Introduction

The coprinoid fungi are a highly diverse and polyphyletic group of the order Agaricales, which species show a unique set of characters: presence of dark spores, deliquescent basidiocarps that often undergo autolysis, and pseudoparaphyses in the hymenium (Uljé 2005). These species were previously classified in one genus, Coprinus Pers., but molecular studies revealed that the genus is split into four main lineages assigned to four genera: Coprinus s. s., Coprinellus P.Karst., Coprinopsis P.Karst., and Parasola Redhead, Vilgalys & Hopple (Hopple and Vilgalys 1999; Moncalvo et al. 2002; Padamsee et al. 2008; Larsson and Örstadius 2008; Nagy et al. 2009, 2010a, b, 2011, 2012, 2013a). The coprinoid fungi with a veil composed of smooth, (sub)globose elements, and lacking pileocystidia belonged to subsection Nivei of Coprinus s.l. (Citérin 1992; Uljé 2005), now included in the genus Coprinopsis (Hopple and Vilgalys 1999; Moncalvo et al. 2002). In Europe, about 20 species from this subsection were reported so far (Uljé 2005; Vesterholt 2012). It cannot be ruled out that at least some of them are probably collective species, e.g., Coprinopsis cortinata (J.E.Lange) Gminder (Uljé 2005). This view may be supported by the molecular analyses of Keirle et al. (2004), which revealed significant genetic diversity among the collections of C. cordispora (T.Gibbs) Gminder originating from Europe and Hawaii.

In this work, a distinct species of Coprinopsis is described on the basis of morphological and molecular analyses that differ from all other known species of the subsection Nivei of Coprinus s.l. (Citérin 1992; Uljé 2005) as well as from remaining recognized Coprinopsis species. This new species was collected several times in two different regions of Poland, indicating that unknown coprinoid fungi can still be found, even in relatively well-studied regions such as Europe.

Materials and methods

Specimen sampling, morphological analyses, and nomenclature of subsections

The study is based on dried herbarium specimens. The holotype is deposited in the fungal collection of the W. Szafer Institute of Botany,  Polish Academy of Sciences, Kraków, Poland (KRAM F), while isotype and remaining specimens are in the personal collection of the first author (BG). The material was studied by light microscope (LM) and scanning electron microscope (SEM). For LM, samples were rehydrated in aqueous ammonia solution (10%) and stained with Congo red (0.5% in aqueous ammonia). Observations, measurements, and micrographs were made using a Bresser Science TRM 301 light microscope. Drawings of microcharacters were made from micrographs taken with a Bresser MicroCam 5.0 digital camera. All measurements were taken directly through the light microscope under an oil immersion objective (100×). The spore dimensions were established from measurements of 50 randomly selected, well-formed spores (the deformed or atrophied spores were excluded from analysis) per collection. The 95% population limits for the mean were calculated, and the lower and upper values are given. For basidia and cystidia, the extreme size values were presented. For these structures, dimensions were obtained after measuring 25 elements. For SEM, fragments of dried fungal hymenium were glued onto carbon tabs and fixed to an aluminum stub with double-sided transparent tape. The stubs were sputter-coated with carbon using a Cressington sputter coater and viewed under a Hitachi S-4700 scanning electron microscope, with a working distance of ca. 12 mm. SEM micrographs were taken in the Laboratory of Field Emission Scanning Electron Microscopy and Microanalysis at the Institute of Geological Sciences of Jagiellonian University (Kraków). Color notation used in the description is according to Kornerup and Wanscher (1984). In the following text, the names of subsections (Alachuani, Lanatuli, Narcotici, Nivei) refer to morphological subsections of Coprinus s.l. as delineated by Uljé (2005).

DNA extraction, PCR amplification, and sequencing

Fungal DNA was extracted from a small part of basidiocarp representing the holotype of Coprinopsis rugosomagnispora. DNA was extracted using the GeneMATRIX Plant and Fungi DNA Purification Kit (EURx, Poland), following the manufacturer’s protocols. The final PCR reaction volume was 10 µl composed of 4 µl DNA template, 0.5 µl of each primer ITS5 and ITS4 (White et al. 1990), and 5 µl Type-it Microsatellite PCR Kit (Qiagen, Germany). PCR amplification was performed using the following thermocycling conditions: an initial denaturing step at 95 °C for 15 min, followed by 35 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final extension of 10 min at 72 °C. PCR product was visualized by running 5 µl DNA amplicon on 1.5% agarose gel for 30 min. The PCR product was sequenced at the Laboratory of Molecular Biology of Adam Mickiewicz University in Poznań. The obtained sequences fragments were checked, assembled, and manually edited using Geneious Pro, version 8.0. (Biomatters Ltd). The ITS sequence obtained in this study was deposited in the NCBI’s GenBank database (http://www.ncbi.nlm.nih.gov/genbank) with accession number KX276859.

Phylogenetic analyses

The ITS sequence from the holotype of Coprinopsis rugosomagnispora generated in this study was checked by a Blast search (Altschul et al. 1997) against the ITS sequences deposited in GenBank to reveal closely related species. Subsequently, the sequence was analyzed within a dataset covering all Coprinopsis species available in GenBank, reduced to one sequence per species (with the exception of Coprinopsis lagopus (Fr.) Redhead, Vilgalys & Moncalvo s.l.), and including sequences generated by experts of Coprinopsis taxonomy (Keirle et al. 2004; Navarro-González et al. 2006; Garcia and Vellinga 2010; Nagy et al. 2010a, 2011, 2013a, b; Geml et al. 2012; Osmundson et al. 2013; Ruiz et al. 2013; Örstadius et al. 2015; Elwess et al. 2016; and some unpublished sequences available in GenBank). A total of 85 sequences were aligned using MUSCLE algorithm and default settings (Edgar 2004) to obtain preliminary hypothesis on the phylogenetic placement of Coprinopsis rugosomagnispora (data not shown). For the final phylogeny, we reduced the dataset to 40 sequences representing lineage containing a new species and most closely related Coprinopsis species (from the subsection Lanatuli) and some more distantly related species (from other subsections, among others from the subsection Nivei). The three sequences of Agrocybe Fayod were chosen as outgroup following Nagy et al. (2013b). GenBank accession numbers are given in Fig. 1 and in Table 1. The alignment was deposited in TreeBASE (S20797).

Fig. 1
figure 1

Phylogenetic relationship among sampled Coprinopsis species, including the newly described Coprinopsis rugosomagnispora, based on Bayesian inference analyses of ITS sequences. The support values (ML—BP ≥ 60%, Bayesian analysis—PP ≥ 0.7) are placed above branches. Thickened branches represent clades well supported at least in one of the analyses (ML—BP ≥ 70%, Bayesian analysis—PP ≥ 0.9). The branch lengths denoted by double slash (//) were shortened (2×, 7×, 2×, 4×, respectively, starting from the top to the bottom of the tree) to improve visualization. The morphological subsections of Coprinus s.l. are demarcated with colored squares, and three Agrocybe species were included as outgroup. The species assigned to the so-called Atramentarii clade by Nagy et al. (2013a) are demarcated within lineage containing species showing characters of the morphological subsection Lanatuli

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Table 1 List of species used in the molecular analyses, with countries of origin, collector, voucher information, GenBank accession numbers (ITS), and references
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Phylogenetic analyses were carried out using maximum likelihood (ML) implemented in RaxmlGUI version 0.9 beta 2 (Silvestro and Michalak 2012) and Bayesian inference in MrBayes version 3.1.2 (Huelsenbeck and Ronquist 2003). The nucleotide substitution models were separately selected for each part of the ITS region (ITS1, 5.8S, ITS2) inferred with PartitionFinder version 1.0.1 (Lanfear et al. 2012) using the Bayesian Information Criterion (BIC) as optimality model criterion in a greedy search. In the likelihood analyses, a GTRGAMMAI substitution model and 1000 bootstrap resamples in separate partitions were performed, and for MCMC analyses, three independent parallel runs were started each with four incrementally heated (temperature = 0.15) chains. This Markov chain was allowed to run for 100 M generations sampling every 1000th tree and a 50% as the burn-in factor. Resulting phylogenetic trees were visualized in TreeView software (Page 1996).

Results

Morphological analyses

The morphological characteristics of new species, Coprinopsis rugosomagnispora, are included in the species description and depicted in section Taxonomic treatment.

Phylogenetic analyses

The current phylogenetic studies included 43 sequences: 40 sequences of Coprinopsis species and three outgroup sequences of Agrocybe species (following Nagy et al. 2013b), with 514 unambiguously aligned nucleotide positions. The consensus tree topology and branch supports were assessed under maximum likelihood (ML) and Bayesian inference analyses. Although the consensus tree topology under ML and Bayesian inference yielded a similar pattern, the Bayesian phylogenetic tree is presented in this study, in which several major Coprinopsis clades are clearly delimitated, and the ML bootstrap values and posterior probabilities are placed above moderately or well-supported branches (Fig. 1).

The molecular analyses revealed Coprinopsis rugosomagnispora as sister species to C. insignis (Peck) Redhead, Vilgalys & Moncalvo and C. fusispora L.Nagy, Vágvölgyi & T.Papp, members of the Atramentarii clade (Nagy et al. 2013a), which altogether formed a lineage showing strong support in the Bayesian analysis (PP = 0.99) and moderate support in ML analysis (BP = 64%). The clade is sister and basal to the large assemblage of 18 Coprinopsis species belonging to the subsection Lanatuli of Coprinus s.l. (Lange 1915; Uljé 2005). Coprinopsis goudensis (Uljé) Redhead, Vilgalys & Moncalvo, a member of the subsection Alachuani of Coprinus s.l., is nested inside this assemblage and therefore probably should be reassigned to subsection Lanatuli. The remaining sampled Coprinopsis species (sister to the lineage containing members of the subsection Lanatuli) were split into distinct groups: (1) Coprinopsis filamentifer (Kühner) Redhead, Vilgalys & Moncalvo (subsection Alachuani) that forms a long branch by its own, (2) a highly supported paraphyletic clade containing species with morphologies similar to members of the genus Psathyrella (Fr.) Quél. (we informally call this lineage ‘Psathyrella-like’), and (3) a not supported lineage including species from the subsection Nivei (Fig. 1).

Discussion

Many of the recently described coprinoid fungi differ only slightly from the earlier recognized species or are even indistinguishable without molecular data (Raut et al. 2011; Nagy et al. 2013a). By contrast, Coprinopsis rugosomagnispora described here has a unique and distinct combination of characters (Figs. 2, 3). Its macromorphological characters, such as small dimensions of basidiocarps and granular veil forming conical warts, are similar to that of Coprinopsis cordispora and morphologically similar species in the subsection Nivei. Likewise, the smooth veil elements suggest affinity of C. rugosomagnispora to members of this subsection. On the other hand, the structure (arrangement and shape) of veil elements in C. rugosomagnispora is intermediate between that of members in the subsections Nivei and Lanatuli. Specifically, veil elements are arranged in chains as in the subsection Lanatuli (in the subsection Nivei, veil elements are composed of spherical elements emerging from thin hyphae), but particular cells of veil elements are (sub)globose in shape, as in the subsection Nivei (in the subsection Lanatuli, the veil elements are cylindrical to fusiform). Remarkably, the molecular phylogenetic analyses indicate a relationship of C. rugosomagnispora with the species possessing morphological traits typical for the subsection Lanatuli. None of the described species of the subsection Nivei have ornamented spores. In the subsection Lanatuli, ornamented spores are characteristic only for C. insignis and C. calospora (Bas & Uljé) Redhead, Vilgalys & Moncalvo. Indeed, the molecular phylogenetic analyses indicated that C. rugosomagnispora and C. insignis are closely related (Fig. 1). In contrast, C. calospora is related to C. cinerea (Schaeff.) Redhead, Vilgalys & Moncalvo as shown by Nagy et al. (2013a). The well-developed episporium in Coprinopsis rugosomagnispora is not typical for species assigned to the subsections Lanatuli and Nivei but is characteristic for Coprinopsis species from the subsection Narcotici of Coprinus s.l. (Uljé 2005); however, ornamented spores are rare in that subsection (Uljé 2005). It is noteworthy that Coprinopsis rugosomagnispora is phylogenetically related to C. fusispora and C. insignis that were assigned to the so-called Atramentarii clade (Nagy et al. 2013a), despite of their morphological similarity to members of the subsection Lanatuli. Since the sectional and subsectional taxonomy within Coprinopsis is still not clarified, we tentatively retain with assignment of these species to the morphological subsection Lanatuli (Fig. 1).

Fig. 2
figure 2

Basidiocarps of Coprinopsis rugosomagnispora from the Puszcza Knyszynska forest, NE Poland: a, b general view; c lamellae; d, e young basidiocarps; f old specimen

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Fig. 3
figure 3

Microcharacters of Coprinopsis rugosomagnispora (holotype: KRAM F-58717): a spores; b, c ornamentation of immature spore surface; d basidia; e cheilocystidia; f, g veil from pileus surface; h veil from stipe surface. Scale bars 10 µm

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The spore ornamentation pattern of Coprinopsis rugosomagnispora is unique among coprinoid fungi. Some of the recently described or redescribed species have warts (C. echinospora (Buller) Redhead, Vilgalys & Moncalvo and related taxa, C. insignis) or nodules (C. calospora) (Uljé 2005; Raut et al. 2011; Fukiharu et al. 2011, 2013, 2014). Spores of Coprinellus species may have warts (Coprinellus silvaticus (Peck) Gminder) or low crest and rows composed from minute warts (Coprinellus verrucispermus (Joss. & Enderle) Redhead, Vilgalys & Moncalvo) (Uljé 2005; Nagy et al. 2012), while spores of Coprinopsis rugosomagnispora are densely pitted. This pattern is poorly visible on mature spores, due to their dark color, but it is easy to observe on immature spores when they are just red-brown in color. The ornamentation is also hardly visible on SEM micrographs, as the episporium is smooth and persistent, so only some darken areas or rarely hollows are present on the spore surface (Fig. 4). The spore size of C. rugosomagnispora is larger than in all known coprinoid taxa. Only Coprinus xerophilus Bogart and Coprinus sterquilinus (Fr.) Fr. have spores, which may be longer than 20 µm, but both these taxa differ by having a smooth spore surface and different veil structure (Uljé 2005). This makes the species newly described here easy to recognize, even if the collection is incomplete, namely autolysed and decayed.

Fig. 4
figure 4

SEM micrographs of the spores of Coprinopsis rugosomagnispora (holotype: KRAM F-58717): a, d general view; b ornamentation pattern; c germ pore close-up. Scale bars: 10 μm

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Taxonomic treatment

Coprinopsis rugosomagnispora Gierczyk, Pietras, Piątek, Gryc, Czerniawski & Rodriguez-Flakus, sp. nov.—TYPE: Poland, Podlaskie Province: Puszcza Knyszyńska forest, S from Supraśl village, on soil in mixed forest, 24 Aug 2013, M. Gryc s.n. (holotype: KRAM F-58717!; isotype: BGF/BF/MG/130824/0005!; GenBank: KX276859). [MycoBank no. MB 820057].

Etymology: The Latin rugoso refers to the ornamentation of the spores, magni refers to their large dimensions, and spora refers to spore.

Description: Basidiocarps small (Fig. 2). Pileus 5–10 × 4–8 mm, first subglobose to broadly ellipsoid, expanding to convex campanulate, up to 15 mm broad; surface radially striate to plicate, covered by mealy to granulose veil forming squarrose, conical to nipple-shaped warts, veil whitish (1B2) to ocher-brown (6D6). Lamellae free, crowded (L = 40–60), first whitish, then gray, finally black, deliquescent and undergoing autolysis with age. Lamellar edge strongly pruinose (Fig. 2c), white. Stipe 10–30 × 1–1.5 mm, white, covered by white fibrils, rather fragile, hollow. Stipe base equal, not rooting. Flesh very thin, white, odorless. Basidiospores (19.5–)21.0–21.5(–24.0) × (12.0–)13.0–13.5(–15.0) × (12.0–)12.5–13.0(–15.0) µm (av. 21.3 × 13.1 × 12.9 µm), Q = 1.56–1.64 (av. 1.60), black in mass, very dark red-brown (10G8) to almost black under light microscope, limoniform with prominent apiculus (apiculus length 1.3–2.0 µm) and with central, papillate germ pore 2.0–2.5 µm broad, not flattened (Figs. 3a, 4, 5a). Ornamentation of spore surface prominent, up to 0.5 µm high, covered with smooth, tight, persistent episporium. Ornament composed of hollows that make rugulose or pitted appearance of the spore wall (pitted spores; Figs. 3b, c, 5a). Basidia 4-spored, dimorphic, long type 52–56 × 16–17 µm, short type 34–38 × 15–16 µm (measured without sterigmata; Figs. 3d, 5b). Sterigmata 5.0–5.5 µm long. Cheilocystidia 50–100 × 32–60 µm, subglobose, broadly ellipsoid to broadly clavate (Figs. 3e, 5d). Pleuro-, caulo- and pileocystidia absent. Veil on the pileus composed of (sub)globose to broadly ellipsoid, smooth, thin-walled, colorless elements, 24–60 × 24–45 µm, and with scattered cylindrical hyphae up to 25 µm broad (Figs. 3f, g, 5e). Stipe covering made up of cylindrical hyphae, 35–85 × 10–30 µm, forming chains, not or moderately constricted at septa. On upper part of the stipe veil consists also of broadly ellipsoid elements up to 35 µm broad. Terminal elements of the stipe veil conically tapering with obtuse, 5–8 µm broad apex (Figs. 3h, 5c). Clamp-connections present.

Fig. 5
figure 5

Drawings of microcharacters of Coprinopsis rugosomagnispora (holotype: KRAM F-58717): a spores; b basidia; c veil from the stipe surface; d cheilocystidia; e veil from pileus surface. Scale bars 10 µm

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Diagnosis: Species characterized by a veil composed of smooth, globose elements in chains, 4-spored, large basidia and rugulose (pitted), limoniform spores up to 24 μm long, with a tight episporium.

Phenology: Specimens found between July and September.

Distribution area: Poland—Podlaskie Province and Małopolska Province. Coprinopsis rugosomagnispora-known localities in Poland are over 450 km apart (Fig. 6); therefore, it is not a local species and should occur in other parts of Poland as well as in other European countries. Further studies are needed to determine the distribution of Coprinopsis rugosomagnispora.

Fig. 6
figure 6

Known localities of Coprinopsis rugosomagnispora in Poland (∙)

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Habitats: Coprinopsis rugosomagnispora grows in forests. It has been found in both coniferous forest and mixed forest dominated by deciduous trees. At all three known stands, it was found on wet, muddy soils.

Additional specimens examined: Poland, Podlaskie Province: Puszcza Knyszyńska forest, 2 km S from Supraśl village, near Krasny Las forest ranger’s lodge, on muddy soil on dirt road side in mixed forest (Picea abies (L.) H.Karst., Alnus glutinosa (L.) Gaertn.), 7 Jul 2013, M. Gryc s.n. (BGF/BF/MG/130707/0002!), 24 Aug 2013, M. Gryc s.n. (BGF/BF/MG/130824/0005!), 1 Sep 2013, M. Gryc s.n. (BGF/BF/MG/130901/0002!); Poland, Podlaskie Province: Puszcza Knyszyńska forest, 6 km NE from Supraśl village, near on muddy soil on dirt roadside in mixed forest, Sep 2013, M. Gryc s.n. (BGF/BF/MG/130901/0002!); Poland, Małopolska Province: Pogórze Wielickie foothills, Jasienica near Sułkowice village, on clayed soil mixed with sandstone rubble in silver fir (Abies alba Mill.) forest, Aug 2010, W. Czerniawski s.n. (BGF/BF/WCz/100801/0002!).