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.

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.

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 (   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.

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.
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.

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.

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.

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.  Etymology: abundans (Lat.) refers to the species with abundant swellings below the sporangia.
Maximum growth temperature: 31 • C.     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     Etymology: yunnanensis (Lat.) refers to the species found in Yunnan Province, southwest China.

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.
Sporangiospores two or more types 7 6.
Sporangiophores neither in pairs nor in whorls A. panacisoli 16.
Sporangiophores in pairs and in whorls 17 17.
Sporangiophores never swollen A. medulla

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 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).
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.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8050471/s1, Supplementary File S1: The aligned dataset for 62 strains, representing 45 species of Absidia and two species of Cunninghamella; Supplementary Table S1: Top hits for the new species based on BLAST search for ITS sequences from type materials.   (Table 2).