Morphological and Molecular Characterization of Nothotylenchus medians and N. similis (Nematoda: Anguinidae) from Southern Alberta, Canada

The nematode family, Anguinidae, is a diverse group of polyphagous nematodes, generally known as fungal feeders or parasites of aerial plant parts. Here, we present the morphological and molecular characterization of adult females of two Nothotylenchus species, N. medians and N. similis, along with host association and geographical distribution data of the genus. Both species are recorded as new reports from Canada and designated as reference populations for future studies. Morphological or morphometrical variation was not observed in the Canadian population of N. medians and N. similis, in comparison with the original description. Phylogenetic analyses based on 18S and D2–D3 of 28S genes placed both species within Anguinidae. Since the biology of the genus Nothotylenchus has not been rigorously characterized, the habitat and distribution information presented in this study will shed some light on the ecology of these nematodes. Notably, the detection of N. medians and N. similis in our nematode inventory survey indicates that considerable Nothotylenchus diversity is hidden in these soils. Consequently, increased surveys and more in-depth research are needed to explore the full diversity of anguinids inhabiting these cultivated areas.


Introduction
Family Anguinidae Nicoll [1] is a diverse group of stylet-bearing nematodes generally known as fungal feeders or parasites of the aerial parts of plants [2]. The family contains about 15 genera, however only Ditylenchus Filipjev [3] and Anguina Scopoli [4] species have received significant attention due to their parasitic potential and quarantine regulations [5]. Among other genera of Anguinidae, Nothotylenchus Thorne [6] was formulated for those Ditylenchus-like species that do not possess a valvular median pharyngeal bulb [2,6]. Based on similarity, Nothotylenchus has been synonymized with Ditylenchus in several studies [7][8][9]. According to the new classification system described by Decraemer and Hunt [5], Nothotylenchus is regarded as a valid genus. Recently, Hashemi and Karegar [10] reviewed the entire genus and improved the species identification by providing a compendium of Nothotylenchus nominal species.
The genus Nothotylenchus is composed of ectoparasitic nematodes that have adapted to a diverse range of ecological niches [2,[11][12][13]. This genus is the second-largest of the family Anguinidae after Ditylenchus, and currently comprises 41 nominal species [10,14]. These species have been reported from both agricultural and natural systems, but no significant plant damage has been associated with any Nothotylenchus spp., except for N. acris, which was reported to cause stunting and deformation of strawberry plants [15]. Biogeographically, the majority of Nothotylenchus species originate from Asian countries,

Isolation and Morphological Studies
The nematode inventory survey was conducted near village of Barnwell (Alberta, Canada) to examine the wide spectrum of PPNs associated with cultivated plants. We collected several soil and root samples from different headland areas of a cultivated field, and maintained them in the cold storage (4 • C) at the University of Lethbridge (Alberta, Canada) until processing. Nematodes were extracted from the soil samples using the modified Cobb sieving and flotation-centrifugation method [29]. Individual anguinid taxa were collected from the mixture of soil nematodes and assigned the population numbers 42A and 42B. For preliminary examination, fresh adults of each species were transferred to a drop of distilled water, heat relaxed, and observed under a Zeiss Axioskope 40 microscope. For morphometric studies, the nematodes were fixed, and permanent slides were prepared according to the methods of Seinhorst [30] and De Grisse [31]. Images of each specimen were acquired using a Zeiss Axioskope 40 microscope equipped with a Zeiss Axiocam 208 camera (Carl Zeiss, Jena, Germany). Measurements from the images were performed using ZEN blue 3.1 imaging software (Carl Zeiss).

DNA Extraction, PCR, and Sequencing
After preliminary microscopic examination, a single nematode of each taxon was transferred to a 0.2 mL PCR tube, and the DNA was extracted as described in Maria et al. [32]. Briefly, the single nematodes were crushed in 6 µL Milli-Q ultrapure water using a sterile pipette tip. The tubes containing crushed nematodes were briefly spun and immediately incubated at −80 • C for at least 30 min. Then, the samples were heated to 85 • C for 2 min, briefly spun, followed by the addition of 2 µL proteinase K and 2 µL 10× PCR buffer. The tubes were incubated at 56 • C for 2 to 3 h, followed by a 10-min period at 95 • C. These tubes, containing crude nematode DNA, were cooled to 4 • C and used in the subsequent PCR analyses. Three sets of DNA primers (Integrated DNA Technologies, Coralville, IA, USA) were used to amplify the 18S, 28S, and ITS1 of the ribosomal RNA (rRNA) genes. The partial 18S rRNA gene was amplified with 1813F and 2646R primers [33]. The 28S rRNA gene was amplified using D2A and D3B primers [34] and the ITS1 gene was amplified using F194 [35] and AB28-R primers [36]. For the 18S, 28S, and ITS1 genes, the PCR conditions were as described in Holterman et al. [33], De Ley et al. [34], and Ferris et al. [35], respectively. Amplified PCR products were resolved by electrophoresis in 1% agarose gels and visualized by staining with GelRed (Biotium, Fremont, CA, USA). PCR products containing amplified DNA fragments of interest were sent to Azenta Life Sciences for DNA sequencing (South Plainfield, NJ, USA).

Phylogenetic Analyses
In the present study, we obtained DNA sequences for the 28S rRNA (D2-D3 domains), ITS1 rRNA, and 18S rRNA genes of both Nothotylenchus populations. These sequences and additional anguinid taxa DNA sequences present in GenBank were used for phylogenetic analysis. The selection of outgroup taxa for each dataset was based on previously published studies [13,14,37]. Multiple nucleotide sequence alignments for the different genes were performed using the heuristics progressive method FFT-NS-2 algorithm of MAFFT v.7.450 [38]. The BioEdit v7.2.5 program [39] was used for sequence alignment visualization. For alignment editing, we used Gblocks v0.91b [40] on the Castresana Laboratory server (available online: http://molevol.cmima.csic.es/castresana/Gblocks_server.html (accessed on 10 November 2021) with options for a less stringent selection (minimum number of sequences for a conserved or a flanking position: 50% of the number of sequences +1; maximum number of contiguous non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half)). Phylogenetic analyses were performed using Bayesian inference (BI) in MrBayes v3.1.2 [41]. The best-fit model of DNA evolution was achieved using JModelTest v2.1.7 [42] with the Akaike information criterion (AIC). Accordingly, the selected models were the general time-reversible model with invariable sites and a gammashaped distribution (GTR + I + G), and GTR + G for the D2-D3 segments of the 28S rRNA and partial 18S, respectively. The best-fit model, base frequency, proportion of invariable sites, gamma distribution shape parameters, and substitution rates in the AIC were then used in MrBayes for the phylogenetic analyses, which run with four chains for 4 × 10 6 generations in all datasets. A combined analysis of the three ribosomal genes was not undertaken due to several sequences not being available for all species. The sampling for Markov chains was carried out at intervals of 100 generations. For each analysis, two runs were conducted. After discarding 30% of the samples for burn-in and evaluating convergence, the remaining samples were retained for more in-depth analyses. The topologies were used to generate a 50% majority-rule consensus tree. On each appropriate clade, posterior probabilities (PP) were calculated. FigTree software v1.42 [43] was used for the visualization of phylogenetic trees from all analyses.

Description of Nothotylenchus medians
Female: Body ventrally arcuate after heat relaxed, general appearance cylindroid except at extremities (Figure 1). Cuticle finely annulated, lateral field with six equidistant incisures. Lip region low, anteriorly flattened, with rounded margins, continuous with the body contours, having two or three fine annuli. Labial framework slightly sclerotized, outer margin of the basal plate extending into two to three annuli inside the body. Stylet delicate, conus 35-40% of the total stylet length ( Table 1). Stylet knobs small, rounded, anteriorly sloping. Dorsal pharyngeal gland orifice (DGO) situated close to the stylet knobs. Median pharyngeal bulb valveless, indiscernible, and fusiform in shape. Isthmus slender, encircled with nerve ring.     Hemizonid streak-like two to three annuli anterior to the excretory pore. Excretory pore located slightly anterior to or in range of the anterior level of the pharyngeal basal bulb. Basal pharyngeal bulb pyriform to slightly elongated, abutting intestine. Ovary outstretched with oocytes in a single row, spermatheca and crustaformeria well developed, the columnar arrangement of crustaformeria well discernable. Spermatheca elongate, vulva a transverse slit occupying less than half of the corresponding body width. Vulval lips prominent, sometimes protruding slightly. Post-vulval uterine sac pouch-like, present along the ventral body wall extending halfway to the anus. Anus a minute slit. Tail conical tapering uniformly to a finely rounded to pointed terminus.
Male: Not found. Juveniles: Present but not studied.
Remarks: This species was first described by Thorne and Malek [28] from cultivated fields of South Dakota, USA. Brzeski [8] described two more populations of the same species from Poland and Syria. Except for the shorter pharynx length, the morphology and morphometry of the Canadian population are consistent with the original description of N. medians and the subsequent populations. The populations from Poland and Syria showed longer pharynx lengths (131 (111-144) µm and 124 (119-130) µm, respectively, vs. 98.0 (88.0-116.0) µm) compared with the Canadian population. In terms of habitat, the USA and Canadian populations of N. medians are similar; both populations were found in cultivated areas. Specifically, the Canadian population of N. medians was recovered in the rhizosphere of Artiplex sp. growing on the headland of a cultivated potato field. The presence of males was reported in the original description; however, we did not observe any males in the Canadian population of N. medians.

Description of Nothotylenchus similis
Female: Body C-shaped after heat relaxed, general appearance cylindroid except at extremities ( Figure 2). Cuticle finely annulated, lateral field with six incisures, inner lines are weaker than the outer ones. Lip region low, anteriorly flattened, with rounded margins, continuous with the body contours having two or three fine annuli. Labial framework slightly sclerotized, outer margin of basal plate extending into two to three annuli inside the body. Stylet delicate, conus 25-30% of total stylet length ( Table 2). Stylet knobs small, rounded, anteriorly sloping. Dorsal pharyngeal gland orifice (DGO) situated close to the stylet knobs. Median pharyngeal bulb valveless, fusiform in shape. Isthmus slender encircled with nerve ring. Hemizonid streak-like, two to three annuli anterior to the excretory pore. Excretory pore located at the anterior level of the pharyngeal basal bulb. Basal pharyngeal bulb cylindroid to slightly elongate abutting the intestine. Ovary outstretched with oocytes in a single row, spermatheca and crustaformeria well developed, the columnar arrangement of crustaformeria well discernable. Spermatheca elongate, irregular-shaped, vulva a transverse slit occupying less than half of the corresponding body width. Vulval lips simple, not protruding. Post-vulval uterine sac small along the ventral body wall, same size as maximum body width. Anus, a minute slit. Tail cylindrical, wider at the anal region tapering uniformly to a finely rounded to slightly pointed terminus.
Male: Not found. Juveniles: Present but not studied.
Remarks: This species was first described by Thorne and Malek [28] from South Dakota, USA, in the rhizosphere of pasture grass. Here, we found N. similis in the rhizosphere of undetermined grasses growing on the headland of a cultivated potato field. After the formal description, this species was never reported outside its type locality. The general appearance and dimensions of the Canadian population of N. similis are consistent with the original description. Both the USA and Canadian populations of N. similis have been found from grasses, indicating their host preference in the family Poaceae Barnhart. The authors of the original description likely provided the measurements of holotype female, which is why we cannot determine intraspecific variation range. Consequently, we refer to the Canadian population of N. similis as the reference population for future studies, until topotypes of this species can be sequenced. Moreover, the male was described in the original description; however, we did not observe any male in the Canadian population of N. similis.

Host Association and Distribution of the Species in Genus Nothotylenchus
Genus Nothotylenchus is recognized as the second-largest genus of the family Anguinidae with 41 nominal species [2,10]. The majority of Nothotylenchus species were described decades ago, with some original descriptions in languages other than English and often difficult to access. To study the host associations and distribution of Nothotylenchus species, we collected all original descriptions from web resources and through personal communication. Through detailed literature review, we found that the food preference of Nothotylenchus species is very diverse; the species were either reported from the rhizosphere of cultivated crops (alfalfa, rice), vegetables (cabbage, eggplant, pea, potato, and sugar beet), fruit trees (apple, citrus, grapevine, mango, palm, and pear), grasses (bermudagrass, Sesleria sp., and turfgrasses), and some very unusual hosts (mosses, dead and decaying wood, insect frass, periphytons, and deciduous shrubs; Table 3). Regarding distribution, of 41 nominal species 28 were reported from Asian countries (e.g., India (14 spp.), Iran (8 spp.), Russia (1 sp.), Japan (1 sp.), and Pakistan (1 sp.)), 8 from USA, and 10 from European countries including Russian regions (Table 3). Since the feeding preference and biology of Nothotylenchus species are poorly understood, here we present a brief review of host associations and their place of discovery with the hope that this information will be useful to shed some light on the biology and biogeography of Nothotylenchus species.

Molecular Characterization and Phylogenetic Relationships of Nothotylenchus medians and Nothotylenchus similis with Related Anguinid Species
We used partial 18S, 28S, and ITS1 sequencing to characterize the two anguinid species recovered in this study. The DNA sequence fragment lengths of D2-D3 of 28S and ITS1 genes for N. medians are 718 bp and 743 bp, respectively, whereas the sequence fragment lengths of 18S, D2-D3 of 28S and ITS1 genes for N. similis are 549 bp, 435-625 bp and 743 bp, respectively. The newly obtained sequences were submitted to NCBI under the following accession numbers: partial 18S (OL622072 for N. similis); D2-D3 of 28S (OL622064-OL622066 for N. medians; and OL622061-OL622063 for N. similis); ITS1 (OL622071 for N. medians; OL622068-OL622070 for N. similis).
For Nothotylenchus species very limited DNA sequence-based information is available in NCBI (10 sequences only). Therefore, the 18S and D2-D3 of the 28S trees were constructed with the closest anguinid species sequences obtained through a BlastN search. Figure 3 presents an 18S Bayesian phylogenetic tree constructed with the most highly matched anguinid species and the sequence of N. similis. Here, the Canadian population of N. similis grouped with N. adasi, Anguina pacificae Cid del Prado Vera and Maggenti [66], and an unidentified Ditylenchus sp. from the Netherlands. This clade is further grouped with D. gigas Vovlas, Troccoli, Palomares-Rius, De Luca, Liébanas, Landa, Subbotin, and Castillo [67], Subanguina radicicola (Greeff) Paramonov [68,69], A. agrostis (Steinbuch) Filipjev [3,70], and A. tritici. (Steinbuch) Chitwood [70,71]. The sequence identity of N. similis with aforementioned species is 96-99% with 7-23 nucleotide difference. The other two Nothotylenchus species, namely N. andrassy and N. phoenixae, occupied the middle position in the tree. Another unidentified Nothotylenchus sp. from Iran [72] grouped further away from known Nothotylenchus species. Figure 4 presents D2-D3 of 28S Bayesian phylogenetic tree constructed with the highly matched anguinid species and the sequences of the Canadian populations of both Nothotylenchus species. In this tree, N. medians and N. similis formed a clade with a few species of Ditylenchus and an identified Tylenchidae sp.1 HMM2018 from Mexico City [73]. This clade further shares a branch with N. persicus and N. phoenixae. The sequence identity of N. medians with related Ditylenchus and Nothotylenchus species is 83-97% with 23-140 nucleotide difference. Whereas the sequence identity of N. similis with related Ditylenchus and Nothotylenchus species is 80-92% with 22-129 nucleotide difference. The ITS phylogenetic tree was not carried out because of the low sequence identity and poor coverage with other anguinids in the NCBI database. However, to highlight the relevance of these sequences, we searched the BlastN homology tool for the N. medians ITS sequence, which found Ditylenchus persicus and D. askenasyi to have 88-89% identity and 47-95% sequence coverage. Similarly, the BlastN search for the N. similis ITS sequence found several populations of Ditylenchus destructor to have 85% identity and 50% sequence coverage. [66], and an unidentified Ditylenchus sp. from the Netherlands. This clade is further grouped with Ditylenchus gigas Vovlas, Troccoli, Palomares-Rius, De Luca, Liébanas, Landa, Subbotin, and Castillo [67], Subanguina radicicola (Greeff) Paramonov [68,69], Anguina agrostis (Steinbuch) Filipjev [3,70], and A. tritici. (Steinbuch) Chitwood [70,71]. The sequence identity of N. similis with aforementioned species is 96-99% with 7-23 nucleotide difference. The other two Nothotylenchus species, namely N. andrassy and N. phoenixae, occupied the middle position in the tree. Another unidentified Nothotylenchus sp. from Iran [72] grouped further away from known Nothotylenchus species.

Discussion
Understanding the existing biodiversity of PPNs is important because of their wide range of survival adaptations and dispersal routes [24,25,27]. In our prior nematode inventory surveys, Nothotylenchus species were never detected because some field areas The results of both phylogenetic trees support the inclusion of Nothotylenchus species in the family Anguinidae and indicate that Nothotylenchus species are not monophyletic. However, at this point, the phylogenetic relationships of Nothotylenchus species are unresolved due to the lack of enough available molecular data.

Discussion
Understanding the existing biodiversity of PPNs is important because of their wide range of survival adaptations and dispersal routes [24,25,27]. In our prior nematode inventory surveys, Nothotylenchus species were never detected because some field areas (headland vegetation) had not been surveyed for nematode infestation. The detection of Nothotylenchus species in the headland areas of cultivated fields indicated that weeds or field vegetation on headlands are potential reservoirs and sources of harboring PPNs.
Our genus-wide literature review highlighted that Nothotylenchus species occur in a wide variety of habitats, mostly in the rhizospheres of plants and less frequently in mosses, periphyton, and decaying wood materials, indicating that the species in this genus are quite generalized and could survive in a multitude of environments and plant hosts. Unlike the closest relative, Ditylenchus, Nothotylenchus species do not have a cosmopolitan distribution; so far, only N. acris, N. acutus, N. affinis, N. adasi, N. hexaglyphus, N. medians, and N. similis have been found outside of their type locality [8,10,17,18,28].
The majority of Nothotylenchus species are amphimictic (i.e., male and female are required for reproduction); however, there are 8 species (N. antricolus, N. attenuatus, N. clavatus, N. hexaglyphus, N. loksai, N. thornei, N. truncatus, and N. tuberosus) that were described without males. Among these species, some were reported with empty spermathecae, and some with or without sperms. It appears that Nothotylenchus males are important in reproduction; however, during our survey, no Nothotylenchus males were encountered in the examined collections, suggesting that they are not very common for these species.
Integrative taxonomic identification covering morphological and molecular aspects of a species offers adequate data to properly assign species identification to the relevant taxa [74]. Regarding Nothotylenchus, only recently described species were characterized with molecular markers. Of 41 nominal species, the molecular characterization is only available for N. andrassy, N. persicus, and N. phoenixae. The lack of molecular information prohibited us from constructing a more complete and conclusive phylogenetic relationship of Nothotylenchus species. Our findings are in line with several researchers who reported that insufficient molecular data is the limiting factor in studying Nothotylenchus species' relationships with each other and with related anguinids [13,14,37]. Based on the available phylogeny results, it may be concluded that Nothotylenchus species are composed of divergent lineages. This has been reflected in their feeding habits as well, e.g., N. antricolus was found in the rotten pieces of decaying wood in a cave [47], N. boroki and N. tenuis, in the deadwood materials of pine and Birch [12], and N. drymocolus and N. petilus, in insect frass [11,54]. Considering this, we anticipate that a genus-wide phylogenetic analysis would shed more light on the ecology and biology of these nematodes.
Taxonomic records presently list N. acris, N. acutus, and N. attenuatus from Canada, indicating there are gaps in our knowledge of this complex group that ought to be studied in more detail [17]. The discovery of N. medians and N. similis from cultivated areas in southern Alberta illustrate that considerable anguinid diversity is hidden within these soils. Consequently, increased surveys and more in-depth research are needed in order to further our understanding of the full diversity of anguinids inhabiting Canada's cultivated soils.

Conclusions
Our study aimed to update and summarize the current state of knowledge regarding the distribution and host associations of Nothotylenchus species. Understanding the occurrence and distribution of plant-parasitic nematodes is crucial-some are economical pests, and some are important to soil function. In the present study, we describe two anguinid species, namely N. medians and N. similis, as the first such records from Canada.
The presence of Nothotylenchus species in our cultivated areas does not indicate that yield losses are inevitable-not all nematode species are destined to harm or significantly reduce crop production. However, we assert that the recognition and accurate identification of detected species are important to assess if these nematodes can pose any potential threat in the future and to predict if these species may eventually require appropriate control strategies and regulatory measures. Importantly, we found that the headland areas of cultivated fields are the main sources of nematode infestation. Finally, our discovery of PPNs hitherto unreported from a cultivated region indicates that exploration of the full diversity of inhabiting anguinids warrants increased research attention.