﻿Morphology and multigene phylogeny reveal three new species of Samsoniella (Cordycipitaceae, Hypocreales) from spiders in China

﻿Abstract The genus Samsoniella was erected based on orange cylindrical to clavate stromata, superficial perithecia and conidiophores with Isaria-like phialides and to segregate them from the Akanthomyces group. In this study, based on morphological features and multigene (SSU, LSU, TEF, RPB1 and RPB2) phylogenetic analysis six Samsoniella species parasitizing spiders were collected in China. Three of them belong to known species S.alpina, S.erucae and S.hepiali. Three new species S.anhuiensissp. nov., S.araneasp. nov. and S.fusiformisporasp. nov. are illustrated and described. They are clearly distinct from other species in Samsoniella occurring in independent subclades. Furthermore, among the four insect-pathogenic fungi specimens collected from similar sites, three of them were identified as the new species described below. Our study significantly broadens the host range of Samsoniella from Insecta to Arachnida, marking a noteworthy expansion in understanding the ecological associations of these fungi. Additionally, the identification of both mononematous and synnematous conidiophores in our study not only expands the knowledge of Samsoniella species but also provides a basis for future research by comparing the ecological significance between these conidiophore types. In conclusion, our study enhances the understanding of Samsoniella diversity, presenting a refined phylogenetic framework and shedding light on the ecological roles of these fungi in spider parasitism.

Over the past seven years, there has been extensive research on the species diversity within the genus Samsoniella, possibly driven by the significant medical and ecological value associated with certain species in the genus.In a follow-up study, Wang et al. (2020a) documented nine new species within the genus Samsoniella.Specifically, Paecilomyces hepiali Chen, formerly misconstrued as the asexual counterpart of Ophiocordyceps sinensis, demonstrated the ability to produce Isaria-like phialides.The perplexing taxonomic status of P. hepiali prompted taxonomists to reconsider its classification.Wang et al. (2020a) determined that the most suitable systematic position for P. hepiali is within the genus Samsoniella.Consequently, they proposed the new taxonomic combination S. hepiali for this species.Subsequently, Chen et al. (2020) described three additional species of Samsoniella.Furthermore, phylogenetic analysis led to the repositioning of strains previously identified as I. farinosa.Notably,strains CBS 240.32 and CBS 262.58 were integrated into the genus Samsoniella and redesignated as S. alboaurantia (Mongkolsamrit et al. 2018;Chen et al. 2021).Similarly, strains OSC 111005 and OSC 111006 were reassigned to S. farinosa Wang (Wang et al. 2020b).More recently, Chen et al. (2021Chen et al. ( , 2022Chen et al. ( , 2023)), Wang et al. (2022), Wang et al. (2023) and Crous et al. (2023) contributed descriptions of fifteen additional novel Samsoniella species.Consequently, the genus Samsoniella now comprises a total of thirty-one recognized species.
We carried out a series of surveys for spider pathogenic fungi in China.A total of seven spider cadavers infected by Samsoniella were collected and isolated.Based on morphological and molecular phylogenetic analyses, three were identified as S. alpina, S. erucae, and S. hepiali.However, the other four strains represented four new species, which are described here as S. anhuiensis sp.nov., S. aranea sp.nov.and S. fusiformispora sp.nov.Among the four insect-pathogenic fungi specimens collected from the same sites, three of them were identified as the new species described below.Our study enhances the understanding of Samsoniella diversity, presenting a refined phylogenetic framework and shedding light on the ecological roles of these fungi in spider parasitism.

Sample collection, isolation and morphological observations
The majority of spider specimens infected by fungi were collected from all over China.Four specimens were collected from the Jingting Mountains National Forest Park, Anhui Province, southeastern China.Four specimens were collected from the Jinggang Mountains National Nature Reserve, Jiangxi Province, southeastern China.One specimen was collected from the Maiji National Forest Park, Gansu Province, northwestern China.One specimen was collected from the Yaoluoping National Forest Park, Anhui Province, southeastern China, and one specimen was collected from the Wanfo Mountains, Anhui Province, southeastern China.Several insect specimens infected by fungi were collected from sites similar to those where spider specimens were collected.The collections were noted and photographed in the field, then carefully deposited in plastic boxes and returned to the laboratory.Fungal cultures were isolated from fresh conidia or mycelia from spider cadavers.Pure cultures were established and incubated on fresh potato dextrose agar (PDA) plates and grown at 25 °C for 2 weeks.The fresh structures of specimens and isolated strains were mounted in water for measurements and lactophenol cotton blue solution for microphotography following Wang et al. (2020a).Features such as size and shape of conidia, colony color in culture, were made from squash mounts and sections made from fresh specimen and culture grown on oatmeal agar (OA, Difco), PDA and one quarter strength SDAY (SDAY/4, Difco) (Bischoff et al. 2009).The color of the cultures was characterized using the Naturalist's Color Guide (Smith 1975).Microscopic observations were made from squash mounts and sections made from fresh material using a ZEISS Axiolab 5 microscope.All samples and strains studied here were deposited in the Research Center for Entomogenous Fungi (RCEF) of Anhui Agricultural University.

Sequence alignment and phylogenetic analyses
The sequences in this study were uploaded to BLAST and searched in the GenBank database to determine probable taxa.DNA sequences generated in this study were assembled and edited using version 6.0.DNASTAR.Generated SSU, LSU, TEF, RPB1 and RPB2 sequences were aligned with those published by Chen et al. (2020) and Wang et al. (2020a) and others downloaded from Gen-Bank were used as a dataset of taxa in Samsoniella and closely related Samsoniella groups (Table 1).Sequences of the genus Akanthomyces (A. aculeatus HUA772 and HUA 186145) were chosen as the outgroup.Multiple sequence alignment was conducted with MAFFT 7.3.13(Katoh and Standley 2013).The final sequence alignment of the combined dataset was used for analyses using Maximum Likelihood (ML) and Bayesian Inference (BI) to infer their phylogenetic relationships.
Phylogenetic inference was done according to Maximum Likelihood (ML) using RAxML version 8 (Stamatakis 2014) and Bayesian Inference (BI) using MrBayes v.3.2 (Ronquist et al. 2012).For the ML analysis, we used the GTR-CAT model for all partitions, in accordance with recommendations in the RAx-ML manual against the use of invariant sites and 1000 rapid bootstrap replicates.The GTR+I+G model was selected by MrModeltest 2.2 (Darriba et al. 2012) as the best nucleotide substitution model for the Bayesian analysis.Four MCMC chains were executed simultaneously for 2000,000 generations, sampling every 100 generations.Finally, phylogenetic trees were visualized using the Interactive Tree of Life (iTOL) (https://itol.embl.de)online tool (Letunic and Bork 2016).Boldface: data generated in this study.

Phylogenetic analysis
To determine the phylogenetic relationship between these fungi and allied species from NCBI we constructed a phylogenetic tree based on Maximum Likelihood (ML) and Bayesian analysis, based on concatenated sequences of five genes included 89 taxa, comprising 4491 characters (SSU: 1047bp, LSU: 849 bp, TEF: 945bp, RPB1: 717 bp, RPB2: 933bp).The multi-gene phylogenetic tree consisted of four genera belonging to the family Cordycipitaceae, including Akanthomyces, Beauveria, Cordyceps and Samsoniella, with strong support (100%).Statistical support (≥75%/0.75) is shown at the nodes for ML bootstrap support/BI posterior probabilities and the strains' numbers are noted after each species' name (Fig. 1).
Habitat.Occurring on spider attached to the upperside of tree leaf.Notes.Samsoniella anhuiensis was easily identified as belonging to Samsoniella based on the phylogenetic analyses (Fig. 1).Based on the combined multigene dataset, S. anhuiensis has an independent branch and has a close relationship with S. tiankengensis.However, colonies of S. tiankengensis exhibit a faster growth rate on PDA compared to S. anhuiensis, displaying white to light pink colonies with a light yellowish reverse.In contrast, colonies of S. anhuiensis appear light yellowish and take on a flesh-pink hue at 30 days on 1/4 SDAY, with a yellowish center in reverse.Notably, S. anhuiensis distinguishes itself from S. tiankengensis through the presence of larger spherical, elliptical to ovoid conidia (Table 2).
Habitat.Occurring on spiders in the litter layer.Notes.Samsoniella aranea was readily classified within the genus Samsoniella through phylogenetic analyses (Fig. 1).Analysis of the combined multigene dataset unveiled that S. aranea forms an independent branch and shares a close relationship with S. yunnanensis.However, notable distinctions were observed between the two species.Unlike S. yunnanensis, where synnemata arise from insect cocoons, synnemata of S. aranea were not observed.Additionally, distinct growth characteristics were noted, with colonies of S. yunnanensis exhibiting a faster growth rate on PDA compared to S. aranea.Morphological differences were evident in the colonies on PDA, with S. aranea colonies being floccose, white to cream-yellowish, and having a yolk-yellowish reverse.On the other hand, colonies of S. yunnanensis were described as loose and hairy, appearing white with a reddish-brown reverse.Description.Sexual morph: Undetermined.Asexual morph: Isaria-like.Synnemata multiple, unbranched, arising from the whole body of spider, 3-6 mm long, Stipes cylindrical or clavate, 0.5-1.0mm wide, pale yellowish, white conidia produced from the synnemaya and hyphal layer (Fig. 4A).Phialides verticillate, in whorl of 2-5, 5.0-12.0× 1.9-2.8μm, with basal portion swollen to ellipsoidal, tapering into a distinct neck, 2.3 -3.8 × 0.5-1.2μm.Conidia in chains, fusiform, aseptate, hyaline, 2.1-3.5 × 1.6-2.2μm.
Habitat.Occurring on spider attached to the underside of tree leaf.Notes.Samsoniella fusiformispora was unequivocally identified as a member of the Samsoniella genus through phylogenetic analyses (Fig. 1) and was found to share a close relationship with S. coleopterorum.However, upon further investigation and comparison of the morphological characteristics of the three new species with other related Samsoniella species (Table 2), distinct differences emerged.Colonies of S. fusiformispora were noted to be white to yellowish, with a pale yellowish edge transitioning to an orange center in reverse.In contrast, colonies of S. coleopterorum were observed to be white, with a yellowish reverse.

Discussion
The typical characteristics of Samsoniella were oval to fusiform conidia, bright red-orange stromata of the sexual morphs and synnemata of the asexual morphs (Chen et al. 2020).In this study, we present a phylogenetic investigation of cordycipitaceous Isaria-like fungi pathogenic on spiders.Combined with microscopic characteristics and phylogenetic analysis based on multi-locus sequence data, S. fusiformispora, S. aranea and S. anhuiensis were described and illustrated as new species in Samsoniella.It was found that the hosts of most reported Samsoniella species are Lepidoptera larvae or pupae, while the host of S. coleopterorum is a snout beetle (Curculionidae), and the host of S. hymenopterorum is a bee (Mongkolsamrit et al. 2018;Chen et al. 2020;Wang et al. 2020a).However, it should be noted that Wang et al. (2020a) described the host of S. hymenopterorum as being "Bee, family Vespidae".The family Vespidae are wasps, not bees.Our study has expanded the hosts of Samsoniella from Insecta to Arachnida.
Generally, the phialides of S. fusiformispora were longer and thinner than those of the closely-related S. coleopterorum while they also had bigger typical fusiform conidia with greater length to width ratio.In the ML and BI phylogenetic trees, S. aranea was inferred as a phylogenetic sister of S. yunnanensis with strong support (93%/1.00) and distinct from other related species in Samsoniella.The synnemata of S. aranea was not observed, but S. yunnanensis has gregarious, flexuous and fleshy synnemata arising from the limacodid cocoons (Wang et al. 2020a).Furthermore S. yunnanensis has smaller fusiform to oval conidia than S. aranea and the colonies on PDA grow faster than S. aranea.Similarly, S. anhuiensis was easily separated by the phylogenetic analyses with independent branches in the phylogenetic tree.Kepler et al. (2017) found that sequences of Isaria sp.spat 09-050 and Isaria sp.spat 09-051 were firstly obtained, and two strains were clustered as the phylogenetic sister of Isaria spp. with 100 bootstrap proportion in the weighted parsimony (WP) analytic tree based on five genes (SSU, LSU, TEF, RPB1 and RPB2), which was classified as Akanthomyces group.Then Wang et al. (2020a) constructed the multigene phylogenetic tree studied the new taxa of the family Cordycipitaceae and the new systematic position of the Chinese cordycipitoid fungus Paecilomyces hepiali.In this multigene phylogenetic tree, Isaria sp.spat 09-050 and Isaria sp.spat 09-051 were clustered in genus Samsoniella as sister group of S. vallis but in two independent branches.In this study, we obtained the same results.We convinced that Isaria sp.spat 09-050 and Isaria sp.spat 09-051 is an unpublished new species of the Samsoniella, should be revised to Samsoniella sp.spat 09-050 and Samsoniella sp.spat 09-051.
In this study, based on morphological characteristics and five loci phylogenetic analysis, S. anhuiensis, S. aranea and S. fusiformispora were separated from other Samsoniella species, which are described here as new species.The strain RCEF0643 was identified as S. alpina, the strain RCEF1481 was named as S. hepiali, and the strains RCEF2592 and RCEF 2590 was identified as S. erucae.Furthermore, our study significantly broadens the host range of Samsoniella from Insecta to Arachnida, marking a noteworthy expansion in understanding the ecological associations of these fungi.Additionally, the identification of both mononematous and synnematous conidiophores in our study not only expands the knowledge of Samsoniella species but also provides a basis for future research by comparing the ecological significance between these conidiophore types.

Table 1 .
Species, strain numbers, accession numbers and origins of Samsoniella and related taxa used in this study, new sequences were shown in bold.

Table 2 .
(Wang et al. 2022parison of three new species with other related Samsoniella species(Wang et al. 2022).