First Report of Three Tylenchidae Taxa from Southern Alberta, Canada

Members of the family Tylenchidae are highly abundant in soil habitats, including agricultural settings, where they play key ecological roles. In the present study, we identified three Tylenchidae species, namely Basiria bhabi, Coslenchus acceptus, and Filenchus vulgaris, using integrative taxonomy. The detailed morphological and morphometric characteristics, distribution, and host associations of each species were also discussed. Phylogenetic analyses of these populations with other Tylenchidae nematodes indicated the presence of divergent lineages in Filenchus and Basiria, whereas Coslenchus appeared to be a monophyletic genus. Herein, we aim to grow awareness about this common but least studied group of nematodes. The species reported in this study are new records for Canada, revealing that the identified nematode diversity in our cultivated areas is relatively underrepresented. Our analyses also provided greater taxonomic resolution and captured rare taxa that might have been missed or misidentified in prior nematode inventory surveys. These findings will add to our understanding of the nematofauna of southern Alberta, thereby providing a more complete picture of existing nematode diversity present in the fields of this highly cultivated region.


Introduction
The nematode family Tylenchidae Örley [1] is exceptionally diverse in soil habitats. Members of this family have high ecological significance and prominence in both agricultural and natural systems [2][3][4]. Based on their life cycle strategies, Tylenchidae species have been given a colonizer-persister (cp) value of 2 [5]. Nematodes in the cp-2 category have ecological relevance as soil health indicators [5]. The taxonomic placement of Tylenchidae in terms of feeding behavior is controversial [6,7], since the feeding habits and food preferences of the majority of tylenchid genera are unknown. Thus, members of this family are either regarded as root hair/epidermal feeders [3], fungivores [8], or herbivores [9], or are generally referred to as plant associates [10].
The majority of Tylenchidae nematodes have similar general appearances, slender bodies, high intraspecific variation, and insufficient molecular data, posing great challenges to their classification and taxonomy [6]. Among plant-parasitic nematodes, Tylenchidae members have what is considered a "primitive morphology", including weak stylets, weakly developed pharyngeal components, and long filiform tails. Subsequently, this family of nematodes has been preconceived to be economically insignificant and therefore often excluded from nematode surveillance and management programs [3,6,11]. In contrast, we believe that the identification of these nematodes is important, as they share a habitat with plant-parasitic nematodes [12,13]. Therefore, we focused on this neglected group of nematodes and found three species belonging to the genera Basiria Siddiqi [14], Coslenchus Siddiqi [15], and Filenchus Andrássy [16] from cultivated areas of southern Alberta, Canada. Previous studies of the family Tylenchidae from Canada were limited to genus level identification of Basiria and Coslenchus [17,18], and to a few Filenchus species from high arctic areas [19][20][21]. This indicates that Tylenchidae nematodes are underrepresented and rarely documented in Canada. In the present study, we carefully examined the three Tylenchidae species found in southern Alberta and performed morphological and molecular analyses to confirm their identities. Detailed morphological examination revealed the presence of B. bhabi Siddiqi [22], C. acceptus Andrássy [23], and F. vulgaris (Brzeski) Lownsberry and Lownsberry [24,25]. Since all three species are first reports from Canada, the objectives of the present study were to (1) provide detailed morphometric and molecular characterizations of these species; (2) examine their phylogenetic relationships with related tylenchid nematodes; and (3) update the taxonomic records of these species from the new location of southern Alberta.
Our study provides increased taxonomic resolution and captures rare taxa that may have been missed or misidentified in prior nematode inventory surveys. Furthermore, our report will aid in filling the gaps of the virtually unknown nematofauna of southern Alberta, thereby providing a more complete picture of the existing nematode diversity present in the region.

Isolation and Morphological Studies
Continuing our wider program of uncovering soil nematofauna of southern Alberta, we collected several root and soil samples from the Vauxhall-Bow Island region. Nematodes were extracted from soil samples using the modified Cobb sieving and flotationcentrifugation method [26]. Basiria, Coslenchus, and Filenchus taxa were collected individually from the mixture of soil nematodes and assigned the population numbers 55, 60, and 70. For preliminary examinations, 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, nematodes were fixed, and permanent slides were prepared according to the methods of Seinhorst [27] and De Grisse [28]. Photomicrographs of each specimen were acquired using a Zeiss Axioskope 40 microscope equipped with a Zeiss Axiocam 208 camera (Carl Zeiss Microscopy, Jena, Germany). Measurements from the images were performed using ZEN blue 3.1 imaging software (Carl Zeiss Microscopy).

DNA Extraction, PCR, and Sequencing
After microscopic examination, a single nematode of each taxon was transferred to a 0.2 mL PCR tube, and DNA was extracted as described in Maria et al. [29]. Three sets of DNA primers (Integrated DNA Technologies, Coralville, IA, USA) were used to amplify the 18S (small subunit or SSU), 28S (large subunit or LSU), and ITS1 of ribosomal RNA (rRNA) genes. The partial 18S rRNA gene was amplified with 1813F and 2646R primers [30]. The 28S rRNA gene was amplified using D2A and D3B primers [31], and the ITS1 gene was amplified using F194 [32] and AB28-R primers [33]. For 18S, 28S, and ITS1 genes, the PCR conditions were as described in Holterman et al. [30], De Ley et al. [31], and Ferris et al. [32], 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 Genewiz, Inc. 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 three tylenchid populations. These sequences and additional Tylenchidae DNA sequences from GenBank were used for phylogenetic analysis. The selection of outgroup taxa for each dataset were based on previously published studies [34][35][36][37]. Multiple nucleotide sequence alignments for the different genes were performed using the heuristics progressive method FFT-NS-2 algorithm of MAFFT  [38]. The BioEdit v7.2.5 program [39] was used for sequence alignment visualization. For alignment edition, we used Gblocks v0.91b [40] on the Castresana Laboratory server (available online: http://molevol.cmima.csic.es/castresana/Gblocks_server.html (accessed on 24 August 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 TIM2 + I + G, TIM3 + I + G, and GTR + G for the D2-D3 segments of the 28S rRNA, partial 18S, and ITS1, 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 ran 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 burn-in samples of 30% 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 visualization of phylogenetic trees from all analyses.

Description of Basiria bhabi
Female: Body shape slightly ventrally arcuate when heat relaxed. Cuticle finely annulated with lateral field having four lines, the outer lines not crenated. Lip region continuous, without striations, narrow with a weakly sclerotized framework. Stylet straight, slender, with fine lumen and small rounded knobs. Orifice of dorsal esophageal gland (DGO) close to stylet knobs. Median bulb oval and weakly developed, with inconspicuous valve plates situated at ca. 42-52% of pharyngeal length. Isthmus slender, surrounded with nerve ring gradually expanding into a cylindrical basal pharyngeal bulb. Excretory pore distinct, situated at the anterior region of pharyngeal bulb. Deirids present in the anterior region of basal pharyngeal bulb, only discernable in inverted specimen. Reproductive system mono-prodelphic, composed of a single, anteriorly outstretched gonad, with oocytes in one row; vulva smooth, vagina straight, weakly sclerotized; spermatheca not offset, irregular squarish-shaped, and mostly empty; post-vulval uterine sac shorter than vulval body diameter. Anus a minute pore, tail elongated and slender, ending in a rounded or clavate tip. Phasmid was not observed in any of the studied specimens ( Figure 1, Table 1). 6.4 ± 0.3 (5.9-6.9) 7 c 7.8 ± 0.5 (7.1-9.0) 8 c' 8.1 ± 0.6 (7.

Characters Canadian Population Bajaj and Bhatti [44]
Pharynx length 124.2 ± 6.5 Male: Not found. Juveniles: Present but not studied. Remarks Basiria bhabi was first described as B. indica by Bajaj and Bhatti in 1979 [44]; however, Siddiqi [22] recognized it as a junior homonym (i.e., a name for a taxon that is identical in spelling to another such name, which belongs to a different taxon) of B. indica described by Chawla et al. [45], and therefore renamed it B. bhabi. The species was described from India and was never reported outside of its type locality. The morphology and morphometry of the Canadian population agreed well with the original description of B. bhabi, except for the longer body length and longer tails. Since the original description of B. bhabi is based on the characteristics of a single specimen, we cannot precisely determine intraspecific variation. Consequently, we refer to the Canadian population of B. bhabi as the reference population for future studies, until topotypes of this species can be sequenced. The species was first discovered in the rhizosphere of Mangifera indica. In the present study this species was found in the rhizosphere of dandelion and grass growing on the headland of a cultivated potato field.

Description of Coslenchus acceptus
Female: Body open C-shaped to ventrally bent when heat relaxed. Cuticle widely annulated with 14 longitudinal ridges. Lateral field with four incisures. Lip region slightly offset, squarish, anteriorly flattened, with three to four annuli. Stylet straight, slender, with rounded knobs. Median bulb oval with refractive valve plates, situated at ca. 42-50% of pharyngeal length. Isthmus slender, encircled with nerve ring gradually expanding into a small pyriform basal pharyngeal bulb. Excretory pore at anterior end of basal bulb. Reproductive system mono-prodelphic, composed of an outstretched ovary with oocytes mostly in two rows, reflexed ovary was observed in some specimens; vulva sunken in body with large vulval flaps, vagina straight, weakly sclerotized; spermatheca irregular round-shaped, filled with few sperm; post-vulval uterine sac shorter than vulval body diameter. Anus a minute pore. Tail elongated, filiform, ending in a finely attenuated tip ( Figure 2, Table 2).     Male: Not found. Juveniles: Present but not studied. Remarks This species was first described by Andrássy [23] from California, USA, in the rhizosphere of strawberry. The same author reported it from two other localities, namely Colorado, USA, and Újszentmargita, Hungary, in the rhizosphere of Carex sp. and Artemisia sp. Here, we found C. acceptus in the rhizosphere of grass growing on the headland of a cultivated potato field. To the best of our knowledge, after the formal description, the species was never reported again. The apparent morphology and morphometry of the C. acceptus original description agree well with the Canadian population except for the longer tail length (100-128 vs. 92-105 µm) and the presence (vs. absence) of a post-vulval uterine sac (PUS). In fresh specimens of the Canadian population, we were not able to observe any PUS; however, in permanent mounts we observed a small rudimentary PUS. The absence of a PUS in the original description could be related to the fixation technique used on specimens [23]. As for the longer tail of Canadian specimens, we agree with Brzeski [46], who stated that tail length in tylenchid nematodes cannot be accepted as a specific character because the filiform portion of the tail is attenuated and could easily break off during handling and processing of specimens.

Description of Filenchus vulgaris
Female: Body slightly ventrally arcuate when heat relaxed. Cuticle finely annulated with four lateral lines. Lip region hemispherical anteriorly flattened, continuous with body contour. Stylet straight, delicate, with rounded knobs. Median bulb oval with refractive valve plates, situated at ca. 39-45% of pharyngeal length. Isthmus slender, encircled with nerve ring gradually expanding into a small pyriform basal pharyngeal bulb. Excretory pore at anterior end of basal bulb. Reprod+uctive system mono-prodelphic, composed of an outstretched ovary with oocytes mostly in a single row; vulva smooth, vagina straight to slightly inclined anteriorly; spermatheca offset, irregular rounded shaped filled with few sperm; post-vulval uterine sac shorter than vulval body diameter. Anus a minute pore. Tail elongated, filiform, ending in a finely attenuated tip (Figure 3, Table 3).
Horticulturae 2021, 7, x FOR PEER REVIEW 9 of 18 Because of the widespread distribution of F. vulgaris, it has a broad host association. The species is known to occur in various biotypes, e.g., agricultural and forest soils and rhizospheres of grasses and vegetables [46,49]. Because F. vulgaris is a common species, morphometric studies were not always performed for the detected populations of this nematode (Table 3). Based on the available data, the morphological and morphometric details of the Canadian population are within the species boundaries of F. vulgaris.  Male: Not found. Juveniles: Present but not studied. Remarks This species was described by Brzeski [24] in the rhizosphere of a vegetable crop from Poland. Filenchus vulgaris is considered to be a cosmopolitan species; it has been reported from Belgium, Germany, Iran, Korea, Poland, Russia, Slovakia, and the USA [9,24,25,[47][48][49][50]. In the present study, the Canadian population of F. vulgaris was recovered from the rhizosphere of dandelion and grass growing on the headland of a cultivated potato field. Because of the widespread distribution of F. vulgaris, it has a broad host association. The species is known to occur in various biotypes, e.g., agricultural and forest soils and rhizospheres of grasses and vegetables [46,49]. Because F. vulgaris is a common species, morphometric studies were not always performed for the detected populations of this nematode (Table 3). Based on the available data, the morphological and morphometric details of the Canadian population are within the species boundaries of F. vulgaris. Table 3. Female morphometrics of Filenchus vulgaris from Canada and retrieved from the original and subsequent published descriptions. All measurements are in µm and presented as mean ± standard deviation (range).
The majority of studies conducted to understand the phylogenetic relationships of the family Tylenchidae is based on D2-D3 domains of the 28S gene. In contrast, very few Tylenchidae members were characterized by ITS1 sequences. Notably, no ITS1 sequence was found for the genus Basiria, and only a single ITS1 sequence was available for each of Filenchus and Coslenchus species. We constructed the ITS tree ( Figure 5) with the available ITS sequences of Cephalenchus Goodey [77] and the Canadian populations of tylenchid nematodes. Basiria bhabi appeared independently and occupied a basal position in the tree, whereas C. acceptus clustered with C. rhombus, and F. vulgaris finally grouped with the F. vulgaris from China, initially identified as sp1 CX-2018. Due to the lack of ITS1 sequences, prediction of the phylogenetic relationships is uncertain; therefore, we omitted calculation of the sequence identity of the Canadian populations of tylenchid nematodes with related species. An 18S tree was constructed with the single sequence of C. acceptus and related Tylenchidae species sequences ( Figure 6). As with the 28S and ITS trees, C. acceptus grouped within the Coslenchus clade. The sequence identity of C. acceptus with the Coslenchus clade species was 99%, with 4-9 nucleotide differences and 0-1% indels.
The Canadian populations of B. bhabi, C. acceptus, and F. vulgaris obtained in this study were identified using an integrative taxonomical approach. Hence, the sequences of these populations could serve as reference sequences for future studies, until topotype specimens of these species can be sequenced. An 18S tree was constructed with the single sequence of C. acceptus and related Tylenchidae species sequences ( Figure 6). As with the 28S and ITS trees, C. acceptus grouped within the Coslenchus clade. The sequence identity of C. acceptus with the Coslenchus clade species was 99%, with 4-9 nucleotide differences and 0-1% indels.

Discussion
Members of the family Tylenchidae are highly abundant in agricultural soil, representing at least 30% of soil nematofauna in any given sample [6]. In the present study, the Canadian populations of B. bhabi, C. acceptus, and F. vulgaris were present at higher densities in headland vegetation (grasses and flowering plants) than those in field soils. The lower densities of these nematodes in field samples may be due to sampling depth, as Tylenchidae nematodes dominated in the upper 10 cm of soil, with their numbers declining with depth [78]. Another reason may simply be that these nematodes have adapted better to the headland vegetation because the headland area is less disturbed-not vigorously tilled as are the cultivated fields.
The family Tylenchidae is notorious for its complicated taxonomy and morphology, whereby phenotypic convergence of morphologically similar but genetically distant gen- The Canadian populations of B. bhabi, C. acceptus, and F. vulgaris obtained in this study were identified using an integrative taxonomical approach. Hence, the sequences of these populations could serve as reference sequences for future studies, until topotype specimens of these species can be sequenced.

Discussion
Members of the family Tylenchidae are highly abundant in agricultural soil, representing at least 30% of soil nematofauna in any given sample [6]. In the present study, the Canadian populations of B. bhabi, C. acceptus, and F. vulgaris were present at higher densities in headland vegetation (grasses and flowering plants) than those in field soils. The lower densities of these nematodes in field samples may be due to sampling depth, as Tylenchidae nematodes dominated in the upper 10 cm of soil, with their numbers declining with depth [78]. Another reason may simply be that these nematodes have adapted better to the headland vegetation because the headland area is less disturbed-not vigorously tilled as are the cultivated fields.
The family Tylenchidae is notorious for its complicated taxonomy and morphology, whereby phenotypic convergence of morphologically similar but genetically distant genera cause problems in the delimitation of taxa [6,35]. However, implementation of molecular tools allows nematologists to better study the characters and utilize them to distinguish the species [6,7,11,34,35,79]. In our phylogenetic analysis, B. bhabi exhibited a sister relationship with B. ritteri and clustered distant from the other species of Basiria. Such divergent phylogenetic placement suggests that Basiria is not a monophyletic genus. Currently, the genus contains over 40 nominal species [21,80], and the molecular information is only available for few species. In our view, genus-wide sequencing is needed to validate the true positioning and phylogenetic relationships of Basiria. Andrássy [23] did a thorough review of Coslenchus and concluded that the genus is homogenous. This close morphological affinity is also reflected in several phylogenetic studies [7,36,81] where all the Coslenchus species form a monophyletic clade. Our results are also in line with these studies, i.e., the Canadian population of C. acceptus was nested within the Coslenchus clade with very little sequence divergence. Filenchus is one of the most diverse genera in the subfamily Tylenchinae, currently representing over 90 species [21]. Phylogenetic studies have indicated the presence of divergent lineages in Filenchus; therefore, superficial observation of morphologically similar species can lead to misidentifications [82]. We also observed questionable sequences of F. vulgaris deposited in the NCBI database. Moreover, no morphological or morphometric details were associated with these supposed sequences. Therefore, future studies are well advised to meticulously examine species morphological characters and use the same specimen for morphological and molecular studies. Nevertheless, molecular studies of Tylenchidae genera are important to expand our knowledge of these microscopic, morphologically-reduced organisms [6].
The feeding habits of the majority of Tylenchidae nematodes are barely known; the species we detected in this study were all isolated from the rhizosphere of plants. We did not observe significant root damage or mycelial growths at the time of sampling, which suggests that Canadian populations of B. bhabi, C. acceptus, and F. vulgaris are plant-feeding rather than plant-parasitic nematodes. The detection of these species in our cultivated areas updates the biogeography of these nematodes; however, there is still lack of knowledge on the diversity of tylenchid species from southern Alberta, Canada. It is likely that tylenchid species not directly implicated in plant diseases will remain undocumented for years to come.

Conclusions
In southern Alberta, the presence of phytoparasitic nematodes has been examined in several studies [12,13,83], but detection and identification of Tylenchidae-related nematodes had never been addressed. As a result, we know almost nothing about insignificant or mild parasitic nematodes that share the same habitat with economically important plantparasitic nematodes. Herein, we aim to grow awareness about a less studied group of nematodes. We believe that including more species in nematode surveillance programs is both a step forward in understanding the nematofauna of agro-ecosystems and an essential component for the assessment of soil biodiversity in any management plan. It is also a promising direction for both taxonomy and education. In addition, our results suggest that the nematode diversity in our cultivated areas is underrepresented, since the species reported in this study are all new records for Canada. This study updates the biogeography of B. bhabi, C. acceptus, and F. vulgaris from Canada.