Advances in the knowledge of the Inocybe mixtilis group (Inocybaceae, Agaricomycetes), through molecular and morphological studies.

Inocybe mixtilis constitutes a complex of species characterized by nodulose-angulose spores, absence of cortina and a more or less bulbous marginate stipe that is not darkening when desiccated. In order to elucidate species limits within the I. mixtilis complex, an ITS-RPB2 phylogeny was performed and interpreted using morphological and ecological characters. Six supported clades were obtained in our analyses that correspond to I. mixtilis, I. subtrivialis, and four new species to science: I. ceskae, I. johannis-stanglii, I. nothomixtilis and I. occulta. Species within this complex can be morphologically recognized through a unique combination of morphological characters, such as the spore shape, cystidial length and shape, presence and development of the velipellis and pileus colour and viscidity. Nevertheless, those characters overlap, especially among I. mixtilis, I. ceskae and I. occulta, and intermediate collections are therefore more reliably identified through ITS-sequencing. Two species, I. ceskae and I. occulta are present in both North America and Europe, while the rest are so far only known in Europe, or Europe and Asia (I. mixtilis). All species, except I. johannis-stanglii, seem to be able to establish ectomycorrhizal association both with conifers and angiosperms. Descriptions, colour illustrations and a key to all known species in the I. mixtilis group are provided.

INTRODUCTION

Inocybe (Agaricales, Basidiomycota) is one of the most diverse genera of fungi, with around 500 (Kirk et al. 2011) or even considerably more (Matheny 2009) species worldwide and is also one of the most common and abundant in ectomycorrhizal communities. For this reason, Inocybe is one of the most cited genera in mycological and ecological studies of ectomycorrhizal communities and also with a large number of unidentified or misidentified sequences in GenBank (Ryberg et al. 2008). This is due to serious taxonomic and nomenclatural problems that persist in Inocybe, mainly due to the existence of cryptic or semi-cryptic species that can be separated by molecular data, or subtle morphological, ecological or chorological differences. On the other hand, there are many species yet to be described in Europe, especially in the Mediterranean region (Esteve-Raventós et al. 2016).

Molecular data have importantly contributed to infer phylogenetic relationships and to disentangle targeted species complexes within Inocybe. Multigene phylogenetic analyses have shown that Inocybe nests in the Agaricoid clade and is classified in the family Inocybaceae by some authors (Matheny et al. 2006), or in Crepidotaceae when Inocybaceae is merged with its sister family (Petersen et al. 2010). In a study assessing traditional taxonomic schemas through multilocus molecular data, Matheny (2009) discussed phylogenetic relationships within the whole genus. This author placed the genus Inocybe into the more restricted family concept of Inocybaceae. Other approaches for the study of the Inocybaceae were based on other genomic regions like RPB1 and RPB2 (Matheny et al. 2002, Matheny 2005), and even on analysing large sets of unidentified ITS sequences assigned to Inocybe and deposited in public databases (Ryberg et al. 2008). Further studies employing also molecular data have addressed related genera like Auritella (Matheny & Bougher 2006), subgenera like Mallocybe (Cripps et al. 2010) or sections like Marginatae (Kropp et al. 2010, Esteve-Raventós et al. 2016) or Rimosae (Larsson et al. 2009). At species level, several studies have integrated distributional and/or morphological data together with the ITS region to disentangle species complexes and to describe new species of Inocybe (e.g., Cripps et al. 2010, Larsson et al. 2014, 2017, Esteve-Raventós et al. 2016, La Rosa et al. 2017, Matheny & Bougher 2017). This integrative approach has proved useful to unravel species complexes.

This study targets a species complex that is in need of revision, the I. mixtilis group. Inocybe mixtilis, described by Britzelmayr (1885) and consensually placed in section Marginatae (Kühner 1933), is characterized by its nodulose to angular basidiospores, lacking a cortina, and a more or less bulbous marginate stipe which is not darkening when desiccated. The epitype material proposed for I. mixtilis by Marchetti et al. (2014) allows for a more accurate phylogenetic placement and a stable interpretation of I. mixtilis. Examination and sequencing of further material by us suggest that I. mixtilis is actually a species complex containing several undescribed species. Thus, the aim of this work is to revise the I. mixtilis complex based on sequence data of two genetic markers (ITS and RPB2) and morphological data and to contribute to a better understanding of the genus Inocybe.

MATERIALS AND METHODS

Morphological study

Specimens studied were collected across Europe and were mostly deposited in KR (Karlsruhe, Germany), AH (Alcalá de Henares, Spain) and ARAN herbaria (Aranzadi Science Society). Additional collections were loaned from the Johann Stangl (M; Munich, Germany), Jukka Vauras (TUR-A; Turku, Finland) and Guillaume Eyssartier (PC; Paris, France) herbaria, while other collections were loaned from private herbaria: Erminio Ferrari (EF; Italy), Roberto Fernández-Sasia (RFS; Bizkaia, Spain), Jordi Vila García (JVG; Barcelona, Spain) and Agustín Caballero Moreno (AC; La Rioja, Spain).

Microscopical features were described from mountings in water and 3–5 % ammonium hydroxide solution and observed by light microscopy with the aid of an oil immersion objective. Photomicrographs were taken with a Nikon (Eclipse 80i) microscope and a digital camera Nikon (DS-5 M) and (in the case of D. Bandini) with a Leica DM-750-microscope and a Zeiss AxioCam ERc5s. Scanning electron micrographs were obtained using a Zeiss DSM-950. For ultramicroscopic studies, the samples were rehydrated in concentrated ammonium hydroxide (28–30 %) for 30 min, dehydrated in aqueous ethanol (70 %) for 30 min, fixed for 2 hours in pure ethylene glycol dimethylether (= 1.2-dimethoxymethane), and finally immersed in pure acetone for at least 2 hours. This was followed by critical point drying and sputtering with gold-palladium. This technique allows the use of very little material (small portions of lamellae).

Colours of basidiomata, either in fresh or dry condition, were standardized following Munsell. Most of the terminology used for microscopic features follows Kuyper (1986) and Vellinga (1988). Lm, Wm and Qm refer to the mean spore length, width and ratio, respectively. Herbarium acronyms follow Thiers (2012). General bibliography, monographs or contributions to specific groups of Inocybe were used in the determination of the samples, i.e., Kauffman (1924), Heim (1931), Malençon & Bertault (1970), Stuntz (1978), Alessio & Rebaudengo (1980), Kuyper (1986), Nishida (1989), Stangl (1989), Bon (1998), Kobayashi (2002), Ferrari (2006, 2010), Jacobsson & Larsson (2012), Matheny (2016) and Bandini (2017).

DNA extraction, amplification and sequencing

Total DNA from BAN (Bandini) samples was extracted from dry herbarium specimens employing a modified protocol based on Murray & Thompson (1980). A portion of each sample was blended with the aid of a micropestle in 600 μL CTAB buffer (CTAB 2 %, NaCl 1.4 M, EDTA pH 8.0 20 mM, Tris-HCl pH 8.0 100 mM). The resulting mixture was incubated for 15 min. at 65 °C. A similar volume of chloroform : isoamylalcohol (24 : 1) was added and carefully mixed with the samples until their emulsion. Samples were then centrifuged for 10 min at 13 000 g, and the DNA in the supernatant was precipitated with a volume of isopropanol. After a new centrifugation of 15 min at the same speed, the pellet was washed in cold ethanol 70 %, centrifuged again for 2 min and dried. Genomic DNA was finally resuspended in 200 μL ddH2O.

For the rest of the specimens, genomic DNA was extracted from dried herbarium specimens using the Prepman Ultra Sample Preparation Reagent (Thermo Scientific, USA). One hundred μL of extraction solution were added to 1.5 ml Eppendorf tubes containing small portions of lamellae and context and vortexed for 1 min. These tubes were incubated for 10 min at 100 °C in a heated block and then centrifuged at 12 000 rpm for 2 min. Fifty μL of the supernatant were transferred to a new tube and stored at –20 °C. DNA concentration was determined using a NanoDrop ND-1.000 spectrophotometer (Thermo Scientific, USA).

We sequenced two regions: the nuclear ITS1-5.8S-ITS2 region using the primers ITS1F-ITS4 (White et al. 1990) and the RNA polymerase II subunit (RPB2) between primers RPB2-6F-bRPB2-7R for the RPB2 region (Matheny 2005). PCR reactions were performed in 25 μL per reaction under a program consisting of a hot start at 95 °C for 5 (6 in the case of Spanish isolates) min, followed by 35 cycles at 94 °C, 54 °C and 72 °C (45, 30 and 45 s, respectively) and a final 72 °C step 10 min. PCR products were checked in 1 % agarose gels, and positive reactions sequenced in both directions. Sequencing primers were the same as employed for PCR amplification. Raw sequence data was manually edited using Chromas 2.5.1 software (Technelysium Pty Ltd., Southport, Queensland, Australia). The identity of obtained consensus sequences was verified through the BLASTn query algorithm in GenBank (http://www.ncbi.nlm.nih.gov) and UNITE (https://unite.ut.ee/) databases.

Phylogenetic analyses

Two genomic regions were sequenced for phylogenetic reconstruction and two alignments were thus assembled. A first ITS-RPB2 alignment aimed to test species boundaries and the monophyly of the Inocybe mixtilis group using representative sequences of each species. This dataset comprised 1 410 characters divided into two partitions (ITS, 707 characters; RPB2, 703) and 22 taxa. Inocybe calopedes (KJ778839, KJ756415) and I. subfibrosoides (KP636879, KM656117) were selected as outgroup because sequences of both markers were available for it in GenBank and blast searches indicated similarity to sequences of the I. mixtilis group. Gene congruence was evaluated manually by comparing supported clades among single-gene genealogies (Mason-Gamer & Kellogg 1996). Each locus was subjected to a Maximum Likelihood (ML) analysis as described below. A supported clade for one marker was considered to be in conflict when contradicted with significant support by another (bootstrap support > 70 %). As no conflict was detected, the ITS and RPB2 regions were concatenated. A second ITS alignment was constructed to place specimens sequenced only for the ITS region. This alignment comprised 771 characters and 120 sequences, of which 67 were generated by us, while the rest were downloaded from GenBank and UNITE databases (Table 1). Sequences were aligned in MUSCLE software (Edgar 2004) of MEGA6 (Tamura et al. 2013) and MAFFT (http://mafft.cbrc.jp/alignment/server/; Katoh et al. 2005) and adjusted manually. An ITS sequence of Crepidotus calolepis was used as outgroup. Ambiguously aligned positions and sequence ends were trimmed at both ends.

Table 1

ITS sequences of the taxa employed in the study, indicating present and original identifications, geographical origin, ecology, voucher numbers, GenBank and UNITE accession numbers. Bold entries refer to newly generated sequences.

Identification (I. = Inocybe)	Original identification	Geographical origin	Ecology	Voucher specimen	GenBank/UNITE accession number
					ITS	RPB2
Crepidotus calolepis	–	Hungary, Szabolcs-Szatmar-Bereg	Unknown	WU 28902/CR04A	KF879617	–
I. ceskae	–	Finland, Koillismaa	Betula sp., Picea abies, Pinus sylvestris	TUR-A 203414 (holotype)	KX290789	MH496023
	–	Finland, Koillismaa	Betula sp., Picea abies, Pinus sylvestris	KR-M-0042321	KY393432	–
	I. mixtilis	Canada, British Columbia	Unknown	UBC F18984	HQ604481	–
	I. mixtilis	Canada, British Columbia	Pseudotsuga menziesii	UBC F19182	HQ604482	–
	I. mixtilis	Canada, British Columbia	Unknown	UBC F19107	HQ604478	–
	I. sindonia	Canada, British Columbia	Unknown	UBC F19221	HQ604484	–
	I. mixtilis	Canada, British Columbia	Arbutus menziesii, Pseudotsuga menziesii	UBC F19202	HQ604483	–
	I. mixtilis	Canada, British Columbia	Unknown	UBC F18974	HQ604478	–
	I. mixtilis	Canada, British Columbia	Unknown	UBC F19079	HQ604479	–
	I. umbratica	Canada, British Columbia	Unknown	UBC F18954	HQ604477	–
	I. praetervisa	Canada, British Columbia	Unknown	UBC F19237	HQ604486	–
	I. mixtilis	USA, Oregon	Pseudotsuga menziesii	OSC 1064046	EU525952	–
	I. mixtilis	Canada, British Columbia	In mixed forest with Arbutus menziesii	UBC F19412	HQ604487	–
	I. glabrodisca	Canada, British Columbia	Unknown	UBC F19553	HQ604480	–
	I. mixtilis	Canada, British Columbia	Unknown	UBC F18974	HQ604485	–
I. flavobrunnescens	–	Portugal, Estremadura	Quercus faginea	AH 29883 (holotype)	KJ938784	–
I. hirculus	–	Finland, Varsinais-Suomi	Betula pubescens, Picea abies, Pinus sylvestris, Salix sp.	TUR-A 2577/ K51 (holotype)	FJ531872	–
I. johannis-stanglii	I. pallida	Germany, Baden-Wurttemberg	Betula pendula, Picea omorica (holotype)	KR-M-0038039	KX290791	MH496018
	–	Austria, Oberosterreich	Abies alba, Larix decidua, Picea abies	KR-M-0043320	KX290792	–
	–	Austria, Oberosterreich	Abies alba, Larix decidua, Picea abies	KR-M-0043321	KX290793	MH496019
	I. pallida	Germany, Bayern	Betula sp., Picea omorika	M-0021483	KX290835	–
I. krieglsteineri	–	Spain, Andalucia	Pinus pinea	AH 44479	KJ938770	–
I. mixtilis	–	Germany, Bavaria, Horbach	Picea abies	M-0219661 (epitype)	KM873369	–
	I. viscossisima var. bulbosovelata	Italy, Piemonte	Pinus strobus	EF 45/2000 (paratype of I. viscossisima var. bulbosovelata)	KX290790	MH496026
	–	Finland, Etela-Hame	Betula sp., Picea abies, Pinus sylvestris, Salix sp.	TUR-A 145111	KX290839	–
	–	Finland, Etela-Hame	Pinus sylvestris, Quercus robur, Tilia sp.	TUR-A 182874	KX290841	–
	I. tabacina	Germany, Schleswig-Holstein	Alnus sp., Betula sp.	M-0022249	KX290836	–
	I. cf. tabacina	Germany, Baden-Wurttemberg	In forest	M-0022251	KX290837	–
	–	Finland, Varsinais-Suomi	Picea abies	TUR-A 199995	KX290842	–
	–	Germany, Sachsen-Anhalt	Picea abies	KR-M-0043295	KX290794	–
	–	Germany, Sachsen-Anhalt	Picea abies	KR-M-0043296	KX290795	–
	–	Spain, Canary Islands	Pinus canariensis	KR-M-0043297	KX290796	–
	–	Germany, Rheinland-Pfalz	Quercus robur	KR-M-0043298	KX290797	–
	–	Germany, Baden-Wurttemberg	Pinus sylvestris	KR-M-0043299	KX290798	–
	–	Germany, Baden-Wurttemberg	Betula pendula, Pinus mugo, Salix caprea	KR-M-0043300	KX290799	–
	–	Austria, Oberosterreich	Abies alba, Larix decidua, Picea abies	KR-M-0043301	KX290800	–
	–	Germany, Baden-Wurttemberg	Picea abies	KR-M-0043302	KX290801	–
	–	Germany, Baden-Wurttemberg	Picea abies	KR-M-0043303	KX290802	–
	–	Germany, Rheinland-Pfalz	Alnus glutinosa, Corylus avellana, Pinus sylvestris, Quercus robur	KR-M-0043304	KX290803	MH496021
	–	Germany, Rheinland-Pfalz	Picea abies	KR-M-0043305	KX290804	–
	–	Germany, Rheinland-Pfalz	Picea abies	KR-M-0043306	KX290805	–
	–	Austria, Salzburg, Tamsweg	Alnus incana, Picea abies	KR-M-0043307	KX290806	MH496020
	–	Germany, Bayern	Picea abies	KR-M-0043308	KX290807	–
	–	Germany, Baden-Wurttemberg	Picea abies	KR-M-0043309	KX290808	–
	–	Germany, Baden-Wurttemberg	Picea abies	KR-M-0043310	KX290809	–
	–	Germany, Baden-Wurttemberg	Picea abies	KR-M-0043311	KX290810	–
	–	Germany, Baden-Wurttemberg	Picea abies	KR-M-0043325	KX290811	–
	–	Germany, Hessen	Pinus sylvestris, Quercus robur	KR-M-0043326	KX290812	–
	–	Spain, Basque Country	Picea abies, Fagus	ARAN-Fungi 04711	MH500842	MH496022
	–	France, Nord,Cyzoing	Pseudotsuga menziesii	LIP PAM05103003	HQ586870	–
	–	Italy	Unknown	MCVE 21534	JF908218	–
	–	Sweden	Unknown	EL13104	AM882838	–
	–	Sweden	Unknown	EL5904	AM882839	–
	–	Sweden	Unknown	EL2604	AM882837	–
	–	Sweden	Unknown	EL9604	AM882835	–
	–	Denmark	Unknown	EL16107	FN550894	–
	–	Estonia, Polva	Unknown	TU118169	UDB015355	–
	–	Estonia, Hiiu	Unknown	TU118144	UDB015338	–
	–	Italy	Unknown	MCVE 13860	JF908151	–
I. nothomixtilis	–	Spain, Basque Country	Pinus radiata, Quercus robur	AH 24510 (holotype)	KX290780	–
	–	Spain, Balearic Islands, Mallorca	Cistus monspeliensis, Pinus halepensis	AH 46558	KX290786	MH496025
	–	Spain, Cataluña	Cistus monspeliensis, C. salviifolius	AH 26921	KX290784	–
	I. tabacina var. pseudovolvata ad int.	France, Île-de-France	Carpinus betulus, Fagus sylvatica, Quercus sp.	PC 0738072	KX290838	–
	I. mixtilis var. aurata nom. inval.	Italy	Unknown	Herb. Alessio, without number	KX290813	–
	–	Italy, Toscana	Quercus sp.	KR-M-0043322	KX290814	–
	–	Germany,Baden-Württemberg	Betula pendula, Fagus sylvatica, Quercus petraea	KR-M-0038133	KX290815	–
	–	Germany, Hessen	Pinus sylvestris, Quercus rubra	KR-M-0043319	KX290816	–
	I. obtusiuscula	–	France, Savoie	Salix herbacea	LIP PAM02081710	HQ586869	–
I. ochracea	–	Germany, Bavaria, Augsburg	Alnus incana, Fagus sylvatica, Salix sp.	M Stangl 88/K65 (holotype)	FJ755803	–
I. occulta	I. mixtilis	Spain, La Rioja	Quercus ilex subsp. ballota	AH 36443 (holotype)	KX290787	MH496017
	–	Spain, Castilla-Leon	Pinus pinea	AH 30697	KX290785	–
	–	Spain, Andalucia	Quercus suber	AH 22108	KX290779	–
	–	Spain, Castilla-La Mancha	Cistus ladanifer, Pinus pinaster	AH 40499	KX290788	–
	–	Spain, Castilla-Leon	Quercus robur	AH 24987	KX290781	MH496016
	I. mixtilis	Spain, Cataluña	Salix pyrenaica	AH 21576	KX257359	–
	–	Finland, Enontekion Lappi	Betula pubescens subsp. czerepanovii	TUR-A 145165	KX290840	–
	–	Spain, Castilla-Leon	Fagus sylvatica	AH 24989	KX290782	–
	I. mixtilis	Spain, Cataluña	Dryas octopetala, Salix retusa	AH 26731	KX290783	MH496015
	–	Germany, Bayern	Larix decidua, Picea abies	KR-M-0038028	KX290817	–
	–	Germany, Rheinland-Pfalz	Picea abies	KR-M-0038125	KX290818	–
	–	Germany, Baden-Wurttemberg	Helianthemum nummularium, Pinus sylvestris, Quercus robur	KR-M-0043312	KX290819	–
	–	Austria, Tirol	Picea abies	KR-M-0043313	KX290820	–
	–	Germany, Bayern	Larix decidua, Pinus mugo	KR-M-0043314	KX290821	–
	–	Austria, Kärnten	Picea abies	KR-M-0043315	KX290822	–
	–	Austria, Salzburg, Tamsweg	Picea abies	KR-M-0043316	KX290823	–
	–	Germany, Baden-Wurttemberg	Picea abies	KR-M-0043323	KX290824	–
	–	Germany, Baden-Wurttemberg	Fagus sylvatica, Picea abies	KR-M-0043318	KX290825	–
	–	Germany, Baden-Wurttemberg	Abies procera	KR-M-0043317	KX290826	–
	–	Germany, Rheinland-Pfalz	Betula pendula, Larix decidua, Quercus robur	KR-M-0043230	KX290827	–
	–	Netherlands, Friesland	Pinus sylvestris	KR-M-0038029	KX290828	–
	–	Germany, Bayern	Betula pendula, Larix decidua, Pinus sylvestris	KR-M-0043324	KX290829	–
	–	Finland, Koillismaa	Betula sp., Picea abies, Pinus sylvestris	KR-M-0046554	KX290830	–
	–	Finland, Koillismaa	Betula sp., Picea abies, Pinus sylvestris	KR-M-0046555	KX290831	–
	–	Finland, Koillismaa	Betula sp., Picea abies, Pinus sylvestris	KR-M-0046558	KX290832	–
	–	Finland, Koillismaa	Betula sp., Picea abies, Pinus sylvestris	KR-M-0046557	KX290833	–
	–	Finland, Koillismaa	Betula sp., Picea abies, Pinus sylvestris	KR-M-0046556	KX290834	–
	I. mixtilis	Estonia, Saare	Unknown	TU120084	UDB023622	–
	I. praetervisa	Canada, British Columbia	Unknown	UBC F19174	HQ604492	–
	I. jacobi	Canada, British Columbia	Mixed forest with Arbutus menziesii and Dodecatheon hendersonii	UBC F19302	HQ604491	–
	I. mixtilis	Australia, Australian Capital Territory	Pinus sp., Ulmus sp.	CANB 574539	KP308781	–
	I. mixtilis	USA, Oregon	Pseudotsuga menziesii	OSC 1064111	EU525968	–
	I. mixtilis	USA, Oregon	Pseudotsuga menziesii	OSC 1064112	EU525941	–
	I. mixtilis	USA, Oregon	Pseudotsuga menziesii	OSC 1064115	EU525969	–
	I. mixtilis	Italy	Unknown	MCVE 5052	JF908121	–
	I. mixtilis	France, Pas-de-Calais	Betula sp., Pinus pinaster, Populus tremula	LIP PAM 07110104	HQ586872	–
	I. mixtilis	Sweden	Unknown	BJ920916	AM882840	–
	I. mixtilis	Canada, British Columbia	Unknown	UBC F19341	HQ604490	–
	I. mixtilis	Canada, British Columbia	Unknown	UBC F19030	HQ604488	–
	I. mixtilis	Italy	Unknown	MCVE 91	JF908089	–
	I. mixtilis	Mexico, Morelos	Unknown	GO-2010-169	KC152132	–
	I. xanthomelas	Italy	Unknown	MCVE 6549	JF908127	–
	I. mixtilis	Italy	Unknown	MCVE 9844	JF908141.2	–
I. phaeocystidiosa	–	Spain, Segovia	Pinus sylvestis	AH 9154 (holotype)	KT203789	–
I. praetervisa	–	Italy, Rabbi	In coniferous wood	SF229598 (epitype)	KT203792	–
I. praetervisoides	–	Spain, Madrid	Quercus ilex subsp. ballota	AH 29863 (holotype)	KT203794	–
I. saliceticola	–	Finland, Pohjois-Karjala	Alnus incana, Betula sp., Salix sp .	TUR-A 19738F/ K11 (holotype)	FJ755796	–
I. subtrivialis	–	Spain, Castilla-La Mancha	Quercus ilex, Cistus	AH 19225 (holotype)	MH496013 / MH496014	–
–	Spain, Madrid	Cistus ladanifer, Pinus pinaster	AH 26789	KX354977	MH496024
I. tabacina	–	France, Wannehin	Populus nigra	LIP PAM05071302	HQ586865	–
I. urbana	–	Italy, Torino	Tilia sp.	AMB 17142 (holotype)	KT229613	–
I. xanthomelas	–	Spain, Madrid	Betula pendula	AH 29895	KJ938774	–

Bayesian inference of phylogenetic relationships was performed using MrBayes v. 3.2.3 (Ronquist et al. 2012), applying a Gamma+P-Imvar model of rate heterogeneity and a GTR substitution model. Markov Monte Carlo Chains (MCMC) were run for 2 M generations; two independent runs with four chains were performed for trees sampled every 1 000 steps, setting the heating temperature as 0.05. Burnin was set at 25 %. ML phylogenetic analysis was conducted using the dataset alignment with the E-INS-i strategy of MAFFT, employing default settings in RAxML v. 8.2.9 (Stamatakis 2006), via CIPRES platform (https://www.phylo.org/; Miller et al. 2010). To assess branch confidence 1 000 non-parametric bootstraps were performed.

RESULTS

The 50 % majority-rule consensus phylogenetic tree obtained in the Bayesian analysis of the ITS-RPB2 alignment is shown in Fig. 1. The inferred tree was fully supported in both analyses of this alignment. The I. mixtilis group is monophyletic (PP 1; ML-BP 100 %), being I. glabrodisca sister to it. Five of the species attributed here to the I. mixtilis group received strong support, namely I. johannis-stanglii (PP 1; ML-BP 100 %), I. mixtilis (PP 1; ML-BP 100 %), I. nothomixtilis (PP 1; ML-BP 100 %), I. occulta (PP 1; ML-BP 100 %) and I. subtrivialis (PP 1; ML-BP 100 %), while I. ceskae was only supported in the Bayesian analysis (PP 0.99; ML-BP 59 %), I. johannis-stanglii and I. subtrivialis form a strongly supported clade that is basal to the rest of the species of the I. mixtilis group (PP 0.98; ML-BP 99 %).

Fig. 1

The Bayesian 50 % majority-rule consensus tree inferred from ITS-RPB2 regions. Numbers above and below nodes represent Bayesian posterior probabilities and ML bootstrap values, respectively. Only nodes significantly supported in one of the analyses bear values. Sequences of Inocybe stellata were used as outgroup. Coloured boxes represent different taxa recognized for the Inocybe mixtilis species complex.

The 50 % majority-rule consensus tree of the Bayesian analysis of the ITS region shows a similar topology, but I. subtrivialis and I. johannis-stanglii do not form a monophyletic clade (Fig. 2). The six species of the I. mixtilis group receive support in at least the Bayesian analysis. Intraspecific sequence variability was extremely low for all treated taxa.

Fig. 2

The Bayesian 50 % majority-rule consensus tree inferred from ITS region. Numbers above and below nodes represent Bayesian posterior probabilities and ML bootstrap values, respectively. Only nodes significantly supported in one of the analyses bear values. Phylogram was rooted with an ITS sequence of Crepidotus calolepis. Coloured boxes represent different taxa recognized for the Inocybe mixtilis species complex together with the I. xanthomelas / phaeocystidiosa group. Sequences are coloured according to their geographical origin: Europe (black), North America (blue), Australia (red), Canary Islands (green). Host associations are given for each collection when known.

Each of the clades could be morphologically characterized based on a unique combination of several characters, such as the spore shape, the cystidial length, the pileus colour and viscidity, and the presence and development of the velipellis. Nevertheless, these characters were overlapping. Ecology and host specificity showed some patterns (Fig 2). While I. ceskae and I. subtrivialis are restricted to taiga forests and Mediterranean forests, respectively, other species like I. mixtilis exhibited a broader ecological amplitude and associates with both conifers and angiosperms. Likewise, a species seems to be restricted to Europe (I. mixtilis), while I. occulta and I. ceskae have a transoceanic distribution and were also present in North America.

TAXONOMY

(Britzelm.) Sacc., Syll. Fung. 5: 780. 1887. —Fig. 3, 4

Fig. 3

Inocybe mixtilis KR-M-0043296: microscopic features. Sp = spores, Pa = paracystidia, Cpa = cauloparacystidia, Ch = cheilocystidia, Pl = pleurocystidia, Ca = caulocystidia. — Drawn by Ditte Bandini.

Fig. 4

Inocybe mixtilis. a. M-0219661 (epitype designated by Marchetti et al. 2014); b. KR-M-0043296, basidiomata in situ; c–h. hymenial cystidia; i. spores under optical microscope; j. spores under SEM (all microphotos from KR-M-0043296). — Scale bars: a–b = 1 cm; c–i = 10 μm; j = 2 μm.

≡ Agaricus mixtilis Britzelm., Ber. Naturhist. Vereins Augsburg 28: 152, 1885. MycoBank number MB141839.

Lectotypus. Britzelmayr (1882), Dermini aus Südbayern: f. 21, sub nomen Agaricus rimosus, designated by Marchetti et al. (2014:149). Epitypus: Germany, Bayern, Hörbach, Haspelmoor, Landkreis Fürstenfeldbruck, Fichtenwald (Picea forest), auf Nadelstreuhumus wachsend, gesellig, 26 Sept. 1981, leg. J. Stangl, M-0219661, designated by Marchetti et al. (2014: 149; previously as neotype by Stangl 1984: 332). ITS barcode GenBank KM873369.

Selected descriptions and iconography — Stangl (1984), Marchetti et al. (2014: 149).

Basidiomes often gregarious, rarely solitary. Pileus 10–35(–45) mm diam, 5–20 mm high; shape when young subconical, paraboloid or campanulate, then hemispherical, broadly convex to expanded, often showing a broad distinct umbo since young, usually depressed around umbo in mature specimens and uplifted towards the margin; margin slightly inflexed when young, later somewhat deflexed to straight, initially even, often becoming somewhat undulate, irregular or torn in old specimens, not striate, rarely subhygrophanous (e.g., KR-M-0043299), occasionally crenate to appendiculate in young specimens by fugacious velipellis remnants; colour somewhat variable, but most often golden yellow or shiny yellow-ochre with orange, copper or brownish hue (Mu 10YR 8/8, 7.5YR 8/6, 7/6–7/8, 6/6–6/8 ), somewhat darker in centre (Mu 5YR 6/6; 7.5YR 6/6), but in some collections also uniformly brownish, mostly with faint orange or reddish tinge (Mu 5YR 6/4–6/6; 10YR 6/4–6/6), sometimes with darker umbo; surface entirely glabrous and smooth, very often with a lardy or greasy appearance, very finely radially fibrillose to slightly rimose, especially at the margin, where the paler flesh underneath is sometimes exposed; in a few collections (e.g., KR-M-0043325) the surface may also appear minutely verrucose or even cracked around the umbo (KR-M-0043306), or very finely felty (KR-M-0043310); velipellis apparently absent or, if present, fugacious and only visible in very young fruitbodies, leaving delicate appendiculate whitish remnants on the pileus margin (KR-M-0043301), or very unusually (as commented by Ferrari 2006 for I. viscosissima var. bulbosovelata), covered by a thin velar patch (‘bianco-grigiognolo’, greyish white); surface distinctly lubricous and sticky in wet conditions and hence agglutinating soil particles or humus remains. Lamellae subventricose, but almost linear in some collections (e.g., KR-M-0043302), 2–5 mm deep, almost free to adnexed, sometimes also adnate with a decurrent tooth, usually rather crowded, L = 50–60(–70), l = 1–2(–3); colour initially white or whitish, then greyish, quite often showing a faint violet hue, brownish grey, sometimes pale rusty-brown or pinkish rusty-brown when old; edge even to crenulate, whitish or paler than faces, but in old specimens sometimes concolorous or even somewhat darker. Stipe 15–50(–60) × (2–)3–5(–6) mm, solid, fibrose; shape cylindrical or tapering upwards, sometimes more or less curved near the base, which is always distinctly marginately bulbous; bulb 7–12 mm broad, edge mostly sharp or even volviform, occasionally showing a fibrillose rim from velipellis reminiscent of a ‘pseudovolva’; colour pure white, whitish to beige or pale straw to pale ochraceous, more rarely lemon yellow (KR-M-0043301, Mu 10YR 8/6), never darkening; surface evenly and densely pruinose all over. Cortina not seen, even in young primordia. Context firm, fibrose, whitish in pileus, whitish or beige to very pale straw in stipe, whitish in the bulb; smell faint, acidulous or more or less spermatic, especially when cut; taste not recorded. Colour of exsiccata: pileus generally uniformly brownish (Mu 10YR 4/3–4/6 or 6/6–6/8), also reddish brown (Mu 7.5YR 4/4–4/6); stipe concolorous or somewhat lighter than pileus (Mu 10YR 5/6–5/8), never showing any distinct darkening (either browning or blackening) upon drying.

Basidiospores (6.7–)7.1–9.8(–10.5) × (5.1–)5.2–7.9(–8.1) μm, Q = (1–)1.1–1.4(–1.5), Lm = 8.2–9, Wm = 6.1–6.7, Qm = 1.3–1.4 [n = 100, from 4 collections], mostly isodiametric to subisodiametric, more rarely subheterodiametric, with nodulose profile which is often ‘margaritiform’ or ‘pseudoasteriform’ (star-like arrangement), usually presenting quite distinct (7–)9–13(–16), obtuse to subconical knobs, (0.75–)1–1.5(–2) μm high, (1.2–)1.4–1.7(–2) μm broad at the base; apicula distinct, often curved; yellow in ammonia. Basidia 20–32 × 8–11.5 μm, clavate, (2–)4-spored, with sterigmata 3–5(–7) μm long. Pleurocystidia abundant, metuloid, (38–)40–48–66(–70) × (12–)14–18–25(–26) μm, Q = (1.8–)1.9–2.6–3.4(–3.5) [n = 40], variable in shape among collections, mostly sublageniform, subfusiform to subutriform, often ventricose and extending upwards into a defined neck (see Marchetti et al. 2014: 151), base rounded, not pedicellate or with short rounded pedicel; content hyaline or rarely with orange-yellowish, or yellow greenish amorphous matter; mostly crystalliferous at apex; walls (1–)1.5 μm thick (at the bulge), –3.5(–5) μm thick (at the apex), sometimes coalescing along the neck, yellowish in ammonia. Lamellar edge homogeneous and sterile, composed of abundant cheilocystidia and numerous clavate to subovoid paracystidia. Cheilocystidia abundant, similar in shape and size to pleurocystidia (35–)38–50–65(–67) × (12–)13–17–21(–26) μm [n = 40], with similar thick walls and crystalliferous apex. Hymenophoral trama regular to subregular, consisting of parallel to interwoven hyphae, 4–15(–20) μm wide, cylindrical to subfusiform in shape, often constricted at the septa, hyaline. Subhymenium poorly developed, consisting of 2–3 layers of small, subisodiametric, irregular cells, 4–6 μm diam. Pileipellis constituted by an ixocutis, made up of parallel hyphae, 3–5 μm wide, with finely encrusting and parietal golden to orange yellowish pigment; subcutis with wider and paler elements, up to 20 μm broad; epicutis in young fruitbodies sometimes covered by thin hyaline hyphae, with scattered free ends (belonging to velipellis remains). Stipitipellis consisting of a cutis bearing numerous bundles of caulocystidia similar to hymenial cystidia, sometimes with tapering neck, (35–)42–49–64(–65) × (13–)14–19–27(–33) μm [n = 40], mostly sublageniform, subfusiform or subutriform, rarely narrowly clavate or nearly cylindrical, often crystalliferous at apex, intermixed with extremely abundant globose, ovoid to subclavate paracystidia; both elements forming the dense and conspicuous pruina that extends to the bulbous stipe base. Clamp-connections present in all tissues.

Habitat & Distribution — Inocybe mixtilis has a broad ecological range, but shows a marked preference for acid soils, sometimes with a sandy or gravelly texture, being also rather frequent in boggy or peaty soils in forests, where the epitype was encountered. It can be often found in nitrified locations, such as parks, roadsides, cemeteries or gardens in urban areas and also conifer reforestations (e.g., in northern France under Pseudotsuga menziesii, GenBank HQ586870, leg. & det. P.A. Moreau). Most collections are associated with pure stands of coniferous trees, especially in Picea abies forests, but also with Pinus (P. mugo, P. pinea, P. sylvestris). One collection from Germany grew under Quercus robur, and several included in the UNITE database (Kõljalg et al. 2013) are associated with broadleaf trees. All information available suggests that I. mixtilis shows a broad host range.

Although certainly mistaken for I. occulta and I. ceskae, I. mixtilis is widespread in Europe, but much less common in southern Europe. For instance, after extensive sampling in the Iberian Peninsula, only a specimen could be collected in an area of Eurosiberian affinity. Interestingly, the southernmost collection of I. mixtilis was made in La Palma (Canary Islands, Spain) under Pinus canariensis in laurisilva forest (Macaronesian biogeographical Region). Unlike I. occulta, I. mixtilis is unknown above the timberline in alpine areas, although its presence there is probable.

Up to date, I. mixtilis is unknown from North America. Current information indicates that deposited sequences from North American records of I. mixtilis correspond to I. ceskae and I. occulta, in spite that the name I. mixtilis appears commonly cited in the United States (Smith 1938, Grund & Stuntz 1980, 1983, Cripps 1997, Matheny 2005, Cripps & Horak 2008). Not recorded by Kobayashi (2002) from Asia, a sequence obtained from an ectomycorrhizal root, nearly identical to our sequences, suggests that I. mixtilis is present in Iran (UNITE UDB005301).

Additional specimens examined. Austria, Oberösterreich, Bezirk Braunau am Inn, Kobernausser Wald, near Schalchen, alt. 525 m, in acid boggy soil with Sphagnum in mixed forest with Picea abies, Abies alba and Larix decidua, 17 Aug. 2014, D. Bandini, J. Christan & L. Quecke (KR-M-0043301); Salzburg, Bezirk Tamsweg, Tamsweg, near Prebersee, alt. c. 1500 m, in boggy acid soil with Picea abies and Alnus incana, 14 Aug. 2013, H. Prelicz & B. Haberl (KR-M-0043307). – Finland, Etelä-Häme, Lammi, Biological Station, in park near Quercus robur, Tilia and Pinus sylvestris, 13 Aug. 2008, J. Vauras (TUR-A 182874; duplicate KR-M-0046560); Somero, Häntälä, W of the road to Kerkola, NW of Haapasaari farm house, former field planted with Pinus sylvestris, with scattered young trees of Picea abies, Betula, Salix, partly mossy and grassy, with abundant litter of Pinus, 11 Oct. 1995, J. Vauras (TUR-A 145111; duplicate KR-M-0046559); Varsinais-Suomi, Salo, Helisnummi, cemetery, S part, on lawn near Picea abies, 16 Sept. 2014, J. Vauras (TUR-A 199995; duplicate KR-M-0046561). – Germany, Baden-Württemberg, Alb-Donau-Kreis, Illerrieden, TK 7726, auf Wiese an Waldrand (grassy soil in forest), 10 Oct. 1978, M. Enderle, det. J. Stangl (as I. cf. tabacina) (M-0022251); Landkreis Calw, Neuweiler, Agenbach, TK 7217/4, alt. c. 950 m, under Picea abies, 19 July 2012, D. Bandini & B. Oertel (KR-M-0043302); Kreis, Freudenstadt, Baiersbronn, Nature Reserve ‘Wilder See - Hornisgrinde’, TK 7415/1, alt. 1031 m, acid soil of mixed forest with Pinus mugo, Salix caprea and Betula pendula, 12 Sept. 2014, D. Bandini (KR-M-0043300); Heidelberg, Königstuhl, TK 6618/1, alt. c. 460 m, acid soil with Picea abies, 25 Oct. 2014, D. Bandini, (KR-M-0043303); ibidem, alt. c. 460 m, in acid soil with Picea abies, 25 Sept. 2014, D. Bandini (KR-M-0043325); ibidem, at about 5 km distance from the former location, alt. c. 500 m, with Picea abies, 25 Oct. 2014, D. Bandini (KR-M-0043310); Rhein-Neckar-Kreis, Schönbrunn, TK 6519/4, alt. c. 360 m, boggy acid soil with Picea abies, 2 Nov. 2014, D. Bandini (KR-M-0043309); Rhein-Neckar-Kreis, Sandhausen, TK 6617/4, alt. c. 110 m, sandy soil with Pinus sylvestris, 21 Oct. 2012, D. Bandini & B. Oertel (KR-M-0043299); Bayern, Kreis Rottal-Inn, Julbach, TK 7743/2, alt. c. 360 m, with Picea abies, 15 Aug. 2014, leg. A. Hussong, KR-M-0043308; Schwarzwald-Baar-Kreis, Schonach im Schwarzwald, Rensberg, TK 7815/1, alt. c. 950 m, with Picea abies, 20 July 2012, D. Bandini & B. Oertel (KR-M-0043311); Hessen, Landkreis Bergstraße, Bürstadt, Waldfriedhof Lampertheim, TK 6317/3, alt. 95 m, in acid sandy soil under Pinus sylvestris and Quercus rubra, 26 Oct. 2014, D. Bandini (KR-M-0043326); Rheinland-Pfalz, Landkreis Bad-Kreuznach, Bad Sobernheim, Meddersheim, Dornberg, TK 6211/2, alt. c. 400 m, acid ground with Quercus robur, 2 Nov. 2013, D. Bandini & B. Oertel (KR-M-0043298); Rhein-Pfalz-Kreis, Böhl-Iggelheim, TK 6615/1, alt. c. 110 m, with Quercus robur, Pinus sylvestris, Corylus avellana and Alnus glutinosa, 27 Sept. 2014, D. Bandini (KR-M-0043304); Landkreis Trier-Saarburg, Kell am See, TK 6306/4, alt. c. 360 m, boggy acid soil with Sphagnum and Picea abies, 2 Oct. 2011, D. Bandini (KR-M-0043306); ibidem, at about 5 km from the former location, alt. c. 400 m, with Picea abies, 6 Oct. 2011, D. Bandini & B. Oertel (KR-M-0043305); Sachsen-Anhalt, Landkreis Harz, Nature Reserve ‘Kramershai’, TK 4229/4, alt. c. 600 m, boggy acid soil with Sphagnum and Picea abies, 1 Oct. 2012, D. Bandini & B. Oertel (KR-M-0043295); ibidem, alt. 680 m, boggy acid soil under Picea abies, 2 Oct. 2012, D. Bandini & B. Oertel (KR-M-0043296); Schleswig-Holstein, Lübeck, TK 2130, Friedhof bei Weide, Erle (cemetery with willows and alders), 26 Sept. 1981, H. Glowinski, det. J. Stangl (as I. tabacina) (M-0022249). – Italy, Piemonte, Novara, Fara Novarese, alt. c. 210 m, in clay soil under Pinus strobus, 9 Nov. 2000, E. Ferrari (EF45/2000, paratypus of I. viscosissima var. bulbosovelata, Ferrari 2006). – Spain, Basque country, Gipuzkoa, Amezketa, Ondarreko langa, alt. 595 m, under planted Picea abies and Fagus sylvatica on calcareous ground, 20 Oct. 2016, I. Olariaga (ARAN-Fungi 04711); Islas Canarias, La Palma, Breña Baja, Parque Natural de Cumbre Nueva, Montaña de la Venta, alt. c. 1450 m, in Pinus canariensis forest, 15 Dec. 2013, J. Christan (KR-M-0043297). – Switzerland, Graubünden, Ramosch, alt. c. 1300 m, in Picea abies forest, 14 Sept. 1995, F. Esteve-Raventós, E. Horak & G. Moreno (AH 40432).

Notes — Inocybe mixtilis is undoubtedly one of the most cited nodulose-spored species in the genus and has always been considered as a common well known representative of section Marginatae Kühner (1933). Sequence data from collections deposited as I. mixtilis in GenBank and UNITE are very numerous and reflect this fact, as well as the extensive iconography and descriptions available in literature (see Materials and Methods).

Stangl (1984) neotypified I. mixtilis using a collection from the Bavarian location where Britzelmayr first collected and described it, but this choice is not correct as Britzelmayr's original plate (sub A. rimosus) was available for lectotypification, as Marchetti et al. (2014) proposed. These authors chose as epitype the ‘neotype’ selected by Stangl (M-0219661). ITS sequence data obtained from the epitype have been fundamental to start to unravel this taxon complex and to prove the existence of some other closely related species like I. occulta and I. ceskae.

The description of I. mixtilis given here is based on extensive material and complements Stangl's (1984). The holotype and further material show several distinct diagnostic characters:

the rather viscid pileus with a greasy or lardy appearance (mostly in wet conditions);

spores with protruding knobs arranged often in a ‘margaritiform’ appearance (hence isodiametric to subisodiametric); and

cystidial length of about 50 μm in average, their shape being normally ventricose and tapering upwards into a very often clearly differentiated neck (lageniform to sublageniform).

However, non-typical collections of I. mixtilis are difficult to morphologically distinguish from I. ceskae and I. occulta, which have more heterodiametric spores. All three species can be found in boreal taiga forests of the Northern Hemisphere.

Two examined collections determined by J. Stangl as I. tabacina from (M) had an identical ITS region compared to the epitype of I. mixtilis. This fact provides reliable information regarding the cromatic variation of the pileus in I. mixtilis (see description) and its final colour in dry specimens (exsiccata). Specimens of I. mixtilis with a more brownish orange pileus are mistaken for I. tabacina, especially in dry conditions. However, I. tabacina shows a tobacco brown to buff brown pileus, without any orange or yellow colour (in fresh condition), and microscopically both species show very different cystidia, being generally slender and fusiform in I. tabacina and ventricose to utriform in I. mixtilis. A sequence obtained from a typical specimen of I. tabacina fell outside the I. mixtilis complex in our ITS analyses (Fig. 2), and also in previous published analyses (Fig. 1; clade B in Esteve-Raventós et al. (2016).

Our analyses (Fig. 1, 2) also confirm that the paratypes of I. viscosissima var. bulbosovelata (Ferrari 2006) belong to I. mixtilis, but a synonymy is not proposed here since the holotype was not studied (see Specimens examined).

Bandini, Esteve-Rav. & B. Oertel, sp. nov. — MycoBank MB819790, ITS barcode GenBank KX290789; Fig. 5, 6

Fig. 5

Inocybe ceskae TUR-A 203414 (holotype). a–c. Basidiomata in situ and in dry condition; d–i. hymenial cystidia; j. spores under optical microscope; k. spores under SEM. — Scale bars: a–c = 1 cm; d–j = 10 μm; k = 2 μm.

Fig. 6

Inocybe ceskae TUR-A 203414 (holotype): microscopic features. Sp = spores, Pa = paracystidia, Cpa = cauloparacystidia, Ch = cheilocystidia, Pl = pleurocystidia, Ca = caulocystidia. — Drawn by Ditte Bandini.

Etymology. Named after Oluna Ceska who has made numerous mushroom collections and contributed to the knowledge of fungal diversity in Canada (see notes).

Holotypus. Finland, Koillismaa, Kuusamo, Oulanka National Park, W of the Research Station, N of the river Oulanganjoki, Liikasenvaarantie 135, next to gravelled parking place and houses, alt. 166 m, ETRS89 N66°22'10.2" E29°18'37.2", on sandy and mossy soil, near Pinus sylvestris, Betula sp. and Picea abies, 21 Aug. 2015, D. Bandini, J. Vauras & B. Oertel (TUR-A 203414). Isotypes in KR-M-0046013 and AH 46568.

Diagnosis: Inocybe ceskae is characterised by a yellowish beige, straw-coloured to pale ochre, smooth and sticky pileus, a thin whitish evanescent velipellis in young basidiomata, shortly heterodiametrical nodulose spores with rather protruding knobs, and short cystidia (average ≤ 50 μm long). Inocybe ceskae has been found in one locality in boreal forest in Northern Europe (Pinus, Betula, Picea) and also occurs in humid and subtemperate coastal regions of the Pacific North (North America).

Basidiomes usually gregarious, rarely solitary. Pileus 15–30 mm diam; shape campanulate or obtusely conical to hemispherical, then convex applanate when expanded, sometimes undulate when young, often showing a low and broad umbo, sometimes not or hardly umbonate; margin straight or deflexed, somewhat undulate in expanded specimens, not striate, not distinctly hygrophanous; colour beige-ochre, straw-yellow to pale ochre (Mu 10YR 8/2–8/6, 2.5Y 8/2–8/4), sometimes more intense in colour or with faint orange hue towards the centre, paler and whitish at the extreme margin because of the velipellis; surface radially appressed-fibrillose, smooth and glabrous when young, sometimes becoming finely felty fibrillose when old, lubricous and sticky in wet conditions, adhering soil and humus particles; velipellis present, distinct in young fruitbodies, as a very thin whitish coating especially at margin but also over the entire pileus, soon disappearing and nearly absent in washed or aged specimens. Lamellae ventricose to subventricose, 2–5 mm deep, nearly free to adnate or adnexed with decurrent tooth, moderately crowded to rather crowded, L = 40–60, l = 1–3; colour initially whitish, beige or greyish, showing a rather distinctive pinkish or purplish hue, especially towards the lamellar edge, then becoming brownish grey or even deep brown to orange or rusty brown when old; edge crenulate, whitish when young, sometimes concolorous with age. Stipe 20–45 × 2–5 mm, solid, fibrose; shape cylindrical to slightly tapering upwards, base most often abruptly marginately bulbous; bulb 6–9 mm broad, agglutinating abundant soil and humus material and sometimes hidden and difficult to perceive; colour whitish when young, then becoming yellowish with age (Mu 2.5Y 8/1–8/4) but not especially browning or darkening; surface evenly and densely pruinose all over, also discernible under the lens in the lower third of stipe and near the bulb. Cortina not seen, even in young primordia. Context fibrose, firm, whitish in pileus, stipe and bulb, concolorous to the surface in the stipe cortex; smell more or less spermatic when cut, otherwise sometimes indistinctive. Colour of exsiccata: pileus shiny brownish to brownish orange at the centre (10YR 7/4–7/6), pale brown, beige or straw-coloured towards the margin; stipe similar to pileus (10YR 8/4–8/6), not showing evident darkening (deep brown to blackish) upon drying; lamellae brown, deep brown to rusty brown.

Basidiospores (6.7–)7–11.1(–11.5) × (5–)5.2–7.6(–7.7) μm, Q (1.2–)1.3–1.6(–1.7), Lm = 8.6–8.9, Wm = 6.2–6.4, Qm = 1.4 [n = 100, from 2 collections], heterodiametrical to subheterodiametrical, seldom subisodiametrical, often with a curved adaxial face in profile, presenting (8–)9–12(–14) distinct and rounded to conical-obtuse knobs, these often irregularly protruding, 0.7–1.4 μm high, 1.1–1.6(–2) μm broad at base; apicula distinct, curved; without germ pore and sometimes with a large central guttula. Basidia 18–30 × 7.5–11.5 μm, clavate, sometimes with small guttulae, (2–)4-spored, with sterigmata 2.5–5(–7) μm long. Pleurocystidia abundant, metuloid, (38–)40–48–60(–65) × (16.5–)18–22.7–30.5(–31) μm, Q = 2.1–2.3–3.3 [n = 40]; shape mostly rather ventricose and broadly subutriform with wide neck, but also broadly fusiform, generally not pedicellate or tapering into a short rounded base, rarely with a short pedicel; rather crystalliferous at apex; walls (1–)1.5–2 μm, often thickened towards the neck or apex, 2–4.5 μm thick, but not coalescing at the neck, pale yellowish in ammonia; content hyaline, occasionally pale yellowish. Lamellar edge nearly homogeneous and sterile, composed of numerous cheilocystidia and often rather small subclavate or subovoid paracystidia. Cheilocystidia abundant, similar in shape and size to pleurocystidia, (36–)37–46–54(–55) × (16–)18–22–25(–29) μm [n = 40], with similar thick walls, rather crystalliferous at apex and sometimes showing a pale yellowish content. Hymenophoral trama subregular, constituted by more or less interwoven hyphae, 4–26 μm wide, subcylindrical, subfusiform to almost ovoid in shape, mostly constricted at septa, hyaline. Subhymenium poorly developed, consisting of 2–3 layers of small subisodiametric or irregular cells, 4–6.5 μm diam, hyaline. Pileipellis consisting of an epicutis, composed of slightly gelatinized parallel hyphae, 4–12 μm wide, with incrusting and intracellular yellowish pigment; hyphae somewhat paler to almost hyaline in deeper layers (subcutis), up to 25 μm wide; epicutis sometimes covered by thin hyaline hyphae with scattered free ends, from remnants of velipellis. Stipitipellis consisting of a cutis of subcylindrical hyphae, constricted at septa, bearing numerous bundles of caulocystidia similar to hymenial cystidia, (29.5–)27.5–47.2–63.5(–67.5) × (17.5–)18.5–21.9–26.5 μm [n = 40], crystalliferous at apex, intermixed with abundant hyaline subclavate paracystidia, similar to those of lamellar edge; these elements constitute the dense and abundant pruina reaching the bulb. Clamp connections present in all tissues.

Habitat & Distribution — The holotype collection was found in Finland on sandy soil, next to a gravelled parking place in an anthropogenic area, but it is unknown to us whether the soil had calcareous profile. Several Canadian collections (British Columbia) were also gathered in human-influenced locations (see Notes), while our two European collections (holotype and paratype) were made in taiga forest, suggesting that I. ceskae is associated with conifers (Pinus or Picea) with the presence of Betula species. In the Pacific Coast of Canada (British Columbia) and north-western USA (Oregon), I. ceskae is associated with Pseudotsuga menziesii (Douglas-fir) and probably also with Tsuga heterophylla (western hemlock). Inocybe ceskae occurs further south along the Western Cordillera, and reaches Sierra Nevada Foothills (Northern California), where it is probably associated with Arbutus menziesii and Quercus. (Smith et al. 2002, Morris et al. 2008). Kennedy et al. (2012) have assessed the potential for the existence of common mycelial networks between A. menziesii and P. menziesii; Arbutus menziesii hosts a highly diverse mycorrhizal fungal community with similar composition to those found on other angiosperm and Pinaceae hosts (including P. menziesii). This fact could explain why I. ceskae has been found forming ectomycorrhiza with A. menziesii and some oaks in more southern and subtemperate areas of Northern California (Morris et al. 2008).

Due to its transoceanic distribution, it is very probable that I. ceskae extends throughout boreal to subboreal taiga forests in both continents and also Siberia.

Additional specimen examined. Finland, Koillismaa, Kuusamo, Oulanka National Park, west of the Research Station, N of the river Oulanganjoki, Liikasenvaarantie 135, next to gravelled parking place and houses, alt. 166 m, ETRS89 N66°22'10.2" E29°18'37.2", on sandy and mossy soil, near Pinus sylvestris, Betula sp. and Picea abies, 26 Aug. 2016, J. Vauras (paratype KR-M-0042321; isoparatype TUR-A 203863 (Vauras 31668F)).

Notes — Our description is based on the holotype and the paratype, both gathered in the same spot in consecutive years. These materials are conspecific with eleven collections at UBC and all form a strongly supported monophyletic clade (Fig. 1). The UBC materials were mostly determined as I. mixtilis from Canada (see Table 1) by Oluna Ceska, after whom we name this species. Photographs, drawings and notes of these Canadian vouchers are available on the internet (www.biodiversity.ubc.ca/museum/herbarium/). All of them were gathered in different localities in British Columbia, alt. 30–240 m, in the so-called Marine West Coast Forests, characterized by a temperate and moist climate. In all UBC material in which the ecology is noted the ectomycorrhizal host is Pseudotsuga menziesii, except for two specimens collected under Tsuga heterophylla (F19812) and Arbutus menziesii (F19202), respectively.

Seven of these collections at UBC were identified as I. mixtilis and actually belong to I. ceskae. The other four were originally determined as I. glabrodisca, I. praetervisa, I. sindonia and I. umbratica. These identifications suggest that the pileus colour can be pale and interestingly, the fruitbody named I. glabrodisca (F19553) is, as explicitly told, a very aged one, and apparently brownish.

Unfortunately, the examination of these UBC materials was not authorized while undertaking this study and this has refrained us from designating a North American collection as the holotype.

According to Oluna Ceska's drawings and annotations, there must be a certain range of variation in macro- and micromorphological features, but the resemblance with the holotype is obvious:

the range of pileus size in the Canadian vouchers is 25 (F23607) to 55 mm diam (F18984);

most exsiccata show a light yellowish to pale straw-coloured pileus, mostly broadly umbonate, and the surface is often shiny and glabrous, with adhered soil particles suggesting that they were lubricous or viscid;

fresh pileus colour indicated by Oluna Ceska ranges from ochre (F18954) to yellow-ochre or yellow-brown, or darker yellowish brown or even very pale (F18984). The centre of the pileus in one collection is described as ‘with an ochre-orangy patch’ (F18974);

an obtusely umbonate pileus is present in photos and sketches of most fruitbodies;

lamellar colour is whitish brown to greyish brown turning to dark olive (F18974);

the colour of the stipe is white, ‘white silky’ or whitish with a yellow tinge (F18984);

the stipe is always depicted pruinose over the entire length, and a marginate bulbous base is distinct in F19221 (photograph of fresh specimens) and also in Ceska's sketches (e.g., F18984); and

the stipe length is variable among these collections, up to 80 mm long (F18954) and –8 mm wide, with a bulb up to 11 mm (F18984). On the contrary, basidiomata in F23607 are very slender, with pileus up to 30 mm diam, with a prominent umbo and stipe up to 40 × 2.5–4 mm.

Unfortunately, microscopic features are roughly drawn. Spores show distinct, often prominent knobs, with measurements 8–10(–11) × 6–7 μm (F18954), 8–9(–10) × 6–7 μm (F18974), 7–9.5(–10) × 6.5–7(–8) μm (F18984) and 8–11 × 6–7 μm (F19553). Cystidia are ventricose with an attenuated obtuse neck and thick walls (–3 μm), but their base is never depicted. Measurements are only indicated for three collections: 45–55 × 12–13 μm (F18954), 45–50 × (10–)13–20 μm (F19553) and 45–50 × 15–22 μm (F19553), which reflects the small size and variable width of cystidia.

Although somewhat paler in colour, the habit of I. ceskae is similar to I. mixtilis and I. occulta. Inocybe mixtilis is shown to be sister to I. ceskae in our ITS-RPB2 phylogeny (Fig. 1), but it differs from the latter in showing a more intensely yellow to orange pileus, lubricous or sticky in wet conditions, and, microscopically, a large proportion of the spores have protruding obtuse knobs that give the spores a ‘pseudoasteriform’ or ‘margaritiform’ shape. Some collections of I. ceskae and I. occulta may share a thin whitish velipellis, which is mostly ephemeral and observed in young specimens not washed by rain, especially at pileus margin. Nevertheless, both species are sometimes difficult to recognize morphologically, but I. ceskae shows paler colours and shorter cystidia in average. Inocybe ceskae and I. occulta can occur in similar habitats, being I. occulta more widespread geographically, also extending to warmer and more temperate climates in Europe and North America. Inocybe johannis-stanglii and I. nothomixtilis show distinct heterodiametrical spores with a lower ornamentation. Furthermore, I. johannis-stanglii has a greyish velipellis and larger spores and cystidia, while I. nothomixtilis prefers instead southern, subtemperate to warm ecosystems where it is associated mainly with Fagaceae and Cistaceae.

Based on material from Kamchatka (Eastern Siberia, Russia), Peintner & Horak (2002) attributed to I. mixtilis a pale-coloured pileus, heterodiametric spores provided with distinct knobs and short cystidia, that suggest I. ceskae instead. This fact substantiates our hypothesis of I. ceskae being more widespread over circumpolar regions of the Northern Hemisphere.

Bandini, Esteve-Rav. & G. Moreno, sp. nov. — MycoBank MB819791. ITS barcode GenBank: KX290791; Fig. 7, 8

Fig. 7

Inocybe johannis-stanglii. a. KR-M-0038039 (holotype) in situ; b. Inocybe pallida sensu Stangl M-0021484; c–h. hymenial cystidia; i. spores under optical microscope; j. spores under SEM (all microphotos from KR-M-0038039, holotype). — Scale bars: a–b = 1 cm; c–i = 10 μm; j = 2 μm.

Fig. 8

Inocybe johannis-stanglii KR-M-0038039 (holotype): microscopic features. Sp = spores, Pa = paracystidia, Cpa = cauloparacystidia, Ch = cheilocystidia, Pl = pleurocystidia, Ca = caulocystidia. — Drawn by Ditte Bandini.

Etymology. Named after Johann Stangl, for his great contribution to the knowledge of the genus Inocybe.

Holotypus. Germany, Baden-Württemberg, Heidelberg, TK 6518/3, alt. 120 m, churchyard of Johanneskirche, on lawn with Picea omorika and Betula pendula, 4 July 2012, D. Bandini, KR-M-0038039. Isotype AH 46580.

Misapplied names

– Inocybe pallida Velen. sensu Stangl, Hoppea 46: 308. 1989.

Selected descriptions and iconography: Stangl (1989: 308, Taf. 34/2); Ferrari et al. (2014: 106, 175)

Diagnosis: Inocybe johannis-stanglii is characterized by: a) the distinctly heterodiametric spores, measuring (7.6–)8.4–10.1–12(–13) × 6–6.9–7.8(–8.2) μm, Q = (1.2–)1.3–1.5–1.8(–1.9) μm, provided with numerous (8–16), low and rounded knobs; b) thin greyish velar patches on the pileus; c) abruptly bulbous stipe, not always distinctly marginate; d) intense lilac hues on lamellae when young; and e) cystidia often tapering into a distinct pedicel. Associated with conifers in central Europe in natural to semi-natural woodlands areas.

Basidiomes usually gregarious, rarely solitary. Pileus 10–30 mm diam; shape when young campanulate, subconical to hemi-spherical, then convex to expanded when old, sometimes somewhat undulate or irregular, sometimes with a broad low obtuse umbo; margin mostly straight, occasionally deflexed; colour when young pale ochre, then mostly dull ochre (Mu 10YR 7/6–7/8), brownish ochre or hazel brown (Mu 10YR 6/6–6/8 or 5/6–5/8), or, due to the presence of velipellis greyish brownish, entirely or only at margin and umbo; surface glabrous or finely felty to finely fibrillose with adpressed, not diverging fibres, in some collections slightly hygrophanous (e.g., KR-M-0043321); often lubricous in wet conditions and with adhering soil particles; velipellis distinct, especially in young fruitbodies, as a thin greyish layer covering the pileus centre or as scattered patches. Lamellae ventricose to subventricose, 2–5 mm deep, almost free, adnexed, emarginate-adnexed or emarginate with decurrent tooth, moderately crowded to crowded, L = 40–60, l = 1–2; colour initially whitish, very often with a pale vinaceous grey, greyish lavender or light lilac tinge, finally greyish brownish, sometimes with a lilac tinge; edge fimbriate, whitish to even concolorous in old basidiomata. Stipe 20–40 × 2–4 mm, solid, fibrose; shape cylindrical or slightly tapering upwards, base bulbous or often faintly marginately bulbous; bulb up to 8 mm broad, mostly rounded marginate, in some collections hardly or not marginate and apparently clavate bulbous (e.g., in KR-M-0043320); colour whitish when young, then also wax-coloured or very pale brown at most (Mu 10YR 8/3–8/4), never showing a strong darkening when bruised or with age; surface evenly and densely pruinose all over, reaching the bulbous base. Colour of exsiccata: pileus rather uniform light brown, greyish brown (Mu 10YR 5/4–5/6, 10YR 4/4), stipe beige to straw yellow (10YR 7/4–7/6) or yellowish brown (Mu 10YR 6/4), never showing any distinct darkening upon drying. Cortina not seen, even in young specimens. Context fibrose, firm, whitish in the pileus and also in the stipe when young, then with a brownish tinge, especially in the upper half and the cortex of the stipe; smell indistinct, weakly spermatic when cut.

Basidiospores (7.6–)8.4–12(–13) × 6–7.8(–8.2) μm, Q = (1.2–)1.3–1.8(–1.9), Lm = 8.3–9.1, Wm = 6.6–7.1, Qm = 1.4–1.5 [n = 100, from 4 collections], heterodiametric, with nodulose profile, presenting (8–)10–16 low, rounded to broadly conical knobs, 0.4–1.2(–1.4) μm high, 1–2(–2.2) μm broad at base; yellow in ammonia, without germ pore and sometimes with a large central guttule. Basidia 22.5–32 × 8.5–12 μm, clavate, 4-spored, in some collections mixed with 2-spored, with sterigmata 2.5–6 μm long. Pleurocystidia abundant, metuloid, (35–)45–55–63(–65) × (11–)13–17.5–21(–24) μm, Q = 1.9–3–4.4 [n = 40], subutriform, sublageniform, also subfusiform, occasionally subcylindrical or subclavate, often rather ventricose, mostly with rather short obtuse neck, base often tapering into a more or less distinct, sometimes curved pedicel; apex usually rather crystalliferous; walls 1–2.5 μm, often thickened towards the neck, up to 3.5 μm thick, yellowish in ammonia; content generally hyaline, but sometimes showing a pale yellow to glaucous amorphous content in ammonia solution. Lamellar edge almost homogenous and sterile, consisting of numerous hyaline cheilocystidia intermixed with subclavate paracystidia. Cheilocystidia abundant, similar in shape and size to pleurocystidia, (31–)33–53–63(–70) × (11.5–)12–16–21(–25) μm [n = 40], with similar walls and often crystalliferous. Hymenophoral trama regular to subregular, consisting of parallel-arranged to slightly interwoven hyphae, 4–26(–30) μm wide, subcylindrical to subfusiform in shape, mostly more or less constricted at septa, hyaline. Subhymenium poorly developed, consisting of 1–2 layers of small subisodiametric cells, 3.5–6.5 μm diam. Pileipellis consisting of an epicutis, often slightly gelified, constituted by parallel-arranged hyphae 5–12 μm wide, with encrusting and intracellular brownish orange to yellowish pigment, somewhat wider and paler and almost whitish in the subcutis, up to 20(–25) μm wide, mostly covered by thin hyaline hyphae, with scattered free ends (velipellis remains). Stipitipellis consisting of a cutis of subcylindrical hyphae, slightly constricted at septa, bearing numerous bundles of caulocystidia, (37–)39–46–55(–64) × (12–)13–15.5–20(–21) μm [n = 40], crystalliferous at apex, intermixed with numerous paracystidia, similar to those of the lamellar edge; both elements forming a dense and conspicuous pruina, clearly reaching the bulbous base. Clamp connections present in all tissues.

Habitat & Distribution — Inocybe johannis-stanglii is associated with conifers (Abies, Picea, Larix) in temperate continental forests of Europe, and is also found in urban and man-influenced areas like gardens, parks, churchyards and roadsides, where it thrives near planted or introduced firs or spruces like Picea omorika (= Abies omorika, Serbian spruce), which grows spontaneously in mountainous areas of Serbia, between 800–1400 m alt., and is planted in Germany and Switzerland. Inocybe johannis-stanglii appears to prefer neutral to basic soils, judging from Austrian material and the holotype, but collecting further material is necessary to confirm this assumption.

Inocybe johannis-stanglii is known with certainty from Austria and Germany, but considering its habitat, it could be widespread in continental conifer forests. The collection reported from Switzerland by Breitenbach & Kränzlin (2000, as I. pallida) seems to correspond to I. johannis-stanglii as well. Inocybe pallida has been reported by Solak et al. (2009) from Turkey, but this find cannot be confirmed to be conspecific with I. johannis-stanglii or I. subtrivialis with available information.

Additional specimens examined. Austria, Oberösterreich, Bezirk Braunau am Inn, Kobernausser Wald, near Schalchen, alt. c. 525 m, mixed forest with Picea abies, Abies alba, Larix decidua, 17 Aug. 2014, D. Bandini, J. Christan & L. Quecke (KR-M-0043320); in about 2 km distance from the former location, alt. c. 550 m, mixed forest with Picea abies, Abies alba, Larix decidua, 17 Aug. 2014, D. Bandini, J. Christan & L. Quecke (KR-M-0043321). – Germany, Bayern, Augsburg-Bergheim, TK 7631, garden of A. Sedlmeir, with Betula and Picea omorika, 28 Oct. 1987, J. Stangl (M-0021484, as I. pallida sensu Kuyper); 9 Nov. 1987, J. Stangl (M-0021483, as I. pallida sensu Kuyper); Baden-Württemberg, Heidelberg, TK 6518/3, alt. 120 m, churchyard of Johanneskirche, on lawn with Picea omorika and Betula pendula, 18 July 2012, D. Bandini (KR-M-0046122); 19 July 2012, M. Rave (KR-M-0038082); 31 July 2014, D. Bandini (KR-M-0042290, duplicate in TUR-A 203094).

Notes — Inocybe johannis-stanglii is a well-delimited species within the Mixtilis group, as revealed by our analyses (Fig. 1, 2). Morphologically, the presence of a thin greyish velipellis and the heterodiametric spores, the largest known in this group (often longer than 10 μm), are diagnostic. Spores of I. johannis-stanglii recall those of I. nothomixtilis and I. subtrivialis in shape and ornamentation, with low but distinct numerous knobs. The hymenial cystidia, in average longer than 50 μm, are also very particular because of having a base tapered into a pedicel. Caulocystidia are also abundant near the bulb of the stipe, which is not distinctly marginate in some specimens.

The presence of a distinct velipellis has also been depicted by Stangl (1989, Taf. 34/2) from two collections deposited at M that were seen by T.W. Kuyper and determined as I. pallida by the latter. Both collections have identical ITS sequences compared to the holotype of I. johannis-stanglii. Stangl (1989) named both collections as I. pallida following Kuyper (1985), who revised Velenovský's Inocybe collections at PR and PRC. Velenovský (1920) originally described and depicted I. pallida with a narrow stipe (1–2 mm), progressively enlarged towards the base, but never really bulbous, and angulose spores in outline. The material was gathered in a xerophilous open grassland habitat, with presence of Helianthemum, Thymus, Phleum and Trifolium. This particular habitat suggests a possible relationship with the ectomycorrhiza-forming Helianthemum (Cistaceae), common in heliophilous and warm habitats in Mediterranean and continental European areas. Some years later, Velenovský (1947) gave a Latin diagnosis of I. pallida and added some microscopical data, like spores ‘12-14 μm, cuneatae, angulato-aculeatae’, cystidia measuring 50–60 × 25–40 μm, and habitat ‘in gramine collium, in fruticibus Ruborum non raro’. Kuyper's revision (1985: 394 and f. 33) of the holotype of I. pallida confirmed the angulose outline of the spores (‘with indistinct nodulae’), the very wide cystidia × 20–24(–26) μm, with walls up to 1.5 μm thick and caulocystidia descending over halfway of stipe. Kuyper also suggested that I. pallida is related to I. decipiens, but differed in the ‘smaller, more angular spores’.

The original concept of I. pallida remains difficult to interpret with certainty and sequencing of type material is necessary to infer its position among the nodulose/angulose-spored Inocybe species. Nevertheless, this is probably impossible to achieve, since the holotype is preserved in a liquid solution (Kuyper 1985), which is a mixture of formaldehyde, acetic acid and water (Antonín 1986, 1990), and its DNA is presumably highly degraded. Thus, an epitype for I. pallida should be selected, preferably using material from the type locality, in order to establish a consistent interpretation.

In the meantime, morphological and ecological features of the holotype of I. pallida strongly suggest that this taxon could be a member of the I. decipiens/dunensis complex due to the angular spores (with hardly protruding knobs, if any), large ventricose utriform cystidia without tapering base, an indistinctly bulbous stipe, caulocystidia only descending halfway of the stipe and a xerophilous habitat. All of this suggests a closer relationship with the I. decipiens/dunensis complex, as earlier suggested by Esteve-Raventós & Caballero Moreno (2009: 112). Sequences identified as I. pallida in GenBank show that this name has been consistently employed for species of Inocybe of the I. decipiens/dunensis group.

Esteve-Rav., Bandini & V. González, sp. nov. — MycoBank MB819792; ITS barcode GenBank KX290780; Fig. 9, 10

Fig. 9

Inocybe nothomixtilis. a. AH 24510 (holotype) in situ; b. KR-M-0043322 in situ; c–h. hymenial cystidia; i. spores under optical microscope; j. spores under SEM (all microphotos from AH 24510, holotype). — Scale bars: a–b = 1 cm; c–i = 10 μm; j = 2 μm.

Fig. 10

Inocybe nothomixtilis AH 24510 (holotype): microscopic features. Sp = spores, Pa = paracystidia, Cpa = cauloparacystidia, Ch = cheilocystidia, Pl = pleurocystidia, Ca = caulocystidia. — Drawn by Ditte Bandini.

Etymology. From Latin nothus, which means false, and mixtilis (Inocybe), which represents a different but very close species.

Holotypus. Spain, Basque Country, Araba (Alava), Zuia, Markina, in acid, peaty soil, among Sphagnum spp. and other mosses near Erica tetralix in a Quercus robur stand, with some reforested pines (Pinus radiata) and Sorbus aria nearby, 29 Aug. 1996, R. Fernández-Sasia, RFS no. 960829-01 (AH 24510). Isotypes KR-M-0046551 and in CEME-EMI (Centro de Estudios Micológicos de Euskadi-Euskadiko Mikologia Ikastegia).

= Inocybe tabacina var. pseudovolvata Eyssart. ad int., Bull. Soc. Mycol. France 122: 177. 2006 [not validly published].

Diagnosis: Macroscopically I. nothomixtilis resembles I. mixtilis in habit, size and colours, but it is distinguished by: a) a developed whitish velipellis, easily observed on the pileus and the edge of the bulb in primordia or young specimens; b) the heterodiametric basidiospores 7–8.5–10.1(–11) × 5–5.9–6.8(–6.9) μm, Q = 1.2–1.45–1.7(–1.8) [n = 100, from 4 collections], with 10–13 low, obtuse and well-delimited knobs; c) rather short cystidia of average length ≤ 50 μm; d) pileipellis not viscid or slimy, but sticky to lubricous in wet conditions, or even dry. Distributed in temperate continental and Mediterranean regions of Europe.

Basidiomes usually gregarious, rarely solitary. Pileus 15–30(–35) mm diam, 5–17 mm high, when young broadly conical to hemispherical campanulate, then expanded to broadly convex, convex or more rarely applanate, often with a broad and low umbo; margin slightly inflexed when young, then more straight upon expansion, initially entire or regular, then somewhat wavy to irregular in old specimens, not striate, not or very slightly hygrophanous in old or very soaked fruitbodies; colour somewhat variable, but always showing a range of yellow colours, from golden yellow to brownish yellow or orange-yellow in young specimens (Mu 10YR 6/8; 7.5YR 7/6–7/8), often more brownish to reddish yellow or brownish with a reddish orange hue in older specimens (Mu 7.5YR 6/8), sometimes pale yellowish to ochraceous, especially towards the margin (Mu 10YR 8/6 or 7/8); surface radially fibrillose, most often felty to even woolly-fibrillose with bunches of adpressed fibres, sometimes becoming glabrous towards the centre or in some collections even practically smooth in its entire surface (e.g., in I. tabacina var. pseudovolvata), lubricous and sticky but not viscid in wet conditions, with adhered soil and humus particles; velipellis clearly visible in young fruitbodies as a thin to somewhat thick whitish coating on a yellowish background, rather persistent, sometimes lanose-fibrillose and still visible at centre and/or margin in old specimens. Lamellae ventricose to subventricose, 2–4(–5) mm deep, nearly free or adnexed, moderately crowded, L = 40–48(–50), l = 1–2(–3); colour initially white or whitish, soon becoming beige or pale grey, brownish grey and finally ochre-brown, in some collections showing a typical lilacinous or pinkish lilac reflection (e.g., AH 24510, AH 26925, PC 0738072), never olivaceous at maturity; edge crenulate, whitish or paler than faces, sometimes concolorous by places. Stipe (10–)15–50 × (2–)3–6 mm, solid, fibrose, cylindrical or slightly tapering upwards, base bulbous (AH 24510), most often abruptly and marginately bulbous, in some specimens curved or sinuous (AH 24510); bulb 7–9 mm broad, in young specimens with well-delimited edge covered by a whitish fibrillose coating reminiscent of a ‘pseudovolva’; colour initially whitish to cream (Mu 10YR 8/3), sometimes showing light yellow tinges in adult specimens (Mu 10YR 8/6), never darkening; surface evenly pruinose all over, reaching the bulb, with pruina thin and dense, sometimes pruinose-fibrillose at the lower region, close to the bulb. Colour of exsiccata: pileus brownish (Mu 7.5YR 5/4); stipe beige to brownish yellow (Mu 10YR 6/4), never showing any distinct darkening (either browning or blackening) upon drying. Cortina not seen, even in young primordia. Context fibrose, firm, whitish in pileus centre, white to concolorous to the surface in the stipe, white in the bulb; smell weakly to strongly spermatic, especially when cut; taste mild.

Basidiospores 7–10.1(–11) × 5–6.8(–6.9) μm, Q = 1.2–1.7(–1.8), Lm = 8.3–9.1, Wm = 6.1–6.2, Qm = 1.4–1.5 [n = 100, from 4 collections], heterodiametric, rarely subheterodiametric, with nodulose profile, presenting low but distinctive, 10–13 rounded to conical obtuse knobs, 0.5–0.7 μm high, (0.7–)0.9–1.1 μm broad at base, mostly distributed in abaxial position, sometimes presenting an apical knob up to 1.2 μm high and hence the spore outline showing an attenuated apex; yellow in ammonia, without germ pore. Basidia 22–30 × 8–11(–12) μm, clavate, 4-spored, with sterigmata 2–4(–5) μm long. Pleurocystidia moderately abundant to scattered, metuloid, (35–)38–46.5–55(–60) × 10–15.5–22(–28) μm, Q = 2.1–3–4 [n = 40], variable in shape depending on collections, often ventricose and subutriform with broad and short neck, also subfusiform, occasionally subcylindrical to subovoid, base attenuate-truncate (rather frequent in the holotype AH 24510) or tapering in a more or less distinct pedicel (e.g., in I. tabacina var. pseudovolvata voucher); generally rather crystalliferous at apex (hardly or not crystalliferous in Alessio's collection of I. mixtilis var. aurata); walls 1.5–2.5 μm, more or less uniformly thickened, in some collections (e.g., in the holotype) up to 3–4 μm at the neck and the extreme apex, and then sometimes walls coalescing (like a ‘plug’), hyaline to very pale yellow in ammonia; content hyaline, never showing dark (brown to dark brown) pigments, but sometimes filled with a hyaline to pale yellowish amorphous content. Lamellae edge homogeneous and sterile, composed of numerous to scattered cheilocystidia and very abundant clavate to ovoid paracystidia, mixed with intermediate elements in shape, hyaline, occasionally pale yellow mostly in aged specimens. Cheilocystidia similar in shape and size to pleurocystidia, (26–)30–44–57(–60) × (10–)12–16–21(–24) μm [n = 40], with similar thick walls and crystalliferous apex. Hymenophoral trama regular to subregular, constituted of more or less parallel hyphae, 4–20(–25) μm wide, cylindrical to fusiform in shape, often constricted at the septa, hyaline or with pale yellow intracellular pigment. Subhymenium poorly developed, consisting of 1(–2) layers of small, subisodiametric cells, 4–6 μm diam. Pileipellis consisting of an epicutis, somewhat gelatinized (but not a distinct ixocutis), constituted by parallel hyphae up to 4 μm wide, with finely encrusting and intracellular parietal yellowish pigment; hyphae somewhat wider and paler in the subcutis, up to 20(–25) μm wide; the epicutis is sometimes covered by thin hyaline hyphae, with scattered free ends (velipellis remains). Stipitipellis consisting of a cutis bearing numerous bundles of caulocystidia similar to hymenial cystidia, (26–)30–41.5–53(–60) × (12–)13–15.5–19(–20) μm [n = 40], crystalliferous at apex, and intermixed with numerous paracystidia, similar to those of the lamellar edge (both elements forming a dense and conspicuous pruina), and clearly reaching the bulbous stipe base. Clamp connections present in all tissues.

Habitat & Distribution — In humus of warm or temperate natural forests of Fagaceae (Fagus sylvatica, Quercus spp.) and Cistaceae stands (i.e., Cistus monspeliensis, C. salviifolius), sometimes mixed with Pinus spp. Inocybe nothomixtilis is also found in parks or urban areas and then in mixed forests with broad-leaved trees (e.g., Betula, Carpinus, Tilia) or conifers (e.g., Pinus, Abies). It prefers acid to decalcified soils with sandy texture. Its current known distribution includes France, Germany, Italy and Spain, but considering its habitat preferences, it is probably more widespread in Mediterranean and sub-Mediterranean European areas, and also in warm and temperate locations in continental Europe.

Additional specimens examined. France, Île-de-France, Seine-Saint-Denis, Parc du Sausset, under mixed broadleaved trees (Fagus sylvatica, Carpinus betulus, Quercus spp.), 25 Oct. 2006, G. Eyssartier, GE 06-110 (PC 0738072, as I. tabacina var. pseudovolvata). – Germany, Baden-Württemberg, Karlsruhe, TK 6916/3, alt. 118 m, in a lawn with Quercus petraea, Fagus sylvatica and Betula pendula, 1 June 2013, D. Bandini & B. Oertel (KR-M-0038133); Hessen, Landkreis Bergstraße, Bürstadt, Waldfriedhof Lampertheim, TK 6317/3, alt. 95 m, in acid sandy soil under Pinus sylvestris and Quercus rubra, 26 Oct. 2014, D. Bandini (KR-M-0043319, duplicate in TUR-A 203452). – Italy, Toscana, Castiglion Fiorentino, St. Stefano, alt. approx. 500 m, in Mediterranean forest near Quercus spp. (possibly Q. pubes-cens or Q. cerris), in presumably decalcified soils, 18 Oct. 2010, M. Dondl (KR-M-0043322). Locality unknown (probably Piemonte, Pamparato according to Alessio & Rebaudengo (1980)), under Abies alba (?), 24 June 1986, C.L. Alessio, (private herbarium of C.L. Alessio, as I. mixtilis var. aurata). – Spain, Cataluña, Barcelona, Collserola, Can Ferrer, in Cistus bushes stand (C. monspeliensis and C. salviifolius), in acid soil, 23 Oct. 2000, J. Vila & X. Llimona (AH 26921, duplicate in JVG 1001023-3); 31 Oct. 2000 (AH 26924, duplicate in JVG 1001031-3); Islas Baleares, Mallorca, Escorca, Predio Marjanor, alt. 500–700 m, among mosses in Mediterranean mixed forest with Pinus halepensis, Cistus monspeliensis and Pistacia lentiscus, in decalcified washed soils, 26 April 1994, J.L. Siquier, JLS 715B (AH 46558).

Notes — Habit and size of I. nothomixtilis fruitbodies are very similar to other taxa in the Mixtilis group, but I. nothomixtilis is characterized by its well-developed and rather persistent veil, at least in primordia and young specimens, as well as by its regularly felty to rather woolly-fibrillose pileus in most collections. This whitish veil, when not washed out by heavy rainfall, is easily observed on the pileus and on the bulb edge, forming here a ‘pseudovolva’. In wet conditions, this velipellis covers a sticky but not really viscid pileus, which has often adhering soil particles and humus remains. In some collections the cuticle becomes cracked radially, forming adpressed-fibrillose scales, when withered or upon ageing. All specimens assigned here to I. nothomixtilis form a supported monophyletic clade in all analyses (Fig. 1, 2).

Furthermore, microscopical characters of I. nothomixtilis distinguish reliably from the other species. The spores in I. nothomixtilis are rather small, clearly heterodiametric (Qm = 1.45), and show numerous (usually 10–13) knobs. These protuberances are small, low and rounded (defined as ‘patatiform’ by Bon (1998: 4), referring to I. umbrina). Spore shape and ornamentation remind of those of I. johannis-stanglii or I. subtrivialis, but these species have larger (often exceeding 10 μm) and more rectangular spores. A distinct thin velipellis is also present in I. johannis-stanglii, but this species shows larger cystidia mostly tapering into a distinct attenuate base. Inocybe nothomixtilis is characterized by its rather short cystidia, in average ≤ 50 μm, variable and ranging from truncate to shortly pedicellate at the base. Both taxa also differ by a different distribution and habitat preferences (see comment on I. johannis-stanglii).

Our analyses support that both I. tabacina var. pseudovolvata and a collection of I. mixtilis var. aurata are contaxic with I. nothomixtilis. Regarding I. tabacina var. pseudovolvata, Eyssartier (2006: 177) based his description on a single collection with tobacco-brown pilei (‘paille brunâtre terne à beige-brun terne ou un peu roussâtre ou encore beige ochracé’), and practically devoid of any yellow tone. The pileus surface was described as nearly smooth, somewhat sticky and agglutinating some soil and humus particles (Eyssartier 2006: Planche V). Eyssartier named this taxon I. tabacina var. pseudovolvata because of a well-developed velipellis forming a ‘pseudovolva’ around the bulb edge. As mentioned above, this character is very helpful to recognise I. nothomixtilis, especially when basidiomata are young and in good condition. Eyssartier also provided some interesting macrochemical reactions not tested by us: KOH 5 % yellow on pileus flesh and orange brown on pileus surface; silver nitrate chocolate brown after 10–15 min on pileus flesh.

Inocybe tabacina var. pseudovolvata was never validly published, but a detailed description was provided. We confirm here by examining the collection kept at PC that it matches I. nothomixtilis in most characters, especially in the heterodiametric and small spores (Lxlm = 8.4 × 6; Qm = 1.4, n = 21) and in having short cystidia (Lm < 50 μm). Eyssartier also pointed out some similarities between his variety and I. mixtilis but stated that I. tabacina var. pseudovolvata was distinguished by a well-developed veil and a darker pileus colour. However, no comparison of microscopic characters was made by him.

Inocybe mixtilis var. aurata was introduced by Alessio & Rebaudengo (1980: 302) to include some collections with a more yellow golden colour and a drier pileus (‘pilei cuticulam aridiorem . . .’). Alessio described the pileus as practically dry, hence devoid of the characteristic slimy cuticle which is evident in wet conditions in I. mixtilis. This var. aurata was not validly published because two different gatherings were designated as the holotype (Art. 40.1). Moreover, no original material (Pamparato, Cabanet in 1972 and Roburentello in 1976) is located in E. Rebaudengo's private herbarium (Ceva, Italy), where it was said to be deposited.

Although most Alessio's Inocybe collections used for his monograph are lost (M. Candusso and E. Bizio, pers. comm.), we were able to study three authentic (but not original) collections of var. aurata kept in Alessio's private herbarium, sent to us by M. Candusso. The material is poorly preserved, mouldy and lacks accurate collection data. Two collections could be ITS-sequenced, one of them belonging to I. nothomixtilis (24 June 1986) and the other one conspecific with I. mixtilis (15 Sept. 1984). DNA extraction was successful and the ITS sequence obtained and analysed. The specimen dated 24 June 1986, ‘prato nostro sotto abete bianco’ is conspecific with I. nothomixtilis, while the one dated 15 Sept. 1984, belongs to I. mixtilis. A third collection collected also on 24 June 1986 is a leiosporeous species. From these facts, we conclude that Alessio's concept of var. aurata was heterogeneous.

Esteve-Rav., Bandini, B. Oertel & G. Moreno, sp. nov. — MycoBank MB819793; ITS barcode GenBank KX290787; Fig. 11, 12

Fig. 11

Inocybe occulta. a. AH 36443 (holotype) in situ; b. KR-M-0046556 in situ; c–h. hymenial cystidia; i. spores under optical microscope; j. spores under SEM (all microphotos from AH 36443, holotype). — Scale bars: a–b = 1 cm; c–i = 10 μm; j = 2 μm.

Fig. 12

Inocybe occulta AH 36443 (holotype): microscopic features. Sp = spores, Pa = paracystidia, Cpa = cauloparacystidia, Ch = cheilocystidia, Pl = pleurocystidia, Ca = caulocystidia. — Drawn by Ditte Bandini.

Etymology. From Latin occultus, which means hidden or concealed, due to the likely confusion with similar taxa of the Inocybe mixtilis group.

Holotypus. Spain, La Rioja, Villarroya, 800 m, 30T WM76, on washed and decalcified sandy soil, in a Quercus ilex subsp. ballota forest, 17 May 2008, A. Caballero, AC3805 (AH 36443). Isotypus KR-M-0046550 and A. Caballero's private herbarium.

Selected description and iconography: Esteve-Raventós & Caballero Moreno (2009: 37, f. 11, photos 27–31 – as I. mixtilis).

Diagnosis: Inocybe occulta is morphologically rather close to I. ceskae, differing in the more intense yellow-orangish to orange-brown pileus colour and the slightly longer cystidia in average. Inocybe occulta is sister to I. nothomixtilis in our ITS-RPB2 analyses (Fig. 1), but it differs primarily from the latter in having more isodiametric spores with lower knobs. Inocybe ceskae and I. mixtilis, both with spores of similar shape (Qm = 1.3–1.4) to I. occulta, cluster in a different clade and are phylogenetically distinct from I. occulta (Fig. 1).

Basidiomes usually gregarious, rarely solitary. Pileus (12–)15–30(–45) mm diam, shape initially campanulate or obtusely conical to hemispherical, then convex applanate when expanded, often with a low and broad umbo, but occasionally not or hardly umbonate; margin straight to slightly deflexed, sometimes wavy in expanded specimens, not striate, only slightly appendiculate by fugacious velar remnants when present; colour variable, often yellow to orange, dull to more or less bright, e.g., beige, yellowish ochre, buff yellow to golden yellow, orange-yellow to orange-brown (Mu 10YR 8/4–8/8, 7/6–7/8, 7.5YR 7/6–7/8 or 6/6–6/8), in some collections also light brown (Mu 10YR 5/6–5/8), at least towards the margin (e.g., KR-M-0046554 or KR-M-0046555), uniform or very often slightly darker at the centre or the umbo; surface radially appressed-fibrillose, smooth and glabrous, sometimes with a micaceous and/or a falsely hygrophanous appearance (e.g., KR-M-0046556), even not clearly rimose towards the margin, with lubricous aspect in wet conditions, somewhat sticky and hence often with adhering soil and humus particles; velipellis absent or scarcely developed, in this case fugacious but still observable in young specimens of some collections, especially towards the periphery and margin, forming small fibrillose patches or squamules, but mostly absent in many collections when washed by rainfall or aged. Lamellae ventricose to subventricose, 2–6 mm deep, practically free or hardly adnexed, often crowded, sometimes moderately crowded, L = 45–55, l = 1–2; colour initially whitish to beige or greyish, often showing a lilac to violet reflection (‘Psathyrella-like’), when old becoming ochraceous to brownish grey and finally yellow-brown to orange-brown; edge finely fimbriate to crenulate, whitish or paler than the sides. Stipe 20–50(–55) × (2–)3–6(–8) mm, solid, fibrose; shape cylindrical to tapering upwards, base distinctly marginately bulbous, often agglutinating soil or humus debris; bulb 7–10(–12) mm broad, edge often clear-cut and even volviform, but velar rests absent or ephemeral in young stages; colour white or dirty white when young, often turning to ochraceous, ochraceous yellow or even bright yellow in some collections (e.g., AH 36148), or fawn with age (Mu 2.5Y 8/1–8/4), never browning nor blackening; surface evenly and densely pruinose all over, up to the bulb. Cortina not seen, even in young primordia. Context fibrose, firm, whitish, concolorous towards the cortex in pileus and stipe, hardly becoming yellowish with age or when drying; smell generally faint in fresh specimens, often indistinct, in some collections more or less spermatic when cut, exceptionally aromatical or even honey-like (AH 36443); taste not recorded. Colour of exsiccata: pileus brownish to orange brown (Mu 7.5YR 6/6–6/8, 5/6–5/8) to brown (7.5YR 4/6); stipe dirty whitish to ochraceous brown, paler than pileus (10YR 7/3–7/4 or 6/4–6/6), not darkening; lamellae deep brown orange.

Basidiospores (7.2–)7.5–10(–10.5) × (5.2–)5.3–7.5 μm, Q = (1.1–)1.15–1.57(–1.63), Lm = 8–9.6, Wm = 5.9–7, Qm = 1.3–1.5 [n = 100, from 4 collections], variable in shape (even within a specimen), from heterodiametrical to shortly heterodiametrical and less frequently subisodiametrical; spore ornamentation somewhat variable, mostly presenting 8–13 conical obtuse knobs, (0.9–)1.1–1.4(–1.7) μm high, 1.2–1.6 μm broad at base (but in KR-M-0043312 many spores show low knobs or even a polyhedrical angulose outline, ‘sub-entolomatoid’); apicula distinct; without germ pore and sometimes with a central oil guttule. Basidia 22–28(–30) × 8–10 μm, clavate, 4-spored, with sterigmata 3–5 μm long. Pleurocystidia abundant, metuloid, (37–)39–51.5–64(–67) × (11.2–)11.5–17–22(–24) μm, Q = 2–3–4.1 [n = 40]; of variable shape among collections, normally ventricose fusiform to subutriform, occasionally ventricose sublageniform with obtuse apex and short neck, but also not distinctly ventricose in some collections; base normally not pedicellate, tapering into an obtuse base, seldom truncate; rather crystalliferous at apex in most collections; walls (1–)1.5–2.5 μm, often thickened towards the apex up to 3–4 μm, not coalescing at the neck, pale yellow in ammonia; content hyaline or pale yellowish, not showing dark pigments. Lamellar edge homogeneous and sterile, composed of numerous cheilocystidia and rather abundant small clavate to subovoid paracystidia, 7–12 μm broad. Cheilocystidia abundant, similar in shape and size to pleurocystidia, 33–47–61(–65) × 11.5–17.5–23 μm [n = 40], with similar thick walls, rather crystalliferous at apex, hyaline or showing a pale yellowish content. Hymenophoral trama subregular, consisting of more or less parallel to interwoven hyphae, 3–12 μm wide, subcylindrical to subfusiform, hyaline. Subhymenium poorly developed, consisting of 2–3 layers of small branched to subisodiametric or irregular cells, 3.5–5 μm diam, hyaline. Pileipellis consisting of an epicutis of slightly gelified parallel hyphae, 3.5–15 μm wide, with incrusting and intracellular yellowish pigment; hyphae somewhat paler to almost hyaline in deeper layers (subcutis), up to 20(–25) μm wide. Stipitipellis consisting of a cutis of subcylindrical hyphae, often constricted at septa, bearing numerous bundles of caulocystidia similar to hymenial cystidia, (27–)30–48.5–66.5(–69) × (11–)11.5–17–22.5 μm [n = 40], crystalliferous at apex, intermixed with abundant hyaline, ovoid to subclavate paracystidia (× 8.5–18 μm), similar to those of lamellar edge; these elements constitute the dense and abundant pruina covering the whole stipe. Clamp connections present in all tissues.

Habitat & Distribution — Inocybe occulta occurs in a broad range of habitats and is distributed from cold boreal areas to warm Mediterranean regions of the Northern Hemisphere. Many collections were made in boreal to subboreal taiga forests in Europe (mixed forests including Picea, Larix, Betula, Pinus, etc.), but also in warmer and temperate habitats in central and southern Europe in association with conifers (Picea, Larix, Pinus) or broad-leaved trees like Fagaceae (Fagus sylvatica, Quercus robur, etc.). Inocybe occulta also occurs in more xeric Mediterranean habitats, in association with Pinaceae (Pinus pinaster, P. pinea, etc.), Fagaceae (evergreen oaks like Quercus ilex, Q. suber, etc.) and also Cistaceae (Cistus spp.). Interestingly, I. occulta is also present at the alpine stage in the Pyrenees, in Dryas octopetala and dwarf Salix communities on slightly calcareous soils. These data suggest that, unlike I. ceskae and I. mixtilis, I. occulta has probably a more extensive distribution and broader range of hosts. The known distribution of I. occulta in North America overlaps that of I. ceskae, being known in subtemperate coastal Pseudotsuga forests from the Northern Pacific (British Columbia, Canada) to Oregon (USA), here in young P. menziesii forests (Smith et al. 2002). According to sequences available in GenBank, I. occulta is found in Mexico (Morelos, Volcán El Pelado), presumably in native Pinus forests (KC152132) and also in Australia (Canberra), introduced with Pinus (KP308781).

Inocybe occulta does not show specific soil pH requirements. Many of the collections from montane areas in northern and continental Europe were gathered in calcareous soils, but it also occurs in decalcified, neutral to slightly acid soils with sandy texture in Mediterranean areas, (e.g., in evergreen-oak or Mediterranean pine forests, like Pinus pinea in Italy, ref. GenBank JF908141, Bizio pers. comm.), often with presence of Cistus bushes.

Additional specimens examined. Austria, Kärnten, Bezirk Spittal an der Drau, Reißeck, Penk, alt. 1100 m, under Picea abies, 16 Aug. 2013, D. Bandini & B. Oertel (KR-M-0043315); Salzburg, Bezirk Tamsweg, Tamsweg, near Prebersee, alt. 1500 m, in Picea abies forest, 15 Aug. 2013, D. Bandini & B. Oertel (KR-M-0043316); Tirol, Bezirk Landeck, Fließ, Pillerhöhe, alt. 1540 m, in Picea abies forest, 28 Aug. 2014, B. Oertel & G. Schmidt-Stohn (KR-M-0043313). – Finland, Enontekiön Lappi, Enontekiö, Kilpisjärvi, along the small river Tšahkaljohka, in rather moist forest of Betula pubescens subsp. czerepanovii, 15 Aug. 1990, J. Vauras (TUR-A 145165, duplicate in KR-M-0046553); Koillismaa, Kuusamo, Oulanka National Park, near the Research Station, mixed forest with Pinus sylvestris, Picea abies and Betula sp., 17 Aug. 2015, J. Vauras, D. Bandini & B. Oertel (KR-M-0046554); Kuusamo, Oulanka National Park, Ampumavaara, roadside with Pinus sylvestris, Picea abies and Betula sp., 20 Aug. 2015, J. Vauras, D. Bandini & B. Oertel (KR-M-0046555); Kuusamo, Oulanka National Park, Pähkänä, wayside with Pinus sylvestris, Picea abies and Betula sp., 21 Aug. 2015, J. Vauras, D. Bandini & B. Oertel (KR-M-0046556); Kuusamo, Oulanka National Park, foot of Ampumavaara, roadside with Pinus sylvestris, Picea abies and Betula sp., 22 Aug. 2015, J. Vauras, D. Bandini & B. Oertel (KR-M-0046557); Kuusamo, Oulanka National Park, Liikasenvaara, lawn of a house of Iso Sirkkalampi, in mixed forest with Pinus sylvestris, Picea abies and Betula sp., 25 Aug. 2015, J. Vauras, D. Bandini & B. Oertel (KR-M-0046558). – Germany, Baden-Württemberg, Landkreis Breisgau-Hochschwarzwald, Titisee-Neustadt, Schwärzenbach, Black Forest, TK 8015/3, alt. 930 m, wayside near Picea abies, 18 Aug. 2014, D. Bandini (KR-M-0043323); Rhein-Neckar-Kreis, Sandhausen, TK 6617/4, alt. 100 m, Nature Reserve ‘Sandhausener Düne, Pflege Schönau-Galgenbuckel’, in calcareous sand dune with Pinus sylvestris, Quercus robur and Helianthemum nummularium, 13 Oct. 2012, D. Bandini, B. Oertel & W. Winterhoff (KR-M-0043312); Rhein-Neckar-Kreis, Wiesenbach, TK 6618/2, alt. 190 m, mixed forest with Fagus sylvatica and Picea abies, 17 Oct. 2012, D. Bandini (KR-M-0043318); at about 4 km distance from the former location, alt. 170 m, under Abies procera, 20 Oct. 2012, D. Bandini (KR-M-0043317); Bayern, Landkreis Berchtesgadener Land, Schönau am Königssee, Gotzenalm, TK 8443/4, alt. 1680 m, in calcareous soil with Picea abies, Larix decidua and Juniperus communis, 14 Aug. 2011, D. Bandini & B. Oertel (KR-M-0038028); Landkreis Garmisch-Partenkirchen, Garmisch-Partenkirchen, Riffelriss, TK 8531/4, alt. 1600 m, in calcareous soil with Larix decidua, Pinus sylvestris and Betula pendula, 1 Aug. 2012, B. Oertel (KR-M-0043324); ibidem, alt. 1660 m, in calcareous soil with Larix decidua and Pinus mugo, 20 Aug. 2012, D. Bandini & B. Oertel (KR-M-0043314, duplicate in TUR-A 203441); Rheinland-Pfalz, Landkreis Mainz-Bingen, Bingen, Stadtwald, TK 6013/1, alt. 300 m, mixed forest with Quercus robur, Larix decidua and Betula pendula, 10 Sept. 2011, D. Bandini & B. Oertel (KR-M-0043230); Landkreis Trier-Saarburg, Kell am See, TK 6306/4, alt. 460 m, under Picea abies, 1 Oct. 2011, D. Bandini (KR-M-0038125). – Netherlands, Friesland, Ameland, Hollum, alt. 5 m, in calcareous sand dune, wood with Pinus sylvestris, 19 Sept. 2011, D. Bandini (KR-M-0038029). – Spain, Andalucía, Cádiz, Los Barrios, road from Los Barrios to Facinas (km. 5), Parque Natural de los Alcornocales, alt. 180 m, in acid soil under Quercus suber, 14 Dec. 1990, F. Esteve-Raventós & A. Ortega (AH 22108); Castilla-La Mancha, Guadalajara, Tamajón, El Chortal, alt. 1000 m, in sandy slightly acid soil in a reforested Pinus pinaster forest with Cistus ladanifer, 31 Oct. 2014, G. Moreno (AH 40499); Castilla-León, Burgos, Nava de Mena, Embalse de Ordunte, alt. 300 m, under Quercus robur in slightly acid and sandy soils, 14 Oct. 1998, J.M. Barrasa & F. Esteve-Raventós (AH 24987); Castilla-León, Burgos, Valle de Mena, Vivanco, Santuario de Cantonad, alt. 480 m, in Fagus sylvatica forest, 14 Oct. 1998, J.M. Barrasa & F. Esteve-Raventós (AH 24989); Valladolid, Tudela de Duero, in slightly acid and sandy soil in Pinus pinea forest, 23 Nov. 2002, P. Juste (AH 30697); Cataluña, Gerona, Queralbs, Val de Núria, Coma de l’Embut, alt. 2220 m, in calcareous soil among Dryas octopetala and Salix retusa, 8 Aug. 1996, J. Vila (AH 21573, duplicate in JVG 960808-26); 12 Aug. 1997, J. Vila & X. Llimona (AH 23481, duplicate in JVG 970812-8); 23 July 1999, J. Vila (AH 26731, duplicate in JVG 990723-15; published by Esteve-Raventós & Vila (1997), as I. mixtilis); Gerona, Setcases, Refugio de Ulldeter, alt. 2300 m, among Salix retusa in slightly calcareous soils, 2 Aug. 1997, F. Esteve-Raventós & J. Vila (AH 23480, duplicate in JVG 970802-4); Lérida, Espot, Estany de Sant Maurici, La Roca de l’Estany, alt. 2150 m, under Salix pyrenaica in calcareous soil, 12 Sept. 1996, J. Vila & X. Llimona (AH 21576, duplicate in JVG 960912-25; published by Esteve-Raventós & Vila (1997), as I. mixtilis); Extremadura, Cáceres, Jarandilla de la Vera, in acid soil under Quercus pyrenaica, 14 Nov. 2014, J. Muñoz (AH 44531); La Rioja, Santa Lucía, Valle de Ocón, alt. 800 m, in decalcified sandy soil in Quercus ilex subsp. ballota forest, 3 June 1997, A. Caballero (AH 36110, duplicate in AC 2008); 6 June 2009 (AH 40279, duplicate in AC 4102); Villarroya, alt. 800 m, in decalcified sandy soils of Quercus ilex subsp. ballota forest, 30 April 2006, A. Caballero (AH 36148, duplicate in AC 3189; published by Esteve-Raventós & Caballero Moreno (2009), as I. mixtilis ).

Notes — This study suggests that I. occulta is the species with the broadest, transoceanic distribution within the Mixtilis group, and probably the most common one. Its epithet refers to the fact that this taxon has been overlooked and has probably been misinterpreted for I. mixtilis or other species of this group.

Inocybe occulta forms a well-supported clade in our analyses (Fig. 1) but it is sometimes difficult to recognize morphologically. Typical collections are characterized by the heterodiametrical spores, pleurocystidia 34–69 μm long and a pileus of intense colour. Nevertheless, those characters are somewhat overlapping in I. mixtilis, I. ceskae and I. occulta.

The spore shape in I. occulta and I. ceskae shows a clear tendency to be heterodiametric to shortly heterodiametric, while spores are generally isodiametric to subisodiametric in I. mixtilis. The three species are characterized by producing spores with a prominent ornamentation constituted by high knobs, in contrast to those of I. nothomixtilis, I. johannis-stanglii and I. subtrivialis, which have considerably lower knobs.

Esteve-Rav., M. Villarreal & Heykoop, Rev. Catalana Micol. 20: 157. 1997 — Fig. 13, 14

Fig. 13

Inocybe subtrivialis AH 19225 (holotype): microscopic features. Sp = spores, Pa = paracystidia, Cpa = cauloparacystidia, Ch = cheilocystidia, Pl = pleurocystidia, Ca = caulocystidia. — Drawn by Ditte Bandini.

Fig. 14

Inocybe subtrivialis. a. AH 19225 (holotype) in situ; b. AH 26783 in situ; c–h. hymenial cystidia; i. spores under optical microscope; j. spores under SEM (all microphotos from AH 19225, holotype). — Scale bars: a–b = 1 cm; c–i = 10 μm; j = 2 μm.

Holotypus. Spain, Castilla-La Mancha, Toledo, La Iglesuela, El Bonal, 30TUK525575, alt. 450 m, in acid sandy soil of Quercus ilex subsp. ballota forest with Cistus ladanifer and Lavandula pedunculata bushes, 16 Jan. 1996, J.L. Aguirre, M. Heykoop & M. Villarreal (AH 19225).

Selected descriptions and iconography: Esteve-Raventós et al. (1997: 157, f. 5). ITS barcode GenBank MH496013, MH496014.

Habitat & Distribution — Inocybe subtrivialis is currently known only in the Iberian Peninsula, but its ecology suggests it is more widespread and has been overlooked in Mediterranean Europe. Some records of I. pallida from Mediterranean environments might actually belong to I. subtrivialis, as that by Solak et al. (2009) in pine forests in southern Turkey. The typical habitat of I. subtrivialis are thermophilous Mediterranean evergreen oak forests (Quercus ilex subsp. ballota) or pine forests (Pinus pinea, P. pinaster), with presence of Cistus.

Specimens examined. Spain, Toledo, La Iglesuela, El Bonal, 30TUK525575, alt. 450 m, in acid sandy soil of Quercus ilex subsp. ballota forest with Cistus ladanifer and Lavandula pedunculata bushes, 16 Jan. 1996, J.L. Aguirre, M. Heykoop & M. Villarreal (AH 19225, holotype); 5 April 1996, M. Villarreal & M.A. Jiménez (AH 20435); 24 April 2000, F. Esteve-Raventós & M. Villarreal (AH 26783); AH 26784. — Inocybe cf. subtrivialis. Spain, Madrid, Rozas de Puerto Real, Embalse de los Morales, alt. 780 m, in acid sandy soil in a mixed forest with Pinus pinaster and Cistus ladanifer bushes, 14 May 2000, F. Esteve-Raventós & M. Villarreal (AH 26789).

Notes — Inocybe subtrivialis was described in detail by Esteve-Raventós & Caballero Moreno (2009), who commented on the similarities with I. pallida in the sense of Stangl (1989) (see comments under I. johannis-stanglii). Our ITS-RPB2 analyses (Fig. 1) revealed that both are close but independent species. Furthermore, both show a different distribution, being I. subtrivialis a characteristic thermophilous species associated with Mediterranean evergreen oaks (Quercus spp.), Cistus spp.and Pinus spp., in acid sandy soil (see also notes on I. johannis-stanglii for comparison).

Inocybe subtrivialis shows the following diagnostic characteristics: 1) pileus covered by whitish veil in young specimens, usually sticky to viscid in wet conditions, with a warm brownish yellow (Mu 10YR 5/6–5/8, 6/6–6/8), orange (Mu 7.5YR 6/6–6/8) to dark yellow-orange brown or ‘bronze’ colour (Mu 7.5YR 5/6–5/8, 4/6), smooth or often broken into appressed scales; 2) stipe in most cases with a napiform or subbulbous base, in some collections with a slightly marginate bulb, white and typically becoming fuscous yellow with age (especially evident in exsiccata), densely pruinose all over but sometimes sparsely pruinose in the lower third; 3) lamellae without distinct violet or lilac shades when young; 4) spores heterodiametric, (8–)8.2–10.5–12.7(–12.8) × (5.2–)5.5–6.4–7.3(–7.5) μm, Q = (1.25–)1.3–1.65–2(–2.2) [n = 100, from 4 collections], provided with numerous (10–)12–16(17) low and obtuse knobs (0.35–0.75 μm high, 0.7–2 μm broad at base), but in some occasional collections without distinct knobs and hence subangulose in outline; 5) pleurocystidia mostly narrowly lageniform to subfusiform, also sublageniform provided with a differentiated obtuse neck and base often tapering into a pedicel, 46–55.5–65 × 9.5–16–22 μm [n = 40], walls (1–)1.2–2(–2.5) μm thick, yellowish (not very intense) in ammonia solutions, content often showing a pale yellow-fuscous intracellular pigment; and 6) caulocystidia similar to hymenial cystidia, often more slender or even hair-like near the base, mixed with clavate to sphaeropedunculate or subovoid paracystidia.

Specimen AH 26789 is morphologically similar to I. subtrivialis but its ITS sequence deviates from the one obtained from the holotype specimen (11 changes and several insertions/deletions). Our material needs to be examined to more appropriately assess its identity. In addition to its habitat and distribution, I. subtrivialis differs from I. johannis-stanglii in the darker, brownish orange pileal colour, a strong tendency of the fruitbodies to become yellow fuscous to bronze on ageing or upon drying, slightly narrower spores and slenderer cystidia.

1. Spores with Qm = 1.4–1.65, provided with low knobs (0.5–1 μm high) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2

1. Spores with Qm = 1.3–1.4, provided with clear and distinct protruding knobs (1–1.5 μm high) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

2. Spore width Wm ≥ 6.5 μm; velipellis present, buff to greyish; lamellae with a violet to lilac reflection when young; cystidia bulgy and mostly pedicellate. — In temperate Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I. johannis-stanglii

2. Spore width Wm < 6.5 μm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

3. Spores with Qm = 1.6, more or less rectangular heterodiametric; cystidia not very short (Lm = 55 μm), often pedicellate and with thin walls (1–2 μm); velipellis whitish to buff, often ephemeral; basidiomata generally becoming yellow-copperish to orange or brown copperish with age; lamellae without lilac or violet reflection when young. — In Mediterranean sandy forests, mainly under evergreen oaks (Quercus spp.), sometimes with the presence of Cistus and/or Pinus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I. subtrivialis

3. Spores with Qm = 1.4–1.5, polygonal heterodiametric, often tapering to an apical knob; cystidia short (Lm = 45–50 μm), not pedicellate, often with thick walls 2–3(–4) μm; velipellis rather developed and persistent, sometimes leaving a rim around the bulb margin; pileus sometimes fibrillose-shaggy, dry to weakly sticky in humid conditions. — In temperate to warm areas of Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I. nothomixtilis

4. Spores mostly subisodiametric to isodiametric, with Qm = 1.3–1.4, often with a ‘margaritiform’ shape; pileus in wet conditions remarkably lubricous and sticky, with a lardy aspect; hymenial cystidia often showing a differentiated neck (lageniform to sublageniform). — Only known with certainty from Europe, especially in north temperate to subboreal cold areas, rare in southern countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I. mixtilis

4. Spores with Qm = 1.4–1.5, generally more heterodiametrical; pileus without a lardy aspect, slightly sticky in humid conditions, sometimes subhygrophanous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

5. Pileus pale straw-coloured, yellowish beige, pale ochre, slightly sticky in wet conditions; cystidia ≤ 50 μm, fusiform, with attenuate but not pedicellate base. — Known only from cold subboreal areas of Europe and certainly also from temperate coastal forests of Pacific Canada extending to Oregon (USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I. ceskae

5. Pileus darker, buff yellow, golden-yellow, orange-yellow or light brown, often with a subhygrophanous appearance; cystidia often with a sublageniform shape, often extending into a distinct neck, though sometimes variable, often > 50 μm long. — Widespread all over continental Europe, and also known from North America and Australia. . . . . . . . I. occulta

DISCUSSION

Inocybe is a species-rich genus of ectomycorrhizal fungi. Due to lack of reference sequences and lack of comparative taxonomic studies, identification of Inocybe species is often challenging and many sequences and material remain unidentified or misidentified (Ryberg et al. 2008). Previous ITS-based phylogenies integrating comparative morphological studies and distributional data have proven to be a good approach to resolve several species complexes (e.g., Larsson et al. 2009, 2017, Cripps et al. 2010, Esteve-Raventós et al. 2015, 2016) and six species are recognized here within the I. mixtilis group. The addition of RPB2 sequences to the ITS data yielded a more robust phylogeny, especially concerning deeper nodes. Our analyses confirmed also that a considerable amount of sequences remain being erroneously identified in GenBank (Ryberg et al. 2008). Thus, sequences of at least I. ceskae and I. occulta have been named as I. mixtilis in GenBank (Table 1).

The six species recognized in this study are rather well delimited using morphological, ecological and distributional characters. Among morphological characters, spore shape and cystidial length/shape, pileus colour and viscidity and the presence of velipellis were useful in species identification, but some characters overlap between close species. Thus, I. mixtilis, I. ceskae and I. occulta, distinct species according to our phylogenetic analyses (Fig. 1), cannot always be reliably identified through morphological species recognition and we consider them ‘semicryptic’ species. From a molecular point of view, these taxa are regarded as distinct phylospecies (Wilkins 2009), constituting three diagnosable, monophyletic entities that represent unique lineages in the complex. The term semicryptic has been applied to species subsumed under a collective epithet and having overlapping morphological characters (Bickford et al. 2006), as in the I. mixtilis group and also used in similar cases concerning some ‘telamonioid’ Cortinarius groups by Brandrud et al. (2015).

Interestingly, our phylogenetic analyses (Fig. 2) show certain patterns in host-specificity and distribution, but conclusions should be taken cautiously as our sampling includes a small representation of the potential diversity of the I. mixtilis group. Two species, I. ceskae and I. occulta have a transoceanic distribution, the former being apparently restricted to subboreal and boreal forests. On the contrary, I. mixtilis is so far only known in Europe and Asia, but examination of further specimens is needed to confirm this. In general, species of the I. mixtilis group seem to have a broad host-specificity since all species but I. johannis-stanglii seem to establish ectomycorrhizal association with both conifers and angiosperms, as earlier observed for other groups of Inocybe (Ryberg et al. 2011, Esteve-Raventós et al. 2016).

This study represents one more example of cryptic speciation in Inocybe. The use of the ITS region and comparative morphological studies were useful to disentangle the I. mixtilis complex also in this case and allowed us to make a step further towards a better understanding of this ecologically important and diverse genus of ectomycorrhizal fungi.