Three new species of Conidiobolus sensu stricto from plant debris in eastern China

Abstract The genus Conidiobolus Bref. is widely distributed and the Conidiobolus sensu lato contained three other genera, Capillidium, Microconidiobolus and Neoconidiobolus. A molecular phylogeny based on the nuclear large subunit of rDNA (nucLSU), the mitochondrial small subunit of rDNA (mtSSU) and the translation elongation factor 1-alpha gene (TEF1) revealed three novel species within the clade of Conidiobolus s.s., i.e. C. bifurcatussp. nov., C. taihushanensissp. nov. and C. variabilissp. nov. These three species were isolated from plant debris in eastern China. Morphologically, C. bifurcatussp. nov. is characterised by its secondary conidiophores often branched at the tip to form two short stipes each bearing a secondary conidium. C. taihushanensissp. nov. is different from the others in its straight apical mycelia and the production of 2–5 conidia. C. variabilissp. nov. is distinctive because of its various shapes of primary conidia. All these three new taxa are illustrated herein with an update key to the species of the genus Conidiobolus s.s.

During the past decade, Bo Huang's research group have carried out a comprehensive study on the taxonomy of Conidiobolus sensu lato in China and proposed five new species, five Chinese new records and 23 new combinations (Wang et al. 2010a, b, Nie et al. 2012, 2016, 2017, 2020, Chen and Huang 2018. Recent collections by this research group in eastern China resulted in the discovery of three unique species within the Conidiobolus sensu stricto lineage, which are described and illustrated herein with a multi-locus molecular phylogeny on the nuclear large subunit of rDNA (nucLSU), the mitochondrial small subunit of rDNA (mtSSU) and the translation elongation factor 1-alpha gene (TEF1).

Isolates and morphology
Plant debris was collected from Taihushan and Jilongshan National Forest Parks, Anhui Province, China and Laoshan National Forest Park, Jiangsu Province, China. Isolations were carried out using the canopy-plating approach (Drechsler 1952, King 1976a. A Petri dish with potato dextrose agar (PDA; potato 200 g, dextrose 20 g, agar 20 g, H 2 O 1000 ml) was inverted over the plant debris and incubated at 21 °C for daily examining for one week. When entomophthoroid fungi on the PDA canopy were detected, they were quickly transferred to new PDA and 2% water agar (agar 20 g, H 2 O 1000 ml) plates for purification and description. Morphological features were measured with an Olympus BX51 research microscope for 35 primary conidia and conidiophores each and photographed by an Olympus DP25 microscope-camera system. The descriptions were made with the method of King (1976a). Cultures were deposited in the Research Center for Entomogenous Fungi of Anhui Agricultural University, Anhui Province, China (RCEF) and the China General Microbiological Culture Collection Center, Beijing, China (CGMCC). Dried cultures were deposited in the Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS). In order to infer the phylogeny of the genus Conidiobolus s.s., a total of 21 ex-types of species in Conidiobolus s.l., serving as outgroup, were obtained from the American Type Culture Collection, Manassas, USA (ATCC).

Phylogenetic analyses
In addition to the sequences obtained in this paper, nucLSU, mtSSU and TEF1 sequences of 20 strains in Conidiobolus sensu stricto were downloaded from GenBank.
Three genera Capillidium, Microconidiobolus and Neoconidiobolus, each represented by two species, were selected as outgroups. The nucLSU, mtSSU and TEF1 sequences were aligned with Clustal X (Thompson et al. 1997) and deposited at TreeBase (submission ID 26063). Phylogenetic analyses with Bayesian Inference (BI), Maximum Parsimony (MP) and Maximum Likelihood (ML) were carried out according to Nie et al. (2018Nie et al. ( , 2020. BI phylogeny was estimated using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). The best-fit model selected with the Akaike Information Criterion (AIC) in Modeltest 3.7 (Posada and Crandall 1998) was used to evaluate Posterior Probabilities (PP) and the critical value for the topological convergence diagnostic was set to 0.01 of the average standard deviation of split frequencies. Four Markov chains ran simultaneously from random starting trees for 0.5 million generations and trees were sampled every 100 th generation. MP analyses were performed using a heuristic search with PAUP* 4.0b10 (Swofford 2002). All characters were weighted and gaps were treated as missing data. Tree bisection-reconnection (TBR) was set as the branch swapping algo- rithm. Branch robustness was estimated with bootstrapping 1,000 replicates (Felsenstein 1985). ML analyses were performed with the RAxML (Stamatakis 2006), implemented in raxmlGUI 1.5b1 (Silvestro and Michalak 2012). Branch reliabilities were determined by 1,000 ML rapid bootstrap replicates with the GTRGAMMA substitution model. Phylogenetic trees were checked and modified in FigTree 1.4 (Rambaut 2012).

Phylogenetic analyses
The combined nucLSU+TEF1+mtSSU dataset was composed of 29 taxa representing 27 species and 1949 characters including 986 constant, 276 parsimony-uninformative and 687 parsimony-informative. The most parsimonious tree was generated with a tree length (TL) of 2716 steps, a consistency index (CI) of 0.5497, a homoplasy index (HI) of 0.4503, a retention index (RI) of 0.6191 and a rescaled consistency index (RC) of 0.3403. The best model applied in the BI analysis was GTR+I+G. The final average standard deviation of split frequencies was 0.0086 and the final likelihood value was -14423. The three phylograms resulted in similar topologies and the ML tree was presented along with MP/ML bootstrap and BI posterior probability values at relative branches ( Fig. 1). Three clades can be seen to form for the Conidiobolus s.s. The three species, described here, were located in clade I.  Etymology. bifurcatus (Lat.), referring to secondary conidiophores often branched at the tip to form two short stipes, each bearing a secondary conidium.
Ecology and distribution. Plant debris in Jiangsu Province, China. Description. Colonies on PDA at 21 °C for 3 d, opaque, white, reaching ca. 2 mm in diameter, with many small colonies around the periphery due to discharged conidia. Mycelia colourless, 8-11 μm wide, rarely branched and non-septate when young, often septate and distended to a width of 10-27 μm after 5 d. Primary conidiophores arising from the hyphal segments, colourless, 38-254 × 7.5-12 μm, unbranched and producing a single globose conidium, without widening upwards near the tip. Primary conidia for-cibly discharged, globose to subglobose, 2-40 × 2-33 μm, with a papilla more or less tapering and pointed, 7-11 μm wide at the base, 3-12 μm long. Secondary conidiophores arising from the primary conidia, often branched almost at the tip, forming two short stipes each bearing a secondary conidium. Secondary conidia similar to, but smaller than the primary ones, mostly forcibly discharged, occasionally falling off and leaving a relic of the secondary conidiophores. On 2 % water agar, microconidia produced readily, globose to ellipsoidal, 7-12 × 6-9 μm. Zygospores homothallic, usually formed between adjacent segments of the same hypha after an incubation of 5-7 d at 21 °C on PDA, smooth, mostly globose, 25-40 μm in diameter, with a 1.5-3 μm thick wall.
Notes. Conidiobolus bifurcatus sp. nov. is characterised by its secondary conidiophores, which are often bifurcated near the tip and bear a secondary conidium on each  stipe. Morphologically, it is allied to Conidiobolus mycophilus Srin. & Thirum., which has smaller primary conidia (Srinivasan and Thirumalachar 1965). It appears to be similar to C. incongruus Drechsler and C. mycophagus Srin. & Thirum. in the size of primary conidia and zygospores and the formation of microconidia, but different in its longer primary conidiophores (Drechsler 1960;Srinivasan and Thirumalachar 1965). However, it is distantly related to these two species in the molecular phylogenetic tree. Instead, it is phylogenetically closely related to C. brefeldianus Couch (Figure 1: MP 71/ML 89/BI 1.00), but morphologically distinct by its larger primary conidia and zygospores (Couch 1939).  Etymology. taihushanensis (Lat.), referring to the region where the fungus was isolated.
Notes. Conidiobolus taihushanensis sp. nov. is morphologically highly distinct with its straight apical mycelia and the production of 2-5 conidia from the hyphal body. Conidiobolus taihushanensis sp. nov. is similar to C. polytocus Drechsler in the structure of several short branches at the top of conidiophores, but the latter is distinguished by smaller primary conidia (12-25 × 14-29 μm) and slightly curved mycelia (Drechsler 1955c). Conidiobolus taihushanensis sp. nov. is related to C. margaritatus B. Huang, Humber & K.T. Hodge and C. megalotocus Drechsler by the size of primary conidia, but C. margaritatus forms a chain of undischarged repetitional conidia (Huang et al. 2007) and C. megalotocus lacks zygospores (Drechsler 1956). Phylogenetically, C. taihushanensis sp. nov. is closely related to C. megalotocus (Figure 1: MP 73/BI 1.00) and distantly related to C. polytocus, though no molecular data are available for C. margaritatus. Phylogenetically, C. taihushanensis sp.
Notes. Considering the large size of primary conidia, Conidiobolus variabilis sp. nov. is allied to C. coronatus (Cost.)  μm), C. macrosporus Srin. & Thirum. (38-45 × 48-54 μm) and C. utriculosus . It is distinguished from C. coronatus by its various shapes of primary conidia and the absence of villose spores. It differs from C. macrosporus by its longer primary conidiophores and the absence of resting spores (Batko 1964, Srinivasan andThirumalachar 1967). It is differentiated from C. utriculosus by the shapes of primary conidia and the absence of zygospores. Phylogenetically, C. variabilis sp. nov. is basal in clade I and distantly related to C. coronatus and C. macrosporus.

Discussion
The genus Conidiobolus has recently been divided into four lineages and one of them was treated as Conidiobolus sensu stricto on the basis of a synapomorph, namely microspores (Nie et al. 2020). The three new species C. bifurcatus sp. nov., C. taihushanensis sp. nov. and C. variabilis sp. nov. are located in the clade of Conidiobolus s.s. (Fig. 1). Conidiobolus taihushanensis sp. nov. was paraphyletic to C. megalotocus Drechsler, C. lamprauges Drechsler and C. coronatus (Cost.) Batko with a robust support of BI posterior probability of 1.00. Conidiobolus bifurcatus sp. nov. was a sister group to C. brefeldianus, which was supported by all three inferring methods (MP 71/ML 89/BI 1.00). Conidiobolus variabilis sp. nov. was basal in clade I with a relatively high confidence (MP 97/ML 96/BI 1.00). Conidiobolus bifurcatus sp. nov. and C. variabilis sp. nov. ghan, C. lamprauges and C. parvus Drechsler were not reported to produce microspores either. This may be due to the need for particular conditions, such as growth temperature and nutritional supply. For example, the microspores of C. khandalensis Srin. & Thirum. were only observed on 2% water-agar at 16 °C (Nie et al. 2020).
Except microspores, species of the Conidiobolus s.s. clade are morphologically diverse, particularly the secondary conidia. For instance, C. iuxtagenitus produces single fusiform discharged secondary conidia (Waters and Callaghan 1989) and C. margaritatus forms a necklace-like chain of up to seven undischarged conidia (Huang et al. 2007). Although these special characteristics provide good identification, most members of this lineage are difficult to distinguish phenotypically. Sequence data of nucLSU and TEF1 have provided a better understanding of species circumscription or inter-and intraspecific variations (Nie et al. 2012). In this study, morphology and molecular data support C. bifurcatus sp. nov., C. taihushanensis sp. nov. and C. variabilis sp. nov. as new species in the Conidiobolus s.s. clade. Although the microspores of C. taihushanensis sp. nov. were not observed, its straight apical mycelium and the production of 2-5 conidia from the hyphal body make it easily distinguishable from other species of Conidiobolus s.s.
With the proposal of the three new species herein, 17 species are currently accepted in the genus Conidiobolus s.s. and only five were found distributed in China (King 1976a, b, 1977, Wang et al. 2010a, b, Nie et al. 2017, 2020. For updating, the key to all these 17 species are provided as follows.
Key to the species of Conidiobolus s.s.