Abstract
Myxomycetes (true slime molds) are amoebozoan protists involved in decomposition and nutrient cycling in terrestrial ecosystems. Recent molecular studies have led to major taxonomic revisions in Physaraceae, including the establishment of the genus Nannengaella for highly calcified species previously placed in Physarum. To refine the taxonomy and distribution of Nannengaella in China, we used an integrative approach combining multilocus phylogenetics and morphological examination. A total of 149 specimens from 14 provinces and autonomous regions in China were studied, and phylogenetic analyses were conducted using five loci: nSSU, EF-1α, mtSSU, α-tubulin, and COI. Results confirmed that Nannengaella is a distinct and monophyletic lineage within Physaraceae. One new species, Nannengaella luteotestacea, is described as a well-supported lineage in the genus, and three former Physarum species (N. herbatica, N. cremilutea, N. conglomerata) are recombined into Nannengaella based on molecular and morphological evidence. New distribution records in China are provided for several species: N. herbatica from Jilin Province, N. cremilutea from Heilongjiang, Shaanxi, and Henan Provinces, and N. conglomerata, N. contexta, N. sulphurea, and N. leucopus from Sichuan Province. Detailed morphological descriptions, voucher information, ecological notes, and an identification key are also presented. These findings improve the taxonomic framework of Nannengaella and enhance understanding of its diversity and biogeographic distribution in China.
Keywords: multilocus phylogeny, Myxomycetes, Nannengaella, new combinations, new species, taxonomy
Introduction
Myxomycetes (Myxogastria; informally referred to as “slime molds”) are a distinctive lineage of amoebozoan protists. Taxonomically, they are members of the class Myxomycetes (or Myxogastrea) in biological classification, being amoeboid eukaryotes that produce fungus-like sporocarps (Keller et al., 2022) and a monophyletic group within the phylum Amoebozoa of the kingdom Protozoa (Wijayawardene et al., 2020). Their systematic position is also supported by relevant taxonomic and systematic studies (Adl et al., 2012; Cavalier-Smith et al., 2014; Adl et al., 2019; Fiore-Donno et al., 2019). Ecologically, myxomycetes play important roles in terrestrial ecosystems, particularly in the decomposition of organic matter and nutrient recycling, as documented in studies focusing on their biological and ecological characteristics (Stephenson and Stempen, 1994). Beyond their ecological significance, myxomycetes have attracted considerable interest due to their complex life cycles and morphological diversity, which make them valuable models for studying microbial evolution and phenotypic plasticity.
Within this group, the family Physaraceae Rostafiński (1873) is characterized by the presence of lime (calcium carbonate) deposits and a wide range of sporocarp morphologies. However, these features exhibit substantial variation and convergence, posing long-standing challenges for taxonomy and phylogenetic inference. The genus Physarum Pers. exemplifies this difficulty, having historically been treated as a broad and heterogeneous assemblage encompassing species with considerable morphological variability.
Recent advances in molecular systematics have substantially reshaped our understanding of relationships within Physaraceae. Multigene phylogenetic analyses, coupled with detailed morphological reassessment, have demonstrated that Physarum in its traditionally circumscription is polyphyletic (García-Martín et al., 2023). As a result, the genus Nannengaella was established to accommodate a distinct lineage characterized by highly calcified sporocarps and the presence of a true pseudocolumella (García-Martín et al., 2023). Although this revision has improved generic delimitation, morphological overlap among closely related genera, including Fuligo, Badhamia, and Physarum continues to complicate species-level identification.
The integration of morphological and molecular data has significantly enhanced taxonomic resolution in myxomycetes. Phylogenetic analyses conducted by García-Martín et al. (2023) strongly support the monophyletic status of Nannengaella; this conclusion is robustly inferred from sequence data of nSSU, EF-1α, mtSSU, α-tubulin.
Nevertheless, important gaps remain. The global diversity of Nannengaella is still incompletely resolved, and cryptic diversity may be underestimated due to limited taxon sampling and reliance on a restricted set of molecular markers. Beyond cryptic diversity, most described Physarum species have not yet been sequenced for even a single molecular marker, leaving their phylogenetic positions unclear; some of these species are likely to belong to Nannengaella. In addition, ecological traits, life-cycle dynamics, and responses to environmental gradients remain poorly characterized. Ultrastructural features, such as the ontogeny of the capillitium and lime knot formation, are also insufficiently studied, limiting their utility as diagnostic characters.
In this study, we address these gaps by integrating multigene phylogenetic analyses (nSSU, EF-1α, mtSSU, α-tubulin, COI) with detailed morphological observations to refine the taxonomy of Nannengaella in China. We describe one new species, propose three new combinations, and provide updated distributional records for the genus. Furthermore, we present comprehensive morphological descriptions, voucher information, ecological data, and an identification key to facilitate accurate species delimitation. These findings contribute to a more robust taxonomic framework for Nannengaella and improve our understanding of its diversity, evolution, and biogeographic distribution.
Materials and methods
Sampling and specimens
A total of 149 specimens of Nannengaella were collected from multiple provinces and regions of China, including Heilongjiang, Henan, Hubei, Gansu, Jiangsu, Jiangxi, Jilin, Liaoning, Shaanxi, Shandong, Sichuan, Yunnan, Guangxi Zhuang Autonomous Region, and the Inner Mongolia Autonomous Region. In addition, previously collected materials preserved in the Herbarium of Mycology, Jilin Agricultural University (HMJAU), were examined. All voucher specimens generated in this study are deposited in HMJAU.
Morphological study
Macroscopic characteristics, including sporocarp color and texture, stalk morphology, and columella structure, were examined using a Zeiss Axio Zoom V16 dissecting microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany). Photographs were obtained using a Leica M165 stereomicroscope (Leica Microsystems, Wetzlar, Germany).
For microstructural observations, dried specimens were rehydrated in 3% KOH and examined under a Zeiss Axio Imager A2 light microscope equipped with a Zeiss Axiocam 506 camera. Diagnostic features included spore size and ornamentation, capillitium morphology and pigmentation, and the presence of lime knots. For each specimen, at least 30 mature spores were measured.
Ultrastructural feature of spores and the capillitium were further investigated using a JSM-IT800 scanning electron microscope (JEOL, Tokyo, Japan). Color terminology follows the Flora of British Fungi Colour Identification Chart (Royal, 1969). The range of variation in size for sporocarps and spores is described as (minimum–)25% quartile–75% quartile(–maximum), following the latest literature standards (Leontyev et al., 2023).
DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from sporocarps using the TIANamp Micro DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China), following the manufacturer’s instructions. Five loci were targeted for phylogenetic analyses: nuclear small subunit rDNA (nSSU), elongation factor 1-alpha (EF-1α), mitochondrial small subunit rDNA (mtSSU), α-tubulin, and cytochrome c oxidase subunit I (COI). Standard primer pairs were employed: S2(F)/SR4Dark(R) for nSSU (Fiore-Donno et al., 2008, 2012); PB1F/PB1R for EF-1α (Novozhilov et al., 2013); Kmit-F/Kmit-R for mtSSU (Lado et al., 2022); COMF/COMRs for COI (preferred longer fragment) (Liu et al., 2015; Novozhilov et al., 2019), with COIF1/COIR1 used as an alternative (shorter fragment) (Feng and Schnittler, 2015); and KTub-F2/KTub-R1 for α-tubulin (longer fragment) (García-Martín et al., 2023), with KTub-F3/KTub-R1 as a backup (shorter fragment) (García-Martín et al., 2023).
PCR reactions were performed in 25 μL volumes containing 12.5 μL of 2 × EasyTaq® PCR SuperMix (TransGen Biotech Co., Ltd., Beijing, China), 1 μL of each primer (10 μM), 3 μL of DNA template, and 7.5 μL of ddH2O. Amplifications were carried out under locus-specific thermocycling conditions.
For nSSU and mtSSU, the cycling protocol consisted of an initial denaturation at 94°C for 1 min; 30 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 3 min; and a final extension at 72°C for 10 min. EF-1α amplification was performed with an initial denaturation at 95°C for 5 min; 36 cycles of 95°C for 30 s, 65.4°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. For the COI, two primer pairs were used. The COMF/COMRs pair targeted a longer fragment (95°C for 5 min; 36 cycles of 95°C for 30 s, 52°C for 20 s, and 72°C for 1 min; and a final extension at 72°C for 10 min), while COIF1/COIR1 amplified a shorter fragment under similar conditions with an annealing temperature of 50.7°C. For α-tubulin, KTub-F2/KTub-R1 and KTub-F3/KTub-R1 were used, with annealing temperatures of 52 and 54°C, respectively.
PCR products were verified by electrophoresis on 1% agarose gels stained with ethidium bromide, purified, and sequenced using Sanger sequencing. All newly generated sequences were deposited in GenBank. For each newly described species, sequences were obtained from at least two independent collections (Table 1).
TABLE 1.
Taxa included in the phylogenetic analyses and their corresponding GenBank accession numbers for the five loci (nSSU, EF-1α, mtSSU, α-tubulin, and COI).
Bold fonts indicate the newly generated sequences in this study.
Phylogenetic analyses
Phylogenetic datasets were constructed by integrating morphological identifications with BLAST-based sequence retrieval across the five loci (nSSU, EF-1α, mtSSU, α-tubulin, COI). The final dataset comprised newly generated sequences, 167 representative sequences of Nannengaella retrieved from GenBank, and additional sequences from related genera used as outgroups. Each locus was aligned using MAFFT v7.490 (Katoh and Standley, 2013) with the “—auto” strategy and normal alignment mode, and manually adjusted in BioEdit v7.1.3 (Hall, 1999). Ambiguously aligned regions were inspected and excluded where necessary. Individual alignments were concatenated into a combined dataset using PhyloSuite.
Model selection and partitioned alignment evaluation were implemented using ModelFinder (Kalyaanamoorthy et al., 2017) within IQ-TREE v1.6.12 (Nguyen et al., 2015). Maximum likelihood (ML) analyses were conducted under the best-fit models identified for each partition (TIM2 + F + I + G4 for nSSU, GTR + F + I + G4 for EF-1α/α-tubulin, TPM3u + F + R3 for mtSSU, TVM + F + G4 for COI), with 1,000 ultrafast bootstrap replicates to assess nodal support.
Bayesian inference (BI) was performed via MrBayes integrated in PhyloSuite v1.2.2 (Zhang et al., 2020; Xiang et al., 2023), utilizing the same partition-specific substitution models as ML analyses. Two independent runs with four Markov chains were executed for 2 million generations, with sampling conducted every 1,000 generations. Convergence was verified by effective sample size (ESS) values (> 200 for all parameters) and an average standard deviation of split frequencies converging to 0.0053. The initial 25% of samples were discarded as burn-in, with remaining posterior samples retained for downstream phylogenetic interpretation. Phylogenetic trees were visualized and annotated using iTOL v6.9.
Results
Phylogenetic analyses
Phylogenetic relationships were inferred from a concatenated dataset of five loci (nSSU, EF-1α, mtSSU, α-tubulin, and COI) comprising 99 samples representing 45 taxa. The final alignment included 263 sequences, of which 86 were newly generated in this study (21 nSSU, 27 EF-1α, 21 mtSSU, 5 α-tubulin, and 12 COI). The combined dataset consisted of 9,800 aligned characters, including gaps (nSSU: 6,429; EF-1α: 1,139; mtSSU: 436; α-tubulin: 1,049; COI: 747) (Supplementary material). Twelve taxa from the families Stemonitaceae and Lamprodermataceae were designated as outgroups. Phylogenetic tree comparisons were conducted, and both Maximum Likelihood (ML) and Bayesian Inference (BI) analyses yielded congruent topologies. Consequently, the ML tree (Figure 1) is presented, with support values manually mapped to the corresponding nodes; nodal support is indicated by bootstrap values (BS) and BI posterior probabilities (PP).
FIGURE 1.
Maximum likelihood phylogenetic tree inferred from the concatenated dataset of five loci (nSSU, EF-1α, mtSSU, α-tubulin, and COI) for Nannengaella species. Support valuesat the nodes represent maximum likelihood bootstrap (UBS) percentages and Bayesian posterior probabilities (PP). Taxa newly generated in this study are shown in bold, whereas newly described species and new combinations are highlighted in red.
The phylogeny resolved two major lineages within Physaraceae corresponding to Nannengaella and Physarum (Figure 1). The Nannengaella clade was recovered as sister to Physarum with strong support (BS = 100%, PP = 1) (Figure 1). Within Nannengaella, a distinct lineage corresponding to Nannengaella luteotestacea sp. nov., was recovered as distinct and well supported (BS = 92.1%, PP = 0.94). In addition, three previously described species, N. conglomerata, N. cremilutea, and N. herbatica, were nested within the Nannengaella clade, supporting their transfer to this genus. New distributional records were identified for several taxa. Nannengaella herbatica was recorded for the first time from Jilin Province, while N. cremilutea was newly documented from Heilongjiang, Shaanxi, and Henan Provinces. Furthermore, N. conglomerata, N. contexta, N. sulphurea, and N. leucopus were newly recorded from Sichuan Province.
Taxonomy
Nannengaella luteotestacea X.F. Li, B. Zhang & Y. Li, sp. nov.
MycoBank: MB855045
FIGURE 2.
Habitat and microstructure of Nannengaella luteotestacea (HMJAU M10278 holotype). (A,B) Sporocarps and plasmodiocarps. (C,E) Capillitium and lime nodes by SEM. (D) Peridium by SEM. (F) Spores by SEM. Scale bars: (A) = 2 mm; (B) = 1 mm; (C) = 100 μm; (D,E) = 40 μm; (F) = 5 μm.
Etymology: The epithet “luteotestacea” refers to the deep yellow to orange-yellow color of the plasmodiocarps.
Diagnosis: Distinguished by dark-yellow, sessile plasmodiocarps that are circular to reticulate in habit, a double-layered peridium, irregular dehiscence lacking a preformed line, absence of surface wrinkles, and spores 8–9 μm in diameter.
Type: China, Jiangxi Province, Fuzhou City, Tang Xianzu Memorial Hall, on decaying leaves, 17 June 2013, B. Zhang (HMJAU M10278, holotype).
Description: Fructifications mainly plasmodiocarps, curved-linear, circular to reticulate, occasionally forming spherical sporangia, dark-yellow, often fading with age, sessile, readily dehiscence, often persisting as remnants after dehiscence. Columella absent. Peridium thin, double-layered; dark yellow, outer surface covered with calcareous particles, often brown due to lack of calcium at the plasmodiocarp base; the inner layer membranous, colorless, and transparent. Capillitium dense, colorless, with expanded membranous areas. Lime nodes white, yellowish by transmitted light, large, 25–95 × 15–75 μm, circular to polygonal, sometimes forming a central pseudocolumella. Spores dark brown in mass, light brown by transmitted light, globose, (7.5–) 8–9 μm in diam., warted.
Habitat: On decaying leaves.
Distribution in China: Jiangxi Province.
Global distribution: Known only from China.
Notes: Nannengaella luteotestacea is morphologically and phylogenetically distinct from P. bogoriense, P. hongkongense, P. serpula, and N. plicata. In multigene phylogenies, the new species forms a distinct, well-supported lineage. Morphologically, it is characterized by dark-yellow, sessile plasmodiocarps (easily fading, with a brownish calcium-free base), a double-layered peridium (vs. three-layered peridium in P. bogoriense and P. hongkongense), irregular dehiscence without preformed lines or longitudinal folds (vs. stellate dehiscence with persistent reflexed lobes in P. bogoriense, and dehiscence along pre-formed lines in P. hongkongense), and abundant, large polygonal lime nodes (25–95 × 15–75 μm) that frequently aggregate to form a pseudocolumella (vs. small, scattered lime nodes in the two Physarum species). Sporophores of N. luteotestacea are non-compressed, in contrast to the laterally strongly compressed sporophores of P. hongkongense and the non-compressed ochre-brown sporophores of P. bogoriense; its spores are light brown, (7.8–) 8.0–9.0 μm in diameter, with uniform warts, differing from the violet-brown, clustered-wart spores (7.5–10 μm) of P. bogoriense and the smaller light brown, uniformly warted spores (7.5–8 μm) of P. hongkongense. Compared with P. serpula, the new species differs in having a double-layered peridium (vs. single-layered) and white lime nodes with smaller spores (8–9 μm vs. 10–13 μm). In contrast to N. plicata, N. luteotestacea lacks longitudinal surface wrinkles, exhibits irregular dehiscence (vs. dehiscence via preformed lines), and has darker yellow plasmodiocarps. The detailed morphological features are compared in Table 2.
TABLE 2.
Comparison of morphological characteristics between N. luteotestacea and its related species.
| Characteristic | N. luteotestacea | P. bogoriense | P. hongkongense | P. serpula | N. plicata |
|---|---|---|---|---|---|
| Phylogenetic status | Forms a distinct and well-supported lineage in multigene phylogeny | Known species of the Physarum | Known species of the Physarum | Known species of the Physarum | Known species of the Nannengaella |
| Sporophore morphology | Plasmodiocarps, dark yellow, non-compressed, easily fading, brownish at the calcium-free base | Plasmodiocarps, light reddish brown,reddish brown, non-compressed | Plasmodiocarps, bright yellow, or ochre yellow, strongly laterally compressed, constricted at the base | Plasmodiocarps, orange-yellow | Plasmodiocarps, light yellow or bright yellow |
| Peridium | Double-layered | Three-layered | Three-layered: | Single-layered | Double-layered |
| Dehiscence pattern | Irregular dehiscence, without preformed lines or longitudinal folds | Polygonal dehiscence at the upper part, stellate dehiscence at the side with persistent, reflexed lobes | Dehiscence along pre-formed lines | Irregular dehiscence | Dehiscence along pre-formed lines, with longitudinal surface wrinkles |
| Lime nodes | Abundant, large, polygonal, white, often form a pseudocolumella | Small, rounded to polygonal, white, scattered | Small, rounded to polygonal, white, scattered | Yellowish-white, scattered | Polygonal, white |
| Spores | Dark brown in mass, light brown under transmitted light | Dark brown in mass, violet-brown under transmitted light | Blackish-brown in mass, light brown under transmitted light | Light brown under transmitted light | Light brown under transmitted light |
| Spore size | (7.8–)8.0–9.0 μm | 7.5–10 μm | 7.5–8 μm | 10–13 μm | 8–9 μm |
| Spore ornamentation | Verrucose (uniform warts) | Verrucose (warts sometimes clustered) | Verrucose (uniform warts) | Verrucose | Verrucose |
Nannengaella cremilutea (Y.F. Chen & C.H. Liu) X.F. Li, B. Zhang & Y. Li, comb. nov.
MycoBank: MB855053
FIGURE 3.
Habitat and microstructure of Nannengaella cremilutea (HMJAU M10217). (A,B) Sporocarps. (C,D) Sporocarp by SEM. (E) Peridium by SEM. (F) Capillitium and lime nodes by SEM. (G) Spores by SEM. Scale bars: (A) = 1 mm; (B) = 500 μm; (F) = 20 μm; (G) = 5 μm.
Basionym: Physarum cremiluteum Y.F. Chen & C.H. Liu, in Liu & Chen, Taiwania 43(3):186 (Liu and Chen, 1998).
Description: Sporocarps gregarious, stipitate, globose or subglobose, cream-yellow, lemon-yellow or bright yellow-green, 0.4–0.5 mm in diam., and 0.8–0.9 mm in total height; basal part of the sporangium non-calcareous, with a blue or purple iridescence. Stalk white, calcareous, stout, tapering upwards, 0.4–0.5 mm long. Columella absent. Hypothallus small, membranous. Peridium membranous, covered with light yellow lime granules, dehiscing petaloidally. Capillitium netted, with abundant cream-yellow lime nodes that are polygonal to fusiform; connecting threads colorless and transparent. Spores dark in mass, violaceous brown by transmitted light, globose, (7.5–) 8–10 (–11) μm in diam., minutely warted.
Habitat: On decaying leaves.
Distribution in China: Taiwan Province, Sichuan Province, Hubei Province, Jilin Province, Shaanxi Province, Henan Province, Heilongjiang Province.
Global distribution: China and Japan.
Specimens examined: China, Jilin Province, Jian City, on decaying leaves, 5 Oct. 2000, Tolgor (HMJAU M20974, HMJAU M20975); China, Jilin Province, Antu County, Erdaobaihe Town, Changbai Mountain Scenic Area, Hunting Ground, on decaying leaves, 22 July 2012, B. Zhang (HMJAU M10162, HMJAU M21012); China, Jilin Province, Changchun City, Jingyuetan national forest park, on decaying leaves, 8 Sept. 2015, B. Zhang (HMJAU M21009); China, Jilin Province, Baishan City, Fusong County, Songjianghe National Forest Park, on decaying leaves, 8 July 2018, B. Zhang (HMJAU M20966); China, Jilin Province, Shulan City, Jiulongshan National Forest Park, on decaying leaves, 29 July 2022, X.F. Li, X.Y. Yang (HMJAU M21028, HMJAU M21029); China, Jilin Province, Jiaohe City, Red Leaf Valley Scenic Area, General’s Altar, on decaying leaves, 31 July 2022, X.F. Li (HMJAU M21013, HMJAU M21014); China, Jilin Province, Tonghua City, Huinan County, Sanjiaolongwan Nature Reserve, on decaying leaves, 10 Aug. 2022, X.F. Li (HMJAU M21011). China, Sichuan Province, Garze Tibetan Autonomous Prefecture, Gexigou National Nature Reserve, on decaying leaves, 3 Aug. 2012, B. Zhang (HMJAU M21049, HMJAU M21050, HMJAU M21051, HMJAU M21052, HMJAU M21053, HMJAU M21054, HMJAU M21055); China, Sichuan Province, Liangshan Yi Autonomous Prefecture, Mianning County, Yihai Scenic Area, on decaying leaves, 6 July 2013, B. Zhang (HMJAU M21026, HMJAU M21027); China, Sichuan Province, Liangshan Yi Autonomous Prefecture, Mianning County, Lingshan Temple, on decaying leaves, 12 Julu 2013, B. Zhang (HMJAU M10177, HMJAU M10200, HMJAU M10198, HMJAU M10168, HMJAU M10159, HMJAU M10150, HMJAU M10210, HMJAU M10190, HMJAU M10195, HMJAU M10128, HMJAU M10207, HMJAU M10182, HMJAU M10152, HMJAU M10122, HMJAU M10216, HMJAU M10137, HMJAU M21030, HMJAU M21031, HMJAU M21032, HMJAU M21033, HMJAU M21034, HMJAU M21035, HMJAU M21036, HMJAU M21037). China, Hubei Province, Shiyan City, Fang County, on decaying leaves, 18 Sept. 2013, B. Zhang (HMJAU M10187). China, Shaanxi Province, Shangluo City, Niubeiliang National Forest Park, on decaying leaves, 21 July 2014, B. Zhang (HMJAU M20971, HMJAU M20972, HMJAU M20973). China, Henan Province, Nanyang City, Neixiang County, Baotianman National Nature Reserve, on decaying leaves. 30 June 2015, B. Zhang (HMJAU M21006, HMJAU M21007, HMJAU M21008). China, Heilongjiang Province, Heihe City, Sunwu County, Shengshan Fortress, on decaying leaves, 13 Aug. 2022, X.F. Li (HMJAU M20969); China, Heilongjiang Province, Heihe City, Sunwu County, Near Yijiazi Mountain, on decaying leaves, 15 Aug. 2022, X.F. Li (HMJAU M20967).
Notes: The transfer of this species to Nannengaella is supported by its placement within the Nannengaella clade in the multigene phylogeny and by its concordant morphology, including calcified structures and typical lime-node development. Morphologically, N. cremilutea is distinguished by cream-yellow sporocarps and lime nodes, and by a short, white, calcareous stalk. It resembles N. mellea, but the latter has light yellow to dark orange sporocarps and a conical columella, whereas N. cremilutea lacks a columella and has uniformly cream-yellow sporocarps. It also differs from Physarum tenerum, which lacks the characterstic short calcareous stalk and polygonal lime nodes of N. cremilutea. Compared to other congeners, this species is relatively easy to recognize. Due to the uncertainty of environmental changes, after observing a large number of specimens, we found that the spore diameter of this species is slightly larger than the original description.
Nannengaella herbatica (Shuang L. Chen & Yu Li) X.F. Li, B. Zhang & Y. Li, comb. nov.
MycoBank: MB855054
FIGURE 4.
Habitat and microstructure of Nannengaella herbatica (HMJAU M20342). (A,B) Sporocarps. (C,D) Capillitium and lime nodes by TL. (E–G) Spores by TL. Scale bars: (A) = 1 mm; (B) = 500 μm; (C) = 100 μm; (D) = 20 μm; (E–G) = 10 μm.
Basionym: Physarum herbaticum Shuang L. Chen & Yu Li, Mycosystema 19(3):332 (Chen and Li, 2000).
Description: Fructifications mainly plasmodiocarps, curved or linear, occasionally producing a single sporocarp, yellow to yellowish-green, sessile. Columella absent. Peridium single-layered, membranous, covered with yellow-green calcareous particles, irregularly dehiscent. Capillitium dense, composed of slender, transparent threads bearing white, polygonal to irregular lime nodes. Spores dark in mass, yellowish-brown by transmitted light, globose, (8.5–) 9–10 μm in diam., minutely warted.
Habitat: On decaying leaves.
Distribution in China: Guangxi Zhuang Autonomous Region, Jilin Province.
Global distribution: China.
Specimens examined: China, Jilin Province, Dunhua City, Hongye Valley Scenic Area, Hancong Ridge, on decaying leaves, 26 July 2022, X.F. Li, X.Y. Yang (HMJAU M20342).
Notes: The transfer of this species to Nannengaella is supported by its placement within the Nannengaella clade in the multigene phylogeny, together with its concordant morphological features, especially the calcified peridial surface and characteristic lime nodes. Morphologically, N. herbatica is distinguished by yellow to yellowish-green plasmodiocarps, a single-layered peridium, white polygonal lime nodes, slender capillitium threads, and the absence of a columella. It resembles N. lakhanpalii and Physarum plicatum in overall coloration, but differs from N. lakhanpalii in lacking fascicled spores and from P. plicatum in lacking the wrinkled outer peridium and preformed dehiscence line. It is also comparable to P. serpula A.P. Morgan, but that species has larger spores (10–13 μm), whereas N. herbatica has smaller spores (8.5–10 μm).
Nannengaella conglomerata ((Fr.) Rostaf.) X.F. Li, B. Zhang & Y. Li, comb. nov.
MycoBank: MB855055
FIGURE 5.
Habitat and microstructure of Nannengaella conglomerata (HMJAU M20366). (A,B) Sporocarps. (C) Plasmodiocarp by SEM. (D) Peridium by SEM. (E) Capillitium and lime nodes by SEM. (F) Spores by SEM. Scale bars: (A,B) = 1 mm; (C) = 100 μm; (D,E) = 40 μm; (F) = 5 μm.
Basionym: Diderma conglomeratum Fr., Syst. mycol. 3(1):111 (1829).
Description: Sporocarps or short plasmodiocarps, sessile, densely crowed, often angular due to mutual pressure, pale yellow to yellow, with orange-yellow calcareous particles on the surface. Sporocarps subglobose, 0.3–0.5 mm in diam., plasmodiocarps up to 1 mm. Columella absent. Peridium double-layered, outer layer calcareous, inner layer pale yellow, translucent, membranous, and tightly adherent to the outer layer, dehiscent irregular. Capillitium abundant, transparent, with membranous expansions. Lime nodes white to pale yellow, large, angular or rounded, often aggregated centrally to form a pseudocolumella. Spores dark brown in mass, purple-brown by transmitted light, (8.5–) 9–10 μm in diam., with minutely spinulose.
Habitat: On decaying leaves.
Distribution in China: Xinjiang Uygur Autonomous Region, Yunnan Province, Sichuan Province.
Global distribution: China, France, Germany, Spain, the United States, the United Kingdom, Japan, Antigua and Barbuda, the Netherlands, Russia, Ukraine, Finland, India, Romania, Australia.
Specimens examined: China, Sichuan Province, Garze Tibetan Autonomous Prefecture, Gexigou National Nature Reserve, on decaying leaves, 14 Aug. 2012, B. Zhang (HMJAU M20366, HMJAU M20332).
Notes: The new combination is supported by the placement of this species within the Nannengaella clade in the concatenated phylogeny and by its agreement with the morphological circumscription of the genus. Nannengaella conglomerata closely resembles N. contexta in having pale -yellow plasmodiocarps, a double-layered peridium, white lime nodes, and no columella. However, N. conglomerata differs in producing subglobose sporocarps smaller spores ((8.5) 9–10 μm), and frequent pseudocolumella formation. By contrast, N. contexta typically forms denser aggregations approaching pseudoaethalia and has larger spores (10–14 μm) ornamented with spinules or warts.
Nannengaella mellea (Berk. & Broome) J.M. García-Martín, J.C. Zamora & Lado, Persoonia 51:110 (García-Martín et al., 2023).
FIGURE 6.
Habitat and microstructure of Nannengaella mellea (HMJAU M10204). (A,B) Sporocarps. (C) Sporocarp by SEM. (D,E) Peridium by SEM. (F) Capillitium and lime nodes by SEM. (G) Spores by SEM. Scale bars: (A) = 2 mm; (B) = 500 μm; (E) = 10 μm; (F) = 40 μm; (G) = 5 μm.
Description: Sporocarps, globose to subglobose, usually stipitate and only rarely sessile, orange-yellow to brownish-orange against an olivaceous-gray background, 0.4–0.6 mm in diam., 0.7–1 mm in total height. Stalk stout, white, calcareous, tapering toward, 0.7–1 mm. Columella present, small, conical, white. Peridium membranous, roughened by a layer of white calcareous particles, dehiscing petaloidally. Hypothallus membranous, transparent. Capillitium reticulate, with large white polygonal lime nodes, 27–95 × 10–40 μm; connecting threads colorless and transparent, with membran ous expansions. Spores dark in mass, light brown by transmitted light, subglobose7–9 (–10) μm in diam., ornamented with clusters of darker warts.
Habitat: On decaying leaves.
Distribution in China: Beijing City, Hebei Province, Jilin Province, Heilongjiang Province, Jiangsu Province, Anhui Province, Fujian Province, Hubei Province, Hunan Province, Suchuan Province, Yunnan Province, Taiwan Province, Gansu Province, Guangdong Province, Liaoning Province, Shandong Province, Henan Province, Guangxi Zhuang Autonomous Region, Inner Mongolia Autonomous Region, Xizang Autonomous Region, and Hong Kong Special Administrative Region.
Global distribution: Widely distributed around the worldwide.
Specimens examined: China, Sichuan Province, Liangshan Yi Autonomous Prefecture, Mianning County, Lingshan Temple, on decaying leaves, 12 July 2013, B. Zhang (HMJAU M10106, HMJAU M10295); China, Sichuan Province, Chengdu City, Tazishan Park, on decaying leaves, 15 July 2013, B. Zhang (HMJAU M10158, HMJAU M10206, HMJAU M10100, HMJAU M10108, HMJAU M10166, HMJAU M10104, HMJAU M20875, HMJAU M20876); China, Sichuan Province, Garze Tibetan Autonomous Prefecture, Gexigou National Nature Reserve, on decaying leaves, 14 Aug. 2012, B. Zhang (HMJAU M20863). China, Hubei Province, Suizhou City, Tianhekou Township, Hetao Gou, on decaying leaves, 20 July 2010, B. Zhang (HMJAU M10114, HMJAU M10213). China, Jiangxi Province, Fuzhou City, Junfengshan National Forest Park, on decaying leaves, 19 June 2013, B. Zhang (HMJAU M10171); China, Jiangxi Province, Fuzhou City, Linchuan District, Zhanping Town, on decaying leaves, 9 July 2010, B. Zhang (HMJAU M20880, HMJAU M20881, HMJAU M20882, HMJAU M20883, HMJAU M20884). China, Jilin Province, Changchun City, Jilin Agricultural University Campus, on decaying leaves, 7 Sept. 2015, B. Zhang (HMJAU M20896, HMJAU M20897, HMJAU M20898, HMJAU M20899, HMJAU M20900, HMJAU M20901, HMJAU M20902, HMJAU M20903); China, Jilin Province, Changchun City, Jingyuetan national forest park, on decaying leaves, 1 Aug. 2013, B. Zhang (HMJAU M10169, HMJAU M10165, HMJAU M10139, HMJAU M10175, HMJAU M10172, HMJAU M10116, HMJAU M20871, HMJAU M20872). China, Gansu Province, Tianshui City, Dangchuan Forest Farm, on decaying leaves, 15 Aug. 2010, B. Zhang (HMJAU M20890). China, Yunnan Province, Lijiang City, Zhishan, on decaying leaves, 20 Aug. 2012, B. Zhang (HMJAU M10199, HMJAU M10148, HMJAU M10197, HMJAU M10157, HMJAU M20329). China, Henan Province, Zhumadian City, Biyang County, Tongshan Lake, on decaying leaves, 12 June 2023, B. Zhang (HMJAU M20319, HMJAU M20346, HMJAU M20355). China, Heilongjiang Province, Heihe City, Sunwu County, on decaying leaves, 15 Aug. 2022, X.F. Li (HMJAU M20862, HMJAU M21064, HMJAU M21094). China, Shaanxi Province, Liuba Garden, on decaying leaves, 14 Aug. 2012, B. Zhang (HMJAU M20864). China, Liaoning Province, Fuxin City, Fuxin County, Haitang Mountain Scenic Area, on decaying leaves, 1 Sept. 2012, B. Zhang (HMJAU M20884, HMJAU M20885, HMJAU M20886, HMJAU M20887). China, Guangxi Zhuang Autonomous Region, Baise City, on decaying leaves, 13 July 2017, B. Zhang (HMJAU M20894). China, Shandong Province, on decaying leaves, 24 Jan. 1905, Y. Li (HMJAU M20895). China, Jiangsu Province, Nanjing City, Nanjing Agricultural University, on decaying leaves, 8 June 2016, B. Zhang (HMJAU M20913).
Notes: Nannengaella mellea is a common and variable species, especially in sporocarp coloration, which ranges from honey yellow and orange-yellow to grayish-yellow. It may be confusion with Physarum citrinum, owing to because of the similar sporocarp color and small columella. However, N. mellea differs in having polygonal lime nodes and a shorter, stouter stalks, whereas P. citrinum typically has rounded lime nodes and a more slenderer stalks.
Nannengaella sulphurea (Alb. & Schwein.) J.M. García-Martín, J.C. Zamora & Lado, Persoonia 51:110 (García-Martín et al., 2023).
FIGURE 7.
Habitat and microstructure of Nannengaella sulphurea (HMJAU M20341). (A,B) Sporocarps. (C–F) Capillitium and lime nodes by TL and SEM. (G) Spores by SEM. Scale bars: (A) = 2 mm; (B) = 1 mm; (C,D) = 20 μm; (E) = 2 μm; (F) = 4 μm; (G) = 1 μm.
Description: Sporocarps gregarious, stipitate, cylindrical to clavate, pale yellow to pale ochraceous. Stalk short, calcareous, usually thicker at the base, white or yellowish brown. Columella present as a white conical protrusion. Hyporhallus white, calcareous. Peridium membranous, semi-transparent, covered with orange-yellow calcareous scales, irregularly dehiscent. Capillitium reticulate, colorless and transparent, with membranous and elongated expansions. Lime nodes large, angular, yellowish to white, sometimes forming a pseudocolumella. Spores dark in mass, crineous under cinereous by transmitted light, 9–10 (–11) μm in diam., warted, with warts occasionally arranged in lines.
Habitat: On decaying leaves.
Distribution in China: Beijing City, Hebei Province, Hubei Province, Anhui Province, Hunan Province, Sichuan Province.
Global distribution: China, the United States, Canada, India, Mexico, Norway, Brazil, Japan, Germany, Russia, Argentina, Puerto Rico, Sierra Leone.
Specimens examined: China, Sichuan Province, Garze Tibetan Autonomous Prefecture, Gexigou National Nature Reserve, on decaying leaves, 3 Aug. 2014, B. Zhang (HMJAU M20333, HMJAU M20334, HMJAU M20341); China, Sichuan Province, Garze Tibetan Autonomous Prefecture, Gexigou National Nature Reserve, on decaying leaves, 14 Aug. 2012, B. Zhang (HMJAU M21278, HMJAU M21279, HMJAU M20288).
Notes: This species is readily recognized by its pale yellow to ochraceous, cylindrical to clavate sporocarps, and a peridium roughened with orange-yellow calcareous scales, and distinct conical columella. It resembles Physarum auriscalpium in sporocarp color and spore size, but differs in its more robust calcareous structures and in the presence of a conical columella.
Nannengaella leucopus (Link) J.M. García-Martín, J.C. Zamora & Lado, Persoonia 51:110 (García-Martín et al., 2023).
FIGURE 8.
Habitat and microstructure of Nannengaella leucopus (HMJAU M20301). (A,B) Sporocarps. (C) Peridium by SEM. (D,E) Capillitium and lime nodes by SEM. (F) Spores by SEM. Scale bars: (A) = 2 mm; (B) = 500 μm; (C,E) = 20 μm; (D) = 100 μm; (F) = 5 μm.
Description: Sporocarps gregarious, erect, short-stalk or sessile, light yellow, rough, subglobose, sometimes slightly umbilicate below, 0.3–0.4 mm in diam., and about 0.5 mm in total height. Stalk thick, cylindric, slightly tapering upwards, white to yellowish-white, calcareous, 0.2–0.25 mm long. Columella present, small, sometimes expressed as a short conical protuberance, yellowish-white. Hypothallus membranous, transparent, yellow-brown to light yellow. Peridium single-layered, membranous, roughened with white granular scales, light yellow, irregularly dehiscent, lacking calcium at the junction with the stalk and often showing a iridescence. Capillitium loosely reticulate, colorless and transparent, with large light-yellow to white lime nodes, that are elongate to angular, sometimes forming an irregular pseudocolumella. Spores dark in mass, light brown by transmitted light, 9–10 (–11) μm in diam., warted.
Habitat: On decaying woods.
Distribution in China: Beijing City, Hebei Province, Jilin Province, Jiangsu Province, Zhejiang Province, Anhui Province, Fujian Province, Yunnan Province, Taiwan Province, Heilongjiang Province, Inner Mongolia Autonomous Region, Hainan Province, Gansu Province, Guangdong Province, Liaoning Province, Sichuan Province.
Global distribution: China, the United States, Germany, Mexico, Russia, the Netherlands, France, Canada, Spain, Argentina, and the United Kingdom.
Specimens examined: China, Sichuan Province, Garze Tibetan Autonomous Prefecture, Gexigou National Nature Reserve, on decaying woods, 14 Aug. 2012, B. Zhang (HMJAU M20301). China, Heilongjiang Province, Heihe city, Sun Wu County, near Yijia Mountain, on decaying woods, 15 Aug. 2022, X.F. Li (HMJAU M21058, HMJAU M20364, HMJAU M21088, HMJAU M20327). China, Inner Mongolia Autonomous Region, Motianling, on decaying woods, 15. Aug. 1985, Y. Li, S.L. Chen (HMJAU 9123).
Notes: Nannengaella leucopus is a widespread species characterized by its short, robust calcareous stalk, pale-yellow sporocarps, and larger spores relatively [9–10 (–11) μm]. It is morphologically similar to N. globulifera and Physarum citrinum. However, N. globulifera has smaller spores, whereas P. citrinum differs in having smaller, rounded lime nodes and a golden stalk, rather than the elongate to angular lime nodes and white to yellowish-white stalk typical of N. leucopus.
Nannengaella contexta (Pers.) J.M. García-Martín, J.C. Zamora & Lado, Persoonia 51:110 (García-Martín et al., 2023).
FIGURE 9.
Habitat and microstructure of Nannengaella contexta (HMJAU M20354). (A,B) Sporocarps. (C) Peridium by SEM. (D,E) Capillitium and lime nodes by SEM. (F) Spores by SEM. Scale bars: (A,B) = 1 mm; (C) = 2 μm, (D) = 40 μm, (E) = 20 μm, (F) = 1 μm.
Description: Sporocarps or short plasmodiocarps, gregarious, pale yellow to yellow, densely grouped and often angular due to mutual pressure, but not superimposed, ovoid to reniform, sessile. Columella absent. Peridium double-layered, outer layer thick, calcareous, pale yellow, inner layer membranous, pale to yellowish. Capillitium dense, with white to yellowish, angular lime nodes, sometimes confluent in the center to form a pseudocolumella. Spores dark in mass, reddish-brown by transmitted light, 10–11.0 (–11.5) μm in diam., with densely warted to spinulose.
Habitat: On decaying leaves.
Distribution in China: Heilongjiang Province, Jilin Province, Henan Province, Gansu Province, Shandong Province, Yunnan Province, Xizang Autonomous Region, Inner Mongolia Autonomous Region, Sichuan Province.
Global distribution: China, the United States, Canada, France, Germany, the United Kingdom, the Netherlands, Spain, Russia, Mexico, Sweden, India, Japan, Pakistan.
Specimens examined: China, Sichuan Province, Garze Tibetan Autonomous Prefecture, Gexigou National Nature Reserve, on decaying leaves, 3 Aug. 2014, B. Zhang (HMJAU M20354); China, Sichuan Province, Garze Tibetan Autonomous Prefecture, Gexigou National Nature Reserve, on decaying leaves, 14 Aug. 2014, B. Zhang (HMJAU M21297, HMJAU M20340, HMJAU M20289, HMJAU M20339).
Notes: Nannengaella contexta is distinguished by pale-yellow sporocarps that become angular by mutual pressure, the absence of a columella, double-layered peridium, and angular lime nodes. It resembles Physarum conglomeratum in having yellow sporocarps and a two-layered peridium, but differs in its flatter to concave sporocarps with angular margins, larger spores (10–11.0 (–11.5) μm), and more regular dehiscence. By contrast, P. conglomeratum has round to convex plasmodiocarps, irregular dehiscence, and smaller spores (8–10 μm). Physarum tessellatum differs further in having white sporocarps, rounded lime nodes, and a distinctly tessellated calcareous peridium.
Nannengaella plicata (Nann. - Bremek. & Y. Yamam.) J.M. García-Martín, J.C. Zamora & Lado, Persoonia 51:110 (García-Martín et al., 2023).
FIGURE 10.
Habitat and microstructure of Nannengaella plicata (HMJAU M20348). (A,B) Plasmodiocarps. (C) Peridium by SEM. (D,E) Capillitium and lime nodes by SEM. (F) Spores by SEM. Scale bars: (A) = 2 mm; (B) = 1 mm.
Description: Fructifications mainly plasmodiocarps, scattered to gregarious, reticulate or branched, cylindric, curved, and distinctly wrinkled; yellow, orange-yellow or greenish yellow, sessile. Columella absent. Peridium double-layered; both layer membranous, light yellow, with white calcareous particles deposited between them, dehiscence occurring along a preformed longitudinal line, irregular, the remaining areas smooth. Hypothallus membranous, transparent, yellowish. Capillitium dense, reticulate, with numerous small and white angular lime nodes, 25–65 × 15–40μm. Spores dark brown in mass, light brown by transmitted light, subglobose, (8.5–) 9–10 μm in diam., warted.
Habitat: On decaying leaves.
Distribution in China: Taiwan Province, Henan Province, Jilin Province, Sichuan Province.
Global distribution: China, Japan, Democratic Republic of Congo, Equatorial Guinea, Nepal.
Specimens examined: China, Henan Province, Zhumadian City, Biyang County, Wanfeng Temple, on decaying leaves, 12 June 2023, B. Zhang (HMJAU M20348, HMJAU M21290, HMJAU M20371, HMJAU M21102, HMJAU M21103); China, Henan Province, Zhumadian City, Biyang County, Tongshan Lake, on decaying leaves, 12 June 2023, B. Zhang (HMJAU M21101); China, Henan Province, Zhumadian City, Biyang County, botanical garden, on decaying leaves, 11 June 2023, B. Zhang (HMJAU M20320, HMJAU M20321, HMJAU M21124, HMJAU M21125, HMJAU M21126, HMJAU M21127, HMJAU M21128, HMJAU M21129, HMJAU M21130, HMJAU M21131, HMJAU M21132, HMJAU M21133, HMJAU M21134). China, Sichuan Province, Garze Tibetan Autonomous Prefecture, Gexigou Nature Reserve, on decaying leaves, 14 Aug. 2012, B. Zhang (HMJAU M20303).
Notes: This species is characterized by its yellow plasmodiocarps with distinct longitudinal folds, a double-layered peridium with a preformed dehiscence line, and numerous small white angular lime nodes. It resembles Physarum aeneum in having yellow plasmodiocarps and a two-layered peridium, but differs in its larger spores ((8.5–) 9–10 μm vs. 7–9 μm), white angular lime nodes rather than pale yellow rounded ones, and a membranous rather than cartilaginous outer peridium.
Key to the species of Nannengaella
Note: This key includes all currently recognized species of Nannengaella, including taxa not treated in detail in the present study (N. alpestris, N. alpina, N. globulifera, N. lakhanpalii, and N. laevis).
1. Fructifications mainly plasmodiocarps or aethalia…………2
1. Fructifications mainly sporocarps…………°……………3
2. Fructification forming aethalia.……………..Nannengaella laevis
2. Fructification forming plasmodiocarps.…………………..4
3. Columella present……………………………………5
3. Columella absent……………………………………..6
4. Plasmodiocarps lacking a preformed dehiscence line……….…….°……………7
4. Plasmodiocarps with preformed dehiscence line, producing longitudinal irregular folds.……………………………………8
5. Sporocarps pale yellow to pale ochraceous, cylindrical or clavate.….……………………………..Nannengaella sulphurea
5. Sporocarps rounded, not as above ……………….…….………….…….9
6. Sporocarps cream to lemon yellow, stipitate……………….Nannengaella cremilutea
6. Sporocarps yellow to pale yellow, sessile……………………….….….………0.10
7. Peridium double-layered; sporocarps dark yellow.……0.11
7. Peridium single-layered; sporocarps yellow to yellowish-green……………………………Nannengaella herbaticum
8. Lime nodes white, polygonal…….…. Nannengaella plicata
8. Lime nodes yellow, circular to fusiform.Nannengaella lakhanpalii
9. Stalk 0.6–1.0 mm long; spores < 9 μm……….….…12
9. Stalk 0.2–0.25 mm long; spores > 9 μm…….….…….…Nannengaella leucopus
10. Sporocarps with orange-yellow surface scales, spores 9–10 μm.……………….…………….…… Nannengaella conglomeratum
10. Sporocarps without orange-yellow surface scales; spores 10–14 μm.…13
11. Spores <10 μm.Nannengaella luteotestacea
11. Spores >10 μm.Nannengaella alpestris
12. Sporocarps light yellow, honey yellow, or dark yellow.Nannengaella mellea
12. Sporocarps white.Nannengaella globulifera
13. Lime nodes white or yellowish.Nannengaella contexta
13. Lime nodes yellow or light yellow.Nannengaella alpina
Discussion
This study refines the taxonomy of Nannengaella, a recently established genus within Physaraceae, through an integrative framework combining multilocus phylogenetics and detailed morphological analyses of specimens collected across a broad geographic range in China. Our analyses consistently recovered Nannengaella as a distinct and well-supported lineage separate from Physarum and related genera, thereby supporting the generic circumscription proposed by García-Martín et al. (2023). By incorporating five molecular markers (nSSU, EF-1α, mtSSU, α-Tub, COI), the present study provides a robust basis for evaluating taxa in a group where traditional classification has long been complicated by overlapping morphological characters, particularly sporocarp architecture, peridial structure, and lime deposition patterns (Nandipati et al., 2012; Chen and Li, 2000; Ronikier et al., 2022; García-Martín et al., 2023; Prikhodko et al., 2023). These findings further highlight the central role of molecular phylogenetics in resolving taxonomic complexity in morphologically variable myxomycete groups.
The recognition of Nannengaella luteotestacea expands the currently known diversity of the genus. In the concatenated phylogeny, this taxon formed a distinct lineage (Figure 1), and its morphology was likewise diagnostic, especially the dark-yellow plasmodiocarps, double-layered peridium, absence of a preformed dehiscence line, and small warted spores (Figure 2). Its distinction from morphologically similar taxa such as Physarum serpula and N. plicata illustrates the value of combining morphology with multilocus evidence in species delimitation. This integrative approach is particularly important in myxomycetes, where morphological convergence and intraspecific variation can obscure taxonomic boundaries and potentially conceal cryptic diversity.
Our results also support the transfer of three previously described Physarum species, N. herbatica, N. cremilutea, and N. conglomerate to Nannengaella (Tice et al., 2016; Liu and Chen, 1998). These recombinations are supported not only bytheir phylogenetic placement within the Nannengaella clade, but also by morphological features consistent with the genus, including calcified fructifications, characteristic lime nodes, and, in some taxa, pseudocolumella formation. Together, these changes strengthen the ongoing revision of Physaraceae initiated by García-Martín et al. (2023) and contribute to a more stable delimitation of Nannengaella. Rather than merely increasing the number of recognized taxa, these findings help reduce the taxonomic ambiguity historically associated with the broad and heterogeneous concept of Physarum.
The present study also expands the known distribution of Nannengaella in China. We documented the first report of N. herbatica from Jilin Province, significant substantial range extensions for N. cremilutea into Heilongjiang, Shaanxi, and Henan Provinces, and new records of N. conglomerata, N. contexta, N. sulphurea, and N. leucopus from Sichuan Province. These findings indicate that some species are more widely distributed than previously recognized especially N. cremilutea and N. mellea. At the same time, the current distributional pattern likely still reflects uneven sampling intensity rather than the full extent of species ranges. Myxomycete species exhibit different substrate selectivity, but most prefer decaying wood and litter (Lin et al., 2024). Most collections were made in forested habitats rich in decaying plant material, such as most specimens collected from Sichuan’s Gexigou Reserve and Jilin’s Changbai Mountains in this study. Nevertheless, ecological interpretation remains limited because environmental variables such as substrate type, humidity, pH, and microclimatic conditions were not systematically quantified (Schnittler and Stephenson, 2000; Schnittler, 2001; Stephenson et al., 2003; Lado et al., 2007; Novozhilov and Schnittler, 2008; Tice et al., 2016; Dagamac et al., 2017). Future studies integrating taxonomic with habitat metadata will be important for understanding ecological preferences, local persistence, and range limits within the genus.
Although the overall phylogenetic framework was well resolved, some internal relationships within Nannengaella received only moderately support. This may reflect recent divergence, incomplete lineage sorting, insufficient resolution of the currently sampled loci. Limited taxon coverage for some markers may also have contributed to reduced support at deeper or intermediate nodes. These results suggest that, while the present multilocus dataset is sufficient for delimiting major lineages and supporting several taxonomic decisions, additional data will be needed to clarify finer-scale relationships within the genus. Expanding genomic sampling, including phylogenomic approaches or broader locus representation, may help improve resolution and reveal overlooked diversity (Leontyev et al., 2019; Shchepin et al., 2019), particularly in morphologically variable taxa such as N. mellea (Figure 6), in which color variation may obscure underlying lineage structure.
The detailed taxonomic treatments provided here, including SEM observations of peridium, capillitium, and spore ornamentation (Figures 2–10), also highlights the continuing importance of morphology in myxomycete systematics. Features such as spore ornamentation, lime-node shape, and peridial architecture proved especially informative when interpreted in combination with phylogenetic evidence (Nandipati et al., 2012; Leontyev et al., 2019; García-Martín et al., 2023). In this study, these characters were critical for distinguishing closely related taxa and for diagnosing the new species and new combinations. At the same time, the taxonomic utility of some structures remains incompletely understood because their developmental variation has rarely been studied in detail. Further work on ontogenetic changes in lime deposition, peridium differentiation, and capillitium development could improve character interpretation and provide additional insight into evolutionary trends within Physaraceae (Everhart, 2008).
In summary, this study provides an expanded and better-resolved taxonomic framework for Nannengaella by integrating multilocus phylogenetic evidence, comparative morphology, and new distributional data from China. The recognition of a new species, the proposal of three new combinations, and the documentation of multiple range extensions collectively improve current understanding of the genus. Although ecological and developmental questions remain open, the present results provide a stronger basis for future studies on the diversification, biogeography, and evolutionary ecology of Nannengaella and related physaraceous myxomycetes.
Funding Statement
The author(s) declared that financial support was received for this work and/or its publication. This work was financed by the Natural Science Foundation of China (No. 32570017), Jilin Agricultural University Doctoral Initiation Fund (202020353), the 2024 Science and Technology Support Project of the Inner Mongolia Innovation Center of Biological Breeding Technology (2024NSZC01), and 111 Program (No. D17014).
Footnotes
Edited by: Yusufjon Gafforov, National University of Uzbekistan, Uzbekistan
Reviewed by: Oleg Shchepin, University of Greifswald, Germany
Mandela Elorm Addrah, Inner Mongolia Agricultural University, China
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.
Author contributions
XL: Methodology, Formal analysis, Software, Investigation, Writing – review & editing, Writing – original draft, Data curation, Resources. JW: Validation, Investigation, Writing – original draft, Software, Data curation. FS: Formal analysis, Data curation, Writing – review & editing, Software. JW: Writing – original draft, Investigation, Formal analysis. BZ: Conceptualization, Supervision, Writing – review & editing, Investigation, Funding acquisition, Project administration. XL: Writing – review & editing, Conceptualization, Project administration, Funding acquisition. YL: Project administration, Conceptualization, Supervision, Writing – review & editing.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2026.1733966/full#supplementary-material
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Supplementary Materials
Data Availability Statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.