Cryptic Diversity of Isaria-like Species in Guizhou, China

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.


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][7][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 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

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 • C. 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).

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. 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.    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    Notes: Samsoniella pupicola was identified as belonging to Samsoniella, based on the phylogenetic analyses ( Figure 2) and has a close relationship with S. alboaurantium, S. alpina, and S. cardinalis. However, S. pupicola (Figure 4) is distinguished from S. alboaurantium by having larger fusiform conidia, distinguished from S. alpina by having white colony and fusiform conidia, and distinguished from S. cardinalis by having shorter phialides.   Note: DY10951 and DY10952 strains were identified as belonging to Samsoniella, based on the phylogenetic analyses (Figure 2), and clustered with Samsoniella aurantia in a clade. The characteristics of DY10951 and DY10952 ( Figure 5) strains are similar to that of S. aurantia, which had fusiform conidia (2-4 × 1-2 µm) and larger phialide (5-13 × 2-3 µm). Besides, the pairwise dissimilarities of ITS sequences show no difference within 554 bp between DY10951 and S. aurantia. Thus, molecular phylogenetic results and morphologically based conclusions support the idea that DY10951 and DY10952 strains were S. aurantia.

Discussion
The taxonomic delimitation of Isaria was originally based on morphological characteristics. However, Isaria shares many morphological characters with other genera in Hypocreales, which has resulted in a turbulent taxonomic history [10,29]. D'Alessandro et al. [30] noted that the morphological characteristics used to classify the genus Isaria frequently do not resolve new isolates into clearly defined species and need additional molecular markers in phylogenetic analyses. In the present study, Isaria-like strains collected from Guizhou Province, China, and previously identified by morphological characteristics, were reanalyzed using multi-gene (ITS, LSU, RPB1, RPB2, TEF) phylogenetic methodology. We proposed two new species of Samsoniella in this study.
The species Isaria farinosa is a well-known entomopathogenic fungi with worldwide distribution and a wide host range [31]. Kepler et al. [9] transferred Isaria farinosa to the genus Cordyceps as C. farinosa (Holmsk.) Kepler, B. Shrestha, and Spatafora based on a phylogenetic analysis of the CBS 111113 strain. We analyzed several strains of Isaria farinosa in the present study. Some properly belonged in the genus Samsoniella. CEP 004, CEP 005, CEP 029, YJ06171, and YJ06172 strains were identified as S. hepiali. Strains DY10951 and DY10952 were identified as S. aurantia. OSC 111005 and OSC 111006 strains were identified as new species but are absent in delineating morphological characteristics. Our results reveal cryptic diversity present in Isaria farinosa (now treated as Cordyceps farinosa) and illustrated that more attention should be paid on cryptic intraspecific diversity across different fungi isolates and genotypes.

Discussion
The taxonomic delimitation of Isaria was originally based on morphological characteristics. However, Isaria shares many morphological characters with other genera in Hypocreales, which has resulted in a turbulent taxonomic history [10,29]. D'Alessandro et al. [30] noted that the morphological characteristics used to classify the genus Isaria frequently do not resolve new isolates into clearly defined species and need additional molecular markers in phylogenetic analyses. In the present study, Isaria-like strains collected from Guizhou Province, China, and previously identified by morphological characteristics, were reanalyzed using multi-gene (ITS, LSU, RPB1, RPB2, TEF) phylogenetic methodology. We proposed two new species of Samsoniella in this study.
The species Isaria farinosa is a well-known entomopathogenic fungi with worldwide distribution and a wide host range [31]. Kepler et al. [9] transferred Isaria farinosa to the genus Cordyceps as C. farinosa (Holmsk.) Kepler, B. Shrestha, and Spatafora based on a phylogenetic analysis of the CBS 111113 strain. We analyzed several strains of Isaria farinosa in the present study. Some properly belonged in the genus Samsoniella. CEP 004, CEP 005, CEP 029, YJ06171, and YJ06172 strains were identified as S. hepiali. Strains DY10951 and DY10952 were identified as S. aurantia. OSC 111005 and OSC 111006 strains were identified as new species but are absent in delineating morphological characteristics. Our results reveal cryptic diversity present in Isaria farinosa (now treated as Cordyceps farinosa) and illustrated that more attention should be paid on cryptic intraspecific diversity across different fungi isolates and genotypes.
Samsoniella hepiali (otherwise known as Paecilomyces hepiali) is isolated from a field collection of natural Ophiocordyceps sinensis insect-fungi complex [33], and is widely used as a medicinal and edible cordycipitoid fungus, creating a great economic value [20]. Lin et al. [32] reported six isolates of Samsoniella hepiali from Anhui Province, China, which were isolated from leafhopper, larva, and cicada. CEP 004, CEP 005, CEP 029 strains from Buenos Aires, Argentina, were isolated from whitefly and soil [30]. YJ06171 and YJ06172 strains from Guizhou Province, China were isolated from ant. It is interesting that Samsoniella hepiali and its hosts are widely distributed in China and Argentina. This result will help us to assess the extent and distribution of genetic diversity of Samsoniella hepiali on a large scale, understand its biology and demographic history, and guide biodiversity conservation programs.