Three New Species of Gongronella (Cunninghamellaceae, Mucorales) from Soil in Hainan, China Based on Morphology and Molecular Phylogeny

The genus Gongronella is important in agriculture and industry by secreting various natural bioactive metabolites such as chitosanases and organic acids. During the most recent 8 years, a total of 14 new species have been described, remarkably enriching the diversity of this genus. In this study, we added three more new species to this valuable genus, based on a combination of morphological traits and phylogenetic information. Six strains of the genus Gongronella were isolated from soil collected in Hainan Province, China. Phylogenetic analyses of ITS and LSU rDNA sequences grouped these strains into three independent clades. According to their unique morphological characteristics, they were classified as G. multiramosa sp. nov., G. qichaensis sp. nov. and G. oleae sp. nov. The G. multiramosa was characterized by multiple branched sporangiophores and was closely related to G. pedratalhadensis. The G. qichaensis was characterized by obscure collars and closely related to G. butleri, G. hydei and G. banzhaoae. The G. oleae was characterized by the presence of oil droplets in the sporangiospores and was closely related to G. chlamydospora and G. multispora. Their descriptions and illustrations were provided, and their differences from morphological allies and phylogenetic-related species are discussed.

It was established in 1952 to accommodate the type species only, G. urceolifera Ribaldi [11].Three years later, the second species was proposed in this genus, namely G. butleri (Lendn.)Peyronel & Dal Vesco, which was recombined from Absidia butleri Lendn.Another seven years passed, and the third species was reported as G. lacrispora Hesselt.& J.J. Ellis.Since then, no more species in this group of fungi have been described for half a century.Traditionally, the main diagnostic criteria for the genus Gongronella have been the presence of distinct swollen apophyses, globose sporangia and constricted columellae [12].Currently, a consensus has been reached that fungi are classified on the basis of a combination of multiple molecular phylogenetic analyses and morphological features [13][14][15].Consequently, the new species of Gongronella have been increased.These newly found species include the following: G. Shivas 2023.The genus Gongronella currently consists of 16 species, with the type species G. urceolifera being treated as a synonym of G. butleri [16].
Six strains of Gongronella were isolated from soils in Hainan Province, China.According to rDNA molecular phylogenetic analysis and morphological comparison, these strains were classified into three new species.

Isolation and Morphological Observation
In 2023, soil samples were collected in Hainan Province, and pure strains were isolated from these samples by combining soil dilution plates and single spore isolation methods.Exactly 10 g of soil samples were transferred into a conical bottle containing 90 mL sterile water and mixed with a shaker at 120 rpm for 20 min to prepare soil suspension.One milliliter of the suspension was pipetted into 9 mL sterile water to obtain 10 −2 soil suspension.The previous step was repeated to obtain 10 −3 and 10 −4 soil suspensions.A 200 µL of 10 −3 and 10 −4 soil suspensions were pipetted onto the center of Rose Bengal Chloramphenicol agar (RBC: peptone 5.00 g/L, Glucose 10.00 g/L, KH 2 PO 4 1.00 g/L, MgSO 4 •7H 2 O 0.50 g/L, Rose Bengal 0.05 g/L, chloramphenicol 0.10 g/L, agar 15.00 g/L) [17], dispersed evenly with a sterilized triangle glass spatula and cultivated at 25 • C in the dark for 2-5 days.Subsequently, the agar with fungal mycelia at the edge of the colony was transferred to a new Potato Dextrose Agar (PDA: glucose 20.00 g, potato 200.00 g, agar 20.00 g, sterilized water 1000.00mL and pH7) and macroscopically photographed on the 7th day with a digital camera (Canon PowerShot G7X, Canon, Tokyo, Japan).
Microscopic morphological characteristics of fungi were observed with a stereoscope (Olympus SZX10, OLYMPUS, Tokyo, Japan) and a light microscope (Olympus BX53, OLYM-PUS, Tokyo, Japan), and photographed with a high-definition color digital camera (Olympus DP80 OLYMPU, Tokyo, Japan).All strains were stored in 10% sterilized glycerin at 4 • C. Morphologies were statistically calculated from 30 measurements per character [18].Living cultures (including ex-types) were deposited in the China General Microbiological Culture Collection Center, Beijing, China (CGMCC) and the Shandong Agricultural University Culture Collection, Taian, China (SAUCC).Dried type specimens were deposited in the Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS).Taxonomic information for the new taxa has been registered in the Fungal Name repository (https://nmdc.cn/fungalnames/,accessed on 25 October 2023).

Phylogenetic Analyses
The obtained DNA sequences were searched using BLAST with default parameters for closely related items against the NCBI GenBank nucleotide database [24].Newly generated sequences and their related sequences retrieved from GenBank (Table 1) were aligned using MAFFT 7 online services (http://mafft.cbrc.jp/alignment/server/,accessed on 20 October 2023) [25], employing default parameters for accurate alignment.Each marker was first analyzed individually (ITS or LSU) and then jointly (ITS-LSU), with maximum likelihood (ML) and Bayesian inference (BI) algorithms integrated with the CIPRES science portal (https://www.phylo.org/,accessed on 20 October 2023) [26].ML was performed with RaxML-HPC2 on XSEDE (8.2.12) [27] and 1000 fast bootstrap repeats were performed using the GTRGAMMA model of nucleotide evolution.For BI, the optimal evolutionary model for each partition was determined using MrModeltest v.2.3 (accessed on 12 May 2022) [28] and included in the analysis.BI was performed with MrBayes on XSEDE (3.2.7a) [29][30][31].For ML analysis, the default parameters were used and BI was performed using a fast boot algorithm with an automatic stop option.Bayesian analysis consisted of 5,000,000 generations of four parallel runs with stop rule options and a sampling frequency of 100 generations.The burnin score was set to 0.25 and the posterior probability (PP) was determined from the remaining trees.All resulting trees were drawn using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree, accessed on 20 October 2023), and the layout of the trees was carried out with Adobe Illustrator CC 2019 (https://adobe.com/products/illustrator/,accessed on 20 October 2023 ).

Phylogenetic Analyses
The sequence matrix included 26 strains from 16 species of Gongronella with Cunninghamella echinulata CBS 156.28 selected as an outgroup for phylogenetic comparison.A total of 1888 characters comprised ITS rDNA (1-909) and LSU rDNA (910-1888).Among them, there were 1207 constant, 426 variable but parsimony non-informative and 255 parsimony informative characters (Supplementary File S1).MrModelTest suggested that the Dirichlet fundamental frequency and GTR+I+G evolution pattern for both partitions were adopted in Bayesian inference.The topology of the Bayesian tree was consistent with that of the ML tree and therefore was used as a representative to summarize the evolutionary history within the genus Gongronella (Figure 1).The G. multiramosa was closely related to G. pedratalhadensis with a well support (MLBV = 100, BIPP = 1.00).The G. qichaensis was closely related to G. butleri (BIPP = 0.74), G. hydei and G. banzhaoae.The clade of G. oleae was a sister clade to G. chlamydospora/G.multispora.Etymology-The epithet "multiramosa" (Latin) refers to the multiple branched sporangiophores.

Discussion
Located on the northern edge of the tropics, China's Hainan Province falls into the Indo-Burma biodiversity hotspots and is known as a natural greenhouse with extraordinary fungal diversity.Based on molecular and morphological data, three new species of Gongronella isolated from soil habitats in southern Hainan are introduced herein.
Gongronella was initially erected to accommodate the Absidia-like fungus, G. urceolifera (currently a synonym of G. butleri), which is characterized by its spherical protrusions [34].However, this genus has remained one of the most understudied taxa of fungi for dozens of years.There has been an increase in research activity since 2015 and more and more new species have been described in this genus.Currently, there are 19 species in this genus including the 3 new species proposed herein, all of which were listed in Table 1.All members of this group of fungi were isolated from soil samples (Table 1), and therefore this genus is probably called soil-born fungus.The 26 Gongronella strains used in this study are distributed all over the world, including Australia (2 strains), Brazil (5), China (11), Korea (5), Portugal (2) and the UK (1), and, consequently, this genus is most likely a worldwide fungus, which was confirmed by 3341 samples and 25,840 sequence variants filtered from the GlobalFungi database (https://globalfungi.com/, accessed on 6 December 2023; Asia, 72.13%, North America, 12.57%, South America, 6.55%, Europe, 3.32%, Africa, 2.87%, Australia, 2.39% and Pacific Ocean, 0.15%).According to these statistics, the investigation of this genus in China seems to be more detailed than in other regions.
It is worth noting that the ITS and LSU sequences of Gongronella pamphilae were not found in NCBI, so the phylogenetic evolutionary tree was constructed in this study without the inclusion of G. pamphilae sequences.Gongronella taxonomy used to be studied primarily on the basis of morphological features and the phylogeny of ITS rDNA sequences [35,36].When G. sichuanensis was published in 2019, the LSU rDNA sequences were added, resulting in a consistency with previous ITS phylogeny [13].Since then, taxonomists studying this genus have adopted the practice of combining ITS-LSU rDNA with morphology.
Phylogenetic analysis of the six strains using ITS and LSU rDNA sequences revealed three robust monophyletic clades.Compared with Gongronella pedratalhadensis, the new species G. multiramosa has longer sporangiophores, more branching frequency, smaller sporangia and larger apophyses.No chlamydospores are found in G. pedratalhadensis, but G. multiramosa found.The new species G. qichaensis forms rhizoids, which are not found in its relatives G. hydei and G. banzhaoae.The sporangial size of G. qichaensis is larger than that of G. hydei, and the apophyses of G. qichaensis are significantly different from those of G. hydei and G. banzhaoae.Lastly, G. qichaensis lacks obvious collars, while G. butleri and G. hydei exhibit remarkable collars.The sporangial diameter of G. oleae is larger than that of G. chlamydospora, while the apophysis width of G. oleae is smaller than that of G. chlamydospora.Giant cells have also been found in G. oleae but not described in G. chlamydospora.These notable morphological dissimilarities plus those phylogenetically independent clades ensure their novelty.
In summary, the molecular phylogenetic and morphological results support that the six strains described in this paper represent three new species, G. multiramosa, G. qichaensis and G. oleae.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof9121182/s1,Supplementary File S1: the combined ITS-LSU sequence matrix used in this study.

Figure 1 .
Figure 1.A maximum likelihood (ML) phylogenetic consensus tree inferred from ITS and LSU rDNA sequences, showing relationships among species of Gongronella with Cunninghamella echinulata CBS 156.28 as outgroup.The maximum likelihood bootstrap value (MLBV) and Bayesian inference posterior probability (BIPP) are successively shown at the nodes and separated by a slash "/".Strains marked with a star "*" and bolded represented are ex-types or ex-holotypes.The strains isolated and sequenced in this study are shown in red.Branches shortened to fit the page are represented by double slashes "//" and folds "×".The scale in the bottom center indicates 0.06 substitutions per site.

Table 1 .
Information of specimens used in this study.
Notes: New species established in this study are in bold.Ex-type or ex-holotype strains are labeled with a star mark "*".The abbreviation of "n.a." stands for "not available".