2024 Volume 66 Issue 1 Pages 58-66
Diderma microsporum is a taxonomically controversial species of Myxomycetes. Currently, it is treated as a synonym for Diderma effusum. In this study, two specimens from Lafa Mountain National Forest Park in Jilin Province of China were researched concerning morphology with the light microscope and scanning electronic microscope and compared with the holotype from Japan to confirm that the species was first discovered in China. It is mainly characterized by cushion-shaped plasmodiocarps; single peridium, ornamented with dense flake-like lime crystals on the outer surface; columella absent; capillitium (0.99-)1.29-2.05(-2.30) μm diam, with spindle-shaped enlargement forms at branching points, decorated with dense small warts; spores (5.70-)6.49-6.87(-7.61) μm diam, warted and with clusters of large warts. In addition, the partial nucleotide sequences of nuclear 18S rDNA and elongation factor-1 alpha genes of D. microsporum differed clearly from those of other Diderma species. The results supported the rationality that D. microsporum is an independent species. Light microscopic photos, scanning electron microscopic photos, and an emended description of morphological characters are provided.
Myxomycetes, also known as Myxogastria, are a group of fungus-like organisms providing enough morphological characters belonging to Eumycetozoa of Amoebozoa (Adl et al., 2019). The genus Diderma Pers. was established and described by Persoon in 1794 (Martin & Alexopoulos, 1969), and belongs to the family Didymiaceae, the order Physarales (García-Martín et al., 2023; Lado & Eliasson, 2022; Leontyev et al., 2019; Prikhodko et al., 2023). The key characters of the genus Diderma are dark-colored spores, the absence of lime in capillitial threads (very rarely some threads may contain several amorphous granules), and granulated lime in a one-, or two- even three-layered peridium (García-Martín et al., 2023; Martin & Alexopoulos, 1969). Currently, more than 89 valid species are recognized in the genus Diderma (Lado, 2005-2024; Li et al., 2024). In China, 31 species of the genus Diderma have been reported (Chen et al., 2013; Gao et al., 2018; Li et al., 2024; Liu & Chang, 2011; Song et al., 2022; Song et al., 2024; Zhao et al., 2022). Among them, five species are only known from China up to the present (Li et al., 2024; Rao & Chen, 2023).
Diderma microsporum (Nann.-Bremek. & Y. Yamam.) Y. Yamam. is a unique and questionable species. Its scientific name can be traced back to Nannenga-Bremekamp and Yamamoto (1987) who first proposed a variety of Diderma effusum (Schwein.) Morgan based on the specimen from Japan, namely Diderma effusum var. microsporum Nann.-Bremek. & Y. Yamam. Then, Yamamoto (1998) upgraded it to a species as D. microsporum (Nann.-Bremek. & Y. Yamam.) Y. Yamam., and drew its morphological characters. In recent years, Yamamoto still treated it as D. microsporum (Nann.-Bremek. & Y. Yamam.) Y. Yamam. in Biota of Japanese Myxomycetes (Yamamoto, 2021). This species has not been reported from countries other than Japan. However, according to “An Online Nomenclatural Information System of Eumycetozoa” (Lado, 2005-2024), D. microsporum was treated as a synonym for D. effusum.
In our recent studies on the taxonomy of Myxomycetes of China, the unique morphological characters of two Diderma specimens from Lafa Mountain National Forest Park in the Jiaohe City of Jilin Province reminded us of D. microsporum according to the original description of D. microsporum by Yamamoto (1998). However, this species had not been discovered and reported in China before this study. To verify whether our specimen belongs to D. microsporum, we borrowed the holotype of D. microsporum from the National Museum of Nature and Science (TNS, Tsukuba, Japan) for a morphological study. To further demonstrate the rationality of D. microsporum as an independent species, we conducted a detailed analysis and comparison of D. microsporum with related species from a morphological perspective and determined the phylogenetic position of D. microsporum based on two genetic markers obtained from our two specimens: nuclear 18S rDNA (18S) and elongation factor-1 alpha (EF-1α). We also provided an emended description of D. microsporum.
Macroscopic characters were observed and measured using a JSZ6 dissecting microscope (Jiangnan Yongxin Optics Co., Jiangsu Province China). Microscopic slides with capillitium and spores were prepared using 3% KOH. Observations and measurements of microscopic characters were conducted with an Axio Imager A1 light microscope (Carl Zeiss AG, Göttingen, Germany). The diameter of 10 capillitium threads and 50 spores including ornamentation from each specimen were determined using an oil immersion lens with a magnification of 100× (Ronikier et al., 2022). The range of variation diameter for capillitium and spores is given in descriptions as (minimum-)25th percentile-75th percentile(-maximum) (Yatsiuk et al., 2023). For SEM observations, air-dried specimens were mounted on aluminum stubs with mucous membranes and sputtered with gold by a GVC-2000 Ion sputtering instrument (Gewei Instrument Co., Beijing, China). Microscopic characters of capillitium, spores, and peridium were examined using an Apreo 2S scanning electron microscope (Thermo Scientific, Massachusetts, USA) at 5 kV (Song et al., 2022).
The difference between the spore diameter of Chinese specimens and the holotype of D. microsporum was checked using the T-tests in Prism 10. The colors are described using the system by Ridgway (1912). The two specimens of D. microsporum from China are respectively deposited in the Herbarium Mycologicum, Academiae Sinicae (HMAS, Beijing, China) and the Herbarium of Fungi of Nanjing Normal University (HFNNU, Nanjing, Jiangsu Province, China). The holotype of D. microsporum was browned from the National Museum of Nature and Science.
2.2. DNA extraction, amplification, and sequencingFruiting bodies from each specimen (approximately 10 mg) were picked up with forceps under a dissecting microscope and placed in a sterile 1.5 mL Eppendorf tube containing three glass beads with 2.5 mm diam. DNA extraction adhered to the instructions provided in the DP316 TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China).
To demonstrate the independence of D. microsporum and explain its phylogenetic position, two unlinked genetic markers (18S and EF-1α) were sequenced. The 5′ end of the 18S combined with EF-1α recovered statistically supported clades at various taxonomic ranks and is preferred to study phylogenetic relationships in Myxomycetes (Prikhodko et al., 2023; Shchepin et al., 2024). Approximately 550 base pairs (bp) from the 5′ end of the 18S, a fragment that is free of introns, were obtained with S1 (Fiore-Donno et al., 2008)/SU19R (Fiore-Donno et al., 2011) or S2 (Fiore-Donno et al., 2008)/SSU_rev (Prikhodko et al., 2023). An exon fragment of approximately 800 bp from EF-1α was obtained with PB1F/PB1R (Novozhilov et al., 2013). The polymerase chain reaction (PCR) protocol for 18S was as follows: initial denaturation at 98 ℃ for 2 min; 30 cycles of denaturation at 98 ℃ for 10 s, annealing at 56 ℃ for 10 s, and elongation at 72 ℃ for 15 s; and final elongation at 72 ℃ for 5 min. For EF-1α, the PCR protocol was somewhat different: 98 ℃ for 2 min; 30 cycles of denaturation at 98 ℃ for 10 s, annealing at 65.4 ℃ for 10 s, and elongation at 72 ℃ for 15 s; and final elongation at 72 ℃ for 5 min. Each PCR contained 25 μL 2× T8 High-Fidelity Master Mix (Tsingke Biotech, Beijing, China), 1 μL of each forward and reverse primers (10 mM), 1 μL template DNA, and completed with water up to a final volume of 50 μL. All PCR products were sequenced by Sangon Biotech (Shanghai, China).
2.3. Sequence alignment and phylogenetic analysesGenBank was searched for all 18S and EF-1α sequences from the family Didymiaceae. The final dataset consists of a total of 179 sequences, including 24 sequences newly obtained from 13 specimens (Table 1), and 155 sequences (78 sequences of 18S and 77 sequences of EF-1α) downloaded from Genbank (Supplementary Table S1). 18S and EF-1α sequences were aligned using MAFFT 7.471 (Katoh & Standley, 2013) with the E-INS-i option for 18S and the G-INS-i option for EF-1α with default gap penalties (Prikhodko et al., 2023) in PhyloSuite 1.2.3 (Xiang et al., 2023; Zhang et al., 2020). Ambiguously aligned fragments, priming binding sites, gaps, and low-quality ends were removed using Globcks (Talavera & Castresana, 2007) with the following parameter settings: minimum number of sequences for a conserved/flank position (49/49 for 18S and 46/46 for EF-1α), maximum number of contiguous non-conserved positions (8), minimum length of a block (10), allowed gap positions (with half). A two-gene phylogeny was built from a concatenated dataset consisting of 18S and EF-1α. Four partitions were defined: 18S and one partition for each codon position of EF-1α. The best-fit model of nucleotide substitution was determined by ModelFinder (Kalyaanamoorthy et al., 2017). The best-fitting substitution models for 18S and EF-1α suitable for Maximum likelihood (ML) analysis were TIM2e+I+G4 and GTR+F+I+G4, respectively. ML analyses were conducted with IQ-TREE 1.6.8 (Minh et al., 2013; Nguyen et al., 2015). One thousand ultrafast bootstraps (UBS) pseudoreplicates were performed to obtain confidence values for the clades. The best-fitting substitution models for 18S and EF-1α suitable for Bayesian inference (BI) analysis were SYM+I+G4 and GTR+F+I+G4, respectively. BI analyses were conducted using MrBayes 3.2.6 (Ronquist et al., 2012), with four separate chains each 10×106 generations long (sampling every 1000th generation). The convergence of Markov Chain Monte Carlo (MCMC) simulations was estimated using Tracer 1.7.2 (Rambaut et al., 2018) and by the average standard deviation of split frequencies. Consequently, the first 19% of sampled data and trees were discarded as burn-in. Bayesian Posterior probabilities (PP) for clades were mapped onto the ML tree. ML and BI trees were visualized in FigTree 1.4.4 (available from: http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator 2021 (San Jose, CA, USA). Members of the genus Meriderma were used to root the tree.
| Species | Voucher | GenBank accession | Collected date | Geographic location | Substrate | |
| 18S | EF-1α | |||||
| Diachea subsessilis | HFNNU 8863 | PP506320 | PP528888 | Jul 7, 2023 | Zijin Mountain, Jiangsu Province | Dead leaves |
| Diachea subsessilis | HFNNU 8865 | PP506321 | PP528889 | Nov 2, 2022 | Zijin Mountain, Jiangsu Province | Dead leaves |
| Diderma chondrioderma | HFNNU 8508 | PP506325 | - | Dec 1, 2023 | Wuxiang Mountain, Jiangsu Province | Living bark |
| Diderma effusum | HFNNU 9863 | PP506322 | PP528890 | Dec 29, 2019 | Houhe Nature Reserve, Hubei Province | Dead leaves |
| Diderma effusum | HFNNU 9787 | PP506326 | - | Aug 31, 2016 | Tiantangzhai Nature Reserve, Anhui Province | Dead wood |
| Diderma effusum | HFNNU 9867 | PP506571 | PP528891 | Sep 30, 2000 | Tiejia Mountain, Yunnan Province | Dead leaves |
| Diderma globosum | HFNNU 9866 | PP506572 | PP528892 | Oct 12, 2000 | Cang Mountain, Yunnan Province | Dead wood |
| Diderma hemisphaericum | HFNNU 9869 | PP506573 | PP528893 | Aug 22, 2000 | Birong Canyon, Yunnan Province | Dead leaves |
| Diderma microsporum | HFNNU 9845 | PP506319 | PP528897 | Aug 30, 1991 | Lafa Mountain National Forest Park, Jilin Province | Dead leaves |
| Diderma microsporum | HMAS 74976 | PP506318 | PP528898 | Aug 30, 1991 | Lafa Mountain National Forest Park, Jilin Province | Dead leaves |
| Diderma tigrinum | HFNNU 9868 | PP506574 | PP528894 | Aug 20, 2000 | Tianchi, Yunnan Province | Dead wood |
| Didymium difforme | HFNNU 9864 | PP506323 | PP528895 | Oct 20, 2019 | Houhe Nature Reserve, Hubei Province | Dead leaves |
| Didymium squamulosum | HFNNU 1305 | PP506324 | PP528896 | Mar 24, 2019 | Qixia Mountain, Jiangsu Province | Dead leaves |
Note. - refer to data not availed.
Diderma microsporum (Nann.-Bremek. & Y. Yamam.) Y. Yamam. Myxomycete Biota Japan 279 (1998), emend. W.-L. Song, S.-Z. Yan & Shuang L. Chen, stat. nov. Figs. 1, 2.
≡ Diderma effusum var. microsporum Nann.-Bremek. & Y. Yamam., Proc. Kon. Ned. Akad. Wetensch., C. 90(3):321 (1987)
MycoBank no.: MB 450448
Diagnosis: Plasmodiocarps cushion-shaped, densely clustered; peridium single, ornamented with dense flake-like lime crystals on the outer surface, dehiscence irregularly; columella absence; capillitium abundant, hyaline, (0.99-)1.29-2.05(-2.30) μm diam, branched abundant, spindle-shaped enlargement forms at branching points, decorated with dense small warts; spore flesh-ocher in transmitted light, (5.70-)6.49-6.87(-7.61) μm diam, 6.71 μm on average, warted with clusters of large warts.
Emended description: Sporocarps sessile to plasmodiocarps cushion-shaped, densely clustered , 1.05-5.45 mm diam, 0.48-0.87 mm in thickness, white or aniline yellow, glossy; hypothallus inconspicuous, membraneous, hyaline; peridium single, compacted with lime granules of ca. 1 μm diam, brittle, ornamented with dense flake-like lime crystals on the outer surface, dehiscence irregularly, the peridium is often divided into small pieces, occasionally with metallic luster fragments; columella absence; capillitium abundant, hyaline, about (0.99-)1.29-2.05(-2.30) μm diam, rising from base and attached to the inner peridium, branches abundant, spindle-shaped enlargement often forms at branching points, short lines connecting the tubular lines occasionally, no obvious ornamentations under transmitted light, threads surface densely warted under SEM; spores blackish mouse gray in mass, flesh-ocher in transmitted light, (5.70-)6.49-6.87(-7.61) μm diam, 6.71 μm on average, minute warts, some of the warts in clusters; plasmodium unknown.
Habitat: On living herbaceous plants.
Distribution: Japan: Kochi Pref of Shikoku (Nannenga-Bremekamp & Yamamoto, 1987), Honshu Island (Yamamoto, 1998); China: Lafa Mountain National Forest Park, Jiaohe City, Jilin Province (This study).
Specimens examined: JAPAN. Shikoku: Kochi Pref, Gohoku-son, Kamiyakawa, on the living herbs, 3 Aug 1986, Yukinori Yamamoto (holotype, TNS-M-Y-21980). CHINA. Jilin Province: Jiaohe City, Lafa Mountain National Forest Park, 127°12′35″ E, 43°25′12″ N, on the living herbs, 30 Aug 1991, Shuang-Lin Chen (HMAS 74976; HFNNU 9845).
3.2. Phylogenetic analysisThe final two gene alignments included 179 sequences, with 16 sequences of eight specimens from the genus Diachea, 18 sequences of nine specimens from the genus Didymium, 18 sequences of nine specimens from the genus Polyschismium, and 122 sequences of 67 specimens from the genus Diderma, and five sequences of three specimens from the genus Meriderma (order Stemonitales) serving as the outgroup. The topologies of ML and BI trees were similar. Accordingly, only the ML tree is shown in Fig. 3. In the two-gene phylogenetic tree, the clade represented the genera Polyschimium and Didymium all received strong support in ML and BI analysis (UBS = 100, PP = 1), and the clade represented the genus Diachea only received partial support in ML and BI analyses (UBS = 79, PP = 0.843).
Species of the genus Diderma were scattered throughout the two-gene phylogenetic tree (Fig. 3) and appeared in three clades. The clade of the genus Diderma was composed of the vast majority of species of the genus Diderma and received partial support (UBS = 96, PP = 0.997), including two subclades. The subclade Ⅰ occupied a more basal position and received strong support in ML and BI analysis (UBS = 100, PP = 1), including Diderma acanthosporum, Diderma chondrioderma, Diderma deplanatum, Diderma effusum, Diderma hemisphaericum, Diderma microsporum, Diderma pseudotestaceum, Diderma teataceum, and Diderma yucatanense. The subclade Ⅱ only received partial support in ML and BI analysis (UBS = 92, PP = 0.995) including the type taxa Diderma globosum, a nivicolous Diderma species, and some other Diderma species. Two more Diderma clades were small and occupied an unresolved position within the family Didymiaceae in the present two-gene phylogenetic tree (Fig. 3). Diderma cor-rubrum assumed a sister position to the clade representing the genus Diachea (UBS = 62, PP = 0.836). Diderma dalatense and Diderma ochraceum were closely related to the clade representing the genus Polyschismium and only received partial support in ML analysis (UBS = 29).
In the two-gene phylogenetic tree (Fig. 3), two specimens representing D. microsporum formed a strongly supported clade (UBS = 100, PP = 1) together with D. hemisphaericum MA-Fungi 90974 and D. effusum MA-Fungi 91604. Thus D. microsporum as an independent myxomycete species also can be strongly supported in the present phylogenetic analysis based on 18S and EF-1α.
Two specimens of Diderma species collected from the Lafa Mountain National Forest Park in Jilin Province were detailed studied through light microscope and scanning electron microscope and found that the main morphological characters including plasmodiocarps cushion-shaped, densely clustered, dense flake-like lime crystals decorated in the single peridium outer surface, columella absence; capillitium branched abundant, spindle-shaped enlargement forms at branching points, decorated with dense small warts; spores (5.70-)6.49-6.87(-7.61) μm diam, warted with clusters of large warts.
Compared with the original description of D. microsporum (Yamamoto, 1998), our specimen has slight differences in spore diameter and the color of the sporocarps. Firstly, the spore diameter of D. microsporum in the original description was 5-6 μm (Yamamoto, 1998), while the spore diameter of the Chinese specimen is (5.70-)6.49-6.87(-7.47) μm. However, this slight difference disappeared after observing the holotype of D. microsporum. The spore diameter of the holotype of D. microsporum (TNS-M-Y-21980) is (6.07-)6.50-6.97(-7.61) μm, decorated with warts (Fig. 1M-O), and no significant difference in spore diameter between Chinese specimen and the holotype (Fig. 4, P = 0.7502). Secondly, there is indeed a little difference in the color of the sporocarps between the Chinese specimen and the holotype. After careful observation and comparison, it was found that when the surface of the plasmodiocarp has more and denser calcareous scales, the color it exhibits is aniline yellow (Fig. 1D, 2A-D). When there are fewer calcareous scales on its surface, white will be more pronounced (Fig. 1B, 1G, 2C). The color, shape, and other differences caused by differences in calcareous matter cannot be considered to have taxonomic significance, as seems to have been demonstrated. For example, the gray matter of Lepidoderma crassipes and L. tigrinum is spherical and scaly, but L. crassipes is considered a transitional form to L. tigrinum by Ronikier et al. (2022).
Therefore, these subtle differences are still within a reasonable range of changes in species characters. In addition, we noticed that both Chinese specimens and the holotype from Japan occurred on living herbaceous plants. This can more or less serve as evidence that the two are of the same kind. It is believed that our specimens do indeed belong to D. microsporum.
4.2. Is Diderma microsporum a variant of Diderma effusum or an independent species?The independence of D. microsporum as a species has always been controversial. Nannenga-Bremekamp and Yamamoto (1987) described and reported a variety, Diderma effusum var. microsporum Nann.-Bremek. & Y. Yamam. Then, it was promoted as an independent species (Yamamoto, 1998) and continued to introduce it as an independent species in his monography (Yamamoto, 2021). However, in the nomenclatural database of Lado (2005-2024), D. microsporum still is a synonym of D. effusum. Because there were only specimens of D. microsporum from Japan before, the description was relatively simple and written in Japanese and lacked molecular evidence, making it a questionable and controversial species.
Comparing the morphological characters with D. effusum (Morgan, 1894), we believe that D. microsporum and D. effusum have clear morphological differences. According to our observation and comparison, these two species can be distinguished by the following characters (Table 2): (ⅰ) peridium (single layer, the outer surface decorated with dense flake-like lime crystals vs. double layers, the outer surface of the outer layer smooth); (ⅱ) spore diameter ((5.70-)6.49-6.87(-7.61) μm vs. 8-10 μm); (ⅲ) columella (absent vs. flat-pulvinate, sometimes scarcely more than a thickened base); (ⅳ) capillitium (branches abundant, spindle-shaped enlargement often forms at branching points vs. few branch). Thus, D. microsporum is significantly different from D. effusum in connecting morphological characters, supporting the treatment of D. microsporum as an independent species.
| Diderma microsporum | Diderma effusum | Diderma saundersii | Diderma deplanatum | Diderma pseudotestaceum | ||
| Source literature | This study | Morgan (1894) | Nannenga-Bremekamp (1966) | Martin & Alexopoulos (1969) | Novozhilov et al. (2014) | |
| Fruiting bodies | Shape | Plasmodiocarps cushion-shaped, occasional presence of sessile sporocarps | Plasmodiocarp very much flattened | Plasmodiocarps, sessile, scattered, very thin | Sporangia pulvinate, sessile, or forming curved or ring-shaped plasmodiocarps | Sporocarps sessile, flat-pulvinate, rounded, slightly umbilicate above |
| Color | White or aniline yellow | White or cinereous | White | White or pale cream-colored or lilaceous | White | |
| Peridium | Single, brittle, ornamented with dense flake-like lime crystals on the outer surface | Double, the outer surface of the outer layer smooth | Double | Double, the outer layer smooth, brittle, the inner layer membranous, iridescent, deep orange below | Double, outer layer white smooth, polished, porcelain-like with white inner surface, fragile | |
| Dehiscence form | Irregularly | Irregularly | Irregular rupturing of the inner peridium | NA | Apical and frequently breaking to leave a ridge | |
| Columella | Absent | Flat-pulvinate, sometimes scarcely more than a thickened base | Absent | Laking or represented by a thickened orange-brown base | Indefinite as a thickened light yellowish pink or light orange base of sporocarp | |
| Capillitium | Diameter | (0.99-)1.29-2.05(-2.30) μm | NA | NA | NA | 0.5 μm |
| Color (LM) | Hyaline | Colorless | Nearly colorless | Dark purple | Hyaline | |
| Ornamentation | Spindle-shaped enlargement often forms at branching points, dense warts | NA | NA | Spiny or nodular enlargements | Sparingly branching with membranous expansions | |
| Spores | Color (LM) | Flesh-ocher | Pale violaceous | Pale rosy gray | Dark yellow-brown | Pale yellowish pink or brownish pink |
| Diameter | (5.70-)6.49-6.87(-7.61) μm | 8-10 μm | 10-11 μm | (9-)9.5-10(-11) μm | 6.2-7.2 μm | |
| Ornamentation | Minute warts, some of the warts in clusters | NA | Minute warts, some of the warts in clusters | Minute spinule | Unevenly covered by warts, some of the warts in clusters | |
Note. NA refers to data not availed.
From a phylogenetic perspective, D. microsporum and D. effusum are separated and do not aggregate into one clade in the two-gene phylogenetic tree (Fig. 3). Six specimens (HFNNU 9787, LE302408, HFNNU 9867, MYX7994, MA-Fungi 51760, and MA-Fungi 90984) representing D. effusum form a stable and independent clade (UBS = 100, PP = 1), Two specimens (HFNNU 9863 and MYX11342) representing D. effusum form an independent clade (UBS = 100, PP = 1) sister to the clade composed of D. pseudotestaceum and D. yucatanense. This clustering pattern is also reflected in the phylogenetic tree constructed by García-Martín et al. (2023), which may indicate that the taxon D. effusum seems to encompass distinct, non-sister species. Two specimens representing D. microsporum have the closest phylogenetic relationship with D. hemisphaericum MA-Fungi 90974 (UBS = 100, PP = 1) followed by D. effusum MA-Fungi 91604 (UBS = 100, PP = 1). The reciprocal monophyletic of the gene trees is a very stringent criterion for delimiting species (Shchepin et al., 2024). However, the specimens representing D. microsporum and the specimens representing D. effusum don’t form monophyletic clades. Thus, D. microsporum and D. effusum cannot be considered the same species.
4.3. Morphological differences between Diderma microsporum and other Diderma speciesDiderma microsporum is located in the subclade Ⅰ in the present two-gene phylogenetic tree (Fig. 3) with a spore diameter of (5.70-)6.49-6.87(-7.61) μm diam. According to García-Martín et al. (2023), spore diameter of all species in the subclade Ⅰ are relatively small (6-9 μm). This important character distinguishes this species from the vast majority of species in the genus Diderma. To our knowledge, only D. pseudotestaceum Novozh. & D.W. Mitch. (Novozhilov et al., 2014) has a similar spore diameter (6.2-7.2 μm) to D. microsporum among Diderma species. However, D. pseudotestaceum and D. microsporum can be easily distinguished on fruiting body shape (sporocarps vs. plasmodiocarps cushion-shaped, densely clustered, occasional presence of sessile sporocarps), peridium (double, outer layer white smooth, polished vs. single, brittle, ornamented with dense flake-like lime crystals on the outer surface), capillitium (0.5 μm diam, sparingly branching with membranous expansions vs. (0.99-)1.29-2.05(-2.30) μm diam, spindle-shaped enlargement often forms at branching points, dense warts under SEM), and columella (apical and frequently breaking to leave a ridge vs. absent) (Table 2).
Additionally, most species of the genus Diderma have a double peridium and a porcelain-like outer surface (Martin & Alexopoulos, 1969). However, the peridium of D. microsporum is single, and the outer surface is covered with relatively large lime scales. Scaly lime deposits used to be the main distinguishing character of Lepidoderma (Martin & Alexopoulos, 1969), but with the migration of L. tigrinum (Schrad.) Rostaf. to the genus Diderma (Prikhodko et al., 2023) and the genus Polyschismium resurrection (Ronikier et al., 2022), this character makes it difficult to clearly distinguish between the genera Diderma and Polychisium, individual lime scales may form in the peridium outer surface of Diderma species in some case. Therefore, the differences in the shape and size of lime deposits on the peridium cannot be considered taxonomically meaningful (Ronikier et al., 2022).
In addition to the discussed above, there are still some species that are easily confused with D. microsporum in terms of macroscopic characters, such as Diderma saundersii (Berk. & Broome ex Massee) E. Sheld. and Diderma deplanatum Fr. (Martin & Alexopoulos, 1969). However, the plasmodiocarps of D. saundersii are very thin, the peridium double, the outer layer thin, the inner layer separate, but close, membranous, brown, and translucent, spores 10-11 μm diam (Table 2). Diderma deplanatum has sporangia scattered or in small groups, pulvinate, sessile, or forming curved or ring-shaped plasmodiocarps, peridium double, the outer layer smooth, crustose, brittle, thick, the inner layer membranous, iridescent, deep orange below, capillitium composed of dark purple, often bearing spiny or nodular enlargements, spores (9-)9.5-10(-11) μm diam (Table 2). These characters make D. microsporum significantly different from D. saundersii and D. deplanatum.
The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.
This research was supported by the National Natural Science Foundation of China (no. 32070007 & no. 32270005) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (no. KYCX24_1849). We sincerely thank the anonymous reviewers for their comments and suggestions. We thank Dr. Yin-Ping Zhang and Mr. You-Sheng Liu of Nanjing Normal University for their assistance with scanning electronic microscopy. We especially appreciate Dr. Zhuo Du of the Herbarium Mycologicum, Academiae Sinicae (HMAS), and Dr. Tsuyoshi Hosoya of the National Museum of Nature and Science (TNS) for their assistance in borrowing specimens. We also thank Dr. Carlos Lado of Real Jardín Botánico, Consejo Superior de Investigaciones Científicas, for providing references.
