixed forest with Pinus yunnanensis and Quercus aquifolioides, 5 Aug. 2005, T.-Z. Wei & F.-Q. Yu Gm1082, HMAS 145537 (GenBank ID: KX513582); Deqin County, Baima Mountains, alt. 3,100 m, in mixed forest with P. densata and Q. aquifolioides, 19 Aug. 2008, T.-Z. Wei, X.-Q. Zhang & F.-Q. Yu 258, HMAS 250509 (GenBank ID: KX513583); Xizang, Nyingchi City, Bayi District, Bayi Town, 29°38′02.58″N, 94°23′44.72″E, alt. 3,360 m, in mixed forest with P. densata and Q. aquifolioides, 14 Sept. 2014, T.-Z. Wei, J.-Y. Zhuang, X.-Y. Liu & H. Huang 5281, HMAS 270305 (GenBank ID: OR538888); Lulang Town, alt. 3,481 m, 22 Sept. 2014, W.-L. Lu & Q.-M. Wang, 3017, HMAS 272520 (GenBank ID: OR538887).

Notes: Cortinarius hengduanensis is characterized by its distinct olive tinged basidiomata, fibrillose-squamulose pileus and subglobose to broadly ellipsoid basidiospores (5.2–7.5 × 4.8–6.5 μm). The new taxon is close to C. flavifolius, the latter also has a fibrillose to squamulose pileus and subglobose basidiospores (Ammirati et al., 2021). Cortinarius flavifolius is distributed in eastern North America. It does not have any olive or greenish tint on the pileus, and its basidiospores are 6.7–8.9 × 4.8–5.9 μm (Ammirati et al., 2021) larger than that of C. hengduanensis. The new species shows high morphological similarity to C. venetus, the latter has obvious olive basidiomata, subglobose basidiospores and distributed in Europe of mixed forest (Breitenbach & Kränzlin, 2000; Bidaud et al., 2005; Soop, 2018). The stipe of C. venetus is fibrillose when mature (Breitenbach & Kränzlin, 2000; Bidaud et al., 2005), and only with ephemeral veil annulate zone (Bidaud et al., 2005) or thin girdle (Soop, 2018) when young. However, the stipe surface of C. hengduanensis is obviously annulate and banded from persistent veil remains. Cortinarius melanotus Kalchbr. also has olive or olive tinged basidiomata and fibrilloso-squamulose pileus (Breitenbach & Kränzlin, 2000), but differs from the subglobose basidiospores of C. hengduanensis, its basidiospores are ellipsoid (6.2–8 × 4.3–5.3 μm) (Breitenbach & Kränzlin, 2000).

Cortinarius yadingensis P. Hong, Ke Wang, Z. Du, M.L. Xie, Di Liu & T.Z. Wei, sp. nov. Fig. 3.

Index Fungorum: IF901374.

Holotype:—CHINA, Sichuan Province, Daocheng County, Yading Scenic Spot, alt. 4,034 m, in mixed forest of Abies sp., Larix potaninii, Picea sp. and Q. aquifolioides, 18 Aug. 2016, T.-Z. Wei, L.-H. Sun, Z.-X. Wu & R.-C. Zhang, 7168, HMAS 254819 (GenBank ID: OR538892).

Etymology:—“yading”, Chinese, referring Yading Scenic Spot of Sichuan Province, China, the locality of the type collection.

Diagnosis:—Cortinarius yadingensis has distinct brown squamules on the basidiomata surface and subglobose to broadly ellipsoid basidiospores (6.5–9 × 6.3–7.6 μm). It differs from C. cotoneus, C. hughesiae, C. selinolens and C. subcotoneus by its lack of olivaceous coloration. ITS sequences of the new species (GenBank ID: OR538889–OR538893) are distinct from other members of sect. Leprocybe and deviating from them by at least 11 substitutions and indel positions. In mixed forest with Abies sp., Larix potaninii, Picea sp. and Q. aquifolioides.

Description:—Pileus 2–6 cm diam., hemispherical at first, later becoming convex to applanate, mostly with a shallow and obtuse umbo when mature, margin decurrent to straight, sometimes split when mature, surface pale brown (RAL 8025), grayish brown (RAL 8019) to rust brown (RAL 8012), covered brown (RAL 8000) to dark brown (RAL 8022) fibrillose squamules, radially striate, silky and shining. Context up to 5 mm thick at pileus center, pale brown (RAL 8025) to grayish brown (RAL 8019), fleshy, soft; odor indistinct, smell mild. Lamellae emarginate-adnate, up to 5 mm wide, brownish (RAL 8000) when young, rust-brown (RAL 8012) when mature, moderately crowded, edges margin paler. Stipe 3.5–8 × 0.5–1 cm, subcylindrical, with base up to 1.5 cm in diam., surface brownish (RAL 8000) to pale brown (RAL 8025), covered with brown (RAL 8000) to dark brown (RAL 8022) fibrillose squamules, with annular zone from partial veil, longitudinally striate; at first solid then soft to hollow. Partial veil arachnoid, grayish (RAL 7035) when young, forming a rust brown (RAL 8012) ring from basidiospores when mature. Universal veil forming fibrillose brown (RAL 8000) to dark brown (RAL 8022) squamules on the surface of the stipe. Basidiospore deposit rust brown. Chemical reaction with 20% KOH dark brown to blackish brown on pileus, lamella and stipe surface, and reddish brown on context. Fluorescence reaction under ultraviolet light bright yellow on lamellae, and weaker on surfaces of pileus and stipe.

Basidiospores (6.5–) 7–8.5 (–9) × (6.3–) 6.5–7.2 (–7.6) μm, Q = (1.03–) 1.08–1.2 (–1.24) (av. = 1.16); subglobose, yellow-brown, distinctly verrucose. Basidia 28–35 × 8–10 μm, clavate, thin-walled, mostly subhyaline, with four sterigmata. Lamella edges heterogeneous, with sterile hyphae, 20–30 × 6–9 μm, clavate, subhyaline, thin-walled. Pleurocystidia absent. Subhymenial layer up to 10 μm thick, of narrow and branched hyphae, hyaline, thin-walled, 2–4 μm diam. Hymenophoral trama regular, 80–100 μm wide, of hyaline and thin-walled hyphae, 5–20 μm diam. Pileipellis: epicutis well developed, hyphae 9–15 μm wide, subcylindrical, colourless, yellowish to brownish, thin-walled, smooth; Hypodermium present, hyphae 3–8 µm wide, irregular, with yellowish intracellular pigment in 5% KOH. Hyphae of the cortina 3–6 μm diam., subhyaline to yellowish, thin-walled. Clamp connections present.

Additional Specimens Examined: CHINA, Sichuan, Litang County, road side to Daocheng County, alt. 3,937 m, in conifer forest with Abies sp. and Picea sp., 17 Aug. 2016, T.-Z. Wei, L.-H. Sun, Z.-X. Wu & R.-C. Zhang, 7117, HMAS 254811 (GenBank ID: OR538889); Daocheng County, Yading Scenic Spot, alt. 4034 m, in mixed forest of Abies sp., Larix potaninii, Picea sp. and Q. aquifolioides, 18 Aug. 2016, T.-Z. Wei, L.-H. Sun, Z.-X. Wu & R.-C. Zhang, 7169, HMAS 254820 (GenBank ID: OR538893); 7123, HMAS 280697 (GenBank ID: OR538890); 7127, HMAS 280698 (GenBank ID: 254819).

Notes: Cortinarius yadingensis is characterized by its grayish brown squamulose to brown pileus and stipe and subglobose, distinctly verrucose basidiospores (6.5–9 × 6.3–7.6 μm). The species is phylogenetically close to four species of sect. Leprocybe, C. cotoneus (Breitenbach & Kränzlin, 2000, Soop, 2018), C. hughesiae Ammirati, Matheny, Liimat. & Niskanen (Ammirati et al., 2021), C. selinolens Bidaud & Bellanger (Bidaud et al., 2021) and C. subcotoneus (Bidaud et al., 2005). Compared with C. yadingensis, all the four related species have similar finely tomentose to squamulose pileus and strongly verrucose basidiospores. Unlike C. yadingensis, all the related species have olive tinged basidiomata. Besides, C. cotoneus, C. selinolens and C. subcotoneus inhabit in broad-leaf forest associated with Fagaceae trees, but C. yadingensis are reported in mixed forest with Abies sp., Larix potaninii, Picea sp. and Q. aquifolioides. Phylogenetically, C. yadingensis can be distinguished from its sister species by at least 11 substitutions and indels in their full ITS sequences.