Fungus-infected insect specimens were collected from seven locations in 2016 and 2020, including three different locations within Yunnan Province, China, two locations within Lao Cai Province, Vietnam, one location within Oudomxay Province, Laos, and one location in Chiang Mai, Thailand. Teleomorph specimens were collected by carefully unearthing their hosts with a scoop and placing the samples in sterile bags. Conidia developing on insect cadavers were transplanted onto plates of potato dextrose agar (PDA; potato 200 g/L, dextrose 20 g/L, agar 20 g/L) and cultured at 25 °C. Colonies of the isolated filamentous fungi appearing in the culture were transferred onto fresh PDA media. The purified fungal strain was transferred to PDA slants and cultured at 25 °C until hyphae spread across the entire slope. Emerging fungal spores were washed with sterile physiological saline and made into a spore suspension of 1 × 10³ cells/mL. To obtain monospore cultures, a portion of the spore suspension was placed on PDA using a sterile micropipette, and then incubated at 25 °C. Teleomorph specimens were rinsed with tap water, washed with sterile distilled water, and then dried on sterile filter paper. A mass of ascospores and asci was removed from perithecia with a fine needle and placed in a drop of sterile water that was stirred with a different needle to distribute the elements on the slide. A portion of the drop containing ascospores was placed on PDA using a sterile micropipette, and then incubated at 25 °C. The purified fungal strains were maintained in a culture room at 25 °C or transferred to PDA slants and stored at 4 °C. Voucher specimens and the corresponding isolated strains were deposited in the Yunnan Herbal Herbarium (YHH) and the Yunnan Fungal Culture Collection (YFCC), respectively, of Yunnan University, Kunming, China.

Macro-morphological characteristics, including the host, geographical location, color and shape of the stromata, and perithecial orientation (superficial, immersed, or semi-immersed; ordinal or oblique) were examined under a dissecting microscope (SZ61, Olympus Corporation, Tokyo, Japan). For morphological evaluation, microscope slides were prepared by placing mycelia from the cultures on PDA blocks (5 mm in diameter) and then overlaid with a coverslip. Medan dye solution was used to stain asci and ascospores. Other structures were mounted in water. The sizes and shapes of the microcharacteristics (e.g., asci, ascospores, conidiogenous cells, and conidia) were determined using a light microscope (CX40, Olympus Corporation, Tokyo, Japan) and a scanning electron microscope (Quanta 200 FEG, FEI Company, Hillsboro, USA). Individual length and width measurements were taken for 30–100 replicates, including the absolute minima and maxima.

Specimens and live axenic cultures were prepared for DNA extraction. Genomic DNA was extracted using a Genomic DNA Purification Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The phylogenetic positions of unknown Samsoniella isolates were evaluated with phylogenetic inferences based on five genes, namely, nr SSU, nr LSU, 3P_TEF, RPB1, and RPB2. The primer pair nrSSU-CoF and nrSSU-CoR (Wang et al. 2015) was used to amplify nr SSU; the primer pair LR5 and LR0R (Vilgalys and Hester 1990; Rehner and Samuels 1994) was used to amplify nr LSU, and the primer pair 983F and 2218R (Rehner and Buckley 2005) was used to amplify 3P_TEF. For amplification of the RPB1 and RPB2 genes, PCR primer pairs RPB1-5’F / RPB1-5’R and RPB2-5’F / RPB2-5’R (Bischoff et al. 2006; Sung et al. 2007) were employed. In total, seven DNA regions were evaluated as potential DNA barcodes for establishing species boundaries in Samsoniella because of their wide usage in phylogenetic studies of the sac-fungi. The markers included a fragment of the ITS region using primers ITS5 and ITS4 (White et al. 1990), a fragment of the 5P_TEF region using primers EF1T and EF2T (Rehner and Buckley 2005; Bischoff et al. 2006), a fragment of the 3P_TEF, RPB1 and RPB2 region with the same primer sets mentioned previously, a fragment of the ACT region using primers Act-1 and Act-4R (Voigt and Wöstemeyer 2000), a fragment of the TUB region using primers Bt2a and Bt1b (Glass and Donaldson 1995), and a fragment of the MCM7 region using primers Mcm7-709for and Mcm7-1348rev (Schmitt et al. 2009). All of the PCR reactions were performed in a final volume of 50 μL containing 25 μL 2 × Taq PCR Master Mix (Tiangen Biotech Co., Ltd., China), 0.5 μL of each primer (10 μM), 1 μL of genomic DNA, and 23 μL of RNase-Free water. PCR products were sequenced by Beijing Sinogenomax Co., Ltd., China.

Amplified fragments were sequenced in both directions using the same primer pairs used for amplification. All retrieved sequences from GenBank were combined with those generated in our study. The taxonomic information and GenBank accession numbers are provided in Suppl. material 1 and Table 1. Sequences were aligned using MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/). The aligned sequences were then manually corrected where necessary. Following alignment, the sequences of the five genes, namely, nr SSU, nr LSU, 3P_TEF, RPB1, and RPB2, were concatenated. Conflicts among the five genes were resolved using PAUP* 4.0b10 (Swofford 2002). The results showed that the phylogenetic signals for the five loci were congruent (P = 0.02). The best-fitting substitution model for these 11 partitions was calculated by using jModelTest version 2.1.4 (Darriba et al. 2012). The model GTR+G+I was used for partitions of nr SSU, nr LSU, 3P_TEF_pos1, 3P_TEF_pos2, 3P_TEF_pos3, RPB1_pos3, and RPB2_pos3, and the model GTR+I was used for partitions of RPB1_pos1, RPB1_pos2, RPB2_pos1, and RPB2_pos2. Bayesian posterior probabilities (BP) were estimated with the same partition parameters conducted in MrBayes v3.2.7a (Ronquist et al. 2012). Four Markov Chain Monte Carlo chains were run, each beginning with a random tree and sampling, one tree every 100 generations of 2,000,000 generations, and the first 25% of samples were discarded as burn-in. Maximum likelihood (ML) phylogenetic analyses were conducted in RAxML 7.0.3 (Stamatakis et al. 2008) with the recommended partition parameters, and 1000 rapid bootstrap replicates were performed on the dataset.

We applied a (phylo-) genetic distance matrix calculation for the candidate markers, namely, ITS, 3P_TEF, 5P_TEF, RPB1, RPB2, ACT, TUB, and MCM7, to assess species boundaries of 11 Samsoniella spp. (Suppl. material 2) because their sequence data for the eight loci were complete. The pairwise genetic distances of the 11 Samsoniella lineages were measured based on the Kimura two-parameter model using MEGA v6.06 software (Tamura et al. 2013). Only candidate markers with the mean threshold criteria (p-distances) > 0.01 were used to examine phylogenetic relationships between Samsoniella spp. and to assess species boundaries within the genus. As a result, four markers, namely, 3P_TEF, 5P_TEF, RPB1, and MCM7, were singled out. Phylogenetic analyses were based on the combined four-locus (5P_TEF + 3P_TEF + RPB1 + MCM7) sequences. The best-fitting nucleotide substitution model was determined using PartitionFinder V1.1.1 (Lanfear et al. 2012), resulting in three partitions (5P_TEF + 3P_TEF, RPB1, and MCM7). The following models were implemented in the Bayesian phylogenetic analyses: GTR + I + G for 5P_TEF + 3P_TEF and GTR + I for partitions of RPB1 and MCM7. The BI analysis was run on MrBayes v3.2.7a for five million generations. GTR + FO + G was selected as the optimal model for ML analysis, and 1000 rapid bootstrap replicates were performed on the dataset. ML phylogenetic analyses were conducted in RAxML 7.0.3 (Stamatakis et al. 2008). Additional ML analyses were performed using IQ-TREE v. 2.1.3 with ultrafast bootstrapping for the estimation of branch support (Minh et al. 2020). Further, ML analysis (RAxML) was applied to single-locus genealogies for 5P_TEF, 3P_TEF, RPB1, and MCM7.

The host insects of Samsoniella spp. were identified on the basis of morphological characteristics and further identified using molecular analyses based on the mitochondrial cytochrome oxidase I gene (COX1) and mitochondrial cytochrome b gene (CYTB). Genomic DNA was extracted from the head and leg areas of the cadavers of the host insects using the CTAB method (Liu et al. 2001). The COX1 and CYTB loci were amplified using primer pair Hep-cox1F / Hep-cox1R and primer pair Hep-cytbF / Hep-cytbR, respectively (Simon et al. 1994). Sequences were analyzed using MEGA v6.06 software and run through Standard Nucleotide BLAST (Genbank, NCBI nucleotide database) to assess similarity with reported insect sequences.

The 11 DNA loci were readily amplified and sequenced, and there was a fairly high success rate in this study. Phylogenetic analyses based on the combined five-gene (nr SSU + nr LSU + 3P_TEF + RPB1 + RPB2) sequences from 120 fungal taxa confirmed the presence and positions of Samsoniella and related genera within Cordycipitaceae. The concatenated five-gene dataset consisted of 4994 bp (nr SSU, 1134 bp; nr LSU, 901 bp; 3P_TEF, 1044 bp; RPB1, 759 bp; RPB2, 1156 bp). Eleven well-supported clades were recognized based on both BI and ML analyses of the combined dataset from Cordycipitaceae and Trichoderma, corresponding to the genera Akanthomyces, Amphichorda, Ascopolyporus, Beauveria, Blackwellomyces, Cordyceps, Gibellula, Hevansia, Samsoniella, Simplicillium, and Trichoderma as the outgroup (Fig. 1). A collection of eight isolates of unknown identity were shown to resolve in Samsoniella and to likely represent two known species and two new species of Samsoniella. Sequenced strains resolving in aforementioned distinct clades are thus proposed to represent the hereby newly erected species S. asiatica and S. sapaensis. The phylogenetic analyses suggested the existence of distinct species in the Samsoniella clade that we accordingly propose as new species: S. asiatica and S. sapaensis. These results also showed some ambiguous positions, including those of S. inthanonensis / S. lanmaoa, S. cristata / S. tortricidae, and S. coleopterorum / S. pseudogunii (Fig. 1). Although morphological observations revealed some differences in the characteristics between the three pairs of ambiguous species (Mongkolsamrit et al. 2018; Wang et al. 2020a; Chen et al. 2021), they were practically indistinguishable in the phylogeny based on the sequences of the five genes. This suggests that their status as distinct species is subjective and warrants further critical analyses, including the necessity for more DNA molecular markers.

The genetic divergence comparisons showed that: (1) the mean thresholds (p-distances) of ITS, RPB2, ACT, and TUB were lower than 0.01, indicating that neither were qualified as DNA markers; (2) the mean thresholds (p-distances) of 3P_TEF, 5P_TEF, RPB1, and MCM7 were > 0.01, and (3) the highest number of species was delimited in the genetic distance analysis for the 3P_TEF sequence data, followed by 5P_TEF, MCM7, and RPB1 sequences (Suppl. material 2).

The analyzed data matrix used to construct the phylogeny of Samsoniella species included sequences from 56 fungal taxa (Table 1). The final dataset consisted of 3130 bp of sequence data, including gaps (5P_TEF, 743 bp; 3P_TEF, 1023 bp; RPB1, 735 bp; and MCM7, 629 bp). Both BI and ML analyses produced trees with similar topologies that resolved most of the Samsoniella lineages in separate terminal branches (Fig. 2). The conservative results from phylogenetic analyses suggested that Samsoniella is composed of 26 species, namely, S. alboaurantium, S. alpina, S. antleroides, S. asiatica, S. aurantia, S. cardinalis, S. coccinellidicola (= S. guizhouensis), S. coleopterorum (= S. pseudogunii), S. cristata, S. erucae, S. farinospora, S. haniana, S. hepiali, S. hymenopterorum, S. inthanonensis, S. kunmingensis, S. lanmaoa, S. neopupicola, S. pseudotortricidae, S. pupicola, S. ramosa, S. sapaensis, S. sinensis, S. tiankengensis, S. tortricidae, and S. yunnanensis. The trees based on the combined five-locus sequences (nr SSU + nr LSU + 3P_TEF + RPB1 + RPB2) and the combined four-locus (5P_TEF + 3P_TEF + RPB1 + MCM7) sequences showed topological differences, especially in the clades composed of S. inthanonensis / S. lanmaoa and S. cristata / S. tortricidae (Figs 1, 2). However, the latter resolved these clades, suggesting that they should be regarded as different species.

Figure 2. 

Phylogenetic tree of Samsoniella based on Bayesian inference and Maximum Likelihood analyses of a 4-locus (5P_TEF, 3P_TEF, RPB1 and MCM7) dataset. No significant differences in topology are observed between BI and ML (IQ-TREE) phylogenies. Numbers at the branches indicate support values (BI-PP/RAxML-BS/IQ-TREE-BS) above 0.7/70%/70%. Isolates in bold type are those analyzed in this study.

The tree topologies for the individual loci (5P_TEF, 3P_TEF, RPB1, and MCM7) did not show congruence (Suppl. material 3). However, in all of the analyses, the newly discovered species S. asiatica had a close genetic relationship with S. yunnanensis. The RPB1 gene was unable to distinguish the two species (Suppl. material 3: fig. S3). However, they were regarded as different species with strong support from 5P_TEF, 3P_TEF and MCM7 (Suppl. material 3: figs S1, S2, S4). Phylogenetic analyses based on the combined data revealed that the distinctive species S. sapaensis was closely related to S. haniana, which is in agreement with the results obtained for 3P_TEF and RPB1.

Based on the results of the phylogenetic analyses and the morphological data, we add two new descriptions to the record of two known species and propose to erect two new species of Samsoniella.

 Samsoniella asiatica H. Yu bis, Y. Wang & Z.Q. Wang, sp. nov.

MycoBank No: 848022

Fig. 3

Etymology

Named after Asia (China, Vietnam and Laos), where the species was originally collected.

Figure 3. 

Samsoniella asiatica A fungus-infected lepidopteran pupa B stipes producing a mass of conidia at the apex C synnemata of fungus arising from lepidopteran larva D colony as obversed on PDA medium E–M conidiophores, phialides and conidia on PDA N conidia on PDA. Scale bars: 10 mm (A, C); 5 mm (B); 20 mm (D); 15 μm (E); 10 μm (F, G, H); 5 μm (I, J, K, N); 3 μm (L, M).

Type

China, Yunnan Province, Yuanyang County, Xinjie Town, Duoyishu Village (23°4′50″N, 102°48′34″E, 1866 m above sea level), on a pupa of Lepidoptera in a dead twig, 10 December 2021, Yao Wang (holotype: YHH 869; ex-type living culture: YFCC 869).

Description

Teleomorph: Undetermined. Anamorph: Synnemata arising from lepidopteran insects. Synnemata erect, flexuous, white or pale orange, 4–26 × 0.4–1.5 mm. Stipes cylindrical, producing a mass of conidia at the branches of synnemata, powdery and floccose. Colonies on PDA moderately fast-growing, 41–45 mm diameter in 14 days at 25 °C, white, cottony, generating several concentric rings at the centrum, sporulating abundantly, reverse white to pale yellow. Hyphae smooth-walled, branched, septate, hyaline, 1.3–2.0 µm wide. Conidiophores smooth-walled, cylindrical, solitary or verticillate, 4.6–10.3 × 0.8–1.9 µm. Phialides on conidiophores verticillate, usually in whorls of two to four, or solitary on hyphae, 2.7–8.6 µm long, basal portion cylindrical to narrowly lageniform, tapering gradually or abruptly toward the apex, from 0.7–1.7 µm wide (base) to 0.6–1.1 µm wide (apex). Conidia smooth and hyaline, fusiform or oval, one-celled, 1.1–1.8 × 0.8–1.2 µm, often in chains. Size and shape of phialides and conidia similar in culture and on natural substratum.

Distribution

Yunnan Province, China; Lao Cai Province, Vietnam; Oudomxay Province, Laos.

Additional materials examined

Vietnam, Lao Cai Province, Sa Pa District, Hoang Lien Mountains (22°21′4″N, 103°46′29″E, 1931 m above sea level), on a larva of Noctuidae buried in soil, 31 October 2016, collected by Hong Yu (YHH 871; living culture: YFCC 871); Laos, Oudomxay Province, Muang Xay County, Nagang Village (20°42′51″N, 102°5′44″E, 698 m above sea level), on a larva of Spilosoma, 29 July 2019, Yao Wang (YHH 870; living culture: YFCC 870).

Commentary

Morphologically, S. asiatica resembles the phylogenetically closely related sister species S. yunnanensis in producing orange to pink stipes, a mass of conidia toward the apex synnemata and Isaria-like asexual conidiogenous structure. Additionally, both of the fungal sexual morphs have not been determined yet. However, S. asiatica can be distinguished from S. yunnanensis by its shorter phialides (2.7–8.6 µm) and smaller conidia (1.1–1.8 × 0.8–1.2 µm). Ecologically, S. asiatica has been found to parasitize larvae and pupae of Lepidoptera, whereas S. yunnanensis is associated with pupae of Limacodidae in cocoons and Cordyceps spp. (Wang et al. 2020a). Both morphological and phylogenetic analyses support the idea that this fungus is a distinct species in the genus Samsoniella.

 Samsoniella aurantia Mongkolsamrit, Noisripoom, Thanakitpipattana, Spatafora & Luangsa-ard

MycoBank No: 823786

Fig. 4

Type

Thailand (holotype: BBH 33739; ex-type living culture: TBRC 7271).

Figure 4. 

Samsoniella aurantia A, B perithecial stromata as encountered in the field C, D fungus on the pupae of Limacodidae inhabiting cocoons E, F synnemata arising from lepidopteran larvae G, H Perithecia I–K asci L colony as obversed on PDA. Scale bars: 10 mm (C, D, F); 5 mm (E); 200 µm (G); 100 µm (H); 10 µm (I, J, K); 20 mm (L).

Description

Teleomorph: Stromata arising from lepidopteran insects, gregarious, branched or unbranched, up to 8.8–63.8 mm long. Stipes fleshly, flexuous, yellowish to orange, cylindrical to clavate, 4.1–49.1 × 0.2–2.3 mm. Fertile parts reddish orange, clavate, lateral side usually have a longitudinal section without producing perithecia, 3.8–17.7 × 0.8–4.1 mm. Perithecia crowded, superficial, narrowly ovoid to fusiform, 302.7–449.7 × 105.3–164.9 µm. Asci hyaline, cylindrical, 8‐spored, 92–190 × 1.8–3.6 μm. Apical caps prominent, hemiglobose, 2.1–3.4 µm wide, 1.2–2.3 µm high. Ascospores not observed. Anamorph: See Mongkolsamrit et al. (2018). The following descriptions are based on other specimens examined from China. Synnemata arising from lepidopteran larvae. Synnemata erect, flexuous, irregularly branched, white or pale orange, 7.7–32.6 × 0.2–2.1 mm. Stipes cylindrical, producing a mass of conidia at the branches of synnemata, powdery and floccose. Colonies on PDA moderately fast-growing, 26–30 mm diameter in 14 days at 25 °C, light orange to orange, consisting of a basal felt and cottony, sporulating abundantly at the centrum, reverse yellowish, turning deep yellow. Synnemata emerging after 25 days, solitary, unbranched. Size and shape of phialides and conidia similar to that of S. aurantia ex-type isolate (TBRC 7271).

Distribution

Chiang Mai Province, Thailand; Guizhou and Yunnan Province, China; Lao Cai Province, Vietnam.

Materials examined

China, Yunnan Province, Zhaotong City, Shuifu County, Taiping Town, Tongluoba National Forest Park (28°24′36″N, 104°9′0″E, 1750 m above sea level), on larvae of Hepialidae living in Qiongzhuea tumidinoda forests, 20 June 2015, collected by Hong Yu (YHH 874, YHH 890–YHH 893; living culture: 874). Vietnam, Lao Cai Province, Sa Pa District, Hoang Lien Mountains (22°21′8″N, 103°46′29″E, 1900 m above sea level), on a pupa of Limacodidae in a cocoon buried in soil, 31 October 2016, Hong Yu (YHH 880, YHH 894; living culture: YFCC 880). Thailand, Chiang Mai Province, Chiang Mai City, Queen Sirikit Botanic Garden (536 m above sea level), on lepidopteran larvae in leaf litter, 26 August 2018, Yao Wang (YHH 895–YHH 896).

Commentary

Numerous species of Samsoniella were described originally from asexual morphs, including S. aurantia from Thailand (Mongkolsamrit et al. 2018; Wang et al. 2020a; Chen et al. 2022; Wang et al. 2022). Chen et al. (2021) reported S. aurantia isolated from a lepidopteran pupa as a new record for China. However, its sexual morph was undetermined in these studies. The present study is the first to report the teleomorph reproductive stage for S. aurantia. Samsoniella aurantia has fleshy stromata, clavate fertile parts, superficial perithecia, and cylindrical asci with bola‐shaped ascospores. However, S. aurantia differs from other teleomorph species by its phylogenetic placement in the genus by having abundant long stromata extruded from the entire body of lepidopteran insects and by shorter asci measuring 92–190 µm.

 Samsoniella hepiali (Q.T. Chen & R.Q. Dai ex R.Q. Dai et al.) H. Yu, R.Q. Dai, Y.B. Wang, Y. Wang & Zhu L. Yang

MycoBank No: 833114

Fig. 5

Type

China (holotype: IMM 82-2 = CHICMM 82-2; ex-type living culture: ICMM 82-2).

Figure 5. 

Samsoniella hepiali A stromata of fungus arising from lepidopteran pupa B stromata and synnemata arising from lepidopteran pupa C, D perithecia E, F asci G colony as obversed on PDA medium H–L conidiophores, phialides and conidia on PDA. Scale bars: 10 mm (A, B); 300 µm (C); 100 µm (D); 20 µm (E, F); 20 mm (G); 10 µm (H, I); 5 µm (J, K).

Description

Teleomorph: Stromata from the whole body of lepidopteran pupae, gregarious, generally unbranched, up to 5–23 mm long. Stipes fleshly, flexuous or erect, yellowish to orange, cylindrical to clavate, 2.5–15.5 × 0.9–4.6 mm. Fertile parts orange, clavate, lateral side usually have a longitudinal section without producing perithecia, 1.3–8.5 × 0.8–5.2 mm. Perithecia crowded, superficial, narrowly ovoid to fusiform, 277.9–355.3 × 116.3–199.6 µm. Asci hyaline, cylindrical, 8‐spored, 145–300 × 3.5–5 μm. Apical caps prominent, hemiglobose, 2.5–4 µm wide, 2.4–3.2 µm high. Ascospores hyaline, bola‐shaped, septate, 120–240 × 0.8–1.5 μm. Anamorph: See Wang et al. (2020a). The following descriptions are based on other specimens examined from Vietnam. Synnemata arising from the whole body of lepidopteran pupae, branched or unbranched, 5–20 mm long. Stipes cylindrical or clavate, 0.6–4.2 mm wide, with powdery conidia at the apex, white to yellowish. Colonies on PDA moderately fast-growing, 32–45 mm diameter in 14 days at 25 °C, white to yellowish, cottony, with high mycelial density, reverse white to pale yellow, turning orange when old. Hyphae smooth-walled, branched, septate, hyaline, 0.9–2.3 µm wide. Conidiophores smooth-walled, cylindrical, solitary, 3.9–10.2 × 1.5–1.9 µm. Phialides on conidiophores verticillate, usually in whorls of two to five, or solitary on hyphae, 5.7–10.9 µm long, basal portion cylindrical to narrowly lageniform, tapering gradually or abruptly toward the apex, from 1.4–1.9 µm wide (base) to 0.5–0.9 µm wide (apex). Conidia smooth and hyaline, fusiform or oval, one-celled, 1.9–2.8 × 1.0–1.6 µm, often in chains. Size and shape of phialides and conidia similar in culture and on natural substratum.

Distribution

Yunnan, Qinghai, Anhui and Guizhou Province, China; Lao Cai Province, Vietnam; Buenos Aires City, Argentina.

Materials examined

Vietnam, Lao Cai Province, Sa Pa District, Hoang Lien Mountains (22°21′10″N, 103°46′29″E, 1989 m above sea level), on pupae of Hepialidae buried in soil, 30 October 2016, collected by Hong Yu (YHH 868, YHH 897–YHH 899; living culture: YFCC 868, YFCC 897–YFCC 899).

Commentary

The strain (YFCC 868) isolated from the pupa of Hepialidae from Vietnam formed a well-supported clade with S. hepiali ex-type isolate (ICMM 82-2) (Fig. 2). Based on microscopic observation, the strain YFCC 868 displayed typical morphological characteristics of anamorphs found in species of Samsoniella. For YFCC 868, the size and shape of phialides and conidia were similar to those of S. hepiali as described by Wang YB et al. (2020b). Both morphological study and phylogenetic analyses supported the isolate YFCC 868 as being S. hepiali.

In the current study, the sexual morph of S. hepiali was first reported. As for other teleomorph species of Samsoniella, S. hepiali has fleshy stromata, clavate fertile parts, superficial perithecia, and cylindrical asci with bola‐shaped ascospores. Among these species, only three, namely, S. cardinalis, S. hepiali, and S. kunmingensis, have short stromata (Wang et al. 2020a; Wang et al. 2022). However, S. hepiali differs from S. cardinalis and S. kunmingensis by having abundant stromata extruded from the entire body of lepidopteran insects and by having clavate fertile parts with orange color.

 Samsoniella sapaensis H. Yu bis, Y. Wang & Z.Q. Wang, sp. nov.

MycoBank No: 848023

Fig. 6

Etymology

Named after the location Sa Pa District where the species was collected.

Figure 6. 

Samsoniella sapaensis A stromata of fungus arising from lepidopteran larva B, C stromata and synnemata arising from lepidopteran insects D synnemata of fungus E, F perithecia G, H asci I, J colony as obversed and its backside on PDA K–M conidiophores, phialides and conidia on PDA N conidia on PDA. Scale bars: 10 mm (A, C); 5 mm (B); 20 mm (D); 100 µm (E, F, G); 50 µm (H); 20 mm (I); 30 mm (J); 5 µm (K, N); 10 µm (L, M).

Type

Vietnam, Lao Cai Province, Sa Pa District, Hoang Lien National Park (22°19′30″N, 103°46′50″E, 2178 m above sea level), on a larva of Lepidoptera buried in soil, 26 October 2017, collected by Hong Yu (holotype: YHH 873; ex-type living culture: YFCC 873).

Description

Teleomorph: Stromata arising from the whole body of lepidopteran pupae or larvae, gregarious, generally unbranched, up to 22–38 mm long. Stipes fleshly, flexuous, yellowish to orange, cylindrical to clavate, 7.5–14.5 × 0.7–4.6 mm. Fertile parts yellowish to reddish orange, clavate, lateral side usually have a longitudinal section without producing perithecia, 1.5–21.3 × 1.0–2.8 mm. Perithecia crowded, superficial, narrowly ovoid to fusiform, 383.2–412.1 × 125.4–156.9 µm. Asci cylindrical, hyaline, 282.5–444.5 × 2.6–3.9 µm, with a hemispheric apical cap of 1.8–2.2 × 2.6–3.0 µm. Ascospores not observed. Anamorph: Synnemata arising from lepidopteran insects. Synnemata flexuous, irregularly branched, white or pale orange, 9–58 × 0.2–1.6 mm, Isaria-like morph producing a mass of conidia at the branches of synnemata, powdery and floccose. Colonies on PDA moderately fast-growing, 36–40 mm diameter in 14 days at 25 °C, white to pale pink, cottony, sporulating abundantly, reverse yellow to orange. Hyphae smooth-walled, branched, septate, hyaline, 1.0–1.9 µm wide. Conidiophores smooth-walled, cylindrical, solitary or verticillate, 6.5–17.5 × 1.0–1.6 µm. Phialides verticillate, usually in whorls of two to three, or solitary on hyphae, 2.8–7.6 µm long, basal portion cylindrical to narrowly lageniform, tapering gradually or abruptly toward the apex, from 0.8–1.5 µm wide (base) to 0.6–0.9 µm wide (apex). Conidia smooth and hyaline, fusiform or oval, one-celled, 1.2–1.5 × 0.8–1.0 µm, often in chains. Size and shape of phialides and conidia similar in culture and on natural substratum.

Distribution

At present, known only in Sa Pa District, Lao Cai Province, Vietnam.

Additional materials examined

Vietnam, Lao Cai Province, Sa Pa District (22°21′4″N, 103°46′29″E, 1931 m above sea level), on a pupa of Limacodidae in a cocoon buried in soil, 31 October 2016, collected by Hong Yu (YHH 872; living culture: YFCC 872). Vietnam, Lao Cai Province, Sa Pa District, Hoang Lien National Park (22°19′30″N, 103°46′50″E, 2178 m above sea level), on larvae of Lepidoptera buried in soil, 26 October 2017, collected by Yuan-Bing Wang (YHH 900–YHH 906).

Commentary

Samsoniella sapaensis was identified as belonging to Samsoniella based on the phylogenetic analyses and was shown to resolve closely to S. haniana (Fig. 2). Morphologically, S. sapaensis is similar to S. haniana in sharing Isaria-like asexual conidiogenous structure which produces phialides with cylindrical to narrowly lageniform basal portion, fusiform or oval conidia (Wang et al. 2022). However, two samples of S. sapaensis were clustered together and formed a separate clade from S. haniana with strong statistical support (BI posterior probabilities = 1, ML bootstrap = 100%). Our morphological observation revealed some differences between them. Phialides on PDA of S. sapaensis (2.8–7.6 µm) are shorter than those of S. haniana (5.4–12.1 μm). Furthermore, conidia on PDA of S. sapaensis (1.2–1.5 × 0.8–1.0 μm) are smaller than those of S. haniana (2.3–3.7 × 1.2–2.8 μm).

The authors have declared that no competing interests exist.

No ethical statement was reported.

This work was supported by the National Natural Science Foundation of China (grants 32200013, 31870017 and 32160005).

Conceptualization: YW. Formal analysis: YW. Funding acquisition: HY. Investigation: VMD, CT, YW, ZQW, HY, YBW. Methodology: YW. Software: ZQW. Writing - original draft: YW. Writing - review and editing: HY.

Yao Wang https://orcid.org/0000-0002-1262-6700

Hong Yu https://orcid.org/0000-0002-2149-5714

All of the data that support the findings of this study are available in the main text or Supplementary Information.