Molecular and morphological diversity in the /Rhombisporum clade of the genus Entoloma with a note on E. cocles

A combined morphological and molecular genetic study of the European species within the /Rhombisporum clade of the genus Entoloma reveals a high species diversity. This group comprises typical grassland species with pronounced and well-differentiated cheilocystidia, and a wide range of spore shapes varying from rhomboid to five-angled. To fix the concept of the classical species E. rhombisporum, a neotype is designated. Nine species are described as new to science based on the result of nrDNA ITS phylogeny with additional gap coding, and morphological characterization: E. caulocystidiatum, E. lunare, E. pararhombisporum, E. pentagonale, E. perrhombisporum, E. rhombiibericum, E. rhombisporoides, E. sororpratulense, and E. subcuboideum. The ITS sequences of the holotypes of previously described species belonging to the /Rhombisporum clade, viz., E. laurisilvae and E. pratulense have also been generated and are published here for the first time. Since many of the above-mentioned species have been misidentified as E. cocles, it seemed opportune to also study this species and to designate a neotype to fix its current concept. A key including European species is presented. As most of the species are potentially important indicators for threatened grassland communities, the 130 ITS barcodes newly generated for this study may be useful as a reference in conservation and metabarcoding projects.


Introduction
described Leptonia rhombispora, a new entolomatoid species from a subalpine meadow in the French Alps, with rather peculiar spores with a more or less rhomboid outline; and it was incorporated in the famous Flore analytique des Champignons supérieurs de France (Kühner and Romagnesi 1953). Since then, this species has been accepted in mycological literature (e.g., Horak 1976; N o o r d e l o o s 1 9 8 0 , 1 9 8 7 , 1 9 9 2 ) . R h o d o p h y l l u s prismatospermus, another species with more or less similar spores, was added by Romagnesi (1974), and Noordeloos & Wölfel (Noordeloos 1987) described a variety of E. rhombisporum with cystidia all over the length of the stipe as E. rhombisporum var. floccipes. The current phylogenetic study based on the nrDNA ITS region revealed that the European species with rhomboid spores cluster together in a well-supported clade but mixed with species with five or sixangled spores. All species, however, are characterized by the presence of well-differentiated cheilocystidia. The /Rhombisporum clade can therefore be characterized as follows: habit mycenoid or collybioid, with a conicalcampanulate to convex pileus, often with a truncate-slightly depressed centre, hygrophanous or not hygrophanous, glabrous or radially fibrillose to rugulose pileus; adnateemarginate lamellae, often with a distinct decurrent tooth (becoming sometimes arcuate-decurrent in expanded pilei); and a polished, pruinose or fibrillose stipe. Spores (irregularly) rhomboid or 5 (-6) angular in side-view; cheilocystidia always present, typically lageniform, sometimes partly clavate-mucronate to fusiform, often thin-walled and easily collapsing, sometimes, however, rather large and conspicuous, and with thickened walls. The pigment in the pileipellis is exclusively intracellular. Brilliant granules are absent. Caulocystidia are sometimes present in form of fusiform to lageniform cystidia; otherwise, no caulocystidia could be found or just some undifferentiated, subcylindrical terminal elements of the hyphae of the stipe surface. Clampconnections are present or absent. According to Pegler and Young (1979), the spores of E. rhombisporum are 7-faceted, and not cuboid, as they are sometimes incorrectly called. The /Rhombisporum clade, however, is phylogenetically distant from the true cuboidspored /Cubospora clade (Karstedt et al. 2019), as well as from the Entoloma conferendum group in the /Nolanea clade with cruciform spores. The name E. cocles very often has been applied to specimens of the /Rhombisporum clade, and for that reason it has been decided to fix this species here with a neotype and give a full description. Nine new species are described in the present study which reveals the remarkable diversity in the /Rhombisporum clade. Many species treated here are characteristic for old, semi-natural grasslands, a critically endangered vegetation type all over Europe (e.g., Nitare 1988;Griffith et al. 2013), and therefore we discuss some ecological and conservational aspects.

Morphological study
The base of this study is formed by material collected in the field by the authors, and the sequenced exsiccates from various institutions. Freshly collected specimens were photographed in the field, where also much attention was paid to characterize the surrounding vegetation and ecology. The material was described straight after collecting to fix the ephemeral macroscopic characters, then dried and stored. The state of the sequenced exsiccates varied, some were well-annotated, others not. All the available material was studied microscopically, either by the collectors, or by the first author (MEN) during visits to the botanic garden and herbarium in Oslo. The second author (GMJ) studied a large set of exsiccates with a Leica DMLS microscope, using a drawing tube and a Touptek Phototronics camera and a Zeiss Axiophot microscope with DC controlled Cree XP-G3 R3 CRI 90+ LED illumination, Plan Neofluar objectives 40×/1.30 Oil, 100×/1.30 Oil, differential interference contrast (DIC) and 12MP Touptek video camera with SONY Exmor IMX226 CMOS sensor, Toupview advanced video & image processing application. Spores, basidia and cystidia were observed in squash preparations of small parts of the lamellae in 5 % KOH or 1 % Congo Red in concentrated NH 4 OH. The pileipellis was studied by examining a radial section or a scalp in water, 5% KOH or ammonia. Basidiospore dimensions are based on observing 20 spores, and cystidia and basidia dimensions on observing at least 10 structures per collection. Basidia were measured without sterigmata, and the spores without hilum. Spore length to width ratios are reported as Q.

Molecular genetic study and phylogenetic reconstruction
DNA extraction, PCR amplifications and sequencing were performed in the Norwegian Barcode of Life (NorBOL) or followed Larsson and Jacobsson (2004), Larsson et al. (2018), Alvarado et al. (2015), Dima et al. (2016), Morozova et al. (2018), Reschke et al. (2018), and Voitk et al. (2020a). The primer pairs ITS1F/ITS4, ITS1F/ITS4B, ITS1F/ITS2 and ITS3/ITS4 (White et al. 1990; Gardes and Bruns 1993) were used to amplify and sequence the complete or partial nrDNA ITS barcoding region. Chromatograms were checked and edited with the CodonCode Aligner package (CodonCode Corp., Centerville, Massachusetts, USA). Sequence comparison with public and own databases followed Noordeloos et al. (2017). Our dataset is composed of 141 nrDNA ITS sequences, carefully selected after an initial analysis using published and all our unpublished ITS sequences (data not shown). Newly generated sequences were submitted to GenBank (Table 1). The dataset was aligned in MAFFT online v7 (http://mafft.cbrc.jp/alignment/server) choosing the E-INS-I method (Katoh and Standley 2013) with setting the scoring matrix for nucleotide sequences to 1PAM / K=2, gap opening penalty to 1.0 and offset value to 0.123. The alignment was checked and edited in SeaView 4 (Gouy et al. 2010). Based on the work of Nagy et al. (2012), phylogenetically informative indels were coded as presence/absence data with FastGap 1.2 (Borchsenius 2009) following the simple indel coding algorithm (Simmons et al. 2001). Maximum Likelihood (ML) phylogenetic reconstruction was performed in raxmlGUI (Silvestro and Michalak 2012) using rapid bootstrap analysis with 1,000 replicates. Three nucleotide partitions (ITS1, 5.8S, ITS2) were set to the GTRGAMMA substitution model in addition to one binary partition (indel characters) that was set to default.
MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) was used to perform Bayesian Inference (BI) phylogeny. The alignment was divided into four partitions (ITS1, 5.8S, ITS2, and indels): the GTR + Γ substitution model was applied to the nucleotide characters, while the two-parameter Markov model was set for the indels. Two independent runs of four Markov Chain Monte Carlo (MCMC) were performed each for 5 000 000 generations, sampling every 1000th generation. The first 35% of the trees was discarded as burn-in. For the remaining trees, a 50% majority rule consensus phylogram with posterior probabilities as nodal supports was computed. The phylogenetic trees were edited in MEGA 7 (Kumar et al. 2016) and Adobe Illustrator CS4.   The /Rhombisporum clade received strong to high support (MLBS=89 / BPP=1) in our analyses with Entoloma cocles placed basally. The /Rhombisporum clade consists of 16 lineages of which most of them can be regarded as species supported by phylogenetic and additional morphological evidence. The exception is E. aff. caulocystidiatum represented by a single sequence which only slightly differs from E. caulocystidiatum and needs further study. Two single branches represent North American species yet without a name. The remaining thirteen lineages represent European species of which nine are described here as new to science. All species are monophyletic and gained high statistical supports ( Figs. 1 and 2), except E. caulocystidiatum which was supported only moderately by the ML analysis (MLBS=70).

Characters used for species delimitation
The macromorphology of the species in the clade is rather uniform: most species share a more or less conico-truncate pileus, usually with a slight umbo, a slightly to distinctly hygrophanous pileus which is indistinctly or distinctly translucently striate, and not entirely smooth, but covered with innate fibrils, that sometimes give the surface a lustrous to slightly fluffy appearance; the adnate-decurrent, arcuate lamellae, and a stipe that is often much paler than the pileus, with either a hyaline, polished or innate silvery fibrillose surface. The spores differ in size and shape, ranging from either Fig. 3 Entoloma rhombisporum. a habit; b spores; c-d cheilocystidia (all from epitype); e habit (from LE 312533)); f habit (from KaiR1208). Bars = 10 μm rhomboid, 4 to 5(-6)-angled in side view, more or less isodiametrical to heterodiametrical. Cheilocystidia are always present, and generally in majority lageniform. They can be very thin-walled, slender and rather inconspicuous and easily collapsing, or robust, with relatively thick walls, and with a rather broad basal part, very obvious and protruding from the hymenium. Rarely there are also pleurocystidia, similar to cheilocystidia or substantially smaller, but usually only near the lamella edge. Some species lack clamp-connections, in other species either only the basidia have clamp-connections, or they are found in several tissues. In a search for new diagnostic characters, a very distinct character was discovered during our study: in E. pararhombisporum the stipe covering has very abundant and distinct congophilous plaques in the hyphal wall, a seemingly constant and rather striking character that was not encountered in any of other species within this clade, nor is it known from other Entoloma species or other agarics. It is therefore considered of potential diagnostic value, and supports the phylogenetically distinct position of that species.  yellowish red-brown, absolutely glabrous or even shiny, finely fibrillose with sometimes rugulose at centre. Context very thin. Smell weakly spermatic or reminiscent of onion. Lamellae, L = 19-23, l = 3, moderately distant, pinkish flesh-colour (not brown), not decurrent, ventricose, sinuate, adnate but not broadly so. Stipe 35-50 × 1.5-3.5 mm, equal, with white mycelium at base, whitish, hyaline, glabrous or slightly white pruinose at apex, almost stuffed and very fragile. Spores short, 9.2-11.5 × 8-10, average 10 × 8.7 μm, more or less cuboid or 5-angled.
Habitat and distribution: In small groups in semi-natural grasslands and in the margin of subalpine, calcareous bogs, in boreal and subalpine regions of Europe. Distribution poorly known, due to the unexpected diversity in this group. Sequenced material is known from Norway, Sweden and Russia.
Comments: We have interpreted Entoloma rhombisporum here as closest to the original description as possible, i.e., as a species with predominantly rhomboid (4-sided) spores, and thin-walled, easily collapsing, scattered to fairly abundant lageniform cheilocystidia. In his comments on the species, Kühner in Kühner and Romagnesi (1953) added some more information that helped us to interpret this species correctly: spores 8.5-12.2 × 7-9.7 μm, seemingly 4-sided in profile and side-view (rhomboid sensu lato). Cheilocystidia are inconspicuous, but rather frequent, hidden among the basidia and not protruding from the hymenium, ventricose-fusiform or clavate-mucronate with rather blunt and short appendix, thin-walled. Stipe finely powdered at apex, downwards grey-brown, horn brown or almost white; pileus rather sordid greyish brown, sometimes with slight yellow tinge, distinctly translucently striate, blunt, subumbilicate; pigment brown, intracellular. Clamp-connections present, but not at all septa. Lamellae pallid, without grey tinge. Smell spermatic. Taste distinctly farinaceous. Praz de St Bon, Savoie, France. No type has been indicated, nor is original material preserved.
The following characters are therefore considered distinctive for E. rhombisporum: a translucently striate pileus, almost white, hyaline stipe, spores 4-angled in profile and side-view, and inconspicuous cheilocystidia. As such, the description above fits best with the original diagnosis. The interpretation of E. rhombisporum by Horak (1976), based on a collection by Favre, and accepted by Noordeloos (1980) refers to another species in the clade with a non-translucently striate pileus, yellow to brown fibrillose stipe, slightly smaller spores and conspicuous cheilocystidia, and is described below as a new species (see E. perrhombisporum). In the ITS region, E. rhombisporum differs from E. rhombiibericum and E. subcuboideum by 28 and 29 substitution and indel positions, respectively, with a similarity of 95%.
Additional specimens examined: Norway, Nordland, Grane, Holmvassdalen, on shallow soil over limestone at the edge of a rich fen, 3 Etymology: Refers to the resemblance to Entoloma rhombisporum Diagnosis: Entoloma perrhombisporum is characterized by the rather regularly shaped, mostly quadrangular spores in sideview and the rather pronounced, thick-walled cheilocystidia.
Habitat and distribution: Terrestrial in extensively grazed meadows on calcareous soil. Sequenced collections are from the county of Nordland in Norway, and Jämtland in Sweden, in hot-spot sites with many calciphilous Entoloma species.
Comments: Entoloma perrhombisporum fits very well with the interpretation of E. rhombisporum by Horak (1976) and Noordeloos (1980), but, as discussed above, this does not agree with the protologue of that species. Entoloma perrhombisporum differs from its closest species E. caulocystidiatum by 36 substitution and indel positions in the ITS region, with a similarity of 94%.
Additional Etymology: Refers to the spores appearing partly almost cuboid.
Habitat and distribution: In small groups in various types of habitats, most records are from semi-natural grasslands (pasture grazed by sheep, slightly to distinctly calcareous), but also found in rich, open and swampy fir (Abies alba) forest vegetation, calcareous herb rich forest and rich fen margins. Except for a coastal, lowland find in SW Norway and Teletskoye Lake shore (Altai Republic), the species was up to now only found at higher altitudes, in C and S Europe in montane-subalpine zones, and in N Europe mainly in northern boreal (and low alpine) zone(s). Widespread species, known from Austria, Germany, Norway, Spain (Pyrenees), Sweden and the Altai Republic in Asian Russia.
Comments: This is apparently the most frequent species within the /Rhombisporum clade, with a wide distribution and a wide habitat spectrum. The species can be distinguished  Etymology: Refers to the resemblance with Entoloma rhombisporum Diagnosis: Entoloma pararhombisporum is differentiated from E. rhombisporum and E. subcuboideum particularly by the combination of a more greyish tinged pileus, fibrillose stipe, the voluminous and easily collapsing cheilocystidia, and the congophilous plaques in the stipitipellis.
Habitat and distribution: In semi-natural grasslands, roadsides and lawns on calcareous soil, and low herb and tall herb vegetation in calcareous forests. So far verified from mainly northern regions in Norway (many sequences), Austria, Russia, and Sweden.
Comments: A particular feature observed in some collections of this species is the presence of minute congophilous plaques (red spots) in the hyphal walls of the stipitipellis, a phenomenon not known to exist in other species of this clade. One collection (KaiR1224) resembles E. caulocystidiatum, because of the abundant caulocystidia, but these are present only at apex, and differently shaped and less voluminous than in that species. Apart from the congophilous plaques seen in some collections, E. pararhombisporum is very similar to E. subcuboideum. Entoloma pararhombisporum differs from its closest species Entoloma sp. from North America (GenBank: MK580835) by 25 substitution and indel positions in the ITS region, with a similarity of 95%.
Habitat and distribution: in a calcareous grassland, Norway. Comments: Entoloma cf. prismatospermum has by far the smallest spores of the /Rhombisporum clade, which tend to be more or less rhomboid (and sometimes more or less triangular in the holotype). The holotype has been studied morphologically (Noordeloos 1980), but DNA extraction and PCR amplification of the ITS region have so far failed. We think, however, that the Norwegian collection fits well with the original concept of the species. We refer to this collection as E. cf.
prismatospermum. It differs from its closest species E. rhombisporoides by 28 substitution and indel positions in the ITS region, with a similarity of 95%.
Habitat and distribution: In open, dry, calcareous grasslands, often grazed or mown, often in vicinity of sea-shores or along riverbanks. Known from Germany, Norway, Russia and the Netherlands (from environmental DNA sample).
Comments: Entoloma rhombisporoides differs from its closest species E. caulocystidiatum by the presence of caulocystidia at apex only, and a rather polished stipe surface, and at least 13 substitution and indel positions in the ITS region, with a similarity of 97%. Holotypus : Germany, Thuringia, Warza, 13 Oct 1984, F. Gröger, 211/1984. GenBank No. OL853707 (ITS).
Etymology: The name refers to the cystidiate surface of the stipe.
Diagnosis: The species is characterized by the flocculose stipe (apex) with well-developed caulocystidia.
Habitat and distribution: Gregarious in pastures, in road sides, the type in xerophytic grassland on calcareous soil. Known to occur in Austria, Estonia, Germany, Norway and the Netherlands (from environmental DNA sample).
Comments: Entoloma caulocystidiatum resembles E. rhombisporum, but differs by the floccose stipe with abundant caulocystidia, darker colours, abundant cheilocystidia, and slightly larger spores. The ITS sequence of holotype of Entoloma rhombisporum var. floccipes belongs to this wellseparated species, but the name cannot be used as a basionym for a combination to species level, since two herbaria were indicated in the protologue (Noordeloos 1987) and therefore, based on ICN Art 40.7 (Turland et al. 2018), the name of this variety is invalid. A full description and line drawings can be found in Noordeloos (1987Noordeloos ( , 1992. Entoloma caulocystidiatum differs by six substitution and indel positions from its sister lineage, tentatively named as E. aff. caulocystidiatum, represented by a single sequence. Whether this latter belongs to E. caulocystidiatum needs further detailed study. Otherwise Entoloma rhombiibericum Vila, Dima & Noordel.,sp. nov. Fig. 10 MycoBank: MB 842132 Holotype: Spain, Catalonia, Barcelona, Osona, Vidrà, near Turó del Pla de la Creu, 20 Sept 2008, J. Vila, F. Caballero, JVG 1080920-6 (L-0607540). GenBank No. OL853779 (ITS).
Etymology: Refers to the placement in the /Rhombisporum clade and the fact that it is the only species originally described from Southern Europe, specifically from the Iberian Peninsula.
Diagnosis: Entoloma rhombiibericum differs from E. caulocystidiatum by the darker, greyish brown colour of the pileus, which is only indistinctly translucently striate, and the considerably shorter cheilo-and caulocystidia, which are present at apex of stipe only. Its phylogenetic position is rather distant from E. caulocystidiatum.
Habitat and distribution: Terrestrial in montane (1340 m asl.), subatlantic shrub vegetation with Buxus sempervirens, Rubus caesius, and Sorbus aria, on calcareous soil. So far only known from NE Spain.
Comments: This species is well-supported phylogenetically and has some distinct micromorphological features, and is thus here described as new, although it is so far only known from the type locality with box (Buxus) shrubs. Entoloma rhombiibericum differs from its closest species E. caulocystidiatum by at least 22 substitution and indel positions in the ITS region, with a similarity of 96%.
Habitat and distribution: terrestrial in extensively grazed semi-natural grassland on calcareous soil. Only known from two collections at the type-locality in a coastal site in northwestern Norway.
C o m m e n t s : E n t o l o m a l u n a r e f i t s w e l l i n t h e /Rhombisporum-clade. The basidiomata seem to be paler than those of E. pentagonale. Microscopically both species are rather similar, except for the occurrence of caulocystidia in E. lunare. Entoloma lunare differs from its closest species E. pentagonale by 22 substitution and indel positions in the ITS region, with a similarity of 96%.
Habitat and distribution: In semi-natural and natural grasslands, hayfields and meadows, preferably on calcareous soil, but also found once in a calcareous forest, fruiting from August to October. Rare, but widespread, in subalpine (northern boreal) and alpine regions, rarely in the lowlands, apparently with a transatlantic distribution. Sequenced records are from Norway, Sweden (type) and Canada, nonsequenced records are from Austria, Italy, Great Britain, and Sweden.
Comments: Entoloma pratulense differs in habitat preferences from the other members of the group, being concentrated to alpine and subalpine regions. Entoloma pratulense and E. sororpratulense make up a sister clade/lineage to the /Rhombisporum clade in the strict sense. Although genetically close, the pratulense-sororpratulense lineage is distinguished from the rest of the /Rhombisporum clade by the normally non-rhomboid spores (usually 5-6 angled). Furthermore, the two are characterized by very long cheilocystidia. For distinguishing between E. pratulense and E. sororpratulense, see comments on the latter. Entoloma pratulense differs from its sister species E. sororpratulense by 20 substitution and indel positions in the ITS region, with a similarity of 96%.
Comments: Morphologically, E. sororpratulense can apparently be distinguished by the heterogeneous lamellae edge (not completely sterile), and the more wavy-irregular cheilocystidia. Apart from the small differences in morphology, E. sororpratulense could be distinguished from its sister E. pratulense on habitat-preferences. Entoloma sororpratulense is widely distributed in the coastal and lowland regions of Norway, and lower altitude habitats in Sweden and Germany, whereas E. pratulense is a predominantly boreal/subalpine/alpine species. Entoloma sororpratulense has formerly been treated as part of E. pratulense s.l., but is well-separated genetically, differing from E. pratulense by 20 substitution and indel positions in the ITS region, with a similarity of 96%.
Norway, DD (data deficient) in Sweden, and CR (critically endangered) in Austria and Denmark (see http://www.eccf.eu/). The species treated here thus deserve high attention in conservation biology and habitat management.