Description of Longidorella (Saevadorella) caspica n. sp. (Dorylaimida: Nordiidae) from north Iran

Abstract This contribution provides the morphological and molecular identification of a new species of the genus Longidorella (Saevadorella). L. (S.) caspica n. sp., was recovered from the rhizospheric soil of grasses in Mazandaran province in the seashore of the Caspian Sea. It is characterized by females with a length of 788 to 874 μm and a cephalic region with prominent papillae; and separated from the rest of the body by a remarkable constriction, an odontostyle of 32 to 33 μm, vulva at 52.5 to 59.0%, and a tail of 33 to 38 μm with a rounded tip. Males have 32 to 35 μm long spicules of dorylaimoid form and five to seven ventral supplements ending at 48 to 55 μm distance from cloacal pair. The new species was morphologically compared with seven nominal species viz. L. (S.) arenicola, L. (S.) cuspidata, L. (S.) magna, L. (S.) perplexa, L. (S.) saadi, L. (S.) saevae, and L. (S.) tharensis. It was further compared with similar species under the subgenus Longidorella (Enchodorella) viz. L. (E.) deliblatica and L. (E.) murithi. Besides morphological studies, molecular phylogenetic studies using partial sequences of D2 to D3 expansion fragments of 28S rDNA were performed for the new species and its phylogenetic relationships with other species and genera were discussed.

presented a taxonomic history of the family Nordiidae (Jairajpuri and Siddiqi, 1964). According to him, the main characteristics of the family are a needle-like odontostyle with a fine aperture, often flanged odontophore and similar tail in sexes. The needle-like odontostyle probably makes the members capable to parasitize lower or higher plants (Peña-Santiago, 2021). Although some nordiids are recovered from rhizosphere of higher plants (e.g. Longidorella (Actinolamoides) foveola Siddiqi, 2007, L. (Actinolamoides) ecuadorica Siddiqi, 2007, and L. (Longidorella) miniparva Siddiqi, 2007), these nematodes have not been reported as economically important crop pests, and the studies dealing with their plant parasitism ability are poor.
The genus Longidorella (Thorne, 1939), currently, with 39 known species, belongs to the subfamily Nordiinae (Jairajpuri and Siddiqi, 1964), with L. parva (Thorne, 1939) as its type species. The small and stout body, large amphids, long needle-like odontostyle, and small pharyngeal bulb are characteristics of this genus (Andrássy, 2009). Siddiqi (2007) reviewed the taxonomy of the genus, listed its synonym genera, added several species, and categorized the species under seven subgenera. The subgenus Saevadorella (Siddiqi, 1982) is one of them, being characterized mostly by its offset cephalic region, amphidelphic female reproductive system, a usually transverse vulval slit and usually sclerotized vagina.
The history of taxonomic studies on nordiids in Iran is given by Heydari et al. (2020). During the present study, one population of a nordiid nematode was recovered from a sandy soil sample collected in seashore of Caspian Sea in Mazandaran province. Its preliminary studies revealed it belongs to Longidorella. The detailed comparisons revealed that the recovered species belongs to the subgenus Saevadorella based upon its morphological characters (see Discussion) and was described and illustrated herein as L (S.) caspica n. sp. Thus, the present study aims to describe and illustrate the new species, and unravel its phylogenetic relationships with other spices.

Materials and methods
Sampling, extracting, mounting, and drawing Several soil samples were collected from north Iran close to Caspian Sea during 2014. The new species was recovered from soil of grasses, using the tray method (Whitehead and Hemming, 1965). They were handpicked under a Nikon SMZ1000 stereomicroscope, heat-killed by adding boiling 4% formalin solution, transferred to anhydrous glycerin according to De Grisse (1969), mounted on permanent slides, and examined using a Nikon Eclipse E600 light microscope. Photographs were taken using an Olympus DP72 digital camera attached to an Olympus BX51 microscope powered with differential interference contrast. Drawings were made using a drawing tube attached to the microscope and were redrawn using CorelDRAW software version 17. The location of pharyngeal glands' nuclei was calculated following Andrássy (1998). The proposed taxonomic frame by Siddiqi (2007) as followed by Nasira et al. (2010) was followed in this study.

DNA extraction, PCR, and sequencing
One live female nematode of the new species was used for DNA extraction. The specimen was washed and observed under a temporary slide, transferred to a small drop of TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) on a clean slide and squashed using a clean cover slip, and the pressure of a plastic pipette tip. The suspension was collected by adding 15 μ l TE buffer. The DNA sample was stored at −20°C. Primers for 28S rDNA D2-D3 amplification/sequencing were forward primer D2A (5´-ACAAGTACCGTGAGGGAAAGT-3´) and reverse primer D3B (5´-TCGGAAGGAACCAGCTACTA-3´) (Nunn, 1992). The PCR cycles and sequencing of amplified fragments were according to Jahanshahi  and sequenced directly for both strands using the same primers with an ABI 3730XL sequencer (Bioneer Corporation, South Korea). The newly generated sequence for the new species was deposited in the GenBank database under the accession number MH346475.

Phylogenetic analysis
The newly generated sequence in this study was compared with those of other relevant sequences from other nematodes deposited in the GenBank database using the BLAST homology search program. Several 28S rDNA D2-D3 sequences of nordiid and other dorylaimid taxa were downloaded. Two sequences of mononchid species were used as outgroups (for species names, accession numbers, and related plants, see Table 1). The sequences were aligned using the Q-INS-i algorithm of the online version of MAFFT version 7 (http://mafft.cbrc.jp/alignment/ server/) (Katoh and Standley, 2013). The poorly aligned positions and divergent regions were eliminated using the online version of Gblocks 0.91b (Castresana, 2000) using all three less stringent options (Mobasseri et al., 2019;Panahandeh et al., 2018). The model of base substitution was selected using MrModeltest 2 (Nylander, 2004). The Akaike-supported model, a general time-reversible model including amongsite rate heterogeneity and estimate of invariant sites (GTR + G + I), was selected for phylogenetic analysis. Bayesian analysis was performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), running the chains for two million generations. Burn-in phase was set at 25% of the converged runs. The Markov chain Monte Carlo (MCMC) method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic tree (Larget and Simon, 1999) using the 50% majority rule. To visualize the results of each run in order to check the effective sample size of each parameter, Tracer v1.5 (Rambaut and Drummond, 2009) was used. The output file of MrBayes was visualized using Dendroscope v3.2.8 (Huson and Scornavacca, 2012) and was drawn in CorelDRAW version 17.

Measurements
Measurements of the new species are given in Table 2.

Etymology
The specific epithet was derived from Caspian Sea, from where the new species was recovered in its vicinity.

Diagnosis and relationships
Longidorella (Saevadorella) caspica n. sp. is mainly characterized by its cephalic region separated from the rest body by a deep constriction and prominent labial papillae. It was further characterized by 788 to 874 μ m long females having 92 to 94 μ m long odontostyle in females and 80 to 91 μ m in males, with didelphic-amphidelphic reproductive system, vulva at 52.5 to 59.0%, 33 to 38 µm long dorsally convex tail in females, males with 32 to 35 µm long dorylaimoid spicules, and five to seven ventral supplements.

D2-D3 fragments of 28S rDNA phylogeny
Sequencing of D2-D3 expansion segments of 28S rDNA of the new species yielded a single fragment of 800 nt long. A total number of 53 sequences of 28S rDNA D2-D3 of Longidorella spp., other nordiids as well as some other dorylaimids (ingroup sequences) were retrieved from GenBank for phylogenetic analyses. The outgroup sequences belonged to two mononchid species (Table 1). Figure 4 represents the Bayesian phylogenetic tree reconstructed using this dataset. The nordiid genera occupied separate placements in this tree. The new species fell into a highly supported major clade including five other sequences of the genus Longidorella and two other dorylaimid genera. The clade including two sequences with accession numbers AY593042 and AY593042 assigned to Longidorella sp. and Longidorella cf. macramphis, represents the tentative sister clade to the new species.

Discussion
In this study, one species of the genus Longidorella was recovered in north Iran. It was assigned to the subgenus Saevadorella sensu (Siddiqi, 2007) mainly by having offset cephalic region, sclerotized vagina, amphidelphic reproductive system of females and not twisted posterior body region; and was described and illustrated using morphological and molecular approaches. The genus has been divided into seven subgenera by Siddiqi (2007). Such an artificial grouping can be helpful for species identification under this specious genus, but, the qualitative traits delimiting the subgenera, sometimes fail to well distinguish them, and as the result, species under the similar subgenera need to be compared with all similar forms. Thus, the new species was compared with species under both subgenera Longidorella (Saevadorella) and L. (Enchodorella). In the proposed framework by Andrássy (2009), however, all species have unified under the genus Longidorella. Lacking of molecular data of type populations of most species of the genus or poor descriptions of the species and inaccessibility of the type specimens are the main obstacles in taxonomy of the genus.
Currently, GenBank database is poor for molecular data of nordiids, and based on the currently available ribosomal RNA sequences, the family is not monophyletic (present study; Pedram et al., 2011;Álvarez-Ortega, 2020;Peña-Santiago et al., 2015).
The newly described species in present study has been characterized using both traditional and molecular data, and further future molecular data will help better clarifying the phylogeny of the genus.