Lamproderma vietnamense: a new species of myxomycetes with reticulate spores from Phia Oắc - Phia Đén National Park (northern Vietnam) supported by molecular phylogeny and morphological analysis

Abstract

A new species of Lamproderma (Myxomycetes), described herein as L. vietnamense, was recovered in the field on ground litter from mountain subtropical forests (Phia Oắc - Phia Đén National Park) of northern Vietnam. Morphological details were examined by light and scanning electron microscopy. The species is characterized by a distinct and unique combination of morphological features, including a bright blue, shiny and very thin membranous peridium, a small dome-shaped columella, rigid, straight, branched, brown capillitial threads which gradually become pale at the periphery and finally colorless at the tips and small-meshed, banded-reticulate spores with 9-12 meshes across the spore diameter and solid walls without perforations 0.3-0.5 µm high. The stability of the taxonomic characters of L. vietnamense is supported by two well-developed collections found in 2018 and 2019. Partial sequences of three molecular markers (SSU, EF1α, COI) for both collections are identical. A two-gene phylogeny of the first two markers displays the two known accessions as a well-separated entity and indicates affinity of the new species with L. columbinum (the type taxon of the genus), L. violaceum, and several nivicolous Lamproderma species.

1. Introduction

The genus Lamproderma was erected by Rostafiński (1873) for Physarum columbinum Pers. (= Lamproderma columbinum [Pers.] Rostaf.). Based on the morphological species concept, 56 species are currently known worldwide (Lado, 2005-2022). In addition, there are numerous forms and varieties described within the genus. Lamproderma is characterized by a capillitium mainly arising from the columella apex, an epihypothallic stalk connected with columella and the persistent peridium, not evanescent as in Comatricha and Stemonitis but also not attached to the capillitium as is the case in Meriderma (Poulain, Meyer, & Bozonnet, 2011; Fiore-Donno et al., 2012).

The genus was traditionally (Macbride, 1922; Dennison, 1945a, 1945b; Kowalski, 1968, 1970) included in the order Stemonitidales (= Stemonitales). In the phylogeny of Fiore-Donno et al. (2012), members of the genus Lamproderma belong to the dark-spored clade of myxomycetes and group with other taxa of the traditional order Physarales. These relationships were reproduced in the study of Leontyev, Schnittler, Stephenson, Novozhilov, and Shchepin (2019), where the authors proposed to transfer Lamproderma and the re-erected and emended family Lamprodermataceae to the order Physarales.

Many species of Lamproderma are associated with coarse woody debris and ground litter and about 25 species belong to the distinctive ecological guild of nivicolous species that fruit on soil, ground litter and living branches of bushes and trees near melting snowbanks, mostly in subalpine and alpine zones in the mountains (Ronikier & Ronikier, 2009).

The key macroscopic characters used to recognize species of Lamproderma are the shape of the sporotheca, the ratio sporotheca length to stalk length, peridium color and structure, and columella size and shape. Microscopic characters include capillitium shape and color, especially those of capillitial thread tips, spore surface ornamentation and spore size and color.

During intensive studies targeting myxomycete diversity and ecology in mountain deciduous monsoon subtropical forests of northern Vietnam, in the Phia Oắc-Phia Đén National Park (POPD), two large colonies of Lamproderma with reticulate spores were found in two different years (2018 and 2019). Both localities were situated nearby, and in both cases the substrate was ground litter of Quercus spp. Spore ornamentation and sporocarp morphology are very distinctive and stable, with features not encountered for other species of Lamproderma and related genera, which suggests that our collections belong to a species new to science.

2. Materials and methods

2-1. Collection sites and field sampling

The reserve is situated in Nguyên Bình District, Cao Bằng Province of Vietnam, covering a region between 22°32′-40′N and 105°49′-57′E. The study area is dominated by a range of mountains from 900 to almost 2,000 m a.s.l., including the summit Phia Oắc Mt. (1,931 m a.s.l.). POPD occupies 10,245.6 ha covered by undisturbed subtropical broadleaf evergreen mountain forests, making up for ca. 77% of the territory. Detailed information about vegetation, climate and landscape of POPD is given in Fedorova, Novozhilov, and Gmoshinskiy (2020). Localities for all collections were georeferenced with a portable GPS device (WGS 84 mapping data). The two specimens described in this paper were recorded during two different field surveys in Nov 2018 and 2019.

2-2. Morphology

Air-dried sporocarps were studied with a Zeiss Axio Imager A1 light microscope (LM) with differential interference contrast (DIC), a Stemi 2000 dissecting microscope (DM), a Zeiss motorized stereomicroscope ZEISS Axio Zoom.V16 and a JSM-6390 LA scanning electron microscope (SEM) at the Core Facility Center of the Komarov Botanical Institute of the Russian Academy of Sciences. For microscopy, sporocarps were preserved as permanent slides in polyvinyl-lactophenol. Microscopic measurements were made using Zeiss Zen 3.2 software (Carl Zeiss Microscopy GmbH, free license, blue edition). The average spore diameter (including spore ornamentation) was calculated from 30 spores measured for each collection. Specimens for scanning electron microscopy were mounted on copper stubs with a double-sided tape and sputter-coated with gold.

2-3. Molecular phylogeny

2-3-1. DNA extraction, amplification, sequencing and sequence alignment

Extraction of genomic DNA was performed from matured air-dried fructifications without a trace of fungal contamination. Approximately 2-5 sporocarps were placed in 2 mL safe-lock tubes with addition of 3 mm diam. steel balls and frozen at -20 °C for at least 30 min. Afterwards, the samples were crushed in a TissueLyser LT homogenizer (QIAGEN, Hilden, Germany) for 1 min at 30 Hz. DNA was extracted with a magnetic bead-based DNA extraction kit PhytoSorb (Sintol, Moscow, Russia) according to the manufacturer's protocol with minor modifications: spore homogenate was eluted with 450 μL of extraction buffer; lysis buffer was added without a preliminary precipitation step and transfer of the supernatant into a new sterile tube; final elution volume was 100 μL.

To determine the phylogenetic position of the new species, two unlinked genetic markers were sequenced. The first part of the 18S rRNA gene (SSU), which is free of introns, was obtained with the primers S2 (Fiore-Donno, Meyer, Baldauf, & Pawlowski, 2008) and SU19R (Fiore-Donno, Novozhilov, Meyer & Schnittler, 2011). A fragment of the protein-coding gene for the translation elongation factor 1-alpha (EF1α) was amplified by the primer pair PB1F/PB1R (Novozhilov et al., 2013a). In case of unsuccessful amplification of EF1α, a set of primers for a semi-nested PCR named EF03(EF04)/KEF R3 (Ronikier, García-Cunchillos, Janik, & Lado, 2020) was used. This set of primers was also used to obtain longer gene fragments with two overlapping reads from the type material of the new species. Thus, three different amplification protocols were applied: denaturation for 5 min at 95 °C, 36 cycles (30 at 95 °C, 20 sec at 56 °C, 50 at 72 °C) and 5 min at 72 °C with S2/SU19R; denaturation for 5 min at 95 °C, 36 cycles (30 at 95 °C, 30 at 65.4 °C, 1 min at 72 °C) and 10 min at 72 °C with PB1F/PB1R; denaturation for 5 min at 95 °C, 35 cycles (30 sec at 95 °C, 30 sec at 60 °C, 90 sec at 72 °C) and 10 min at 72 °C with EF03/KEF R3 or EF04/KEF R3.

In addition, partial sequences of the cytochrome c oxidase subunit 1 (COI) gene were obtained with the primer combinations COMF/COMRs (Liu, Yan, & Chen, 2015; Novozhilov, Prikhodko, & Shchepin, 2019; gene fragment ca. 800 bp) for the holotype and COIF1/COIR1 (Feng & Schnittler, 2015; gene fragment ca. 650 bp) for the paratype, since the first primer pair did not produce a PCR product with the latter specimen. For amplification of these fragments, annealing temperatures were set to 52.0 °C and 50.7 °C, respectively.

PCR reactions were prepared with 10 μl of 2× BioMaster HS-Taq PCR-Color reaction mix (Biolabmix, Novosibirsk, Russia) containing 100 mM KCl, 0.4 mM dNTPs, 4 mM MgCl₂, 0.06 U/μl TaqDNA polymerase, 0.2% Tween20 and several dyes (xylene cyanol, bromphenol blue, OrangeG, tartrazine) with addition of 3 nmol of each primer, 1-3 μl of template DNA and diH₂O up to a total volume of 20 µl. The amplification was carried out with thermal cycler C1000 Touch (Bio-Rad, Hercules, CA, USA). Products of amplification were stained with GelRed (Biotium, San Francisco, CA, USA), separated by 1.2% agarose gel electrophoresis, observed in Gel Doc XR+ System (Bio-Rad, Hercules, CA, USA), and then purified using the CleanMag DNA (Evrogen, Moscow, Russia) purification kit before being sequenced with the BrilliantDye Terminator v3.1 Cycle Sequencing Kit (NimaGen, Nijmegen, the Netherlands). Sequencing products were purified with the Nimagen D-Pure DyeTerminator Cleanup kit, and then analyzed on ABI 3500 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA).

For all of the abovementioned steps we used the equipment of the Core Facility Center ‘Cell and Molecular Technologies in Plant Science’ at the Komarov Botanical Institute RAS (St. Petersburg). Quality-checked sequences were deposited into NCBI GenBank. The list of the newly obtained sequences with accession numbers and details on specimens as well as the list of sequences retrieved from GenBank (Supplementary Table S1) can be seen in FigShare (DOI: 10.6084/m9.figshare.15073515).

SSU and EF1α sequences were combined in two multiple alignments in UNIPRO UGENE (Okonechnikov, Golosova, Fursov, & the UGENE team, 2012) and aligned using MAFFT online service (Katoh & Standley, 2013; Katoh, Rozewicki, & Yamada, 2019) with E-INS-i or G-INS-i options, respectively, and default gap penalties. Exon parts of EF1α sequences were delimited according to the known protein and nucleotide EF1α sequence of Physarum polycephalum Schwein. (Genbank AF016243; Baldauf & Doolittle, 1997). After manual editing and removal of introns from the protein-coding alignment, two sets of nucleotide sequences were merged into a single alignment with two partitions using SEQUENCEMATRIX 1.8. (Vaidya, Lohman, & Meier, 2011). The final alignment consists of 91 sequences (91 sequences in the 18S rDNA partition and 87 in the EF1α partition) with 1,781 sites, 915 distinct patterns, 148 singleton sites and 1,014 non-informative (constant) sites.

2-3-2. Phylogenetic analyses

Maximum likelihood (ML) analyses were performed using IQ-TREE 1.6.12 (the last stable release; Nguyen, Schmidt, von Haeseler, & Minh, 2015) launched on the local machine. The TIM2e+I+G4 and GTR+F+I+G4 models were selected for SSU and EF1α partitions respectively according to the ModelFinder tool implemented in the program (Kalyaanamoorthy, Minh, Wong, von Haeseler, & Jermiin, 2017). One thousand ultrafast bootstrap replicates (Hoang, Chernomor, von Haeseler, Minh, & Vinh, 2018) were performed to obtain confidence values for the branches. Bayesian analysis was performed with the same dataset using MRBAYES 3.2.7a (Ronquist et al. (2012) https://academic.oup.com/sysbio/article/61/3/539/1674894?login=false ) run on CIPRES Science Gateway (Miller, Pfeiffer, & Schwartz, 2010); the GTR+I+gamma model was applied. The phylogenetic analysis was run 4 times as 4 separate chains for 15 million generations (sampling every 1000). The convergence of MCMC chains was estimated using TRACER 1.7.2 (Rambaut, Drummond, Xie, Baele, & Suchard, 2018); based on the estimates by TRACER, the first 3.75 million generations were discarded for burn-in. Posterior probabilities for clades were exported to the ML-tree. Phylogenetic tree with combined supports was visualized using FigTree 1.4.4 (Rambaut, 2014) and edited using CorelDRAW 24.0. The final tree can be seen in Fig. 1.

Fig. 1 - Two-gene phylogenetic tree obtained with a Maximum Likelihood analysis of concatenated SSU and EF1α sequences from Lamproderma vietnamense (red font), 15 morphospecies of the genus Lamproderma, species of the genera Meriderma, Collaria, Diacheopsis, and members of the Didymiaceae, rooted with Barbeyella minutissima and Echinostelium bisporum (Echinosteliales). Labels depict species names and herbarium numbers of the studied specimens. Bold font indicates sequences obtained in this study; coloured font indicates Lamprodermataceae species with reticulated spores. Branch supports are shown only for ultrafast bootstrap replicates/Bayesian posterior probabilities ≥ 80/0.8; black dots indicate maximum supports in both analyses (= 100/1); the scale bar represents the mean number of nucleotide substitutions per site.

3. Results

3-1. Taxonomy

Lamproderma vietnamense Novozh., Prikhodko, Fedorova, Shchepin & Schnittler, sp. nov. Fig. 2B-I.

MycoBank no.: MB 840234.

Fig. 2 - Lamproderma vietnamense (LE 317740, holotype). A: Habitat of the type specimen. B: Part of a colony of sporocarps as seen under a dissecting microscope (DM). C: Sporocarp (DM). D: Sporocarp seen with a compound microscope (LM). E: Columella (marked with arrow, LM). Scanning electron micrograph (SEM) of capillitial threads at the periphery. G: Sporocarp with membranous peridium (DM). H: Spore ornamentation as seen under compound microscope (DIC, oil immersion, 100x, top and median view). I: Spore ornamentation (SEM). Bars: B 1000 µm; C, D, G 200 µm; E 50 µm; F 10 µm; H 5 µm; I 2 µm.

Diagnosis: Differs from other Lamproderma species by the following combination of characters: a bright blue, shiny and very thin membranous peridium, a small dome-shaped columella and small-meshed, banded-reticulate spores, with 9-12 meshes across the spore diameter and solid walls without perforations emerging 0.3-0.5 µm from the surface.

Type: VIETNAM, Cao Bằng Province, Nguyên Bình District, Phia Oắc-Phia Đén National Park, closed evergreen subtropical monsoon (seasonal) lower montane broad-leaved forest (Fig. 2A), 22°36′25.8′′N, 105°52′17.3′′E, ca. 1,650 m a.s.l., on ground litter and mosses at the bottom of a large tree of Quercus sp., 8 Nov 2018, coll. Y.K. Novozhilov (holotype LE 317740), deposited in the Mycological Herbarium of the Komarov Botanical Institute RAS (Saint Petersburg, Russia).

Gene sequences ex-holotype: MZ241460 (SSU), MZ234254 (COI), MZ234289 (EF1α).

Etymology: Refers to the geographical region where the type specimen was found.

Description: Sporocarps stalked, 1.5-2.0 mm in total height, forming large, gregarious colonies (Fig. 2B). Sporotheca subglobose, flattened and slightly umbilicate at the base, 0.5-0.8 mm in diameter (Fig. 2C, G). Peridium membranous, very thin, blue with violet and purple iridescence under dissecting microscope (Fig. 2C, G), pale brown in transmitted light (Fig. 2E), irregularly breaking at the apex into small plates (Fig. 2G), persisting at the base as a more or less petaloid cup (Fig. 2D, F). Stalk 1.3-1.5 mm long, around 2 times longer than the sporotheca diameter (Fig. 2C), cylindrical, broader towards the base, black, opaque, with fibers at the base (Fig. 2C, D). Columella concolorous with the stalk, short cylindrical or dome-shaped, very small, 50-100 µm high, occupying approximately 1/8 of the sporotheca height (Fig. 2D-F). Capillitial threads rigid, straight, brown, branched (Fig. 2E) and bifurcate at the ends (Fig. 2F), gradually becoming pale at the periphery and finally colorless at the tips, whitish under dissecting microscope (Fig. 2G). Spores black in mass (Fig. 2C), (10.6-)12.1±0.6(-13.7) µm diam (n = 30) when the ornamentation is included, brown in LM, small-meshed, banded-reticulate (Fig. 2H, I), with 9-12 meshes of variable size across the spore diameter and solid walls without perforations, ridges 0.3-0.5 µm tall (Fig. 2I). Plasmodium unknown.

Distribution: Lamproderma vietnamense is currently known only from two localities in northern Vietnam.

Additional specimen examined: VIETNAM, Cao Bằng Province, Nguyên BìnhDistrict, Phia Oắc-Phia Đén National Park, closed evergreen subtropical monsoon (seasonal) lower montane broad-leaved forest (22°36′41.0′′N, 105°52′31.3′′E), ca. 1,600 m a.s.l., closed evergreen subtropical monsoon (seasonal) lower montane broad-leaved forest, on ground leaf litter of Quercus sp., 5 Oct 2019, coll. Y.K. Novozhilov, O.N. Shchepin (paratype LE 326172a), deposited in the Mycological Herbarium of the Komarov Botanical Institute RAS (Saint Petersburg, Russia). Gene sequences ex-paratype: MZ241461 (SSU), MZ234255 (COI), MZ234290 (EF1α).

Note: Morphological comparisons (Supplementary Table S2) between our new species L. vietnamense and the eight Lamproderma species with banded-reticulated spores demonstrate that L. vietnamense differs from other species either in size and shape of columella or/and in size and ornamentation of spores. The most important discriminative character of L. vietnamense is the very short and dome-shaped columella (occupying 10-15% of sporotheca heght).

Lamproderma clynense Ing & K. Lawson (Ing, 2020) found in South Wales (United Kingdom) differs from L. vietnamense by its columella reaching center of the sporotheca and by ornamentation of spores (a reticulum with 4-5 meshes vs 9-12 across the spore diameter in L. vietnamense).

Lamproderma magniretisporum G. Moreno, C. Rojas, S.L. Stephenson & H. Singer was described from Costa Rica (described from Costa Rica, 2009) and differs from L. vietnamense by larger sporocarps (2.2-3 mm vs 1.5-2.0 mm), larger spores (16-18 µm vs 11-13 µm) and grey or steel-grey peridium vs blue one of the latter species.

Lamproderma reticulosporum Gilert was found in western Java in Indonesia (Gilert & Neuendorf, 1991) and can be distinguished from L. vietnamense by the peridium covered with distinct elevated patches and a capillitium with regularly distributed fusiform to globose swellings with yellow to orange oily contents. Due to the last character, this species, as well as L. australiensis S.L. Stephenson, G. Moreno & H. Singer, was transferred by Moreno, Singer, and Stephenson (2008) to the genus Elaeomyxa. Similar to the case of L. retisporum (Dhillon & Nann.-Bremek.) T.N. Lakh. & K.G. Mukerji, this taxonomic view has yet to be confirmed by phylogenetic analyses, so currently we prefer to consider both species as Lamproderma.

Lamproderma retisporum resembles L. vietnamense in spore ornamentation, sporocarp size and ratio of the sporotheca height to the stalk length, but differs from the latter by its brown peridium and much smaller spores (6-8 µm vs 11-13 µm in L. vietnamense). This species was found in Uttar Pradesh province (northern India, northwestern part of the Himalaya) and firstly described as Collaria retispora Dhillon & Nann.-Bremek. (Dhillon & Nannenga-Bremekamp, 1977). Further it was transferred to the genus Lamproderma as L. retispora (Lakhanpal & Mukerji, 1981), and then this name was corrected by Moreno et al. (2009) to L. retisporum. However, in the nomenclatural information system of the Eumycetozoans (Lado, 2005-2022) it is validated as Comatricha retispora (Dhillon & Nann.-Bremek.) H. Neubert, Nowotny & K. Baumann (Neubert, Nowotny, & Baumann, 2000). Molecular research is urgently needed to reveal the correct taxonomic position of this species, so due to the absence of material we will consider it as a species of Lamproderma for now.

Lamproderma vietnamense differs from all the nivicolous Lamproderma species with reticulate spores (L. australiensis, L. lycopodiicola Kuhnt, L. meyerianum (Y. Yamam.) G. Moreno, and L. retirugisporum G. Moreno, H. Singer, Illana & A. Sánchez) not only by its different ecology, but also by the very small columella, spore size and details of spore ornamentation (Supplementary Table S2).

3-2. Molecular phylogeny

Using two independent markers (SSU and EF1α), a two-gene phylogeny of representatives of the genera Meriderma Mar. Mey. & Poulain, Collaria Nann.-Bremek. and Lamproderma with Didymiaceae (Diachea Fr., Diderma Pers., Didymium Schrad., and Lepidoderma de Bary ex Rostaf.) as the closest clade was constructed and rooted with the genera Barbeyella Meyl. and Echinostelium de Bary (Echinosteliales) (Fig. 1).

As in all hitherto published phylogenies of dark-spored myxomycetes (e.g. Fiore-Donno et al., 2012; Cainelli, de Haan, Meyer, Bonkowski, & Fiore-Donno, 2020), species of the genus Meriderma together with Collaria rubens (Lister) Nann.-Bremek. assumed a basal position. The next clade was formed by Collaria arcyrionema Rostaf. Relationships of the families Didymiaceae and Lamprodermataceae remained unresolved, since the corresponding deep branches were poorly supported. Lamproderma cacographicum Bozonnet, Mar. Mey. & Poulain formed a sister clade to all other species of Lamproderma and all Didymiaceae. Within the remaining Lamproderma, several strongly supported groups appeared, but their relationships were not resolved.

The two specimens of Lamproderma vietnamense had identical sequences of SSU, COI and EF1α, and thus clustered together in the two-gene phylogeny with maximal support. They assumed a poorly resolved position within the clade that united Lamproderma columbinum (Pers.) Rostaf., which is the type taxon of the genus Lamproderma, together with L. arcyrioides (Sommerf.) Rostaf., L. cristatum Meyl., L. pulveratum Mar. Mey. & Poulain, and L. violaceum (Sommerf.) Torrend (Fig. 1). Three other species of Lamprodermataceae with reticulate spores that were included in the two-gene analysis (L. retirugisporum, L. lycopodiicola, and Diacheopsis reticulospora) Mar. Mey. & Poulain did not group together with L. vietnamense or with each other, but were scattered throughout the Lamproderma clade.

As an additional result, it was found that the codon structure of EF1α gene in some species differs from that of Physarum polycephalum (GenBank AF016243; Baldauf & Doolittle, 1997). One newly obtained sequence from Didymium quitense (Pat.) Torrend (LE205082; GenBank MZ234265) has an insertion of three nucleotides between positions 1184-1185 according to AF016243 (positions 1282-1284 in the EF1α alignment) provided by us in FigShare (DOI: 10.6084/m9.figshare.15073515 ), which leads to the insertion of one additional amino acid. In addition, several SNPs cause substitutions in the amino acid sequence. Besides, all three studied specimens of C. arcyrionema seem to possess a large intron close to the landing site of the KEF_R3 reverse primer. All attempts to amplify a longer gene fragment were unsuccessful, so we cannot resolve whether the position of the intron in C. arcyrionema is different from the known one (Fiore-Donno, Berney, Pawlowski, & Baldauf, 2005), or if the gene sequence has two or more introns.

4. Discussion

Reticulate spores are rare in the genus Lamproderma (Lado, 2005-2022; Moreno et al., 2009), and only eight previously described species show this trait. These are L. australiensis S.L. Stephenson, G. Moreno & H. Singer, L. clynense Ing & K. Lawson, L. lycopodiicola Kuhnt, L. magniretisporum G. Moreno, C. Rojas, S.L. Stephenson & H. Singer, L. meyerianum (Y. Yamam.) G. Moreno, Singer & Illana, L. reticulosporum Gilert, L. retirugisporum G. Moreno, H. Singer, Illana & A. Sánchez, and L. retisporum (Dhillon & Nann.-Bremek.) T.N. Lakh. & K.G. Mukerji. Our results have shown that the specimens of L. vietnamense sp. nov. differ from these species either in morphology (L. clynense, L. magniretisporum, L. reticulosporum, and L. retisporum) or in both morphology and ecology (nivicolous species: L. australiensis, L. lycopodiicola, L. meyerianum, and L. retirugisporum), with the short columella being the most prominent distinguishing character.

Species with reticulate spores previously regarded as L. cribrarioides (Fr.) R.E. Fr. and L. anglicum (G. Lister & H. J. Howard) Ing appeared to belong to another genus - Meriderma - based on the presence of evanescent peridium and expanded, funnel-shaped capillitium ends (Poulain et al., 2011). Although in the description of L. anglicum (Ing, 1999) the author stated that “the weak stalks and faintly reticulate spores and lack of funnel-like expansions at the junction of the capillitium tips and the peridium are diagnostic”, these expansions are clearly visible in his figure of this species (Ing, 1999) as well as in the figure of L. atrosporum Meylan var. anglicum G. List. & Howard (Lister, 1919), which is the basionym of L. anglicum. The author notes that “the capillitium consists of a network of slender, flexuose, dark brown threads, radiating from all parts of the columella, and attached by their expanded tips to the sporangium-wall”. This morphology of capillitium is typical for Meriderma and it clearly differentiates these species from L. vietnamense, which shows thin bifurcating capillitial tips that are not attached to the peridium.

Molecular approaches have proved to be effective in elucidating the systematic position of some myxomycete taxa at both genus and species levels (e.g., Fiore-Donno, Clissmann, Meyer, Schnittler, & Cavalier-Smith, 2013; Novozhilov et al., 2013b; Leontyev et al., 2019; Cainelli et al., 2020). However, at the present time sequences of DNA barcodes such as SSU have been obtained and submitted to GenBank from only a small fraction of all morphologically described myxomycete species (Schnittler, Shchepin, Dagamac, Borg Dahl, & Novozhilov, 2017; Shchepin et al., 2019). Here we provide partial sequences of three molecular markers of the new species L. vietnamense (SSU, EF1α, COI) in addition to the morphological description.

Our two-gene phylogenetic analysis supported L. vietnamense as a separate species and has shown that the four studies species of Lamprodermataceae with reticulate spores do not form a monophyletic clade. This suggests that the reticulate spore ornamentation have developed independently several times within Lamprodermataceae.

Additionally, our results support late-autumn species L. violaceum as a distinct taxon not synonymous to the nivicolous L. arcyrioides. Although we did not find any prominent differences in morphological characters between our collections of L. violaceum and L. arcyrioides, the two taxa differ in sporulation phenology and in both SSU and EF1α sequences (for supporting information see FigShare DOI: 10.6084/m9.figshare.15073515 ) and form a separate fully supported clade in our two-gene phylogeny. Thus, we support Rostafiński's (1875) original intention - he treated both species separately in his monograph. However, an in-depth taxonomic study is necessary to reach a final decision.

The ultimate delimitation of the species-rich genus Lamproderma by a robust phylogeny lies beyond the aim of this study. The data presented herein mount clear evidence that the current delimitation of the genus, based on morphological characters, does not correspond to a monophyletic clade. To develop a delimitation based on phylogenies, more gene markers and a broader taxon sampling should be employed.

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the countries where they were performed.

Acknowledgments

We acknowledge the use of equipment of the Core Facility Center ‘Cell and Molecular Technologies in Plant Science’ at the Komarov Botanical Institute of the Russian Academy of Sciences (BIN RAS, St. Petersburg) and send personal thanks to Ludmila Kartzeva and Elena Krapivskaya (lead engineers of the Core Facility Center). The authors are grateful to Marianne Meyer and Edwin Johannesen for providing specimens.

The field and molecular work of YKN, ISP, NAF and ONS was supported by the state task “Biodiversity, ecology, structural and functional features of fungi and fungus-like protists” (BIN RAS, 122011900033-4) and the project “Taxonomic and ecological diversity of mycobiota of tropical forests in Vietnam, Ecolan E-1.5” of the state task “Tropical Ecology” of the Joint Russian-Vietnamese Tropical Research and Technological Centre. The molecular work for YKN, ISP, and ONS were supported by the Ministry of Science and Higher Education of the Russian Federation (agreement No. 075-15-2021-1056) and for MS by the German Research Foundation (DFG, grants RTG 2010, SCHN 1080/2-1).

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