A taxonomic revision of the genus Conidiobolus (Ancylistaceae, Entomophthorales): four clades including three new genera

Abstract The genus Conidiobolus is an important group in entomophthoroid fungi and is considered to be polyphyletic in recent molecular phylogenies. To re-evaluate and delimit this genus, multi-locus phylogenetic analyses were performed using the large and small subunits of nuclear ribosomal DNA (nucLSU and nucSSU), the small subunit of the mitochondrial ribosomal DNA (mtSSU) and the translation elongation factor 1-alpha (EF-1α). The results indicated that the Conidiobolus is not monophyletic, being grouped into a paraphyletic grade with four clades. Consequently, the well-known Conidiobolus is revised and three new genera Capillidium, Microconidiobolus and Neoconidiobolus are proposed along with one new record and 22 new combinations. In addition, the genus Basidiobolus is found to be basal to the other entomophthoroid taxa and the genus Batkoa locates in the Entomophthoraceae clade.


Introduction
More than 250 species of entomophthoroid fungi were isolated from insects, soil and litter throughout the world (Gryganskyi et al. 2013). For a long time, this group has been considered to be polyphyletic (Nagahama et al. 1995;Jensen et al. 1998;James et al. 2006;Liu and Voigt 2010) and was classified into a subphylum Entomophthoromycotina and a pending taxon Basidiobolus (Hibbett et al. 2007). However, a recent phylogeny using the multi-gene dataset, 18S rDNA, 28S rDNA, mtSSU and RPB2, indicated that this group formed a monophyletic lineage including Basidiobolus and it was consequently reclassified as a new fungal phylum Entomophthoromycota. More recently, a phylogenomic analysis (192 clusters of orthologous proteins) has divided traditional zygomycotan into two phyla Mucoromycota and Zoopagomycota and the entomophthoroid fungi have been re-assigned into the subphylum Entomophthoromycotina under the latter phylum (Spatafora et al. 2016). This taxonomic scheme was supported by the phylogeny of mitochondrial genomes (Nie et al. 2019).
Three subgenera -Capillidium, Conidiobolus and Delacroixia -were proposed within the Conidiobolus, based on shape of the secondary conidia and, amongst them, the subgenus Delacroixia was reduced from generic rank (Ben-Ze'ev and Kenneth 1982). This subgeneric criterion provided a valuable contribution for the taxonomy of the genus Conidiobolus (Humber 1989). Since the 1990s, molecular analysis has become an increasingly important tool for fungal taxonomy (Bruns et al. 1991;Taylor et al. 2000). The nucLSU rDNA and EF-1α regions proved to be distinguishable amongst Conidiobolus species (Nie et al. 2012), while nucSSU rDNA indicated the genus Conidiobolus might be a polyphyletic group (Jensen et al. 1998). The subgeneric circumscription was not defined because of instability to form a certain type of secondary conidia for each phylogenetic clade (Callaghan et al. 2000;Gryganskyi et al. 2013;Nie et al. 2018). Besides, the phylogenetic relationships amongst species of Conidiobolus have not been fully resolved due to the absence of types. The genus Batkoa, morphologically similar to Conidiobolus, was phylogenetically closely related to Entomophthoraceae rather than Ancylistaceae (Gryganskyi et al. 2012(Gryganskyi et al. , 2013. In the present study, a reclassification of the entomophthoroid fungi, including as many as available Conidiobolus types, was constructed based on four loci (nucSSU, nu-cLSU, EF-1α and mtSSU) to present the taxonomic delimitation of the genus Conidiobolus and to re-evaluate the phylogenetic relationship between Basidiobolus and Batkoa.

Isolates and morphology
A total of 26 ex-types of Conidiobolus were purchased from the American Type Culture Collection, Manassas, USA (ATCC) and collected from the China General Microbiological Culture Collection Center, Beijing, China (CGMCC) and the Research Center for Entomogenous Fungi of Anhui Agricultural University, Anhui Province, China (RCEF). Dried cultures were deposited in the Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS). Morphology was observed with an Olympus BX51 research microscope and photographed by an Olympus DP25 microscope-camera system. Growth diameter on PDA (potato 200 g, dextrose 20 g, agar 20 g, H 2 O 1 l), Mycelia, primary conidiophores, primary conidia, microconidia, capilliconidia and resting spores were measured and described with the method of King (1976a).

DNA extraction, PCR amplification and sequencing
Fungal strains were incubated on PDA for 7 d at 21 °C. Total genomic DNA was extracted from the fresh fungal mycelia by using modified CTAB method (Watanabe et al. 2010). Four gene portions from cell nuclei and mitochondria and one protein coding gene were used in this study: the large subunit of nuclear ribosomal RNA genes (nucLSU), the small subunit of nuclear ribosomal RNA genes (nucSSU), the small subunit of mitochondrial ribosomal RNA genes (mtSSU) and the translation elongation factor 1-alpha gene (EF-1α). The nucLSU region was amplified with the primers LR0R and LR5 (Vilgalys and Hester 1990), the nucSSU region with nucSSU-0021-5' (Gargas and DePriest 1996) and nucSSU-1780-3' (DePriest 1993) and EF-1α region with the primers EF983 and EF1aZ-1R (http://www.aftol.org/primers.php). These PCR reactions have been described by Liu et al. (2005), Jensen et al. (1998) and Nie et al. (2012). The primers used for the mtSSU region were mtSSU1 and mtSSU2R and the PCR reaction was performed using the following cycling parameters: denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, extension at 72 °C for 1 min and finalised with an extra extension at 72 °C for 7 min (Zoller et al. 1999). PCR products were purified and sequenced by Shanghai Genecore Biotechnologies Company (Shanghai, China) with the same primers as relative PCR. The nucleotide sequence data have been deposited in the GenBank (Table 1).

Phylogenetic analyses
More available nucLSU, nucSSU, mtSSU and EF-1α sequences of 14 Conidiobolus species and 47 other entomophthoroid fungi were obtained from GenBank. Ten species of Glomeromycotina, Mortierellomycotina, Mucoromycotina, Kickxellomycotina, Zoopagomycotina, Blastocladiomycota, Chytridiomycota and Cryptomycota, were chosen as outgroups. Alignments were constructed separately for each locus with MUSCLE 3.8.31 (Edgar 2004) and the concatenated matrices were assembled by SequenceMatrix 1.7.8 (Vaidya et al. 2011). The best model for the phylogenetic analysis was selected with Akaike Information Criterion (AIC) by using Modeltest 3.7 (Posada and Crandall 1998). Phylogenetic relationships were inferred using Maximum Likelihood (ML) and Bayesian Inference (BI). The best-scoring ML tree analysis was performed using raxmlGUI 1.5b1 with GTRGAMMA model and 1000 replicates (Silvestro and Michalak 2012). The BI analysis was performed using MrBayes 3.2.2 (Ronquist and Huelsenbeck 2003). Markov Chain Monte Carlo (MCMC) chains ran until the convergences met and the standard deviation fell below 0.01. The first 25% of trees were discarded as burn-in. The combined dataset was deposited at TreeBase (No. S25064). Phylogenetic trees were checked and modified in FigTree 1.4 (Rambaut 2012).

Phylogenetic analyses
The combined dataset contained 4521 characters of nucLSU (1-1326), nucSSU (1327-3424), EF-1α (3425-4062) and mtSSU (4063-4521) after alignment. With the optimal model GTR+I+G and random starting trees, four Markov chains were run for 7 million generations and every 100 th generation was sampled once. ML and BI analyses of the combined dataset resulted in phylogenetic reconstructions with almost similar topologies and the average standard deviation of split frequencies was 0.006721 (BI). In the ML phylogenetic tree (Figure 1), the Basidiobolaceae lineage (88/0.94) is located at the base of the entomophthoroid fungi and is closely related to the Ancylistaceae group (56/0.91). The Batkoa lineage is grouped within the Entomophthoraceae Clade (60/0.89). All Conidiobolus lineages are clustered into a paraphyletic grade and therefore cannot be considered congeneric. Moreover, the Conidiobolus grade consists of four well supported clades. In detail, there are 7, 10, 16 and 3 species in Clade I (100/1.00), II (77/1.00), III (100/1.00) and IV (99/1.00), respectively.

Taxonomy
In order to provide a more natural taxonomic classification, four genera (Capillidium, Conidiobolus, Microconidiobolus and Neoconidiobolus) and their type species (Ca. heterosporum, C. utriculosus, M. paulus and N. thromboides) are described here in this paper.  Description. Mycelia colourless. Primary conidiophores simple, bearing a single primary conidia. Primary conidia forcibly discharged multinucleate, colourless, globose, pyriform to obovoid. Two kinds of replicative conidia, the first one is similar and smaller than primary conidia, the second one (capilliconidia) arises from elongate and slender conidiophores. Zygospores present or absent, formed in axial alignment with conjugating segments, globose to subglobose, often smooth, sometimes rough, colourless or yellowish.
Notes. The species was firstly reported from America (Drechsler 1953a). The extype living culture is ATCC 12589 isolated by Drechsler (1953a). It is mainly characterised and differs from other Capillidium species by its ability to form both microconidia and capilliconidia (Callaghan et al. 2000). The Chinese specimen CGMCC 3.15888 clusters completely (100/1.00) with an isotype ARSEF 451 (98% sequence similarity in nucLSU) and fits well with its morphological descriptions. It is reported in China for the first time.
Notes. C. utriculosus, the type species of the genus Conidiobolus, has not been re-collected since Brefeld isolated it in 1884 and most taxonomists working on entomophthoroid fungi now universally recognised it as C. coronatus (Gryganskyi et al. 2013). However, the smaller pear-shaped conidia of C. utriculosus are different from the larger globose conidia of C. coronatus and villose spores in C. coronatus are not observed in C. utriculosus (Brefeld 1884;King 1977). Consequently, C. coronatus is not synonymised with C. utriculosus in this study. Instead, this study agrees with Srinivasan and Thirumalachar (1967) and King (1977) to place C. minor in synonymy with C. utriculosus because the small conidia of C. minor were probably replicative conidia of C. utriculosus. Nevertheless, neither C. utriculosus nor C. minor has available living cultures. Therefore, we have not yet designated an epitype and thus no DNA sequences for explaining this type. Fortunately, we are able to recognise clade III (Fig. 1) as Conidiobolus on the basis of its synapomorph, namely microspores.
Notes. According to the original morphological description (Srinivasan and Thirumalachar 1962b) and the re-examination by King (1977), microconidia have not been reported. However, we observed the microconidia produced from global conidia on 2% water-agar at 16 °C. Moreover, this specimen was located in the Conidiobolus lineage ( Figure 1 Description. Mycelia colourless. Primary conidiophores simple and short, bearing a single primary conidia. Primary conidia forcibly discharged, multinucleate, colourless, globose to obovoid, usually small, mostly less than 20 μm. Only globose repli- Figure 5. a-g Conidiobolus iuxtagenitus h Conidiobolus khandalensis a colony on PDA after 3 d at 21 °C b primary conidiophores bearing primary conidia c primary conidia d tertiary fusiform conidium from a globose spore e zygospore formation with the beak almost emptied of protoplasm f production of secondary conidia g zygospores h microconidia produced from global conidia. Scale bars: 10 mm (a); 20 μm (b-h). cative conidia produced, similar and smaller than primary conidia. Chlamydospores globose, formed terminally on hyphae or from globose cells by thickening of the wall. Zygospores formed in axial alignment with two conjugating segments, globose to ellipsoidal, smooth and yellowish.
Notes. This genus includes three species producing smaller primary conidia (mostly less than 20 μm) without microspores or capilliconidia compared to other Conidiobolus spp. These three species are C. nodosus, C. paulus and C. terrestris. According to the taxonomic scheme of Conidiobolus by King (1977), C. undulatus is a synonym of C. paulus, which is supported herein by molecular evidence (Figure 1). However, the phylogeny does not support C. nodosus and C. terrestris as synonyms of C. lachnodes, since the former two were located in clade IV and the latter in clade II ( Figure 1). Therefore, we accept the taxonomic status at species level for C. nodosus and C. terrestris, based on the morphological and phylogenetic analyses. ( Description. Mycelia colourless. Primary conidiophores simple, sometimes branched from hyphal knots or differentiated from aerial hyphae, positively phototropic, bearing a single primary conidium. Primary conidia forcibly discharged, multinucleate, colourless, globose, pyriform to obovoid. Replicative conidia similar and smaller than primary conidia. Chlamydospores globose, formed terminally on hyphae or from globose cells by thickening of the wall. Zygospores formed in axial alignment with two conjugating segments, globose to ellipsoidal, smooth, colourless, rarely pale yellowish.

Microconidiobolus paulus
Notes. The genus Neoconidiobolus is strikingly similar to the subgenus Conidiobolus which produces neither microconidia nor capilliconidia. All members in the clade of Neoconidiobolus share the following characteristics: forcibly discharged, colourless, globose, pyriform to obovoid primary conidia. Two kinds of replicative conidia produced. One is discharged, similar and smaller than primary conidia and the other is elongate and forcibly discharged. Two types of resting spores produced: zygospores and chlamydospores. Conidiobolus thromboides Drechsler, J. Wash. Acad. Sci. 43: 38 (1953 Drechsler, Am. J. Bot. 41: 567 (1954

Discussion
The phylogenetic position of Basidiobolus in the Kingdom Fungi has been problematic for a long time. Previous phylogenetic analyses of the rDNA (18S, 28S and 5.8S) sequences grouped Basidiobolus outside or basal in the Entomophthorales (Nagahama et al. 1995;Jensen et al. 1998;White et al. 2006). Combined with the study of other protein-coding molecular markers, Basidiobolus was located inside the Entomophthorales . Recently, according to the phylogeny of much more available molecular data of entomophthoroid fungi in three families, Basidiobolus was grouped basal to other entomophthoroid taxa (Gryganskyi et al. 2012) which was also supported by the phylogenomic analyses of zygomycete fungi (Spatafora et al. 2016) and by the multi-gene analyses in this study. Although the morphological characteristics of Batkoa were similar to Conidiobolus, the Batkoa lineage appeared to be most closely related to the other taxa in the Entomophthoraceae Clade and should be distinguished from Conidiobolus lineage by its obligate pathogenicity for invertebrates and by staining readily, while most members of Conidiobolus are saprobic and non-staining. The phylogenetic relationship of the genus Conidiobolus has been unclear for a long time, because of its high heterology (Gryganskyi et al. 2013). This article used more available ex-type strains to revise this genus, based on phylogeny and morphology. According to Figure 1, four main clades were reconstructed and the results showed that Conidiobolus s.l. is not a monophyletic group but paraphyletic with Macrobiotophthora vermicola. The M. vermicola was originally placed in Entomophthora (Mcculloch 1977) and transferred to Macrobiotophthora, based on the morphological characters of primary spores, secondary spores and zygospores (Tucker 1981). The paraphyletic relationship between Macrobiotophthora vermicola and Conidiobolus s.l. was also revealed by Gryganskyi et al. (2012). In this paper, we treated it as a new combination and, therefore, proposed a monophyletic group of the new genus Neoconidiobolus.
In Clade II of the genus Neoconidiobolus, all 14 strains comprising 10 species produce neither microspores nor capilliconidia. Amongst these, C. antarcticus was identified as a synonym of C. osmodes (Chen and Huang 2018), which was confirmed here as they grouped into a robust clade.
Considering its long history and significant impact, we kept and emended the genus Conidiobolus and the original illustrations of the type species C. utriculosus (Brefeld 1884) were designated as its lectotype. Thus, we were able to recognise clade III under the genus name Conidiobolus on the basis of its synapomorph, namely microspores. In Clade III of the genus Conidiobolus, all species definitely produce microspores, except Conidiobolus dabieshanensis, C. iuxtagenitus, C. khandalensis and C. lichenicolus. Microspores have never been observed in C. dabieshanensis and C. iuxtagenitus (King 1977;Waters and Callaghan 1989;Nie et al. 2017), but cases for C. khandalensis and C. lichenicolus are somewhat different. For C. khandalensis, the protologue did not document any microspores (Srinivasan and Thirumalachar 1962b;King 1977), but they can be observed on 2% water-agar at 16 °C (Fig. 5h). Although the microspore of C. lichenicolus was not mentioned in the original description, the ability to produce microspores has been exhibited in accordance with original illustrations (Srinivasan and Thirumalachar 1968a). The phylogeny also resulted in the following taxonomic treatments. On the one hand, some previously synonymised taxa recover their specific status, for example, C. gonimodes, C. megalotocus and C. mycophagus should be separated from C. incongruus, C. macrosporus and C. mycophilus, respectively. On the other hand, C. chlamydosporus is synonymised with C. firmipilleus.
Phylogenetically, Conidiobolus lamprauges does group with Clade III and received strong bootstrap support (100/1.00). Morphologically, this species produces small primary conidia (12.5-20 × 15-22 μm) without microconidia or capilliconidia and is similar to species within Clade IV. Its taxonomic status remains unclear in the present study.