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Unveiling Species Diversity Within Early-Diverging Fungi from China VI: Four Absidia sp. nov. (Mucorales) in Guizhou and Hainan

A peer-reviewed version of this preprint was published in:
Microorganisms 2025, 13(6), 1315. https://doi.org/10.3390/microorganisms13061315

Submitted:

12 April 2025

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15 April 2025

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Abstract
Absidia represents the most species-rich genus within the family Cunninghamellaceae, with its members commonly isolated from diverse substrates, particularly rhizosphere soil. In this study, four novel Absidia species (A. irregularis sp. nov., A. multiformis sp. nov., A. ovoidospora sp. nov., and A. verticilliformis sp. nov.) were discovered from south and southwestern Chinese soil samples through integrated morphological and molecular phylogenetic analyses. Phylogenetic analyses based on concatenated ITS, SSU, LSU, Act, and TEF1α sequence data reconstructed trees that strongly supported the monophyly of these four new taxa each. Key diagnostic features include A. irregularis (closely related to A. oblongispora) exhibiting irregular colony morphology, A. multiformis (sister to A. heterospora) demonstrating polymorphic sporangiospores, A. ovoidospora (forming a clade with A. panacisoli and A. abundans) producing distinctive ovoid sporangiospores, and A. verticilliformis (next to A. edaphica) displaying verticillately branched sporangiophores. Each novel species is formally described with comprehensive documentation, including morphological descriptions, illustrations, Fungal Names registration identifiers, designated type specimens, etymological explanations, maximum growth temperatures, and taxonomic comparisons. This work constitutes the sixth installment in a series investigating early-diverging fungal diversity in China, expanding the recognized Absidia species to 71. The findings enhance our understanding of mucoralean biodiversity in Asian tropical and subtropical ecosystems.
Keywords: 
Mucoromycota
;  
soil-borne fungi
;  
new species
;  
taxonomy
;  
molecular phylogeny
Subject: 
Biology and Life Sciences  -   Immunology and Microbiology

1. Introduction

The genus Absidia Tiegh. (Mucoromycota Doweld, Mucoromycetes Doweld, Mucorales Dumort., Cunninghamellaceae Naumov ex R.K. Benj.) was established in 1878 and typified with A. reflexa [1, 2]. It is currently the most numerous genus in the family Cunninghamellaceae, with 67 recognized species (https://www.catalogueoflife.org/data/taxon/LSM, accessed on 2 December 2024). Its taxonomic position has been controversial since its establishment. Initially, this genus was classified in Absidiaceae [3]. Then, Benny et al. transferred it to Mucoraceae in 2001 [4]. Finally, Ashton et al. accommodated it in Cunninghamellaceae in 2009 [5]. With the deepening of research, Absidia s.l. was divided into Lichtheimia Vuill. (thermotolerant, optimum growth temperature 37–45 ℃), Absidia s.s. (mesophilic, optimum growth temperature 25–34 ℃), and Lentamyces Kerst. Hoffm. & K. Voigt (parasitic on other mucoralean fungi, optimum growth temperature 14–25 ℃) [6-9].
Strains of Absidia are distributed worldwide, ubiquitous in soil, dung, leaf litter, food, air, etc [10]. In the GlobalFungi database (https://globalfungi.com/, accessed on 22 December 2024), Absidia members are recorded from soil (37,828 records, 44.52 % of all records), root (15,865, 18.67 %), shoot (11,740, 13.82 %), topsoil (6,923, 8.15 %), rhizosphere soil (2,530, 2.98 %), deadwood (2,253, 2.65 %), air (2,080, 2.45 %), water (1,549, 1.82 %), litter (1,406, 1.65 %), mosses (265, 0.41 %), lichen (217, 0.25 %), coral (72, 0.08 %), dust (53, 0.06 %), fungal sporocarp (31, 0.04 %), and glacial ice debris (3, 0 %). In summary, soil and rhizosphere substrates account for approximately 74.32%.
Absidia coerulea, the most common species in this genus, plays an important role in bioengineering. It possesses the capability of transforming spirulina biomass into (-)-α-bisabolol [11]. It is able to catalyze the specific C-3 dehydrogenation for derivatives of ginsenoside-Rg₁, as well as hydroxylation at the 7β and 15α positions. And some metabolites in this species exhibit moderate reversal activity towards multidrug-resistant tumor cells [12].
Recently, eight fungal strains were isolated from soil in Hainan and Guizhou provinces, the south and southwest region of China. According to ITS-SSU-LSU-Act-TEF1α molecular phylogenetic analyses and morphological comparisons, these strains were classified into four new species of Absidia and described herein as A. irregularis sp. nov., A. multiformis sp. nov., A. ovoidospora sp. nov., and A. verticilliformis sp. nov. This is the sixth report of a serial work on diversity of Chinese early-diverging fungi [13-17].

2. Materials and Methods

2.1. Isolation and Morphology

Soil samples were collected in Hainan province in April 2023 and Guizhou province in August 2023, following the methods by Li et al.and Zou et al. [18, 19]. Soil sample collection started with removal of surface contaminants using a sterilized stainless steel shovel. About 100 g of homogenized soil was put into sample bags, labeled with collection date, administrative location, GPS coordinates, and altitude. Strains were obtained from the soil samples by serial dilution spread plate and single spore isolation.
About 1 g of soil samples was mixed with 10 mL sterile water to prepare 10-1 soil suspension. One millilitre of the 10-1 suspension was transferred to 9 mL of sterile water to obtain a 10-2 soil suspension. In the same way, 10-3 and 10-4 soil suspensions were made. Approximately 0.2 mL of the final 10-4 soil suspension were dispersed evenly with sterilised coating rods on Rose-Bengal Chloramphenicol agar (RBC: peptone 5.00 g/L, glucose 10.00 g/L, KH2PO4 1.00 g/L, MgSO4·7H2O 0.50 g/L, rose red 0.05 g/L, chloramphenicol 0.10 g/L, agar 15.00 g/L) [20], and then cultured in the dark at 25 ℃. Once visible, colonies were transferred and further cultured on Potato Dextrose Agar (PDA: glucose 20.00 g/L, potato 200.00 g/L, agar 20.00 g/L, pH 7) [14, 21].
Appropriately 0.1 g of soil samples were evenly sprinkled on the RBC medium with 0.06 mg/mL streptomycin and then incubated in darkness at 25 ℃ for 2–5 d. When sporangia formed, a sterilized inoculation needle was adopted to pick up a sporangium onto the PDA medium supplemented with 0.06 mg/mL streptomycin.
The maximum growth temperature was determined by a gradient method [13]. The strain was initially cultured at 25℃ for 2 d, and then increased by 1℃ per day until no growths.
Pure cultivation was applied for observing anamorphs, and pairing experiments were carried out for observing zygospores by adding 0.1 % lecithin to PDA and sealing Petri dishes to retain moisture. The morphological characteristics were observed with an optical microscope (Olympus BX53) and photographed with a high-definition colour digital camera (Olympus DP80). Each morphological character was statistically calculated against 30 measurements [22]. All strains were stored at 4 ℃ with 20 % sterilised glycerine. Cultures were deposited in the China General Microbiological Culture Collection Center, Beijing, China (CGMCC) and the Shandong Normal University, Jinan, Shandong, China (XG). Strains were deposited in the Herbarium Mycologicum Academiae Sinicae, Beijing, China (Fungarium, HMAS). Taxonomic information for the new taxa was registered in the Fungal Names repository (https://nmdc.cn/fungalnames/).

2.2. DNA Extraction and Amplification

Genomic DNA was extracted from mycelia using the CTAB method and GOMagTM Rapid Plant DNA Kit [21]. Information of the primers for PCR amplification is listed in Table 1. Amplification was performed in a final volume of 20 μL, containing 10 μL 2× Hieff Canace® Plus PCR Master Mix (Cat No.10154ES03; Yeasen Biotechnology, Shanghai, China), 0.5 μL of forward and reverse primers each (10 μM; TsingKe, Beijing, China), 1 μL template genomic DNA (1 μM) and 8 μL distilled deionised water. Molecular loci, PCR primers and programmes used in this study are listed in Table 1. PCR products were electrophoresed with 1 % agarose gel. DNA fragments were stained with TS-GelRed Nucleic Acid Gel Stain (10,000× in Water; TSJ002; Beijing Tsingke Biotech Co., Ltd.) and observed under ultraviolet light. Then a gel extraction kit (Cat# AE0101-C; Shandong Sparkiade Biotechnology Co., Ltd.) was used for gel recovery. Sanger sequencing was carried out by Biosune Co., Ltd. (Shanghai, China). Consensus sequences were assembled using MEGA v.7.0 [23]. Target sequences in some strains could not be obtained by conventional methods due to heterogeneous gene duplications, and thus they were extracted from genomic data [24]. The genomes were sequenced by Singke Biotech Co., Ltd. (Jinan, China). All sequences generated in this study were deposited at GenBank under the accession numbers in Table 2.
Relative sequences were obtained by BLAST search against the NCBI GenBank nucleotide database. SSU, ITS, LSU, Act and TEF1α sequences both generated herein and retrieved from GenBank (Table 2) were individually aligned using MAFFT 7 online service. The aligned matrices were manually proofread and then jointly analyzed. The optimal evolutionary model was determined for each partition and included in the analysis using MrModelTest v.2.3 [29]. Phylogenetic history was reconstructed using maximum likelihood (ML) algorithm with RaxML-HPC2 on XSEDE (8.2.12) and Bayesian inference (BI) algorithm with MrBayes [30-32]. Maximum Likelihood analysis was performed using the best model with 1,000 bootstrap replications. The BI analysis consisted of five million generations with four parallel runs under stopping rules and a sampling frequency of 100 generations. The burn-in score was set to 0.25, and the posterior probability (PP) was determined from the remaining trees. Initial adjustments to the phylogenetic tree were made using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and the finalization was performed using Adobe Illustrator CC 2019 (https://adobe.com/products/illustrator).

3. Results

3.1. Phylogenetic Analyses

The sequence matrix included 87 strains in 64 species of Absidia, with Cunninghamella blakesleeana CBS 782.68 as outgroup. A total of 4,656 characters comprised ITS rDNA (1–975), SSU rDNA (976–2,017), LSU rDNA (2,018–3,030), Act (3,031–3,689) and TEF1α (3,690–4,656). As many as 2,737 characters were constant, while 706 and 1,213 among the variable characters were parsimony-uninformative and informative, respectively (Supplementary File S1). MrModelTest suggested that the Dirichlet fundamental frequency and GTR-I-G evolution pattern for all partitions were adopted in Bayesian Inference. The topology of the Bayesian tree, consistent with that of the ML tree, was used as a representative to summarise the evolutionary history within the genus Absidia (Figure 1), exhibiting the phylogenetic placement of the four new species. A. irregularis is related to A. oblongispora with full supports (MLBV = 100, BIPP = 1.00), A. ovoidrospora is closely related to A. panacisoli and A. abundans with full supports (MLBV = 100, BIPP = 1.00). A. multiformis is most closely related to A. heterospora with high supports (BIPP = 0.99). And A. verticilliformis is closely related to A. edaphica with robust supports (MLBV = 94, BIPP = 1).

3.2. Taxonomy

Absidia irregularisYi Xin Wang & X.Y. Liu, sp. nov. Figure 2
Fungal Names—FN 572283
Etymology—The epithet “irregularis” (Latin) pertaining to irregular colonies.
Type—China, Hainan, Changjiang Li Autonomous County, Bawangling National Forest Park, 19.0859333° N, 109.122752° E, altitude 745.3 m, from soil sample, August 9, 2023, Yi-Xin Wang (Holotype HMAS 353186, ex-holotype strain CGMCC 3.27812 = XG05674-6)
Description—Colonies growing moderately on PDA in darkness at 25 ℃ for 7 d, reaching 43.2–54.8 mm in diameter, initially white, soon becoming gray to brown, irregular and scaly at edge, cottony, reversely white to gray. Hyphae hyaline at first, brownish when mature, branched, irregular, 10.2–14.5 μm in diameter. Stolons hyaline, branched, and smooth. Rhizoids hyaline, branched, irregular or root-like. Sporangiophores on aerial mycelia, hyaline, erect or slightly curved, unbranched or slightly branched, swollen usually below sporangia, umbellately or sympodially branched, often with a septum below apophyses, 26.5–148.1 × 3.4–5.3 μm. Sporangia oval to subglobose, 27.2–32.4 × 26.5–28.9 μm, hyaline at first and then brown, deliquescent-walled, leaving a collar after releasing sporangiospores. Apophyses hyaline, smooth, bowl-shaped, 2.9–9.6 × 8.6–19.0 μm. Collars distinct. Columellae hyaline or brown, hemispherical, 10.0–19.8 × 9.8–21.0 μm, with a protruding (3.5–6.3 × 1.4–2.9 μm) at the top. Protrudings always slightly contracted in the middle. Sporangiospores hyaline, smooth, not uniform, mostly cylindrical, 3.6–4.6 × 2.3–2.9 μm. Chlamydospores present. Zygospores not observed.
Additional strain examined—China, Hainan, Changjiang Li Autonomous County, Bawangling National Forest Park, 19.0859333° N, 109.122752° E, altitude 745.3 m, from soil sample, August 9, 2023, Yi-Xin Wang (living culture XG05674-8).
GenBank accession numbers—CGMCC 3.27812 (ITS, PQ306325; LSU, PQ289020; Act, PQ807209; SSU, PQ799254; TEF1α, PV126019), XG05674-7 (ITS, PQ306326; LSU, PQ289021; Act, PQ807210; SSU, PQ799255; TEF1α, PV126020).
Maximum growth temperature—CGMCC 3.27812 (32 ℃), XG05674-7 (32 ℃).
Notes—Based on phylogenetic analyses of ITS-SSU-LSU-Act-TEF1α sequences, the two isolates of the new species Absidia irregularis formed an independent clade with full supports (MLBV = 100, BIPP = 1; Figure 1), which is closely related to A. oblongispora (BIPP = 1; Figure 1). This new species differs morphologically from A. oblongispora in apophysis and columella [33]. The new species is bigger than A. oblongispora in apophysis, 2.9–9.6 × 8.6–19.0 μm vs. 3.5–6.5 × 3.5–7.5 μm. The new species is also bigger than A. oblongispora in columellae, 10.0–19.8 × 9.8–21.0 μm vs. 7.0–15.0 × 8.5–16.5 μm. Combining the morphological and molecular phylogenetic analyses, the two isolates were classified as a new taxon, allied to A. oblongispora.
Absidia multiformisYi Xin Wang & X.Y. Liu, sp. nov. Figure 3
Fungal Names—FN 572285
Etymology—The epithet " multiformis " (Latin) pertaining to polymorphic sporangiospores.
Type—China, Hainan, Lingshui Li Autonomous County, 18.6958768° N, 109.9407998° E, altitude 151.6 m, from soil sample, April 23, 2023, Yi-Xin Wang (Holotype HMAS 353194, ex-holotype strain CGMCC 3.27807 = XG04016-2).
Description—Colonies growing moderately on PDA in darkness at 25 ℃ for 7 d, reaching 82.2–90.0 mm diameter, initially white, soon becoming gray to brown, irregular at edge, cottony, reversely white to gray. Hyphae hyaline at first, brownish when mature, branched, irregular, 4.2–12.3 μm in diameter. Stolons hyaline, branched, and smooth. Rhizoids hyaline, branched, irregular or root-like. Sporangiophores on aerial mycelia, hyaline, erect or slightly curved, unbranched or slightly branched, swollen usually below sporangia, often with a septum below apophyses, 49.9–125.4 × 2.9–3.7 μm. Sporangia oval to subglobose, 20.1–36.1 × 23.1–36.3 μm, hyaline at first and then brown, deliquescent-walled, leaving a collar after releasing sporangiospores. Apophyses hyaline, smooth, shallow mouth bowl shaped, 2.5–7.9 × 8.0–19.8 μm. Collars distinct. Columellae hyaline or brown, hemispherical, 5.3–19.4 × 8.3–22.9 μm, with long or short cylindrical protrusions at top, 1.7–6.4 × 1.1–4.0 μm. Sporangiospores hyaline, smooth, not uniform, mainly cylindrical, 4.5–6.1 × 2.5–4.4 μm, some ovoid, 4.4–5.8 × 3.9–6.1 μm, occasionally subglobose, 4.9–7.2 μm. Chlamydospores present. Zygospores present, not uniform.
Additional strain examined—China, Hainan, Lingshui Li Autonomous County, 18.6958768° N, 109.9407998° E, altitude 151.6 m, from soil sample, April 23, 2023, Yi-Xin Wang (living culture XG04016-3).
GenBank accession numbers—CGMCC 3.27807 (ITS, PQ306319; LSU, PQ803168; Act, PQ807203; SSU, PQ799260; TEF1α, PV126021), XG0 4016-3 (ITS, PQ306320; LSU, PQ803169; Act, PQ807204; SSU, PQ799261; TEF1α, PV126022).
Maximum growth temperature—CGMCC 3.27807 (32 ℃), XG0 4016-3 (32 ℃).
Notes—Based on phylogenetic analyses of ITS-SSU-LSU-Act-TEF1α sequences, the two isolates of the new species Absidia multiformis formed an independent clade with full supports (MLBV = 100, BIPP = 1; Figure 1), which is closely related to A. heterospora (BIPP = 0.99; Figure 1). This new species differs morphologically from A. heterospora in sporangiophore, sporangium, apophysis and columella [34]. It differs from A. heterospora by narrower and shorter sporangiophores, 49.9–125.4 × 2.9–3.7 μm vs. 230–1700 × 4.3–10.0 µm. It is smaller than A. heterospora in sporangia, 20.1–36.3 μm vs. 15.0–55.0 µm. In columellae, it differs from A. heterospora by more shapes and smaller size. In detail, it is hemispherical and 5.3–19.4 × 8.3–22.9 μm, while A. heterospora is regularly dorsiventrally flattened and 10.5–34 µm in diameter. Combining morphological and molecular phylogenetic analyses, the two isolates were classified as a new taxon, allied to A. heterospora.
Absidia ovoidosporaYi Xin Wang & X.Y. Liu, sp. nov. Figure 4
Fungal Names—FN 572284
Etymology—The epithet “ovoidospora” (Latin) pertaining to ovoid sporangiospores.
Type—China, Hainan, Changjiang Li Autonomous County, Bawangling National Forest Park, 25.905722° N, 107.279063° E, altitude 745.3 m, from soil sample, August 9, 2023, Yi-Xin Wang (Holotype HMAS 353185, ex-holotype strain CGMCC 3.27811 = XG05673-2).
Description—Colonies growing fast on PDA in darkness at 25 ℃ for 7 d, reaching 90 mm diameter, initially white, soon becoming gray to brown, irregular at edge, cottony, reversely white to gray. Hyphae hyaline at first, brownish when mature, branched, irregular, 5.8–13.7 μm in diameter. Stolons hyaline, branched, and smooth. Rhizoids hyaline, branched, irregular or root-like. Sporangiophores on aerial mycelia, hyaline, erect or slightly curved, unbranched or slightly branched, swollen usually present below sporangia, umbellately or sympodially branched, often with a septum below apophyses, 45.3–355.3 × 2.5–4.0 μm. Sporangia oval to subglobose, 13.6–29.0 × 13.3–28.5 μm, hyaline at first and then brown, deliquescent-walled, leaving a collar after releasing sporangiospores. Apophyses hyaline, smooth, bowl shaped and long funnel shaped, 4.1–6.9 × 8.6–10.9 μm. Columellae hyaline or brown, hemispherical with a short or long protruding at the top, 7.9–12.9 × 9.0–11.6 μm. Protrudings always slightly contracted in the middle, 1.9–4.6 × 1.7–2.6 μm. Sporangiospores hyaline, smooth, not uniform, mostly ovoid, 3.2–3.8 × 2.4–3.1 μm, some cylindrical, 3.4–4.6 × 2.2–3.0 μm μm. Chlamydospores present. Zygospores not observed.
Additional strains examined—China, Hainan, Changjiang Li Autonomous County, Bawangling National Forest Park, 25.905722° N, 107.279063° E, altitude 745.3 m, from soil sample, August 9, 2023, Yi-Xin Wang (living culture XG05673-3).
GenBank accession numbers—CGMCC 3.27811 clone1 (ITS, PQ306327; LSU, PQ803164; Act, PQ807207; SSU, PQ799256; TEF1α, PV126015), CGMCC 3.27811 clone2 (ITS, PV069753; LSU, PQ803165; Act, PV126023; SSU, PQ799257; TEF1α, PV126017), XG05673-3 clone 1 (ITS, PQ306328; LSU, PQ803166; Act, PQ807208; SSU, PQ799258; TEF1α, PV126016), XG05673-3 clone 2 (ITS, PV069754; LSU, PQ803167; Act, PV126024; SSU, PQ799259; TEF1α, PV126018).
Maximum growth temperature—CGMCC 3.27811 (30 ℃), XG05673-3 (30 ℃).
Notes—Based on phylogenetic analyses of ITS-SSU-LSU-Act-TEF1α sequences, the new species Absidia ovoidospora formed two sister clades with high supports (MLBV = 96, BIPP = 1; Figure 1), which are closely related to A. panacisoli and A. abundans with full supports (MLBV = 100, BIPP = 1; Figure 1). These two clades were resulted from two clones. This new species differs morphologically from A. oblongispora in sporangiophore and sporangium [35]. The new species differs from A. panacisoli by wider sporangiophore, 2.5–4.0 μm vs. 1.7–2.8 μm. In sporangia, the new species is bigger than A. panacisoli, 13.6–29.0 × 13.3–28.5 μm vs. 13.0–21.5 × 9.4–15.5 μm. This new species differs morphologically from A. abundans in sporangiophore, sporangium and columella [9]. The new species differs from A. abundans by bigger sporangiophores, 45.3–355.3 × 2.5–4.0 μm vs. 35.0–170.0 × 2.0–3.5 μm. The new species is bigger than A. abundans in sproangia, 13.6–29.0 × 13.3–28.5 μm vs. 8.0–16.5 × 8.5–16.0 μm. In columellae, the new species is bigger than A. abundans, 7.9–12.9 × 9.0–11.6 μm vs. 4.5–10.0 × 3.5–8.0 μm. Combining morphological and molecular phylogenetic analyses, the two were classified isolates as a new taxon, allied with A. panacisoli and A. abundans.
Absidia verticilliformis, Yi Xin Wang & X.Y. Liu sp. nov. Figure 5
Fungal Names—FN 572287
Etymology—The epithet “verticilliformis” (Latin) pertaining to verticillate branches of sporangiophores.
Type—China, Hainan, Sanya City, Jiyang District, G224 Haiyu Middle Line, 18.391817° N, 109.641068° E, altitude 193.0 m, from soil sample, April 24, 2023, Yi-Xin Wang (Holotype HMAS 353183, ex-holotype strain CGMCC 3.27810 = XG04088-3)
Description—Colonies growing fast on PDA in darkness at 25 ℃ for 7 days, reaching 90 mm diameter, initially white, soon becoming gray to brown, irregular at edge, cottony, reversely white to gray, growing outward in a petal shape. Hyphae hyaline at first, brownish when mature, branched, 4.4–8.9 μm in diameter. Stolons hyaline, branched, and smooth. Rhizoids hyaline, branched, irregular or root-like. Sporangiophores on aerial mycelia, hyaline, erect or slightly curved, unbranched or slightly branched, mostly umbellately branched, swollens usually present below sporangia, often with a septum below apophyses, 22.3–368.7 × 3.3–4.6 μm. Sporangia oval to subglobose, 15.7–29.0 × 17.5–26.0 μm, hyaline at first and then brown, deliquescent-walled, mostly leaving a collar after releasing sporangiospores. Apophyses hyaline, smooth, shallow mouth bowl shaped, 4.5–14.4 × 11.1–20.4 μm. Collars distinct. Columellae hyaline or brown, hemispherical, tip with short or long cylindrical protrusions, 12.6–25.6 × 14.7–26.2 μm. Protrudings 3.1– 6.3× 1.8–2.2 μm. Sporangiospores hyaline, smooth, not uniform, mostly ovoid, 3.4–4.3 × 2.3–3.1 μm, some cylindrical, 3.9–4.4 × 2.1–3.0 μm. Chlamydospores present. Zygospores not observed.
Additional strain examined—China, Hainan, Sanya City, Jiyang District, G224 Haiyu Middle Line, 18.391817° N, 109.641068° E, altitude 193.0 m, from soil sample, April 24, 2023, Yi-Xin Wang (living culture XG04088-4)
GenBank accession numbers—CGMCC 3.27810 clone 1 (ITS, PQ306315; LSU, PQ803170; Act, PQ807205; SSU, PQ799262; TEF1α, PV126011), CGMCC 3.27810 clone 2 (ITS, PV069755; LSU, PQ803171; Act, PV126025; SSU, PQ799263; TEF1α, PV126013), XG04088-4 clone1 (ITS, PQ306316; LSU, PQ803172; Act, PQ807206; SSU, PQ799264; TEF1α, PV126012), XG04088-4 clone2 (ITS, PV069756; LSU, PQ803173; Act, PV126026; SSU, PQ799265; TEF1α, PV126014).
Maximum growth temperature—CGMCC 3.27810 (34 ℃), XG04088-4 (34 ℃).
Notes—Based on phylogenetic analyses of ITS-SSU-LSU-Act-TEF1α sequences, the two isolates of the new species Absidia verticilliformis formed two sister clades with full supports (MLBV = 100, BIPP = 1; Figure 1), which is closely related to A. edaphica (MLBV = 94, BIPP = 1; Figure 1). This new species differs morphologically from A. edaphica in sporangium, columella and sporangiospore [36]. The new species is smaller than A. edaphica in sporangia, 15.7–29.0 × 17.5–26.0 μm vs. 30.5–35.5 × 24–27 μm. In columellae, the new species is bigger than A. edaphica, 12.6–25.6 × 14.7–26.2 μm vs. 5–9.5 × 6.5–20 μm. The new species is smaller than A. edaphica in sporangiospores, 3.4–4.4 × 2.1–3.1 μm vs. 3.5–5.5 × 2–3.5 μm. Combining morphological and molecular phylogenetic analyses, the two isolates were classified as a new taxon, allied to A. edaphica.

4. Discussion

The genus Absidia was established nearly 150 years ago. Between 1878 and 2010, the taxonomic status of the genus was changed several times, and the species of the genus were divided according to the optimum growth temperature. In 2007, Kerstin Hoffmann et al. divided Absidia s. l. into Absidia (Absidiaceae s. str., mesothermal type) and Mycocladus (Mycocladiaceae, thermophilic type) [7]. In 2009, A. parricida and A. zychae were separated from the genus Absidia by Hoffmann and Voigt and reassigned to a newly described genus Lentamyces [8]. In 2010, Hoffmann et al. investigated and evaluated Absidia s. l., and finally divided it into three genera, namely Absidia s. s., Lichtheimia and Lentamyces [6]. From 2011 to 2019, the discovery of Absidia stagnated, only six new species have been described (https://www.indexfungorum.org/, accessed on 7 February 2025; [35, 37-40]). Since 2020, taxonomic studies on this genus have been extensively carried out, with a total of 49 new species described (https://www.indexfungorum.org/, accessed on 7 February 2025; [9, 10, 13, 33, 36, 41-50]). Nowadays, it is the most species-rich genus in the family Cunninghamellaceae. Together with the four new species proposed in this study, the world diversity of Absidia reaches 71 recognized species.
Absidia species predominantly inhabit soil environments across tropical, subtropical, and temperate climatic zones. In accordance with this ecological distribution pattern, the four novel species described in this study were successfully isolated from soil samples collected in tropical and subtropical geographic regions of China.
Prior to 2020, the phylogenetic tree of this genus was predominantly reconstructed utilizing the combination of ITS and LSU sequences. In 2021, Hurdeal et al. added SSU and Act sequences for reconstructing the phylogenetic [36]. Subsequently, in 2024, Tao et al. employed a more comprehensive approach, utilizing SSU, ITS, LSU, Act, and TEF1α to reconstruct the phylogenetic tree, yielding results that were largely in line with previous findings [13]. In this study, the phylogenetic history was inferred using ITS-SSU-LSU-Act-TEF1α, and newly isolated strains were grouped into four individual clades with high supports, namely Absidia irregularis sp. nov., A. multiformis sp. nov., A. ovoidospora sp. nov., and A. verticilliformis sp. nov.
Absidia irregularis is related to A. oblongispora with high supports (MLBV = 100, BIPP = 1; Figure 1), while distinguished by bigger apophyses and columellae. Absidia multiformis is most closely related to A. heterospora with high support (BIPP = 0.99; Figure 1). Morphologicaly, A. multiformis has narrower and shorter sporangiophore, smaller sporangia size, different columellae shape and size. Absidia ovoidospora is closely related to A. panacisoli and A. abundans. However, compared with A. panacisoli, A. ovoidospora had wider sporangiophore width and larger sporangium. At the same time, compared with A. abundans, the microscopical measurements of the sporangiophore, sporangium and columella of A. oviodrospora were larger. A. verticilliformis is closely related to A. edaphica. A. verticilliformis differs from A. edaphica by smaller sporangia, bigger columellae and smaller sporangiospores.
In this study, thermal tolerance thresholds of fungal strains were determined using a temperature gradient cultivation technique. Growth characterization revealed distinct maximum growth temperatures for the four Absidia species: A. ovoidospora (30℃), A. irregularis (32℃), A. multiformis (34℃), and A. verticilliformis (34℃). These thermal parameters align with the established physiological profile of Absidia s. s. (mesophilic genus with optimal growth at 25-34℃).
In summary, the molecular phylogenetic and morphological results both support the identification of the four new species. These findings further enhance our understanding of mucoralean biodiversity in Asian tropical and subtropical ecosystems.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/doi/s1, File S1: ITS-SSU-LSU- Act-TEF1a.

Author Contributions

Y.-X.W. took charge of the microscopy, DNA sequencing, data analyses and manuscript draft; Z.-Y.D. and Y.-X.W. collected samples and isolated living cultures; X.-Y.J. and Z.-Y.D. made dry cultures; Z.M. revised the manuscript; X.-Y.L. contributed to new species proposal, manuscript revision and financial support; funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (Nos. 32170012, 32470004, 32300011), Ji’nan City’s ‘New University 20 Policies’ Initiative for Innovative Research Teams Project (202228028), Innovative Agricultural Application Technology Project of Jinan City (CX202210) and Key Technological Innovation Program of Shandong Province, China (2022CXGC020710).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The sequences were deposited in the GenBank database.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The Beyesian phylogenetic tree of Absidia based on ITS-SSU-LSU-Act-TEF1α sequences, with Cunninghamella blakesleeana CBS 782.68 as outgroup. The maximum likelihood bootstrap value (MLBV) ≥ 75, and the Bayesian inference posterior probability (BIPP) ≥ 0.85 are shown at the first and second positions and separated by a slash "/" on relevant nodes. The ex-types or ex-holotypes are in bold and marked with an asterisk "*", and strains involved in this study are in red. Branches shortened to fit the page are represented by double slashes “//” and folds “×”. The scale at the bottom centre indicates 0.2 substitutions per site.
Figure 1. The Beyesian phylogenetic tree of Absidia based on ITS-SSU-LSU-Act-TEF1α sequences, with Cunninghamella blakesleeana CBS 782.68 as outgroup. The maximum likelihood bootstrap value (MLBV) ≥ 75, and the Bayesian inference posterior probability (BIPP) ≥ 0.85 are shown at the first and second positions and separated by a slash "/" on relevant nodes. The ex-types or ex-holotypes are in bold and marked with an asterisk "*", and strains involved in this study are in red. Branches shortened to fit the page are represented by double slashes “//” and folds “×”. The scale at the bottom centre indicates 0.2 substitutions per site.
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Figure 2. Absidia irregularis ex-holotype CGMCC 3.27812. a, b, Colonies on PDA (a, obverse; b, reverse); c, d, An unbranched sporangiophore with a sporangium; e, f, Columellae, collars, sporangiospores and apophyses; g, Branched sporangiophores with sporangia; h, Branched sporangiophores with columellae, collars, sporangia and apophyses; i, j, Rhizoids; k, Sporangiospores; Bars: c–k 10 μm.
Figure 2. Absidia irregularis ex-holotype CGMCC 3.27812. a, b, Colonies on PDA (a, obverse; b, reverse); c, d, An unbranched sporangiophore with a sporangium; e, f, Columellae, collars, sporangiospores and apophyses; g, Branched sporangiophores with sporangia; h, Branched sporangiophores with columellae, collars, sporangia and apophyses; i, j, Rhizoids; k, Sporangiospores; Bars: c–k 10 μm.
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Figure 3. Absidia multiformis ex-holotype CGMCC 3.27807. a, b, Colonies on PDA (a, obverse; b, reverse); c, d, An unbranched sporangiophore with a sporangium; e, f, Columellae, collars, sporangiospores and apophyses; g, Branched sporangiophores with columellae, collars, sporangium and apophyses; h, i, Rhizoids; j, k, Sporangiospores; k, m, Zygospores; Bars: c–m 10 μm.
Figure 3. Absidia multiformis ex-holotype CGMCC 3.27807. a, b, Colonies on PDA (a, obverse; b, reverse); c, d, An unbranched sporangiophore with a sporangium; e, f, Columellae, collars, sporangiospores and apophyses; g, Branched sporangiophores with columellae, collars, sporangium and apophyses; h, i, Rhizoids; j, k, Sporangiospores; k, m, Zygospores; Bars: c–m 10 μm.
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Figure 4. Absidia oviodospora ex-holotype CGMCC 3.27811. a, b, Colonies on PDA (a, obverse; b, reverse); c–e, An unbranched sporangiophore with a sporangium; f, Columellae, collars, sporangiospores and apophyses; g, Unbranched sporangiophores with swelling and sporangium; h, Branched sporangiophores with columellae, collars and apophyses; i, Branched sporangiophores with sporangia; j, Rhizoids; k–m, Giant cells; n, Sporangiospores; Bars: c–n 10 μm.
Figure 4. Absidia oviodospora ex-holotype CGMCC 3.27811. a, b, Colonies on PDA (a, obverse; b, reverse); c–e, An unbranched sporangiophore with a sporangium; f, Columellae, collars, sporangiospores and apophyses; g, Unbranched sporangiophores with swelling and sporangium; h, Branched sporangiophores with columellae, collars and apophyses; i, Branched sporangiophores with sporangia; j, Rhizoids; k–m, Giant cells; n, Sporangiospores; Bars: c–n 10 μm.
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Figure 5. Absidia verticilliformis ex-holotype CGMCC 3.27810. a, b, Colonies on PDA (a, obverse; b, reverse); c–e, An unbranched sporangiophore with a sporangium; f, Columellae, collars, sporangiospores and apophyses; g, h, Branched sporangiophores with columellae, collars, sporangiospores and apophyses; i, j, Rhizoids; k, Sporangiospores; Bars: c–k 10 μm.
Figure 5. Absidia verticilliformis ex-holotype CGMCC 3.27810. a, b, Colonies on PDA (a, obverse; b, reverse); c–e, An unbranched sporangiophore with a sporangium; f, Columellae, collars, sporangiospores and apophyses; g, h, Branched sporangiophores with columellae, collars, sporangiospores and apophyses; i, j, Rhizoids; k, Sporangiospores; Bars: c–k 10 μm.
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Table 1. Molecular loci, PCR primers and programmes used in this study.
Table 1. Molecular loci, PCR primers and programmes used in this study.
Loci PCR primers Sequence (5’ – 3’) PCR cycles References
Act ACT-512F ATG TGC AAG GCC GGT TTC GC 95℃ 3 min; (95℃ 1 min, 55℃ 1 min, 72℃ 1 min) × 30 cycles; 72℃ 10 min [25]
ACT-783R TAC GAG TCC TTC TGG CCC AT
ITS ITS5 GGA AGT AAA AGT CGT AAC AAG G 95℃ 5 min; (95℃ 30 s, 55℃ 30 s, 72℃ 1 min) × 35 cycles; 72℃ 10 min [26]
ITS4 TCC TCC GCT TAT TGA TAT GC
LSU LR0R GTA CCC GCT GAA CTT AAG C 95℃ 5 min; (95℃ 50 s, 47℃ 30 s, 72℃ 1.5 min) × 35 cycles; 72℃ 10 min [27]
LR7 TAC TAC CAC CAA GAT CT
SSU NS1 GTA GTC ATA TGC TTG TCT C C 95 °C 5 min; (94 °C 60 s, 54 °C 50 s, 72 °C 60 s) × 37 cycles; 72 °C 10 min [26]
NS4 CTT CCG TCA ATT CCT TTA AG
TEF1α EF1-728F CAT CGA GAA GTT CGA GAA GG 95℃ 5 min; (95℃ 30 s, 55℃ 60 s, 72℃ 1 min) × 30 cycles; 72℃ 10 min [28]
EF2 GGA RGT ACC AGT SAT CAT GTT
Table 2. Information of strains used in this study.
Table 2. Information of strains used in this study.
Species Strains GenBank accession numbers
ITS LSU TEF1α Act SSU
Absidia abundans CGMCC 3.16255* NR_182590 ON074683 n.a. n.a. n.a.
A. abundans XY09274 ON074696 ON074682 n.a. n.a. n.a.
A. aguabelensis URM 8213* NR_189383 NG_241934 n.a. n.a. n.a.
A. alpina CGMCC 3.16104 OL678133 n.a. n.a. n.a. n.a.
A. ampullacea CGMCC 3.16054 MZ354138 MZ350132 n.a. n.a. n.a.
A. anomala CBS 125.68* MH859085 MH870799 n.a. n.a. n.a.
A. anomala FSU5798 EF030523 n.a. n.a. EF030535 n.a.
A. biappendiculata CBS 187.64 MZ354153 MZ350147 MZ357420 MZ357438
A. bonitoensis URM 7889* MN977786 MN977805 n.a. n.a. n.a.
A. brunnea CGMCC 3.16055* MZ354139 MZ350133 MZ357403 MZ357421 n.a.
A. caatinguensis URM 7156* NR_154704 NG_058582 n.a. n.a. n.a.
A. caerulea CBS101.36* MH855718 MH867230 n.a. n.a. n.a.
A. californica CBS 314.78 JN205816 MH872902 n.a. n.a. n.a.
A. chinensis CGMCC 3.16057 MZ354141 MZ350135 n.a. MZ357422 n.a.
A. chinensis CGMCC 3.16056* MZ354140 MZ350134 n.a. n.a. n.a.
A. cinerea CGMCC 3.16062 MZ354146 MZ350140 MZ357407 MZ357427 n.a.
A. cornuta URM 6100* NR_172976 MN625255 n.a. n.a. n.a.
A. crystalloides CGMCC3.27496* PP377803 PP373736 PP790574 PP790582 PP779723
A. crystalloides SAUCC693201 PP377804 PP373737 PP790573 PP790581 PP779722
A. cuneospora CBS 101.59* MH857828 MH869361 n.a. n.a. n.a.
A. cuneospora FSU5890 EF030524 n.a. n.a. EF030533 n.a.
A. cylindrospora CBS 100.08 JN205822 JN206588 n.a. n.a. n.a.
A. digitula CGMCC 3.16058* MZ354142 MZ350136 MZ357404 MZ357423 n.a.
A. edaphica MFLUCC 20-0088 NR_172305 NG_075367 n.a. MT410739 NG_074951
A. frigida CGMCC 3.16201* NR_182565 OM030223 n.a. n.a. n.a.
A. fusca CBS 102.35* NR_103625 NG_058552 n.a. n.a. n.a.
A. gemella CGMCC 3.16202* OM108488 OM030224 n.a. n.a. n.a.
A. glauca CBS 101.08* MH854573 MH866105 n.a. n.a. n.a.
A. glauca FSU660 AY944879 EU736302 EU736248 EU736225 EU736275
A. globospora CGMCC 3.16031* NR_189829 MW671544 MZ357412 MZ357431 n.a.
A. globospora CGMCC 3.16036 MW671539 MW671546 MZ357414 MZ357433 n.a.
A. healeyae UoMAU1 n.a. MT436027 n.a. n.a. n.a.
A. heterospora SHTH021 JN942683 JN982936 n.a. n.a. JQ004928
A.irregularis CGMCC 3.27812* PQ306325 PQ289020 PV126019 PQ807209 PQ799254
A.irregularis XG05674-7 PQ306326 PQ289021 PV126020 PQ807210 PQ799255
A. jiangxiensis CGMCC 3.16105* OL678134 PP780377 n.a. n.a. n.a.
A. jindoensis CNUFC-PTI1-1 MF926622 MF926616 MF926513 MF926510 MF926626
A. koreana EML-IFS45-1* KR030062 KR030056 KR030060 KR030058 KT321298
A. koreana XY00816 OL620083 ON123771 n.a. n.a. n.a.
A. lobata CGMCC 3.16256 ON074690 ON074679 n.a. n.a. n.a.
A. longissima CGMCC 3.16203* NR_182566 OM030225 n.a. n.a. n.a.
A. macrospora FSU4746 AY944882 EU736303 EU736249 AY944760 EU736276
A. medulla CGMCC 3.16034 NR_189832 MW671549 MZ357417 MZ357436 n.a.
A. montepascoalis URM 8218 NR_172995 n.a. n.a. n.a. n.a.
A.multiformis CGMCC 3.27807* PQ306319 PQ803168 PV126021 PQ807203 PQ799260
A.multiformis XG04016-3 PQ306320 PQ803169 PV126022 PQ807204 PQ799261
A. multispora URM 8210* MN953780 MN953782 n.a. n.a. n.a.
A. nigra CBS 127.68* NR_173068 MZ350146 MZ357419 MZ357437 n.a.
A. nigra CGMCC 3.16060 MZ354144 MZ350138 MZ357406 MZ357425 n.a.
A. oblongispora CGMCC 3.16061 MZ354145 MZ350139 n.a. MZ357426 n.a.
A. ovalispora CGMCC 3.16019 NR_176748 MW264131 n.a. n.a. n.a.
A.ovoidospora CGMCC 3.27811 clone1* PQ306327 PQ803164 PV126015 PQ807207 PQ799256
A. ovoidospora CGMCC 3.27811 clone2* PV069753 PQ803165 PV126017 PV126023 PQ799257
A. ovoidospora XG05673-3 clone1 PQ306328 PQ803166 PV126016 PQ807208 PQ799258
A. ovoidospora XG05673-3 clone2 PV069754 PQ803167 PV126018 PV126024 PQ799259
A. pacifica CGMCC3.27497* PP377802 PP373735 PP839793 PP790579 PP779720
A. pacifica SAUCC413601 PP377801 PP373734 PP839794 PP790580 PP779721
A. panacisoli SYPF 7183* MF522181 MF522180 MF624251 n.a. MF522179
A. pararepens CCF 6352 MT193669 MT192308 n.a. n.a. n.a.
A. pateriformis CGMCC3.27495* PP377805 PP373738 PP790575 PP790583 PP779724
A. pateriformis SAUCC634702 PP377806 PP373739 PP790576 PP790584 PP779725
A. pernambucoensis URM<BRA>7219 MN635568 MN635569 n.a. n.a. n.a.
A. pseudocylindrospora EML-FSDY6-2 KU923817 KU923814 n.a. KU923815 KU923819
A. pseudocylindrospora CBS 100.62* NR_145276 MH869688 n.a. n.a. n.a.
A. psychrophilia FSU4745 AY944874 EU736306 EU736252 AY944762 EU736279
A. radiata CGMCC 3.16257 ON074698 ON074684 n.a. n.a. n.a.
A. radiata XY09330-1 ON074699 ON074685 n.a. n.a. n.a.
A. repens CBS 115583* NR_103624 NG_058551 n.a. n.a. n.a.
A. saloaensis URM 8209* MN953781 MN953783 n.a. n.a. n.a.
A. sichuanensis CGMCC 3.16258* NR_182589 ON074688 n.a. n.a. n.a.
A. soli MFLU-20-0414* MT396373 MT393988 n.a. n.a. MT394049
A. soli MFLU 20-0413 MT396371 MT393985 n.a. n.a. MT394046
A. spinosa FSU551 AY944887 EU736307 EU736253 EU736227 EU736280
A. stercoraria EML-DG8-1* KU168828 KT921998 KT922002 KT922000 NG_065640
A. sympodialis CGMCC 3.16063* MZ354147 MZ350141 n.a. n.a. n.a.
A. sympodialis CGMCC 3.16064 MZ354148 MZ350142 MZ357408 n.a. n.a.
A. terrestris FMR 14989* LT795003 LT795005 n.a. n.a. n.a.
A. turgida CGMCC 3.16032* NR_189830 NG_241931 MZ357415 MZ357434 n.a.
A. varians CGMCC 3.16065* MZ354149 MZ350143 MZ357409 MZ357428 n.a.
A.verticilliformis CGMCC 3.27810 clone1* PQ306315 PQ803170 PV126011 PQ807205 PQ799262
A.verticilliformis CGMCC 3.27810 clone2* PV069755 PQ803171 PV126013 PV126025 PQ799263
A.verticilliformis XG04088-4 clone1 PQ306316 PQ803172 PV126012 PQ807206 PQ799264
A.verticilliformis XG04088-4 clone2 PV069756 PQ803173 PV126014 PV126026 PQ799265
A. virescens CGMCC 3.16066* MZ354150 MZ350144 MZ357410 MZ357429 n.a.
A. virescens CGMCC 3.16067 MZ354151 MZ350145 MZ357411 MZ357430 n.a.
A. xinjiangensis CGMCC 3.16107* OL678136 n.a. n.a. n.a. n.a.
A. yunnanensis XY09528 ON074701 ON074686 n.a. n.a. n.a.
A. yunnanensis CGMCC 3.16259* NR_182591 NG_149054 n.a. n.a. n.a.
A. zonata CGMCC 3.16033* NR_189831 MW671548 MZ357416 MZ357435 n.a.
A. zygospora RSPG 214 KC478527 n.a. n.a. n.a. n.a.
A. zygospora ANG28 DQ914420 n.a. n.a. n.a. n.a.
Cunninghamella blakesleeana CBS 782.68 JN205869 MH870950 n.a. n.a. n.a.
Notes: New species proposed in this study are in bold. Ex-type or ex-holotype strains are labelled with an asterisk “*”. The abbreviation “n.a.” stands for “not available”.
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