Phylogeny and Systematics of the Genus Tolypocladium (Ophiocordycipitaceae, Hypocreales)

The taxonomy and phylogeny of the genus Tolypocladium are herein revised based on the most comprehensive dataset to date. Two species-level phylogenies of Tolypocladium were constructed: a single-gene phylogeny (ITS) of 35 accepted species and a multigene phylogeny (nrSSU, nrLSU, tef-1α, rpb1, and rpb2) of 27 accepted species. Three new species, Tolypocladium pseudoalbum sp. nov., Tolypocladium subparadoxum sp. nov., and Tolypocladium yunnanense sp. nov., are described in the present study. The genetic divergences of four markers (ITS, tef-1α, rpb1 and rpb2) among Tolypocladium species are also reported. The results indicated that species of Tolypocladium were best delimited by rpb1 sequence data, followed by the sequence data for the rpb2, tef-1α, and ITS provided regions. Finally, a key to the 48 accepted species of Tolypocladium worldwide is provided.

The taxonomy of Tolypocladium has been discussed extensively for decades. Cordyceps sensu lato was recently reclassified into three families (Clavicipitaceae sensu stricto, Cordycipitaceae, and Ophiocordycipitaceae) and four genera (Cordyceps s. str., Elaphocordyceps, Metacordyceps, and Ophiocordyceps) based on multigene phylogeny [7]. Molecular phylogenetic analyses suggested that Tolypocladium species fall within the Ophicordycipitaceae [7,8]. The genus Elaphocordyceps Sung and Spatafora 2007 was proposed for 23 species of the Cordyceps Fr. (1818: 316); these species parasitize the fungal genus Elaphomyces and some species of arthropods (e.g., cicada nymphs and beetle larvae) [7]. The Elaphocordyceps species within the Ophiocordycipitaceae form a clade sister to those of the genus Ophiocordyceps.
Gams established the Chaunopycnis to accommodate C. alba, which morphologically resembles Tolypocladium species in its conidiogenesis [9]. With the end of dual nomenclature for fungi, the generic name Tolypocladium was chosen over Elaphocordyceps and Chaunopycnis as Tolypocladium is the oldest and most commonly used name [8]. Chaunopycnis was integrated into the genus Tolypocladium. Accordingly, C. alba, C. ovalispora, and C. pustulata were renamed T. album, T. ovalisporum, and T. pustulatum, respectively [8].
Tolypocladium species have been widely studied due to their importance in the medicinal domain. These species can produce cyclosporine A, tolypoalbin, tolypin, cyclosporine D hydroperoxide, cylindromicin, and tolyprolinol [20,21], all of which have significant antitumoral, anti-inflammatory, antifungal, and/or antiparasitic properties [22]. Cyclosporine A, which is naturally isolated from T. inflatum, is widely used in autoimmune disease treatment and to prevent allograft rejection [23][24][25]. Tolypoalbin is a peptide mixture and a tetrameric acid produced by T. album [26]. Tolypin is also a peptide mixture [27]. Like kojic acid, cylindromicin is a significant bioactive inhibitor of tyrosinase [28]. Tolyprolinol, a dipeptide produced by Tolypocladium sp. FKI-7981, contains a rare moiety prolinol and was the first natural product isolated from Tolypocladium species. Tolyprolinol exhibits moderate antimalarial activity without cytotoxicity or any other antimicrobial properties [29].
Recent investigations and phylogenetic analyses have ascribed many new taxa to Tolypocladium. Therefore, the diversity of Tolypocladium may be underestimated. In the present study, we aimed first to investigate and document the worldwide diversity of Tolypocladium fungi using our current collection of specimens and data collected over the last several years. We used comprehensive morphological and molecular phylogenetic reconstructions to identify and reevaluate our specimens. Based on these reconstructions, we herein describe and illustrate three new taxa. We then clarify the phylogenetic affinities of these new taxa using rDNA sequence analyses.

Sampling
Tolypocladium species were collected in Kunming, Pu'er, Yunnan, China. 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.
Tolypocladium strains were isolated from soil samples, as described in our previous publication [30]. In brief, 2 g of soil was added to a flask containing 20 mL of sterilized water and glass beads. The suspension was then shaken for 10 min and diluted 100 times. Finally, 200 µL of diluted soil suspension was spread on petri dishes containing solidified onion garlic agar (OGA: 1 L of distilled water, 20 g of grated garlic, and 20 g of onion were boiled together for 1 h; the boiled biomass was filtered and 2% agar was added to the filtrate). Czapek yeast extract agar (CYA; Advanced Technology and Industrial Co., Ltd., Hong Kong, China) and potato dextrose agar (PDA; Difco, USA) were used. Rose bengal (50 mg/L) and kanamycin (100 mg/L) were added to all media. Conidia grown on insect cadavers were transferred to PDA plates and cultured at 22 • C. The filamentous fungal colonies isolated from the culture were transferred to fresh PDA media. The purified fungal strains were maintained at 22 • C in a culture room or transferred to PDA slants and stored at 4 • C.

Morphological Studies
Morphological studies were performed as described in our previous study [31]. Micromorphological characteristics, such as phialides and conidia, were studied by picking and mounting cultures on glass slides. The sizes and shapes of the microcharacteristics were determined using an Olympus CX40 and BX53 (Olympus Corporation, Tokyo, Japan). Individual length and width measurements were taken for 20-30 replicates, including the absolute minima and maxima. The morphological characteristics were described based on the digital images and the measurement dataset.

DNA Extraction and PCR Amplification
Total DNA was extracted from the fungal mycelia on PDA plates or from herbarium materials using the modified CTAB procedure [32]. The primer pair nrSSU-CoF and nrSSU-CoR [33] was used to amplify nrSSU, the primer pair LR5 and LR0R [34,35] was used to amplify nrLSU, and the primer pair EF1α-EF and EF1α-ER [7,36] was used to amplify the translation elongation factor 1α (tef-1α). The primer pair RPB1-5 F and RPB1-5 R and the primer pair RPB2-5 F and RPB2-5 R [7,36] were used to amplify the largest and secondlargest subunits of RNA polymerase II (rpb1 and rpb2), respectively. The ITS fragment was amplified using the primer pair ITS5 and ITS4 [37].

DNA Sequence Alignments
To investigate the placement of our samples within Tolypocladium, the nucleotide sequences of ITS, nrSSU, nrLSU, tef -1α, rpb1, and rpb2 were compared with sequences from representative Tolypocladium species downloaded from GenBank ( Table 1, Figures 1 and 2). Individual gene sequence datasets (ITS, nrSSU, nrLSU, tef -1α, rpb1, and rpb2) were aligned and manually checked using Bioedit v7.0.9 [38]. To identify possible phylogenetic conflicts among the datasets, the partition homogeneity (PH) test was performed with 1000 randomized replicates of heuristic searches with simple sequence addition in PAUP* 4.0a166 (http://paup.phylosolutions.com, accessed on 28 August 2022) [39]. The results showed that the phylogenetic signals from the five gene markers were in conflict.

Phylogenetic Analyses
Phylogenetic analyses were based on a concatenated five-gene dataset and the ITS sequences alone. nrSSU, nrLSU, tef -1α, rpb1, rpb2, and ITS sequences were retrieved from GenBank, and combined with those generated in this study. Taxon information and GenBank accession numbers are given in Table 1. Sequences were aligned using Clustal X2.0 and MEGA v6.06 [40,41]. Group I introns in the nrSSU sequences of some species were excluded from the phylogenetic analyses, and gaps were treated as missing data. After alignment of the five genes individually, the alignments were concatenated. A partition homogeneity test was conducted in PAUP* 4.0a166 [39], and the results indicated that there were no conflicts among the data partitions. PartitionFinder V1.1.1 identified eleven data partitions: nine corresponding to the three codon positions in each of the protein-coding genes (tef -1α, rpb1, and rpb2) and one each for nrLSU and nrSSU [42,43]. The results showed that the phylogenetic signals of the five genes were congruent (p = 0.02).
Maximum likelihood (ML) phylogenetic analyses were conducted using RaxML 7.0.3 [44] with the recommended partition parameters and 1000 rapid bootstrap replicates. Bayesian posterior probabilities (BP) were estimated with the same partition parameters using MrBayes v3.1.2 [45]. Bayesian inference (BI) analysis ran in MrBayes v3.1.2 for 5 million generations. Maximum parsimony (MP) analysis of the ITS dataset was performed using PAUP v. 4.0a166 [39], adopting the random addition of sequences model (10 replications), with gaps treated as missing data. A bootstrap (MPBS) analysis was performed using the maximum parsimony criterion in 1000 replications.
The following taxa were included in the five-gene concatenated dataset: Drechmeria W. Gams and H.-B. Jansson, Harposporium Lohde, Ophiocordyceps Petch, Purpureocillium Luangsa-Ard, Hywel-Jones, Houbraken and Samson, and Tolypocladium. Two species of Polycephalomyces Kobayasi were used as outgroups. ITS analysis was performed on Tolypocladium taxa only. Phylogenetic trees were visualized with FigTree v1.4.0 [46], edited in Microsoft PowerPoint, saved in PDF format, and converted to JPG format using Adobe Illustrator CS6 (Adobe Systems Inc., San Jose, USA). The finalized alignments and trees were submitted to TreeBASE (multigene submission ID 29808).
We calculated a phylogenetic distance matrix for the markers ITS, tef -1α, rpb1, and rpb2 to assess the species boundaries of the 10 Tolypocladium species (Supplementary Tables S1-S4), because the sequence data were complete for these four loci. The paired distances among the 10 Tolypocladium lineages were measured using the Kimura two-parameter model in MEGA v6.06 [41].

Genetic Distance Analyses
Comparisons of genetic divergence showed that (1) the minimum thresholds (p-distances) required to distinguish species within the Tolypocladium lineages were 0.026, 0.017, 0.013, and 0.008 for tef -1α, rpb1, rpb2, and ITS, respectively (Supplementary Tables S1-S4); and (2) the phylogenetic relationships within Tolypocladium were best resolved by the rpb1 sequence data, followed by those of rpb2, tef -1α, and ITS (Supplementary Tables S1-S4). Sexual morph: Stromata are solitary or several, simple or branched. The stipe is tough, dark-brownish to greenish, cylindrical, and abruptly to enlarging in the fertile part. The fertile part is cylindrical to clavate. Perithecia are superficial, wholly or partially immersed, ordinal or oblique in arrangement. Asci are cylindrical with a thickened ascus apex. Ascospores are usually cylindrical, multiseptate, disarticulate into part spores, and are occasionally non-disarticulating. Part spores are cylindrical.

Discussion
Tolypocladium is one of the most diverse fungal groups in terms of shape, substrate or host, and habitat range. Many new species have recently been added to Tolypocladium [11][12][13][14]73]. The present study described three new species (T. pseudoalbum sp. nov., T. subparadoxum sp. nov., and T. yunnanense sp. nov.) based on phylogenetic analyses and morphological characteristics. Phylogenetically, these three species fell within the Tolypocladium clade, while morphologically all three species possessed cylindrical phialides and ellipsoidal or globose conidia. It is challenging to distinguish species of Tolypocladium based only on morphological characteristics, because several species in this genus are morphologically cryptic [7,8,11]. Sexual morphological features are diverse: the ovoid perithecia may be superficial or completely immersed and part spores size varies [7,10]. However, the asexual morphological features are relatively simple.
Species of Tolypocladium play a significant role in a variety of artificial and wild ecosystems and may participate in antifungal, host-fungi, and insecticidal interactions [10,77]. Many species have been described in Tolypocladium based on host associations or morphology [11,12]. Over the past several decades, the increasing number of new fungal species being discovered globally has dramatically changed the classification of earlydiverging fungi [78]. In most previous studies, the classification of Tolypocladium was developed based on morphological characteristics. However, the advent of molecular biology, which was an important scientific milestone, revolutionized the taxonomic characterization of this genus. Over the last few decades, the number of accepted species in Tolypocladium has doubled. All 48 of the currently accepted species of Tolypocladium were included in the key developed in this study. However, because the sequence loci for many of these taxa were incomplete, only 27 species were included in the multigene phylogenetic analyses (Figure 1). The multilocus phylogenetic approach used in this study of the genus Tolypocladium shed considerable light on this influential group of fungi.
The ITS region is the most commonly used molecular marker for species delimitation in fungi. Schoch et al. proposed ITS as the standard barcode for fungi. That proposal will satisfy most fungal biologists, but not all [57,79,80]. Species-level identification of fungi has long been considered challenging. Carlson et al. reported that ITS has a low molecular variation in Trametes leading to poorly resolved phylogenies and unclear species boundaries, especially in the T. versicolor species complex [80]. The results of this study indicated that the ITS sequences did not help substantially to separate Tolypocladium species. However, the ITS sequences did help to resolve the phylogenetic relationships between Tolypocladium and related genera. The analyses of molecular phylogeny based on ITS sequences used in the current classification of the genus fungus are congruent with the higher genus clades inferred from these analyses. However, ITS sequence data are not likely to resolve species-level relationships or to delimitate closely related species and species complexes. Using the ITS phylogeny, it was still not possible to identify some species of Tolypocladium with confidence in the new classification system; the ITS region alone could not accurately identify species in Tolypocladium. For example, in the ITS phylogeny, T. varium CBS 429.94 was inseparable from T. inflatum OSC 71235 and T. inflatum NBRC 31669, while T. tundrense CBS 569.84 was inseparable from T. cylindrosporum ARSEF 2920 and T. cylindrosporum YFCC 1805001 ( Figure 2). In contrast, relationships among Tolypocladium species were highly resolved in the phylogeny based on the protein-coding gene rpb1. Multilocus sequence analyses provide additional information to better characterize species boundaries [81]. Therefore, we used both morphological and multilocus phylogenetic evidence to support the novelty of the new species described in this study and to ensure accurate species identifications.
Tolypocladium extinguens was first reported from New Zealand by Samson et al. The original description was based on only a single isolate [82]. Tolypocladium extinguens is characterized by its prolonged growth in pure culture and its subglobose to ellipsoidal, sometimes kidney-shaped, conidia [82]. Our phylogenetic analysis did not support the placement of this species in Tolypocladium due to long branch attraction in the phylogenetic tree. More taxa must be added to this analysis in future to clarify the phylogenetic position of this species.
Tolypocladium species are well-known medicinal fungi that are also plant endophytes, soil inhabitants, and insect pathogens [10,12]. Because many of species of fungi are present in the soil environment at some stage of their life cycle, this substrate is preferred by researchers for the isolation of Tolypocladium. At least eight species have been reported from the soil: T. geodes, T. microsporum, T. nubicola, T. pseudoalbum sp. nov., T. subparadoxum sp. nov., T. terricola, T. tundrense, and T. yunnanense sp. nov. In Asia (China, Japan, and Thailand), Tolypocladium species are mainly known from insects [19], and few studies have focused on Tolypocladium species in the soil and in plant roots. Recently, Tolypocladium species in Chinese soils were surveyed, but no new species were identified.