Two New Species of the Genus Diderma (Physarales, Didymiaceae) in China with an Addition to the Distribution

Myxomycetes are an important component of terrestrial ecosystems, and in order to understand their diversity and phylogenetic relationships, taxonomic issues need to be addressed. In our 1985–2021 biodiversity investigations in Shaanxi Province, Jilin Province, the Inner Mongolia Autonomous Region, Hubei Province, and Henan Province, China, Diderma samples were observed on rotten leaves, rotten branches, and dead wood. The samples were studied, based on morphological features coupled with multigene phylogenetic analyses of nSSU, EF-1α, and COI sequence data, which revealed two new species (Diderma shaanxiense sp. nov. and D. clavatocolumellum sp. nov.) and two known species (D. radiatum and D. globosum). In addition, D. radiatum and D. globosum were newly recorded in Henan Province and the Inner Mongolia Autonomous Region, respectively. The paper includes comprehensive descriptions, detailed micrographs, and the outcomes of phylogenetic analyses for both the newly discovered and known species. Additionally, it offers morpho-logical comparisons between the new species and similar ones.


Introduction
Myxomycetes are a unique group of small eukaryotic organisms found in a wide range of terrestrial ecosystems.They are one of the few protist groups that exhibit sufficient morphological characteristics to enable the application of the morphological species concept.These organisms belong to the Eumycetozoa within the Amoebozoa [1,2].
The genus Diderma was established by Persoon in the 18th century (1794) to accommodate species characterized by the absence of lime knots connecting the capillitium, dark brown or black spores in mass, and a peridium with two or three layers covered by calcareous or cartilaginous material [3,4].Diderma globosum Pers.was designated as the type species [3][4][5][6][7][8].Rostafinski placed the genus Diderma in the family Didymiaceae in 1873 [9,10], a classification adopted by subsequent mycologists [3].Historically, this genus has been primarily identified based on morphological characteristics.
In recent years, the advancement of molecular biology has enabled researchers to conduct phylogenetic studies on the genus Diderma using sequences such as SSU and EF-1A [6][7][8].These studies, which also utilized COI sequences, have demonstrated that Diderma forms a monophyletic clade within the family Didymiaceae.
Currently, over 1100 species of myxomycetes are known [11] but only 87 species of Diderma have been identified worldwide, with 30 species recorded in China [12][13][14][15][16].In this study, we investigate the species diversity of Diderma and report the first occurrences of two species, D. shaanxiense and D. clavatocolumellum.Additionally, we document two new provincial records: D. globosum in the Inner Mongolia Autonomous Region and D. radiatum in Henan Province.We provide a morphological description of the new species.

Sampling and Morphological Studies
Habitat details of fresh myxomycetes were recorded at the time of collection.To prevent mixing or crushing, each specimen was wrapped in a single box.Fresh specimens were air-dried in a cool place shortly after returning from the field.Both macroscopic and micromorphological descriptions were based on dried materials.The color terminology used in our descriptions follows the Flora of British Fungi: Colour Identification Chart [17].Macroscopic characteristics were observed using a Zeiss dissecting microscope (Axio Zoom V16, Carl Zeiss Microscopy GmbH, Göttingen, Germany), and photographs were taken with a Leica stereoscopic dissector (Leica M165, Wetzlar, Germany).The prepared specimens were observed using a Lab A1 microscope (Carl Zeiss AG, Jena, Germany) and documented with photographs using ZEN 2.3 software (Carl Zeiss AG).
Twenty mature spores from each species were measured in side view.Spore dimensions are presented as (a) b-c (d), where the b-c range encompasses at least 90% of the measured values, while "a" represents the smallest length and "d" represents the largest value, if present.Additionally, specimens for scanning electron microscopy (SEM) were mounted on copper stubs with double-sided tape or a thin layer of nail polish, sputtercoated with gold, and then observed and photographed using a Hitachi S-4800 SEM (Hitachi, Tokyo, Japan) to examine the ultrastructure of spores and capillitium.The examined collections have been deposited in the Herbarium of Mycology at Jilin Agriculture University (HMJAU), Jilin, China.
The PCR programs for amplifying the nSSU region were as follows: initial pre-denaturation at 95 • C for 6 min, followed by 32 cycles of denaturation at 94 • C for 1 min, annealing at 52 • C for 1 min, an extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min.For the amplification of EF-1α, the PCR program included an initial pre-denaturation at 95 • C for 5 min, followed by 36 cycles of denaturation at 95 • C for 30 s, annealing at 65.4 • C for 30 s, extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min.Similarly, for COI, the PCR parameters consisted of an initial pre-denaturation at 95 • C for 5 min, followed by 36 cycles of denaturation at 95 • C for 30 s, annealing at 52 • C for 20 s, extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min.The PCR products were sequenced using the Sanger method by Kumei Biotechnology Co., Ltd.(Changchun, China).

Data Analysis
The sequences from different loci were auto-strategy aligned using MAFFT 7.490 software [23] and manually edited with BioEdit 7.1.3and Clustal X 1.81 [24,25] separately.Separate phylogenetic analyses were conducted for each marker gene, and the resulting topologies were compared to assess congruence through visual inspection.The partition homogeneity test was then performed using PAUP 4.0 [26] to evaluate the compatibility of the marker sequences.Based on the test results, which indicated sufficient congruence, the marker sequences were concatenated for the final phylogenetic analysis.Both the Bayesian Inference (BI) and maximum likelihood (ML) methods were employed for constructing phylogenetic trees.The best-fit substitution models were determined using Modelfinder [27].For maximum likelihood (ML) analyses, the datasets we were subjected to analysis using IQTREE 1.6.12software [28] with a rapid bootstrap comprising 1000 bootstrap (BS) replicates.For the Bayesian analysis, posterior probabilities (PPs) were computed using Markov Chain Monte Carlo sampling (MCMC) in MrBayes on XSEDE v. 3.2.7a[29,30], employing the estimated evolutionary models.Runs of 4,000,000 generations were conducted, with trees sampled every 1000 generations, using eight heated and one cold Markov chain(s), with the initial 25% of samples discarded as burn-in.The average standard deviation of split frequencies was 0.003222, indicating a significant difference.If the average standard deviation of split frequencies was above 0.01, ESS values were checked using Tracer v1.7.1 software [31], where values greater than 200 indicated convergence.The final trees were visualized using ITOL version 6.9 [32].Detailed information regarding reference strains is provided in Table 1 and Table S1.

Phylogenetic Analysis
The topologies of individual marker sequences were examined through separate phylogenetic analyses for SSU, EF-1A, and COI genes.A visual inspection showed general congruence among the topologies.To quantitatively assess this congruence, a partition homogeneity test was performed using PAUP*.The results for the individual marker genes and the concatenated dataset are shown in Table S2.
The best nucleotide substitution models for the BI phylogeny were GTR+F+G4 for SSU and GTR+F+I+G4 for both EF-1α and COI.The Bayesian analysis ran for two million generations, yielding an average standard deviation of split frequencies of 0.003873.Subsequently, the same dataset and alignment underwent analysis using the ML method.In the ML analysis, the best nucleotide substitution model for SSU and EF-1A is TIM2e+I+G4, while the COI model is TIM2e+F+I+G4.A comparison of the overall topologies between the ML and BI trees indicated nearly identical results across all datasets.Given this congruence, the ML analysis was chosen as the representative phylogeny.Internal nodes of the tree are labeled with values representing ML and BI support.Branches display bootstrap values (BP) of ≥70% from the ML analysis and Bayesian posterior probabilities (BPPS) of ≥0.90 (Figure 1).The phylogenetic tree obtained  Diderma shaanxiense sequences formed a distinct branch with 100/1 support, closely related to D. globosum and D. europaeum.The phylogenetic distance between D. shaanxiense and D. floriforme must be very similar to that of D. aurantiacum and lower than that of D. tigrinum.These data indicate significant differences between them, supporting the classification of these two as independent species.Additionally, two sampled specimens, which clustered with D. radiatum and D. globosum with strong support, were confirmed as new records from Henan Province and the Inner Mongolia Autonomous Region, respectively.
Diderma shaanxiense sequences formed a distinct branch with 100/1 support, closely related to D. globosum and D. europaeum.The phylogenetic distance between D. shaanxiense and D. floriforme must be very similar to that of D. aurantiacum and lower than that of D. tigrinum.These data indicate significant differences between them, supporting the classification of these two as independent species.Additionally, two sampled specimens, which clustered with D. radiatum and D. globosum with strong support, were confirmed as new records from Henan Province and the Inner Mongolia Autonomous Region, respectively.Etymology.The epithet "shaanxiense" refers to Shaanxi, the location of the holotype.Description.Sporangia, sessile, spheroid, gregarious, mutual squeezing deformation.Peridium is double-layered; the outer layer is calcareous, white, smooth; the inner layer is membranous, grey, iridescent, sometimes wrinkled, and separated from the outer layer.Columella is developed, yellowish brown or white, calcareous, pulvinate, or hemispherical, with a shining layer of film.Hypothallus is inconspicuous.Capillitium is crude, branched, and anastomosing, with membranous enlargement, brown or colorless, occasionally with some darker thickenings.Spore-mass dark or dark brown, fawn under transmitted light (TL), 8-11 µm in diam., with warts.
Habitat.On the rotten leaves.Notes.Diderma shaanxiense is characterized by its shining layer of film columella.Diderma shaanxiense shares morphological similarities with D. europaeum and D. globosum in terms of sessile and subglobose sporocarpic, two-layered peridium.However, it can be differentiated by its iridescent columella, spores with warts, and small size (8-11 µm) compared to D. europaeum, while D. europaeum has a pulvinate columella, spores with spines, and larger size (10-12 µm).D. shaanxiense can be distinguished from D. globosum based on its small and pulvinate shining columella.From the phylogenetic tree, we can see that D. shaanxiense is located between D. globosum and D. europaeum, and a separate branch is formed.
Diderma Etymology.The epithet "clavatocolumellum" refers to the club-shaped columella.Description.Sporophores sporocarpic, gregaria, sessile or short-stalk, oblate or hemispherical, grey.Peridium is two-layered; the outer layer is cartilaginous, covered with a layer of lime granules, crustose, white, light brown and light ochre, smooth; the inner layer is tightly attached together with the outer layer, calcareous, white or grey.Irregular dehiscence at the top, basal petaloid dehiscence and eversion.Columella present, either developed or conspicuous, clavate, close to the top of the columella, rough, ochreorange or ivory, 0.5-0.7 mm.Hypothallus is inconspicuous, membranous, translucent, brown.Capillitium is abundant, radiantly extending from the columella, brown or colorless, branching and connecting, with many dark brown swollen knots.Spores dark in mass, purple-brown via transmitted light, subglobose or oval, 9-11 (11.5) µm in diam., with obvious warts, sparse.
Habitat.On the dead woods.Distribution in China.Jilin Province.Distribution in the world.China.Additional specimens examined.China, Jilin Province, Yanji Prefecture, Antu County, Erdaobaihe, on the dead woods, 5 October 2001, H.Z. Liu and Tolgor, HMJAU M20120, HMJAU M20121.Notes.Morphologically, D. clavatocolumellum is similar to D. alpinum (Meyl.)Meyl.and D. floriforme with its double-layer peridium, spore size, and capillitium having swelling.However, D. clavatocolumellum can be distinguished from D. alpinum based on its developed and conspicuous columella (clavate), two layers of peridium that are tightly connected, membranous and brown hypothallus, and spores with warts; meanwhile, D. alpinum has a pulvinate columella; two layers of peridium kept away; easily separated, calcareous, and white hypothallus; and spores with spines.Diderma clavatocolumellum can be distinguished from D. floriforme based on its sessile or short stalk, while D. floriforme has a long stalk.From the phylogenetic tree, we can clearly see that D. clavatocumullum is related to D. floriforme and they are similar, but the genetic distance between the two is relatively far.
Habitat.On rotten branches and moss.Distribution in China.Beijing City, Gansu Province, Hebei Province, Heilongjiang Province, Inner Mongolia Autonomous Region, Jilin Province, Liaoning Province, Shaanxi Description.Sporophores sporocarpic, gregarious or densely crowded, mutual extrusion deformation, sessile, hemispheric to globose.Peridium is two-layered; the outer layer is limy, eggshell-like, smooth, white; the inner layer is membranous, grey-white, separate from the outer layer, iridescent.Columella is developed, hemispheric, white or pale cream, calcareous.Capillitium threads are slender, dense, pale-brown, sometimes with darker swellings, branched and anastomosed at the end, with membranous enlargement.
Spores are dark in mass, reddish brown via transmitted light, with obvious warts, 9-11 µm in diam.
Habitat.On rotten branches and moss.Distribution in China.Beijing City, Gansu Province, Hebei Province, Heilongjiang Province, Inner Mongolia Autonomous Region, Jilin Province, Liaoning Province, Shaanxi Province, and Yunnan Province.
Distribution in the world.Algeria, Australia, Austria, Canada, China, Costa Rica, Finland, France, Germany, Italy, Japan, Karelia, Korea, Latvia, Morocco, Nepal, the Netherlands, New Zealand, Norway, Peru, Russia, Spain, Sweden, Switzerland, the United States, the United Kingdom, Ukraine, and Venezuela.
Notes.Diderma globosum is often confused with D. europaeum and D. crustaceum due to their similar morphology.They are all smooth, white, and hemispheric to globose sporocarp with a two-layered peridium.However, D. globose differs from D. europaeum and D. crustaceum due to its small spores with warts and developed and hemispheric columella.
Diderma radiatum (L.) Morgan, J. Cincinnati Soc.Nat.Hist.16(4):151, 1894.Notes.Diderma globosum is often confused with D. europaeum and D. crustaceum due to their similar morphology.They are all smooth, white, and hemispheric to globose sporocarp with a two-layered peridium.However, D. globose differs from D. europaeum and D. crustaceum due to its small spores with warts and developed and hemispheric columella.
Habitat.On the rotten woods.Description.Fructification sporocarps, scattered, spherical or oblate, concave below the umbilicus, light yellow-brown, with floral spots and lattice concavities, 0.6-1.58mm in diam.Sessile or short-stalked, thick, yellow-brown, sulcate, 0.2-0.6 mm in diam.Columella developed and conspicuous and then calcareous, hemispherical, orange, ivory or auburn.Peridium is two-layered; the outer layer is cartilaginous, brown, latticed; the inner layer is membranous, light reddish brown, tightly fused with the outer layer, petaloid or radially for verifying species.These approaches indicate that the species diversity of myxomycetes is not yet fully understood [34].Phylogenetic trees constructed by incorporating molecular barcodes into numerous morphologically described species have revealed that these species are often complex, comprising several or even dozens of distinct species [6,21,[35][36][37][38][39].
In order to evaluate the compatibility of the marker sequences, the partition homogeneity test was performed.The results indicated sufficient congruence among the marker sequences, justifying their concatenation.The balanced index values for the concatenated dataset suggest a robust and reliable phylogenetic inference, minimizing biases from individual markers.In our constructed phylogenetic tree, Physarum Pers., Badhamia Berk., and Fuligo Haller are sister branches, suggesting that the taxonomic status of Physarum and Badhamia is uncertain and that they may be polyphyletic.This finding is consistent with previous studies by Nandipati et al. [40], Cainelli et al. [41], Prikhodko et al. [7], and Ronikier et al. [42].Prikhodko et al. [7] conducted a phylogenetic analysis of species within Didymiaceae (Myxomycetes) based on three independent genetic markers (18S rDNA gene, translation elongation factor 1-alpha, and cytochrome c oxidase), which supported taxonomic rearrangements in this family.Their study revealed that all studied species of Lepidoderma formed a distinct clade along with Diderma fallax, suggesting that they should be classified under the genus Polyschismium.Additionally, Lepidoderma tigrinum and two related species were transferred to the genus Diderma, leading to the invalidation of the genus Lepidoderma and an increase in species diversity within the genus Diderma.
The current study has expanded the known species diversity of the genus Diderma in China.However, ongoing debates persist regarding the taxonomic classification of this genus, largely due to limited species sampling and insufficient genetic variation in DNA loci.Therefore, additional evidence is required to enhance our understanding of this genus comprehensively.Despite recent discoveries of new Diderma species in China, the full extent of its species diversity remains uncertain, highlighting the need for a comprehensive systematic analysis.

Data Availability Statement:
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Figure 1 .
Figure 1.Maximum likelihood phylogenetic tree generated from the combined nSSU, EF-1α, and COI dataset of the Diderma species.The display on internal nodes indicates maximum likelihood bootstrap (BP) and Bayesian posterior probability (BPPS) values.The study species are marked in bold (known species) and red (new species) font for easy identification.(Notes: the final dataset

Figure 1 .
Figure 1.Maximum likelihood phylogenetic tree generated from the combined nSSU, EF-1α, and COI dataset of the Diderma species.The display on internal nodes indicates maximum likelihood bootstrap (BP) and Bayesian posterior probability (BPPS) values.The study species are marked in bold (known species) and red (new species) font for easy identification.(Notes: the final dataset comprised 6442 bp, including gaps, with 3980 bp (including gaps) from SSU, 1676 bp from EF-1α, and 786 bp from COI.).

Table 1 .
Information for the sequences produced in this study.