Morphological and Phylogenetic Analyses Reveal Three New Species of Entomopathogenic Fungi Belonging to Clavicipitaceae (Hypocreales, Ascomycota)

This study aims to report three new species of Conoideocrella and Moelleriella from Yunnan Province, Southwestern China. Species of Conoideocrella and Moelleriella parasitize scale insects (Coccidae and Lecaniidae, Hemiptera) and whiteflies (Aleyrodidae, Hemiptera). Based on the phylogenetic analyses of the three-gene nrLSU, tef-1α, and rpb1, it showed one new record species (Conoideocrella tenuis) and one new species (Conoideocrella fenshuilingensis sp. nov.) in the genus Conoideocrella, and two new species, i.e., Moelleriella longzhuensis sp. nov. and Moelleriella jinuoana sp. nov. in the genus Moelleriella. The three new species were each clustered into separate clades that distinguished themselves from one another. All of them were distinguishable from their allied species based on their morphology. Morphological descriptions, illustrations, and comparisons of the allied taxa of the four species are provided in the present paper. In addition, calculations of intraspecific and interspecific genetic distances were performed for Moelleriella and Conoideocrella.

Torrubiella Boud.species infect a wide range of arthropods, mainly spiders and scale insects [4,20].The genus currently has about 80 records in the index (according to the Index Fungorum, which is available online at http://www.indexfungorum.org;accessed on 11 December 2023).However, the fact that the previous studies identified species on the basis of their morphological characteristics resulted in a lack of molecular data for most species of Torrubiella.With the advent of molecular technology and the application of multigene phylogenetic analyses, species identification methods based on phylogenetic analyses combined with morphological characteristics have gradually gained recognition.Johnson et al. (2009) found that previous phylogenetic studies had shown that the genus Torrubiella was not monophyletic, but none of them had attempted to resolve this [4,21,22].Subsequently, a multigene phylogenetic tree covering 10 species of Torrubiella was constructed by Johnson et al. to determine the phylogenetic position of these species [4].Phylogenetic analyses showed that these species were distributed in Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae [4].Torrubiella tenuis (Petch) D. Johnson, G.-H. Sung, Hywel-Jones & Spatafora and Torrubiella luteorostrata (Zimm.)D. Johnson, G.H. Sung, Hywel-Jones & Spatafora form a statistically well-supported clade in Clavicipitaceae.Therefore, a new genus, Conoideocrella, was proposed by Johnson et al. to accommodate the species T. tenuis and T. luteorostrata, and T. luteorostrata was designated as the type species [4].The genus Conoideocrella was named thus for its perithecium with a conical shape that is similar to that of Torrubiella [4].It currently contains three species, all of which have elongate, conical perithecia and planar stromata [23,24].Conoideocrella luteorostrata was shown to be distributed in Seychelles, Sri Lanka, Java, Samoa, New Zealand, the far Eastern U.S.S.R., and Thailand [23,25].Conoideocrella tenuis was known to be distributed in Sri Lanka and Thailand, and C. krungchingensis was known only to be in Thailand [23][24][25].All three species have been reported to be able to parasitize scale insects [23,24].
Entomopathogenic fungi are widely distributed in China, and Yunnan Province is one of the richest provinces in China in terms of biodiversity.In this study, we collected some specimens with macro-morphological similarities to Moelleriella and Conoideocrella during an investigation of entomopathogenic fungi in Yunnan.A three-gene phylogenetic analysis revealed two new species of Moelleriella, one new species, and one known species of Conoideocrella.Conoideocrella tenuis is a recently newly recorded species in China.

Fungal Collection and Isolation
The specimens were collected from Bampo village, Jinuo Township, Jinghong City, and the Fenshuiling National Nature Reserve, Jinping County, Yunnan Province, China.In fields, whole leaves with stromata were collected, and some bark from branches with stromata was chipped off with a pocket knife.Then they were placed in sterilized plastic boxes and brought to the laboratory.The detailed procedure to obtain axenic cultures in this study was described in Yang et al. [15].After the isolation of pure cultures, they were transplanted to PDA slant and grown for 10 days before being stored at 4 • C. The specimens were deposited in the Yunnan Herbal Herbarium (YHH) of Yunnan University,

Phylogenetic Analyses
Datasets of three genes (nrLSU, tef-1α, and rpb1) used to construct a phylogenetic tree were downloaded from GenBank and combined with the newly generated data in this study.The sequences downloaded were based on previous studies by Mongkolsamrit et al. [24] and Yang et al. [15].Names, voucher information, and corresponding GenBank accession numbers of the taxa are listed in Table 1.Sequences were aligned, and poorly aligned regions were removed with MEGA v.6.06 [31].The aligned three-gene sequences were concatenated using Phylosuite v1.2.2 [32].Phylogenetic analyses were performed using BI and ML methods [33,34].A maximum likelihood (ML) tree was created using IQ-tree v.2.1.3,and a Bayesian inference (BI) tree was created using MrBayes v.3.2.2 [35,36].Modelfinder was used to select the best-fitting likelihood model [37].The optimal model for the ML analyses was the TIM2+F+I+G4 model, with 5000 rapid bootstraps in a single run [38].The optimal model for the BI analysis was the GTR+F+I+G4 model.The four Markov chain Monte Carlo simulations ran for 2 million generations from a random start tree with a sampling frequency of 100 generations.Twenty-five percent of initial sampled data were discarded as burn-in.Phylogenetic trees were viewed and edited in Figtree v.1.4.3 and visualized in Adobe Illustrator CS6.The interspecies and intraspecies genetic distances for the three genes (tef-1α, rpb1, and nrLSU) in Moelleriella and Conoideocrella were calculated using MEGAE v.6.06.Genetic distances were calculated by selecting the maximum composite likelihood model.

Sequencing and Phylogenetic Analyses
Three-gene (nrLSU, tef-1α, and rpb1) sequences were generated from eight specimens and two living cultures (see Table 1).Three-gene sequences of 129 samples from 14 genera in the family Clavicipitaceae were used for the ML and BI phylogenetic analyses.Pleurocordyceps aurantiaca MFLUCC 17-2113 and Pleurocordyceps marginaliradians MFLU 17-1582 were used as the outgroups.The concatenated three-gene sequences contained 2726 bp (nrLSU: 935 bp, tef-1α: 1024 bp, and rpb1: 767 bp).Both the ML and BI analyses exhibited nearly consistent overall topologies.The results of the phylogenetic analysis showed three highly supported clades, viz., the Pulvinate clade (BP = 100%, PP = 1), the Globose clade (BP = 100%, PP = 1), and the Effuse clade (BP = 100%, PP = 1) (Figure 1 The genetic distances calculated based on the three genes (nrLSU, tef-1α, and rpb1) among interspecies and intraspecies in Moelleriella and Conoideocrella are shown in Tables S1-S6.The intraspecific genetic distances for nrLSU, tef-1α, and rpb1 in Moelleriella were 0-0.0276, 0-0.0428, and 0-0.0168, respectively.The interspecific genetic distances for nrLSU, tef-1α, and rpb1 between the known species and M. longzhuensis were 0.03-0.08,0.09-0.16,and 0.08-0.24,respectively, and those between the known species and M. jinuoana were 0.04-0.08,0.10-0.15,and 0.15-0.25,respectively.In Conoideocrella, the intraspecific genetic distances of nrLSU, tef-1α, and rpb1 were 0-0.0028, 0-0.0052, and 0.0019-0.0027,respectively, and the genetic distances between C. fenshuilingensis and the known species were 0.01-0.02,0.06-0.09,and 0.08, respectively.ord of the size of the asci or ascospores.Hywel-Jones [23] collected specimens of T. tenuis in Thailand.They recorded the size of the asci and ascospores and isolated pure cultures.In our study, specimens of this species were collected in Yunnan, China, and it was found to be distributed in China.Its macromorphology and micromorphology were generally consistent with those described by Petch and Hywel-Jones and Evens, with one difference being that the materials used in this study extend the perithecium (190-900 × 160-270 µm) and asci (190-500 × 3.3-7.0µm) size range of this species.Noteworthy, a hirsutella-like asexual state was observed on the stromatic colonies in the present study, which has not yet been observed in other studies.Unfortunately, as in the case of the Thai material, partspores were not seen in the Chinese collection.≡Torrubiella tenuis Petch, Ann.Perad.7, 323 (1923).MycoBank No: MB 512029.Description.Sexual morph: Teleomorphic stromata pulvinate, flattened pulvinate or almost planar, 2-4 mm in diam, white to orangish-pink, tomentose, rather loose internally, surrounded by a broad, fibrillose margin or hypothallus.Perithecia mostly distributed at the margin of the stroma or on the hypothallus, scattered or clustered, color deepens from the bottom to the top, white to pale brown, covered with hyphae up to two-thirds their height in mature perithecia, dozens of perithecia per stroma, elongated flask shape or elongated conic shape, 190-500 × 160-270 µm.Asci cylindrical, eight-spored, 190-480 × 3.3-5.5 µm, caps 2.5-3.5 µm thick.Ascospores whole, filiform, septate.Asexual morph: Not known.
Culture characteristics.Colonies grow slowly on PDA at 25 • C, attaining a diam of 15-17 mm in 21 days, greyish-white to cream-white mycelium at first, turning lilac with age.Colonies are loose on the surface and compact at the bottom.Hyphae smooth, septate, hyaline, 1.1-3.6 µm wide.Hirsutella-like asexual state arises from hyphae, conidiogenous structures with slender base tapering more or less evenly to a neck, hyaline, produced directly on hyphae of the stromatic colonies from ca. 5 wk onwards, 16.3-149.4× 0.6-2.4µm, and 0.3-1.3µm wide at the apex.Conidia hyaline, smooth, fusiform and slightly curved, produced singly or in a group of two at the neck apex, 6.1-12.5 × 1.3-2.3µm.
Habitat.Parasitic on Aspidiotus destructor on a jungle tree; on a black Aleyrodes on Sarcococca pruniformis; on a scale on Hedyotis lessertianan; on Aleyrodes on Lasianthus walkerianus and Psychotria elongata.
Distribution.Sri Lanka (type locality) and Thailand, China.
Other material examined.China, Yunnan Province, Jinghong City, Jinuo Township, Banpo village, 100 Commentary.The species C. tenuis, formerly in the genus Torrubiella, was reclassified by Johnson et al. [4] to Conoideocrella.In 1923, Petch described the morphological characteristics of T. tenuis, as well as its distribution sites and host insects.But there was no record of the size of the asci or ascospores.Hywel-Jones [23] collected specimens of T. tenuis in Thailand.They recorded the size of the asci and ascospores and isolated pure cultures.In our study, specimens of this species were collected in Yunnan, China, and it was found to be distributed in China.Its macromorphology and micromorphology were generally consistent with those described by Petch and Hywel-Jones and Evens, with one difference being that the materials used in this study extend the perithecium (190-900 × 160-270 µm) and asci (190-500 × 3.3-7.0µm) size range of this species.Noteworthy, a hirsutella-like asexual state was observed on the stromatic colonies in the present study, which has not yet been observed in other studies.Unfortunately, as in the case of the Thai material, part-spores were not seen in the Chinese collection.
Mycobank No: 851868.Etymology.Named after the Fenshuiling National Nature Reserve where the species was collected.

Discussion
The calculation of genetic distances for the three genes showed that the interspecific genetic distances between the new species in this study and other species of the genus were greater than the intraspecific genetic distances (see Tables S1-S6).This study resulted in the discovery of two new species of Moelleriella and one new species of Conoideocrella

Discussion
The calculation of genetic distances for the three genes showed that the interspecific genetic distances between the new species in this study and other species of the genus were greater than the intraspecific genetic distances (see Tables S1-S6).This study resulted in the discovery of two new species of Moelleriella and one new species of Conoideocrella through phylogenetic analyses, morphological observations, and calculations of inter-and intraspecific genetic distances within genera.
All three reported species of Conoideocrella were present in the field as telemorphic stromata with elongated flask-shaped or elongated conic-shaped perithecia [23,24].In this way, it could be easily distinguished from Moelleriella, although it was in good agreement with Moelleriella in terms of growing environment and host category.Two genera, Moelleriella and Conoideocrella, were parasitic on whiteflies (Aleyrodidae, Hemiptera) or scale insects (Coccidae and Lecaniidae, Hemiptera) [3,23,24].The fungus always completely obliterated the host, making it nearly impossible to identify the insect [3,23].
Scale insects and whiteflies are tiny, widely distributed parasites that suck plant sap, and many of these species are serious agricultural pests and act as vectors for viral plant diseases [40][41][42][43].In the face of severe crop infestation by whiteflies, pesticides have been used mainly to suppress the whitefly population [44].However, the overuse of pesticides has led to a certain degree of resistance to pesticides and harmful effects on non-target organisms and the environment [45].The ability of Moelleriella and Conoideocrella species to parasitize large populations of whiteflies and scale insects in the wild gives these species the potential to be developed as green and non-polluting biological control agents.Moelleriella libera (anamorph A. aleyrodis) was the first species of Moelleriella to be applied to control whiteflies in Florida, U.S.A. [46].Subsequently, there have been an increasing number of studies on the control of pests with M. libera [47][48][49][50][51]. Relatively few studies have been conducted on other species of Molleriella for biocontrol materials.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof10060423/s1,Table S1: Interspecific genetic distance matrix and intragroup genetic distances for nrLSU sequences of Moelleriella.Table S2: Interspecific genetic distance matrix and intragroup genetic distances for tef-1α sequences of Moelleriella.Table S3: Interspecific genetic distance matrix and intragroup genetic distances for rpb1 sequences of Moelleriella.Table S4: Interspecific genetic distance matrix and intragroup genetic distances for nrLSU sequences of Conoideocrella.Table S5: Interspecific genetic distance matrix and intragroup genetic distances for tef-1α sequences of Conoideocrella.Table S6: Interspecific genetic distance matrix and intragroup genetic distances for rpb1 sequences of Conoideocrella.
).The Effuse clade was segregated into two sister clades, subclade I and subclade II.Moelleriella contains the Effuse clade and the Globose clade.Two new species of Moelleriella were distributed in the Effuse clade (M.longzhuensis) and the Globose clade (M.jinuoana).Three samples of M. longzhuensis were clustered closely with M. rhombispora (M.Liu & K.T. Hodge) M. Liu & P. Chaverri and formed a monophyletic clade in the Effuse clade with a high level of statistical support (BP = 100%, PP = 1).Three samples of M. jinuoana formed a monophyletic clade in the Globose clade with a high level of statistical support (BP = 91%, PP = 0.8).The genus Conoideocrella was clustered with Orbiocrella and had a new species (C.fenshuilingensis) and one known species (C.tenuis).Two samples of C. fenshuilingensis formed a monophyletic clade in Conoideocrella with a high level of statistical support (BP = 100%, PP = 1).

Figure 1 .
Figure 1.Phylogenetic relationships of 14 genera in Clavicipitaceae are based on the maximum likelihood (ML) and the Bayesian inference (BI) analyses using nrLSU, tef-1α, and rpb1 sequences.Statistical support values greater than 70% are shown at the nodes for the BI posterior probabilities/the ML bootstrap proportions.The new taxa are highlighted in bold and T for ex-type material.

Table 1 .
Names, voucher information, and corresponding GenBank accession numbers of the taxa used in this study.