Meriderma species (Myxomycetes) from the Polish Carpathians: a taxonomic revision using SEM-visualized spore ornamentation

Meriderma represents a recently described genus of nivicolous myxomycetes with high morphological variability. Due to many complications in its taxonomy and species recognition in the past, the group was considered a morphologically variable complex. Recent clarifications and recognition of morphological boundaries into species and morphotypes has fostered a classification revision of specimens found in the Carpathians. Material used in this study was systematically collected in the Polish part of the Carpathians from 2004 to 2009. As a result of microand macroscopic observations of 54 collections, we recorded nine taxa of Meriderma. Seven of these (all but M. carestiae and M. cribrarioides) are the first records for Poland and for the Carpathians overall. Our observations based on analysis of spore ornamentation by SEM are in accordance with recently proposed classification and confirm segregation of taxa based on spore ornamentation pattern.


Introduction
The genus Meriderma Mar.Mey & Poulain is a relatively new taxon described by Poulain et al. [1] that accommodate species of Lamproderma characterized by evanescent peridium and funnel-shaped capillitium ends.Initially, one species, Lamproderma atrosporum Meyl.described by Meylan in 1910 [2] belonged to the group.Later, Meylan [3] distinguished a few varieties and forms of Lamproderma atrosporum Meyl.and described L. fuscatum Meyl.as a separate species differing from black L. atrosporum by ferruginous brown sporocarps.American myxomycetologist Donald T. Kowalski [4] recognized L. cribrarioides as a species with evanescent peridium, closely related to Lamproderma atrosporum, but differing by featuring completely reticulate spores ornamentation.However, his interpretation was not supported by European researchers, who interpreted L. cribrarioides as a taxon with persistent peridium.On the other hand, Kowalski [4] misinterpreted L. fuscatum and considered it as a species with persistent peridium (see Ronikier et al. [5]).Due to high variability of general habit, spore size and ornamentation pattern observed already by Meylan [2,3], Neubert et al. [6] recognized L. atrosporum to be a species complex and this view was followed by other researchers (e.g., [7]).
Recent molecular analysis, based on 18S SSU rRNA sequences, confirmed that Meriderma represents a separate clade clearly divergent from Lamproderma group [12].
This study represents the analysis of Meriderma diversity in the Polish part of the Carpathians.Description and illustration of macro-and micromorphological characteristics, including scanning electron microscopy (SEM) images of spores showing every morphotype are provided as part of this research.At the same time, because of complicated taxonomic interpretations and nomenclatural changes made by different authors, detailed descriptions for each species and morphotypes are also presented.

Material and methods
The Carpathians are relatively a low but vast central European mountain range (the highest peak 2655 m).The northernmost part of this mountain range, studied within a framework of the present project, is situated in southern Poland and includes the Tatra Mts (the highest Carpathian massif) and several lower mountain massifs to the north of the Tatra Mts.The material for this study was collected in lower elevational belts (montane zone) of several Carpathian massifs (Fig. 1) from 2004 to 2009, in spring (April to June), in forests and also in open areas such as glades and meadows (Tab.1).
Included in the examined material there are specimens from the Gorce Mts reported previously by Ronikier et al. [18] as being Lamproderma carestiae and L. cribrarioides.Due to recent changes in taxonomy of this group these collections required revision.In order to include all Carpathian collections we have previously collected, the material from the Gorce Mts was re-examined.All specimens have been identified using the key by Poulain et al. [1].
Material collected in the field was put into carton boxes and air-dried for further examination in the laboratory.Observations and measurements of morphological characters were conducted under a stereoscopic microscope Nikon SMZ 1500.The total height of the sporocarps, the height and the width of the sporothecae and length of the stalk of most mature individuals were measured, usually up to 10 sporocarps per collection.Collections and permanent slides of examined specimens are deposited at KRAM.Analysis of microscopic characters and all microscopic measurements were performed on permanent preparations fixed in Hoyer's medium and observed under a light microscope (LM) Nikon Eclipse E-600, with Nomarski interference contrast, equipped with a digital Nikon DSFi1 camera head for photography.Measurements were made under the oil immersion ×100 objective.For each collection 30 spores were measured.The reported spore sizes include spore ornamentation.Values noted in less than 1% of all measurements are given in parentheses.The scanning electron microscopy (SEM) observations were carried out with a Hitachi S-4700 microscope, using 10 kV voltage and working distance of about 12 mm.The material was prepared in a sequence of acetone dilutions (50-100%) followed by the critical point drying procedure and coating with gold.SEM studies were made in the Laboratory of Field Emission Scanning Electron Microscopy and Microanalysis, at the Institute of Geological Sciences of the Jagiellonian University, Cracow.Spore ornamentation by SEM is given according to terminology of Rammeloo [20,21] with some modifications.

Results
In this study a total of 54 collections yielded nine taxa of Meriderma (two species, two varieties, and five forms).One species, Meriderma fuscatum is new for Poland.Other species or morphotypes are also reported here for the first time in Poland under  Description.Sporocarps mostly gregarious, stalked (Fig. 2a), (1.26-)1.30-1.98(-2.06)mm total height.Sporotheca black, ovoid to broadly ovoid, with conical to obtuse base, occasionally globose to subglobose, 0.96-1.12(-1.18)mm high, 0.70-0.94(-1.20)mm wide.Stalk (0.26-)0.34-0.82(-0.88)mm long, broadened at the base, black, shining.Peridium evanescent with small pieces remaining attached to the tips of the capillitium, usually persistent at the base of the sporocyst, with silvery and golden reflections.Columella reaching the center of the sporotheca.Capillitium dense, uniformly dark brown by LM, originating from the whole length of the columella, threads mostly smooth, with funnel-shaped ends and occasionally with some irregularities.Hypothallus discoid, sometimes common for a group of sporocarps, ferruginous brown to dark brown.Spores black in mass, moderately to dark brown by LM, paler on one side, 10-13 µm in diameter, subreticulate (covered with incomplete reticulum composed of fused spines), reticulate with perforated muri under SEM (Fig. 2b,c).
Description.Differing from the typical variety by almost completely reticulate spores forming almost closed meshed reticulum; meshes small, ornamentation mostly 0.5 µm up to 1 µm high (Fig. 2d,e).

Discussion
The genus Meriderma is one of the most significant nivicolous myxomycetes, because all species occur during snowmelt, and secondly because they are very common at such sites.However, because the taxonomic clarification in the species complex of Lamproderma atrosporum was proposed very recently [1,7], all older records of Lamproderma atrosporum, L. carestiae, and L. cribrarioides available in the literature need to be revised.Here we provide a re-examination of collections reported by Ronikier et al. [18] from the Gorce Mts.Additionally, the new records from other Carpathian massifs resulted in recognition of nine taxa (including varieties and forms).
Currently, the species delimitation of Meriderma is based on a morphological species concept.Characteristics such as general habit (stipitate vs. sessile sporocarps) and spore features (color, ornamentation pattern, size) are key characters in species delimitation.Poulain et al. [1] provide the key to identification of all recognized morphotypes with short descriptions, color images of sporocarps and drawings of spore ornamentation.Our studies based on specimens collected in the area of the Carpathians are in agreement with segregation of morphotypes proposed by these authors.Application of SEM images provide a highly precise and detailed characterization of spore ornamentation, which undoubtedly increase proper species determination.Thus, we provide results of observations of spore ornamentation under scanning electron microscope that supplement spore description given by Poulain et al. [1].Among species with warted spores we found that M. fuscatum (Fig. 5) spore ornamentation under SEM can be described as composed of irregularly distributed, isolated, cylindrical short baculae (Fig. 5c), according to the terminology of Rammeloo [20].Meriderma fuscatum is the only species in the genus forming ferruginous-brown sporophores (Fig. 5a), as described by Poulain et al., Meylan, Moreno et al. [1,3,22] and thus it is easy to distinguish from other morphotypes.All other species are black [1].Among them there are two sessile or subsessile forms: M. echinulatum (Fig. 4a) and M. aggregatum, the latter not yet found in the study area.The two species are easily distinguished from one another by spore ornamentation: of spines (baculae under SEM) isolated or fused into very short ridges composed of a few spines in the case of M. aggregatum [1], for SEM image of spore see Ronikier and Lado [23], and covered by dense labyrinth of fused spines (forming complex cristate subreticulate with some isolated baculae type of ornamentation) in the case of M. echinulatum (Fig. 4b,c).All remaining species are stipitate, with short or long stalk, and macroscopically very similar to one another.Meriderma spinulosporum has spiny spores and spines (baculae) are isolated or fused into very short rows composed of a few spines (Fig. 6b-g; as in the case of a sessile M. aggregatum), M. carestiae has subreticulate spores (or reticulate with small meshes, in the case of var.retisporum), ornamentation is usually up to 1 µm high (Fig. 2b-g), and M. cribrarioides has spores covered with complete reticulum of larger meshes (reticulate with non-perforated and perforated muri under SEM), which often exceed 1 µm and sometimes even reach 2 µm height (Fig. 3b,c).
Macroscopic distinction between sessile morphotypes (M.echinulatum and M. aggregatum) and stalked ones (M.spinulosporum, M. carestiae, and M. cribrarioides) is quite obvious, whereas segregation within group of stiptate specimens appeared to be problematic.Although spore ornamentation is the most reliable characteristic to distinguish morphotypes, we found that shape of sporotheca can be another macroscopic feature (apart from stalk length) helpful in species identification.In the Carpathian specimens Meriderma carestiae has mostly broadly ovoid sporothecae with conical base (Fig. 2a) whereas sporothecae of M. cribrarioides is globose to subglobose, only occasionally broadly ovoid and with umbilicate base (Fig. 3a).Meriderma spinulosporum has obovoid sporothecae with conical or rounded base.Poulain et al. [1] do not describe differences in sporotheca shape for stalked morphotypes, so this character has to be verified based on observations on specimens from other areas.
Apart from M. aggregatum, we have not found in the Polish Carpathians M. verrucosporum and M. atrofuscatum.They are stipitate and have spores covered with warts [1].
Poulain et al. [1] recognize forms of four species according to spore dimensions.Among the Carpathian collections, we found a considerable variability of spore size in M. spinulosporum (Fig. 6b-g) so that three forms recognized by Poulain et al. [1] could be quite clearly distinguished based on this characteristic.On the other hand, the Carpathian specimens of M. cribrarioides have larger spores (up to 18 µm) than provided by these authors [1] for that species.Two from the three identified specimens have spore diameter fully overlapping with those indicated by Poulain [1].Only one specimen has spore size exceeding indicated ranges, nevertheless one collection is not sufficient to distinguish a new form.
Worldwide distribution of Meriderma species is not yet fully known.As a whole, the genus is cosmopolitan and present in the many studied areas; about 553 records could be found for Meriderma at GBIF webpage [24], but many of them are still registered under the former name (Lamproderma atrosporum).All so far recognized Meriderma species are present mainly in Europe, the Alps [1,14].Six of them have recently been reported from the Caucasus [13] and five from the Khibiny Mountains [25].Three species, M. aggregatum, M. carestiae, and M. spinulosporum are known from the Andes in South America [23], however, the first and the last slightly deviating in spore ornamentation.Meriderma carestiae and M. fuscatum are also known from Japan and the first, additionally from the USA [1].Meriderma cribrarioides was also reported from the North American mountains, as L. cribrarioides by Kowalski [4], who correctly interpreted this species (see explanation in the "Introduction").Other species and morphotypes currently recognized in Meriderma are certainly present in many mountain massifs from which L. atrosporum s. l. was reported.
Nivicolous myxomycetes are widely known from their high morphological plasticity, which is often reflected in minor differences between morphotypes.This constantly challenges accurate determination of species, and during last years have led to increased number of newly described taxa [26].Moreover, there are still some doubts, which of morphological characters could be used for reliable distinction between different species.Meriderma genus is an example of taxonomically difficult group of species with high morphological diversity, reflected mainly in the continuum of spore size and ornamentation pattern, the latter character was considered as one of the most important traits.Research of Fiore-Donno et al. [12], supported this idea, indicating that delimitation of M. carestiae, M. cribrarioides, and M. aggregatum seems to correlate with phylogenetic analysis based on the SSU sequences.As well established, molecular analysis provides a promising methodology to clarify as well as verify taxonomic positions of many genera and species [12,13].This combinational approach could successfully resolve taxonomic problems and properly indicate inter-and intraspecific variability within this group.

Fig. 1
Fig. 1 Study area and localities of collection sites.a Location of study area within main massif of the Carpathians.b Sampling sites of specimens of Meriderma; numbers of localities refer to those from Tab. 1 and are cited for each species/morphotypes.

Tab. 1 List of localities of studied collections. No. Locality, habitat, date of collection, collectors Coordinates Elevation (m)
Turbacz range, ridge of the Średni Wierch Mt, above the village Obidowa, surroundings of the glade Polana Stusy; on Vaccinium myrtillus stems; 13 May 2006; leg.AR, MR They were previously identified as Lamproderma carestiae or L. cribrarioides (currently Meriderma carestiae and M. cribrarioides, respectively).Thus all but M. carestiae and M. cribrarioides are the first records for Poland and for the Carpathians overall.The list below contains all taxa (species, varieties, and forms) that could be distinguished using the key by Poulain et al. [1].carestiae (Ces.& de Not.) Mar.Mey.& Poulain var.carestiae Material studied.Loc.14, on stems of Rubus sp., Ron353 KRAM M-1640.Loc. 24, on small twigs, Ron635 KRAM M-1641.