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Three novel insect-associated species of Simplicillium (Cordycipitaceae, Hypocreales) from Southwest China

Abstract In this paper, we introduce three new species of Simplicillium, viz. S. cicadellidae, S. formicidae and S. lepidopterorum, which were isolated from an infected leafhopper, ant and carpenterworm, respectively. Morphological comparisons and phylogenetic analyses based on multigene datasets (LSU+RPB1+RPB2+TEF and ITS+LSU) support the establishment of the three new species. Simplicillium cicadellidae was distinguished from other species in morphological characteristics by having smaller phialides and ellipsoidal conidia, and lacking octahedral crystals. The reverse of colonies were yellowish (#FFBF00), especially in the middle, and radially sulcate. Simplicillium formicidae was morphologically distinguished from other by having longer phialides and filiform to fusoid conidia, and by lacking octahedral crystals. Simplicillium lepidopterorum was morphologically distinguished from other species by having smaller, ellipsoidal to fusiform conidia, and by lacking octahedral crystals. The reverse of the colony was pale white. The three new species are likely to be nourished by plant to animal (especially insect) nutrients based on the evolutionary pattern of the Hypocreales, and they are described herein as being clearly distinct from other species in Simplicillium.


Introduction
The genus Simplicillium W. Gams & Zare was introduced by Zare and Gams (2001) with S. lanosoniveum (J. F. H. Beyma) Zare & W. Gams as the type species. The genus is characterized with its complete lack of verticillate branching; mostly solitary phialides, which are discrete, aculeate and narrow and arise from aerial hyphae; conidia short-ellipsoidal to suglobose or obclavate, and adhering in globose heads or imbricate chains (Zare and Gams 2001). The members of Simplicillium are fungicolous and occur on various substrata (Zare and Gams 2001;Chen et al. 2008;Baiswar et al. 2014;Gauthier et al. 2014;Gomes et al. 2018). Furthermore, Zare and Gams (2001) introduced three additional species, viz., S. lamellicola (F. E. V. Sm.) Zare & W. Gams, S. obclavatum (W. Gams) Zare & W. Gams and S. wallacei H. C. Evans. The typical characteristics of Simplicillium include mostly solitary phialides, conidia adhering in globose, slimy heads or imbricate chains, and commonly present crystals in the agar (Zare and Gams 2001). Later, Zare and Gams (2008) transferred S. wallacei to Lecanicillium W. Gams & Zare based on the phylogenic analysis of internal transcribed spacer (ITS) region and this transfer was confirmed by Sung et al. (2007). Liu and Cai (2012) Zhang et al. (2017), Gomes et al. (2018) and Crous et al. (2018), respectively. Currently, Simplicillium consists of 12 species. Kepler et al. (2017) re-evaluated the Cordycipitaceae based on the multigene dataset (SSU, LSU, TEF, RPB1 and RPB2), and indicated that Simplicillium species group in a clade and are the earliest diverging lineage in Cordycipitaceae. The nuclear ribosomal ITS and LSU were first used to identify cryptic diversification among Simplicillium species by Liu and Cai (2012) and then were widely applied in the identification of Simplicillium species by Nonaka et al. (2013), Zhang et al. (2017), Gomes et al. (2018) and Crous et al. (2018). Zare and Gams (2001) noted that Simplicillium species were found on various substrata and fungi. Other substrata were found later, such as limstone and wood (Liu and Cai 2012;Zhang et al. 2017). Many bioactive compounds were discovered in Simplicillium, such as alkaloids (Fukuda et al. 2014), peptides (Liang et al. 2016;Dai et al. 2018), diketopiperazine , xylanases (Roy et al. 2013), anthraquinones (Huang et al. 2015), antibiotics (Takata et al. 2013;Dong et al. 2018), and especially Simpotentin, which is a new potentiator of amphotericin B activity against Candida albicans (C. P. Robin) Berkhout and has showed great potential ap-plications in medicine (Uchida et al. 2019). Furthermore, the antimicrobial activities and entomopathogenicity has meant that Simplicillium has potential applications in biocontrol (Ward et al. 2012;Zhao et al. 2013;Le Dang et al. 2014;Lim et al. 2014;Chen et al. 2017;Skaptsov et al. 2017). However, as far as we know, there are limited reports of Simplicillium species isolated from infected insects.
Three infected insect specimens were found during a survey of araneogenous fungi and allies from southwestern China. Some fungal strains were isolated and purified from the three specimens. Based on polyphasic approach (morphological, ecological characteristics along with a phylogenetic analysis), they were identified as three new species, Simplicillium cicadellidae sp. nov., S. formicidae sp. nov. and S. lepidopterorum sp. nov.

Culture and identification
The strains were incubated in PDA at 25 °C for 14 d. Macroscopic and microscopic morphological characteristics of the fungi were examined using classical mycological techniques, and the growth rates were determined. The fresh hyphae were observed with an optical microscope (OM, BX35, Olympus, Japan) following pretreatment with lactophenol cotton blue solution or normal saline. The ex-type cultures and dried culture as holotype specimens were deposited in GZAC, Guizhou University, Guiyang, China.
DNA extraction, PCR amplification and nucleotide sequencing DNA extraction was carried out in accordance with Liang et al. (2009). The extracted DNA was stored at −20 °C. The amplification of large subunit ribosomal RNA (LSU) genes was performed using NS1-1/AB28 primers (Curran et al. 1994). Translation elongation factor 1 alpha (TEF) and DNA-directed RNA polymerase II largest subunit 2 (RPB2) were amplified using 983F/2218R and RPB2-5F/RPB2-7Cr primers according to van den Brink et al. (2012). DNA-directed RNA polymerase II largest subunit 1 (RPB1) was amplified with the primer pair CRPB1 and RPB1-Cr (Castlebury et al. 2004). The internal transcribed spacer (ITS) region was amplified using ITS4/ITS5 primers by PCR following the procedures described by White et al. (1990). PCR products were purified using the UNIQ-10 column PCR products purification kit [no. SK1141; Sangon Biotech (Shanghai) Co., Shanghai, China] in accordance with the manufacturer's protocol and sequenced at Sangon Biotech (Shanghai) Co. The resulting sequences were submitted to GenBank.
The new species Simplicillium cicadellidae, S. formicidae and S. lepidopterorum were registered in MycoBank with the numbers MB 831336, MB 831337 and MB 831335, respectively.
Two different analyses have been carried out using Bayesian inference (BI) and maximum likelihood (ML) methods. Analysis 1: To check the relationship between Simplicillium species and its allies in Cordycipitaceae based on the combined dataset of (LSU+RPB1+RPB2+TEF). Analysis 2: To check the relationship among Simplicillium spp. based on the combined dataset of (ITS+LSU). For the BI analysis, two runs were executed simultaneously for 10,000,000 generations, saving trees every 500 generations, with the GTR+G nucleotide substitution model across all the partitions, in MrBayes 3.2 (Ronquist et al. 2012). After the analysis was finished, each run was examined with the program Tracer v1.5 (Drummond and Rambaut 2007) to determine burn-in and confirm that both runs had converged. For the ML analysis in RAxML (Stamatakis 2014), the GTRGAMMA model was used for all the partitions in accordance with recommendations in the RAxML manual against the use of invariant sites. The final alignment is available from TreeBASE under submission ID: 24549 (http:// www.treebase.org)
A phylogenetic tree of Simplicillium species level was generated from the maximum-likelihood (ML) and Bayesian inference (BI) analysis based on a combined data set of ITS and LSU sequence data set. Statistical support (≥ 50%/0.5) are shown at the nodes for ML bootstrap support/BI posterior probabilities. The strain numbers are noted after each species' name. The tree is rooted with Pochonia chlamydosporia (Goddard) Zare & W. Gams (CBS 103.65). The dataset includes 16 taxa and consists of 1,000 characters with gaps (ITS: 489 and LSU: 511).
Analysis 1: family Cordycipitaceae. The RAxML analysis of the combined dataset (LSU+RPB1+RPB2+TEF) yielded a best scoring tree ( Fig. 1) with a final ML optimization likelihood value of -24,337.973328. Parameters for the GTR model of the concatenated data set was as follows: estimated base frequencies; A = 0.242689, C = 0.276532, G = 0.270879, T = 0.209901; substitution rates AC = 0.926706, AG = 2.728719, AT = 0.823168, CG = 0.803225, CT = 6.257555, GT = 1.000000; gamma distribution shape parameter α = 0.410435. The Bayesian analysis resulted in 20,001 trees after 10,000,000 generations. The first 4,000 trees, representing the burn-in phase of the analyses, were discarded, while the remaining 16,001 trees were used for calculating posterior probabilities in the majority rule consensus tree. In the phylogenetic tree ( Fig. 1), Simplicillium cicadellidae, S. formicidae and S. lepidopterorum cluster with other Simplicillium species in a clade, and within the earliest diverging lineage in Cordycipitaceae.
Analysis 2: Simplicillium species. The RAxML analysis of the combined dataset (ITS+LSU) yielded a best scoring tree ( Fig. 2) with a final ML optimization likelihood value of -4,849.039588. Parameters for the GTR model of the concatenated data set was as follows: Estimated base frequencies; A = 0.243952, C = 0.258870, G = 0.268223, T = 0.228956; substitution rates AC = 1.296760, AG = 2.678402, AT = 1.354112, CG = 1.488619, CT = 5.097242, GT = 1.000000; gamma distribution shape parameter α = 0.462419. The Bayesian analysis resulted in 20,001 trees after 10,000,000 generations. The first 4,000 trees, representing the burn-in phase of the analyses, were discarded, while the remaining 16,001 trees were used for calculating posterior probabilities in the majority rule consensus tree. In the phylogenetic tree  ( Fig. 2), Simplicillium species were resolved into four obvious clades. S. cicadellidae, S. formicidae and S. lepidopterorum were nested in a subclade and formed three independent branches, which received maximum statistical support (BI posterior probabilities 1, ML bootsrap 100%).  Etymology. The epithet cicadellidae refers to an insect host in family Cicadellidea. Diagnosis. Characterized by phialides always solitary and rather long and narrow, 12.9-18.3 × 0.8-1.1 μm. Conidia adhering in globose slimy heads, mostly ellipsoidal, 1.8-2.8 × 1.4-1.8 μm. Octahedral crystals absent. Reverse of colony yellowish, especially in the middle, and radially sulcate.
Host. Leafhopper (Hemiptera) Distribution. Huaxi District, Guizhou Province, China Remarks. Zare and Gams (2001) summarized the typical characteristics of Simplicillium as having mostly solitary phialides arising from aerial hyphae, conidia adhering in globose slimy heads or imbricate chains, crystals commonly present, fungicolous and on various other substrata. Simplicillium cicadellidae was easily identified as belonging to Simplicillium because of its solitary phialides, conidia adhering in ellipsoidal slimy heads, and lack of octahedral crystals. Comparing with the typical characteristics of 12 species (Table 2), it was easily distinguished from other species in having the phialides always solitary and rather long and narrow (12.9-18.3 × 0.8-1.1 μm), the conidia adhering in globose slimy heads, which are mostly ellipsoidal (1.8-2.8 × 1.4-1.8 μm), and the octahedral crystals absent. The reverse of colony was yellowish, especially in the middle, and radially sulcate. Based on ITS and LSU rDNA, S. cicadellidae is phylogenetically close to S. formicidae and S. lepidopterorum. However, S. cicadellidae has ellipsoidal conidia and shorter phialides (12.9-18.3 × 0.8-1.1 μm), and the reverse of colony was yellowish. Etymology. The epithet formicidae refers to an insect host in family Formicidae.
Host. Ant (Hymenoptera) Distribution. Rongjiang County, Guizhou Province, China Remarks. Simplicillium formicidae was easily identified as belonging to Simplicillium because of its solitary phialides, conidia adhering in globose slimy heads, and lack of octahedral crystals. Compared with the typical characteristics of 12 species (Table  2), it was easily distinguished from those species by having the phialides always solitary and rather long and narrow (51-70.1 × 0.7-0.9 μm) and the conidia mostly filiform to fusoid (3.9-7.9 × 0.8-1.3 μm), and adhering in globose slimy heads, and in having octahedral crystals absent. Based on ITS and LSU rDNA, S. formicidae is phylogenetically close to S. cicadellidae and S. lepidopterorum. However, S. formicidae has larger filiform to fusoid conidia (3.9-7.9 × 0.8-1.3 μm). Etymology. The epithet lepidopterorum refers to an insect host in order Lepidoptera.

Discussion
Two types of the evolutionary correlation patterns between fungi and hosts are known, co-evolutionary patterns and the more frequent host jump events ). The generation of host jumping is closely related to a common living environment (Vega et al. 2009). Nutritional sources are very important factors in determining whether a host has undergone a host jump. The nutritional model of Hypocreales fungi is from plants (including living plants and plant residues) to animals (especially insects), and finally to fungi. Plants and their residues were the initial sources of nutrition for the common ancestor of Hypocreaceae and Clavicipitaceae. The jumps from plants to animals and then to fungi indicate that the fungal nutrient requirements have changed with the environment ). Prediction of the characteristics and evolutionary placement of any given member should be based on the correlation between molecular-phylogenetic genealogy and nutritional preferences Vega et al. 2009). Additionally, host insect species are an important diagnostic feature in the identification of entomopathogenic fungi. Among the 12 reported Simplicillium species, S. aogashimaense (soil), S. calcicola (calcareous rock), S. chinense (decaying wood), S. cylindrosporum (soil), S. minatense (soil), S. obclavatum (air), S. subtropicum (soil) and S. sympodiophorum (soil) were isolated from soil, marine water, rock, decaying wood and air (Zare and Gams 2001;Liu and Cai 2012;Nonaka et al. 2013;Liang et al. 2017). Simplicillium filiforme and S. coffeanum were isolated as endophytic fungi from plants (Crous et al. 2018;Gomes et al. 2018). Simplicillium lamellicola belongs to the hyperparasite fungi (Shin et al. 2017). Simplicillium lanosoniveum was reported as both an endophytic and hyperparasite fungi (Baiswar et al. 2014). It has been reported that Simplicillium is pathogenic to insects. Unfortunately, there are limited reports of insect-related Simplicillium.
The hosts of Simplicillium cicadellidae and S. lepidopterorum were larvae of Cicadidae and Lepidoptera, which feed through piercing-sucking and chewing. Moreover, S. formicidae was isolated from an infected ant. These three strains are likely to receive nutrients from plants (including living plants and plant residues) and animals (especially insects) based on the evolutionary pattern of Hypocreales. Simplicillium cicadellidae, S. formicidae and S. lepidopterorum represent three new species based on their nutritional preferences. To our knowledge, this is the first report of insect-associated Simplicillium species.
ITS and LSU have been widely used in the identification of Simplicillium (Liu and Cai 2012;Nonaka et al. 2013;Zhang et al. 2017;Sliva et al. 2018). In the present study, the combined dataset (ITS+LSU) was used to analysis of phylogenetic relationships among the new taxa and other Simplicillium species. Additionally, RPB1, RPB2 and TEF loci were added to analysis that the relationship among Simplicillium and its allies. The new species clustered with other Simplicillium species in a clade (Fig. 1), and this was consistent with morphological characteristics based identification. Six strains were clustered into three subclades (Fig. 2) and were distinctly different from other reported Simplicillium spp. Additionally, three species, S. chinense, S. coffeanum and S. filiforme were clustered in a subclade, and these species were associated with plants. This may be because of their nutritional preferences. Therefore, S. cicadellidae, S. formicidae and S. lepidopterorum are based on morphological characteristics, ecological characteristics and a phylogenetic analysis.