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.

Akanthomyces, entomopathogenic fungi, fungal taxonomy, multilocus phylogeny

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 (Gams and Zare 2001; 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.

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 verticillium-like morphologies (Gams and Zare 2001). 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.

The teleomorph comparison between some species within Akanthomyces is shown in Table 3. Akanthomyces noctuidarum and A. pyralidarum differed from A. tuberculatus by the size of ascospores, asci, and perithecia. Akanthomyces tuberculatus has smaller ascospores measuring 2–6 × 0.5–1 µm, whereas A. noctuidarum and A. pyralidarum have larger ascospores at 6–13 × 1 µm and 5–12 × 1 µm, respectively. However, all three of them have the same shape of ascospore and asci. Akanthomyces pyralidarum has the smallest size of asci (A. pyralidarum; 170–360 × 2–4 µm, A. noctuidarum; 170–550 × 2–4 µm, and A. tuberculatus; 300–600 × 4–5 µm). The shape of perithecia from A. noctuidarum is ovoid, while A. pyralidarum is ovoid to obpyriform and A. tuberculata is narrowly ovoid or conoid. Akanthomyes pyralidarum also has the smallest perithecia compared to the other species in the genus (A. pyralidarum; 290–650 × 150–340 µm, A. tuberculatus; 420–900 × 180–370 µm, A. sulphureus; 650–680 × 240–330 µm, A. thailandicus; 700–850 × 300–400 µm, and A. noctuidarum; 530–1000 × 290–425 µm). Moreover, A. sulphureus and A. thailandicus are found on spiders (Araneae) while the others were found on moths.

All strains from these species did not produce conidia or reproductive structures when grown on PDA and OA for 14 days at 25 °C. Nevertheless, one strain from A. pyralidarum (BCC 29197) started to produce a synnemata-like structure on OA after 28 days. However, this synnemata-like structure was sterile and did not produce any phialides or conidia. Overall, fungal growth was faster in OA than in PDA.

This research was supported by the Platform Technology Management Section, National Center for Genetic Engineering and Biotechnology (BIOTEC), Grant No. P19-50231, National Science and Technology Development Agency (NSTDA), Thailand. We thank Natnapha Phosrithong for insect identification. We are indebted to the Department of National Parks, Wildlife and Plant Conservation of Thailand for their cooperation and support for our project research. Arifah Nur Aini and Anto Budiharjo thank to Diponegoro University - Indonesia for providing RPIBT grant No. 387–03/UN7.P4.3/PP/2018.