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Research Article
Three new species of Conidiobolus sensu stricto from plant debris in eastern China
expand article infoYong Nie‡§, Yue Cai|, Yang Gao, De-Shui Yu‡, Zi-Min Wang#, Xiao-Yong Liu¤, Bo Huang‡
‡ Anhui Agricultural University, Hefei, China
§ Anhui University of Technology, Ma’anshan, China
| Hefei University, Hefei, China
¶ Jiangxi Agricultural University, Nanchang, China
# Anhui University of Technology, Ma'anshan, China
¤ Chinese Academy of Sciences, Beijing, China

Corresponding author: Bo Huang ( bhuang@ahau.edu.cn )

Academic editor: Christian Wurzbacher
© 2020 Yong Nie, Yue Cai, Yang Gao, De-Shui Yu, Zi-Min Wang, Xiao-Yong Liu, Bo Huang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Nie Y, Cai Y, Gao Y, Yu D-S, Wang Z-M, Liu X-Y, Huang B (2020) Three new species of Conidiobolus sensu stricto from plant debris in eastern China. MycoKeys 73: 133-149. https://doi.org/10.3897/mycokeys.73.56905
Open Access

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. bifurcatus sp. nov., C. taihushanensis sp. nov. and C. variabilis sp. nov. These three species were isolated from plant debris in eastern China. Morphologically, C. bifurcatus sp. nov. is characterised by its secondary conidiophores often branched at the tip to form two short stipes each bearing a secondary conidium. C. taihushanensis sp. nov. is different from the others in its straight apical mycelia and the production of 2–5 conidia. C. variabilis sp. 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.

Keywords

basal fungi, Entomophthorales, taxonomy, molecular phylogenetics, new species

Introduction

The genus Conidiobolus Bref. (Ancylistaceae) was established to accommodate the type C. utriculosus Bref. and a second species C. minor Bref. (Brefeld 1884). This genus was characterised by simple conidiophores, globose to pyriform conidia and resting spores formed in the axis of the hypha (mostly as zygospores) (Humber 1997). Until 1968, a total of 41 species occurring saprotrophically in soil and plant debris had been assigned to this genus (Martin 1925, Couch 1939, Drechsler 1952, 1953a, b, 1954, 1955a, b, c, 1956, 1957a, b, c, 1960, 1961, 1962, 1965, Srinivasan and Thirumalachar 1961, 1962a, b, 1965, 1967, 1968a, b). In a review of these taxa with the numerical technique, 27 definitive species were recognised (King 1976a, b, 1977). On the basis of the shape of secondary conidia, Ben-Ze’ev and Kenneth (1982) classified the genus Conidiobolus into three subgenera, including Capillidium Ben-Ze’ev & Kenneth, Conidiobolus Brefeld and Delacroixia Tyrrell & Macleod. Until 2018, no remarkable taxonomic treatments had been made for this genus, although additional species were reported continuously (Bałazy et al. 1987, Waters and Callaghan 1989, Bałazy 1993, Tosi et al. 2004, Huang et al. 2007, Waingankar et al. 2008, Nie et al. 2012, 2016, 2017, 2018). Meanwhile, higher-rank molecular phylogenetic studies on entomophthoroid fungi suggested Conidiobolus to be polyphyletic (Jensen et al. 1998, Gryganskyi et al. 2013, Nie et al. 2020). Consequently, the three genera Capillidium, Microconidiobolus and Neoconidiobolus were separated from Conidiobolus sensu lato and Conidiobolus sensu stricto was characterised by microspores arising from conidia (Nie et al. 2020).

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, 2018, 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).

Materials and methods

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, H2O 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, H2O 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).

DNA extraction, PCR amplification and sequencing

Fungal biomass was collected from the plate surface and ground in liquid nitrogen with a pestle and mortar. Genomic DNA was extracted using the CTAB method (Watanabe et al. 2010). The extracted DNA was stored in 100 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at -20 °C. Universal primer pairs LR0R (5'-ACC CGC TGA ACT TAA GC-3') and LR5 (5'-TCC TGA GGG AAA CTT CG-3') (Vilgalys and Hester 1990), mtSSU1 (5'-GCW GCA GTG RGG AAT NTT GGR CAA T-3') and mtSSU2R (5'-GTR GAC TAM TSR GGT ATC TAA TC-3') (Zoller et al. 1999) and EF983 (5'-GCY CCY GGH CAY CGT GAY TTY AT-3') and EF1aZ-1R (5'-ACA TCW CCG ACA CCC TTG ATC TTG -3') (Nie et al. 2012) were used for the amplification of the partial region of nucLSU, mtSSU and TEF1, respectively. The PCR reactions followed those in Liu et al. (2005) and Nie et al. (2012, 2020). A 50 μl mixture contained 200 μM dNTPs each, 1 × Mg-free buffer, 2.5 mM MgCl2, 0.5 μM primers each, 50 ng genomic DNA and 2 U Taq polymerase (Super Pfx DNA Polymerase, Cowinbioscience Co. Ltd., Shanghai, China). The programme consisted of an initial denaturation at 100 °C for 5 min without Taq polymerase, an extra denaturation at 95 °C for 5 min after the Taq polymerase was added, then 34 cycles of 94 °C for 1 min plus 55/54/57 °C (nucLSU / mtSSU / TEF1) for 2 min plus 72 °C for 2 min and a final extension at 72 °C for 10 min. The amplification products were sequenced by Shanghai GeneCore BioTechnologies Co. Ltd. (Shanghai, China), with the same primers as used in relative PCR reactions. All sequences were assembled with BioEdit (Hall 1999) and deposited at GenBank (Table 1).

Table 1.
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The taxa used in phylogenetic analyses.

Species Strains* GenBank accession numbers References
nucLSU EF-1α mtSSU
Capillidium adiaeretum CGMCC 3.15888 MN061284 MN061481 MN061287 Nie et al. 2020
Ca. lobatum ATCC 18153 (T) JF816218 JF816233 MK301187 Nie et al. 2012, 2020
Conidiobolus bifurcatus sp. nov. CGMCC 3.15889 (T) MN061285 MN061482 MN061288 This article
C. brefeldianus ARSEF 452 (T) EF392382 – EF392495 Genbank
C. chlamydosporus ATCC 12242 (T) JF816212 JF816234 MK301178 Nie et al. 2012, 2020
C. coronatus NRRL 28638 AY546691 DQ275337 – Lutzoni et al. 2004
C. coronatus RCEF 4518 JN131537 JN131543 – Nie et al. 2016, 2018
C. dabieshanensis CGMCC 3.15763 (T) KY398125 KY402206 MK301180 Nie et al. 2017, 2020
C. firmipilleus ARSEF 6384 JX242592 – JX242632 Gryganskyi et al. 2012
C. gonimodes ATCC 14445 (T) JF816221 JF816226 MK301182 Nie et al. 2012, 2020
C. humicolus ATCC 28849 (T) JF816220 JF816231 MK301184 Nie et al. 2012, 2020
C. incongruus NRRL 28636 AF113457 – – Voigt et al. 1999
C. iuxtagenitus ARSEF 6378 (T) KC788410 – – Gryganskyi et al. 2013
C. khandalensis ATCC 15162 (T) KX686994 KY402204 MK301185 Nie et al. 2012, 2020
C. lamprauges ARSEF 2338 DQ364206 – DQ364226 Genbank
C. lichenicolus ATCC 16200 (T) JF816216 JF816232 MK301186 Nie et al. 2012, 2020
C. macrosporus ATCC 16578 (T) KY398124 KY402209 MK301188 Nie et al. 2017, 2020
C. megalotocus ATCC 28854 (T) MF616383 MF616385 MK301189 Nie et al. 2018, 2020
C. mycophagus ATCC 16201 (T) JX946694 JX946698 MK301190 Nie et al. 2018, 2020
C. mycophilus ATCC 16199 (T) KX686995 KY402205 MK301191 Nie et al. 2016, 2020
C. parvus ATCC 14634 (T) KX752051 KY402207 MK301192 Nie et al. 2016, 2020
C. polyspermus ATCC 14444 (T) MF616382 MF616384 MK301193 Nie et al. 2018, 2020
C. polytocus ATCC 12244 (T) JF816213 JF816227 MK301194 Nie et al. 2012, 2020
C. taihushanensis sp. nov. CGMCC 3.16016 (T) MT250086 MT274290 MT250088 This article
C. variabilis sp. nov. CGMCC 3.16015 (T) MT250085 MT274289 MT250087 This article
Microconidiobolus nodosus ATCC 16577 (T) JF816217 JF816235 MK333388 Nie et al. 2012, 2020
M. terrestris ATCC 16198 (T) KX752050 KY402208 MK301199 Nie et al. 2016, 2020
Neoconidiobolus stromoideus ATCC 15430 (T) JF816219 JF816229 MK301198 Nie et al. 2012, 2020
N. thromboides ATCC 12587 (T) JF816214 JF816230 MK301200 Nie et al. 2012, 2020

*ARSEF, ARS Entomopathogenic Fungus Collection (Ithaca, U.S.A.). ATCC, American Type Culture Collection (Manassas, U.S.A). CGMCC, China General Microbiological Culture Collection Center (Beijing, China). NRRL, ARS Culture Collection (Peoria, U.S.A). RCEF, Research Center for Entomogenous Fungi (Hefei, China). T = ex-type.

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. (2018, 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 100th 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 algorithm. 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).

Results

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).

Figure 1. 

Phylogenetic tree of Conidiobolus s.s. reconstructed by maximum likelihood analyses of nucLSU, mtSSU and TEF1 sequences, with six Conidiobolus s.l. species as outgroups. Three new species of Conidiobolus are shown in bold. Maximum parsimony bootstrap values (≥ 70%) / Maximum likelihood bootstrap values (≥ 70%) / Bayesian posterior probabilities (≥ 0.95) of each clade are indicated along branches. Scale bar indicates substitutions per site.

Three clades can be seen to form for the Conidiobolus s.s. The three species, described here, were located in clade I.

Taxonomy

Conidiobolus bifurcatus B. Huang & Y. Nie, sp. nov.

MycoBank No: 831599
Facesoffungi: FoF 08142
Figure 2

Typification

China, Jiangsu: Nanjing, Laoshan National Forest Park, 32°6'7"N, 118°36'17"E, from plant debris, 1 Dec 2018, Y. Nie and Y. Gao (holotype HMAS 248359, ex-holotype culture CGMCC 3.15889 = RCEF 6551, GenBank: nucLSU = MN061285; TEF1 = MN061482; mtSSU = MN061288).

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 forcibly 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).

Figure 2. 

Conidiobolus bifurcatus sp. nov. a Colony on PDA after 3 d at 21 °C b mycelium c septate mycelium and distended segments d, e primary conidiophores bearing primary conidia f, g primary conidia h, i a single secondary conidium produced from primary conidia j two secondary conidia arising from a branched conidiophore k secondary conidia falling from primary conidia l the relic of secondary conidiophores on secondary conidia (arrows) m microconidia arising from a conidium n, o globose microconidia p, q ellipsoidal microconidia r zygospores formed between adjacent segments of the same hypha s zygospores. Scale bars: 10 mm (a); 100 μm (b); 20 μm (c–s).

Conidiobolus taihushanensis B. Huang & Y. Nie, sp. nov.

MycoBank No: 835124
Facesoffungi: FoF 08143
Figure 3

Typification

China, Anhui: Ma’anshan City, Hanshan County, Taihushan National Forest Park, 31°30'53"N, 118°2'49"E, from plant detritus, 12 Jan 2019, Y. Nie and Y. Cai (holotype HMAS 248724, ex-holotype culture CGMCC 3.16016 = RCEF 6559, GenBank: nucLSU = MT250086; TEF1 = MT274290; mtSSU = MT250088).

Etymology

taihushanensis (Lat.), referring to the region where the fungus was isolated.

Ecology and distribution

Plant debris in Anhui Province, China.

Description

Colonies on PDA at 21 °C after 3 d, white, reaching ca. 11–14 mm in diameter. Mycelia colourless, straight and unbranched when young, 8.5–12 μm wide; distended and non-contiguously segmented when old, 10–20 μm wide. Primary conidiophores arising from the older mycelia without an upward widening near the tip, colourless, 44–180 × 7–13 μm, usually unbranched and often producing a single globose primary conidium, at the initial growth stage 2–5 short branches bearing a primary conidium each. Primary conidia forcibly discharged, mostly subglobose, 27–42 × 19–32 μm, with tapering and pointed papilla, 4–10 × 8–12 μm. Secondary conidia arising from primary conidia, similar to, but smaller than the primary ones, forcibly discharged. On 2% water agar, microconidia not observed. Zygospores usually formed between adjacent segments of the same hypha after 5 d, 34–48 × 23–40 μm, with a 2–4 μm thick wall, ellipsoid and rich in content when young, smooth, mostly globose, subglobose to ovate when mature.

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. nov. is also closely related to C. lamprauges Drechsler and C. coronatus Batko, but it differs from C. lamprauges by larger primary conidia (27–42 × 19–32 μm vs. 12.5–20 × 15–22 μm) and from C. coronatus by the absence of villose resting spores (Drechsler 1953a).

Figure 3. 

Conidiobolus taihushanensis sp. nov. a colony on PDA after 3 d at 21 °C b mycelia unbranched at the colony edge c young mycelia d, e primary conidiophores arising from mycelia segments f two branches germinated from hyphal bodies and each bearing a primary conidium (arrows) g– j two, three, four or five branches germinated from hyphal bodies and each bearing a primary conidium k globose to subglobose primary conidia l secondary conidia arising from primary conidia m zygospores formed between adjacent segments of the same hypha n young zygospores o mature zygospores. Scale bars: 10 mm(a); 100 μm (b, c, f); 20 μm (d, e, g–o).

Conidiobolus variabilis B. Huang & Y. Nie, sp. nov.

MycoBank No: 835125
Facesoffungi: FoF 08144
Figure 4

Typification

China, Anhui: Ma’anshan City, Hexian County, Jilongshan National Forest Park, 31°48'1"N, 118°12'19"E, from plant debris, 23 Dec 2017, Y. Nie (holotype HMAS 248723, ex-holotype culture CGMCC 3.16015 (= RCEF 6540), GenBank: nucLSU = MT250085; TEF1 = MT274289; mtSSU = MT250087).

Etymology

variabilis (Lat.), referring to producing various shapes of primary conidia.

Ecology and distribution

Plant debris from Anhui Province, China.

Description

Colonies on PDA at 21 °C after 3 d white, reaching ca. 41–48 mm in diameter. Mycelia colourless, 6–11 μm wide, rarely branched at the colony edge. Primary conidiophores unbranched and producing a single globose conidium, colourless, 60–200 × 9–15 μm, without an upward widening near the tip. Primary conidia forcibly discharged, globose, subglobose, pyriform to oboviod, 31–55 × 25–40 μm, with tapering and pointed papilla, 3.5–9 × 8–13 μm. Secondary conidia arising from primary conidia, similar to, but smaller than primary ones, forcibly discharged. On 2% water agar, microconidia rarely observed, globose, subglobose to ellipsoidal, 10–12 × 9–14 μm. Resting spores not observed.

Notes

Considering the large size of primary conidia, Conidiobolus variabilis sp. nov. is allied to C. coronatus (Cost.) Batko (14.5–38.5 × 17–48.5 μm), C. macrosporus Srin. & Thirum. (38–45 × 48–54 μm) and C. utriculosus Brefeld (25–35 × 37.5–51 μm). 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 and Thirumalachar 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.

Figure 4. 

Conidiobolus variabilis sp. nov. a Colony on PDA after 3 d at 21 °C b mycelia rarely branched at the colony edge c mycelia d, e primary conidiophores bearing primary conidia f–i primary conidia with different shapes j secondary conidia arising from primary conidia k microconidia arising from conidia l globose microconidia m ellipsoidal microconidia. Scale bars: 10 mm (a); 100 μm (b); 20 μm (c–m).

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. morphologically produce microspores. However, C. taihushanensis sp. nov. lacks this synapomorph. Besides C. taihushanensis sp. nov., four other species in the Conidiobolus s.s., i.e. C. dabieshanensis Y. Nie & B. Huang, C. iuxtagenitus S.D. Waters & Callaghan, 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

1 Villose resting spores produced Conidiobolus coronatus
– Villose resting spores not produced 2
2 Microspores produced 3
– Microspores not observed 4
3 Two types of sexual reproduction, zygospores formed in axial alignment with one or both conjugating segments 5
– One type of sexual reproduction, zygospores formed in one of the conjugating segments 6
5 Primary conidia larger, up to 51 μm C. utriculosus
– Primary conidia smaller, less than 36 μm C. brefeldianus
6 2–4 branches germinated at the top of primary conidiophores 7
– Unbranched at the top of conidiophores 8
7 Only 2 primary conidia arising from 2 branches, larger, up to 44 μm C. megalotocus
– 2–4 primary conidia arising from 2–4 branches, smaller, less than 29 μm C. polytocus
8 Secondary conidiophores branched 9
– Secondary conidiophores unbranched 10
9 Secondary conidiophores branched almost at the tip, primary conidia larger, up to 40 μm C. bifurcatus sp. nov.
– Secondary conidiophores branched at the tip or base, primary conidia smaller, less than 30 μm C. mycophilus
10 Primary conidia larger, up to 55 μm 11
– Primary conidia smaller, maximum not over 42 μm 12
11 Primary conidia globose to pyriform, zygospores globose, 26–40 μm C. macrosporus
– Primary conidia globose, subglobose, pyriform to oboviod, zygospores not observed C. variabilis sp. nov.
12 Primary conidia smaller, less than 21 μm C. khandalensis
– Primary conidia larger, more than 33 μm 13
13 Two types of resting spores produced: zygospores or chlamydospores C. humicolus
– One type of resting spores produced 14
14 Only chlamydospores produced C. firmipilleus
– Only zygospores produced 15
15 Primary conidiophores shorter, less than 80 μm C. gonimodes
– Primary conidiophores longer, more than 130 μm 16
16 Zygospores globose or elongate, larger, 15–40 × 18–45 μm C. incongruus
– Zygospores globose, smaller, 30–36 μm C. mycophagus
4 Fusiform secondary conidia produced, each zygospore in a position separated by a short, but relatively constant distance from a lateral conjugation outgrowth or beak C. iuxtagenitus
– Fusiform secondary conidia not produced, each zygospore in a position not separated by a short, but relatively constant distance from a lateral conjugation outgrowth or beak 17
17 A chain of up to seven undischarged repetitional conidia produced C. margaritatus
– No chains of undischarged repetitional conidia produced 18
18 Primary conidiophores produced from cushion mycelia C. lichenicolus
– Primary conidiophores not produced from cushion mycelia 19
19 Apical mycelia straight, 2–5 conidia arising from hyphal body, no chlamydospores, zygospores produced C. taihushanensis sp. nov.
– Apical mycelia slightly curved, unbranched at the top of conidiophore, chlamydospores produced, no zygospores C. dabieshanensis

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Nos. 30770008, 31900008 and 31670019).

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