The genus Simplicillium

Abstract Simplicillium species have a wide host range and an extensive distribution. Some species are associated with rusts, as well as other plant pathogenic fungi and play an important role in biological control. In this study, two specimens of Simplicillium were collected from Chiang Mai Province, Thailand. Simplicillium formicae sp. nov. was isolated from an infected ant and S. lanosoniveum from Ophiocordyceps unilateralis which is a new host record. Species were initially identified using ITS gene sequences and confirmed using morphology coupled with phylogenetic analyses of a combined nrLSU, nrSSU, TEF and RPB1 dataset. Simplicillium formicae differs from other species in the genus by the presence of flask-shaped synnemata and phialides with intercalary nodes. Simplicillium lanosoniveum resembles other collections of the species by its completely solitary, tapering phialides and globose to ellipsoidal conidia which adhere in a slimly head. A key to species of Simplicillium is also provided.


Introduction
introduced Simplicillium to accommodate four taxa including the type species S. lanosoniveum and three other species, S. lamellicola, S. obclavatum and S. wallacei. Simplicillium species were historically placed in Verticillium sect. Prostrata which was described by Gams (1971) for prostrate conidiophore-producing species. Later, most of the species of Verticillium sect. Prostrata were reported as members in Clavicipitaceae, based on molecular data (including SSU, LSU and ITS sequences), whereas Simplicillium species consistently formed a monophyletic group apart from the other described taxa in this family (Zare et al. 2000;Gams and Zare 2001;Sung et al. 2001;Zare and Gams 2001). Recently, Clavicipitaceae was divided into three families, based on multi-gene phylogenetic analyses and Simplicillium was assigned to Cordycipitaceae (Hypocreales, Hypocreomycetidae, Sordariomycetes) Maharachchikumbura et al. 2016;Wijayawardene et al. 2018). Zare and Gams (2008) excluded Simplicillium wallacei from Simplicillium and transferred it to Lecanicillium due to the basal position being closer to the latter genus than to the former genus in the cladogram of ITS data. Subsequently, ten species viz. Simplicillium chinense (Liu and Cai 2012), S. aogashimaense, S. cylindrosporum, S. minatense, S. subtropicum, S. sympodiophorum , S. lanosoniveum var. tianjinensis (Dong et al. 2014), S. calcicola (Zhang et al. 2017), S. coffeanum (Gomes et al. 2018) and S. filiforme (Crous et al. 2018) were restricted to Simplicillium, based on the phylogenetic analyses of ITS sequence data and strong morphological evidence. Its sexual-asexual connection has been established with S. lanosoniveum linked to a Torrubiella sp. .
Simplicillium species have a wide distribution and are considered as mammal and plant-parasitic, symbiotic, entomopathogenic, fungicolous and nematophagous fungi, as they have a broad spectrum of hosts and substrates, such as insects, plants, rusts, nematodes, human nails, canine tissues and mushrooms, Chroococcus sp., soil, freshwater, marine and terrene environments Guo et al. 2012;Liu and Cai 2012;Dong et al. 2014;Liang et al. 2016;Sun et al. 2019). Several studies have been shown that Simplicillium species have a high ecological and economical value for biocontrol and bioactive compounds (Takata et al. 2013;Yan et al. 2015;Hyde et al. 2019). For example, Simplicillium lanosoniveum can be a phytopathogen, causing brown spots and lesions on flowers (Chen et al. 2008) or a mycoparasite on soybean rust (Ward et al. 2012;Gauthier et al. 2014) or a pathogen on aphids and other phytopathogens (Chen et al. 2017) or an anti-Trichomonas vaginalis agent (Scopel et al. 2013). Simplicillium chinense can be a biological control agent against plant parasitic nematodes Luyen 2017). Simplicillium lamellicola can suppress plant bacterial diseases and grey mould diseases of tomato (Solanum lycopersicum) and ginseng (Panax ginseng) (Dang et al. 2014;Shin et al. 2017). Simplicillium obclavatum has the ability to produce multiple xylanases and endoglucanases that have the potential to be used in biofuels, animal feed and food industry applications (Roy et al. 2013). Bioactive compounds with anti-fungal and anti-bacterial profiles and pharma-ceutical exopolysaccharides have been isolated from S. lanosoniveum (Yu et al. 2013;Fukuda et al. 2014;Xing et al. 2016;Dong et al. 2018). Linear and cyclic peptides with anti-fungal and anti-viral properties have also been discovered from the secondary metabolites of S. obclavatum (Liang et al. 2016(Liang et al. , 2017.
Recent studies have shown that Thailand supports an amazing fungal diversity with numerous new species that have the potential for biotechnological application . In this study, we introduce a novel species, Simplicillium formicae from northern Thailand and a new record of S. lanosoniveum with evidence from a combination of molecular analyses and morphological characteristics to reserve a natural resource for future studies regarding biocontrol in the forestry, agricultural and pharmaceutical industries.

Sample collection and isolation
The Mushroom Research Centre (MRC) is a disturbed rainforest located in Chiang Mai Province, Thailand (Aung et al. 2008). The forest consists of various tall tree and lower shrubs. The climate of Chiang Mai is controlled by tropical monsoons and the weather is typically hot and humid with temperatures often close to or above 30 °C. Frequent rain and thunder showers usually last from June to late October (Chiang Mai Buddy website: https://chiangmaibuddy.com/welcome-to-chiang-mai/weather-andclimate/, accessed 26.8.2019). Two ant fungi were found anchored to the underside of two different shrubby leaves in the forest at the Mushroom Research Centre. These two fresh specimens; HKAS 102459 and HKAS 102447 were collected and placed in plastic containers and transported to the laboratory for subsequent study. Interestingly, the ant fungus HKAS 102447 was already dead and was colonised by a saprobic fungus. The isolate MFLUCC 18-1385 was separated from this saprobe which occurred on the surface of specimen HKAS 102447 via single spore isolation. The isolate MFLUCC 18-1379 was separated from specimen HKAS 102459 by directly cultivating the hyphae which covered the surface of the ant host. These two isolates were cultured with potato dextrose agar (PDA, 1% w/v peptone) and incubated at room temperature (25 °C).

Morphological studies
For long-term deposit, these two specimens were dried with allochroic silica gel to protect them from contamination of opportunistic fungi and to retain the informative taxonomic characters. The macro-morphological characters were observed with a stereoscope (Olympus SZ61) and the micro-morphological features were examined with a compound microscope (Nikon ECLIPSE Ni). Important characteristics such as myce-lium, phialides and conidia were captured with a digital camera (Canon EOS 600D). Measurements of perithecia, synnemata, phialides and conidia were taken using the Tarosoft (R) Image Frame Work programme and the images used were processed with Adobe Photoshop CS3 Extended v. 10.0 (Adobe, San Jose, CA).
DNA extraction, PCR amplification and sequencing DNA was extracted from fresh mycelium of isolates MFLUCC 18-1379 and MFLUCC 18-1385 and from stromal tissue of ant fungus HKAS 102447 (the host of isolate MFLUCC 18-1385) using a DNA extraction kit (Biospin Fungus Genomic DNA Extraction Kit, BioFlux, China), following the instructions of the manufacturer. Extracted DNA was stored at 4 °C for use in regular work and duplicated at -20 °C for long-term storage. The internal transcribed spacer (ITS1-5.8S-ITS2, ITS) was amplified with primer ITS4 and ITS5 (White et al. 1990) and was used for individual gene phylogenetic analyses. The large subunit (LSU), small subunit rDNA (SSU), translation elongation factor 1-alpha gene (TEF1-α) and RNA polymerase II largest subunit 1 (RPB1) were also amplified as described in Wei et al. (2018) and used for multi-gene phylogenetic analyses. The PCR products were sent to Sangon Company, Kunming City, Yunnan Province, China for sequencing using the above primers. Newly generated sequences, used in the study, were submitted to GenBank to be assigned their accession numbers.

Sequence alignments and phylogenetic analyses
The raw sequences were verified with Finch TV version 1.4.0 (Mccredden 2016) and assembled with BioEdit v. 7.0.9.1 (Hall 1999). Sequence data were downloaded from GenBank based on BLAST searches of ITS sequences and with reference to the recent publications (Table 1). Most Simplicillium species are lacking protein-coding genes, but ITS sequences are available for all the species that are useful in understanding the intraspecific relationships within Simplicillium (Liu and Cai 2012, Dong et al. 2014and Crous et al. 2018. Therefore, phylogenetic analyses, based on ITS regions, were generated throughout Simplicillium for the primary identification. Multi-gene phylogenetic analysis of the combined SSU, LSU, TEF and RPB1 sequences from representative species in Hypocreales was afterwards performed to confirm the taxonomic placements of our isolates. The generated sequences of each gene region were aligned separately with representative sequences using MAFFT v. 7 web server (http://mafft.cbrc.jp/alignment/ server) (Kuraku et al. 2013;Katoh et al. 2017). The uninformative gaps and ambiguous regions were manually removed and different gene regions were concatenated using BioEdit v. 7.0.9.1 (Hall 1999). The maximum Likelihood (ML) analyses was performed using RAxML-HPC2 on XSEDE (8.2.10) at CIPRES Science Gateway V. 3.3 (https://www.phylo.org/portal2/home.action), with default setting, except the boot-  (Swofford 2002) with the heuristic search option and Tree-Bisection-Reconnection (TBR) branch-swapping algorithm for 1000 random replicates. Branches that have a minimum branch length of zero were collapsed. Gaps were treated as "missing" and starting tree(s) were generated via stepwise addition (Hillis and Bull 1993 PAUP v. 4.0b10 (Ronquist and Huelsenbeck 2003). Bayesian analysis was performed using MrBayes v. 3.1.2 (Rannala and Yang 1996;Zhaxybayeva and Gogarten 2002) to evaluate posterior probabilities (BYPP) with the Markov Chain Monte Carlo sampling (MCMC) method. Trees were sampled and printed to output at every 1000 generations. The first 25% of sampled trees were discarded as part of a burn-in procedure, the rest of the trees were used to create the consensus tree and the average standard deviation of split frequencies was set as 0.01. Phylogenetic trees were visualised with FigTree v1.4.0 (Rambaut 2006) and edited in Microsoft PowerPoint, then saved as a PDF format and finally altered to JPG format using Adobe Illustrator CS6 (Adobe Systems Inc., United States). The finalised alignments and trees were submitted in TreeBASE (http://www.treebase.org/), with submission ID 24238 (ITS) and 24240 (multi-gene).
Culture characters. The colonies on PDA medium were rapid-growing, reaching a diam. of 5.5 cm in 30 days at 22 °C, white, entire margin, velvety, with radial cracks and primrose-yellow on the reverse. Host and distribution: Saprophytic on fungi, endophytic or symbiotic or pathogenic on plant, parasitic on rust, nematode and insect, occurring on soil, animal hair or human bronchoalveolar lavage fluid, with a cosmopolitan distribution (see Table 2 Note. Our isolate MFLUCC 18-1385 colonised on a decayed Ophiocordyceps unilateralis with white hyphae. In a thorough examination of the Ophiocordyceps unilateralis host, we found the phialides and conidia of our isolate grown on the surface of the host ( Figure 5). Phylogenetically, our isolate grouped with the strains of Simplicillium lanosoniveum with high bootstrap support (85% ML, 0.99 BYPP, 67% MP, Figure 2). The nucleotides comparison between our isolate and the type strain of Simplicillium lanosoniveum (CBS123.42) showed only 5 bp differences out of 539 in the ITS region. This evidence proves that our isolate is a strain of S. lanosoniveum, according to Jeewon and Hyde (2016). Morphologically, it resembles S. lanosoniveum with solitary phialides without verticillate branches and conidia adhering on a slimy head. Most of the previous descriptions of this species were given in hand-drawings and scanning electron microscopy (SEM) patterns Ward et al. 2012;Gauthier et al. 2014). Simplicillium lanosoniveum has been reported from Enhalus acoroides (seagrass) in Trang Province, Thailand. In this study, we introduce our isolate MFLUCC 18-1385 as a new host record of Simplicillium lanosoniveum from Ophiocordyceps unilateralis and provide the updated morphological features for a better understanding of this species. Simplicillium lanosoniveum has been frequently reported as a hyperparasite of rust and plant pathogenic fungi. Therefore, this species has a high potential of being a natural source of microbial agents against microbiological diseases in commercial agriculture (Baiswar et al. 2014;Berlanga-Padilla et al. 2018). At first, we included all available sequences of S. lanosoniveum from GenBank in the individual gene tree. Some strains did not group with other strains but distributed throughout the genus in primary analyses (data not shown), so we excluded those strains from the final phylogenetic analysis. Most of the reported strains of S. lanosoniveum, including the invalid strains, are listed in Table 2 to show their distribution and host range, as well as the sequence data availability. Description. Parasitic on ants (Formicidae). Sexual morph: Stromata up to 14 mm in length, 0.5 mm wide in the broadest part, cylindrical, brown, slightly tapering towards the apex, single, piercing through the dorsal neck region of the ant host.  Ascomatal cushion hemisphere, up to 1.2 mm in diam., laterally attaching to the erect stroma stalk, dark brown, with ostioles protruding from the cushions. Perithecia 200-400 × 50-120 (x = 294 × 81, n = 10) µm, sub-immersed, flask-shaped. Asci and ascospores were too old to observe their features. Asexual morph: Undetermined.
Note. This collection was already decayed and was colonised by other fungi which we introduced as a new host record of Simplicillium lanosoniveum from Thailand. The outline of this specimen was intact, while its asci and ascospores were too old to analyse. We retrieved DNA through direct sequencing from the stromal tissue.

Conclusion
A new species Simplicillium formicae and a new host record species Simplicillium lanosoniveum from Ophiocordyceps unilateralis were introduced, based on phylogenetic analyses and morphological evidence. The host and distribution of S. lanosoniveum was summarised and a key to Simplicillium was provided.