Morphological and phylogenetic characterisations reveal three new species of Samsoniella (Cordycipitaceae, Hypocreales) from Guizhou, China

Abstract Samsoniella species have been found on lepidopteran larvae or pupae buried in soil or leaf litter. Three new species, Samsoniella hymenopterorum, S. coleopterorum and S. lepidopterorum, parasitic on hymenopteran larvae, coleopteran larvae and lepidopteran pupae, respectively, are reported. Morphological comparisons with extant species and DNA-based phylogenies from analysis of a multigene (ITS, RPB1, RPB2 and TEF) dataset supported the establishment of the new species. Unusually, all three new species have mononematous conidiophores. The new species are clearly distinct from other species in Samsoniella occurring in separate subclades.


Specimen collection and identification
Three fungus-infected insect specimens were collected from Xishui County (28°29'56.70"N, 106°24'31.04"E) (A1950 and A1952) and Dali,Rongjiang County (26°01'58.70"N,108°24'48.06"E) (DL1007), Guizhou Province, on 20 July and 1 October 2018, respectively. Isolation of the fungi was done as described by Chen et al. (2019). The surface of the specimens was rinsed with sterile water, followed by surface sterilisation with 75% ethanol for 3-5 sec. A part of the insect body was cut off and inoculated with haemocoel on potato dextrose agar (PDA) and PDA, to which 1% w/v peptone (PDAP) had been added. Fungal colonies emerging from specimens were isolated and cultured at 22 °C for 14 d under 12 h light/12 h dark conditions following protocols described by Zou et al. (2010). Accordingly, strains A19501, A19502, A19521, A19522, DL10071 and DL10072 were obtained. The specimens and the isolated strains were deposited in the Institute of Fungus Resources, Guizhou University (formally Herbarium of Guizhou Agricultural College; code, GZAC), Guiyang City, Guizhou, China.
Macroscopic and microscopic morphological characteristics of the fungi were examined and growth rates determined from PDA cultures incubated at 25 °C for 14 d. Hyphae and conidiogenous structures were mounted in lactophenol cotton blue or 20% lactate solution and observed with an optical microscope (OM, DM4 B, Leica, Germany).

DNA extraction, PCR amplification and nucleotide sequencing
DNA extraction was carried out in accordance with Liang et al. (2011). The extracted DNA was stored at −20 °C. Translation elongation factor 1 alpha (TEF) and RNA polymerase II largest subunit 2 (RPB2) genes were amplified using 983F/2218R and RPB2-5F/RPB2-7Cr primers, according to van den Brink et al. (2012). The RNA polymerase II largest subunit 1 (RPB1) gene was amplified with the primer pair CRPB1 and RPB1-Cr (Castlebury et al. 2004). The internal transcribed spacer (ITS) region was amplified by PCR using ITS4/ITS5, which was described by White et al. (1990). PCR products were purified using the UNIQ-10 column PCR products purification kit (no. SK1141; Sangon Biotech (Shanghai) Co., Shanghai, China) in accordance with the manufacturer's protocol and sequenced at Sangon Biotech (Shanghai) Co. The resulting sequences were submitted to GenBank.
Maximum Likelihood (ML) analyses were constructed with RAxMLGUI (Silvestro and Michalak 2012). The GTRGAMMA model was used for all partitions, in accordance with recommendations in the RAxML manual against the use of invariant sites. For Bayesian Inference (BI), a Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities using MrBayes v.3.2 (Ronquist et al. 2012) for the combined sequence datasets. The selection of the best-fit nucleotide substitution model for each locus was calculated by the Akaike Information Criterion (AIC) with jModelTest 2 (Darriba et al. 2012). The TIM+I+G model was selected for the concatenated ITS+RPB1+RPB2+TEF sequences. The Bayesian analysis resulted in 20,001 trees after 10,000,000 generations. The first 4,000 trees, representing the burn-in phase of the analyses, were discarded, while the remaining 16,001 trees were used for calculating posterior probabilities in the majority rule consensus tree. After the analysis was finished, each run was examined using the programme Tracer v1.5 (Drummond and Rambaut 2007) to determine burn-in and confirm that both runs had converged. The final alignment is available from TreeBASE under submission ID: 24710 (http://www.treebase.org).
Analysis 2: Samsoniella species and closely-related species. The RAxML analysis of the combined dataset (ITS+RPB1+RPB2+TEF) yielded a best scoring tree (Fig. 2) with a final ML optimisation likelihood value of -9,722.503130. Parameters for the GTR model of the concatenated data set were as follows: estimated base frequencies;   (Fig. 2), Samsoniella species were clustered in a clade and easily distinguished with Akanthomyces species. S. coleopterorum and S. lepidopterorum clustered in a clade (Fig. 2) and formed two independent branches. S. hymenopterorum was phylogenetically close to S. inthanonensis and S. aurantia. Description. Colonies on PDA, 3.6-4.0 cm diam. in 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose hyphal overgrowth, reverse yellowish. Prostrate hyphae smooth, septate, hyaline, 1.1-1.8 μm diam. Erect conidiophores usually arising from aerial hyphae, Isaria-like with phialides in whorls of two to four. Phialides 5.4-9.7 × 1.2-1.8 μm, with a cylindrical to ellipsoidal basal portion, tapering into a short distinct neck. Conidia in chains, hyaline, fusiform, ellipsoidal or subglobose, one-celled, 1.7-2.5 × 1.2-1.8 μm. Chlamydospores and synnemata not observed. Size and shape of phialides and conidia similar in culture and on natural substratum. Sexual state not observed.
Host. Snout beetle, family Curculionidae. Distribution. Xishui County, Guizhou Province, China. Etymology. Referring to its insect host, order Coleoptera. Remarks. Samsoniella coleopterorum was easily identified as belonging to Samsoniella based on the phylogenetic analyses (Fig. 1). Comparing with the typical characteristics of three species (Table 2), S. coleopterorum has a close relationship with S. aurantia by having cylindrical to ellipsoidal phialides and similar in size. However, it differs from S. aurantia by having shorter conidia and snout beetle host in the family Curculionidae. Based on the combined dataset of ITS, RPB1, RPB2 and TEF sequences, S. coleopterorum has a close relationship with S. lepidopterorum (Fig. 2). However, S. coleopterorum has cylindrical to ellipsoidal phialides, smaller fusiform to ellipsoidal conidia and a different host.  Description. Colonies on PDA, 6.2-6.4 cm diam. in 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose hyphal overgrowth, reverse yellowish. Prostrate hyphae smooth, septate, hyaline, 1.1-1.6 μm diam. Erect conidiophores usually arising from aerial hyphae, Isaria-like with phialides in whorls of three to four. Phialides 6.5-10.6 × 1.2-2.0 μm, with a cylindrical basal portion, tapering to a distinct neck. Conidia in chains, hyaline, fusiform to ovoid, 1-celled, 1.9-2.5 × 1.5-2.1 μm. Chlamydospores and synnemata not observed. Size and shape of phialides and conidia similar in culture and on natural substratum. Sexual state not observed.

Samsoniella lepidopterorum
Host. Pupa, order Lepidoptera Distribution. Rongjiang County, Guizhou Province, China Etymology. Referring to its insect host, order Lepidoptera Remarks. Samsoniella lepidopterorum was easily identified as belonging to Samsoniella, based on the phylogenetic analyses (Fig. 1). Based on the combined dataset of ITS, RPB1, RPB2 and TEF sequences (Fig. 2) and the typical characteristics of Samsoniella species (Table 2), S. lepidopterorum has a close relationship with S. coleopterorum. However, S. lepidopterorum has larger, ellipsoidal phialide conidia and its pupa host is in the order Lepidoptera.

Discussion
Phylogenetic analyses, based on the combined datasets of (ITS+RPB1+RPB2+TEF), suggest that the three new species are members of the Cordycipitaceae and belong to the genus Samsoniella (Fig. 1). Mongkolsamrit et al. (2018) noted that the typical characteristics of Samsoniella were oval to fusiform conidia, bright red-orange stromata of the sexual morphs and synnemata of the asexual morphs. The phialides in this genus range from cylindrical to possessing a swollen basal portion. S. coleopterorum, S. hymenopterorum and S. lepidopterorum all have cylindrical phialides and fusiform conidia. However, the three new species had mononematous conidiophores rather than synnemata. Synnematous entomopathogenic fungi (such as Gibellula spp.) can be found on abaxial leaf surfaces of shrubbery, forest floors and shallow soil layers (Hywel-Jones 1996). As air flow under the forest canopy is slow and humid, the dispersal of conidia through airflow diffusion may be difficult. Therefore, these entomopathogenic fungi may employ a particular strategy, such as producing synnemata and sticky conidia, to accommodate various arthropod activities and facilitate conidium spread (Abbott 2002). The three new species were located in the more open portion of the forest and this may favour the dispersal of dry conidia. Thus, we could speculate that the mononematous conidiophores of the three new species may be the result of a convergent evolution to adapt to the ecological environment.
The evolutionary dynamics of fungi and their hosts are usually described either by co-evolution or by host shifts. Shifts often occur to new hosts that are evolutionarily distant, but which occupy a common ecological niche (Vega et al. 2009). Nutrient requirements often determine whether host shifts occur (Vega et al. 2009). Relationships between insects and fungi have been described as biotrophy, necrotrophy and hemibiotrophy, inter alia. The common ancestor of Hypocreaceae and Clavicipitaceae corresponds to a departure from plant-based nutrition to one that specialises on animals and fungi (Spatafora et al. 2007). Prediction of the characteristics and evolutionary placement of any given member should be based on the correlation between molecular-phylogenetic genealogy and nutritional preferences (Spatafora et al. 2007;Vega et al. 2009). Species of Samsoniella were originally found on lepidopteran larvae or pupae buried in soil or leaf litter (Mongkolsamrit et al. 2018). Mongkolsamrit et al. (2018) also reported that the true range of host affiliations of Samsoniella in nature may not be currently represented. Here, we report Samsoniella spp. from coleopteran, hymenopteran larvae and lepidopteran pupae. The presence of different hosts indicates that the nutrient requirements of Samsoniella spp. can change with the environment (Spatafora et al. 2007).
In the present study, a four loci phylogenetic analysis showed that S. coleopterorum, S. lepidopterorum and S. hymenopterorum clustered in separate subclades from other Samsoniella species. They represent new taxa, based on morphological characteristics, nutritional preferences and phylogenetic analyses.  (2015) 337). We also thank Lesley Benyon, PhD, from Liwen Bianji, Edanz Group China (www. liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.