Morphological and Phylogenetic Characterization of Five Novel Nematode-Trapping Fungi (Orbiliomycetes) from Yunnan, China

Nematode-trapping fungi are widely studied due to their unique morphological structure, survival strategy, and potential value in the biological control of harmful nematodes. During the identification of carnivorous fungi preserved in our laboratory, five novel nematode-trapping fungi were established and placed in the genera Arthrobotrys and Drehslerella based on morphological and multigene (ITS, TEF, and RPB2) phylogenetic analyses. A. hengjiangensis sp. nov. and A. weixiensis sp. nov. are characterized by producing adhesive networks to catch nematodes. Dr. pengdangensis sp. nov., Dr. tianchiensis sp. nov., and Dr. yunlongensis sp. nov. are characterized by producing constricting rings. Morphological descriptions, illustrations, taxonomic notes, and phylogenetic analysis are provided for all new taxa; a key for Drechslerella species is listed; and some deficiencies in the taxonomy and evolution study of nematode-trapping fungi are also discussed herein.


Introduction
Nematode-trapping fungi (NTF) are a group of fungi that can produce unique structures (trapping structures) to capture nematodes [1][2][3]. They have attracted much attention for over 180 years since Corda (1839) reported the first species (Arthrobotrys superba Corda) because of their unique survival strategy, excellent application potential in nematode control, and significance of maintaining the balance of nematode populations in the ecosystem [4][5][6][7][8]. Orbiliomycetes NTF is the research focus of NTF due to their abundant species, diversified trapping structures, and mature research methods [3,9,10]. Currently, 119 Orbiliomycetes NTF species have been reported and divided into Arthrobotrys, Dactylellina, and Drechslerella based on their trapping structures according to modern molecular biology research [11][12][13][14].
Arthrobotrys, the most widespread and diverse (67 species) genus among Orbiliomycetes NTF, was established by Corda (1839) with A. superba Corda, which is characterized by 1-septate conidia growing in clusters on the nodes of the conidiophores [4]. With the improvement in the isolation method, more species were discovered, and the characteristic of Arthrobotrys was revised as producing obovoid, elliptic, pyriform 0-3-septate conidia on the nodes or short denticles of the conidiophores [2,[15][16][17]. However, the taxonomy system based on these characteristics still needs to be clarified due to confusion caused by scholars attaching different importance to morphological features. The development of

Sample Collection
Terrestrial soil and freshwater sediment samples involved in this study were collected from Yunnan Province, China. The detailed collection methods are the same as Zhang et al.'s [22].

Fungal Isolation
The soil sprinkling technique and baited plates method [3,[23][24][25] were used to incubate nematode-trapping fungi (NTF) in the soil samples. The single-spore isolation method was used to obtain the pure culture of NTF. The details of the above three methods are the same as Zhang et al. [22].

Morphological Observation
The observation well and nematode baiting methods [26] were used to induce the trapping structure of NTF in accordance with Zhang et al. [22]. All micromorphological features, such as conidia, conidiophore, trapping structure, and chlamydospores, were photographed and measured with an Olympus BX53 differential interference microscope (Olympus Corporation, Tokyo, Japan).

DNA Extraction, PCR Amplification, and Sequencing
The total genomic DNA of isolates was extracted from the mycelium grown on potato dextrose agar (PDA) plates using a rapid fungal genomic DNA isolation kit (Sangon Biotech Company, Limited, Shanghai, China). The ITS, TEF, and RPB2 regions were amplified with the primer pairs ITS4-ITS5 [27], 526F-1567R [28], and 6F-7R [29], respectively. The PCR amplification was performed according to Zhang et al. [22]. A DiaSpin PCR Product Purification Kit (Sangon Biotech Company, Limited, Shanghai, China) was used to purify the PCR products according to the user manual. The purified PCR products of the ITS and RPB2 regions were sequenced in the forward and reverse directions using PCR primers, and TEF genes were sequenced using the primer pair 247F-609R [11] (BioSune Biotech Company, Limited, Shanghai, China).
Sequences were checked, edited, and assembled via SeqMan v. 7.0 [30]. The sequences generated in this study were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/; accessed on 20 March 2023).
The best-fit optimal substitution models of ITS, TEF, and RPB2 were selected as GTR+I+G, TrN+I+G, and GTR+I+G via jModelTest v2. 1.10 [37] under the Akaike Information Criterion (AIC).
Maximum likelihood (ML) analysis was implemented using IQ-Tree v1. 6.5 according to Zhang et al. [22]. The statistical bootstrap support values (BS) were computed using rapid bootstrapping with 1000 replicates [38].
Bayesian inference (BI) analysis was conducted with MrBayes v. 3.2.6 [39] according to Zhang et al. [22]. The remaining 75% of trees were used to calculate the posterior probabilities (PP) in the majority rule consensus tree. FigTree v1. 3.1 [40] was used to visualize the trees. The backbone tree was edited and reorganized using Microsoft PowerPoint (2013) and Adobe Photoshop CS6 software (Adobe Systems, San Jose, CA, USA).
A best-scoring maximum likelihood tree was performed with a final ML optimization likelihood value of −6158. 611237. Within the Bayesian analysis (BI), the Bayesian posterior probabilities were evaluated with a final average standard deviation of the split frequency of 0.009264. The trees inferred by ML and BI showed slightly different topologies in some clusters, but both trees showed that all tested nematode-trapping fungi were clustered into two large clades, and five new species showed distinct divergence from known species. The best-scoring ML tree was selected to present herein (Figure 1), and the Bayesian majority rule consensus tree (BI) was also attached in the Supplementary Materials ( Figure S1).    (Figure 1 and Figure S1).

Key to Known Species of Drechslerella
We do not update the species key of Arthrobotrys in this study because it has been updated in Zhang et al. [22], and no more new species have been reported except the two new species reported in this study. Super-cell in the species key refers to the cell in the conidia significantly larger than other cells.

Discussion
Both the phylogenetic analysis in this study and previous studies divided NTF into two main clades based on the mechanisms by which they catch nematodes (the genus Drechslerella produces constricting rings to capture nematodes with mechanical force, and the genera Arthrobotrys and Dactylellina catch nematodes with adhesive traps) [11][12][13][14]. These results again emphasized the significance of trapping structure for species division and evolution. Different from previous studies, this study failed to cluster Dactylellina species into a stable cluster, possibly due to insufficient DNA data. We believe that as more DNA data are used, we will find more morphological or physiological features that match phylogenetic studies.
The evolution of nematode-trapping fungi (NTF) is one crucial node to understanding the history of fungal evolution because of its unique morphological characteristics and survival strategy [2][3][4][5]. Currently, the main focus of the evolution research on NTF is the evolution of the trapping structure [9,11,43]. However, on the one hand, the phylogenetic clade of Drechslerella in this study showed that some species with similar conidia morphology cluster stably into one branch, such as species in clade I producing fusiform conidia and species in clade II producing ellipsoidal conidia ( Figure 1). Moreover, in the whole NTF, species that produce the same trapping structure can easily be divided into different groups according to their conidia. For example, Drechslerella species can be divided into two groups according to the presence or nonpresence of super-cell in their conidia, and all Arthrobotrys species can be divided into three groups according to their conidia shape [2]. In addition to the law above, as the most critical reproductive structure in the asexual generation of fungi, conidia should have crucial evolutionary significance in theory. Based on the above, conidia may also be an essential evolutionary feature for NTF and an important basis for the NTF classification. Similarly, are other structures or physiological characteristics of NTF experiencing the same problems as conidia (which have important evolutionary or taxonomic significances but have been neglected)? In conclusion, the evolution of organisms is a process of interaction between organisms and the environment. The evolution of a single structure (trapping structure) cannot represent the evolution of the NTF species. The excessive focus on the evolution of a single structure while ignoring the characteristics of the whole species may lead to the mistake of the blind man feeling the elephant.
The compilation logic of the key of Drechslerella species is that species are first roughly classified by those features that can be used to identify species and are easily distinguishable, such as whether the conidia produce a super-cell or not, the shape of the conidia (fusiform, elliptical, cylindrical, digitate, etc.), whether the conidiophore is branched or not, and the number of conidia on the conidiophore. Then, species are further classified by those features that can be used for species identification but require further measurement and observation, such as the detailed feature of macroconidia (number and position of the septum and the size of the macroconidia). Finally, morphologically similar species are distinguished by those characteristics that are uncertain whether they can be used for species identification but are differences between different species, such as the presence and features of microconidia, the detailed feature of the apex of the conidiophore, and the features of the chlamydospore. Even identifying Drechslerella species requires those morphological features that are not known to be valid, so how difficult would it be to identify the more complex Arthrobotrys and Dactylellina species based on these features alone? Therefore, follow-up research needs to systematically study all potential morphological characteristics to find more reliable characteristics for species identification.
Most of the sexual generations of Orbiliomycetes nematode-trapping fungi are members of Orbilia [3]. However, due to the morphological conservation of the sexual generations, there exists a phenomenon wherein one sexual species corresponds to several morphologically different asexual species [44]. Additionally, with the implementation of the one fungus, one name policy [45,46], asexual NTFs need to use sexual names when discovering their sexual generation (Orbilia sp. ). This results in these different asexual species sharing the same sexual species name [44], which further leads to confusion in the classification system and relevant data in some databases (such as Genebank, https://www.ncbi.nlm.nih.gov/nuccore/?term=Orbilia+auricolor (accessed on 3 April 2023)). For this reason, we suggest that when reporting a pair of sexual and asexual species, it is necessary to discuss the difference between the sexual generation and known sexual species and, more importantly, consider the distinction between the asexual generation and known asexual generation. The naming of this pair of sexual and asexual species should be carefully evaluated separately, giving sexual and asexual generations different species names if necessary.

Supplementary Materials:
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof9070735/s1, Table S1: GenBank accession numbers involved in this study. Ex-type strains are in bold. The newly generated sequences are indicated in blue. Figure S1: Bayesian majority rule consensus tree based on a combined ITS, TEF and RPB2 sequence data from 87 species of Orbiliaceae nematode-trapping fungi. Bayesian posterior probabilities values equal or greater than 0.90 are indicated above the nodes. The new isolates are in blue, type strains are in bold. The tree is rooted by Vermispora fusarina YXJ02-13-5 and V. leguminacea AS 6.0291.

Data Availability Statement:
The data that support the finding of this study are contained within the article.