Introduction

The genus Infundibulicybe Harmaja was initially proposed for Clitocybe sect. Infundibuliformes (Fr.) in the restricted delimitation used by Harmaja [1, 2]. The species of this genus are mainly characterized as slightly depressed to infundibuliform pileus, decurrent lamellae, smooth, inamyloid spores that are mostly lacrymoid in shape, abundant clamp connections and the lack of hymenial cystidia, and in particular, mycelia that cannot reduce nitrate and cyanophobic spore walls [2, 3]. Matheny et al. [4] recognized the genus in the Agaricales and as a sister group of the Tricholomatoid clade based on multilocus analysis of a six-gene region supermatrix was re-certified by Binder et al. [5]. Subsequently, Dentinger et al. proposed the suborder Tricholomatineae Aime, Dentinger & Gaya for four families, including Infundibulicybe species as “Incertae sedis” within it [6]. He et al. [7] also treated the genus as “Incertae sedis” suggesting it remained with an uncertain familial placement.

Recent molecular analysis has increased the accuracy of fungal identification and the accumulated sequence data also provide an advantage of promptly discovering new or rare species [8,9,10,11]. Presently, 26 species have been recognized in Infundibulicybe [2, 3, 12, 13]. In China, only 8 of those species have been identified (with some species under Clitocybe): Infundibulicybe alkaliviolascens (Bellù) Bellù, I. altaica (Singer) Harmaja (≡Clitocybe altaica Singer), I. geotropa (Bull.) Harmaja (=Clitocybe geotropa (Bull.) Quél.), I. gibba (Pers.) Harmaja [=Clitocybe gibba (Pers.) P. Kumm.], I. hongyinpan L. Fan & H. Liu, I. rufa Q. Zhao, K.D. Hyde, J.K. Liu & Y.J. Hao, I. subsalmonea (Lamoure) N. Schwab (≡Clitocybe subsalmonea Lamoure), I. trulliformis (Fr.) Gminder {=Clitocybe trulliformis (Fr.) P. Karst. [as 'trullaeformis']} [12,13,14,15,16,17,18]. The present study describes a new species, Infundibulicybe trachyspora, from northeastern China based on morphology and molecular phylogenetic analyses and documents Infundibulicybe species reported in China.

Materials and Methods

Sampling and Morphological Analysis

The basidiocarps were collected in Sep 2020 in the forests dominated by Larix olgensis of northeastern China (Jilin Province). Specimens were preserved and deposited in the Herbarium Mycology of Jilin Agricultural Science and Technology University (HMJU). Conventional macro-morphological characters and detailed anatomy of the specimens were recorded from fresh collections and photographic illustrations. Color names are given in parentheses according to the color chart by Kornerup and Wanscher [19]. For microscopic studies, sections of dried basidiocarps were rehydrated in 3% KOH, subsequently stained in Congo red solution, Melzer’s reagent and Cotton blue, and then observed under an Olympus BX 53 (Tokyo, Japan) light microscope (Nikon, Tokyo, Japan) at either 40, 100, 400, 600, and 1000 magnifications. To evaluate the range of basidiospore size, 20 basidiospores each from one specimen of every collection were measured. The notation [n/m/p] indicates that measurements were made on “n” randomly selected spores from “m” basidiocarps of “p” collections. Q = the quotient of length and width in any one basidiospore; Qm = average of Q. The procedure for scanning electron microscopy followed that of Xu et al. [20], and an FEI Quanta 200FE-SEM (JEOL Ltd., Japan) was used at an accelerating voltage of 5–10 kV.

DNA Extraction, PCR and Sequencing

Genomic DNA was isolated from dried specimens using an M5 Fungal Genomic DNA Kit (Mei5 Biotechnology Co., Ltd, Beijing, China) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) and DNA sequencing were performed with the primers ITS1 and ITS4 [21] for the ITS region; LROR and LR7 [22] for the nrLSU region. The cycling parameters were as follows, ITS: 5 min at 94 ℃ for one cycle; 30 s at 94 ℃, 30 s at 53 ℃, 30 s at 72 ℃ for 33 cycles, 5 min at 72 ℃ for one cycle; nrLSU: 4 min at 94 ℃ for one cycle; 45 s at 94 ℃, 40 s at 46 ℃, 40 s at 72 ℃ for 30 cycles, 4 min at 72 ℃ for one cycle. The PCR amplification products were examined on a 1% agarose gel and checked by a JY 600 electrophoresis apparatus (Beijing JUNYI Electrophoresis Co., Ltd, Beijing, China). Sequencing was completed by BGI Co., Ltd, Beijing, China.

Phylogenetic Analyses

The newly generated sequences in this study were compared with the representative sequences in the GenBank database using the BLASTn algorithm. Since the accessible gene markers of Infundibulicybe species are limited in GenBank databases, this study mainly focuses on the ITS and nrLSU regions for phylogenetic analyses to obtain a representative topological structure. Based on the BLASTn results and outcomes of recent phylogenetic studies focused on Infundibulicybe [3, 12, 13, 18, 23,24,25], sequences were retrieved from GenBank databases, then were aligned automatically with MAFFT 7.0 [26] using ‘–auto’ strategy and normal alignment mode. Aligned sequences were visually inspected and manually adjusted using MEGA 7.0. ModelFinder [27] determined the best-fitted substitution model using the Akaike information criterion (AIC). GTR + F + I + G4 was chosen as the best model for both ITS and ITS-LSU analyses. Phylogenetic analyses using Bayesian Inference (BI) analyses and Maximum Likelihood (ML) analyses were subsequently conducted on MrBayes 3.2.2 [28] and IQ-TREE [29], respectively. Four incrementally heated simultaneous Monte Carlo Markov Chains (MCMC) were run over 10 M generations for the BI analyses. Trees were sampled every 1000 generations resulting in a broad sampling of 10,001 trees. The first 2500 trees were discarded as burn-in (25%). For the ML analyses, statistical supports were obtained using nonparametric bootstrapping with 1000 replicates. The resulting consensus trees were visualized using FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). The significance threshold was set at ≥ 0.90 for Bayesian posterior probability (PP) and ≥ 70% for ML bootstrap proportions (BP). All the sequences used in this study are listed in Table 1.

Table 1 Species list and DNA sequence information employed for phylogenetic analysis
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Results

Phylogenetic Analyses

The ITS data matrix comprised 49 sequences (including 41 from GenBank). This dataset was 860 bp long and contained 404 (47.0%) variable sites. The combined ITS-LSU dataset matrix comprised 33 sequences (including 26 from GenBank). Both BI and ML approaches resulted in the same tree topology, as such only the ML trees are shown with Bayesian PP values (left) and MLBP values (right) for each node (Figs. 1, 2). Phylogenetic analysis based on ITS and ITS-nrLSU sequences showed similar results, the new species grouped in the genus Infundibulicybe and represented a distinct monophyletic lineage.

Fig. 1
figure 1

50% majority rule Bayesian phylogenetic analysis of Infundibulicybe based on ITS sequences, with Singerocybe species as outgroup taxa. Nodes were annotated if supported by ≥ 0.90 Bayesian PP (left) or ≥ 70% ML BP (right) values. For each sequenced taxon, the Genbank number is given. The new species are in red (Color figure online)

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Fig. 2
figure 2

50% majority rule Bayesian phylogenetic analysis of Infundibulicybe based on ITS-nrLSU sequences, with Mycena species as outgroup taxa. Nodes were annotated if supported by ≥ 0.90 Bayesian PP (left) or ≥ 70% ML BP (right) values. For each sequenced taxon, the collection number/Specimen voucher number is given. The new species are in red (Color figure online)

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Taxonomy

Infundibulicybe trachyspora J. Z. Xu, J. C. Qin & Yu Li, sp. nov.

Figures 3, 4, and 5.

Fig. 3
figure 3

Habitat and basidiocarps of Infundibulicybe trachyspora (HMJU 744, holotype)

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Fig. 4
figure 4

Microscopic characteristics of Infundibulicybe trachyspora (HMJU 744, holotype). a Pileipellis. b Stipitipellis. c Basidia. d Basidiospores. Bars: a, b 10 μm; c, d 5 μm

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Fig. 5
figure 5

Scanning electron microscope images of basidiospores of Infundibulicybe trachyspora (HMJU 744, holotype)

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Mycobank No. MB 842,075.

Type China, Jilin Province, Jilin City, Zuojia Town, (44°3′55′′ N, 126°6′8′′ E, elevation 349 m), on the ground under mixed forests dominated by Larix olgensis Henry, 1 Sep 2020, J.Z. Xu (holotype, HMJU 744, Genbank accession numbers: ITS, MW736885; LSU, MW880692; RPB1, OL677427; RPB2, OL677428).

Etymology The epithet derives from the non-smooth spores.

Diagnosis Distinguished by the clitocyboid to omphalioid habit, a carneous, greyish-yellow to brownish pileus, a brown to dark reddish-brown, longitudinally fibrillose-striate stipe, non-amyloid, non-smooth spores and the presence of clamp connections.

Description Basidiocarp clitocyboid to subomphalioid. Pileus 4.5–8 cm diam., at first plane to slightly depressed in the center, finally subinfundibuliform to umbilicate, sagging at the margin, carneous (3B3) then greyish-yellow to brownish (4B3), and dark brown (6D5) at the center. Surface dry, smooth, glabrous, some with a ring stripe, slightly appearing hygrophanous when water-soaked. Margin always involute, irregular, eroded. Lamellae decurrent, up to 0.3 cm broad, dark cream to yellowish-white (1A2, 1A3), moderately crowded, with lamellulae of 1 or 3 lengths, edges concolorous, entire. Stipe 3.5–7 × 0.5–0.75 cm, cylindrical, concolorous with the center of pileus or slightly lighter, brown to dark reddish-brown (6D5, 7E8), solid, some slightly twisty, surface with fibrous longitudinal stripes.

Basidiospores [60/6/3] 6.5–8.1(8.5) × (3.6)3.8–4.4(4.7) μm, Q = (1.69)1.71–1.87(1.95), Qm = 1.78, cyanophilous, lacrymoid to shortly ellipsoid, apiculate, non-amyloid, surface non-smooth under a light microscope, hyaline. Basidia (20.7)21.5–29.3(33.2) × (4.4)4.7–5.9 μm, subclavate to clavate, hyaline, infertility numerous, 4-spored, sterigmata up to 3.5 μm long. Hymenial cystidia absent. Hymenophoral trama regular to subregular, consisting of parallel, cylindrical to clavate, thin-walled hyphae, hyphae 3.1–9.6 μm wide. Pileipellis as a cutis composed of repent, cylindrical, subparallel, septate, thin-walled hyphae, hyphae 3.9–11.0 μm wide. Stipitipellis similar to the pileipellis. Clamp connections are present.

Habitat Scattered on the ground under mixed forests dominated by Larix olgensis.

Distribution Currently, only known from northeastern China (Jilin Province).

Additional specimen examined China, Jilin Province, Jilin City, Zuojia Town, on the ground under mixed forests dominated by Larix olgensis, 1 Sep 2020, J.Z. Xu, HMJU 850, GenBank accession numbers: ITS, MW880701; LSU, MW880707; same location, 1 Sep 2020, J.Z. Xu, HMJU 851, Genbank accession numbers: ITS, MW880702; LSU, MW880708; same location, 1 Sep 2020, J.Z. Xu, HMJU 758, Genbank accession numbers: ITS, MW913424.

Notes Infundibulicybe trachyspora, characterized by the clitocyboid to subomphalioid habit, a carneous, greyish-yellow to brownish pileus, moderately crowded, narrow lamellae, a brown to dark reddish-brown, longitudinally fibrillose-striated stipe, cyanophilous, non- amyloid and smooth spores and the presence of clamp connections. Morphologically, I. trachyspora shares similarities with Infundibulicybe gigas (Harmaja) Harmaja in crowed and narrow lamellae and lacrymoid to short ellipsoid spores. But I. gigas showed bigger size in pileus and stipe as compared in I. trachyspora (I. gigas, pileus diameter 9–28 cm, stipe 4–11 × 1.2–3.8 cm; I. trachyspora, pileus diameter 9–28 cm, stipe 3.5–7 × 0.5–0.75 cm) [2, 30]. Infundibulicybe lapponica resembles I. trachyspora having a medium-sized pileus, but it differs in the minutely scaly to the areolate surface of the pileus, which is smooth in I. trachyspora [1]. The new species recently described from China, I. rufa, resembles I. trachyspora in the smooth pileus and the longitudinally striated stipe. However, I. rufa produces a reddish pileus and stipe, I. trachyspora has a carneous, greyish-yellow to brownish pileus and a brown to dark reddish-brown stipe, I. rufa shows ixocutis hyphae in pileipellis, but I. trachyspora shows cutis hyphae. In addition, I. rufa differs from I. trachyspora by the incurved, wavy to undulate pileus margin (appeared to the species I. kotanensis M. Ishaq, M. Fiaz & A.N. Khalid, also described from Pakistan) [12, 25]. Infundibulicybe hongyinpan is also a new species from China growing on the ground under mixed forests dominated by Larix olgensis in autumn, just like I. trachyspora. But the pileus color of I. hongyinpan is reddish-brown, of I. trachyspora is calcareous, greyish-yellow to brownish. There is a more remarkable feature that I. trachyspora produces non-smooth spores absent in all Infundibulicybe species.

Discussion

The Status of Infundibulicybe trachyspora

Based on morphology and phylogenetic analyses, I. trachyspora was maintained as a new taxon in the genus Infundibulicybe. In ITS and combined ITS-nrLSU phylogenetic analyses, Infundibulicybe group showed close relationships with Ampulloclitocybe clavipes (Pers.) Redhead, Lutzoni, Moncalvo & Vilgalys and Trichocybe puberula (Kuyper) Vizzini, which is consistent with previous studies [12, 23,24,25]. Ampulloclitocybe clavipes and I. trachyspora both have non-smooth spores and lack cystidia. However, A. clavipes produces bulbose-base stipe, I. trachyspora produces cylindrical stipe [31]. Trichocybe puberula and I. trachyspora share the same characteristics in the presence of clamp connections, but T. puberula differs from I. trachyspora by having cystidia and smooth spores [32].

So far, this genus has no subdivisions and there is no established opinion on the infrageneric classification of Infundibulicybe. Meanwhile, the non-smooth spores and the distance of I. trachyspora from the other Infundibulicybe species suggested that the non-smooth spored species may deserve its section. Additional specimens from different areas are needed to evaluate the status of I. trachyspora and reveal the infrageneric classification of Infundibulicybe.

Key to Chinese Species of Infundibulicybe

1a. The color of pileus is red-brown……………………………………………………………………0.2

1b. The color of pileus is not red-brown………………………………………………………………3.

2a. Pileus hygrophanous…………………………………………………………….. I. hongyinpan.

2b. Pileus not hygrophanous………………………………………………………………….. I. rufa.

3a. Pileus surface smooth………………………………………………………………. I. trachyspora.

3b. Pileus surface not smooth…………………………………………………………………………….0.4

4a. The base of stipe with a few rhizoids…………………………………….. I. trullaeformis.

4b. The base of stipe without rhizoids………………………………………………………………5.

5a. Cystidia present………………………………………………………………………………. I. altaica.

5b. Cystidia absent……………………………………………………………………………………………6.

6a. Stipe diameter > 2 cm …………………………………………………………………. I. geotropa.

6b. Stipe diameter < 2 cm…………………………………………………………………………………0.7

7a. Stipe fibrillose…………………………………………………………………… I. alkaliviolascens.

7b. Stipe not fibrillose………………………………………………………………….. I. subsalmonea.