Diversity of Akanthomyces on moths (Lepidoptera) in Thailand

Abstract Akanthomyces is a genus of invertebrate-pathogenic fungi from the family Cordycipitaceae (Ascomycota, Hypocreales). Its species occurs on two different types of hosts, spiders and insects, and in the latter case specifically Lepidoptera adults. Three new species of Akanthomyces, A. noctuidarum, A. pyralidarum, and A. tortricidarum occurring on adult moths from Thailand are proposed based on the differences of their morphological characteristics and molecular data. Phylogenetic analyses using a combined dataset, including the internal transcribed spacer regions, the large subunit of the ribosomal DNA, translation elongation factor 1-α, the largest subunit of RNA polymerase II, and the second largest subunit of RNA polymerase II, support the delimitation of these new species in Akanthomyces.


Introduction
Cordycipitaceae is one of the families of the order Hypocreales with entomogenous nutritional habit. Many of the species in this family have been originally isolated from dead insects and spiders that are buried in the soil, leaf litter, or attached to the undersides or upper sides of a leaf. Some species, especially in Beauveria, could be found in the soil (Rehner and Buckley 2005) or as endophytes (Mantzoukas and Lagogiannis 2019;Afandhi et al. 2019). Cordycipitaceae is validated based on the type of Cordyceps, Cordyceps militaris, and it has initially included pyrenomycetes that possess pallid to brightly colored, fleshy stromata (Kepler et al. 2017). It is also characterized by producing superficial to completely immersed perithecia, cylindrical asci with thickened apex, and multi-septate filiform ascospores that disarticulate into part-spores or remain intact at maturity (Sung et al. 2007). Well-known for its use in traditional Chinese medicine, C. militaris produces some polysaccharides and cordycepin that have been used for anti-inflammatory, antioxidant, anti-tumor, anti-metastatic, and immunomodulatory functions (Das et al. 2010). The recent study of C. militaris shows that this fungus has an anti-hypertension and neuroprotective effect to delayed neural death (Takakura et al. 2017;Kim et al. 2018).The most popular anamorph in this family is Beauveria, notably with its type species, Beauveria bassiana, which has been used globally as a mycoinsecticide since the 1960s (Vega et al. 2012). Spider pathogens are mostly found within Cordycipitaceae (Shrestha et al. 2019). Their anamorph are found in Akanthomyces, Gibellula, or Hevansia (Kepler et al. 2017).
Akanthomyces was established by Lebert (1858) with Akanthomyces aculeatus, the type species, found on a moth in Europe (Mains 1950). Gibellula differs from Akanthomyces in the production of aspergillus-like conidiophores and the host range (Kepler et al. 2017). Gibellula is only found on spiders, while Akanthomyces can be found on both, spiders and insects. Akanthomyces was known attacking some insect orders such as Hemiptera (Akanthomyces lecanii), Coleoptera (Akanthomyces neocoleopterorum), Lepidoptera (Akanthomyces pistillariiformis), and Orthoptera (Akanthomyces fragilis) (Hodge et al. 2003;Mongkolsamrit et al. 2018;Chen et al. 2020). In general, the host range of Akanthomyces for both, teleomorph and anamorph are similar. The genus includes Cordyceps tuberculata found on adult moths, which is linked to the anamorph Akanthomyces pistillariiformis. Akanthomyces has taxonomic priority by date over Lecanicillium, one of the anamorphs in Cordycipitaceae with verticillium-like morphologies Kepler et al. 2017). The type species of Lecanicillium, L. lecanii (Cephalosporium lecanii, now regarded as Akanthomyces lecanii) is found on lice and scale insects and is known as the anamorph of Cordyceps confragosa. On the basis of previous studies on Akanthomyces in Thailand, Mongkolsamrit et al. (2018) proposed four new species of Akanthomyces on spiders, namely, A. kanyawimiae, A. sulphureus, A. thailandicus, and A. waltergamsii. Here, we describe three new Akanthomyces species found on adult moths (Lepidoptera) from Thailand based on morphological and molecular studies.
Species complexes or cryptic species are common in the kingdom Fungi. Given the simplicity of the phenotypic characters and the overlap of the size and shapes of important diagnostic features, species in many genera cannot be easily classified and identified. Cryptic species refers to taxa that are morphologically similar, yet evidence has shown that they are on different evolutionary paths as revealed by molecular phylogenetic methods and can only be recognized by their DNA sequences. Entomopathogenic fungi from Thailand are commonly encountered in the forests and constitute a huge number in our collections (Kobmoo et al. 2012;Luangsa-ard et al. 2018;Mongkolsamrit et al. 2018;Tasanathai et al. 2019).
In surveys of entomopathogenic fungi in national parks and community forests, collections of pathogens on adult moths were found on the underside of leaves of dicotyledonous forest plants. The phenotypic characters of the collections in having cylindrical to narrowly clavate synnemata and superficial perithecia scattered on the body and wings of the moth identify them primarily to be members of Akanthomyces in Cordycipitaceae, mostly as Akanthomyces cf. tuberculatus. The aims of this study were (1) to elucidate the relationships of these collections to known members of Cordycipitaceae, (2) to uncover hidden species in A. tuberculatus species complex, and (3) to describe new taxa to accommodate species diversity in Akanthomyces.

Fungal materials and isolation
The specimens used in this study were obtained from BIOTEC Culture Collection (BCC) and BIOTEC Bangkok Herbarium (BBH), Thailand. Fungal specimens were collected from several national parks in Thailand. Soil from the forest floor, leaf litter, undersides, and upper sides of the leaves were scanned for fungal growth on dead insects. Collected specimens were stored in plastic boxes, returned to the laboratory, and examined under a stereo microscope (Olympus SZ61). Isolation from the teleomorphs followed the method described by Luangsa-ard et al. (2018).
Isolation from the anamorphs was carried out using a sterilized inoculation needle to pick the conidia out from sporulating structures and then transfer them on to a PDA plate. These plates were stored in a plastic box chamber at room temperature, left overnight until the conidia germinated, and treated the same way as described in Luangsa-ard et al. (2018).

Colony growth and morphology
Fungal structures of both, anamorph and teleomorph, such as perithecia, asci, ascospores, synnemata, phialides, and conidia were mounted on glass slides with a drop of lactophenol cotton blue solution. Microscopic measurements of 50 individual fungal structures were obtained using a light microscope (Olympus CX31). Variability was provided as the mean ± standard deviation with absolute minima and maxima in parentheses. Detailed colony descriptions and morphological comparisons of some fungal structures were determined from cultures grown on PDA and OA for 14 days at 25 °C (Mongkolsamrit et al. 2018). The colors of specimens and cultures incubated were described and codified following the Online Auction Color Chart (www.boletales.com/2011/01/new-colour-chart-for-mycologists; abbreviated "OAC" herein). For DNA extraction purposes, starter cultures were grown on PDA for 2 weeks at 25 °C.

DNA extraction
Genomic DNA was extracted from fungal cultures on PDA using a modified CTAB method (Sung et al. 2001). About 600 µL of CTAB buffer was added to the microcentrifuge tube that contained fungal mycelium, which was ground with pestles and incubated at 65 °C for 1 h. Once the suspension had cooled down, 600 µL of chloroform:isoamyl alcohol (24:1) was added. The supernatant was gently mixed until an emulsion was obtained and centrifuged at 12,000 rpm for 20 min. The aqueous phase was transferred to a new sterile microcentrifuge tube. About 300 µL of cold isopropanol alcohol was added to precipitate DNA and left at -20 °C for 1 h. DNA was then separated from the solution by centrifugation at 4 °C and 12,000 rpm for 20 min. The pellet was washed in 200 µL of 70% cold ethanol and air-dried at room temperature. The DNA pellet was then dissolved in 50 µL of TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA pH 8.0) (Laessøe et al. 2013). The extracted DNA was stored at -20 °C before amplification (Chen et al. 2018).

PCR amplification and sequencing
Five nuclear loci regions, namely, internal transcribed spacers 1 and 2 along with the 5.8S rDNA (ITS), large subunit of the ribosomal DNA (LSU), translation elongation factor 1-α (TEF), the largest subunit of RNA polymerase II (RPB1), and the second largest subunit of RNA polymerase II (RPB2), were amplified and sequenced. PCR amplifications were conducted in a 25 µL volume consisting of 1× PCR buffer, 0.4 M betaine, 200 µM of each of the four dNTPs, 1 U Taq DNA polymerase (Thermo Scientific, USA), and 0.2 µM of each primer. The primer pairs used in this study were ITS5 and ITS 4 for ITS (White et al. 1990), LROR and LR5 for LSU (Vilgalys et al. 1994), 983F and 2218R for TEF (Rehner and Buckley 2005), CRPB1 and RPB1Cr for RPB1 (Castlebury et al. 2004), and 5F2 and 7cR for RPB2 (Liu et al. 1999). PCR amplifications were performed using a BioRad T100 thermal cycler following the procedure described in Luangsa-ard et al. (2005) for ITS and Sung et al. (2001) for the other gene regions. PCR products were visualized by ethidium bromide staining after gel electrophoresis of 4 µL of the product in 1% agarose gel (Luangsa-ard et al. 2004). The PCR products were quantified using a standard DNA marker of known size and weight.

Sequence alignment and phylogenetic analysis
Each DNA sequence was checked for ambiguous bases and assembled in BioEdit v.7.0.5.3 (Hall 2005). Additional sequences from previous studies (Kepler et al. 2017;Mongkolsamrit et al. 2018) were used as a dataset of taxa in Cordycipitaceae. Multiple sequence alignment was conducted with MUSCLE 3.6 software (Edgar 2004) and manually adjusted. The DNA sequences were compared to sequences in the GenBank database by BLAST search to determine the closest matches with Akanthomyces. The final sequence alignment of the combined dataset was used for analyses using maximum parsimony (MP), Bayesian inference, and maximum likelihood to infer their phylogenetic relationships.
MP analysis used PAUP4.0a116 (Swofford 2019), and heuristic searches were performed with 100 replicates of random sequence addition and tree bisection reconnection swapping algorithm. Bootstrap analysis was performed using the MP criterion with 1000 replications. MrModeltest 2.2 (Nylander 2004) was used to choose the best model of DNA substitution that fit the data. MrBayes (Ronquist and Huelsenbeck 2003) was used to determine the Bayesian phylogenetic inference with a general timereversible plus proportion-invariant plus gamma (GTR+I+G) model of DNA substitution as the best model. Maximum likelihood analysis was performed with RAxML-HPC2 on XSEDE in CIPRES Science Gateway 3.3 (https://www.phylo.org/) using a GTRCAT model of evolution with 1000 bootstrap replicates (Stamatakis 2014).

Multilocus phylogeny
A total of 55 new sequences from 11 specimens were obtained in this study (Table 1). ITS sequences were used in a preliminary study to select 11 specimens that represent new species. The combined dataset included 101 taxa and four loci consisting of 3511 bp (LSU 850 bp, TEF 1041 bp, RPB1 732 bp, and RPB2 888 bp). Purpureocillium lilacinum in Ophiocordycipitaceae was used as the outgroup for this dataset.
The phylogenetic analyses were run using a combined dataset comprising four loci: LSU, TEF, RPB1, and RPB2. The combined dataset included 3511 characters, of which 2053 characters were constant, 231 were parsimony-uninformative, and 1227 were parsimony-informative. Gaps were treated as missing data. The maximum parsimony analyses resulted in 31 equally most parsimonious trees, of which one is shown in Figure 1   - Romaleidae; Tropidacris cristata JN049843 Air above sugarcane field -AF339517 c EF468798 c -- Note. The accession numbers in bold font refer to sequences generated in this study. Strain numbers with T are type species.
References. a Chaverri et al. (2005) Etymology. Referring to the host (Noctuidae, Lepidoptera) where the fungus was found.
Culture characters. Colony on PDA growing with a diameter of 20-24 mm in 14 days, circular, flat to raised, entire edges, white (OAC909) and fluffy mycelium. Colony reverse cream (OAC814). Colony on OA growing with a diameter of 20-25 mm in 14 days, circular, flat to raised, entire, white (OAC 909) and fluffy mycelium. Colony reverse uncolored. Conidia and reproductive structures not observed on both, PDA and OA in 14 days.
Distribution. Thailand, known from various national parks throughout the country. Ecology. All specimens are found on the underside of leaves of plants. Notes. Akanthomyces tortricidarum is found only in its anamorph state. This species differs from A. noctuidarum by having smaller conidia (2-3 × 1 µm) than A. noctuidarum (3-6 × 1 µm). Furthermore, the shape of conidia of A. tortricidarum is fusoid, while conidia of A. noctuidarum is cylindrical with a round end.

Discussion
The genus Akanthomyces established by Lebert (1858) was revised by Mains (1950). This genus is characterized by cylindrical synnemata covered by a hymenium-like layer of phialides producing single-celled catenulate conidia (Samson 1974). Presently, 20 Akanthomyces species have been formally described (Kepler et al. 2017;Mongkolsamrit et al. 2018), while eight species of Akanthomyces on spiders were transferred to the genus Hevansia. Hevansia includes the type species Hevansia novoguineensis (previously described as Akanthomyces novoguineensis), which differs from Akanthomyces by the immersed perithecia in a disk sitting at the top of a well-formed stipe. However, now it has to be an akanthomyces-like teleomorph (Kepler et al. 2017). Akanthomyces is considered as a synonym of Lecanicillium, an anamorph within Cordycipitaceae with verticilliumlike morphologies . Lecanicillium does not form a single monophyletic clade and species within this genus are distributed throughout Cordycipitaceae (Sukarno et al. 2009). Based on the molecular analyses from five nuclear genes (SSU, LSU, TEF, RPB1, and RPB2), Kepler et al. (2017) proposed that Lecanicillium should be rejected and Akanthomyces has priority by date over this genus. The type species of Lecanicillium, L. lecanii as well as some other species (L. attenuatum, L. muscarium, and L. sabanense) have phylogenetic affinities to Akanthomyces (Chiriví-Salomón et al. 2015) The type species of Akanthomyces, A. aculeatus and another Akanthomyces species on moth, A. pistillariiformis (= A. tuberculatus), were the closest related species to the three new species described here. Two of three new species were found in their anamorph state. Fortunately, in A. noctuidarum both, teleomorph and anamorph are present in the same specimen. The anamorph comparison between some species within Akanthomyces is shown in Table 2. The conidia of A. noctuidarum and A. aculeatus are almost in the same size (A. noctuidarum; 3-6 × 1 µm, A. aculeatus; 3-6 × 2-3 µm). However, the conidial shape of A. noctuidarum is cylindrical with a round end while A. aculeatus is ellipsoid or obovoid. Akanthomyces noctuidarum has the smallest synnemata compared to all the others (A. noctuidarum; 650-1500 µm, A. aculeatus; 1-8 × 0.1-0.5 mm, A. tuberculatus; 1-6 mm × 50-300 µm). Akanthomyces noctuidarum also has smaller phialides than both aforementioned species (5-10 × 2-3 µm, A. aculeatus; 6-16 × 2.5-4 µm, A. tuberculatus; 7-10.5 × 2.7-3.5 µm) with cylindrical shape and papillate at the end.
Akanthomyces tortricidarum was distinguished from the others species by having two different types of synnemata. The long synnemata of A. tortricidarum are cylindrical to clavate with acute or blunt ends. The hyphae diverged in the upper portion of the synnema and repeatedly branched more or less dichotomously, whereas the phialides were terminal on the branches. At the lower portion of synnema, the phialides were produced either as lateral cells or frequently as terminal cells of short lateral branches produced along the entire length of the outer hyphae of the synnema. The production of phialides was abundant at the upper portion of the synnema, resulting in a compact hymenial layer, whereas the phialides at the lower portion of the synnema were scattered and well separated from each other. Unlike the long synnemata, the hymenium-like layer of phialides on the short synnemata was limited to its upper part and the lower portion was sterile, forming a stipe. In the upper portion of the short synnema, the hyphae diverged and repeatedly branched more or less dichotomously and terminated with phialides. However, at the lower portion, the outer longitudinal hyphae did not produce any lateral phialides or lateral branches bearing phialides. This character was similar to the genus Insecticola proposed by Mains (1950). However, Samson and Evans (1974) transferred all members of this genus to Akanthomyces because variations in these characters did not support the distinction. The shape of synnemata and arrangement of phialides from A. noctuidarum and long synnemata from A. tortricidarum were similar. Nevertheless, A. tortricidarum differs from A. noctuidarum by having smaller conidia (2-3 × 1 µm) than A. noctuidarum (3-6 × 1 µm). Furthermore, the shape of conidia in A. tortricidarum is fusoid, while the conidia of A. noctuidarum is cylindrical with rounded ends.