Morphological and Phylogenetic Characterization Reveals Five New Species of Samsoniella (Cordycipitaceae, Hypocreales)

Samsoniella is a very important fungal resource, with some species in the genus having great medical, economic and ecological value. This study reports five new species of Samsoniella from Yunnan Province and Guizhou Province in Southwestern China and Dole Province in Vietnam, providing morphological descriptions, illustrations, phylogenetic placements, associated hosts and comparisons with allied taxa. Based on morphological observations and phylogenetic analyses of combined nrSSU, nrLSU, tef-1α, rpb1 and rpb2 sequence data, it was determined that these five new species were located in the clade of Samsoniella and different from other species of Samsoniella. The five novel species had morphologies similar to those of other species in the genus, with bright orange cylindrical to clavate stromata (gregarious). The fertile part lateral sides usually had a longitudinal ditch without producing perithecia, and superficial perithecia. The phialides had a swollen basal portion, tapering abruptly into a narrow neck and oval or fusiform one-celled conidia, often in chains. The morphological characteristics of 23 species in Samsoniella, including five novel species and 18 known taxa, were also compared in the present study.


Introduction
Samsoniella was established by Mongkolsamrit et al. (2018) based on morphological and molecular evidence to accommodate three isaria-like species: the type species S. inthanonensis and two other species, S. alboaurantia and S. aurantia [1]. Samsoniella species were characterized by the formation of bright orange, cylindrical to clavate stromata (gregarious). The fertile part lateral sides usually had a longitudinal ditch without producing perithecia, superficial perithecia. Or the formation of anamorphic synnemata, the phialides had a swollen basal portion, tapering abruptly into a narrow neck, conidia oval to fusiform, one-celled, often in chains [1,2]. In order to account for the phylogenetic diversity of isaria-like species and to segregate these isaria-like fungi from the Akanthomyces group, the genus Samsoniella was established [1]. Isaria Pers. was one of the oldest names for asexually typified genera in Cordycipitaceae; however, many entomogenous fungi morphologically similar to Isaria could be found distributed throughout Hypocreales [3]. In 2017, Kepler et al. revealed a polyphyletic distribution of Isaria species within Cordycipitaceae, proposed the rejection of Isaria and combined 11 species of Isaria into Cordyceps Fr., owing to the confusion surrounding the application of Isaria [4]. Two isolates of I. farinosa (CBS 240.32 and CBS 262.58) that remained genetically distant from CBS 111113 were renamed S. alboaurantium [1].
The species of Samsoniella have diverse biological characteristics. The genus currently contains 18 species (http://www.indexfungorum.org, accessed on 1 May 2022), among which S. hepiali is an essential medicinal fungus [1,2]. The chemical profile of S. hepiali is very similar to the profiles of Ophiocordyceps sinensis, and recent studies show that S. hepiali performs various biological pharmacological activities such as anti-cancer, analgesic and hypoglycaemic activity, and is a good substitute for O. sinensis [5]. The related species of S. hepiali may have similar pharmacological activities. However, no research on other members of the genus has yet been reported.
During surveys of entomopathogenic fungi from different regions in Yunnan Province, Guizhou Province of Southwestern China and Dole Province of Vietnam, five Samsoniella species were found and identified. Based on morphological evidence together with multigene (nrSSU, nrLSU, tef-1α, rpb1 and rpb2) sequence analyses, it was shown that these five new Samsoniella species were distinguished from other species of the genus. They were named S. coccinellidicola, S. farinospora, S. haniana, S. pseudotortricidae and S. sinensis. Furthermore, the morphological characteristics of 23 species in Samsoniella, comprising 5 novel species and 18 known taxa, were also compared.

Sample Collection and Isolation
The majority of the specimens used in this study were collected from Yunnan Province in China. Some specimens were collected from the Chu Yang Sin National Park of Dole Province in Vietnam. The specimens were noted and photographed in the fields. The sample was placed in an ice box and brought to the laboratory for preservation at 4 • C. To obtain axenic cultures, the stromata or synnemata were removed from the insect bodies and divided into 3-4 segments, each 2 mm long. The segments were immersed in 30% H 2 O 2 for 30 s and then soaked in sterilized water for 1 minute. After drying on sterilized filter paper, the segments were inoculated onto potato dextrose agar (PDA: fresh potato 200 g/L, dextrose 20 g/L, and agar 18 g/L) plates. The conidia of cordycipitoid fungi at the conidial masses were picked using an inoculating loop and spread on PDA plates containing 0.1 g/L streptomycin and 0.05 g/L tetracycline [2]. Pure cultures were incubated at room temperature (about 25 • C). After isolation into pure cultures, they were transplanted to a PDA slant and stored at 4 • C. The specimens were deposited in the Yunnan Herbal Herbarium (YHH) at the Institute of Herb Biotic Resources, Yunnan University. The strain was deposited at the Yunnan Fungal Culture Collection (YFCC) of the Institute of Herb Biotic Resources, Yunnan University.

Morphological Observations
For descriptions of the sexual morph, fruiting bodies were photographed and measured using an Olympus SZ61 (Tokyo, Japan) stereomicroscope. Stromata were sectioned at a thickness of ca. 40 µm with a freezing microtome and mounted in water or lactic acid cotton blue on a slide for microscopic studies and photomicrography. The micromorphological characteristics of the fungi, such as the perithecia, asci and ascospores, were examined using Olympus CX40 (Tokyo, Japan) and BX53 (Tokyo, Japan) microscopes. The circular agar blocks, circa 5 mm in diameter, from a colony were removed and placed on new PDA plates to observe the colony morphology. The colonies on PDA plates were cultured at 25 • C for 2 weeks, and the colony characteristics (size, texture and colour) were photographed with a Canon 700D camera. To observe the asexual morphological characteristics (e.g., conidiophores, phialides and conidia), Olympus CX40 and BX53 microscopes were employed.

DNA Extraction, PCR and Sequencing for Nuclear Genes
Total genomic DNA was extracted from axenic living cultures using the MiniBEST Plant Genomic DNA Extraction Kit (TaKaRa, Beijing, China), following the manufacturer's instructions. The nuclear ribosomal small subunit (nrSSU) was amplified with the primer pair nrSSU-CoF and nrSSU-CoR [6]. The nuclear ribosomal large subunit (nrLSU) was amplified with the primer pair LR5 and LR0R [7,8]. The translation elongation factor 1α (tef-1α) was amplified with the primers EF1α-EF and EF1α-ER [9,10]. The largest and second largest subunits of RNA polymerase II (rpb1 and rpb2) were amplified with the primers RPB1-5 F and RPB1-5 R, RPB2-5 F and RPB2-5 R, respectively [9,10]. In this study, five nuclear gene loci of all the samples were amplified, and the primers used were shown in Table 1. The above five pairs were synthesized by Kunming Xiuqi Technology Co., Ltd. Each 50 µL PCR included 25 µL of 2 × Taq PCR Master Mix (Tiangen Biotech Co., Ltd., Beijing, China), 0.5 µL of each forward and reverse primer (10 µM), 1 µL of genomic DNA and 23 µL of sterilized distilled water. The polymerase chain reaction (PCR) assay was performed as described by Wang et al. [11]. The PCR products were separated by electrophoresis in 1.0% agarose gels, purified using a Gel Band Purification Kit (Bio Teke Co., Ltd., Beijing, China) and then sequenced on an automatic sequence analyser (BGI Co., Ltd., Shenzhen, China). When the PCR products could not be sequenced directly, cloning was performed using a TaKaRa PMDTM18-T vector system (TaKaRa Biotechnology Co., Ltd., Dalian, China).

Phylogenetic Analyses
Phylogenetic analyses were performed based on the nrSSU, nrLSU, tef-1α, rpb1 and rpb2 sequences. The DNA sequences generated in this study were submitted to Gen-Bank. Reference sequences were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/, accessed on 1 May 2022). The specimen information and GenBank accession numbers were provided in Table 2. The sequences were aligned using the Clustal X2.0 (developted by European Bioinformatics Institute, Cambridge, the United Kingdom) and MEGA v6.06 (developted by Tokyo Metropolitan University, Tokyo, Japan) software with manual adjustment [12,13]. The aligned sequences of five genes were concatenated after sequence alignment and specific processing according to Wang et al. [2]. Phylogenetic analyses were conducted using the Bayesian Inference (BI) and the Maximum Likelihood (ML) methods employing MrBayes v3.1.2 and RAxML 7.0.3 [14,15]. The BI analysis was run on MrBayes v3.1.2 for five million generations using a GTR + G + I model determined by the jModelTest version 2.1.4 (developted by The University of Vigo, Vigo, Spain) [16]. The GTR + I was selected as the optimal model for the ML analyses, with 1000 rapid bootstrap replicates performed on the five-gene datasets.

Sequencing and Phylogenetic Analyses
The 92 taxa of eight genera-Akanthomyces, Amphichorda, Beauveria, Blackwellomyces, Cordyceps, Samsoniella, Simplicillium and Trichoderma-were used for the ML and BI phylogenetic analyses. Two Trichoderma strains (Trichoderma deliquescens ATCC 208838 and Trichoderma stercorarium ATCC 62321) were designated as the outgroup. The concatenated sequence dataset of the five genes consisted of 4642 bp of sequence data (1055 bp for nrSSU, 897 bp for nrLSU, 969 bp for tef-1α, 756 bp for rpb1 and 965 bp for rpb2). Both phylogenetic trees from the BI and ML analyses exhibited similar topologies that had seven recognized, statistically well-supported clades in Cordycipitaceae, designated as Akanthomyces, Amphichorda, Beauveria, Blackwellomyces, Cordyceps, Samsoniella and Simplicillium ( Figure 1). Most of the well-resolved genera and lineages in Cordycipitaceae shared similar relationships with previous analyses [1, 4,10]. The 12 samples of five undescribed species also clustered in the genus Samsoniella clade based on the phylogenetic analyses of the combined dataset and were clearly distinct from S. hepiali and 16 described species, viz., S. alboaurantia, S. alpina, S. antleroides, S. aurantia, S. cardinalis, S. coleopterorum, S. cristata, S. hymenopterorum, S. inthanonensis, S. kunmingensis, S. lanmaoa, S. pseudogunii, S. pupicola, S. ramosa, S. tortricidae and S. yunnanensis ( Figure 1). Similarly, phylogenetic relationships between the genus Samsoniella and closely related species, based on multigene dataset (nrLSU, nrSSU, tef-1α, rpb1 and rpb2) (see Figure 2). Both phylogenetic trees from the BI and ML analyses exhibited similar topologies and the five undescribed species also clustered in the genus Samsoniella clade that were clearly distinct from S. hepiali and 16 described species.

Taxonomy
The key morphological characteristics that distinguish the current Samsoniella species were summarized in the literature (Tables 3 and 4). Including the five new species, there were 23 species of Samsoniella involved in the current study, among which we compared 9 species of the sexual morphs in Samsoniella (Table 3) and 22 species of the asexual morphs in Samsoniella (Table 4).
Host: Larvae of Lepidoptera, Dermaptera. Habitat: On the larvae of Lepidoptera clinging to fallen leaves or on Dermaptera clinging to fallen leaves. Previous studies of cordycipitaceous isaria-like fungi showed that species of Samsoniella were globally distributed generalist entomopathogen that were soilborne and had relatively complicated hosts, including Lepidoptera (Hepialidae, Noctuidae, Limacodidae, Saturniidae and Tortricidae), Coleoptera (Curculionidae), Hymenoptera (Formicidae and Vespidae) and two fungi (O. sinensis and C. cicadae) [1, 2,41]. Here, an extension of the host range was identified, also including Araneae, Dermaptera and Coccinellidae of Coleoptera, as shown in Figure 1 and Table 2. Among the hosts of Samsoniella species, Lepidoptera was the major order ( Table 2). Because of their broad host range and wide geographical distribution, some species of Samsoniella may have high potential for the interspecific transmission and biological control of pest insects. Additional research is needed to determine the effectiveness of isolates in the field.