﻿Unveiling species diversity within the family Conidiobolaceae (Entomophthorales) in China: Descriptions of two new species and reassessment of the taxonomic position of Conidioboluspolyspermus

﻿Abstract In the present study, two new Conidiobolus s.s. species were described relying on the morphological studies and phylogenetic analysis utilizing nuclear large subunit of rDNA (nucLSU), mitochondrial small subunit of rDNA (mtSSU), and elongation-factor-like gene (EFL) sequences. Conidiobolusjiangxiensissp. nov. is distinguished by its short primary conidiophores, a feature not commonly observed in other Conidiobolus s.s. species. Conversely, Conidiobolusmarcoconidiussp. nov. is characterized by larger primary conidia and the emergence of 2–5 secondary conidia from each branched secondary conidiophores. Additionally, the taxonomic reassessment of C.polyspermus confirms its distinct status within the genus Conidiobolus s.s. Moreover, molecular analyses, incorporating the nucLSU, mtSSU, and EFL sequences, provide robust support for the phylogenetic placement of the two newly described species and the taxonomic identity of C.polyspermus. This investigation contributes valuable insights into the species diversity of Conidiobolaceae in China, enhancing our understanding of the taxonomy within this fungal family.


Introduction
The reclassification of conidiobolus-like fungi into three families based on phylogenetic and morphological evidence led to the establishment of the family Conidiobolaceae (Entomophthorales), housing three genera i. Huang & Y. Nie (Gryganskyi et al. 2022).Members of this family primarily exhibit a saprobic lifestyle, thriving in soil and plant debris.However, exceptions exist, with C. coronatus distinguished for its capacity to infect both insects and humans, and C. lunulus being isolated from leafcutter ants in Argentina (Goffre et al. 2020;Möckel et al. 2022) A comprehensive taxonomy of conidiobolus-like fungi has identified Conidiobolus s.s. as distinct from four other genera (Nie et al. 2020a(Nie et al. , b, 2023;;Cai et al. 2021).However, the synonymy of C. megalotocus (= C. polyspermus, = C. eurypus), introduced by King (1976b), remains unexplored, and requiring re-evaluation.Additionally, our previous study confirmed the synonymy of C. firmipilleus (= C. chlamydosporus).Thus, this study aims to add taxonomic clarity by re-evaluating the aforementioned synonym and introducing additional taxonomic taxa.
Previous phylogenetic analyses revealed two main clades within Conidiobolus s.s.when Capillidiaceae and Neoconidiobolaceae species were used as outgroups (Nie et al. 2020a, b;Gryganskyi et al. 2022).However, distinctive traits within each clade were not elucidated.Notably, both clades included species lacking microspore observations, such as C. iuxtagenitus, C. margaritatus, C. lichenicolus, C. taihushanensis, C. dabieshanensis, and C. longiconidiophorus (Srinivasan and Thirumalachar 1968;Waters and Callaghan 1989;Huang et al. 2007;Nie et al. 2017Nie et al. , 2020bNie et al. , 2023)).Particularly, C. iuxtagenitus, potentially forming a separate lineage, produces fusiform secondary conidia, and its zygospores form via a short beak near lateral conjugation (Waters and Callaghan 1989;Nie et al. 2023).Regrettably, molecular data for C. margaritatus are currently unavailable, leaving unanswered questions about the relationships among these morphologically distinct species and the possibility of undiscovered lineages within this fungal group.Resolving these issues requires additional members for phylogenetic and morphological studies.
Over the last decades, only six new Conidiobolus s.s.species and three new records were reported from China (Wang et al. 2010;Nie et al. 2017Nie et al. , 2020bNie et al. , 2023)).Additionally, the understanding of phylogenetic relationships within the accepted species of Azygosporus and Microconidiobolus remains limited.Consequently, a comprehensive exploration of the species diversity within Conidiobolaceae is imperative to unravel the relationships within this intricate fungal group.This article aims to identify two new Conidiobolus s.s.species using morphological characters and phylogenetic analyses of nucLSU, mtSSU, and EFL sequences.Simultaneously, the taxonomic status of C. polyspermus will be clarified.

Isolation and morphology
Plant debris and soil samples were collected from Dashushan and Binhu National Forest Park, Hefei City, Anhui Province, and Aixihu Forest Wetland Park, Nanchang City, Jiangxi Province, during 2022.For isolation of conidiobolus-like fungi, we are following the previous described methods (King 1976a;Nie et al. 2012).All samples were preserved in sterilized plastic bags and transported to the laboratory as soon as possible.Plant debris samples were cut into several approximately 2 cm sized fragments and placed evenly on the Petri dishes cover.Then, using a Petri dish with potato dextrose agar (PDA; potato 200 g, dextrose 20 g, agar 20 g, H 2 O 1 L) inverted over the treated samples to obtain discharged conidia, and incubating at 21 °C for daily examining by a stereomicroscope (SMZ1500, Nikon Corporation, Japan) for 7 days.When conidiobolus-like fungi observed, they were transferred to new PDA plate for purification and morphological observation.
The micro-morphological structure of mycelium, primary conidia and conidiophores, secondary conidia, and resting spores at 400× magnification was observed under a BX51 microscope (Olympus Corporation, Tokyo, Japan) and imaged using a DP25 microscope-camera system (Olympus Corporation, Tokyo, Japan) under differential interference contrast (DIC) condition.Each character was made more than 35 measurements and the description was made with the method by King (1976a).The purification isolates were deposited at the Engineering Research Center of Biofilm Water Purification and Utilization Technology of Ministry of Education at Anhui University of Technology, Anhui Province, China (BWPU), and duplicated at Research Center for Entomogenous Fungi at Anhui Agricultural University, Anhui Province, China (RCEF).In addition, 14 ex-types of Conidiobolus s.l. were purchased from the American Type Culture Collection, Manassas, VA, USA (ATCC).

Phylogenetic analyses
DNA sequences of three loci (nucLSU, mtSSU, and EFL) originated from Conidiobolus s.s.species were downloaded from GenBank database (Table 1) with two Azygosporus and three Microconidiobolus species served as outgroups.Sequence alignment of each locus was performed with MUSCLE 3.8.31(Edgar 2004).Then, the alignments were checked and manually adjusted in BioEdit 7.0.1 (Hall 1999).The concatenated matrices were assembled by SequenceMatrix 1.7.8 (Vaidya et al. 2011).The obtained matrix was deposited in TreeBase (https://treebase.org) with the submission ID 31051.The best-fit likelihood models were estimated for each partition with MrModeltest v.2.3 (Nylander 2004).Maximum likelihood (ML) analyses were conducted with RAxML 8.1.17using 1000 bootstrap replicates (Stamatakis 2014).Bayesian Inference (BI) analyses were calculated with MrBayes 3.2 (Ronquist and Huelsenbeck 2003).Bayesian posterior probabilities (PP) were estimated by the Metropolis-coupled Markov chain Monte Carlo method (Geyer 1991).Four simultaneous Markov chains were run starting from random trees for 1 million generations, keeping one tree every 100 th generation until the average standard deviation of split frequencies was below 0.01.The value of burn-in was set to discard 25% of trees and posterior probabilities (PP) were determined from the remaining trees.The phylogenetic tree was visualized in FigTree 1.4 (Rambaut 2012) and improved with Adobe Illustrator CS6.0.

Phylogenetic analyses
The concatenated dataset comprised 1883 nucleotide sites, with specific contributions of 981 for nucLSU, 501 for SSU, and 401 for EFL.Within this dataset, 964 characters remained constant, 656 were parsimony-informative, and 308 were parsimony-uninformative. Model selection for individual data from each partition in both ML and BI phylogenetic analyses resulted in the application of the GTR+I+G model.The ML optimization likelihood reached a final value of -13813.01,and the average standard deviation of the split frequencies at the end of the analyses was 0.00619.The resulting phylogram from the ML analysis is depicted in Fig. 1.Etymology.jiangxiensis (Lat.), referring to the region where the fungus was isolated.
Description.Colonies on PDA at 21 °C after 3 d white, reaching ca 8 mm in diameter.Mycelia colorless, unbranched at the edge of colony, distended to a width of 9-20 μm segment after 5 d.Primary conidiophores unbranched, slightly curved at the tip, producing a single primary conidium, without widening upward near the tip, 105-230 × 10-16 μm.Primary conidia forcibly discharged, mostly globose, sometimes obovoid, 45-67 × 42-58 μm, with a sharp or round papilla, 13-22 μm wide, 4-13 μm long.Secondary conidia arising from primary conidia, with a short or long secondary condiophore, similar and smaller to the primary conidia.Secondary conidiophores branched at the base or tip, thus bearing 2 secondary conidia at each tip.Sometimes form 2-5 secondary conidia like "tomatoes on sticks" from small to large at each branch.Microconidia not observed on the PDA culture and on the 2% water agar.Zygospores formed between adjacent segments after 7 days, smooth, globose, 30-45 μm in diameter, with a 2-4 μm thick wall.
Notes.Conidiobolus marcoconidius is distinguished morphologically by its larger primary conidia compared to other Conidiobolus s.s.species, with the exception of C. coronatus (King 1977).Notably, it can be readily differentiated from C. coronatus by the absence of villose spores (Batko 1964).Additionally, C. marcoconidius is characterized by secondary conidiophores that branch at the base, giving rise to 2-5 secondary conidia resembling "tomatoes on sticks" at each branch, varying in size from small to large.In the phylogenetic tree, it forms a discrete clade, setting it apart from other Conidiobolus s.s.species.Description.Refer to Drechsler (1961).

Conidiobolus polyspermus
Notes.In accordance with King's numerical taxonomy of Conidiobolus (King 1976b), C. polyspermus was initially identified as a synonym of C. megalotocus.However, upon a thorough comparison of morphological traits based on the original descriptions, it became evident that C. polyspermus (15-55 × 12-48 μm) produces larger conidia than those of C. megalotocus (12-44 × 10-42 μm).Notably, C. polyspermus is not reported to form microconidia, distinguishing it from C. megalotocus (Drechsler 1956;1961).Furthermore, the phylogenetic tree (Fig. 1) revealed a distinct relationship between C. polyspermus and C. megalotocus.In light of these findings, we propose the separation of C. polyspermus from C. megalotocus, affirming its taxonomic status at the species level.

Discussion
Over an extended period, DNA-based techniques have played a pivotal role in uncovering both inter-and intra-species phylogenetic variations, essential for describing new species (Kidd et al. 2023).While the ITS region stands as a universal barcode marker for fungal identification, its applicability to entomophthoroid fungi is hindered by high intragenomic variation (Schoch et al. 2012;Hyde et al. 2023).Fortunately, the development of the full ribosomal operon and additional gene loci encoding proteins as fungal barcodes has addressed some of these challenges (James et al. 2006;Wurzbacher et al. 2019;Voigt et al. 2021;Zhao et al. 2023).In understanding the phylogeny of entomophthoroid fungi, reclassifications based on molecular sequences of nucLSU-SSU, mtSSU, and RPB2 have led to an updated taxonomic system proposed by Humber (2012), building upon the work of Gryganskyi et al. (2012Gryganskyi et al. ( , 2013)).However, with only 31 fungal taxa having molecular data, constituting a mere fraction of entomophthoroid fungi, further phylogenetic analyses are imperative for a comprehensive understanding.
In light of the aforementioned phylogenetic framework, our study employed the same loci (excluding EFL instead of RPB2 for ease of amplification) to investigate the phylogeny of conidiobolus-like fungi.(Nie et al. 2020a;Cai et al. 2021).The evolution of phylogenomic studies in various fungal groups (Spatafora et al. 2016;Vandepol et al. 2020;Li et al. 2021)  Hodge, grounded in morphological evidence and ancestral lifestyle considerations (Gryganskyi et al. 2022).
Furthermore, our molecular analyses underscored the high sensitivity of both nucLSU and EFL sequences in delineating conidiobolus-like fungi (Nie et al. 2012).Considering the balance between amplification efficiency, data integrity, and diversity, three loci of nucLSU, mtSSU, and EFL were selected for species recognition within Conidiobolus s.s.(Nie et al. 2020b(Nie et al. , 2023)).
In this study, we recovered the species status of C. polyspermus, while C. eurypus was synonymized with C. megalotocus.This synonymy will be subject to re-evaluation with the inclusion of molecular data for C. eurypus.Notably, C. polyspermus was also not reported to produce microconidia, a trait shared with six other Conidiobolus s.s.species.With the addition of descriptions for two new species in this manuscript, the count of Conidiobolus s.s.species lacking observation of microconidia has risen to nine.The morphological variation or genetic mutation behind this phenomenon remains a question that could be addressed not only through phylogenomic analyses but also by conducting comparative genomics analyses within a broader spectrum of Conidiobolus s.s.species.
With the introduction of two new Conidiobolus s.s.species, namely C. jiangxiensis and C. marcoconidius in the family Conidiobolaceae herein, the number of known Conidiobolus s.s.species are up to 22.However, limited reports of new species within the genera Azygosporus and Microconidiobolus in China underscore the need for an in-depth exploration of advanced species diversity within Conidiobolaceae from China in our future studies.

Figure 1 .
Figure 1.Maximum likelihood (ML) tree obtained by phylogenetic analyses of the combined nucLSU, EFL and mtSSU sequences.Two Azygosporus and three Microconidiobolus species were served as outgroups.The proposed new species is in boldface.Maximum Likelihood bootstrap values (≥70%) / Bayesian posterior probabilities (≥0.95) of clades are provided alongside the branches.The scale bar at the bottom left indicates substitutions per site.

Figure 2 .
Figure 2. Conidiobolus jiangxiensis RCEF 7484 a colony on PDA after 3 d at 21 °C b mycelia unbranched at the edge of the colony c hyphal segments d-g primary conidiophores bearing a single primary conidia h-k primary conidia l, m primary conidia bearing a single secondary conidium n, o zygospores formed between adjacent segments of the same hypha p young zygospores q mature zygospores.Scale bars: 100 μm (b); 20 μm (d-q).

Figure 3 .
Figure 3. Conidiobolus marcoconidius RCEF 6918 a colony on PDA after 3 d at 21 °C b mycelia unbranched at the edge of the colony c hyphal segments d-f primary conidiophores g-j primary conidia k, l primary conidia bearing a single secondary conidium m secondary conidiophore branched at the base and bearing two secondary conidia at each tip n secondary conidiophore branched at the tip o secondary conidiophore branched at the base bearing 2-5 secondary conidia at each branch p, q zygospores formed between adjacent segments of the same hypha r mature zygospores.Scale bars: 100 μm (b, n); 20 μm (c-m, o-r).