Comprehensive Review of Tolypocladium and Description of a Novel Lineage from Southwest China

Tolypocladium, a diverse genus of fungicolous fungi belonging to Ophiocordycipitaceae, includes saprotrophic soil inhabitants, plant endophytes and pathogens of insects, nematodes, rotifers, and parasites of truffle-like fungi. Here, we review the research progress achieved for Tolypocladium regarding its taxonomy, species diversity, geographic distribution, host affiliations and ecological diversity. Furthermore, an undescribed taxon from China was established using morphology and multi-gene phylogeny. Tolypocladium inusitaticapitatum is introduced as a new species parasitizing ectomycorrhizal Elaphomyces species. It is diagnosed by its irregularly enlarged fertile heads and lemon, yellow-to-dark-brown, smooth and nearly cylindrical stipe. Phylogenetic analyses based on SSU, LSU, ITS, TEF1-α and RPB2 sequence data showed T. inusitaticapitatum to be an independent lineage separated from T. flavonigrum in the clade comprising T. capitatum, T. fractum and T. longisegmentatum. A key for identifying the sexual Tolypocladium species is also provided.

Tolypocladium W. Gams was established based on three soil-inhabiting asexual species: Tolypocladium cylindrosporum W. Gams, T. geodes W. Gams and T. inflatum W. Gams (the type species) [4]. Hodge and colleagues linked the asexual T. inflatum to the sexual species Table 1. Species diversity, hosts/habitats and geographic distribution of Tolypocladium species.

Phylogenetic Placement
The combined SSU, LSU, ITS, TEF1-α and RPB2 sequence dataset comprised 35 species, containing 5384 nt (SSU: 1-1536, LSU: 1537-2441, ITS: 2442-3306, TEF1-α: 3307-4264, RPB2: 4265-5384) after the alignment (including gaps). Among them, 3731 bp (base pairs) were conserved, 378 variable, parsimony-uninformative, and 1275 parsimony-informative. The ML and BI analyses resulted in phylogenetic trees with a similar topology. The ML tree with a final log-likelihood of −27186.604 is shown in Figure 1. Specimens HKAS 112152 and HKAS 112153 clustered together and formed a distinct clade with strong support values (SH-aLRT = 100, UFB = 100 and BIPP = 1), indicating a conspecific relationship. These two specimens separated from other Tolypocladium species with SH-aLRT = 90.2 and BIPP = 0.98 support values. However, their LSU sequences showed an 11 bp difference (1.28%) across the 862 bp region, contributing to the different branch lengths in the phylogenetic tree. Based on the available molecular data for Tolypocladium species, some differences are known to occur due to intraspecific variations in the LSU sequences, ranging from 0.25 to 1.28% (Table 2).  C  T  A  T  T  T  C  A   Species   Locus  8  37  44  51  81  96  110  124  204  210  402 Ratio The locus numbers refer to the base-pair positions of the gene sequences, and " # " represents the reference sequences. Gaps are indicated with '-'.

Taxonomy
Tolypocladium W. Gams  Specimens of the current study are given in red. Type specimens are in bold and the superscript 'ex' indicates ex-type.

Taxonomy
Tolypocladium W. Gams  Morphological characterization: Sexual morph: Stromata arise directly from the host and are sometimes indirectly connected to the host through rhizomorph-like structures. They range from solitary to several and can be simple or branched. Stipe is fibrous to tough, rarely fleshy, dark-brownish to greenish with an olivaceous tint, rarely whitish, cylindrical and enlarges near the fertile part. The fertile part is clavate-to capitate-shaped and varies in color. Perithecia are partially to completely immersed, or superficial, or produced on a highly reduced stromatic pad, and ostiolate. Asci are unitunicate and long cylindrical with a thickened apical cap. Ascospores are filiform, approximately as long as asci, multi-septate, typically disarticulate into part-spores, and are occasionally non-disarticulating when mature (e.g., T. ramosum). Part-spores are hyaline, fusiform to cylindrical with round to truncate ends [6,8]. Asexual morph: They are Tolypocladium-, Chaunopycnis-, or Verticillium-like. Colonies are white, cottony and grow slowly on artificial media (e.g., potato dextrose agar, Czapek-Dox agar, malt extract agar, Sabouraud Glucose agar and water agar). Conidiophores usually are short and bear lateral or terminal phialides whorls. Phialides usually are swollen at the base and thin, often with bent necks. Conidia are globose to oval, one-celled, hyaline, smooth, and aggregative in small heads at the tips of the phialides [4,23].
Hosts and habits: Found in terrestrial and humid environments. Species of Tolypocladium parasitize hypogeous Elaphomyces (20 species including the novel species described in this study), cicada nymphs (4 species), beetle larvae (T. inflatum), pupa of the bagworm moth (T. fumosum), mosquito larvae (T. extinguens), and even bdelloid rotifers exposed to air (T. lignicola and T. trigonosporum). Their ascospores/conidia and mycelia survive in soil, or on various humus, rotting wood, plant tissues and surfaces, body surfaces of insects and mites, tissues of Cordyceps and lichens (Table 1).
Species diversity and distribution: Tolypocladium currently consists of 42 species (including the novel species described in this study) distributed worldwide [2,3,17]. Seventeen species were recorded from China (Table 1). Etymology: The specific epithet 'inusitaticapitatum' is derived from the combination of two Latin words, 1) adjective inusitata (strange, odd) and 2) noun capitatum (head), pointing to the fertile head, which is irregularly expanded.
Description: Asexual morph Stromata 9-11.5 cm high, solitary and simple, arising directly from the fruiting bodies of Elaphomyces sp. Stipe yellow at base, olive-brown to dark brown at the middle part, and yellowish brown at the terminal part. They are 7.5-11.5 cm long and 7-8.5 mm thick in the widest parts and nearly cylindrical, but the middle part is slightly thicker than the basal and upper parts. The fertile part developed from the terminal of the stipe, and is somewhat ellipsoidal, irregularly barrel-shaped, and sometimes slightly compressed, 1.5-2.0 cm × 1.5-2.0 cm. The surface is decorated with white ascospores released from the mature perithecia, which is olive yellow when immature, and olive to dark brown when mature. The outer layer becomes cracked and the olive internal texture is exposed. Structure of cortex of fertile part: composed of olive brown pseudoparenchymatous tissue and an ectal layer. Perithecia 580-720 µm × 180-270 µm (x = 650 µm × 220 µm, n = 10), crowded, entirely immersed, obovoid, ellipsoidal to pyriform. Ostioles papillate, and are visible (protruding up to 55 µm in high) or invisible, lined with periphyses. Asci is 410-510 µm × 10-15 µm (x = 461 µm × 13 µm, n = 20), hyaline, and long cylindrical, with a conspicuously thickened cap (measuring 6.5-7.5 µm × 6.0-7.0 µm). Ascospores are approximately as long as asci, and extremely easy to break into part-spores. Part-spores 20-32 µm × 3.0-4.5 µm (x = 25 µm × 3.6 µm, n = 20), hyaline, cylindrical with rounded ends. Asexual morph: Unknown. Notes: Based on the multi-gene phylogeny results, our specimens are closely related to Tolypocladium flavonigrum, known only from Thailand. Both species have stromata directly emerging from the surface of Elaphomyces sp., and capitate fertile heads with the perithecia entirely immersed in a well-differentiated valliforme-like structure [30]. However, T. inusitaticapitatum considerably differs from T. flavonigrum for the olive, yellowishbrown to dark brown fertile part, and is yellow to yellowish-brown at both ends of the stipe compared to the yellow-black to black stromata in T. flavonigrum. Tolypocladium Notes: Based on the multi-gene phylogeny results, our specimens are closely related to Tolypocladium flavonigrum, known only from Thailand. Both species have stromata directly emerging from the surface of Elaphomyces sp., and capitate fertile heads with the perithecia entirely immersed in a well-differentiated valliforme-like structure [30]. However, T. inusitaticapitatum considerably differs from T. flavonigrum for the olive, yellowishbrown to dark brown fertile part, and is yellow to yellowish-brown at both ends of the stipe compared to the yellow-black to black stromata in T. flavonigrum. Tolypocladium inusitaticapitatum produces obovoid, ellipsoidal to pyriform perithecia, which are markedly distinguished from the elongate-ovoid perithecia produced by T. flavonigrum. Asci and partspores of T. inusitaticapitatum (410-510 µm × 10-15 µm, 20-32 µm × 3.0-4.5 µm) are larger than those of T. flavonigrum ((318-)330-416(-482) µm × 7-8 µm, 2-5 µm × 1.5-2 µm) [30].

. inusitaticapitatum (This Study)
Fertile part Dark reddish brown Olive brown, yellowish-brown to dark brown

Discussion
Tolypocladium, a generalist genus, has been reported to have diverse lifestyles on a wide range of hosts and environments, including soil, insects, plants, lichens and hypogeal fungi [6,8]. The current pattern of host affiliation of Tolypocladium fungi is inferred to be an evolutionary product of intra-and inter-kingdom host shifts [57]. In the last two decades, researchers aimed to infer the evolution of host affiliation within the Tolypocladium, either using a handful of gene loci from dozens to hundreds of taxa, or using genomescale data from fewer taxa [11,12,15,58]. To date, the studies on the host-jumping of Tolypocladium have been performed with multigene phylogeny (seven genes from 202 taxa of Hypocreales) [12] and genome-scale phylogeny (1350 genes from 20 taxa of Hypocreales) [15]. The multigene phylogenies supported three hypotheses for Tolypocladium, as follows: (1) the ancestral hosts were fungi (false truffles) [11,12,57,58]; (2) there were multiple switches to insect pathogenesis from a mycoparasitic ancestor [8,12,13]; (3) the endophytic lineage has arisen with the contact of plant hosts via mycorrhizal associations or plant-associated insects [12]. However, these conclusions, made from multigene phylogenies, conflict with those made from genome-scale phylogenies, which suggested a single ecological transition from insects to fungi within Tolypocladium [15]. Our phylogenic tree, inferred from five genes of 35 species (Figure 1), resulted in consistent conclusions, similar to those from previous multigene phylogenies. Similarly, we encountered several problems, such as phylogenetic conflicts among genetic data partitions and moderate to low support values for some important nodes [8,12,13]. Although whole-genome data provide insights that can further resolve the phylogenetic relationships of Tolypocladium [15,59,60], it is still unknown whether those conclusions will be limited by the few available species.
In this study, a novel Tolypocladium species occurring on Elaphomyces sp. is known from its sexual morph. A taxonomic key is also provided for 26 Tolypocladium species. The shape of the fertile part, the connection between the stipe and host, the structure of the cortex of the fertile part, size of part-spores and host affiliation are thought to be characteristic of taxonomic significance for interspecific identification [8][9][10]. However, there are 16 species whose sexual morphs are still unknown. In addition, the phylogenetic relationships among Tolypocladium species are very sensitive to taxa sampling and loci information [8,15]. Further studies should focus on obtaining more samples from different geographic regions and/or ecological niches, sequencing more markers and even genomic data, building a more robust phylogenetic relationship, and establishing their sexual-asexual morph connections. (Table 4).

Collections and Morphology
Tolypocladium specimens, including their underground host Elaphomyces sp., were collected in an evergreen broad-leaved forest in Lijiang Alpine Botanic Garden, Lijiang City, Yunnan Province, China. The specimens were examined as described in Senanayake and colleagues with the following modifications [61]. Colour codes were recorded following those of Kornerup and Wanscher [62]. Specimens were deposited at the Herbarium of Cryptogams Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China (HKAS, KUN).

Sequence Alignment and Phylogenetic Analyses
Phylogenetic trees were constructed using the sequencing data of T. inusitaticapitatum and the allied reference sequences of closely related Ophiocordycipitaceae species obtained from the GenBank (Table 5). Aschersonia confluens (BCC 7961) and A. paraphysata (BCC 1467) of Clavicipitaceae were used as outgroup taxa. All sequences were assembled and aligned using MAFFT v 6.8 [69] and manually edited where necessary in BioEdit version 7.0.9 [70]. Individual alignments were compiled for SSU, LSU, ITS, TEF1-α and RPB2 genes. The optimal substitution model for each gene dataset was determined using MrModeltest 2.3 [71] under the Akaike information criterion (AIC). The results indicated that the GTR+I+G model was optimal for all the gene regions. Individual datasets were combined to assemble the combined dataset (gene order: SSU, LSU, ITS, TEF1-α and RPB2). The resulted combined dataset was deposited in the TreeBASE database (http://purl.org/phylo/treebase/phylows/study/TB2:S27887?x-access-code= 746eddc746009259527edd3d4c69526b&format=html, accessed on 10 March 2021). Maximum likelihood (ML) analysis was performed using IQ-Tree (http://iqtree.cibiv. univie.ac.at/, accessed on 20 May 2021) [72,73]. The substitution model options for each gene were auto-evaluated according to the provided partition file. Clade support for the ML analysis was assessed using an SH-aLRT test with 1000 replicates [74] and the ultrafast bootstrap (UFB) [75]. In the ML analyses, nodes with support values of SH-aLRT ≥ 80 and UFB ≥ 95 were considered well-supported, those with either SH-aLRT < 80 or UFB < 95 were considered weakly supported, and nodes with SH-aLRT < 80 and UFB < 95 were considered unsupported.
Bayesian Inference (BI) analysis was carried out in MrBayes v3.2.6 [76]. Gaps were treated as missing data. Four simultaneous Markov Chain Monte Carlo (MCMC) chains were run for 10,000,000 generations and were sampled at every 100th generation until the standard deviation of the split frequencies fell below 0.01 and ESS values > 200. Subsequently, phylogenetic trees were summarized and posterior probabilities (PP) were calculated using MCMC by discarding the first 25% generations as the burn-in phase [77]. Phylogenetic trees were viewed in FigTree v. 1.4.4. Nodes with BI posterior probability (BIPP) > 0.90 were considered to be well supported.