Introduction

Crepidotus (Fr.) Staude (Crepidotaceae, Agaricales) is a well-defined fungal genus with typically pleurotoid basidiomata, lamellate hymenophore and brown spore print. Members of this genus are saprotrophs, growing mainly on dead wood, less frequently on other plant remnants or soil, exceptionally on thalli of bryophytes or on fruiting bodies of other fungi (Pilát 1948; Consiglio and Setti 2008; Hausknecht and Krisai-Greilhuber 2010). Early molecular studies suggested that the traditional morphology-based genus concept corresponds to a monophyletic origin of this genus (Aime et al. 2002; Jančovičová et al. 2022). The infra-generic relationships are, however, not fully resolved mainly due to incomplete sampling. The genus has a cosmopolitan distribution with multiple underexplored areas (Singer 1986, https://www.inaturalist.org/taxa/118288-Crepidotus).

Currently, new morphological and molecular Crepidotus profiles providing reliable species identifications are scarce. This prevents estimation of species distribution limits. Recent large-scale phylogenetic studies demonstrated that a part of some saprotrophic phylogenetic lineages include species with broad holarctic or cosmopolitan distributions (Ševčíková et al. 2022; Schünemann et al. 2024). In Crepidotus, some species are probably endemic to a single continent, e.g. the North American species C. brunnescens Hesler & A. H. Sm. (Jančovičová et al. 2017). However, other species originally described from North America have also been reported elsewhere, e.g. C. alabamensis Murrill from India (Kumar et al. 2022) and C. cinnabarinus Peck from Europe (Senn-Irlet 2012). These studies relied on analyses of the LSU or ITS nrDNA regions and morphology.

Our study is focused on C. cinnamomeus Hesler & A. H. Sm. described from Idaho in western North America and published in the first comprehensive North American monograph of the genus Crepidotus by Hesler and Smith (1965). There are 125 accepted taxa in this monograph, including 78 species new to science. However, many of these species are known only from this book and their names are not used. As far as we know, C. cinnamomeus has not been reported or accepted by any other literature in North America or outside the continent after its first publication. The sequence obtained from isotype of this species (TENN-F-026166) is more than 99.2% similar to publicly available sequences from Sweden (Jančovičová et al. 2020) and China (Ge and Bau 2020). The aim of this study is to define taxonomic circumscription of C. cinnamomeus and assess its geographical distribution based on publicly available sequence data.

Materials and methods

Sampling

Sampling included the isotype of Crepidotus cinnamomeus (TENN-F-026166) and additional samples collected by the authors with DNA sequence similarity > 90% to the isotype ITS sequence. The authors' collections originated from Montana (USA), Sweden and Spain. Specimens collected by authors were deposited in the SLO herbarium (Comenius University Bratislava).

DNA extraction, PCR and sequencing

Total genomic DNA was extracted from dried material using the EZNA Fungal DNA Mini Kit (Omega Bio-Tek Inc., Norcross, GA, USA) following the manufacturer´s instruction. Three nuclear loci were amplified and sequenced: (i) ITS1-5.8S-ITS2 rDNA (ITS); (ii) D1–D2 domains of 28S rDNA (LSU) and (iii) the region between domains 6 and 7 of the nuclear gene encoding the second largest subunit of RNA polymerase II (RPB2). The ITS region was amplified with primer pairs ITS5-ITS4 (White et al. 1990), and for the LSU primers LR0R and LR5 were used (Moncalvo et al. 2000) with PCR conditions according to Jančovičová et al. (2022). Primers bRPB2-6F and bRPB2-7.1R (Matheny 2005) were used to amplify the RPB2 region. PCR conditions were as follows: 1 min at 95 °C, 1.5 min at 55 °C, an increase of 1 °C per 5 s to 72 °C and 2 min at 72 °C, repeated 35 times and finalized for 10 min at 72 °C. Amplification of DNA was performed in a PCR reaction mix consisting of approximately 2 ng/μl of template DNA, forward and reverse primers (10 pmol/μl), 5 × HOT FIREPol® Blend Master Mix (Solis BioDyne, Tartu, Estonia) and molecular grade water added up to 20 μl. The target fragments were purified using ExoSap-IT (Thermo Fisher Scientific, Wilmington, Delaware, USA). Sequencing was performed at the SEQme sequencing company (Dobříš, Czech Republic).

Phylogenetic analyses

A multilocus phylogenetic analysis was supported by samples of species with phylogenetic affinity or morphological similarity to Crepidotus cinamomeus (Jančovičová et al. 2020): C. variabilis (Pers.) P. Kumm., C. variabilis var. trichocystis Hesler & A. H. Sm., C. neotrichocystis Consiglio & Setti, C. kubickae Pilát and C. cesatii (Rabenh.) Sacc. Crepidotus applanatus (Pers.) P. Kumm. and C. malachius (Berk. & M.A. Curtis) Sacc. were selected as representatives of unrelated members of the genus. Two samples of Simocybe P. Karst. were selected as an outgroup (Matheny et al. 2020). Samples used in the phylogenetic analyses are listed in Table 1.

Table 1 The list of ITS, LSU and RPB2 sequences used in the phylogenetic analyses. Sequences generated in this study are in bold
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Raw sequence files were edited in Geneious version R10 (Kearse et al. 2012). Intra-individual polymorphic sites having more than one signal were marked with NC-IUPAC ambiguity codes. Datasets for each marker were aligned separately by MAFFT version 7 using the strategy E-INS-i (Katoh and Standley 2013), manually edited in Geneious version R10 (Kearse et al. 2012) and concatenated using SeaView v.4.5.1 (Gouy et al. 2010). The final alignment was analysed with Maximum Likelihood (ML) and Bayesian inference (BI). For ML, the aligned dataset was loaded as a fasta file at the Cipres Science Gateway (Miller et al. 2010) and analysed using RAxML-HPC2 on XSEDE 8.2.12 (Stamatakis 2014). The alignment was partitioned under default settings with a GTR + GAMMA model with 1000 bootstrap iterations. For BI, the aligned fasta files were converted to the nexus format using Mesquite 3.61 (Maddison and Maddison 2023) and further analysed using MrBayes 3.2.6. (Ronquist et al. 2012) on XSEDE at the Cipres Science Gateway (Miller et al. 2010). Bayesian runs were computed independently twice with four MCMC chains for 10 million generations until the standard deviation of split frequencies fell below the 0.01 threshold. The convergence of runs was visually assessed using the trace function in Tracer 1.6 (Rambaut et al. 2014). The results were further edited with TreeGraph 2 (Stöver and Müller 2010) and graphically improved in CorelDRAW X5 (Ottawa, Canada).

The UNITE database (https://unite.ut.ee/) was searched using massBLASTer tool for all sequence variants assigned to C. cinnamomeus by multilocus analysis. Sequences in species hypothesis (SH) and individual sequences with similarity > 95% were included in additional analysis of the ITS region. The final ITS alignment and ML analysis were processed the same way as described for the multilocus dataset. The results of both multilocus and ITS analyses were further edited with TreeGraph 2 (Stöver and Müller 2010) and graphically improved in CorelDRAW X5 (Ottawa, Canada).

Morphological analyses

Macromorphological characters of recent collections of Crepidotus cinnamomeus were observed from fresh material. Colour codes followed Kornerup and Wanscher (1978). Dried specimens were used to examine micromorphological characters. Microscopic structures were prepared in ammoniacal Congo red after a short pre-treatment in 3% aqueous solution of KOH. The structures were measured directly under an Olympus BX41 light microscope using an oil-immersion lens at a magnification of 1000 × . Drawings of microscopic structures were made with a camera lucida using an Olympus U-DA drawing attachment at a projection scale of 2000 × . Statistical calculations were based on 30 (spores, basidia, basidioles, cheilocystidia and terminal cells of pileipellis) and 20 (other microscopic structures) measurements per specimen. Q = ratio of length and width of spores. The range of microscopic characters was given as the minimum, maximum (in the parenthesis), average ± standard deviation, and average values. Morphological groups and frequency of cheilocystidia were adopted from Jančovičová et al. (2020); spore ornamentation was described according to Senn-Irlet (1995); other morphological terminology followed Vellinga (1988).

Results

Phylogenetic analyses

Phylogenetic analysis of combined ITS, LSU and RPB2 included 22 samples (Fig. 1). Three major groups were supported within Crepidotus: C. applanatus and C. malachius formed distinct species-level lineages, whereas all other species were grouped into an inclusive clade (ML = 96, BI = 1.00). Within this larger inclusive clade, all non-singleton species clades received strong ML and BI support, but there was no support among internodes. Within the C. cinnamomeus lineage, there was no support for sample grouping based on geography.

Fig. 1
figure 1

Phylogram generated by ML analysis based on combined sequence data of ITS, LSU and RPB2. ML bootstrap support values greater than 50% and Bayesian posterior probabilities greater or equal to 0.90 are indicated above or below the nodes. Sequences originated from type collections are indicated with “T!”

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A UNITE search resulted in retrieval of five SHs with at least one sequence more similar than 95% to C. cinnamomeus (SH1185163.09FU 99.85%, SH1185183.09FU 96.54%, SH1185159.09FU 96.45%, SH1185148.09FU 95.25%, SH1185185.09FU 95.21%) and one sequence not included in any SH (UDB07673142 96.05%). In the ITS tree (Supplementary file 1), only one new sample OM343180 was placed in the C. cinnamomeus lineage. This sample was published on iNaturalist (https://www.inaturalist.org/observations/90967590) and originated from Arizona, USA. Seven other retrieved sequences were placed in C. cesatii, and two formed a clade that included a molecular annotation of the C. neotrichocystis holotype.

Taxonomy

Crepidotus cinnamomeus Hesler & A. H. Sm., North American species of Crepidotus: 109. 1965. Figs. 2, 3

Original diagnosis: Pileus 8–20 mm latus, sessilis, dimidiatus vel inaequalis, obscuro-albus deinde cinnamomeus, fibrillosus. Lamellae confertae densae, angustae demum medio-latae, pallidae deinde cinnamomeae vel rubido-brunneae. Sporae 5–6.2 × 3.3–4(4.2) μm, ellipsoideae, punctatae. Basidia 22–28 × 4–6 μm, di- et tetraspora. Pleurocystidia desunt; cheilocystidia 32–42 × 5–8 μm. Cuticula sine magno discrimine, hyphas erectas sine colore gerens. Fibulatae adsunt. Specimen typicum in Herb. Univ. Mich.; lectum prope Priest Lake, Idaho, Oct. 1, 1956, A. H. Smith 53816. Holotype: MICH-F-5525. Isotype: TENN-F-026166.

Description of the isotype of Crepidotus cinnamomeus

(TENN-F-026166, Smith 53816, Idaho, isotype).

Basidiomata ca. 10–12 mm wide; pileus surface not scaly, buff to warm buff in colour when dried; lamellae appear subdistant or only moderately close, moderately deep, pale brown to light yellowish brown.

Basidiospores (4.9)5.6–6.1–7(7.3) × (3)3.2–3.4–3.7(3.8) µm, Q = (1.44)1.58–1.79–2.06(2.09) (n = 33/1), ellipsoid, oblong or amygdaliform with rounded or bluntly pointed apices, surface punctate, wall slightly thickened, pale yellowish brown to pale yellowish, hilar appendix very small and not conspicuous. Basidia 23–29 × 5–7 µm, mostly 4-sterigmate, slenderly clavate, not pigmented. Pleurocystidia absent. Cheilocystidia (19)21.7–26–30.3(34) × (4)4.4–6–7.4(8.5) µm, variably or irregularly shaped: mostly rostrate, sometimes clavate, lobate, forked and antler-like, at times flexuous, thin-walled, not pigmented. Lamellar tramal hyphae cylindrical, smooth, thin-walled, lacking pigment, cells 3–12 µm wide. Clamp connections present in all parts.

Description of recent collections of Crepidotus cinnamomeus

(SLO 2407, SLO 2778, SLO 2780, SLO 2811).

Basidiomata pileate, sessile, laterally or dorsally attached to the substrate, or with a rudimental lateral stipe, gregarious or clustered in groups. Pileus 3–17 mm in diameter; when young and mature rounded flabelliform or reniform (as seen from above); when young hemispherical, rarely campanulate, with age convex, plano-convex to applanate (as seen from aside); not hygrophanous; margin long involute, then inflexed, entire or lobed, not translucently striate; surface of young basidiomata white to orange-white (5A2), when mature golden blond (5C4) to brown (5D6-oak brown or 5E6-mustard brown); sericeous to velutinous, in one case (SLO 1096) when mature brownish orange (6C4-red-haired) to light brown (6D4-camel) flocculose; at the point of attachment white mycelial tomentum. Stipe (if present) cylindrical, curved, 1 × 0.5 mm, whitish, pubescent. Lamellae l = (1)3–7, L = 14–24(32), 0.5–1.5 mm wide, ventricose, adnexed, when young white to orange-white (5A2), when mature greyish orange (5B4), light brown (6D6-cinnamon) to brown (6E5, 6E6-leather brown, 6E7); lamellae edges entire or irregularly serrulate, when young concolorous, when mature paler (whitish) than the lamellae sides. Context up to 0.5 μm thick, yellowish white (3A2), smell and taste indistinct. Spore print light brown (6D6-cinnamon).

Basidiospores (5.2)5.8–6.5–7.2(9) × (3)3.1–3.4–3.7(4) μm, Q = (1.71)1.81–1.93–2.26(2.29), oblong, yellowish to yellowish brown, verruculose (punctate under light microscope), hilar appendix not conspicuous. Basidia 4-spored, rarely 2-spored, (16)18.8–20.7–22.7(25) × (5)5.7–6.1–6.5(7.5) μm, clavate, hyaline, thin-walled. Basidioles (10.5)14–17.4–20.7(27) × (3.5)4.5–5.2–6(7) μm, clavate, hyaline, thin-walled. Pleurocystidia absent. Cheilocystidia (13)19.1–24–28.9(40) × (3)4.6–5.9–7.2(10.5) μm, often forked and antler-like, sometimes rostrate, diverticulate and lobate, rarely clavate, at apex obtuse, hyaline, thin-walled. Lamellar trama of 3–11 μm wide, almost parallel, flexuous, irregularly inflated, intricate, occasionally anastomosed or branched, hyaline, thin-walled or up to 0.5 μm thick-walled hyphae. Pileipellis ca. 200 μm deep, composed of ascending to erect, usually unbranched and two-celled, loosely arranged, hyaline, thin-walled hyphae forming a transition from cutis to trichoderm. Terminal cells near pileus centre (38)52.7–83.5–114.3(164) × (3)3.1–3.6–4.1(5) μm, cylindrical, straight or slightly flexuous, occasionally nodulose, at some places fasciculated, apically tapering and ca. 0.5–1 μm narrower than near the septum; near pileus margin (21)29.7–48.5–67.3(78) × (2.5)2.6–3.3–3.9(5) μm, flexuous, angulate, twisted, often branched, with lateral nodes or coralloid. Clamp connectiones present in all parts.

Note: We did not observe any distinct differences among spore and cheilocystidia dimensions. Our study revealed high variability of cheilocystidia shape among collections. These included mostly rostrate, sometimes clavate, lobate, forked and antler-like in TENN-F-026166; mostly rostrate, often forked and antler-like, and never diverticulate in the collection from Sweden (SLO 2407); mostly forked and antler-like, and only sometimes rostrate and diverticulate in the Spanish collections (SLO 2778, SLO 2780); often forked, antler-like and diverticulate and only sometimes rostrate in the collection from Montana (SLO 2811). The pileipellis of all examined collections showed the same variation pattern (Figs. 2 and 3; Supplementary file 2).

Fig. 2
figure 2

Crepidotus cinnamomeus. A Fresh basidiomata in detail (Montana, SLO 2811). B Field aspect of basidiomata (Spain, SLO 2778). C Dried basidiomata of isotype (Idaho, TENN-F-026166). D Basidiospores. E Cheilocystidia. F Terminal cells near pileus centre. G Terminal cells near pileus margin (D–G: Montana, SLO 2811, D stained in KOH, E–G stained in Congo red). Scale bars A–C = 1 cm, D–G = 10 μm. Photos: A: T. B. Wheeler; C: P. B. Matheny; B, D, E, F, G: S. Jančovičová

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Fig. 3
figure 3

Crepidotus cinnamomeus. A Basidia. B Basidioles. C Terminal cells near pileus centre. D Terminal cells near pileus margin. E Cheilocystidia (A–E: Montana, SLO 2811). F Cheilocystidia (Sweden, SLO 2407). G Cheilocystidia (Spain, SLO 2778). Scale bar = 10 μm. Drawings: S. Jančovičová

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Specimens examined: United States, Idaho, Priest Lake, on Betula branches, 1st of October 1956, leg. A. H. Smith Smith 53816 (TENN-F-026166), isotype. United States, Montana, Lake County, Jocko River Canyon, 6606 Jocko Canyon Road, 47°10ʹ52.6″N, 113°37ʹ2.8″W, 1080 m asl., mixed riparian forest with Populus, Thuja, Betula and Picea, on wood/bark of fallen decaying branch of Betula? or Populus?, ca. 5–15 mm in diam., 1st of July 2020, leg. T. B. Wheeler TBW 8159 (SLO 2811). Spain, Pyrénées Mts., Huesca Province, (Javierre) municipality, Valle de Pineta, near Cinca river, 42°38ʹ08″N, 00°10ʹ54″E, 1170 m asl., mixed riparian forest with Pinus sylvestris, Prunus spinosa, Salix, on wood/bark of fallen twig of Rubus, 2–4 mm in diam., 5th of October 2022, leg. S. Jančovičová (SLO 2780); ibidem, on bark of fallen decaying branch of Salix cf. purpurea, ca. 15 mm in diam., 5th of October 2022, leg. S. Jančovičová (SLO 2778). Sweden, Västernorrland County, Âse village, ca. 1 km W of the village or ca. 2 km SW of the Âsetjärnen (lake), 62°31ʹ03″N, 16°02ʹ04″E, 160 m asl., moist brook forest with Alnus, Betula, Picea, Salix, on wood and bark of fallen decaying branch of deciduous tree (Betula?), ca. 5–10 mm in diam., 27th of August 2018, leg. S. Jančovičová (SLO 2407).

Discussion

This study provides the first report and molecular and morphological delimitation of Crepidotus cinnamomeus, a species originally described by Hesler and Smith (1965) and placed in Crepidotus subgen. Dochmiopus (Pat.) Pilát, sect. Dochmiopus Consiglio & Setti. Within this section (in the sense of Hesler and Smith 1965), species were distinguished mainly by the colour of the pilei: white in C. subsphaerosporus (J. E. Lange) Kühner & Romagn., C. variabilis var. variabilis and C. variabilis var. trichocystis; pale cinnamon pileus in C. cinnamomeus; lemon yellow in C. subcroceitinctus Hesler & A. H. Sm.; and orange-buff to warm-buff in C. croceitinctus Peck.

According to our observations, C. cinnamomeaus does not have cinnamon tints on pileus, which can also be seen on the isotype (Fig. 2). On the other hand, yellow and orange tints on pilei of two other species are probably good diagnostic characters such as in case of e.g. C. luteolus Sacc. and C. crocophyllus (Berk.) Sacc. respectivelly (Senn-Irlet 1995).

Morphologically similar species with white pilei include C. variabilis var. variabilis, C. variabilis var. trichocystis, and recently described C. neotrichocystis (Consiglio and Setti 2008). Although Consiglio and Setti (2008) presented C. variabilis var. trichocystis as a misapplied name of C. neotrichocystis, our phylogenetic study confirmed that var. trichocystis deserves a species rank (Fig. 1).

Crepidotus variabilis var. variabilis differs from C. cinnamomeus by consistently larger cheilocystidia (usually in average longer than 30 μm and wider than 7 μm), which is demonstrated and statistically well documented in the study by Jančovičová et al. (2020) that also included one of our collection of C. cinnamomeus (SLO 2407). The cheilocystidia shape of these two species, C. variabilis and C. cinnamomeus, show high variability among collections, but both species have some cystidia types that were rare or absent in the other species (see Fig. 3 and Fig. 7 in Jančovičová et al. 2020). This study confirmed that C. cinnamomeus can be distinguished from C. variabilis var. trichocystis and C. neotrichocystis by more elongated spores (average Q > 1.8), which was also suggested in our previous study (Jančovičová et al. 2020, Fig. 3).

Other species mentioned as similar to C. cinnamomeus by Hesler and Smith (1965) include C. subsphaerosporus, but the species name is invalid and its concept is unclear, probably referring to C. kubickae (Jančovičová and Pennycook 2012). Both C. kubickae and a similar species C. cesatii are clearly different from C. cinnamomeus by spores wider than 4.2 μm (Ripková 2009). Delimitation of C. cinnamomeus and all similar species is presented in the identification key (Supplementary file 3).

One of sequences retrieved from GenBank was MF461345 from China. This sequence number with name “Crepidotus sp.” appeared in the publication by Ge and Bau (2020), in their table of specimens used in the phylogenetic analysis and in the tree. The associated voucher number HMJAU37086, however, refers to the holotype of C. reticulatus T. Bau & Y.P. Ge: “Holotype: CHINA. Guangdong Province: Zhaoqing City, Dinghu Mountain, 17 Jun 2015, Tian, Liu & Zhang HMJAU37086 (HMJAU). Gene sequences ex-holotype: MF461346 (ITS).” As can be seen, authors cited different ITS sequence number MF461346, which is not a sequence similar to C. cinnamomeus. Crepidotus reticulatus is a species described as having a coral red pileus and spores ellipsoid to ovoid, with ridges or partial reticulation. Because of such differences from C. cinnamomeus, we think that the sequence number MF461345, which is placed in our phylogenies in C. cinnamomeus clade (Fig. 1), is incorrectly paired with the type specimen of C. reticulatus and actually represent a record of the former species from China. Crepidotus cinnamomeus was not included in the monographic work by Tolgor et al. (2022).

Boreal and temperate regions are estimated to have lowest endemicity of fungal species, which is in agreement with our discovery of the wide distribution for C. cinnamomeus (Tedersoo et al. 2022). Previous studies reported broad intercontintenal species distributions in Crepidotus based on morphology or phylogenetic analyses. Phylogenetic studies reporting Crepidotus species from different continents were based only on ribosomal DNA regions (ITS and LSU), and either they lacked sequences from continents from where reported species were described (Kumar et al. 2020; Na et al. 2022), or they resulted in an inconclusive outcome (Kasuya et al. 2014). Glacial dynamics have driven widely distributed species of temperate, boreal and arctic environments into isolated refugia with subsequent contact, which resulted in complex genetic structure of geographically distant populations with specific ITS variability. This ITS variability is sometimes non-concordant with other loci in the fungal genome. Multi-locus phylogenetic analysis that include protein-coding regions may help to elucidate differences within and between species in ITS region (Kauserud 2023). The phylogenetic study of Inocybaceae (sister family to Crepidotaceae) confirmed existence of relatively high number of species distributed in Europe and North America (Matheny et al. 2020). This, together with our study, suggests that wide species distributions across the holarctic region of the Northern Hemisphere may be a common distribution pattern also in Crepidotus.

The redescription of C. cinnamomeus is only the first step towards to understand species delimitation, richness and nomenclature of North American species. As the result of technological tools available at the time when the monograph of the genus was published (Hesler and Smith 1965), species diagnoses were brief, which led to incorrect estimation of species richness. For example, in case of studies on North American Hebeloma species described by Hesler and Smith during the 1970s and 1980s, recent studies revealed a high species overestimation (Eberhardt et al. 2023). The holarctic distribution of species described in this study suggests that the nomenclatural assignment in Crepidotus will be even more difficult task because names from other continents must be also considered.

From available material used in this study it seems that Crepidotus cinnamomeus prefers humid habitats such as riparian Salix shrub or riparian Alnus forest. While twigs and branches of Betula, Salix and Rubus were identified as probable substrates, woody remnants of other plants were present at collecting sites, including Populus and Alnus are potential substrates. The substrate varied from 2 mm thick twigs to 15 mm thick branches. The elevation ranged from 160 m in Sweden to 1170 m in Spanish Pyrenees. We think that the absence of the species in warmer temperate and Mediterranean areas suggest that its occurrence is rather limited by a preference of a cooler climate.