Arthrobotrys blastospora sp. nov. (Orbiliomycetes): A Living Fossil Displaying Morphological Traits of Mesozoic Carnivorous Fungi

The evolution of carnivorous fungi in deep time is still poorly understood as their fossil record is scarce. The approximately 100-million-year-old Cretaceous Palaeoanellus dimorphus is the earliest fossil of carnivorous fungi ever discovered. However, its accuracy and ancestral position has been widely questioned because no similar species have been found in modern ecosystems. During a survey of carnivorous fungi in Yunnan, China, two fungal isolates strongly morphologically resembling P. dimorphus were discovered and identified as a new species of Arthrobotrys (Orbiliaceae, Orbiliomycetes), a modern genus of carnivorous fungi. Phylogenetically, Arthrobotrys blastospora sp. nov. forms a sister lineage to A. oligospora. A. blastospora catches nematodes with adhesive networks and produces yeast-like blastospores. This character combination is absent in all other previously known modern carnivorous fungi but is strikingly similar to the Cretaceous P. dimorphus. In this paper, we describe A. blastospora in detail and discuss its relationship to P. dimorphus.


Introduction
The origin and evolution of life are the core of biological research [1]. As primary decomposers in nature, fungi play a vital role in the circulation of matter and energy in ecosystems [2][3][4]. Studying fungal evolution is integral to understanding the origin and evolution of life. In nature, most fungi are saprophytic, symbiotic, and parasitic, while a few fungi feed on micro-animals such as nematodes, rhizopods, rotifers, and mites (carnivorous fungi) [5][6][7][8]. This survival strategy of feeding on other microorganisms is generally considered an adaptive evolution of fungi to allow them to adapt to nitrogen deficiency [8,9]. The study of the origin and evolution of carnivorous fungi is crucial for understanding the history of fungal evolution. More than 80% of carnivorous fungi in the whole fungal kingdom belong to Orbiliaceae (Orbiliomycetes, Ascomycota) [9,10]. These fungi capture nematodes with various trapping structures. Modern molecular phylogenetic and morphological studies have divided all Orbiliomycetes carnivorous species into three genera according to type of trapping structure. Drechslerella captures nematodes using constricting rings, Arthrobotrys produces adhesive networks, and Dactylellina captures nematodes with adhesive branches, adhesive knobs, and non-constricting rings [11][12][13]. Although such studies have revealed the phylogenetic relationship among these fungi, the evolutionary hypothesis of Orbiliomycetes carnivorous fungi is still controversial [9][10][11][13][14][15][16][17].
Fossils hold the key that nature provided us to breakthrough in the study of the origin and evolution of life [18][19][20][21]. However, compared with animals and plants, most fungi are tiny and do not have solid tissue structures to form fossils and be discovered by humans. Therefore, understanding the origin and evolution of fungi is very difficult. Excitingly, a few fossils that might be related to carnivorous fungi have been discovered. Jansson and Poinar found several conidia that resembled modern carnivorous fungi and several nematodes with appendages (the morphology of which is similar to adhesive spores or adhesive knobs produced by carnivorous fungi) attached to their bodies and fungal mycelium in their bodies in approximately 26-million-year-old amber [22]. Schmidt et al. [23,24] discovered the oldest relatively complete and clear fossil of a possible carnivorous fungus (Palaeoanellus dimorphus) in approximately 100-million-year-old amber, which caused a stir in the research on carnivorous fungi. Unfortunately, no species similar to this fossil has been found in the modern ecosystem so far. It has therefore been unclear whether this fossil is an ancestor of modern carnivorous fungi [12,23,25] because the character combination found in this fossil was distinct from any extant taxa.
During our large-scale survey of carnivorous fungi in the three parallel rivers region in China, two extraordinary carnivorous fungal isolates were discovered from 8698 carnivorous fungal strains isolated from 3617 soil samples and identified as a new species of the Orbiliomycetes carnivorous fungi. Fascinatingly, the morphological characteristics of this species are different from other modern carnivorous fungi and are strikingly similar to the carnivorous fossil fungus (Palaeoanellus dimorphus) discovered by Schmidt [23,24], according to which we speculate that this new species is a relict descendant of P. dimorphus. The discovery of this new species suggests that P. dimorphus is a possible ancestor of Orbiliomycetes carnivorous fungi and provides more accurate information for the evolutionary study of this group of fungi.

Sample Collection
The two carnivorous fungal strains were isolated from two freshwater sediment samples in the Nujiang River Basin, the core area of the three parallel rivers. The sample numbers were EOS-1 (N 27 •

Fungal Isolation
Three to five g of freshwater sediment sample was sprinkled on the surface of cornmeal agar plates (CMA) with sterile toothpicks. Roughly 5000 nematodes (Panagrellus redivivus Goodey, free-living nematodes) were added as bait to induce the germination of carnivorous fungi [12,26]. The plates were incubated at 26 • C for three weeks and then observed under a stereomicroscope. A sterile needle was used to transfer a single spore of carnivorous fungi to fresh CMA plates. This step was repeated until a pure culture was attained [10,12].

Morphological Observation
The pure culture was transferred to fresh potato dextrose agar plates (PDA) using a sterile needle and incubated at 26 • C to observe the color and texture of the colony. The pure culture was transferred to the fresh observation well CMA plates (a 2 × 2 cm observation well was created by removing agar from each plate) using a sterile needle. It was incubated at 26 • C until the mycelium overspread the well. Then, approximately 1000 living nematodes (P. redivivus) were added to the well to induce the formation of the trapping structure. The trapping structure in the observation well and the conidiophores extending from the wall of the observation well were photographed with an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan) and Keyence VHX-6000 super deep scene 3D microscope (Keyence Corporation, Osaka, Japan), respectively. A sterile cover glass was obliquely inserted into the fresh CMA plates. Then, strains were inoculated on the plates at 26 • C. The cover glass was removed after the mycelia covered it and was then placed on the glass slide with 0.3% Melan stain to make a temporary slide [12]. The morphological characteristics of conidia and conidiophores were measured and photographed by an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan).

DNA Extraction, PCR Amplification, and Sequencing
The strain was inoculated in PDA plates at 26 • C for ten days. The mycelium was collected using a sterile scalpel. A rapid fungal genomic DNA isolation kit (Sangon Biotech, Limited, Shanghai, China) was used to extract the total genomic DNA. The primer pairs ITS4-ITS5 [27], 526F-1567R [28], and 6F-7R [29] were used to amplify the ITS, TEF, and RPB2 regions. The PCR amplification was performed in a 50 µL reaction system (2 uL DNA template, 3 µL 25 mM MgCl2, 5 µL 10 × PCR buffer, 1 µL 10 µM dNTPs, 2 µL each primer, 1 unit Taq Polymerase, and 34 uL ddH2O) under the following PCR conditions: 4 min of pre-denaturation at 94 • C; followed by 35 cycles of denaturation at 94 • C for 45 s; 1 min of annealing at 52 • C (ITS), 55 • C (TEF), or 54 • C (RPB2); and 1.5-2 min of extension at 72 • C, with a final extension of 10 min at 72 • C. A DiaSpin PCR Product Purification Kit (Sangon Biotech, Limited, Shanghai, China) was used to purify the PCR products. The purified PCR products of the ITS and RPB2 regions were sequenced in the forward and reverse directions using PCR primers, and the primer pair 247F-609R was used to sequence the TEF gene (BioSune Biotech, Limited, Shanghai, China). SeqMan v. 7.0 [30] was used to check, edit, and assemble the sequences. The sequences generated in this study were deposited in the GenBank database (NCBI, https://www.ncbi.nlm.nih.gov/ (accessed on 2 December 2022)).

Phylogenetic Analysis
The sequences generated in this study were deposited in the NCBI Genbank database (Table 1) and compared against the database using BLASTn (https://blast.ncbi.nlm.nih. gov/ (accessed on 1 December 2022)) to determine the attribution of the new isolates. The ITS, TEF, and RPB2 sequences of all reliable taxa of the corresponding genus and partial taxa of the related genus were downloaded (Table 1) according to the BLASTn search results and relevant publications [12,13,31,32]. Three genes were aligned using the online program MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/ (accessed on 3 December 2022)) [33], manually adjusted using BioEdit v7.2.3 [34], and then linked using MEGA6.0 [35]. Vermispora fusarina YXJ02-13-5 was selected as an outgroup. Phylogenetic trees were inferred via maximum likelihood (ML) and Bayesian inference (BI) analyses. The GTR + I + G, SYM + I + G, and GTR + I + G models were selected as the best-fit optimal substitution models of ITS, TEF, and RPB2, respectively, via jModelTest v2.1.10 [47].
IQ-Tree v1.6.5 [48] was used to implement the maximum likelihood (ML) analysis. The dataset was partitioned, and each gene was analyzed with the corresponding optimal substitution model. The statistical bootstrap support values (BS) were computed using rapid bootstrapping with 1000 replicates [49].
A Bayesian inference (BI) analysis was conducted with MrBayes v. 3.2.6 [50]. Fasta Convert [51] was used to convert the multiple sequence alignment file into a MrBayescompatible NEXUS file. The dataset was partitioned, and the optimal substitution models of each gene were equivalently replaced to conform to the setting of MrBayes. Six simultaneous Markov chains were run for 10,000,000 generations, and trees were sampled every 100 generations. The first 25% of the trees were discarded, and the remaining trees were used to calculate the posterior probabilities (PP) in the majority rule consensus tree. The above parameters were edited in the MrBayes block in the NEX file.

Phylogenetic Analysis
Both new fungal isolates were placed in the Arthrobotrys (Orbiliaceae, Orbiliomycetes) genus according to their type of trapping structure [11][12][13] and the BLASTn search results of ITS, TEF, and RPB2 genes. Therefore, all Arthrobotrys species with valid sequence data (62 isolates representing 59 species) [32] and other related taxa in Orbiliomycetes (9 isolates representing 8 Dactylellina species and 5 isolates representing 5 Drechslerella species) were included in this phylogenetic analysis ( Table 1). The final dataset contained 77 ITS, 51 TEF, and 54 RPB2 sequences. The combined DNA dataset comprised 1909 characters (570 for ITS, 531 for TEF, and 708 for RPB2), among which 858 bp are constant, 982 bp are variable, and 770 bp are parsimony informative. After maximum likelihood (ML) analysis, the best-scoring likelihood tree was obtained with a final ML optimization likelihood value of -6867.586931. The Bayesian analysis (BI) evaluated the Bayesian posterior probabilities with a final average standard deviation of the split frequency of 0.009098. The trees generated by maximum likelihood (ML) and Bayesian analysis (BI) showed similar topologies, so the best-scoring ML tree was selected for presentation ( Figure 1). The phylogenetic analysis showed that the tested 72 Orbiliaceae (Orbiliomycetes) carnivorous species were clustered into three clades according to their types of trapping structure. All species catch nematodes with adhesive networks clustered together stably. Both new fungal isolates were placed in Arthrobotrys and formed a basal lineage with A. oligospora and A. superba with 99% MLBS and 1.00 BYPP (Figure 1).

Taxonomy
Arthrobotrys blastospora F. Zhang and X.Y. Yang sp. nov. (Figures 2, 3a, 4b and 5a).            . (a,b) Trapping structure of Palaeoanellus dimorphus: unicellular adhesive hyphal rings, reprinted with permission from Ref. [24]. 2023, John Wiley and Sons; (c,d) The early stages of adhesive networks produced by Arthrobotrys blastospora: single adhesive hyphal rings; (e,f) Trapping structure of some Dactylellina species: non-constricting rings [12].  µm, n = 100) at the apex, hyaline, erect, septate, unbranched, produced by hyphae or directly by spore germination. This species produces hyaline, yeast-like blastospores, which usually cluster on the tuberculous bulges on the upper half of the conidiophores. A conidium is produced on the conidiophore first; then, a second conidium is formed from the right apex, or occasionally the side apex, of the first conidium, thus continuously producing a chain of blastospores. There is a septum between the two conidia, which tend to separate from each

New Species of Arthrobotrys
Arthrobotrys blastospora catches nematodes with adhesive networks, which is consistent with the main characteristics of Arthrobotrys, Orbiliaceae (the largest genus of modern carnivorous fungi) [11][12][13]25]. Our phylogenetic analysis based on ITS, TEF, and RPB2 substantiated that A. blastospora is a member of Arthrobotrys. Phylogenetically, A. blastospora forms the sister lineage to A.oligospora (Figure 1). However, the conidia of all modern carnivorous fungi in Orbiliomycetes are individually born in clusters or singly on the conidiophores, which is significantly different from the catenulate blastospores produced by A. blastospora [12,31]. Therefore, we identified A. blastospora as a new species of Arthrobotrys.

Palaeoanellus Dimorphus Is an Ancient Ancestor of Modern Orbiliomycetes Carnivorous Fungi
Similar to carnivorous plants, as a highly specialized group in the fungal kingdom, carnivorous fungi are a model for the study of adaptive fungal evolution; their evolution is also a critical node in the study of fungal evolution. Such studies rely heavily on the discovery of fossil fungi. Schmidt et al. [23,24] found the earliest and best-preserved fossil of carnivorous fungi (P. dimorphus) in approximately 100-million-year-old amber from Southwestern France. P. dimorphus produced blastospores which were generated in whorls on small projections of conidiophores and also produced unicellular adhesive hyphal rings to trap nematodes (Figures 3 and 4) [23,24]. However, no structure directly connecting the blastospores to unicellular adhesive hyphal rings was illustrated by Schmidt et al. [23,24]. Therefore, whether the blastospores and unicellular adhesive hyphal rings in this fossil actually represent a single fossil species has been a controversial topic due to their unusual combination [53,54]. In addition, the fossil was not considered an ancestor of modern carnivorous fungi but as belonging to an extinct lineage because no blastospore-producing carnivorous fungi were found in modern ecosystems [12,[23][24][25]. A. blastospora reported in this study produced yeast-like blastospores on the small projections of conidiophores and captured nematodes with adhesive networks (a single adhesive hypha ring structure at the initial stage of its formation) (Figure 2) [31]. The discovery of this species confirms the existence of an extant species in nature that produces both specialized nematode-trapping structures and blastospores. Furthermore, A. blastospora and P. dimorphus share strong similarities with regard to the morphological characteristics of conidia, conidiophores, and the manner of catching nematodes with adhesive materials (Figures 3 and 4). Accordingly, we infer that A. blastospora is closely related to P. dimorphus and maintained traits of Mesozoic carnivorous fungi, and P. dimorphus may be an ancient ancestor of modern Orbiliomycetes carnivorous fungi.
Similar to other fungi, convergent evolution has also been observed in carnivorous fungi; for example, some species of Dactylellina (Orbiliaceae, Orbiliomycetes, Ascomycota) and some species of Nematoctonus (Agaricomycetes, Pleurotaceae, Basidiomycota) trap nematodes with stalked adhesive knobs [55]. Therefore, we also cannot rule out the possibility of convergent evolution between A. blastospora and P. dimorphus, which resulted in the sharing of similar characteristics, while having a distant genetic relationship. However, given the scarcity of carnivorous fungi in the fungal kingdom [9,12] and the high similarity of several different structures (conidia, conidiophores, and trapping structures) between A. blastospora and P. dimorphus (Figures 3 and 4) [23,24], we speculate that it is less likely that these two species would have evolved such similar traits with only a distant genetic relationship.

The Possible Ancestral Position of Palaeoanellus Dimorphus
Among Orbiliomycetes carnivorous fungi, all species are divided into two main groups according to their mechanisms of trapping nematodes. One is the genus Drechslerella, which first diverged from other carnivorous species and produces constricting rings to capture nematodes with the mechanical force generated by the expansion of the cells that make up the rings. Another contains all species in the genera Arthrobotrys and Dactylellina, which catch nematodes with adhesive traps (Figure 1) [11][12][13]. P. dimorphus produced unicellular hyphal rings, which possibly produced a sticky secretion used to capture nematodes [23,24]. This structure is similar to those species in Arthrobotrys and Dactylellina in the manner of trapping nematodes (capture of nematodes performed mainly with adhesive material), but it is quite different from the Drechslerella species, which capture nematodes by mechanical force. Therefore, we speculate that P. dimorphus is more related to Arthrobotrys and Dactylellina, and that P. dimorphus may be the common ancestor of Arthrobotrys, Dactylellina, or Arthrobotrys and Dactylellina.
Generally accepted, modern Orbiliomycetes carnivorous fungi originated from saprophytic fungi without a trapping structure [9][10][11][14][15][16][17]. Their evolution from possessing no trapping structure to complex trapping structures, such as modern adhesive trapping structures, was undeniably a course of gradual complexity. The structural complexity of unicellular adhesive hyphal rings produced by P. dimorphus is lower than that of most modern adhesive trapping structures. Therefore, unicellular adhesive hyphal rings may be considered an intermediate stage in the evolution of structural complexity and the common ancestor of all adhesive trapping structures (Arthrobotrys and Dactylellina). However, based on phylogenetic analysis of multiple genes and molecular clock theory, Yang et al. [9] inferred that the adhesive trapping structures of modern Orbiliomycetes carnivorous fungi originated about 246 million years ago and further evolved around 198-208 million years ago. In contrast, P. dimorphus was found in the amber from 100 million years ago [23,24]. Therefore, it can be inferred that the unicellular adhesive hyphal rings produced by P. dimorphus are probably not the ancestor of all the modern adhesive-trapping structures (Arthrobotrys and Dactylellina).
Phylogenetically, A. blastospora forms a sister lineage to A. oligospora and A. superba (Figure 1). Combined with the morphological similarities between A. blastospora and P. dimorphus, we can infer that P. dimorphus is closely related to the Arthrobotrys species. Concerning morphology, the following aspects can also support the close relationship between P. dimorphus and Arthrobotrys: (1) P. dimorphus produced unicellular adhesive hyphal rings to capture nematodes [23,24]. This structure is morphologically similar to the singlering stage of adhesive networks produced by Arthrobotry species (Figure 4) [31]. (2) The formation of the unicellular adhesive hyphal rings produced by P. dimorphus initiated with a branch which was first generated on the vegetative mycelia, then the branch was curved and fused with the mycelia to form a ring [23,24]. This process is highly similar to the formation process of adhesive networks produced by Arthrobotrys species [31] (Figure 4). (3) The blastospores produced by A. blastospora are easily separated from each other to form non-septate and 1-septate elliptic conidia. The blastospores produced by P. dimorphus also had this characteristic. The non-septate or 1-septate conidia formed by the separation of blastospores are morphologically similar to those of many species in Arthrobotrys ( Figure 5) [12,31]. (4) Among Arthrobotrys species, except A. blastospora, a few conidia of A. oligospora and A. conoides also have a similar morphology to blastospores (Figure 6), which suggests that the formation of blastospores may be an ancestral characteristic of Arthrobotrys, or an inherent feature of other Arthrobotrys species, but it is rarely developed or not developed in culture and thus, it has been overlooked so far. This phenomenon further illustrated the close relationship between P. dimorphus and Arthrobotrys. By contrast, Dactylellina demonstrates similarities to P. dimorphus only in the aspect of trapping structure: a few species in Dactylellina produce a single ring covered with adhesive material (non-constricting ring) to capture nematodes, which is formed by producing a branch from the vegetative mycelia, and then the branch is curved and fused to form a ring [31]. This structure is similar to the unicellular adhesive hyphal rings produced by P. dimorphus in the morphology and formation process (Figure 4).
In summary, considering that P. dimorphus and Arthrobotrys share a reproductive structure (conidia) and nutritional structure (trapping structure) morphology, we speculate that P. dimorphus is more likely to be the ancestor of Arthrobotrys.

The Necessity of Strengthening the Research on Carnivorous Fungi in the Three Parallel Rivers
The two A. blastospora strains were isolated from the core area of the three parallel rivers region in China. This region is located in the southwest of the Heng Duan Mountains where mountains alternate with valleys, the terrain is highly diverse, and the region combines tropical, subtropical, temperate, and alpine cold climate types [56,57]. The complex terrain and climate create rich ecosystems and make the region one of the most By contrast, Dactylellina demonstrates similarities to P. dimorphus only in the aspect of trapping structure: a few species in Dactylellina produce a single ring covered with adhesive material (non-constricting ring) to capture nematodes, which is formed by producing a branch from the vegetative mycelia, and then the branch is curved and fused to form a ring [31]. This structure is similar to the unicellular adhesive hyphal rings produced by P. dimorphus in the morphology and formation process (Figure 4).
In summary, considering that P. dimorphus and Arthrobotrys share a reproductive structure (conidia) and nutritional structure (trapping structure) morphology, we speculate that P. dimorphus is more likely to be the ancestor of Arthrobotrys.

The Necessity of Strengthening the Research on Carnivorous Fungi in the Three Parallel Rivers
The two A. blastospora strains were isolated from the core area of the three parallel rivers region in China. This region is located in the southwest of the Heng Duan Mountains where mountains alternate with valleys, the terrain is highly diverse, and the region combines tropical, subtropical, temperate, and alpine cold climate types [56,57]. The complex terrain and climate create rich ecosystems and make the region one of the most biodiverse in the world [58,59]. Glaciers did not cover this region during the Quaternary glaciation due to its unique mountain and deep valley landform, particularly its geographical position, formation and evolutionary process. Therefore, this region is a significant refuge for many ancient species, and it is the center of species distribution and the differentiation of many biological groups [60][61][62][63][64]. According to statistics, 34 species of Chinese national protected plants, 600 species of endemic plants, and 20 species of relict plants are distributed in this region [65,66]. This situation renders it possible to find the relict species of carnivorous fungi in this region and suggests that there may be precious living fossils of other groups living in this region. In addition, P. dimorphus was found in the amber from Southwestern France [23,24] and A. blastospora was isolated from Southwestern China, more than 11,000 km from each other. This indicates that Palaeoanellus-type fungi were widely distributed and numerous in the past, giving rise to the extant genera of Oribiliomycetes.

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