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Research Article
Three novel insect-associated species of Simplicillium (Cordycipitaceae, Hypocreales) from Southwest China
expand article infoWan-Hao Chen, Chang Liu, Yan-Feng Han§, Jian-Dong Liang, Wei-Yi Tian, Zong-Qi Liang§
‡ Guizhou University of Traditional Chinese Medicine, Guiyang, China
§ Guizhou University, Guiyang, China
Open Access

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.

Keywords

Commensal fungi, morphology, nutritional preference, phylogeny

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) reported a new species, S. chinense F. Liu & L. Cai, which was the first Simplicillium species from China. Five new Simplicillium species, S. aogashimaense Nonaka, Kaifuchi & Masuma, S. cylindrosporum Nonaka, Kaifuchi & Masuma, S. minatense Nonaka, Kaifuchi & Masuma, S. subtropicum Nonaka, Kaifuchi & Masuma and S. sympodiophorum Nonaka, Kaifuchi & Masuma were reported by Nonaka et al. (2013) from Tokyo, Japan. Simplicillium calcicola Z. F. Zhang, F. Liu & L. Cai, S. coffeanum A. A. M. Gomes & O. L. Pereira and S. filiforme R. M. F. Silva, R. J. V. Oliveira, Souza-Motta, J. L. Bezerra & G. A. Silva were reported by 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; 2017; Dai et al. 2018), diketopiperazine (Yan et al. 2015), 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 applications 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.

Materials and methods

Collection and isolation

Three infected insect specimens (DL1004, GY1101 and GY2913) were collected from Dali, Rongjiang Country (26°01'58.70"N, 108°24'48.06"E) and Tongmuling (26°23'25.92"N, 106°41'3.35"E), Huaxi District, Guizhou Province, on 1 October, 9 November and 31 July, 2018, respectively. The surface of the specimens were rinsed with sterile water, followed by surface sterilization with 75% ethanol for 3–5 s. A part of the insect body was cut off and used to inoculate a piece of tissue in haemocoel on potato dextrose agar (PDA) and improved potato dextrose agar (PDA, 1% w/v peptone) (Qu et al. 2018). The strain was isolated and cultured at 22 °C for 14 d under 12 h light/12 h dark conditions following protocols described by Zou et al. (2010). Strains DL10041, DL10042, GY11011, GY11012, GY29131 and GY29132 were obtained.

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.

Sequence alignment and phylogenetic analyses

DNA sequences generated in this study were assembled and edited using DNASTAR Lasergene software (version 6.0). Sequences of ITS, LSU, RPB1, RPB2 and TEF were selected based on previously published data by Nonaka et al. (2013), Zhang et al. (2017), Gomes et al. (2018), Crous et al. (2018) and Mongkolsamrit et al. (2018). Multiple sequence alignments for ITS, LSU, RPB1, RPB2 and TEF were carried out using MAFFT v7.037b (Katoh and Standley 2013). Sequence editing was performed with MEGA6 (Tamura et al. 2013), and the resulting output was in Fasta file format. The concatenated LSU+RPB1+RPB2+TEF and ITS+LSU sequences were assembled by SequenceMatrix v.1.7.8 (Vaidya et al. 2011). Gene concordance was assessed with the ‘hompart’ command in PAUP4.0b10 (Swofford 2002).

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)

Results

Phylogenetic analyses

A phylogenetic tree of Simplicillium in Cordycipitaceae was generated from the maximum-likelihood (ML) and Bayesian inference (BI) based on a combined data set of LSU, RPB1, RPB2 and TEF sequence data. Statistical support (≥ 50%/0.5) is shown at the nodes for ML bootstrap support/BI posterior probabilities (Fig. 1). The strain numbers are noted after each species’ name. The tree is rooted with Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson (CBS 284.36 and CBS 431.87). The concatenated sequences including 40 taxa and contained 2,205 characters with gaps (LSU: 447, RPB1: 518, RPB2: 560, and TEF: 680).

Figure 1. 

Phylogenetic relationships among the genus Simplicillium and its allies in Cordycipitaceae based on multigene dataset (LSU, RPB1, RPB2 and TEF). Statistical support values (≥ 0.5/50%) are shown at the nodes for ML bootstrap support/BI posterior probabilities. The tree is rooted with Purpureocillium lilacinum (CBS 284.36 and CBS 431.87). The new species are in bold face. T in the upper right corner indicates the type strains.

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%).

Figure 2. 

Phylogenetic relationships among the new taxa S. cicadellidae, S. formicidae, S. lepidopterorum and other Simplicillium species by ITS+LSU sequences. Statistical support values (≥ 0.5/50%) are shown at the nodes for ML bootstrap support/BI posterior probabilities. The tree is rooted with Pochonia chlamydosporia (CBS 103.65). The new species are in bold face. T in the upper right corner indicates the type strains.

Table 1.

Taxa included in the phylogenetic analyses

Species Strain No. GenBank Accession No.
ITS LSU RPB1 RPB2 TEF
Akanthomyces aculeatus HUA 772 KC519370 KC519366
A. attenuates CBS 402.78 AF339565 EF468888 EF468935 EF468782
A. coccidioperitheciatus NHJ 6709 EU369042 EU369067 EU369086 EU369025
A. farinosa CBS 541.81 JQ425686
A. kanyawimiae TBRC 7242 MF140718 MF140784 MF140808 MF140838
TBRC 7243 MF140717 MF140783 MF140807 MF140837
TBRC 7244 MF140716 MF140836
A. lecanii CBS 101247 AF339555 DQ522407 DQ522466 DQ522359
A. sulphureus TBRC 7247 MF140720 MF140841
TBRC 7248 MF140722 MF140787 MF140812 MF140843
TBRC 7249 MF140721 MF140786 MF140734 MF140842
A. thailandicus TBRC 7245 MF140809 MF140839
TBRC 7246 MF140719 MF140810 MF140840
A. tuberculatus BCC 16819 GQ249987 GQ250037
OSC 111002 DQ518767 DQ522384 DQ522435 DQ522338
A. waltergamsii TBRC 7250 MF140715 MF140835
TBRC 7251 MF140713 MF140781 MF140805 MF140833
TBRC 7252 MF140714 MF140782 MF140806 MF140834
Ascopolyporus polychrous P.C. 546 DQ118737 DQ127236 DQ118745
A. villosus ARSEF 6355 AY886544 DQ127241 DQ118750
Blackwellomyces cardinalis OSC 93609 AY184962 DQ522370 DQ522422 DQ522325
OSC 93610 AY184963 EF469088 EF469106 EF469059
B. pseudomilitaris NBRC 101409 JN941393 JN992482
NBRC 101410 JN941394 JN992481
Cordyceps bifusispora EFCC 5690 EF468806 EF468854 EF468909 EF468746
EFCC 8260 EF468807 EF468855 EF468910 EF468747
C. blackwelliae TBRC 7253 MF140705 MF140774 MF140798 MF140825
TBRC 7254 MF140704 MF140773 MF140797 MF140824
TBRC 7255 MF140703 MF140772 MF140796 MF140823
TBRC 7256 MF140702 MF140771 MF140795 MF140822
TBRC 7257 MF140701 MF140770 MF140794 MF140821
C. ninchukispora EFCC 5197 EF468820 EF468868 EF468760
EFCC 5693 EF468821 EF468869 EF468762
EGS 38.165 EF468846 EF468900 EF468795
EGS 38.166 EF468847 EF468901 EF468794
NHJ 10627 EF468822 EF468870 EF468763
NHJ 10684 EF468823 EF468871 EF468761
Engyodontium aranearum CBS 309.85 AF339526 DQ522387 DQ522439 DQ522341
Gibellula longispora NHJ 12014 EU369055 EU369075 EU369017
G. pulchra NHJ 10808 EU369035 EU369056 EU369076 EU369018
G. ratticaudata ARSEF 1915 DQ518777 DQ522408 DQ522467 DQ522360
Gibellula sp. NHJ 5401 EU369059 EU369079
NHJ 10788 EU369036 EU369058 EU369078 EU369019
NHJ 13158 EU369037 EU369057 EU369077 EU369020
Hevansia arachnophila NHJ 10469 EU369031 EU369047 EU369008
H. cinerea NHJ 3510 EU369048 EU369070 EU369009
H. novoguineensis NHJ 4314 EU369051 EU369071 EU369012
NHJ 11923 EU369032 EU369052 EU369072 EU369013
NHJ 13117 EU369049 EU369073 EU369010
NHJ 13161 EU369050 EU369011
Hyperdermium pulvinatum P.C. 602 AF242353 DQ127237 DQ118746
L. aranearum CBS 726.73a AF339537 EF468887 EF468934 EF468781
L. fusisporum CBS 164.70T AF339549 EF468889 EF468783
L. psalliotae CBS 363.86T AF339559 EF468890 EF468784
CBS 532.81 AF339560 EF469096 EF469112 EF469067
CBS 101270 EF469081 EF469095 EF469113 EF469066
Pochonia chlamydosporia CBS 103.65 MH858504
Purpureocillium lilacinum CBS 284.36 FR775484 EF468898 EF468941 EF468792
CBS 431.87 EF468844 EF468897 EF468940 EF468791
Samsoniella alboaurantium CBS 240.32 JF415979 JN049895 JF415999 JF416019
CBS 262.58 MG665232 JQ425685
S. aurantia TBRC 7271T MF140728 MF140791 MF140818 MF140846
TBRC 7272 MF140727 MF140817 MF140845
TBRC 7273 MF140726 MF140816 MF140844
S. inthanonensis TBRC 7915T MF140725 MF140790 MF140815 MF140849
TBRC 7916 MF140724 MF140789 MF140814 MF140848
TBRC 7270 MF140723 MF140788 MF140813 MF140847
Simplicillium aogashimaense JCM 18167T AB604002
JCM 18168 AB604004
S. calcicola LC 5371 KU746705 KU74675
LC 5586T KU746706 KU746752
S. chinense LC 1342 JQ410323 JQ410321
LC 1345 NR155782 JQ410322
S. cicadellidae GY11011T MN006243 MN006249 MN022271 MN022263
GY11012 MN006244 MN006250 MN022272 MN022264
S. coffeanum COAD 2057T MF066034 MF066032
COAD 2061 MF066035 MF066033
S. cylindrosporum JCM 18169T AB603989
JCM 18170 AB603994
JCM 18171 AB603997
JCM 18172 AB603998
JCM 18173 AB603999
JCM 18174 AB604005
JCM 18175 AB604006
S. filiforme URM 7918 MH979338 MH979399
S. formicidae DL10041T MN006241 MN006247 MN022269 MN022267
DL10042 MN006242 MN006248 MN022270 MN022268
S. lamellicola CBS 116.25T AJ292393 AF339552 DQ522404 DQ522462 DQ522356
UAMH 2055 AF108471
UAMH 4785 AF108480
S. lamellicolab KYK00006 AB378533
S. lanosoniveum CBS 704.86 AJ292396 AF339553 DQ522406 DQ522464 DQ522358
CBS 101267 AJ292395 AF339554 DQ522405 DQ522463 DQ522357
S. lepidopterorum GY29131T MN006246 MN006251 MN022273 MN022265
GY29132 MN006245 MN006252 MN022274 MN022266
S. minatense JCM 18176T AB603992
JCM 18177 AB603991
JCM 18178 AB603993
S. obclavatum CBS 311.74T AJ292394 AF339517 EF468798
JCM 18179 AB604000
S. subtropicum JCM 18180T AB603990
JCM 18181 AB603995
JCM 18182 AB603996
JCM 18183 AB604001
S. sympodiophorum JCM 18184T AB604003
Torrubiella wallacei CBS 101237T AY184967 EF469102 EF469119 EF469073

Taxonomy

Simplicillium cicadellidae W.H. Chen, C. Liu, Y.F. Han, J.D. Liang, Z.Q. Liang, sp. nov.

MycoBank No: 831336
Figure 3

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.

Type

CHINA, Guizhou Province, Huaxi District (26°23'25.92"N, 106°41'3.35"E), 9 November 2018, Wanhao Chen, holotype GZAC GY1101, ex-type culture GZAC GY11011. Sequences from isolated strain GY11011 has been deposited in GenBank with accession numbers: ITS = MN006243, LSU = MN006249, RPB1 = MN022271 and TEF = MN022263.

Figure 3. 

Simplicillium cicadellidae A infected leafhopper (Hemiptera) B–C culture plate, showing the front (B) and the reverse (C) of the colony, cultured on PDA medium D–F phialides solitary, conidia adhering ellipsoidal slimy head and conidia G conidia. Scale bars: 10 mm (B, C), 10μm (D, E, F, G).

Description

Colonies reaching 45–47 mm in diameter in 14 d on PDA; white; reverse yellowish, especially in the middle, and radially sulcate. Hyphae septate, hyaline, smooth-walled, 0.9–1.9 μm wide. Phialides arising from aerial hyphae, gradually tapering towards apex, without basal septa, always solitary and rather long and narrow, 12.9–18.3 × 0.8–1.1 μm. Conidia adhering in ellipsoidal slimy heads, mostly ellipsoidal, hyaline, smooth-walled, 1.8–2.8 × 1.4–1.8 μm. Octahedral crystals absent.

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.

Table 2.

Morphological comparison of three new species with other Simplicillium species

Species Morphological characteristics Notes
Phialide (Conidiogenous cell) (μm) Conidia (μm) Conidia mass Octahedral crystals
S. aogashimaensea (19–)23–53 × 1.2–2.0 cylindrical, 4.2–6.5 × 1.2–2.0 globose heads present Chlamydospores present
S. calcicolab 14–38 × 1–2 micro-: globose, oval or ellipsoidal, 2–3.5 × 1–1.5 absent
macro-: fusiform, 4.5–8 × 1–2
S. chinensec (6.0–)15–30(–68.0) × 1.5 oval, ellipsoidal or cylindrical 3.5–5.0 × 1.0–1.5 branched or unbranched chains present
S. coffeanumd 11–40(–70) × 1.0–2.4 micro-: spindle-shaped, 5.3–8.8 × 1.0–1.6 subglobose to ellipsoidal heads absent
macro-: ellipsoidal to fusiform, 2.2–3.8 × 0.8–1.5
S. cylindrosporuma 17–32 × 1.2–2.0(–2.5) cylindrical, 3.0–4.5(–5.0) × 1.0–2.0 globose heads present
S. filiformee 9–18 × 1 fusoid to filiform, 7.2–12.5 × 1 zigzag chains absent
S. lamellicolaf 15–50 × 0.7–1.0 micro-: spindle-shaped, 4.5–9.0 × 0.8–1.2 subglobose to ellipsoidal heads present
macro-: oval to ellipsoidal, 2.0–3.0 × 0.7–1.2
S. lanosoniveumf 15–35 × 0.7–1.5 subglobose, oval, ellipsoidal 1.5–3 × 0.7–1.3 globose heads present
S. minatensea 11–31(–47) × 1.0–1.7 globose to subglobose, sometimes ellipsoidal, 2.0–3.5 × 1.8–2.5(–2.8) globose heads present
S. obclavatumf 30–52 × 0.8–1.2 obclavate to ellipsoidal, 2.5–3.5 × 1–2 short imbricate chains present
S. subtropicuma (15–)20–42(–50) × 1.0–2.3 subglobose to ellipsoidal, 2.3–4.0(–4.5) × 1.5–3.3 globose heads present
S. sympodiophoruma 20–34(–47) × 0.5–1.3
denticles present
oval to ellipsoidal, 2.2–3.5 × 1.0–2.0 present
S. cicadellidae 12.9–18.3 × 0.8–1.1 ellipsoidal, 1.8–2.8 × 1.4–1.8 ellipsoidal heads absent colonies reverse pale white
S. formicidae 51–70.1 × 0.7–0.9 filiform to fusoid, 3.9–7.9 × 0.8–1.3 globose heads absent
S. lepidopterorum 15.3–26.2 × 0.7–1.4 ellipsoidal, 1.6–2.4 × 1.4–1.7 globose heads absent colonies reverse yellowish

Simplicillium formicidae W.H. Chen, C. Liu, Y.F. Han, J.D. Liang, Z.Q. Liang, sp. nov.

MycoBank No: 831337
Figure 4

Etymology

The epithet formicidae refers to an insect host in family Formicidae.

Diagnosis

Characterized by phialides always being solitary and rather long and narrow, 51–70.1 × 0.7–0.9 μm. Conidia adhering in globose slimy heads, mostly filiform to fusoid, 3.9–7.9 × 0.8–1.3 μm. Octahedral crystals absent.

Type

CHINA, Guizhou Province, Rongjiang County (26°01'58.70"N, 108°24'48.06"E), 1 October 2018, Wanhao Chen, holotype GZAC DL1004, ex-type culture GZAC DL10041. Sequences from isolated strain DL10041 has been deposited in GenBank with accession numbers: ITS = MN006241, LSU = MN006247, RPB1 = MN022269 and RPB2 = MN022267.

Figure 4. 

Simplicillium lepidopterorum A infected carpenterworm (Lepidoptera) B, C culture plate, showing the front (B) and the reverse (C) of the colony, cultured on PDA medium D, E, F phialides solitary and conidia in globose heads D conidia. Scale bars: 10 mm (B, C), 10μm (D, E, F, G).

Description

Colonies reaching 26–32 mm in diameter in 14 d on PDA; white; reverse pale brown to brown, and with brown secretions. Hyphae septate, hyaline, smooth-walled, 1.2–1.8 μm wide. Phialides arising from aerial hyphae, gradually tapering towards the apex, without basal septa, always solitary and rather long and narrow, 51–70.1 × 0.7–0.9 μm. Conidia adhering in globose slimy heads, mostly filiform to fusoid, hyaline, smooth-walled, 3.9–7.9 × 0.8–1.3 μm. Octahedral crystals absent.

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).

Simplicillium lepidopterorum W.H. Chen, C. Liu, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov.

MycoBank No: 831335
Figure 5

Etymology

The epithet lepidopterorum refers to an insect host in order Lepidoptera.

Diagnosis

Characterized by phialides always being solitary and rather long and narrow, 15.3–26.2 × 0.7–1.4 μm, Conidia adhering in globose slimy heads, mostly ellipsoidal, 1.6–2.4 × 1.4–1.7 μm. Octahedral crystals absent. The reverse of colony was pale white.

Type

CHINA, Guizhou Province, Huaxi District (26°23'25.92"N, 106°41'3.35"E), 31 July 2018, Wanhao Chen, holotype GZAC GY2913, ex-type culture GZAC GY29131, sequences from isolated strain GY29131 has been deposited in GenBank with accession numbers: ITS = MN006246, LSU = MN006251, RPB1 = MN022273 and TEF = MN022265.

Figure 5. 

Simplicillium formicidae A isolated substrate an infected ant (Hymenoptera) B–C culture plate, showing the front (B) and the reverse (C) of the colony, cultured on PDA medium D, E phialides solitary, conidia adhering globose slimy head and conidia F conidia. Scale bars: 10 mm (B, C), 10μm (D, E, F).

Description

Colonies reaching 48–51 mm in diameter in 14 d on PDA; white; reverse pale white. Hyphae septate, hyaline, smooth-walled, 1.1–2.2 μm wide. Phialides arising from aerial hyphae, gradually tapering towards the apex, without basal septa, always solitary and rather long and narrow, 15.3–26.2 × 0.7–1.4 μm. Conidia adhering in globose slimy heads, ellipsoidal to fusiform, hyaline, smooth-walled, 1.6–2.4 × 1.4–1.7 μm. Octahedral crystals absent.

Host

Carpenter worm (Lepidoptera)

Distribution

Huaxi District, Guizhou Province, China

Remarks

Simplicillium lepidopterorum was easily identified as belonging to Simplicillium because of its solitary phialides, conidia adhering in globose slimy heads, and lack of octahedral crystals. Comparing with the typical characteristics of 12 species (Table 2), S. lepidopterorum could easily distinguished from other species by having the phialides always solitary and rather long and narrow, 15.3–26.2 × 0.7–1.4 μm. Conidia ellipsoidal (1.6–2.4 × 1.4–1.7 μm), adhering in globose slimy heads, and in having the octahedral crystals absent. Based on ITS and LSU rDNA, S. lepidopterorum is phylogenetically close to S. cicadellidae and S. formicidae. However, S. lepidopterorum has ellipsoidal conidia, longer phialides (15.3–26.2 × 0.7–1.4 μm), and the reverse of colony was pale white.

Key

1 Conidia in globose or subglobose heads 2
Conidia in chains or solitary 11
2 Macro- and microconidia present 3
Only one type of conidia present 4
3 Octahedral crystals present S. lamellicola
Octahedral crystals absent S. coffeanum
4 Octahedral crystals present 5
Octahedral crystals absent 9
5 Conidia cylindrical 6
Conidia subglobose or ellipsoidal 7
6 Chlamydospores present, conidia 4.2–6.5 × 1.2–2.0 μm S. aogashimaense
Chlamydospores absent, conidia 3.0–4.5 (–5.0) × 1.0–2.0 μm S. cylindrosporum
7 Conidia subglobose to ellipsoidal 8
Conidia oval or ellipsoidal to subcylindrical, 1.5–3.0 × 0.7–1.3 μm S. lanosoniveum
8 Conidia subglobose to ellipsoidal, 2.3–4.0 (–4.5) × 1.5–3.3 μm S. subtropicum
Conidia globose to subglobose, sometimes ellipsoidal, 2.5–3.5 × 1.8–2.5 (–2.8) μm S. minatense
9 Conidia ellipsoidal 10
Conidia filiform to fusoid S. formicidae
10 The reverse of colony pale white, phialide 12.9–18.3 × 0.8–1.1 μm S. cicadellidae
The reverse of colony yellowish, phialide 15.3–26.2 × 0.7–1.4 μm S. lepidopterorum
11 Denticles present in conidiogenous cell (phialide) S. sympodiophorum
Denticles absent in conidiogenous cell (phialide) 12
12 Macro- and microconidia present S. calcicola
Only one type of conidia present 13
13 Conidia ellipsoidal 14
Conidia fusoid to filiform, form zigzag chains S. filiforme
14 Conidia in branched or unbranched chains, 3.5–5.0 × 1.0–1.5 μm S. chinense
Conidia in short imbricate chains, 2.5–3.5 × 1.0–2.0 μm S. obclavatum

Discussion

Two types of the evolutionary correlation patterns between fungi and hosts are known, co-evolutionary patterns and the more frequent host jump events (Spatafora et al. 2007). 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 (Spatafora et al. 2007). Prediction of the characteristics and evolutionary placement of any given member should be based on the correlation between molecular-phylogenetic genealogy and nutritional preferences (Spatafora et al. 2007; 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.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31460010, 31860002), the Doctoral Fund of Guiyang University of Chinese Medicine (3043-043170023), the National first-class construction discipline in Guizhou province (Chinese medical science) (GNYL[2017]008), and Engineering Research Center of General Higher Education in Guizhou Province (Qianjiaohe(2015)337). We thank Dr. Lesley Benyon, from Liwen Bianji and Edanz Group China (http://www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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