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Research Article
Morphology and phylogeny of two new species within Cordycipitaceae (Hypocreales) from China
expand article infoYingling Lu‡, Songyu Li‡, Zuoheng Liu‡, Jing Zhao‡, Zhiyong Yu§, Zongli Liang§, Hailong He§, Jianhong Li§, Yun Huang§, Xinming Li§, Hong Yu‡
‡ Yunnan University, Kunming, China
§ Yunnan Jinping Fenshuiling National Nature Reserve, Honghe, China

Corresponding author: Hong Yu ( hongyu@ynu.edu.cn )

Academic editor: Marc Stadler
© 2025 Yingling Lu, Songyu Li, Zuoheng Liu, Jing Zhao, Zhiyong Yu, Zongli Liang, Hailong He, Jianhong Li, Yun Huang, Xinming Li, Hong Yu.
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: Lu Y, Li S, Liu Z, Zhao J, Yu Z, Liang Z, He H, Li J, Huang Y, Li X, Yu H (2025) Morphology and phylogeny of two new species within Cordycipitaceae (Hypocreales) from China. MycoKeys 115: 187-208. https://doi.org/10.3897/mycokeys.115.140683
Open Access

Abstract

Simplicillium and Leptobacillium, sister genera in the family Cordycipitaceae, exhibit a broad range of hosts or substrates. The identification of two novel species, from Simplicillium and Leptobacillium, was achieved by analysing morphological characteristics and phylogenetic data obtained from six molecular markers (ITS, nrSSU, nrLSU, tef-1α, rpb1 and rpb2). The two recently documented species are S. puwenense and L. longiphialidum. Morphologically, S. puwenense possessed slender solitary rod-shaped or columnar phialides with elliptical oval or cylindrical conidia forming small spherical heads at the apex of phialides. On the other hand, L. longiphialidum had solitary columnar phialides with elliptic or subspherical apical conidia while other conidia were narrow columnar or fusiform in shape. Phylogenetic analysis revealed that S. puwenense formed an independent branch as a sister species to S. formicae, whereas L. longiphialidum clustered with L. marksiae exhibiting stable topological structure. The Bayesian inference posterior probability and the maximum likelihood bootstrap-ratio provided robust statistical evidence, indicating the presence of two novel species within the genera of Simplicillium and Leptobacillium. The present study contributes to the discovery of species diversity in Simplicillium and Leptobacillium, while also providing a taxonomic foundation for their rational development and sustainable utilisation.

Key words:

Leptobacillium, morphology, new taxa, phylogenetic analysis, Simplicillium, taxonomy

Introduction

As was well known, many species in the family Cordycipitaceae Kreisel ex G.H. Sung, Hywel-Jones & Spatafora were entomogenous (Mongkolsamrit et al. 2018; Wang et al. 2020). Amongst them, the genera Simplicillium W. Gams & Zare and Leptobacillium Zare & W. Gams were sister genera (Zare and Gams 2001, 2016).

In 2001, Zare and Gams established the genus Simplicillium, which included S. lanosoniveum (J.F.H. Beyma) Zare & W. Gams (type species), S. lamellicola (F.E.V. Sm.) Zare & W. Gams, S. obclavatum (W. Gams) Zare & W. Gams and S. wallacei H.C. Evans. The key distinguishing characteristic of the Simplicillium genus was the solitary presence of phialides, with conidia typically adhering to the apex of phialides in chains that resemble spherical, sticky or tile-like structures, ultimately forming octahedral crystals (Zare and Gams 2001). The solitary phialides enabled the distinction between the genus Simplicillium and its closely-related genus Lecanicillium W. Gams & Zare (Chen et al. 2021). Species belonging to the Simplicillium genus exhibited ecological diversity, including their presence in various environments, such as soil, endophytic fungi of plants, rocks and decaying wood (Liu and Cai 2012; Nonaka et al. 2013; Zhang et al. 2017; Crous et al. 2018, 2021). S. chinense F. Liu & L. Cai was the first Simplicillium species discovered in China (Liu and Cai 2012).

In 2016, the genus Leptobacillium was established by Zare and Gams during the revision of the former Verticillium Nees section Albo-erecta. The name of the genus referred to its characteristic narrow microconidia, with the model species being L. leptobactrum (W. Gams) Zare & W. Gams (Zare and Gams 2016). The genus Leptobacillium comprised species that exhibited two distinct types of conidia. Individual cells aggregated to form chains, with nearly spherical or elliptical conidia located at the apex of long chains, while narrow cylindrical (rod-shaped) to fusiform conidia were found elsewhere within the chain (Zare and Gams 2016; Leplat et al. 2022). Zare and Gams (2016) initially described L. leptobactrum, a species consisting of two varieties, namely L. leptobactrum var. leptobactrum (W. Gams) Zare & W. Gams and L. leptobactrum var. calidius Zare & W. Gams, which were distinguished by their optimal growth temperatures. The species of Leptobacillium exhibited a wide range of host and substrate diversity, having been isolated from various sources including Lepidoptera insects, fungi, plants, fresh water, murals and rocks (Liu and Cai 2012; Zare and Gams 2016; Gomes et al. 2018; Crous et al. 2018; Sun et al. 2019; Okane et al. 2020). The nematophagous properties of Leptobacillium species have been extensively studied (Regaieg et al. 2011; Leplat et al. 2022).

Phylogenetic studies of species in the genera Simplicillium and Leptobacillium have focused on the nuclear ribosomal internal transcribed spacer region (ITS) and the nuclear ribosomal large subunit (nrLSU). Currently, several other DNA loci are frequently used to study species in the Cordycipitaceae family (Kepler et al. 2017; Wang et al. 2020; Leplat et al. 2022). Based on a phylogenetic analysis, S. wallacei was transplanted into the genus Lecanicillium and later Zhang et al. placed L. wallacei in the genus Gamszarea Z.F. Zhang & L. Cai (Zare and Gams 2001, 2008; Zhang et al. 2021). Phylogenetic analysis, based on five locus data, showed that S. coffeanum A.A.M. Gomes & O.L. Pereira, S. chinensis F. Liu & L. Cai and S. filiforme R.M.F. Silva, R.J.V. Oliveira, Souza-Motta, J.L. Bezerra & G.A. Silva were transferred to the genus Leptobacillium (Zare and Gams 2008; Okane et al. 2020; Chen et al. 2021).

Based on a comparative analysis of morphological characteristics and a multi-gene molecular phylogeny, we characterised in this study two newly-identified species from China, namely S. puwenense Hong Yu bis, Y.L. Lu & J. Zhao, sp. nov., from the genus of Simplicillium and L. longiphialidum Hong Yu bis, Y.L. Lu & J. Zhao, sp. nov., from the genus of Leptobacillium, respectively. This investigation has contributed to the expansion of the species diversity within the genera of Simplicillium and Leptobacillium, providing a solid taxonomic foundation to facilitate the rational development and sustainable use of these valuable resources.

Material and method

Material collection and isolation

The specimens of a dead spider infected with fungi were collected in China. One specimen was collected from Puwen Town, Jinghong City, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China and the Xilong Mountains in Jinping County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, China. Another was collected from Limushan National Forest Park, Limushan Town, Qiongzhong City, Hainan Province, China and 511 Township Road, Boluo County, Huizhou City, Guangdong Province, China. The specimens were photographed, assigned numbers and their collection details including habitat, elevation, latitude and longitude were documented. Subsequently, they were placed in freezing tubes within a vehicle-mounted refrigerator set at 4 °C for transportation back to the laboratory. Upon arrival at the laboratory, the specimens underwent initial observation and measurement using an Optec SZ660 stereo dissecting microscope. A select number of fungal conidia were then carefully picked with an inoculation needle and inoculated into PDA solid medium containing 0.05 g tetracycline and 0.1 g streptomycin using the plate streak method (Wang et al. 2020). The pure culture was incubated at a temperature of 25 °C, while the purified strain was transferred to a bevelled test tube containing PDA medium and stored at 4 °C (Wang et al. 2020). The specimens were deposited in the Yunnan Herbal Herbarium (YHH), while the strains were conserved in the Yunnan Fungal Culture Collection (YFCC).

Morphological observations

The pure cultures were transferred to PDA solid medium and incubated at 25 °C for 14 days. Colony diameters were measured, colony characteristics were recorded and photographs of the front and back of the colonies were captured using a Canon camera (Tokyo, Japan). To observe the microscopic morphology of the colonies, filter paper was cut to fit a petri dish and placed inside. A U-shaped glass shelf, a slide and two coverlids that had been sterilised at 121 °C for 30 minutes and then dried were prepared. A layer of PDA medium with a thickness of 1 mm and size of approximately 5 mm was applied onto the slide. A small amount of mycelia was selected from each culture and transferred to the centre of the medium. It was covered with a coverslip, sterile water was added to moisten the medium and sealed in an incubator at 25 °C for cultivation. The microstructure was observed, measured and photographed using fluorescence microscopes CX40 (Tokyo, Japan) and BX53 (Tokyo, Japan).

DNA extraction, polymerase chain reaction (PCR) and sequencing

The total genomic DNA of fungi was extracted using the CTAB method described by Liu et al. (2001). The ITS region was amplified using primer pairs ITS4 and ITS5 (White et al. 1990). The nuclear ribosomal small subunit (nrSSU) and nrLSU were amplified using primer pairs nrSSU-CoF with nrSSU-CoR and LR5 with LR0R, respectively (Vilgalys and Hester 1990; Rehner and Samuels 1994; Wang et al. 2015b). The translation elongation factor 1α (tef-1α) was amplified using primer pairs EF1α-EF and EF1α-ER (Bischoff et al. 2006; Sung et al. 2007). Finally, the largest subunit of RNA polymerase II (rpb1) and the second largest subunit of RNA polymerase II (rpb2) were amplified using primer pairs RPB1-5’F with RPB1-5’R and RPB2-5’F with RPB2-5’R, respectively, as described by Bischoff et al. (2006) and Sung et al. (2007).

The final volume of all PCR reactions was 25 µl, consisting of 17.25 µl of sterile deionised water, 2.5 µl of PCR10 Buffer (2 mmol/l Mg2+) from Transgen Biotech in Beijing, China, 2 µl of dNTP (2.5 mmol/l), 1 µl each of forward and reverse primers, 0.25 µl of Taq DNA polymerase from Transgen Biotech in Beijing, China and 1 µl of DNA template. The polymerase chain reaction (PCR) for the five genes and ITS was conducted using a BIO-RAD T100TM thermal cycler manufactured by BIO-RAD Laboratories in Hercules, CA, United States (Bischoff et al. 2006; Wang et al. 2015a). The PCR products were analysed through electrophoresis on a 1.0% agarose gel and subsequently stored at -20 °C until they were dispatched in dry ice to BGI Co., Ltd, Shenzhen, China for sequencing.

Phylogenetic analyses

After aligning the six-gene sequences of related species obtained from GenBank with those of the present study using the Clustal W programme in MEGA v.5.0 software, we concatenated the six-gene datasets (ITS, nrSSU, nrLSU, tef-1α, rpb1 and rpb2) into a combined matrix comprising all six genes. To both single gene and six-gene datasets, we respectively employed the ModelFinder programme in PhyloSuite v.1.2.2 software to determine the optimal model for the maximum likelihood analysis, based on Corrected AIC (AICc) and IQ-TREE model selection methods. The remaining parameters were set to their default values. Subsequently, we utilised the IQ-TREE programme with 5,000 bootstrap replicates to construct a maximum likelihood tree while selecting appropriate optimal model parameters.

The ModelFinder programme in PhyloSuite v.1.2.2 software was utilised to determine the optimal model for the Bayesian inference using Corrected AIC (AICc) and the MrBayes model, while keeping default settings for other parameters. Subsequently, the MrBayes programme was employed to select appropriate optimal model parameters and run for 2,000,000 generations to construct the BI tree. The constructed phylogenetic trees were visualised in FigTree v.1.4.2 to figure the maximum likelihood method of bootstrap proportion (BP) and the Bayesian inference posterior probability (BPP) and then formatted for editing with Adobe Illustrator CS6.

Results

Phylogenetic analyses

Phylogenetic analysis of single gene molecular fragments

Using single gene fragments of ITS, nrSSU, nrLSU, tef-1α and rpb1 were used to construct Simplicillium and Leptobacillium phylogenetic trees, respectively. Beauveria bassiana ARSEF 1564 and B. brongniartii ARSEF 617 were employed as outgroups (Table 1). Among them, the ITS matrix had 64 sequences, 711 bp of bases, including 783 columns, 381 distinct patterns, 218 parsimony-informative, 64 singleton sites, 500 constant sites. The Best-fit model of the ML tree constructed by the ITS matrix was TIM2+F+I+G4 and the BI tree was GTR+F+I+G4 (Fig. 1). The nrSSU matrix consisted of 21 sequences, 1,122 bp of bases, 2,333 columns, 163 distinct patterns, 30 parsimony-informative, 48 singleton sites and 2,255 constant sites. The Best-fit model for building the nrSSU ML tree was TIM3e+I and the BI tree was SYM+I (Fig. 2). The nrLSU had 52 sequences with 1,126 columns, 325 distinct patterns, 91 parsimony-informative, 340 singleton sites, 695 constant sites and 1,019 bp bases. Based on ML and BI, the Best-fit models used to construct the nrLSU phylogenetic framework were K2P+R5, GTR+F+G4, respectively (Fig. 3). The tef-1α matrix consists of 52 sequences, 1,090 columns, 431 distinct patterns, 289 parsimony-informative, 82 singleton sites, 719 constant sites and 1,154 bp bases. The Best-fit model of the ML tree constructed by the tef-1α matrix was TIM3+F+R8 and the BI tree was GTR+F+I+G4 (Fig. 4). The rpb1 matrix consisted of 20 sequences, 803 bp bases, 2,971 columns, 390 distinct patterns, 294 parsimony-informative, 113 singleton sites and 2,564 constant sites. Based on ML and BI, the Best-fit models used to construct the rpb1 phylogenetic framework were TIM2e+I+G4, SYM+I+G4, respectively (Fig. 5). The tree shapes constructed, based on ML and BI, were basically the same and the topological structure adopted in this study was a phylogenetic tree constructed by the maximum likelihood method (Figs 1–5).

Figure 1. 

The phylogenetic tree of Simplicillium and Leptobacillium was inferred from ITS sequence, based on the Bayesian inference and the maximum likelihood analyses. Each value at a node indicates a bootstrap proportion (the left) and Bayesian posterior probability (the right). The scale bar 0.03 indicates the number of expected mutations per site. The species in bold black font of the Simplicillium and Leptobacillium were from this study. B. bassiana ARSEF 1564 and B. brongniartii ARSEF 617 were designated as outgroups.

Figure 2. 

The phylogenetic tree of Simplicillium and Leptobacillium was inferred from nrSSU sequence, based on the Bayesian inference and the maximum likelihood analyses. Each value at a node indicates a bootstrap proportion (the left) and Bayesian posterior probability (the right). The scale bar 0.005 indicates the number of expected mutations per site. The species in bold black font of the Simplicillium and Leptobacillium were from this study. B. brongniartii ARSEF 617 was designated as outgroup.

Figure 3. 

The phylogenetic tree of Simplicillium and Leptobacillium was inferred from nrLSU sequence, based on the Bayesian inference and the maximum likelihood analyses. Each value at a node indicates a bootstrap proportion (the left) and Bayesian posterior probability (the right). The scale bar 0.3 indicates the number of expected mutations per site. The species in bold black font of the Simplicillium and Leptobacillium were from this study. B. bassiana ARSEF 1564 was designated as outgroup.

Figure 4. 

The phylogenetic tree of Simplicillium and Leptobacillium was inferred from tef-1α sequence, based on the Bayesian inference and the maximum likelihood analyses. Each value at a node indicates a bootstrap proportion (the left) and Bayesian posterior probability (the right). The scale bar 0.05 indicates the number of expected mutations per site. The species in bold black font of the Simplicillium and Leptobacillium were from this study. B. bassiana ARSEF 1564 and B. brongniartii ARSEF 617 were designated as outgroups.

Figure 5. 

The phylogenetic tree of Simplicillium and Leptobacillium was inferred from rpb1 sequence, based on the Bayesian inference and the maximum likelihood analyses. Each value at a node indicates a bootstrap proportion (the left) and Bayesian posterior probability (the right). The scale bar 0.07 indicates the number of expected mutations per site. The species in bold black font of the Simplicillium and Leptobacillium were from this study. B. bassiana ARSEF 1564 and B. brongniartii ARSEF 617 were designated as outgroups.

Table 1.
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Relevant species information and GeneBank accession numbers for phylogenetic research in this study.

Species Strain ITS nrSSU nrLSU tef-1α rpb1 rpb2 Reference
Beauveria bassiana ARSEF 1564 HQ880761 - AF373871 HQ880974 HQ880833 HQ880905 Rehner et al. (2011)
Beauveria brongniartii ARSEF 617 HQ880782 AB027335 - HQ880991 HQ880854 HQ880926 Rehner et al. (2011)
Leptobacillium cavernicola LRMH C212 OM622523 OM628842 OM628781 OM654332 OM677781 OM654321 Leplat et al. (2022)
Leptobacillium cavernicola LRMH C216 OM622524 OM628843 OM628782 OM654333 OM677782 OM654322 Leplat et al. (2022)
Leptobacillium chinense CGMCC 3.14969 JQ410323 - JQ410321 - - - Okane et al. (2020)
Leptobacillium chinense CGMCC 3.14970 JQ410324 - JQ410322 - - - Okane et al. (2020)
Leptobacillium coffeanum COAD 2057 MF066034 - MF066032 - - - Okane et al. (2020)
Leptobacillium coffeanum COAD 2061 MF066035 - MF066033 - - - Okane et al. (2020)
Leptobacillium filiforme URM 7918 - - MH979399 - - - Okane et al. (2020)
Leptobacillium latisporum TBRC 16288 OP856540 OP850838 OP856529 - - - Preedanon et al. (2023)
Leptobacillium leptobactrum ZJ14B02 PP385689 - PP381743 - - - Unpublished
Leptobacillium leptobactrum AH17C05 PP384754 - PP380808 - - - Unpublished
Leptobacillium leptobactrum var. calidius CBS 703.86 EF641866 EF641850 KU382226 - - - Zare and Gams (2016)
Leptobacillium leptobactrum var. leptobactrum CBS 771.69 EF641868 EF641852 KU382224 - - - Zare and Gams (2016)
Leptobacillium longiphialidum YFCC 23039272T PQ509282 PQ508806 PQ508808 PQ560997 PQ567240 - This study
Leptobacillium longiphialidum YFCC 24079491 PQ509281 PQ508805 PQ508807 PQ560996 PQ567239 - This study
L. marksiae BRIP 70307a PQ061114 - PQ047739 - - - Tan and Bishop-Hurley (direct submission)
Leptobacillium muralicola CGMCC 3.19014 MH379983 - MH379997 - - - Sun et al. (2019)
Leptobacillium muralicola CGMCC 3.19015 MH379985 - MH379999 - - - Sun et al. (2019)
Leptobacillium symbioticum NBRC 113865 LC485673 - LC506046 - - - Okane et al. (2020)
Leptobacillium symbioticum OPTF00168 LC485675 - LC506047 - - - Okane et al. (2020)
Simplicillium album LC12442 - - - MK336068 - - Zhang et al. (2021)
Simplicillium aogashimaense JCM 18167 AB604002 - LC496874 LC496904 - - Nonaka et al. (2013)
Simplicillium aogashimaense JCM 18168 AB604004 - LC496875 - - - Nonaka et al. (2013)
Simplicillium araneae DY101811 OM743774 - OM743792 OM818465 - - Chen et al. (2022)
Simplicillium araneae DY101812 OM743840 - OM743846 OM818466 - - Chen et al. (2022)
Simplicillium calcicola LC5586 KU746706 - KU746752 KX855252 - - Zhang et al. (2017)
Simplicillium calcicola LC5371 KU746705 - KU746751 KX855251 - - Zhang et al. (2017)
Simplicillium cicadellidae GY11012 MN006244 - - MN022264 MN022272 - Chen et al. (2019)
Simplicillium cicadellidae GY11011 MN006243 - - MN022263 MN022271 - Chen et al. (2019)
Simplicillium coccinellidae DY101791 MT453861 MT453863 - MT471341 - - Chen et al. (2021)
Simplicillium coleopterorum SD05381 OM743920 - OM743925 OM818467 - - Chen et al. (2022)
Simplicillium coleopterorum SD05382 OM744109 - OM744170 OM818468 - - Chen et al. (2022)
Simplicillium cylindrosporum JCM 18169 AB603989 - LC496876 LC496906 - - Nonaka et al. (2013)
Simplicillium cylindrosporum JCM 18170 AB603994 - LC496877 LC496907 - - Nonaka et al. (2013)
Simplicillium formicae DY09641 OR121054 - OR121057 OR126571 - - Unpublished
Simplicillium formicae DY09642 OR121055 - OR121056 OR126572 - - Unpublished
Simplicillium guizhouense DY10051 OM743225 - OM743226 OM818453 - - Chen et al. (2022)
Simplicillium guizhouense DY10052 OM743241 - OM743252 OM818454 - - Chen et al. (2022)
Simplicillium humicola LC 12494 - - - MK336072 - - Zhang et al. (2021)
Simplicillium humicola CGMCC 3.19573 NR_172845 - MK329041 MK336071 - - Unpublished
Simplicillium hymenopterorum DY101692 MT453851 - - MT471338 - - Unpublished
Simplicillium hymenopterorum DY101691 MT453848 MT453849 - MT471337 MT471344 - Unpublished
Simplicillium lamellicola JC-1 MT807906 MT807908 MT807907 - - - Unpublished
Simplicillium lamellicola CBS 116.25 AJ292393 - - DQ522356 DQ522404 DQ522462 Nonaka et al. (2013)
Simplicillium lanosoniveum CBS 704.86 AJ292396 - - DQ522358 DQ522406 DQ522464 Nonaka et al. (2013)
Simplicillium larvatum DY101731 OM743438 - OM743441 OM818462 OM818460 - Chen et al. (2022)
Simplicillium lepidopterorum GY29132 MN006245 - - MN022266 MN022274 - Chen et al. (2019)
Simplicillium lepidopterorum GY29131 MN006246 - - MN022265 MN022273 - Chen et al. (2019)
Simplicillium minatense JCM 18176 AB603992 LC496893 LC496878 LC496908 - - Nonaka et al. (2013)
Simplicillium minatense JCM 18178 AB603993 LC496894 LC496879 LC496909 - - Nonaka et al. (2013)
Simplicillium neolepidopterorum DY101752 MT453857 - - MT471340 - - Chen et al. (2021)
Simplicillium neolepidopterorum DY101751 MT453854 MT453856 - MT471339 - - Chen et al. (2021)
Simplicillium obclavatum CBS 311.74 AJ292394 - AF339517 EF468798 - - Nonaka et al. (2013)
Simplicillium obclavatum SUF81 - - MK788174 - - - Unpublished
Simplicillium pechmerlense CBS 147188 MW031272 - MW031268 MW033224 MW033222 - Leplat et al. (2021)
Simplicillium puwenense YFCC 23129490T PQ508796 PQ508799 PQ508802 PQ537122 PQ560994 - This study
Simplicillium puwenense YFCC 23089322 PQ508797 PQ508800 PQ508803 PQ537123 - - This study
Simplicillium puwenense YFCC 23069492 PQ508798 PQ508801 PQ508804 PQ537124 PQ560995 - This study
Simplicillium scarabaeoidea DY101392 MT453845 - - MT471336 - - Chen et al. (2021)
Simplicillium scarabaeoidea DY101391 MT453842 MT453843 - MT471335 MT471343 - Chen et al. (2021)
Simplicillium sinense AFMCCC 16a OQ332403 - - OQ352167 - - Yan et al. (2023)
Simplicillium sinense AFMCCC 16b OQ332404 - - OQ352168 - - Yan et al. (2023)
Simplicillium spumae JCM 39054 LC496871 - LC496887 LC496917 - - Kondo et al. (2020)
Simplicillium spumae JCM 39050 LC496869 LC496898 LC496883 LC496913 - - Kondo et al. (2020)
Simplicillium subtropicum JCM 18180 AB603990 - LC496880 LC496910 - - Nonaka et al. (2013)
Simplicillium subtropicum JCM 18181 AB603995 - LC496881 LC496911 - - Nonaka et al. (2013)
Simplicillium sympodiophorum JCM 18184 AB604003 - LC496882 LC496912 - - Nonaka et al. (2013)
Simplicillium yunnanense YFCC 7134 - MN576729 MN576785 MN576955 MN576845 - Wang et al. (2020)
Simplicillium yunnanense YFCC 7133 - MN576728 MN576784 MN576954 MN576844 - Wang et al. (2020)

Based on the phylogenetic framework constructed by single gene fragments, it was found that the resulting topologies were roughly similar and there was no obvious conflict between different gene fragments. The species S. puwenense and L. longiphialidum collected and described in this study were located in roughly the same position in each phylogenetic tree, forming monophyletic, with high support rate and stable topological structure. In the topology constructed, based on ITS, nrLSU and tef-1α matrices, S. puwenense and S. formicae D.P. Wei & K.D. Hyde were closely related. In phylogenetic trees constructed by ITS and nrLSU matrices, L. longiphialidum and L. marksiae Tan, Bishop-Hurley & Marney came together.

Phylogenetic tree reconstructed from multi-gene combined dataset

The phylogenetic framework for the genera Simplicillium and Leptobacillium, comprising 70 taxonomic units, was constructed, based on a six-gene dataset utilising the maximum likelihood method and Bayesian inference. B. bassiana ARSEF 1564 and B. brongniartii ARSEF 617 were employed as outgroups (Table 1). The joint matrix comprised 14,494 columns, 1,873 distinct patterns, 1,190 parsimony-informative, 770 singleton sites and 12,534 constant sites. The most appropriate model for the ML analysis amongst the 286 models simulated by ModelFinder was TIM2+F+R10, which achieved an IQ-TREE best score of -32893.153 and a Total tree length of 2.122. The parameters of the TIM2+F+R10 model used to analyse the dataset were estimated, based on the following nucleotide frequencies: A = 0.243, C = 0.262, G = 0.261, T = 0.233, A–C = 1.15866, A–G = 2.32357, A–T = 1.15866, C–G = 1.00000, C–T = 5.27826 and G–T = 1.00000. The GTR+F+I+G4 model was determined as the most suitable model for the BI analysis using ModelFinder amongst the 24 simulated models. It achieved an IQ-TREE best score of -33090.992 and a total tree length of 1.634. The phylogenetic trees constructed using the maximum likelihood (ML) and the Bayesian inference (BI) methods exhibited a high degree of similarity, as depicted in Fig. 6.

Figure 6. 

The phylogenetic tree of Simplicillium and Leptobacillium was inferred from six-gene dataset (ITS, nrSSU, nrLSU, tef-1α, rpb1, rpb2), based on the Bayesian inference and the maximum likelihood analyses. Each value at a node indicates a bootstrap proportion (the left) and Bayesian posterior probability (the right). The scale bar 0.06 indicates the number of expected mutations per site. The species in bold black font of the Simplicillium and Leptobacillium were from this study. B. bassiana ARSEF 1564 and B. brongniartii ARSEF 617 were designated as outgroups.

The phylogenetic tree of the six-gene joint dataset revealed that the majority of species were grouped in distinct branches with robust support, indicating a stable topology (Fig. 6). The strains YFCC 23129490, YFCC 23069492 and YFCC 23089322, collected and described in this study, formed a well-supported single branch. S. puwenense and S. formicae were identified as sister species, constituting an independent clade with BP and BPP values of 100% and 1, respectively, while maintaining topological stability. YFCC 24079491 and YFCC 23039272 clustered together (BP = 100%, BPP = 1). L. longiphialidum and L. marksiae clustered into a clade, with BP and BPP of 97% and 1, respectively, forming sister species and receiving high support.

Taxonomy

Simplicillium puwenense Hong Yu bis, Y.L. Lu & Jing Zhao, sp. nov.

MycoBank No: 856314
Fig. 7

Etymology.

Named after the location Puwen Town where the pattern material was collected.

Holotype.

China • Yunnan Province, Xishuangbanna Dai autonomous prefecture, Jinghong City, Puwen Town. Specimens were collected from an evergreen broad-leaved forest, alt. 1,062 m, 100°58'60"E, 22°31'20"N, 13 December 2023, Hong Yu (holotype: YHH SP2312001, ex-type living culture: YFCC 23129490).

Description.

Sexual morph. Not found.

Asexual morph. Colonies on PDA medium moderate growth,diameter of 32–35 mm at 25 °C for 14 days, convex in middle surface, white fluffy to cotton like, dense, octahedral crystals absent, reverse brown to light brown with radial emission grooves. Hyphae septate, branched, transparent, with a diameter of 0.67–1.76 µm and smooth-walled. Cultures readily produced phialides and conidia after 14 days on PDA medium at room temperature. Phialides arising were slender, solitary, rod-shaped or columnar, measuring 3.37–52.57 µm in length and 0.5–1.6 µm in width. Conidia, transparent, single celled, smooth-walled, elliptical or oval or cylindrical, 1.19–2.41 × 0.88–1.6 µm. The conidia aggregated into a spherical shape at the top of the phialides, with a size of approximately 3.59–6.59 × 2.6–6.7 µm.

Figure 7. 

Morphology of Simplicillium puwenense a wild material b colonies obverse in PDA at 25 °C c colonies reverse on PDA at 25 °C d–p phialides bearing conidia. Scale bars: 3 mm (a); 3 cm (b, c); 10 µm (d–f); 8 µm (g); 10 µm (h, i); 12 µm (j); 10 µm (k, l); 8 µm (m); 10 µm (n, o); 15 µm (p).

Host.

Spider.

Distribution.

China, Yunnan Province.

Additional material examined.

China • Yunnan Province, Honghe Hani and Yi autonomous prefecture, the Xilong Mountains. Specimens were collected from an evergreen broad-leaved forest, alt. 1,715 m, 102°32'48"E, 22°45'20"N, 1 June 2023, Jing Zhao (paratype: YHH SP2306001, ex-paratype living culture: YFCC 23069492); • Puwen Town,collected from an evergreen broad-leaved forest, alt. 1,019 m, 100°58'42"E, 22°31'10"N, 4 August 2023, Hong Yu (Specimen number: YHH SP2308001, Strain number: YFCC 23089322).

Remarks.

Phylogenetically, three samples of S. puwenense were grouped together on a single branch, forming a monophyletic clade. It was identified as the sister species to S. formicae, supported by robust statistical evidence from both the Bayesian inference (BPP = 1) and the maximum likelihood analysis (BP = 100%). Both S. puwenense and S. formicae exhibited a stable topological structure with BP and BPP values of 100%. Morphologically, the surface of S. puwenense appeared centrally convex and exhibited a white, fluffy or cotton-like texture with densely arranged radial emission grooves ranging from reverse brown to light brown. Additionally, the conidia were observed to aggregate into spherical clusters at the apex of phialides (Table 2).

Table 2.
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Morphological comparisons of asexual morphs in the genus Simplicillium.

Species Colony on PDA Phialides (µm) Conidia (µm) Octahedral crystals References
S. album White, with a yellowish discharge, reverse beige to thick yellow, fluted 2–3 whorls or Solitary, 13.0–40.0 × 1.5–3.0 µm Two conidia: macroconidia sickle-shaped or fusiform, 8.0–11.0 (–13.0) × 2.0–3.5 µm; Microconidia oval or oblong, 3.0–4.0 × 1.5–2.0 µm Present Zhang et al. (2021)
S. aogashimaense White, reverse yellow white Solitary, a few 2–3 whorls, slender and long (19–) 23–53 × 1.2–2.0 µm Cylindrical, 4.2–6.5 × 1.2–2.0 (–2.3), conidia aggregate into spherical small heads at the top of bottle stem Present Nonaka et al. (2013)
S. araneae White fluff, reverse yellow to brown Solitary, slender, tapering from base to top, 32.9–47.1 × 1.2–2.4 µm Subspherical, spherical, or elliptical, 1.8–2.9 × 1.2–1.8 µm Absent Chen et al. (2022)
S. calcicola White or yellow, reverse light yellow to yellow 2–3 whorled or solitary, 14.0–38.0 × 1.0–2.0 µm Two conidia: macroconidial fusiform, 4.5–8.0 × 1.0–2.0 µm; microconidia oval or globose or spherical, 2.0–3.5 × 1–1.5 µm Absent Zhang et al. (2017)
S. cicadellidae White, reverse yellow Solitary, 12.9–18.3 × 0.8–1.1 µm Ellipsoid, 1.8–2.8 × 1.4–1.8 µm Absent Chen et al. (2019)
S. coccinellidae White fluff, reverse yellow to light brown Solitary, 4.9–62.1 × 1.0–1.5 µm Subspherical or cylindrical or elliptical, 2.0–3.4 × 1.6–2.0 µm Absent Chen et al. (2021)
S. coleopterorum White fluff, reverse light brown to brown Solitary, 34.5–64.1 × 0.7–1.2 µm Spherical or subspherical or elliptical, 2.1–3.3 × 1.5–1.9 µm Absent Chen et al. (2022)
S. cylindrosporum White, reverse blond 2–3 whorled or solitary, 17–32 × 1.2–2.0 (–2.5) µm Spherical or cylindrical, 3.0–4.5(–5.0) × 1.0–2.0 µm Present Nonaka et al. (2013)
S. formicidae White, reverse light brown to brown, brown secretions Solitary, 51–70.1 × 0.7–0.9 µm Conidia aggregate into spherical slimy heads, mostly filamentous or fusiform, 3.9–7.9 × 0.8–1.3 µm Absent Chen et al. (2019)
S. guizhouense White, reverse yellow to light yellow Solitary, 1.1–52.2 × 1.0–1.8 µm Oval or spherical, 2.4–2.9 × 1.6–1.8 µm Absent Chen et al. (2022)
S. humicola White, light-yellow secretions, reverse light yellow to brown 2–3 whorled or solitary, 20.0–35.0 (–47.0) × 1.5–3.0 µm Oblong or oval, 3.0–5.0 × 1.5–3.0 µm Present Zhang et al. (2021)
S. hymenopterorum White, reserve light yellow Mainly solitary, rarely whorls, 19.3–46.2 × 1.1–2.3 µm Cylindrical to subellipsoidal, 2.1–2.8 × 1.3–1.9 µm, forming a subspherical small head at the top of the stem Absent Chen et al. (2021)
S. lamellicola White, reserve light yellow 15–50 × 0.7–1.0 µm Two conidia: macroconidia fusiform, 4.5–9.0 × 0.8–1.0 µm; microconidia ovoid to ellipsoid, 2.0–3.0 × 0.7–1.2 µm Present Zare and Gams (2001)
S. lanosoniveum White or cream, reverse brownish cream to light yellow Solitary, 20.0–40.0 × 1.1–2.0 µm Spherical or ellipsoidal, 2.0–4.5 × 1.0–3.0 µm, forming a spherical or ellipsoidal tip at the top of the phialides, Wei et al. (2019)
S. lepidopterorum White, reserve light yellow Solitary, 15.3–26.2 × 0.7–1.4 µm Spindle-shaped or oval, 1.6– 2.4 × 1.4–1.7 µm, forming a slimy spherical head at the top of the phialides Absent Chen et al. (2019)
S. minatense White, no secretion, reverse brown Mainly solitary, rarely in whorls of 2–3, 11.0–31.0 (–47.0) × 1.0–1.7 µm Spherical, 2.0–3.5 × 1.8–2.5 (–2.8) µm, forming a subglobose or ellipsoidal tip at the top of the phialides Present Nonaka et al. (2013)
S. neolepidopterorum White, reverse yellow to light yellow Solitary, 34.1–44.3 × 1.0–1.7 µm
Solitary, 34.1–44.3 × 1.0–1.7 µm		 Solitary, ellipsoidal to cylindrical, occasionally in short imbricate chains, 2.5–3.8 × 1.5–2.1 µm Absent Chen et al. (2021)
S. niveum White 2–5 whorled, 10–20.5 (25.0) × 1–2 µm Top growth, elongated or elliptical in shape, 3.0–4.5 (–6) × 1–2 µm Crous et al. (2021)
S. pechmerlense White, reverse light yellow to orange Solitary, 16.0–31.0 × 0.9–1.2 µm Two conidia: macroconidia fusiform, 5.0–8.0 × 1–1.6 µm; microconidia subglobular or elliptic, 1.8–3.0 × 0.9–1.5 µm, forming a slimy spherical head at the top of the phialides, Absent Leplat et al. (2021)
S. puwenense White fluffy to cotton like, convex in middle surface, reverse brown to light brown with radial emission grooves Slender, solitary, rod-shaped or columnar, measuring 3.37–52.57 µm in length and 0.5–1.6 µm in width Elliptical or oval or cylindrical, 1.19–2.41 × 0.88–1.6 µm. forming a spherical shape at the top of the phialides, 3.59–6.59 × 2.6–6.7 µm in size Absent This study
S. scarabaeoidea White, reverse light yellow Solitary, 18.5–63.4 × 1.1–1.4 µm Ellipsoidal, 1.9–2.9 × 1.4–2.0 µm Absent Chen et al. (2021)
S. subtropicum White, reverse brownish orange to brown (15.0–) 20–42 (–50.0) × 1.0–2.3 µm; Solitary, rarely in whorls of 2–3, (15.0–) 20.0–42.0 (–50.0) × 1.0–2.3 µm Subglobose or ellipsoid, 2.3–4.0 (–4.5) × 1.5–3.3 µm, forming a spherical tip at the top of the phialides, 2.3–4.0 (–4.5) × 1.5–3.3 µm in size Present Nonaka et al. (2013)
S. sympodiophorum White, reverse yellow white 2–4 whorled or solitary, 20.0–34 (–47.0) × 0.5–1.3 µm Oval to ellipsoidal, 2.2–3.5 × 1.0–2.0 µm Present Nonaka et al. (2013)
S. yunnanense White to light yellow, grayish orange to brown on back Solitary, 5.8–16.9 × 1.1–1.5 µm Cylindrical, 2.5–3.4 × 0.7–1.1 µm, conidia usually form chains at the top of the phialides - Wang et al. (2020)

Leptobacillium longiphialidum Hong Yu bis, Y.L. Lu & Jing Zhao, sp. nov.

MycoBank No: 856313
Fig. 8

Etymology.

Referring to its longer phialides than those of the close relationship species in this genus.

Holotype.

China • Hainan Province, Qiongzhong City, Limushan Town, Limushan National Forest Park. Specimens were collected from an evergreen broad-leaved forest, alt. 589.9 m, 109°44'28"E, 19°10'41"N, 8 March 2023, Jing Zhao (holotype: YHH LL2303001, ex-type living culture: YFCC 23039272).

Description.

Sexual morph. Not found.

Asexual morph. The colony was incubated at 25 °C on PDA medium for 14 days, the growth rate was slow, the diameter was 25–27 mm, the middle was fluffy to cotton, dense, convex and radial wrinkles, white and reverse brown to light yellow on the back. Mycelium branches, smooth walls, septate, transparent, with a diameter of approximately 0.97 × 1.72 µm. Cultures readily produced phialides and conidia after 10 days on PDA medium at room temperature. Phialides solitary, columnar, tapering from base to apex, 24.01–205.77 µm long, 1.00–2.24 µm wide. Conidia 2.88–4.54 × 1.18–1.95 µm, transparent, single celled in chains, smooth walls, narrow columnar or spindle-shaped, with apical conidia elliptical or nearly spherical in shape.

Figure 8. 

Morphology of Leptobacillium longiphialidum a wild material b colonies obverse in PDA at 25 °C c colonies reverse on PDA at 25 °C d–p phialides bearing conidia q conida. Scale bars: 2 mm (a); 2 cm (b, c); 20 µm (d); 12 µm (e); 30 µm (f); 20 µm (g); 10 µm (h–k); 9 µm (l); 10 µm (m); 7 µm (n); 8 µm (o); 10 µm (p, q).

Host.

Spider.

Distribution.

China, Hainan Province, Guangdong Province.

Additional material examined.

China • Guangdong Province, Huizhou City, Boluo County, 511 Township Road. Specimens were collected from an evergreen broad-leaved forest, alt. 29.4 m, 114°24'5"E, 23°14'32"N, 23 July 2024, Hong Yu and Y.L. Lu (paratype: YHH LL2407001, ex-paratype living culture: YFCC 24079491.

Remarks.

The key characteristic of L. longiphialidum was its independent, columnar shape and the presence of narrow or fusiform spores. Phylogenetic analyses showed that L. longiphialidum belonged to the Leptobacillium clade and was closest to L. marksiae. However, the host and collection sites of L. longiphialidum were spiders and China, respectively and the host and collection sites of L. marksiae were an unidentified dead insect and Queensland, Australia, respectively. L. longiphialidum and L. marksiae were distinguished by genetic distance. (Table 3).

Table 3.
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Morphological comparisons of asexual morphs in the genus Leptobacillium.

Species Colony on PDA Phialides (µm) Conidia (µm) References
L. cavernicola White, reverse usually dark brown Mainly solitary, slender, tapering toward tip, 5.1–27.2 × 1.2–1.7 µm Forming long, slender chains, narrowly cylindrical to slightly fusiform, some were slightly lemon-shaped, first-formed conidium were usually shorter, obovoid to pyriform with a rounded distal end, 3.1–6.9 × 0.9–1.5 µm Leplat et al. (2022)
L. chinense White, reverse cream to light yellow Solitary, (6.0–) 15–30 (–68.0) × 1.5 µm Ellipsoidal or oval or cylindrical, 3.5–5.0 × 1.0–1.5 µm, the conidia aggregate into chains, with the apex conidia subspherical or obovoid, 1.5–2.5 × 1.5–2.0 µm Liu and Cai (2012)
L. coffeanum White, reverse cream Solitary, few 2–3 whorls, 11.0–44.0 (–70.0) × 1.0–2.4 µm Two conidia, macroconidia spindle-shaped, 5.3–8.8 × 1.0–1.6 µm; microconidia oval to fusiform, 2.2–3.8 × 0.8–1.5 µm Gomes et al. (2018)
L. filiforme White, reverse light yellow Solitary, 9.0–18.0 × 1.0 µm Fusiform to filamentous, chained, sometimes forming zigzag chains, 7.2–12.5 × 1.0 µm Crous et al. (2018)
L. latisporum White, reverse greyish orange to orange white 13.2–40.8 × 2.9–4.8 μm Shuttle shaped to narrow cylindrical, with single cells forming long chains, 3.9–6.3 × 1.9–3.9 μm Preedanon et al. (2023)
L. longiphialidum White, reverse brown to light yellow Solitary, 24.01–205.77 × 1.00–2.24 µm Narrow columnar or spindle shaped, 2.88–4.54 × 1.18–1.95 µm, single celled in chains, with apical conidia elliptical or nearly spherical in shape This study
L. leptobactrum var. calidius White to cream, reverse Light yellow to brown Solitary, few 1–2 whorls, 18.4–60.0 × 0.7–2.0 µm Narrow cylindrical (rod-shaped) to slightly fusiform, 3.0–5.7 × 0.7–1.7 µm Zare and Gams (2016)
L. leptobactrum var. leptobactrum White to cream, reverse Light yellow to yellowish brown 15.8–31.7 × 0.7–1.5 µm
Solitary, few 2–3 whorls, 15.8–31.7 × 0.7–1.5 µm		 Narrow rod-shaped or narrow cylindrical (rod-shaped), 3.0–6.1 × 0.8–2.1 µm Zare and Gams (2016)
L. leptobactrum White, gray white to pinkish white, reverse orange to orange brown, gray white, light yellow, milky white to dark yellow Solitary, few 1–2 branches, 20.0–45.0 µm long, Base width 1–2 µm, top width 0.5–0.7 µm Narrow cylindrical (rod-shaped) to slightly fusiform, 4.5–8.0 × 0.8–1.5 (–2.0) µm Zare and Gams (2016)
L. muralicola White, gray white to green white, reverse light yellow, milky white to dark yellow, orange to orange brown, ochraceous Solitary, few 1–2 branches, 20.0–45.0 µm long, Base width 1.0–2.0 µm, top width 0.5–0.7 µm Narrow cylindrical (rod-shaped) to slightly fusiform, 4.5–6.0 × 1.0–2.0 µm Sun et al. (2019)
L. symbioticum White, reverse orange yellow to orange-brown Solitary, few 2–3 whorls, 7.1–30.6 × 1.6–3.5 µm Slightly fusiform to narrowly cylindrical, 4.0–6.9 × 0.7–1.6 µm Okane et al. (2020)

Discussion

The genera of Simplicillium and Leptobacillium were found to be the most closely related within the family of Cordycipitaceae. They exhibited a wide distribution and were commonly observed on various substrates or hosts, including air, seawater, rocks, leaves, soil, insects, fungi, freshwater environments, murals, rocks and caves (Liu and Cai 2012; Zare and Gams 2016; Crous et al. 2018; Gomes et al. 2018; Sun et al. 2019; Wei et al. 2019; Kondo et al. 2020; Okane et al. 2020; Wang et al. 2020; Leplat et al. 2021). Chen et al. (2019) first reported insect-associated species of Simplicillium while later reporting an additional eight arthropod-related species of the genus Simplicillium (Chen et al. 2021, 2022). Furthermore, Leplat et. al (2022) isolated L. cavernicola Leplat from caves as a representative species of the genus Leptobacillium whereas L. muralicola Z. Sun, Qin Y. Ge, Zhi B. Zhu & Xing Z. Liu was isolated from mural paintings in a Koguryo tomb in China (Sun et al. 2019).

The macroscopic and microscopic morphology of most species in the genera of Simplicillium and Leptobacillium are quite similar and it is difficult to distinguish specific species, based on only morphological features. Thus, it is often necessary to combine morphological and molecular data for species identification. The utilisation of ITS and nrLSU by Liu and Cai (2012) yielded more accurate outcomes in the identification of Simplicillium species. To date, multi-site phylogenies incorporating the combined analysis of ribosomal DNA and functional protein-coding genes have been extensively employed in fungal phylogeny research, yielding numerous significant findings (Sung et al. 2007; Luangsa-ard et al. 2017; Mongkolsamrit et al. 2020; Wang et al. 2020). The results showed that the molecular phylogenies of Simplicillium and Lecanicillium, based on ITS fragment, nrLSU fragment and six-gene combined dataset, were more stable in topology. This was consistent with the results of previous studies. In this study, two novel species, S. puwenense and L. longiphialidum, were identified and characterised through meticulous morphological examination and rigorous phylogenetic analysis.

Through morphological observation, it was found that phialides of species in the genus of Simplicillium were solitary and could be distinguished from those of the genus of Lecanicillium (Chen et al. 2021). It was observed that a prominent characteristic of species within the Simplicillium genus was the solitary nature of phialides, wherein conidia typically adhered to the apex of phialides in chains exhibiting spherical, sticky, or tile-like properties, ultimately resulting in the formation of octahedral crystals (Zare and Gams 2001). The primary distinguishing feature of Leptobacillium species lay in the presence of two conidia; single cells arranged in clusters with near-spherical or elliptical conidia at the apex and other narrow columnar (rod) to fusiform-shaped conidia (Zare and Gams 2016; Leplat et al. 2022). The phialides of S. puwenense collected in this study were slender, solitary, rod-shaped or columnar; the conidia were transparent, single-celled with smooth walls and had an oval or cylindrical shape. They formed aggregates into a spherical structure at the apex of the phialides. These characteristics aligned closely with the primary identification features described for Simplicillium species by Zare and Gams (2001). The phialides of L. longiphialidum appeared as solitary and columnar structures. Two types of conidia were observed, i.e. one type consisted of single cells clustered together in chains, while the other type was oval or nearly spherical and located at the apex. Additionally, there was another type of narrow columnar or spindle-shaped conidium present, which was consistent with previous studies on Leptobacillium species (Zare and Gams 2016; Leplat et al. 2022).

In phylogenetic trees, most species of the genera Simplicillium and Leptobacillium were clustered in their separate clades and were well supported and topologically stable. However, the phylogenetic framework showed that two samples of L. leptobactrum, ZJ14B02 and AH17C05, did not form a monophyletic clade. The ITS sequence and nrLSU sequence of strain ZJ14B02 contained 547 bp and 909 bp, respectively. The ITS and nrLSU sequences of strain AH17C05 contained 557 bp and 929 bp, respectively. It was found that the head and tail bases of ITS sequence of samples ZJ14B02 and AH17C05 were different from those of nrLSU sequences. It was speculated that the two samples of L. leptobactrum did not form a monophyletic clade, which might be caused by the poor processing of the fore-tail primer sequence. Zare and Gams (2016) initially described L. leptobactrum, composed of L. leptobactrum var. leptobactrum and L. leptobactrum var. calidius, which were distinguished by their optimal growth temperature. The optimum temperature for growth of L. leptobactrum var. leptobactrum was 18–21 °C, no growth at 30 °C (Zare and Gams 2016). The optimum temperature for growth of L. leptobactrum var. calidius was 24–27 °C, reduced growth at 30 °C, no growth at 33 °C (Zare and Gams 2016). Phylogenetic studies had placed two strains in unexpected clades, namely L. leptobactrum var. leptobactrum and L. leptobactrum var. calidius. In the phylogenetic framework constructed by nrSSU, L. leptobactrum var. leptobactrum and L. leptobactrum var. calidius clustered into a clade. The findings of phylogenetic frameworks, based on ITS, nrLSU and six-gene datasets, revealed that L. leptobactrum var. calidius and L. chinense formed a cluster, while L. leptobactrum var. leptobactrum and L. cavernicola also clustered together. This was consistent with the findings of Leplat et al. (2022).

S. pechmerlense J. Leplat constituted an independent clade that exhibited slight differences compared to the previously studied phylogenetic framework (Leplat et al. 2021). However, it was the same as the phylogenetic framework reconstructed by Chen et al. (2022). Additionally, Leplat et al. (2021) found that the underside of the colony of S. pechmerlense was light yellow to orange, the phialides was solitary and there were two kinds of conidium, the macroconidia was spindle, 5.0–8.0 × 1.0–1.6 µm. The microconidia were subspherical or elliptic, 1.8–3.0 × 0.9–1.5 µm, forming slimy globular heads at the top of the phialides. S. pechmerlense phialides solitary and conidia attached to the top of the phialides with slimy heads fit the main identification characteristics of Simplicillium (Zare and Gams 2001). S. pechmerlense was morphologically similar to S. calcicola Z.F. Zhang, F. Liu & L. Cai and S. album Z.F. Zhang & L. Cai (Leplat et al. 2021). The phialides of S. calcicola and S. album were 2–3-whorled or solitary (Zhang et al. 2017, 2021), while S. pechmerlense were solitary. The solitary phialides could distinguish S. pechmerlense from S. calcicola and S. album. Species of Simplicillium have frequently been identified using ITS and nrLSU sequences (Liu and Cai 2012). Phylogenetic analyses, based on single gene fragments revealed an unstable systematic position for S. pechmerlense. However, the morphological characteristics of S. pechmerlense align with the primary identification features of Simplicillium. Consequently, it was determined that S. pechmerlense should be retained within the genus Simplicillium. The inclusion of supplementary materials, such as morphological data, would be essential for further verification since only one strain of polygenic sequence data was available for L. leptobactrum var. leptobactrum and L. leptobactrum var. calidius.

Acknowledgements

Many thanks to Peng Ronghua of Guangdong Small Worm Biotechnology Co., Ltd. for his help in this study. At the same time, we also thank the two reviewers and editors for their critiques and suggestions which greatly improved our manuscript.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the National Natural Science Foundation of China (32200013) and supported by the Research and Innovation Fund for Graduate Students of Yunnan University (KC-23235594).

Author contributions

Yingling Lu: Responsible for investigation, article conception, writing and editing, and species identification; Songyu Li: Responsible for investigation, article conception, writing and editing, morphological analysis and phylogenetic analysis; Zuoheng Liu: Collecting the information of specimens and GenBank entry number required for research; Jing Zhao: Responsible for picture editing and processing; Zhiyong Yu, Zongli Liang, Hailong He, Jianhong Li, Yun Huang, Xinming Li: Responsible for investigation; Hong Yu: Responsible for investigation, conceptualisation, writing – review and editing and supervision.

Author ORCIDs

Yingling Lu https://orcid.org/0009-0008-8119-1975

Songyu Li https://orcid.org/0009-0007-9589-0892

Zuoheng Liu https://orcid.org/0000-0003-4118-3694

Jing Zhao https://orcid.org/0000-0001-7871-2209

Zhiyong Yu https://orcid.org/0000-0001-8276-5901

Zongli Liang https://orcid.org/0009-0006-5553-8811

Hailong He https://orcid.org/0009-0000-7862-0865

Jianhong Li https://orcid.org/0009-0001-1234-7816

Yun Huang https://orcid.org/0009-0007-3429-1490

Xinming Li https://orcid.org/0009-0001-7198-281X

Hong Yu https://orcid.org/0000-0002-2149-5714

Data availability

All of the data that support the findings of this study are available in the main text.

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Yingling Lu and Songyu Li contributed equally to this work.
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