Multilocus Phylogeny and Characterization of Five Undescribed Aquatic Carnivorous Fungi (Orbiliomycetes)

The diversity of nematode-trapping fungi (NTF) holds significant theoretical and practical implications in the study of adaptive evolution and the bio-control of harmful nematodes. However, compared to terrestrial ecosystems, research on aquatic NTF is still in its early stages. During a survey of NTF in six watersheds in Yunnan Province, China, we isolated 10 taxa from freshwater sediment. Subsequent identification based on morphological and multigene (ITS, TEF1-α, and RPB2) phylogenetic analyses inferred they belong to five new species within Arthrobotrys. This paper provides a detailed description of these five novel species (Arthrobotrys cibiensis, A. heihuiensis, A. jinshaensis, A. yangbiensis, and A. yangjiangensis), contributing novel insights for further research into the diversity of NTF and providing new material for the biological control of aquatic harmful nematodes. Additionally, future research directions concerning aquatic NTF are also discussed.


Introduction
Nematode-trapping fungi (NTF) are a group of fungi that possess a unique trapping structure to capture nematodes for nutrition [1][2][3][4].NTF in Orbiliomycetes are considered the core representatives of NTF due to their rich species diversity, and intricate and diverse trapping structures, as well as their important role in maintaining ecological balance and their potential value in the bio-control of harmful nematodes [4][5][6][7].Currently, this group of fungi includes 125 species from three genera: Arthrobotrys (73 species) which captures nematodes using adhesive networks; Dactylellina (35 species), the genus that captures nematodes with adhesive branches, non-constricting rings, and adhesive knobs; and Drechslerella (17 species) which catches nematodes using constricting rings [4,7,8].
These fungi are widely distributed in various habitats because of their unique survival strategy.They are commonly found in the soils from farmlands, forests, and even heavy metal-contaminated areas [4,[9][10][11], as well as in sediments from marine, freshwater, and even hot springs [12][13][14].But compared to the well-studied terrestrial ecosystems, the diversity of NTF in freshwater habitats remains insufficiently studied [13,15,16].Previous studies have confirmed the existence of a rich diversity of NTF in freshwater ecosystems, which is reasonable given the abundance of nematodes in aquatic environments [13,17].Meanwhile, the diverse array of nematodes in aquatic habitats includes parasitic species that pose threats to aquatic crops and fisheries [18,19].So, studying aquatic NTF resources is an important part of NTF diversity research and bio-control of harmful aquatic nematodes.Additionally, the study on aquatic NTF also provides a valuable entry point for investigating fungal adaptive evolution, as aquatic NTF originate from their terrestrial counterparts.
In the past 10 years, we have investigated the NTF in the six major watersheds in Yunnan Province and successfully isolated 10 strains, which were identified as five novel members of Arthrobotrys.This paper provides a comprehensive account of these species, offering new material for the bio-control research of harmful nematodes and the study of fungal aquatic adaptive evolution.

Samples Collection
All freshwater sediment samples involved in this study were collected using a Peterson bottom sampler (HL-CN, Wuhan Hengling Technology Company, Limited, Wuhan, China).The samples were placed into plastic zip-lock bags to preserve moisture.Collecting sites, date, and collector were recorded (Table 1).The samples were stored at 4 • C and processed within a week.

Fungal Isolation
Nematodes (Panagrellus redivivus Goodey, free-living nematodes) cultured on oatmeal medium [4] were isolated using the Baermann funnel method [20] and the concentration of the nematodes was adjusted with sterile water to 3000-5000 nematodes per milliliter.The soil sprinkling technique was used to disperse the sediment sample onto the surface of corn meal agar plates (CMA) [4] and 1 mL of nematode suspension was added to promote the germination of NTF.The plates were incubated at room temperature (14-28 • C) for about three weeks and a stereomicroscope was used to observed the plates to search for the NTF spores.The single-spore isolation method was used for the isolation and purification of the NTF [4].

Morphological Observation
The isolates were inoculated onto potato dextrose agar (PDA) [4] plates and cultured at 26 • C for colony observation.The isolate was transferred to CMA observation plates (creating an observation well by removing a 2 × 2 cm piece of agar from the center of the CMA plate and obliquely inserting a sterile cover glass into the surface of the medium) and incubated at 26 • C [21].After the observation well was covered by the mycelium, about 1000 nematodes (P.redivivus) were added as baits to induce the production of traps.The types of traps were checked and photographed using an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan).When the mycelium had spread over the cover glass, the cover glass was removed with tweezers and a temporary slide made with sterile water [22].An Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan) was used to photograph and measure the morphological characteristics such as conidia, conidiophores, and chlamydospores.

Collection of DNA Molecular Data
The mycelium grown on PDA plates was used to extract genomic DNA, as described by Zhang et al. [15].The primer pairs ITS4-ITS5 [23], 526F-1567R [24], and 6F-7R [25] were used to amplify the ITS, TEF1-α, and RPB2 regions, respectively, under the reaction system and conditions described in the previous study [21].The PCR products were sent to BioSune Biotech Company Limited (Shanghai, China) for purification and sequencing (TEF1-α genes were sequenced using the 247F-609R [7] primer pair and ITS and RPB2 regions were sequenced with PCR primers).
The generated sequences were carefully examined, edited, and assembled using SeqMan v. 7.0 [26].All sequences obtained in this study have been submitted to the GenBank database (NCBI; https://www.ncbi.nlm.nih.gov/;accessed on 29 November 2023) for deposition.
The trees were visualized via FigTree v. 1.3.1 [33] and edited using Microsoft Power-Point v. 2016 (Microsoft, Redmond, WA, USA) and Adobe Photoshop CS6 software (Adobe Systems, San Jose, CA, USA).

Phylogenetic Analysis
The combined ITS, TEF1-α, and RPB2 alignment dataset consisted of 88 sequences of ITS, 62 sequences of TEF1-α, and 64 sequences of RPB2 from Arthrobotrys 75 taxa, representing 69 valid species (plus our five new species), other related taxa in Orbiliomycetes (Dactylellina four taxa and Drechslerella seven taxa), and two outgroup taxa.The final dataset comprised 2000 characters (585 for ITS, 8321 for RPB2, and 583 for TEF1-α), among which 900 base pair (bp) are constant, 1087 bp are variable, and 886 bp are parsimony-informative.
The best-scoring ML tree was generated with a final ML optimization likelihood value of −6817.314758.Bayesian analysis (BI) was used to evaluate the Bayesian posterior probabilities with a final average standard deviation of the split frequency of 0.009092.Both ML and BI trees consistently grouped all tested nematode-trapping fungi into three major clades and five new species exhibited distinct divergence from known species.Therefore, the ML tree was chosen for presentation (Figure 1).The phylogenetic tree inferred from the ITS, TEF1-α, and RPB2 combined dataset placed five new species in Arthrobotrys.The phylogenetic position of A. heihuiensis is uncertain but clearly diverges from known species.The two isolates of A. yangbiensis formed a distinct lineage basal to A. gampsospora with 99% MLBS and 0.97 BYPP support.Furthermore, A. yangjiangensis and A. jinshaensis clustered together with A. mangrovispora, A. thaumasia, A. eudermata, and A. janus with 89% MLBS and 0.91 BYPP support.A. cibiensis formed a distinct lineage basal to A. longiphora, A. xiangyunensis, and A. reticulatus with 99% MLBS and 0.96 BYPP support (Figure 1).Culture characteristics: Colonies on PDA white, cottony, reaching 60 mm diameter after 10 days at 26 • C. Hypha composed of septate, branched, smooth, and hyaline.Conidiophores erect, septate, hyaline, unbranched, bearing a single conidium at the apex, 145-315.5 µm long (X = 234.4µm, n = 50), 4.5-7.5 µm (X = 5.6 µm, n = 50) wide at the base, and 2-4 µm (X = 3.1 µm, n = 50) wide at the apex.Conidia smooth-faced and hyaline, rounded at the apex and truncated at the base, 26.5-46 × 13.5-23 µm (X = 37.1 × 17.7 µm, n = 50), immature having drop-shaped, obovate, with a super cell (the cell in the conidia significantly larger than other cells) at the apex and one to three septa (mostly two-septate) at the base of the conidia; mature conidia subfusiform, two-to three-septate (mostly three-septate, one septum at the apex and two septa at the base), with a super cell at the middle of the conidia.Conidia germinate from the small cells at both ends and the super cells never germinate.Catching nematodes with adhesive networks.Chlamydospores 9-35.5 × 6.5-13 µm (X = 16.2 × 10.1 µm, n = 50), smooth-faced and hyaline, cylindrical, globose or ellipsoidal, hyaline, and in chains when present.
Notes: Phylogenetically, A. heihuiensis is sister to A. cystosporia but lacking in statistical support (Figure 1).Of all Arthrobotrys species, the morphological characteristics of A. heihuiensis are relatively unique and only A. scaphoides may be confused with A. heihuiensis but there are several obvious differences between them: (1) the conidia of A. heihuiensis usually scattered on the short denticles of the conidiophores, while the conidia of A. scaphoides are usually clustered on the node of the short branch produced by conidiophores; and (2) A. scaphoides produces five-or six-septate conidia, while the conidia of A. heihuiensis have no more than four septa.Additionally, the conidia of A. heihuiensis are obviously smaller than that of A. scaphoides (A.heihuiensis, 31-56 (45.4) × 6.5-14.5 (11.5) 4) Index Fungorum number: IF901489; Facesoffungi number: FOF14176 Etymology: The species name "jinshaensis" refers to the name of sample collection site: Jinsha River, Jinjiang Town, Shangri-La City, Yunnan Province, China.

Discussion
Among the 130 species (plus our five new species) of Orbiliomycetes nematodetrapping fungi (NTF), 61 species have distribution records in freshwater habitats.Among these species, 17 species were first isolated from freshwater habitats.To date, A. hyrcanus, A. blastospora, A. dainchiensis, A. eryuanensis, A. hengjiangensis and five new species

Discussion
Among the 130 species (plus our five new species) of Orbiliomycetes nematode-trapping fungi (NTF), 61 species have distribution records in freshwater habitats.Among these species, 17 species were first isolated from freshwater habitats.To date, A. hyrcanus, A. blastospora, A. dainchiensis, A. eryuanensis, A. hengjiangensis and five new species reported in this study were exclusively found in freshwater environments [12][13][14][15][16][17]34,35,42].These findings collectively emphasize that freshwater NTF are an important component of NTF diversity.Hence, future studies on NTF diversity should fully consider the significance of freshwater habitats.
The five newly identified species presented were all derived from sediment samples in water depths of less than 2 m.Previous investigations have demonstrated the absence of NTF beyond a water depth of 4 m [13].However, abundant aquatic nematodes still exist in deeper waters [43,44].At such depths, there must be other nematode regulators to perform the function of aquatic nematode population regulation instead of NTF.Accordingly, it is speculated that more novel nematophagous microorganisms may be discovered in deeper waters employing efficacious research methodologies.The exploration and examination of these enigmatic nematophagous microorganisms hold the potential to provide valuable insights into the origins and evolutionary processes of carnivorous microorganisms, while also offering promising prospects for the biological control of detrimental nematodes.
It is generally believed that aquatic NTF originate from terrestrial ecosystems.However, unlike terrestrial environments, NTF face numerous challenges in aquatic habitats.For instance, the interaction between NTF and nematodes relies on processes such as host recognition, generation of trapping structures, invasion and digestion of nematode, etc.These processes are more or less dependent on the transmission of extracellular signaling factors [45][46][47].However, in aquatic environments, these extracellular signaling molecules are likely to be diluted and lose their corresponding functions.Consequently, how aquatic NTF prey on nematodes in water, how they maintain osmotic balance in water, and how they reproduce and spread in water remain unresolved questions in the field.Investigating these questions can not only deepen our understanding of fungal adaptive evolution but also constitutes an important aspect of the study on the origin and evolution of these extraordinary organisms.

Figure 1 .Figure 1 .
Figure 1.The maximum likelihood tree inferred from a combined ITS, TEF1-α, and RPB2 dataset.The black and red numbers in front of the node indicate Bootstrap support values for maximum Figure 1.The maximum likelihood tree inferred from a combined ITS, TEF1-α, and RPB2 dataset.The black and red numbers in front of the node indicate Bootstrap support values for maximum likelihood equal or greater than 70% and Bayesian posterior probabilities values equal or greater than 0.90, respectively.Our new isolates are in blue and the type strains are in bold.

Table 1 .
Samples information involved in this study.