Taxonomy and phylogeny of the Absidia (Cunninghamellaceae, Mucorales) introducing nine new species and two new combinations from China

Absidia is ubiquitous and plays an important role in medicine and biotechnology. In the present study, nine new species were described from China in the genus Absidia , i.e. A. ampullacea , A. brunnea , A. chinensis , A. cinerea , A. digitata , A. oblongispora , A. sympodialis , A. varians , and A. virescens . Besides, two varieties A. cylindrospora var. nigra and A. spinosa var. biappendiculata were elevated to a specific rank as A. nigra comb. nov. and A. biappendiculata comb. nov., respectively. These new taxa were proposed based on a comprehensive investigation of morphological traits (such as shape and size of sporangia, sporangiospores and projections on columellae), physiological feature (maximum growth temperatures), and multi-locus sequences (including internal transcribed spacer, large subunit D1-D2 domains of nuclear ribosomal DNA, partial translation elongation factor 1 alpha gene and actin gene). All species mentioned above are illustrated, and an identification key to all the known species of Absidia in China is included. wide. Stolons branched, hyaline, µm wide. µm clavate projection at the apex, occasionally with two projections, slightly bulbous at the end, 10.5–22.0 × (11.5–)18.0–30.0 µm. Collars distinct if present. Sporangiospores globose, hyaline, smooth, non-uniform, 3.0–5.0 µm diameter. Zygospores not observed. Chlamydospores absent. No growth at 32 ℃ . high, µm at 9.5–18.5 µm wide at the Sporangia globose to pyriform, multispored, deliquescent-walled, Columellae spherical or oval to hemispherical, hyaline, smooth, with a 1.5– µm clavate or slightly swollen projection at the apex, Collars distinct if present. Sporangiospores two types, cylindrical or globose, hyaline, smooth, 3.0–4.0 × 2.0–2.5 µm when cylindrical, 4.5–8.0(–9.0) × 4.0–8.0 when globose. Zygospores not observed. Chlamydospores absent. No growth at 31 ℃


INTRODUCTION
The genus Absidia Tiegh. (Cunninghamellaceae, Mucorales) was established by van Tieghem (1876). Absidia members are ubiquitous, mostly from soil and sometimes also associated with animals and plants. For example, the ex-type of A. spinosa var. biappendiculata Rall & Solheim was isolated from the leaves of Comandra pallida (Rall and Solheim 1964), and the ex-type strains of A. californica J.J. Ellis & Hesselt. and A. stercoraria Hyang B. Lee, H.S. Lee and T.T.T. Nguyen both live on rat dung (Ellis and Hesseltine 1965;Li et al. 2016). Although several species are vital causative agents of mucormycosis, some species are capable of producing chitin, chitosan, chitooligosaccharides (Kaczmarek et al. 2019) and hydrocortisone (Chen et al. 2020). This genus is characterized by rhizoids, stolons bearing single or a bunch of sporangiophores, a septum close to the top of sporangiophores, pyriform apophysate deliquescent-walled sporangia, distinct apophyses, and typically appendaged zygospores (Hoffmann et al. 2007;Hoffmann 2010).
The circumscription and classification of the Absidia have long been disputed.
Before 1964, seven generic names allied to Absidia were successively proposed according to morphology, Tieghemella Berl. & De Toni, Mycocladus Beauverie, Proabsidia Vuill., Lichtheimia Vuill., Pseudoabsidia Bainier, Protoabsidia Naumov and Gongronella Ribaldi (Berlese and de Toni 1888;Beauverie 1900;Vuillemin 1903aVuillemin , 1903bBainier 1903;Naumov 1935;Ribaldi 1952). However, after a study on hundreds of strains comprising these genera, Hesseltine and Ellis (1964) retained Gongronella and Absidia only. The Gongronella has a globose apophysis with a constriction showing the attachment of the sporangial wall. They further divided Absidia into two subgenera, namely subg. Absidia and subg. Mycocladus (Beauverie) Hesselt. & J.J. Ellis. The former produces appendages from the sespensors to envelop zygospores, whereas the latter does not produce any appendages. This classification framework was followed by Schipper (1990) who in turn recognized six groups in the subg. Absidia. However, with the advent of DNA-based molecular phylogenetics, the taxonomic classification of the genus has been refined. Hawksworth et al. (1995) did not accept the above morphologybased subgeneric delimitation, but synonymized all these allied genera with Absidia instead.
Recently, a comprehensive study of molecular phylogenetics, morphology and physiology has provided a more reliable delimitation among Absidia species (Hoffmann et al. 2007), where Absidia was classified into three groups: 1) the thermotolerant species with an optimal growth temperature of 37-45 ℃ ; 2) the mesophilic species with an optimal growth temperature of 25-34 ℃, which have been accepted up to now as Absidia sensu stricto; and 3) the mycoparasitic species, potential to parasitise other mucoralean hosts. Soon, the thermotolerant species were transferred into the genus Lichtheimia (Hoffmann et al. 2009a); and the mycoparasitic species were placed into a new genus Lentamyces Kerst. Hoffm. & K. Voigt (Hoffmann and Voigt 2008).
Currently, 39 species have been reported worldwide in Absidia (Bainier 1889;Hesseltine and Ellis 1961Hesseltine 1965, 1966; Index Liu. (Ariyawansa et al. 2015;Li et al. 2016;Crous et al. 2018Crous et al. , 2020Wanasinghe et al. 2018;Zhang et al. 2018;Cordeiro et al. 2020;Lima et al. 2020;de Lima et al. 2021;Hurdeal et al. 2021;Zhao et al. 2021;Zong et al. 2021). Totally, 13 species are distributed in China (Zheng and Liu 2018;Zhang et al. 2018;Zhao et al. 2021;. Recently, 16 strains of Absidia were isolated from China but could not be assigned to any described species. Morphological, physiological and molecular data (multi-gene locus included ITS, D1-D2 domains of LSU rDNA, TEF-1α and Act) are presented herein to support them within the Absidia sensu stricto, including nine new species and two new combinations. In addition, the synoptic key to all the 23 known species of Absidia in China is revised.

Isolation and Strains
Strains were isolated from the soil collected in China about 20 years ago or even earlier.
Approximately 1 g of soil samples was suspended in 100 mL sterilized water and shaken vigorously. Then, 100 μL of the suspension was added onto a potato dextrose agar (PDA: 20 g/L glucose, 20 g/L agar, 200 g/L potato, and 1000 mL distilled water) with 100 mg / mL each of streptomycin sulfate and ampicillin. The plate was incubated upside down at 27 ℃ and examined once a day with a stereomicroscope (SMZ1500, Nikon Corporation, Japan

Morphology and Growth Experiments
Pure strains were cultivated with malt extract agar (MEA: 20 g/L malt extract, 20 g/L agar, and 1000 mL distilled water). For morphological observation, they were incubated at 27 ℃ for 4-7 d and daily examined under a microscope (Axio Imager A2, Carl Zeiss Microscopy, Germany). For determining maximum growth temperatures, they were initially incubated at 32 ℃ for 4 d, and then at higher temperatures until the colonies stopped growing. The color code of colonies was determined with the chromatography of Ridgway (1912).

DNA Extraction, PCR Amplification and Sequencing
Mycelia were grown at 27 ℃ for 5 d on PDA plates, and then cell DNAs were extracted with GO-GPLF-400 kit (GeneOnBio Corporation, Changchun, China). A span including the internal transcribed spacer (ITS) and large subunit (LSU) D1-D2 domain of rDNA were amplified with primer pair NS5M and LR5M (Wang et al. 2014).
The partial translation elongation factor 1 alpha gene (TEF-1α) was amplified with primer pair EF1-983F (Rehner and Buckley 2005) and TEF1LLErev (Jaklitsch et al. 2005). The partial actin gene (Act) was amplified with primer pair Act-1 and Act-4R (Voigt and Wostemeyer 2000). The PCR procedure for all these gene loci was as follows: an initial temperature at 95 ℃ for 5 min (rDNA and TEF-1α) or 3 min (Act), then 30 cycles of denaturation at 95 ℃ for 30 s (rDNA and TEF-1α) or 60 s (Act), annealing at 55 ℃ for 60 s and extension at 72 ℃ for 60 s, and finally an extra extension at 72 ℃ for 10 min. PCR products were purified and sequenced at BGI Tech Solutions Beijing Liuhe Co., Limited, Beijing, China. All newly generated sequences were deposited at GenBank (Table 1).

Phylogenetic Analyses
The software platform Geneious 9.0.2 (http://www.geneious.com) was used to assemble and proofread DNA sequences. All the sequences were realigned using AliView version 3.0 (Larsson 2014). The sequence alignments and phylogenetic trees were deposited at TreeBase (submission ID 28429). Sequences of Cunninghamella elegans and C. blakesleeana retrieved from GenBank were used as outgroups in multigene analyses following Hoffmann et al. (2007).
Phylogenetic analyses were carried out using algorithms Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). MP phylogenetic analyses followed Zhao and Wu (2017), and the tree construction was performed with PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing data. Trees were inferred using a heuristic search with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap analysis with 1,000 replicates (Felsenstein 1985). Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each maximum parsimonious tree generated.
ML phylogenetic analyses were conducted with raxmlGUI 2.0 beta (Edler et al. 2020). A general time reversible model was used with a gamma-distributed rate variation (GTR+G) and 1000 bootstrap replicates were carried out.
BI phylogenetic analyses were calculated with MrBayes 3.2.7a (Ronquist et al. 2012) by a general time reversible model with an estimate of the proportion of invariant sites and a gamma distribution for variable rates across sites (GTR+I+G). Four Markov chains were run simultaneously from random starting tree for 2,300,000 generations and the tree was sampled every 100 generations. The chains stopped once the average standard deviation of split frequencies decreased lower than 0.01. The first one-fourth of generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches were considered as significantly supported if they received Maximum Likelihood bootstrap (MLB) ≥ 70 %, Maximum Parsimony bootstrap (MPB) ≥ 70 %, or Bayesian posterior probabilities (BPP) ≥ 0.95 (Hillis and Bull 1993;Huelsenbeck and Hillis 1993;Leaché and Reeder 2002).

Phylogenetic Analyses
The multi-gene dataset included sequences from 64 strains representing 44 species of At the end of the inference, the average standard deviation of split frequencies was 0.009960 (BI). All BI, ML and MP phylogenetic trees resulted in similar topologies and consequently were integrated (Fig. 1). Description: Colonies on MEA, irregularly zonate, attaining 46 mm diameter after 5 d at 27 ℃, white at first and then Saccardo's olive (R16). Hyphae hyaline at first, becoming brown when mature, sometimes ampulliform-shaped swollen, 6.0-13.0(-16.5) µm wide. Stolons branched, hyaline, smooth, septate, 4.0-9.0 µm wide. Rhizoids finger-like, mostly twice or more repeatedly, with a septum at the base.

Absidia cinerea appears to be phylogenetically basal to A. pseudocylindrospora
Hesselt. & J.J. Ellis and A. stercoraria, although with relatively low support values.
However, physiologically, A. pseudocylindrospora have a maximum growth temperature of 37 ℃, but neither A. cinerea nor A. stercoraria can grow above 35 ℃.
However, morphologically, A. turgida differs from A. digitata in that it produces sporangiophores singly or in whorls of up to 4, only one projection on the columellae, and such variable sporangiospores as globose, cylindrical or irregular .
A. sympodialis and A. virescens are sister taxa and cluster with A. globospora (100/100/1.00), having similar sporangia. Their relationship is highly supported by the multi-gene tree, with a -/98/1.00 support value. However, physiologically and morphologically, A. globospora is different by no growth at 29°C, whereas the maximum growth temperature of A. sympodialis and A. virescens both are higher than 30 °C. In addition, the sporangiophores of A. globospora are in whorls up to 5, whereas those of A. sympodialis and A. virescens arise in whorls of up to 6 and 4, respectively.
A. virescens produces sporangiospores of non-uniform in size and columellae with two projections, some projection up to 6.5 µm in length, whereas the sporangiospores of A.
sympodialis and A. globospora are uniform in size, and both columellae with only one projection less than 5 µm in length. A. sympodialis differs from A. virescens and A. globospora in the shape of rhizoids and columellae. It has fibrous-root-shaped rhizoids, differing from them in that both have root-like rhizoids. Additionally, columellae of A.
sympodialis are hemispherical, oval and chestnut-shaped, unlike those hemispherical in A. virescens and A. globospora . Absidia varians (100/100/1.00) is phylogenetically next to A. glauca but morphologically similar to A. repens because they both have variable projections on columellae. A. repens is different from A. varians in that it having olive-gray colonies and two types of sporangiospores .
Physiologically, as mentioned in the introduction, growth temperature has been an important characteristic to distinguish Absidia s. str. (optimal growth temperature of 25-34 ℃ ) and Lichtheimia species (optimal growth temperature of 37-45 ℃ , Hoffmann et al. 2007). Most species of Absidia s. str. were within the temperature range, but it was also stated that Absidia cuneospora G.F. Orr & Plunkett, A. idahoensis var. thermophile G.Q. Chen & R.Y. Zheng, A. koreana and A. pseudocylindrospora were able to grow above 37 °C (Orr and Plunkett 1959;Hesseltine and Ellis 1961;Chen and Zheng 1998;Ariyawansa et al. 2015). On the other hand, Lichtheimia sphaerocystis Morphologically, Hoffmann et al. (2007) found that Absidia usually have projections on the apex of columellae. The nine new species and two combinations described here also have this characteristic. Therefore, we believe that projection on columellae is a typical feature of the genus Absidia, except for the species A.
The Absidia genus was proposed to be divided into several groups distinguishable by their sporangiospores (Kwaśna et al. 2006;Hoffmann et al. 2007Hoffmann et al. , 2009bHoffmann and Voigt 2008;Hoffmann 2010). However, with new species of Absidia were increasingly described, some of them were found to have two and more shapes of sporangiospores, such as A. pararepens, A. repens, A. turgida, etc (Fig. 1, Crous et al. 2020;Cordeiro et al. 2020;Zong et al. 2021). Therefore, the clades on the phylogenetic tree should be further divided, especially the clade previously defined as cylindrical spores. Additionally, A. digitata does not goup within the globose spores clade, even though it has globose sporangiospores only, indicating that the shape of sporangiospores in Absidia might polyphyletic evolved.
In recent years, more and more new species, e.g. Absidia globospora, A. medulla, A. ovalispora, A. panacisoli, A. turgida and A. zonata, have gradually been reported from China (Zhang et al. 2018;Zhao et al. 2021;Zong et al. 2021). This study further supplements nine new species, making the list of Chinese Absidia types as long as 16.
As many as eleven new taxa are placed in Absidia, and therefore it is necessary to update the synoptic key for the 23 species distributed in China as follows.

DECLARATIONS Ethics approval and consent to participate
Not applicable.

Adherence to national and international regulations
Not applicable.

Consent for publication
Not applicable.

Availability of data and material
Details of the availability of the data and materials used in this study can be found      (H-L) 20 µm.                     The geographic distribution of Absida types except for A. glauca and A. cylindrospora (neither is available). Triangles represent nine new species and two combinations proposed in this study. Circles represent previously reported species.