Cryptic Diversity of Isaria-like Species in Guizhou, China

Wanhao Chen 1, Jiandong Liang 1, Xiuxiu Ren 1,2, Jiehong Zhao 1, Yanfeng Han 3,* and Zongqi Liang 3

1 Center for Mycomedicine Research, Basic Medical School, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China; chenwanhao052@gzy.edu.cn (W.C.); jdliang317@gzy.edu.cn (J.L.); renxiuxiu207@gzy.edu.cn (X.R.); zhaojiehong020@gzy.edu.cn (J.Z.)

2 College of Ecological Engineering, Guizhou University of Engineering Science, Bijie 551700, China

3 Institute of Fungus Resources, Department of Ecology, College of Life Sciences, Guizhou University, Guiyang 550025, China; zqliang472@126.com* Correspondence: yfhan@gzu.edu.cn

Abstract: Many Isaria-like species have recently been moved into more appropriate genera. However, more robust molecular phylogenetic analyses are still required for Isaria-like fungi to ensure accurate taxonomic identification. We analyzed these Isaria-like strains using multi-gene phylogenetics. Cryptic diversity was discovered in several Isaria farinosa strains, and two new species, Samsoniella pseudogunnii and S. pupicola, are proposed. Our results reveal that more attention needs to be paid to cryptic intraspecific diversity across different isolates and genotypes of the Isaria-like species, some of which will need to be transferred to Samsoniella. Interestingly, S. hepiali, with a very broad host distribution, has been widely used as a medicinal and edible cordycipitoid fungus.

Keywords: cryptic diversity; intraspecific; Isaria-like; multi-gene analysis

Citation: Chen,W.; Liang, J.; Ren, X.;Zhao, J.; Han, Y.; Liang, Z. Cryptic Diversity of Isaria-like Species in Guizhou, China. Life 2021, 11, 1093. https://doi.org/10.3390/life11101093

Academic Editors: Armin Meši´c and Ivana Kušan

Received: 9 September 2021

Accepted: 14 October 2021

Published: 15 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction

The genus Isaria was originally establish based on the species Isaria terrestris Fr. [1].Brown and Smith [2] transferred some species described in Isaria Pers. and Spicaria Harting into Paecilomyces, which possess a conidiogenous structure similar to that of Paecilomyces variotii Bainier. de Hoog [3] redescribed the genus Isaria and chose Isaria felina (DC.) Fr.as the lectotype. Typical characteristics include denticulate conidiogenous cells without elongation that arise in clusters from subtending cells or are solitarily from undifferentiated hyphae; mostly present synnemata; and globose, ellipsoidal, or subcylindrical conidia, mostly with a rounded base [3]. Samson [4] divided the genus Paecilomyces into two sections and all entomogenous species were placed in the section Isarioidea. Hodge et al. [5] reintroduced the genus Isaria with the type species Isaria farinosa (Holmsk.) Fr. and most entomopathogenic mesophilic Paecilomyces species were transferred to Isaria (Hypocreales,Clavicipitaceae) [6–8].

Kepler et al. [9] proposed the rejection of Isaria in favor of Cordyceps and transferred Isaria species into Cordyceps. Mongkolsamrit et al. [10] introduced some Isaria-like species and the new genus Samsoniella Mongkols., Noisrip., Thanakitp., Spatafora, and Luangsaard.Chen et al. [11,12] reported four Isaria-like species: Akanthomyces araneogenus Z.Q.Liang,W.H. Chen, and Y.F. Han; Samsoniella coleopterorumW.H. Chen, Y.F. Han, and Z.Q.Liang; Samsoniella hymenopterorumW.H. Chen, Y.F. Han, and Z.Q. Liang; and Samsoniella lepidopterorumW.H. Chen, Y.F. Han, and Z.Q. Liang. Currently, many species previously placed in the genus Isaria have been transferred to more appropriate genera. However,robust molecular phylogenetic analyses are still needed for Isaria-like fungi to ensure accurate taxonomic identification with comparable results across different isolates and genotypes [10].

We previously collected many Isaria-like morphs of invertebrate-pathogenic fungi from Guizhou Province, China. Some demonstrated close phylogenetic relationships with Isaria farinosa (Holmsk.) Fr. based on the analysis of associated ITS sequences. In the present study, we applied multi-gene (ITS, LSU, RPB1, RPB2, TEF) phylogenetic analysis to reevaluate the taxonomic position of these strains, as well as the cryptic diversity among the different isolates of I. farinosa, and to describe new taxa to accommodate the cryptic diversity of Isaria-like fungi.

2. Materials and Methods

2.1. Fungal Materials and Identification

The strains used in this study were isolated from infected insect and spider specimens collected in different areas of Guizhou Province, China, including Dali Forest in Rongjiang County, Yaorenshan National Forest Park in Sandu County, Mount Fanjing in Yinjiang County, Tongmuling in Guiyang City, and Doupengshan in Duyun City. Isolation of strains was conducted as described by Chen et al. [13]. Fungal colonies emerging from specimens were isolated and cultured at 25 ℃ for 14 days under 12 h light/12 h dark conditions following protocols described by Zou et al. [14]. Accordingly, the living isolates 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 fungal isolates were examined, especially for the arrangement, shape, and measurement of phialides and conidia, and also the growth rates of cultures incubated at 25 ℃ for 14 days were determined in Potato Dextrose Agar (PDA) (Potato powder 6%, Agar 20%, Glucose 20%, Beijing Solarbio Technology Co., Ltd., China). 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).

2.2. DNA Extraction, Polymerase Chain Reaction Amplification and Nucleotide Sequencing

DNA extraction was carried out with a fungal genomic DNA extraction kit (DP2033, BioTeke Corporation) in accordance with Liang et al. [15]. The extracted DNA was stored at -20 ℃. The amplification of the internal transcribed spacer (ITS) region, the large subunit ribosomal RNA (LSU) gene, the RNA polymerase II largest subunit 1 (RPB1), the RNA polymerase II largest subunit 2 (RPB2), and the translation elongation factor 1 alpha (TEF) by PCR was described by White et al. [16], Rakotonirainy et al. [17], Castlebury et al. [18], and van den Brink et al. [19], respectively. PCR reactions for five loci of all strains were performed in a total volume of 25 μL containing 12.5 μL 2×PowerTaq PCR Master Mix (Tiangen Biotech (Beijing) Co., LTD, China), 1 μL of each primer (10 μM), 1 μL of genomic DNA (20–100 ng), and 9.5 μL of sterile water. Primer sequence information is shown in Table 1. PCR products were purified and sequenced at Sangon Biotech (Shanghai) Co.The resulting sequences were submitted to GenBank (the accession number is shown in Table 2).

2.3. Sequence Alignment and Phylogenetic Analyses

Lasergene software (version 6.0, DNASTAR) was applied for the assembling and editing of DNA sequence in this study. The ITS, LSU, RPB1, RPB2, and TEF sequences were downloaded from GenBank, based on Kepler et al. [9], Mongkolsamrit et al. [10,19],Chen et al. [12],Wang et al. [20], and others selected on the basis of BLAST algorithm-based searches in GenBank (Table 2). A single gene data set was aligned and edited by MAFFT v7.037b [21] and MEGA6 [22]. Combined sequences of ITS, LSU, RPB1, RPB2, and TEF were performed by SequenceMatrix v.1.7.8 [23]. The combined datasets (ITS+LSU+RPB2+TEF) and (ITS+LSU+RPB1+RPB2+TEF) were used to determine the family placement of those strains in Hypocreales and the taxonomic position of strains and the cryptic diversity among the different isolates of I. farinosa in Cordycipitaceae.

The combined genes were both analyzed using the Bayesian inference (BI) and maximum likelihood (ML) methods. For BI, the model was selected for Bayesian analysis by ModelFinder [24] in the software PhyloSuite [25]. A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities using Mr-Bayes v.3.2 [26] for the combined sequence datasets. The Bayesian analysis resulted in 20,001 trees after 10,000,000 generations. The first 4000 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 program Tracer v1.5 [27] to determine burn-in, confirming that both runs had converged. ML analyses were constructed with RAxMLGUI [28]. The GTRGAMMA model was used for all partitions, in accordance with recommendations in the RAxML manual against the use of invariant sites.

3. Results

3.1. Phylogenetic Analyses

Gelasinospora tetrasperma Dowding, Neurospora crassa Shear and B.O. Dodge, and Sordaria fimicola (Roberge ex Desm.) Ces. and De Not. were used as the outgroup in analysis 1 (Figure 1) (to determine the family placement of those strains in Hypocreales). Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones, and Samson was used as the outgroup in analysis 2 (Figure 2) (to determine the taxonomic position of strains and the cryptic diversity among the different isolates of I. farinosa in Cordycipitaceae). The concatenated sequences of analysis 1 and 2 included 77 and 62 taxa, respectively, and consisted of 2396 (ITS, 620; LSU, 712; RPB2, 510; and TEF, 554) and 3309 (ITS, 554; LSU, 677; RPB1, 533; RPB2, 671; and TEF, 874) characters with gaps, respectively.

Analysis 1: The selected models for BI analysis were GTR+F+I+G4 parameters for partition ITS and LSU+RPB2, and GTR+F+G4 parameters for partition TEF. The final value of the highest scoring tree was –37,321.078127, which was obtained from an ML analysis of the dataset (ITS+LSU+RPB2+TEF). The parameters of the general time reversible (GTR) model used to analyze the dataset were estimated using the following frequencies:A = 0.230263, C = 0.272892, G = 0.280445, and T = 0.216401; substitution rates AC = 1.451341, AG = 2.441940, AT = 1.532513, CG = 1.182477, CT = 5.701598, and GT = 1.000000; as well as the gamma distribution shape parameter α = 0.381402. In the phylogenetic tree (Figure 1), both analyses of ML and BI trees were largely congruent, and strongly supported in most branches. DY10951, DY10952, DY101681, DY101682, GY407201, GY407202, YJ06171, and YJ06172 strains had a close relationship with Cordyceps Fr., Akanthomyces Lebert, and Simplicillium W. Gams and Zare, and clustered into Cordycipitaceae.

Figure 1. Phylogenetic placement of the new Isaria-like strains in the order of Hypocreales based on multigene dataset (ITS, LSU, RPB2m and TEF). Statistical support values (50%/0.5) are shown at the nodes for ML bootstrap support/BI posterior probabilities.

Figure 2. Phylogenetic placement of the new strains in Cordycipitaceae, based on multigene dataset (ITS, LSU, RPB1, RPB2, and TEF). Statistical support values (50%/0.5) are shown at the nodes for ML bootstrap support/BI posterior probabilities.

Analysis 2: The selected models for BI analysis were GTR+F+I+G4 parameters for partition ITS+LSU+RPB2+TEF and GTR+F+G4 parameters for partition RPB1. The final value of the highest scoring tree was –31,206.916701, which was obtained from an ML analysis of the dataset (ITS+LSU+RPB1+RPB2+TEF). The parameters of the general time reversible (GTR) model used to analyze the dataset were estimated using the following frequencies: A = 0.238319, C = 0.279080, G = 0.271674, and T = 0.210926; substitution rates AC = 1.120096, AG = 2.745044, AT = 0.784066, CG = 0.934312, CT = 6.322628, and GT = 1.000000; as well as the gamma distribution shape parameter α = 0.308970. In the phylogenetic tree (Figure 2),both analyses of ML and BI trees were largely congruent, and strongly supported in most branches. The new strains were all clustered within the genus Samsoniella. GY407201 and GY407202 strains clustered with Samsoniella coleopterorumW.H. Chen, Y.F. Han, and Z.Q.Liang in a subclade. DY10951 and DY10952 strains clustered with Samsoniella aurantia Mongkols., Noisrip., Thanakitp., Spatafora, and Luangsa-ard in a subclade. DY101681 and DY101682 strains had a close relationship with Samsoniella alboaurantia (G. Sm.) Mongkols., Noisrip., Thanakitp., Spatafora, and Luangsa-ard; Samsoniella alpina H. Yu, Y.B.Wang, Y.Wang, and Zhu L. Yang; and Samsoniella cardinalis H. Yu, Y.B. Wang, Y.Wang, Q. Fan, and Zhu L. Yang. YJ06171 and YJ06172 strains clustered with Isaria farinosa (Holmsk.) Fr. in a subclade and had close relationship with Samsoniella hepiali (Q.T. Chen and R.Q. Dai ex R.Q. Dai, X.M. Li, A.J. Shao, Shu F. Lin, J.L. Lan,Wei H. Chen, and C.Y. Shen) H. Yu, R.Q.Dai, Y.B. Wang, Y. Wang, and Zhu L. Yang.