﻿Morphological and phylogenetic analyses reveal two new species in Conidiobolus s.s. (Conidiobolaceae, Entomophthorales) from China

﻿Abstract The genus Conidiobolus s.s. (Conidiobolaceae, Entomophthorales) has been delimited to accommodate members that produce microspores. Herein, morphological studies, combined with phylogenetic analysis based on the nuclear large subunit of rDNA (nucLSU), the mitochondrial small subunit of rDNA (mtSSU), and the elongation-factor-like gene (EFL) revealed two Conidiobolus s.s. species isolated from plant debris in China. Conidioboluslongiconidiophorussp. nov. is mainly characterised by its long primary conidiophores, while Conidioboluspolysporussp. nov. is diagnosed by 2–3 primary conidia arising from branched primary conidiophores. Phylogenetically, the former is grouped into a separate clade, while the latter is closely related to C.incongruus, but is morphologically distinguished by its larger primary conidia and branched conidiophores.

Unfortunately, the type species of Conidiobolus, C. utriculosus Brefeld, had been missing for a long time. Therefore, C. coronatus was proposed as the epitype of Conidiobolus s.s. due to its prominence as a pathogenic fungus, its global distribution, and its usage as a model organism for fungal evolution (Spatafora et al. 2016;Möckel et al. 2022). This genus includes 18 species and is the largest among related genera (Goffre et al. 2020;Nie et al. 2020b).
Notably, not all species of Conidiobolus s.s. produce microspores, making it difficult to recognize them without phylogenetic data. These include C. dabieshanensis (Nie et al. 2017), C. iuxtagenitus (Waters and Callaghan 1989), C. margaritatus (Huang et al. 2007), C. taihushanensis (Nie et al. 2020b) and C. lichenicolus (Srinivasan and Thirumalachar 1968). However, their other unique morphological characters and phylogeny could contribute to their suitable identification. Meanwhile, the key to Conidiobolus s.s. was provided to understand the relationship among this fungal group morphologically (Nie et al. 2020b).
This study aims to describe and illustrate two new species of Conidiobolus s.s. based on their morphology and phylogenetic analyses. This study also details the diagnostic characteristics for species that were not observed to produce microspores, and the diversity of Conidiobolus s.s. found in China.

Isolation and morphology
Plant debris was collected from Guniujiang National Nature Reserve, Qimen County and Shitai County, and Huoli Mountain, Ma , anshan City, Anhui Province, and Yangtianshan National Forest Park, Shandong Province. The strains of Conidiobolus s.s. were isolated from plant debris following the previous described methods (Drechsler 1952;King 1976) and improved by Nie et al. 2012. Plant debris samples were placed into sterilized plastic bags. When they were transferred into the laboratory, the isolation was conducted immediately. Plant debris samples were cut into small pieces with scissors and tiled evenly on the Petri dishes cover, and incubated on inverted Petri dishes containing PDA media (potato 200 g, dextrose 20 g, agar 20 g, H 2 O 1 L) at 21 °C for 7 days.
The inverted Petri dishes were examined daily by a stereomicroscope (SMZ1500, Nikon Corporation, Japan). When a Conidiobolus-like fungus appeared, it was transferred to a new PDA plate to obtain a pure culture for morphological studies. The micro-morphological structure was observed using a light microscope (BX51, Olympus Corporation, Tokyo, Japan) and imaged using a microscope-camera system (DP25, Olympus Corporation, Tokyo, Japan). The morphological traits of the primary conidia and conidiophores, microconidia, resting spores etc. were described using the method by King (1976

Phylogenetic analyses
According to our previous studies (Nie et al. 2020a, b), the sequences of three loci (nucLSU, mtSSU, and EFL) of Conidiobolus s.s. species were retrieved from GenBank. Two Azygosporus and two Microconidiobolus species were chosen as out groups. Newly generated sequences from the three strains were aligned with all reference sequences by MAFFT program (Katoh and Standley 2013) and manually corrected with BioEdit (Hall 1999). The final alignments of three loci were concatenated using SequenceMatrix (Vaidya et al. 2011). The output sequence matrix was deposited in TreeBase (https://treebase.org) with the submission ID 30475. Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic analyses were conducted. The best-fit substitution model of each partition was evaluated by MrModeltest 2.3 (Nylander 2004

Phylogenetic analyses
The concatenated alignment utilized in this study comprised 1899 characters of nucLSU (1-984), EFL (985-1485), and mtSSU (1486-1899), out of which 986 characters are constant, 289 characters were found to be parsimony-uninformative and 624 characters were parsimony-informative. The best substitution model GTR+I+G was chosen for all the partitions during the ML and BI phylogenetic analyses. The final average standard deviation of the split frequencies was 0.00841, and the BI tree topology was found to be similar to that of ML. Therefore, the best scoring RAxML tree was used to represent the phylogenetic relationships among the studied taxa, with a final likelihood value of -13552.35 (Fig. 1) Etymology. Longiconidiophorus (Lat.), referring to the long size of its conidiophores. Description. Colonies on PDA at 21 °C after 3 d white, reaching ca 15 mm in diameter. Aerial hyphae flourishing after 6 d. Mycelia white, 5-10 μm wide, often unbranched at the edge of colony. Primary conidiophores often evolving from aerial hyphae, long, 150-340 × 6-9 μm, unbranched and producing a single primary conidium, without widening upward near the tip. Primary conidia forcibly discharged, globose, obovoid to ellipsoidal, 31-49 × 24-42 μm, papilla tapering and pointed, 7-13 μm wide, 3-7 μm long. Secondary conidiophores short or long, arising from primary conidia, bearing a single similar replicative conidium to primary conidia. Microspores not observed on the 2% water agar, but the structure similar to sterigmatas bearing microspores observed. Resting spores absent after 1 month.
Notes. Conidiobolus longiconidiophorus forms a distinct phylogenetic clade from other Conidiobolus s.s. species. Morphologically, its primary condia are similar in size to those in C. coronatus (Cost.)  μm), C. dabieshanensis Y. ,  . However, it can be distinguished from C. coronatus and C. macrosporus by its longer primary conidiophores and the absence of resting spores (Batko 1964;Srinivasan and Thirumalachar 1967). Additionally, it is differentiated from C. dabieshanensis and C. utriculosus by its obovoid and ellipsoidal primary condia, as well as the absence of resting spores (Brefeld 1884;Nie et al. 2017). While it is closely related to C. megalotocus, it can be differentiated by the shape of its primary condia (Srinivasan and Thirumalachar 1962). Furthermore, in the phylogenetic tree (Fig. 1), C. longiconidiophorus is found to be distantly related to C. megalotocus.

Discussion
Although the family Conidiobolaceae was originally proposed to include three genera, Azygosporus, Conidiobolus s.s., and Microconidiobolus, recent phylogenetic analyses by Gryganskyi et al. (2022) revealed that the genus Microconidiobolus should be placed in a separate clade (Gryganskyi et al. 2022). In addition, we found that this fungal group produces smaller primary conidia, mostly less than 20 μm in size, without microspores, in comparison to most members of Azygosporus and Conidiobolus s.s. (Nie et al. 2020a). Therefore, it may be appropriate to recognize Microconidiobolus as a distinct family rather than a genus in the family Conidiobolaceae. However, additional evidence, including unique morphological characteristics, phylogenetic analyses with more taxa, and more genome data, is necessary to confirm this hypothesis.
C. longiconidiophorus produces long primary conidiophores (over 300 μm) because most of them develop from aerial hyphae. We noticed that C. dabieshanensis (Nie et al. 2017) also produces such long primary conidiophores (up to 287 μm), and they are closely grouped together in the phylogenetic tree (Fig. 1). Coincidentally, these two species were not observed to produce microspores. Nevertheless, we made several attempts, such as culturing at low or high temperatures, on different culture media, and even exposing them to ultraviolet radiation to induce microspore formation. However, we were still unable to observe microspores, and we hypothesized that microspores of these species may only arise under the natural environment. This phenomenon was also observed in four other Conidiobolus s.s. species and may require further investigation.
Conidiobolus polysporus is known to produce 2-3 primary conidia arising from branched primary conidiophores. Similar branched primary conidiophores have also been observed in C. gonimodes (Drechsler 1961), C. margaritatus (Huang et al. 2007), C. polytocus (Drechsler 1955) and C. taihushanensis (Nie et al. 2020b). However, the number of primary conidia borne on these branched primary conidiophores varies: C. gonimodes and C. margaritatus produce 2 primary conidia, C. polytocus produces 2-4 primary conidia, and C. taihushanensis produces 2-6 primary conidia. Notably, the two primary conidia of C. gonimodes arise directly from the top of branched primary conidiophores without short handles (Drechsler 1961). Additionally, C. polysporus produces primary conidia that are larger than those produced by the other four Conidiobolus s.s. species mentioned above.
Interestingly, we found that C. iuxtagenitus was located at the bottom of the phylogenetic tree and was distinct from other Conidobolus s.s. members. C. iuxtagenitus is characterized by an absence of microspore and its zygospores formed by a short beak near a lateral conjugation (Waters and Callaghan 1989). Therefore, it is possible that C. iuxtagenitus represents another potential new lineage.
In this study, we introduce two new species of Conidiobolus s.s. species, namely C. longiconidiophorus and C. polysporus, based on morphological and phylogenetic evidence. These findings expand the number of known Conidiobolus s.s. species to 20.