Cuphophyllus monteverdae and C. hygrocyboides (Hygrophoraceae, Agaricales) in Norway and Sweden

Two poorly known and relatively rare wax-caps Hygrocybe monteverdae and Cupho-phyllus hygrocyboides are described, with notes regarding their distribution and ecological preferences in Norway and Sweden. H. monteverdae is a whitish species originally described from the Canary Islands, characterized by the darkening lamella upon drying. It resembles a slender C. pratensis in habitus but is whitish. C. hygrocyboides also resembles C. pratensis in both colour and habitus but can be separated based on morphology. C. hygrocyboides is found in calcareous semi-natural grasslands, especially pastures, and also in the low alpine zone and higher alpine vegetation. It is not found below the mid-boreal zone even if it has been found at sea level in the northern part of Norway. H. monteverdae seems to be a strict lowland species found in semi-natural grasslands, alvar vegetation, and open grazed forests on calcareous ground. Phylogenetic analysis


INTRODUCTION
Cuphophyllus (Donk) Bon is a genus belonging to Hygrophoraceae Lotzy, with species distributed both in the northern and southern hemispheres.Lodge et al. (2014) showed that Cuphophyllus occupied a relatively isolated phylogenetic position in the family.Several species in the genus have a broad distribution and occur from the nemoral to the arcticalpine zones in Europe (Boertmann 2010).Most European species differ from the ones occurring in North America, but there are exceptions and these are especially found among species with a northern boreal to arcticalpine distribution range, e. g. C. atlanticus (Jordal & Larsson 2021) and C. hygrocyboides (Voitk et al. 2020).
The species in Cuphophyllus are characterised by having clitocyboid basidiomata with thick decurrent lamellae and a white spore print.In micro-morphology they have an interwoven or rarely almost subregular lamellar trama, with or without a regular or subregular central strand; smooth, hyaline, inamyloid basidiospores; very long basidia relative to spore length (usually 7-8, rarely 5-6 times the spore length), and a basal clamp on the basidia (Lodge et al. 2014).An interwoven lamellar trama, together with large basidia to spore length ratio are the most reliable characteristics for separating Cuphophyllus from other white-spored agaric genera.Species of Cuphophyllus are regarded to have a biotrophic mode of nutrition, but the nature of the fungus-plant association is largely unknown (e.g.Halbwachs et al. 2018).
Hygrocybe monteverdae Bañares & Arnolds was described from La Palma, Canary Islands, where it was growing on soil among leaves of Laurus azorica, Persea indica, Ilex can-ariensis and Dryopteris oligodonta in humid "monte-verde" forest (Bañares & Arnolds 2002).In this study, we show that the generated ITS sequence data of the holotype of H. monteverdae is identical with ITS sequence data of collections from Norway and Sweden often labelled as Cuphophyllus pratensis var.pallidus (Berk.& Broome) Bon.
Cuphophyllus hygrocyboides (Kühner) Bon was originally described as Camarophyllus hygrocyboides by Kühner (1977) from environments of Pralognan-la-Vanoise in France, growing among Salix herbacea and S. reticulata in the alpine zone.In association to the work on the volume on Hygrophoraceae of Flora Agaricina Neerlandica it was combined to Hygrocybe by Arnolds (1987).In the study by Voitk et al. (2020) sequence data of the holotype of H. hygrocyboides (Kühner) Arnolds was included in the phylogenetic analyses and confirmed that the species belong in Cuphophyllus.Here we present further data of the species both from Norway and Sweden.

MATERIAL AND METHODS
Fresh basidiomata were photographed in situ and the habitat was noted and described.Detailed observations of macro-morphological characteristics were made on fresh and photographed material together with field notes.Micro-morphological characteristics were observed and measured from dried material dehydrated in 3% KOH and ammoniacal Congo red solution at 1000× magnifications using a Zeiss Axioskop 2 microscope and ZEN imaging software (Zeiss).A minimum of 20 spores were measured from each basidioma, abnormally large or small spores were not considered.Spore measurements exclude apical appendage.Basidial measurements exclude sterigmata, and the sterigmata were measured separately.
The nuc rDNA ITS1-5.8S-ITS2(ITS barcode) and partial LSU sequence data of 24 specimens of Cuphophyllus, including the holotype of H. monteverdae were newly generated for this study.Permission to extract DNA of the holotype was granted by the staff at Herbarium TFC.Methods used for DNA extraction, PCR and sequencing of the ITS region follow Jordal and Larsson (2021).Primers used to amplify the 5´end of the LSU were LR0R and LR7, and primers used for sequencing of the region were Ctb6, LR5 and LR3R (Hopple and Vilgalys 1999).Sequences were edited and assembled using Sequencher 5.1 (Gene Codes, Ann Arbor, Michigan).The sequences have been deposited in GenBank (ON117573-ON117594).
The ITS-LSU data set of Cuphophyllus for this study was compiled based on the results from previous studies of Cuphophyllus (Crous et al. 2021, Jordal and Larsson 2021, Lodge et al. 2014, Voitk et al. 2020).Beside the newly generated data, ITS and LSU data of 15 species in Cuphophyllus was included.Hygrophorus was selected as representatives of Hygrophoraceae and two sequences of Lepista were used as outgroup in the analyses.The ITS of the target species in this study was blasted in GenBank (Clark et al. 2016) and the UNITE database (Kõljalg et al. 2013) to seek additional available sequence data.One additional ITS sequences of C. monteverdae originating from United Kingdom was found and added to the data set.
Alignment of the data set was performed using the L-INS-i strategy implemented in MAFFT 7.017 (Katoh and Standley 2013).The alignment was adjusted using ALIVIEW 1.17.1 (Larsson 2014).For inference of phylogenetic relationships of the dataset, heuristic searches for the most parsimonious trees were performed using PAUP* under the maximum parsimony (MP) criterion (Swofford 2003).All transformations were considered unordered and equally weighted and gaps were treated as missing data.Heuristic searches with 1000 random-addition sequence replicates and TBR branch swapping were performed.Relative robustness of clades was assessed by the bootstrap (BT) method using 1000 heuristic search replicates with 10 random taxon addition sequence replicates and TBR branch swapping, saving 100 trees in each replicate.

RESULTS
The aligned ITS and LSU dataset consisted of 45 sequences and 2386 characters.After exclusion of ambiguous data, mainly from the beginning and the end of the data set, and from the ITS region, 2161 characters remained for the analysis.Of these, 1523 were constant, 127 were variable but parsimony uninformative, and 511 were parsimony informative.The MP analysis yielded 1825 equally most parsimonious trees (length = 1736 steps, CI = 0.5847, and RI = 0.7843).One of these trees is presented in Fig. 1
Ecology: The species is originally described from humid deciduous forest "monte verdae" on the Canary Islands growing among leaves.In Norway it is mainly associated with semi-natural grasslands, growing among mosses, herbs and grasses with the soil at least moderately calcareous.Most localities are situated in the boreonemoral (hemiboreal) to southern boreal vegetation zones near the coast, often on calcareous marine depositsshell beds.In Sweden most collections originate from the calcareous islands of Öland and Gotland where it was found growing among grass and mosses in grazed open coniferous forest, grazed and manured meadows and alvar vegetations.From the mainland of Sweden, it has been found in the southern parts and on the west coast on grazed and semi-natural grasslands with long continuity and more calcareous soils.
Distribution: The species seems to have a wide distribution in the northern parts of Europe, beside Spain (Canary Islands) it is confirmed from Estonia, Norway, Sweden and United Kingdom.

Comments on distribution, ecology and threats
Cuphophyllus monteverdae is a species that we rather recently have been aware of and recognize, so the actual distribution and occurrence is still a bit unclear.One aim with this paper is to recognize the species and make people aware of it and hopefully report it during coming field seasons.The darkening lamellae upon drying and the non-translucent pileus are good discriminating characters that differ it from white forms of C. pratensis and species in the C. virgineus group.It seems mainly to be associated to semi-natural grasslands, grazed and manured meadows and alvar vegetations on calcareous soils.On the calcareous islands Gotland, (Sweden) and Saaremaa (Estonia) it is also found in grazed herb rich Picea forests.Cuphophyllus monteverdae is included as DD (data deficient) in the red list of Sweden (SLU Artdatabanken 2022b) and in the Norwegian red list as VU (vulnerable) (Artsdatabanken 2021).Habitats where it is found is at risk of declining, as old treatments of land use have changed.Nature conservation action by moving meadows and apply grazing by sheep and cows is needed to keep many of these localities open and suitable for the species as well as other grassland fungi.
Below we provide some additional comments base on the Norwegian and Swedish collections: The pileus of C. hygrocyboides is normally brightly to dark orange-brown to apricot, also with bright, golden yellow colours, while C. pratensis has more dull orange to brown colours without yellow or golden tinges.The pileus surface in C. pratensis is normally rather dry, while in C. hygrocyboides it is usually weakly lubricous in fresh specimens and may appear shiny in fresh specimens, like in Fig. 3A.The stipe is warm brownish or orange-brown, while in C. pratensis it is whitish, cream or pale buff yellow.In addition, the basidiomata of C. hygrocyboides are on average smaller than those in C. pratensis.
The photo from the alpine area in Sweden (Fig. 3C) shows a rather compact fungus with a short stipe, resembling the photo in Boertmann (2010) from alpine habitat in France.The photo from boreal grasslands in Norway, Vågå (Fig. 3A) shows a mushroom with a longer and slender stipe than in the alpine specimens, and it is more slender than it usually is in C. pratensis.The spores of C. hygrocyboides are ellipsoid to ovoid and longer (7,5-10 µm) compared to the spores of C. pratensis (5,5-7,5 µm) that are more broadly ellipsioid to subglobose.The Q value is on the average 1,5-1,8 in the former and 1,2-1,5 in the latter.Differences are also found in the pileipellis that is more of a gelatinised cutis in C. hygrocyboides and a dry cutis in C. pratensis (own observations and Borgen & Boertmann 2008).
Ecology: The species was described by Kühner (1977) from the alpine zone in France growing among mosses and dwarf Salix.In Sweden three of the four known localities are in alpine areas associated with base rich/calcareous vegetation, in Pite, Torne and Åsele lappmark, and one from semi-naturalgrasslands.In Norway all collections are from semi-natural grasslands, among mosses, herbs and grasses, on calcareous soil, localities situated from the mid-boreal to low alpine vegetation zones.
Distribution: Sequenced material is confirmed from Canada, France, Greenland, Sweden, and here we also add Norway.Kühner (1977) including the type locality (Salix reticulata is a species of calcareous soil), and by Corriol 2008 the locality in the French Pyrénées ("Arabidion caeruleae" is a basophilous subtype of Salix herbacea snowbeds.There are reasons to believe that the species is a rare.In Sweden four localities are known, three of them in the alpine zone and one from a managed lawn in the northern boreal zone.In Norway also four localities are known, all in semi-natural grasslands in midto northern boreal zone. Cuphophyllus hygrocyboides is included as VU (vulnerable) in the red list of Sweden (SLU Artdatabanken 2022a), as EN (strongly threatened) in the red list of Norway (Artsdatabanken 2021), and as VU (vulnerable) in a regional red list of the Midi-Pyrenées (Corriol 2014).In Fennoscandia we do not know how large part of the population that is living in semi-natural grasslands compared to alpine habitats.Semi-natural grasslands in boreal regions have been strongly declining over the last 100 years, and are still declining.Therefore, it seems reasonable to assume that at least the populations in mid-and northern boreal grasslands are declining and at threat.Measures to maintain boreal semi-natural grasslands will therefore be important both to C. hygrocyboides and other grassland fungi.Alpine medows, heaths and snowbeds may be threatened by climatic change, causing the snow to melt earlier and many snow-beds to dry out and change to other vegetation types.We see already how the tree-line is climbing upwards, even if areas with large population of grazing reindeers are less affected, there is an increased cover of bushes with Salix spp., Betula nana and Betula pubescens, where it some decades ago were open alpine heath and meadow areas.

Figure 1 .
Figure 1.Phylogram showing the phylogenetic position of Cuphophyllus monteverdae and C. hygrocyoboides based on ITS and LSU sequence data, where C. monteverdae comes out strongly supported as a sister species to the clade including C. virgineus, C. russocoreaceus and C. borealis.C. hygrocyboides take a more basal isolated position within Cuphophyllus.Bootstrap values are indicated on branches.Sequences originating from type specimens are marked.