Species Diversity and Ecological Habitat of Absidia (Cunninghamellaceae, Mucorales) with Emphasis on Five New Species from Forest and Grassland Soil in China.

Although species of Absidia are known to be ubiquitous in soil, animal dung, and insect and plant debris, the species diversity of the genus and their ecological habitats have not been sufficiently investigated. In this study, we describe five new species of Absidia from forest and grassland soils in southwestern China, with support provided by phylogenetic, morphological, and physiological evidence. The species diversity and ecological habitat of Absidia are summarized. Currently, 22 species are recorded in China, which mainly occur in soil, especially in tropical and subtropical forests and mountains. An updated key to the species of Absidia in China is also provided herein. This is the first overview of the Absidia ecological habitat.

1. Introduction

The genus Absidia Tiegh., typified by A. reflexa Tiegh., was described nearly 150 years ago [1], belonging to Cunninghamellaceae, Mucorales, Mucoromycetes, Mucoromycota (http://www.indexfungorum.org/, accessed on 1 March 2022). However, it was initially placed in the family Absidiaceae [2]. With the development of molecular biology, it was grouped with Chlamydoabsidia Hesselt. and J.J. Ellis, Cunninghamella Matr., Gongronella Ribaldi, and Halteromyces Shipton and Schipper and Hesseltinella H.P. Upadhyay; this group was nominated as the family Cunninghamellaceae [3,4,5,6,7]. Nine other genera were thought to be allied with Absidia on the basis of morphological similarities, including Rhizopus Ehrenb. 1821, Phycomyces Kunze 1823, Tieghemella Berl. and De Toni 1888, Mycocladus Beauverie 1900, Lichtheimia Vuill. 1903, Proabsidia Vuill. 1903, Pseudoabsidia Bainier 1903, Protoabsidia Naumov 1935, and Gongronella Ribaldi 1952 [8]. As research progressed, Absidia s.l. has been well divided into three genera, i.e., Lichtheimia (thermotolerant, optimum growth temperature 37–45 °C), Absidia s.s. (mesophilic, optimum growth temperature 25–34 °C), and Lentamyces Kerst. Hoffm. and K. Voigt (parasitic on mucoralean fungi, optimum growth temperature 14–25 °C) [9,10,11]. Currently, species of Absidia are characterized by (1) sporangiophores single, in pairs or in groups on stolons, (2) rhizoids at both ends of stolons and never opposite the sporangiophores, (3) sporangia deliquescent-walled and apophysate, (4) columellae bearing one to several projections, (5) zygospores enclosed by appendages, and (6) optimum growth temperatures from 25 °C to 34 °C [1,9,10,11,12,13,14,15].

So far, a total of 46 species of Absidia have been described worldwide, and they are ubiquitous in soil, dung, insect, leaf litter, food, air, etc. (Figure 1; [16]). Among them, 30 species were initially collected from soil, including forest and rhizosphere soil, suggesting that Absidia species are mainly isolated from soil [17,18]. However, a few species are endemic to animal dung and insect remains, such as A. psychrophilia Hesselt. and J.J. Ellis from mycangia of ambrosia beetles [8,19], and A. stercoraria Hyang B. Lee et al. from rat dung [20].

Figure 1

Number of Absidia species in different ecological habitats when they were originally described. These data are from the Index Fungorum (http://www.indexfungorum.org/, accessed on 1 March 2022) and [16].

Absidia glauca Hagem and A. repens Tiegh. are considered as cosmopolitan species, reported in all continents except Antarctica [21,22,23]. Absidia heterospora Y. Ling was reported in China, New Zealand, and France [23,24]; A. idahoensis Hesselt. et al. and A. macrospora Váňová were reported in China, Czechia, and the USA [25,26,27]. Since 2018, 22 endemic species have been described from Korea, China, Thailand, Australia, USA, Mexico, and Brazil [14,15,16,28,29,30,31,32,33,34,35,36]. Type strains were collected from 17 countries, and the two most investigated countries are China and Brazil, with nine and eight type strains, respectively. The studies on Absidia in other countries and regions are obviously insufficient (Table 1).

Table 1

The origin of taxonomic types in Absidia.

Country	Type	Percentage (%)	Country	Type	Percentage (%)
China	9	19.6	Canada	1	2.2
Brazil	8	17.4	Cuba	1	2.2
USA	4	8.7	Egypt	1	2.2
France	3	6.5	Netherland	1	2.2
Korea	3	6.5	Mexico	1	2.2
Czechia	2	4.3	Pakistan	1	2.2
India	2	4.3	Switzerland	1	2.2
Thailand	2	4.3	Tanzania	1	2.2
Australia	1	2.2	Unknown	4	8.7

Note: These data are from the Index Fungorum (http://www.indexfungorum.org/, accessed on 1 March 2022) and [16].

Therefore, there are deficiencies in the studies on species distribution and ecological habitat of Absidia [8,13,14,15,16,18,20,27]. In this paper, we propose five new species from forest and grassland soil in Sichuan, Tibet, and Yunnan in southwestern China. A key to Absidia species in China is consequently updated. Along with the taxonomical study, we also conduct a preliminary investigation on the species distribution and ecological habitat of Absidia.

2. Materials and Methods

2.1. Isolation and Strains

Strains were isolated from forest and grassland soil samples collected in September 2021, in Sichuan, Tibet, and Yunnan in southwestern China. An aliquot of soil samples (1 g) was evenly spread on 15 cm petri dishes containing potato dextrose agar medium (200 g potato, 20 g dextrose, 20 g agar, and 1000 mL distilled water) with streptomycin sulfate and ampicillin 100 mg/mL each, and then cultivated at 20 °C and 25 °C. According to morphological characteristics of Absidia, potential strains were picked out and purified. The purified living cultures (Table 2) were deposited in the China General Microbiological Culture Collection Center, Beijing, China (CGMCC) and the Shandong Normal University (XY), and dry cultures (Table 2) were deposited in the Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS).

Table 2

Taxa information and GenBank accession numbers used in this study.

Species	Strain	GenBank Accession Nos.
		ITS	LSU rDNA
Absidia abundans	CGMCC 3.16255, T	ON074695	ON074683
A. abundans	XY09265	ON074697	ON074681
A. abundans	XY09274	ON074696	ON074682
A. aguabelensis	URM 2813, T	MW763074	MW762874
A. anomala	CBS 125.68, T	NR_103626	NG_058562
A. bonitoensis	URM 7889, T	MN977786	MN977805
A. caatingaensis	URM 7156, T	KT308169	KT308171
A. caerulea	CBS 101.36	MH855718	MH867230
A. caerulea	FSU 767	AY944870	AF113443
A. californica	CBS 314.78	MH861141	MH872902
A. californica	FSU 4747	AY944872	EU736300
A. californica	FSU 4748	AY944873	EU736301
A. cornuta	URM 6100	MN625256	MN625255
A. cuneospora	CBS 101.59, T	NR_159602	NG058559
A. cylindrospora	CBS 100.08	JN205822	JN206588
A. edaphica	MFLU 20-0416	MT396372	MT393987
A. frigida	CGMCC 3.16201, T	OM108487	OM030223
A. fusca	CBS 102.35	NR103625	NG058552
A. gemella	CGMCC 3.16202, T	OM108488	OM030224
A. glauca	CBS 129233	MH865253	MH876693
A. glauca	CBS 101.08, T	NR_111658	NG_058550
A. glauca	FSU 660	AY944879	EU736302
A. globospora	CGMCC 3.16031, T	MW671537	MW671544
A. globospora	CGMCC 3.16035	MW671538	MW671545
A. globospora	CGMCC 3.16036	MW671539	MW671546
A. healeyae	UoMAU1, T	−	MT436027
A. heterospora	SHTH021	JN942683	JN982936
A. idahoensis	CBS 103.91, T	−	NG_058545
A. inflata	NRRL 6591	−	YES
A. jindoensis	CNUFC-PTI1-1, T	MF926622	MF926616
A. koreana	EML-IFS45-1, T	KR030062	KR030056
A. lobata	CGMCC 3.16256 , T	ON074690	ON074679
A. lobata	XY08832-1	ON074691	ON074680
A. longissima	CGMCC 3.16203, T	OM108489	OM030225
A. macrospora	FSU 4746	AY944882	EU736303
A. medulla	CGMCC 3.16034, T	MW671542	MW671549
A. montepascoalis	URM 2818, T	NR_172995	−
A. multispora	URM 8210, T	MN953780	MN953782
A. ovalispora	CGMCC 3.16018, T	MW264071	MW264130
A. panacisoli	SYPF 7183, T	MF522181	MF522180
A. pararepens	CCF 6352	MT193669	MT192308
A. pernambucoensis	URM 7219, T	MN635568	MN635569
A. pseudocylindrospora	CBS 100.62, T	NR_145276	NG_058561
A. pseudocylindrospora	EML-FSDY6-2	KU923817	KU923814
A. psychrophilia	FSU 4745	AY944874	EU736306
A. radiata	CGMCC 3.16257 , T	ON074698	ON074684
A. radiata	XY09330-1	ON074699	ON074685
A. repens	CBS 115583, T	NR103624	HM849706
A. saloaensis	URM 8209, T	MN953781	MN953783
A. sichuanensis	CGMCC 3.16258 , T	ON074692	ON074688
A. sichuanensis	XY09119	ON074693	−
A. sichuanensis	XY09633	ON074694	ON074689
A. soli	MFLU 20-0414, T	MT396373	MT393988
A. spinosa	FSU 551	AY944887	EU736307
A. stercoraria	EML-DG8-1, T	NR_148090	KT921998
A. terrestris	FMR 14989	LT795003	LT795005
A. turgida	CGMCC 3.16032, T	MW671540	MW671547
A. yunnanensis	CGMCC 3.16259 , T	ON074700	ON074687
A. yunnanensis	XY09528	ON074701	ON074686
A. zonata	CGMCC 3.16033, T	MW671541	MW671548
Cunninghamella blakesleeana	CBS 782.68	JN205869	MH870950
C. elegans	CBS 167.53	JN205882	HM849700

Notes: sequences obtained in this study are in bold. “T” after strain number represents strain type. “−” represents the absence of sequence in GenBank. “YES” represents the sequences from the whole-genome sequencing with BioProject accession PRJNA519280.

2.2. Morphology and Maximum Growth Temperature

Pure cultures were incubated with malt extract agar medium (MEA: 30 g malt extract, 3 g peptone, 20 g agar, and 1000 mL distilled water; [37]). For morphological studies, two plates of each strain were cultured at 20 °C and 27 °C, respectively, and then examined under a stereomicroscope (SMZ1500, Nikon, Tokyo, Japan) and a light microscope (Axio Imager A2, Carl Zeiss, Oberkochen, Germany). Maximum growth temperature tests followed the methods in our previous studies [14,15,16,38,39,40,41]. For maximum growth temperature tests, three plates were incubated at 20 °C for 2 d, then incubation temperature was increased by a gradient of 1 °C until the colonies stopped growing. For morphological features, the minimum and maximum sizes based on a statistic of more than 50 measurements were adopted [16]. All cultures were triplicated.

2.3. Molecular Phylogeny

DNA extraction, PCR amplification, and sequencing and phylogenetic analyses followed the methods in our previous studies [14,16,42,43,44]. In brief, total DNAs were extracted using a kit (GO-GPLF-400, GeneOnBio Corporation, Changchun, China) based on the instruction manual. Entire ITS and partial LSU rDNA sequences were amplified with primer pairs NS5M (5′-GGC TTA ATT TGA CTC AAC ACG G-3′) and LR5M (5′-GCT ATC CTG AGG GAA ACT TCG-3′; [14,16]). PCR procedures were as follows: an initial denaturation at 95 °C for 5 min, 30 cycles at 95 °C for 60 s, 55 °C for 45 s, and 72 °C for 60 s, and a final extra extension at 72 °C for 10 min. Sanger sequencing of PCR products were carried out by a company (SinoGenoMax, http://www.sinogenomax.com, accessed on 1 March 2022) with primers ITS1, ITS4, ITS5, and LR5M [14,16,45].

Phylogenetic analyses were conducted with maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) algorithms [39,41] using RAxML (version 8. 1.12; [46]), PAUP (version 4.0b10; [47]) and MrBayes (version 3.2.7a; [48]), respectively. Maximum Likelihood analysis was performed using the GTRGAMMA substitution model with 1000 bootstrap replications. Maximum parsimony analysis was conducted with 1000 bootstrap replications using the heuristic search option with bisection and reconnection. Sequences were randomly added and max-trees were set to 5000. For BI analysis, eight cold Markov chains were run simultaneously for two million generations with the GTR + I + G model, sampling every 1000 generations and with the first 25% sampled tree being removed as burn-in. Obtained sequences and aligned dataset were deposited at GenBank (Table 2) and Supplementary File S1, respectively. Additionally, top hits of the BLAST search for ITS sequences are provided in the Supplementary Table S1.

3. Results

3.1. Phylogenetic Analyses

The ITS and LSU rDNA dataset for 62 strains, representing 45 species of Absidia and two species of Cunninghamella, contains 1642 characters, of which 501, 877, and 264 are constant, parsimony-informative, and parsimony-uninformative, respectively. Maximum parsimony (MP) analyses yielded two equal trees (tree length 6669, consistency index 0.3330, homoplasy index 0.6670, retention index 0.5669, and rescaled consistency index 0.1888). The most optimal model of Bayesian inference (BI) was GTR + I + G, and the average standard deviation of split frequencies was 0.07357. Topology of the ML tree was chosen to represent the phylogenetic relationship (Figure 2). In the phylogenetic tree, the five new species described herein are fully supported with Cunninghamella elegans Lendn. and Cunninghamella blakesleeana Lendn. as outgroup.

Figure 2

Maximum likelihood (ML) phylogenetic tree of Absidia based on ITS and LSU rDNA sequences, with Cunninghamella elegans and C. blakesleeana as outgroup shaded in blue. Five new species are in shade and bold. Maximum likelihood bootstrap values (≥50%)/Bayesian inference (BI) posterior probabilities (≥0.9)/maximum parsimony (MP) bootstrap values (≥50%) of each clade are indicated along branches. “T” after strain number represents type. A scale bar in the upper left indicates substitutions per site.

3.2. Taxonomy

In this paper, five new species of Absidia are proposed from southwestern China, i.e., Sichuan, Tibet, and Yunnan. Besides the ITS and LSU rDNA sequences provided above, all the new species are illustrated along with morphological characteristics and the maximum growth temperature; a physiological trait is also presented.

3.2.1. H. Zhao, Y.C. Dai and X.Y. Liu, sp. nov.

Fungal Names: FN570973. Figure 3.

Figure 3

Morphologies of Absidia abundans ex-holotype CGMCC 3.16255. (a,b). Colonies on MEA ((a), obverse, (b), reverse); (c–e), sporangia; (f), sympodially branched sporangiophores; (g), swellings below sporangia; (h), columellae with projections; (i), columellae with projections and collars; (j), rhizoids; (k), sporangiospores—scale bars: (c–g), (j), 10 μm, (h,i,k), 5 μm.

Etymology: abundans (Lat.) refers to the species with abundant swellings below the sporangia.

Holotype: HMAS 351932.

Description: Colonies on MEA at 27 °C for 7 days, growing moderately fast, attaining 65 mm in diameter, initially white, soon becoming gray to brown, and irregularly and concentrically zonate with ring at reverse. Hyphae are branched, hyaline at first, brownish when mature, aseptate when juvenile, septate with age, and 3.0–15.5 µm in diameter. Stolons branched, hyaline, and smooth. Rhizoids are root-like, often unbranched, and well-developed. Sporangiophores arise from stolons, always erect, unbranched, or simple if branched, monopodial, or sympodial, hyaline, swellings usually present below sporangia, oval to pyriform, hyaline, often with a septum 12.0–17.0 µm below apophyses, 35.0–170.0 µm long, and 2.0–3.5 µm wide. Sporangia are oval to subglobose, hyaline when young, dark green when old, smooth, deliquescent-walled, multi-spored, 8.0–16.5 µm long, and 8.5–16.0 µm wide. Apophyses are distinct, hyaline or subhyaline, 2.5–5.5 µm high, gradually widened upwards, 2.5–4.5 µm wide at the base, and 4.5–8.0 µm wide at the top. Collars absent or present. Columellae are subglobose or oval, hyaline, subhyaline or light brown, 4.5–10.0 µm long, and 3.5–8.0 µm wide. Projections are single if present, subhyaline, and 1.0–2.5 µm long. Sporangiospores are cylindrical, oval or subglobose, light green, smooth, 2.5–3.5 µm long, and 2.0–3.5 µm wide. Chlamydospores are absent. Zygospores are not observed.

Maximum growth temperature: 31 °C.

Materials examined: China. Yunnan, Qujing, from forest soil sample, September 2021, Heng Zhao (holotype HMAS 351932, living ex-holotype culture CGMCC 3.16255, and living cultures XY09265 and XY09274).

3.2.2. H. Zhao, Y.C. Dai and X.Y. Liu, sp. nov.

Fungal Names: FN570974. Figure 4.

Figure 4

Morphologies of Absidia lobata ex-holotype CGMCC 3.16256. (a,b). Colonies on MEA ((a), obverse, (b), reverse); (c,d), columellae with projections; (e,f)c sporangia; (g), rhizoids; (h), sporangiospores—scale bars: (c–e), 10 μm, (f,g), 20 μm, (h), 5 μm.

Etymology: lobata (Lat.) refers to the species with a broadly lobed edge of colonies.

Holotype: HMAS 351933.

Description: Colonies on MEA at 20 °C for 7 days, growing moderately fast, attaining 70 mm in diameter, zonate, broadly lobed at edge, white at first, gradually becoming light to dark brown and greenish, and irregular at reverse. Hyphae are branched, hyaline at first, greenish brown when mature, aseptate when juvenile, septate with age, and 4.0–21.0 µm in diameter. Stolons are branched, hyaline, brownish or light greenish, smooth, and septate. Rhizoids are sometimes unbranched and rootlike when branched. Sporangiophores arising from stolons, in pairs, unbranched, erect or slightly bent, hyaline or light brown, sometimes with a septum 13.0–22.5 µm below apophyses, 50.0–360.0 µm long, and 3.0–7.0 µm wide. Sporangia are pyriform, colorless when young, light to dark brown when old, smooth, deliquescent-walled, multi-spored, 22.0–43.5 µm long, and 18.5–31.0 µm wide. Apophyses are always distinct, hyaline to light brown, 4.0–9.0 µm high, gradually widened upwards, 4.5–8.5 µm wide at the base, and 10.0–16.0 µm wide at the top. Collars are always absent. Columellae are subglobose to depressed globose, rarely irregular, subhyaline or light brown, rough, 12.0–26.5 µm long, and 11.0–25.0 µm wide. Projections are always present, single, hyaline or subhyaline, and 2.0–6.0 µm long. Sporangiospores are mostly globose, occasionally subglobose, light green, smooth, and 2.5–3.0 µm in diameter. Chlamydospores are absent. Zygospores are not observed.

Maximum growth temperature: 26 °C.

Materials examined: China, Yunnan, Lijiang, 27°31′23″ N, 100°44′32″ E, altitude: 3153 m, from rhizosphere soil of Pinus yunnanensis Franch., September 2021, Heng Zhao (holotype HMAS 351933, living ex-holotype culture CGMCC 3.16256, and living culture XY08832-1).

3.2.3. H. Zhao, Y.C. Dai and X.Y. Liu, sp. nov.

Fungal Names: FN570975. Figure 5.

Figure 5

Morphologies of Absidia radiata ex-holotype CGMCC 3.16257. (a,b). Colonies on MEA ((a), obverse, (b), reverse); (c,d), sporangia; (e), columellae with projections; (f), rhizoids; (g), sporangiospores—scale bars: (c–e), 10 μm, (f), 20 μm, (g), 5 μm.

Etymology: radiata (Lat.) refers to the species with a radiate shape of colonies.

Holotype: HMAS 351934.

Description: Colonies on MEA at 27 °C for 7 days, growing moderately fast, attaining 65 mm in diameter, white at first, gradually becoming light to dark brown, with adjoining satellite colonies at the edge at reverse. Hyphae are branched, hyaline when young, light brownish when old, aseptate when juvenile, septate with age, and 3.0–16.0 µm in diameter. Stolons are branched, hyaline to light brown, and smooth. Rhizoids are rootlike, unbranched, and well-developed. Sporangiophores arise from stolons, 1–5 in whorls, erect or slightly bent, unbranched, hyaline to light brown, sometimes with a septum 16.5–24.5 µm below apophyses, 45.0–273.0 µm long, and 3.0–5.0 µm wide. Sporangia are pyriform or subglobose, hyaline when young, brown when old, smooth, deliquescent-walled, multis-pored, 17.5–33.5 µm long, and 18.5–30.0 µm wide. Apophyses are distinct, hyaline to light brown, 5.5–12.5 µm high, with a turning point at the top of sporangiospores, then gradually widened upwards, 4.0–5.5 µm wide at the base, and 9.0–20.0 µm wide at the top. Collars absent. Columellae are mostly oval, depressed globose, occasionally subglobose to globose, subhyaline or hyaline, 13.5–22.5 µm long, and 14.0–24.0 µm wide. Projections are present or absent, single if present, subhyaline, and 2.0–4.5 µm long. Sporangiospores are oval, subhyaline, smooth, 3.0–5.0 µm long and 2.0–3.5 µm wide. Chlamydospores are absent. Zygospores are not observed.

Maximum growth temperature: 32 °C.

Materials examined: China, Yunnan, Yuxi, from forest soil sample, September 2021, Heng Zhao (holotype HMAS 351934, living ex-holotype culture CGMCC 3.16257, and living culture XY09330-1).

3.2.4. H. Zhao, Y.C. Dai and X.Y. Liu, sp. nov.

Fungal Names: FN570977. Figure 6.

Figure 6

Morphologies of Absidia sichuanensis ex-holotype CGMCC 3.16258. (a,b). Colonies on MEA ((a), obverse, (b), reverse); (c), columellae without projections; (d), columellae with a projection; (e), sporangia in whorls; (f), sporangiospores; (g,h). Rhizoids—scale bars: (c–e,g,h), 10 μm, (f), 5 μm.

Etymology: sichuanensis (Lat.) refers to the species found in Sichuan Province, southwest China.

Holotype: HMAS 351935.

Description: Colonies on MEA at 20 °C for 7 days, growing moderately fast, attaining 75 mm in diameter, white at first, soon becoming light brown, irregularly concentrically zonate at reverse. Hyphae are branched, hyaline at first, greenish brown when mature, aseptate when juvenile, septate with age, 5.5–15.5 µm in diameter. Stolons are branched, hyaline to brownish, smooth. Rhizoids are sometimes unbranched, fingerlike or rootlike when branched, and well-developed. Sporangiophores arise from stolons, 1–5 in whorls, unbranched, erect or slightly bent, hyaline, sometimes with a septum 14.0–16.5 µm below apophyses, 30.0–220.0 µm long, and 3.0–5.0 µm wide. Sporangia are pyriform, greenish when old, smooth, deliquescent-walled, multi-spored, 18.0–23.0 µm long and 17.0–21.5 µm wide. Apophyses distinct, hyaline or subhyaline, 2.0–6.5 µm high, gradually widened upwards but with a turning point at the top of sporangiophores, 3.0–5.0 µm wide at the base, and 5.5–12.0 µm wide at the top. Collars are usually absent and rarely present. Columellae are mostly subglobose, occasionally globose, hyaline to greenish, 7.5–13.0 µm long, and 8.0–15.5 µm wide. Projections present or absent, single if present, hyaline or subhyaline, 4.0–6.0 µm long. Sporangiospores are cylindrical, subhyaline, smooth, 3.0–4.5 µm long and 2.0–2.5 µm wide. Chlamydospores are absent. Zygospores are not observed.

Maximum growth temperature: 28 °C.

Materials examined: China. Sichuan, Ngawa, 30°42’18” N, 101°22’17” E, altitude: 3727 m, from grassland soil sample, September 2021, Heng Zhao (holotype HMAS 351935, living ex-holotype culture CGMCC 3.16258), from rhizosphere soil of Picea asperata Mast., Heng Zhao (living culture XY09119). Tibet, Bome, from rhizosphere soil of Pinus yunnanensis, September 2021, Heng Zhao (living culture XY09633).

3.2.5. H. Zhao, Y.C. Dai and X.Y. Liu, sp. nov.

Fungal Names: FN570978. Figure 7.

Figure 7

Morphologies of Absidia yunnanensis ex-holotype CGMCC 3.16259. (a,b). Colonies on MEA ((a), obverse, (b), reverse); (c), sporangia; (d), swellings below sporangia; (e–g), columellae; (h), rhizoids; (i), sporangiospores—scale bars: (c–g), 10 μm, (h), 20 μm, (i), 5 μm.

Etymology: yunnanensis (Lat.) refers to the species found in Yunnan Province, southwest China.

Holotype: HMAS 351936.

Description: Colonies on MEA at 27 °C for 7 days, growing moderately fast, attaining 65 mm in diameter, initially white, soon becoming light brown, and irregularly and concentrically zonate with ring at reverse. Hyphae are branched, hyaline at first, light brown when mature, aseptate when juvenile, septate with age, and 4.0–15.5 µm in diameter. Stolons are branched, hyaline, light brown or green, smooth. Rhizoids rootlike, often unbranched, and well-developed. Sporangiophores arise from rhizoids, 2–5 in whorls, erect or slightly bent, usually unbranched, rarely monopodially branched, hyaline, light brown or green, swellings usually present below sporangia, usually oval, rarely irregular, hyaline, often with a septum 12.0–16.5 µm below apophyses, 29.0–159.0 µm long, and 2.0–5.0 µm wide. Sporangia are pyriform to subglobose, hyaline when young, light green when old, smooth, deliquescent-walled, multi-spored, 16.0–27.5 µm long, and 16.5–27.0 µm wide. Apophyses are distinct, hyaline or subhyaline, 3.0–6.5 µm high, gradually widened upwards, 3.0–7.5 µm wide at the base, and 5.5–14.0 µm wide at the top. Collars are absent or present. Columellae are oval, depressed globose, or occasionally globose, hyaline, subhyaline or light green, 6.5–18.5 µm long, and 7.5–22.0 µm wide. Projections are present or absent, single if present, subhyaline, and 1.5–3.5 µm long. Sporangiospores are cylindrical, light green, smooth, 3.5–5.0 µm long, and 2.0–4.0 µm wide. Chlamydospores are absent. Zygospores are not observed.

Maximum growth temperature: 32 °C.

Material examined: China. Yunnan, Yuxi, from forest soil sample, September 2021, Heng Zhao (holotype HMAS 351936, living ex-holotype culture CGMCC 3.16259). Chuxiong, 25°13’52” N, 101°18’24” E, altitude: 2060 m, from forest soil sample, September 2021, Heng Zhao (living culture XY09528).

3.3. Key to the Species of Absidia in China

Together with the five new species proposed in this study, a total of 51 species of Absidia have been described worldwide. Among these, 22 species are distributed in China. Consequently, we provide a key to the Chinese species of Absidia. Characteristics adopted in the key include maximum growth temperatures, hyphae, rhizoids, sporangiophores, sporangia, collars, columellae, projections, and sporangiospores.

3.4. Species Distribution and Ecological Habitat in China

A total of 22 species of Absidia were recorded in China (Table 3), including the five new species proposed in this study [14,15,16,27,49,50,51]. All species were found in soil, such as forest soil and rhizospheric soil, whereas other habitats, including leaf litter, dung, insect remains, and plant leaves, recorded one to several species. In terms of geographical distribution, Yunnan, Xinjiang, and Taiwan top the list with twelve, six, and five records, respectively.

Table 3

Absidia species distributions and ecological habitat in China.

Species	Location	Habitat	References
A. abundans	Yunnan	forest soil	This study
A. cylindrospora	Jilin, Taiwan, and Xinjiang	Soil and leaf litter	[27,50]
A. frigida	Xinjiang	soil	[16]
A. gemella	Xinjiang	soil	[16]
A. glauca	Beijing, Fujian, Hebei, Hubei, Inner Mongolia, Jilin, Liaoning, Shaanxi, Sichuan, Taiwan, Xinjiang, and Yunnan	soil, forest soil, air, rhizosphere soil of Populus and Pinus, and leaf litter	[27,50]
A. globospora	Hubei and Shaanxi	soil	[15]
A. heterospora	Guizhou, Sichuan, and Taiwan	soil	[27,50]
A. idahoensis	Yunnan	Soil and bee	[24]
A. lobata	Yunnan	rhizosphere soil of Pinus yunnanensis	This study
A. longissima	Yunnan	soil	[16]
A. medulla	Fujian, Jiangxi, and Yunnan	soil	[15]
A. ovalispora	Yunnan	soil	[14]
A. panacisoli	Yunnan	rhizosphere soil of Panax notoginseng	[16]
A. pseudocylindrospora	Taiwan	soil	[27,51]
A. psychrophilia	Heilongjiang and Taiwan	soil, leaf litter, rhizosphere soil of Pinus, and gland of Ambrosia beetle	[27,51]
A. radiata	Yunnan	forest soil	This study
A. repens	Xinjiang and Yunnan	soil, dung, paper, rhizosphere soil of Ambrosia artemisiifolia, and cave depositions	[27]
A. sichuanensis	Sichuan and Tibet	grassland soil, rhizosphere soil of Picea asperata, and rhizosphere soil of Pinus yunnanensis	This study
A. spinosa	Heilongjiang and Taiwan	soil, air, and leaf of Comandra pallida	[27,51]
A. turgida	Xinjiang	soil	[15]
A. yunnanensis	Yunnan	forest soil	This study
A. zonata	Beijing, Chongqing, Fujian, Shaanxi, and Yunnan	soil	[15]

4. Discussion

In this study, five new species are proposed in the genus of Absidia being supported with molecular sequences and morphological and physiological features. Phylogenetically, Absidia abundans is closely related to A. panacisoli T. Yuan Zhang et al. based on the ITS and LSU rDNA sequences (Figure 2). However, A. panacisoli is distinguished from A. abundans by a higher maximum growth temperature (33 °C vs. 31 °C), shape of sporangia (spherical or subpyriform vs. oval to subglobose), sporangiospores (short cylindrical vs. cylindrical, oval or subglobose), and azygospores (present vs. absent; [16]). Moreover, swellings below sporangia are always observed in A. abundans, while absent in A. panacisoli [16].

Absidia lobata is closely related to A. glauca and A. globospora T.K. Zong and X.Y. Liu (Figure 2), while distinguished by a lower maximum growth temperature (26 ℃ in A. lobata, 29 °C in A. glauca, 37 °C in A. globospora) [15,52]. Besides, sporangia are globose in A. globospora, while pyriform in A. lobata and A. glauca [15,52]. Moreover, zygospores and chlamydospores are produced in A. glauca, but not in A. lobata and A. globospora [15,52].

Absidia radiata is related to A. yunnanensis with full support (Figure 3). However, A. yunnanensis differs from A. radiata by colonies (light brown vs. dark brown), shape of sporangiospores (cylindrical vs. oval), and swellings below sporangia (present vs. absent).

Absidia sichuanensis is most closely related to A. frigida H. Zhao et al. and A. psychrophilia (Figure 4), but differs by a higher maximum growth temperature (28 °C in A. sichuanensis, 24 °C in A. frigida, and 25 °C in A. psychrophilia), sporangia (17.0–21.5 µm wide in A. sichuanensis, 12.5–32.0 µm wide in A. frigida, and 20.0–50.0 µm wide in A. psychrophilia), columellae (8.0–15.5 µm wide in A. sichuanensis, 15.5–18.0 µm wide in A. frigida, and 6.5–30.0 µm wide in A. psychrophilia), and number of whorls (1–5 in A. sichuanensis, 1–4 in A. frigida, and 1–8 in A. psychrophilia [8,16]. In addition, collars are always absent in A. sichuanensis and A. frigida but present in A. psychrophilia [8,16]. Moreover, a morphological feature table including six species of Absidia without DNA sequences is listed for distinguishing between the five new species proposed in this study (Table 4).

Table 4

Morphological features of the five new Absidia species and other six related species without DNA evidence.

Species	Colonies	Sporangiophore	Sporangia	Collars	Columellae	Projections	Sporangiospores	Zygospores	Temperature	References
Absidia abundans	on MEA at 27 °C for 7 days, 65 mm in diameter	unbranched, or simple if branched, monopodial, or sympodial	oval to subglobose, 8.0–16.5 × 8.5–16.0 µm	absent or present	subglobose or oval, 4.5–10.0 × 3.5–8.0 µm	single if present	cylindrical, oval or subglobose, 2.5–3.5 × 2.0–3.5 µm	unknown	mesophilic	This study
A. clavata	on MEA rapid growing	2–8 in whorls	pyriform to globose, 10.5–33.0 µm in diameter		globose to subglobose, 9.0–22.5 µm length	present, single	globose to oval, 2.0–5.0 × 1.8–3.2 µm	unknown	−	[53]
A. dubia	−	branched with a whorls	−	−	ovoid to nearly spatulate	−	oval, 2.2–4.2 × 2.2 µm	present	−	[54]
A. egyptiaca	−	−	usually globose, 25.3 µm	−	11.5–20.75 µm	−	ovoid to elliptical, 4.6–4.85 µm	heterozygotes, globose, up to 65 µm	−	MycoBank
A. fassatiae	on MEA for 6 days, 55 mm in diameter	3–9 in whorls	globose, 15–31 µm in diameter	−	hemispherical to coniform, 9–22 µm in diameter	−	cylindrical, 4.7–7.3 × 1.9–3.1 µm	unknown	−	[55]
A. lobata	on MEA at 20 °C for 7 days, 70 mm in diameter	in pairs, unbranched	pyriform, 22.0–43.5 × 18.5–31.0 µm	absent	subglobose to depressed globose, 12.0–26.5 × 11.0–25.0 µm	always present, single	mostly globose, occasionally subglobose, 2.5–3.0 µm in diameter	unknown	mesophilic	This study
A. narayanae	on SMA at 37 °C for 2 days, 75 mm in diameter	in group, up to 8	globose to subglobose,19.5–41.5 × 24.0–41.0 µm	absent or present	globose, hemispherical to mammiform, 14.5–34.0 × 12.0–34.0 µm	−	globose to ovate, 3.0–4.0 µm	unknown	Thermophilic or thermotolerant	[56]
A. radiata	on MEA at 27 °C for 7 days, 65 mm in diameter	1–5 in whorls, unbranched	pyriform or subglobose, 17.5–33.5 × 18.5–30.0 µm	absent	mostly oval, depressed globose, occasionally subglobose to globose, 13.5–22.5 × 14.0–24.0 µm	single if present	oval, 3.0–5.0 × 2.0–3.5 µm	unknown	mesophilic	This study
A. reflexa	−	singly from the stolons	pyriform	present	conical	one or several projections	spherical, 6 p. in diameter	unknown	−	[52]
A. sichuanensis	on MEA at 20 °C for 7 days, 75 mm in diameter	1–5 in whorls, unbranched	pyriform, 18.0–23.0 × 17.0–21.5 µm	absent or present	mostly subglobose, occasionally globose, 7.5–13.0 × 8.0–15.5 µm	single if present	cylindrical, 3.0–4.5× 2.0–2.5 µm	unknown	mesophilic	This study
A. yunnanensis	on MEA at 27 °C for 7 days, 65 mm in diameter	unbranched, monopodially branched, 2–5 in whorls	pyriform to subglobose, 16.0–27.5 × 16.5–27.0 µm	absent or present	oval, depressed globose, or occasionally globose, 6.5–18.5 × 7.5–22.0 µm	single if present	cylindrical, 3.5–5.0 × 2.0–4.0 µm	unknown	mesophilic	This study

Notes: New species proposed in this study are in bold. The “−” represents the absence of features.

Species of Absidia synthesized important metabolites, such as α-galactosidase, laccase, chitosan, and fatty acids [57,58,59,60], and our results provide a basis for their applications. In addition to the five new species proposed in this paper, 51 species of Absidia have previously been described from all around the world. A total of 22 species are recorded in China (https://nmdc.cn/fungarium/fungi/chinadirectories, accessed on 1 March 2022), accounting for 43% of worldwide species of the genus Absidia [14,15,16,18,27,39,40,41,42,43,44,45,46,47,48,49,50,51]. This result suggests that Absidia, a genus of Mucoromycota, is diverse, needing in-depth investigations to discover and describe more potential new species, as in Ascomycota and Basidiomycota [13,61,62,63,64].

The species of Absidia are ubiquitous in soil, dung, and decaying plants, as well as insect remains [1,8,15,16,51]. Most species, including the five new species described herein, are reported from soil and, hence, soil is their main habitat. Some species may be associated with plants (Table 3). For example, A. lobata and A. panacisoli were described in rhizosphere soil with Pinus yunnanensis and Panax notoginseng (Burkill) F. H. Chen ex C. Chow and W. G. Huang, respectively [16].

In China, most species of Absidia are recorded in Yunnan, Xinjiang, and Taiwan (Table 3), located in tropical, subtropical, and temperate zones. At the same time, a number of Absidia are described from Brazil and Thailand, which have a similar climate [17,28,29,30,33,34]. However, species of Absidia are rarely adapted to high temperatures, so that strains in tropical areas are usually described from forest soil or mountains [16,17,28,29,33,34]. Consequently, species diversity of Absidia in tropical and subtropical forest soil needs to be further explored.