Andrássy (2009) 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.
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.
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 μl 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 Afshar et al. (2019) 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.
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 among-site 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.
The used sequences in 28S phylogeny of Longidorella (Saevadorella) caspica n. sp., their accession number, locality, and related plants.
| Species name | Accession number | Location, related plant |
|---|---|---|
| Allodorylaimus sp. | KY942069 | China, unknown |
| Allodorylaimus andrassyi | AY593016 | Unknown |
| Allodorylaimus andrassyi | AY593015 | Unknown |
| Dorydorella bryophila | HM235514 | Unknown |
| Enchodelus sp. | KP190120 | Hamedan province, Iran, mosses |
| Enchodelus longispiculus | KP190119 | Hamedan province, Iran, mosses |
| Enchodelus sp. | EF207240 | Unknown |
| Enchodorus dolichurus | KR184125 | Golestan province, Iran, mosses |
| Enchodorus dolichurus | KR184124 | Golestan province, Iran, mosses |
| Enchodorus yeatsi | KX691911 | East Azarbaijan province, Iran, grasses |
| Eudorylaimus sp. | AY593037 | Unknown |
| Heterodorus brevidentatus | KP963962 | Kerman city, Iran, mosses |
| Heterodorus brevidentatus | KP963960 | Maragheh city, Iran, mosses |
| Heterodorus brevidentatus | KP963963 | Maragheh city, Iran, mosses |
| Heterodorus brevidentatus | KP963964 | Kermanshah city, Iran, mosses |
| Heterodorus brevidentatus | KP963965 | Kermanshah city, Iran, mosses |
| Heterodorus brevidentatus | KP963961 | Tehran city, Iran, mosses |
| Heterodorus morgensis | KX691912 | Mazandaran province, Iran, grasses |
| Heterodorus youbertghostai | KR184126 | Sabalan region, Iran, grasses |
| Heterodorus youbertghostai | KR184127 | Sabalan region, Iran, grasses |
| Longidorella macramphis | AY593042 | Unknown |
| Longidorella sp. | AY593043 | Unknown |
| Longidorella penetrans | HM235515 | Unknown |
| Longidorella sp. | AY593044 | Unknown |
| Longidorella sp. | AY593045 | Unknown |
| Mesodorylaimus sp. | AY593006 | Unknown |
| Mesodorylaimus sp. | AY593005 | Unknown |
| Microdorylaimus miser | AY593046 | Unknown |
| Microdorylaimus modestus | HM235513 | Unknown |
| Microdorylaimus modestus | AY593049 | Unknown |
| Mononchus truncatus | AY593064 | Unknown |
| Nordiidae sp. | KP202362 | Tehran city, Iran, grasses |
| Nordiidae sp. | KP202361 | Tehran city, Iran, grasses |
| Nordiidae sp. | AY593054 | Unknown |
| Nordiidae sp. | MH346478 | Tehran city, Iran, mosses/pine tree |
| Prionchulus punctatus | MG994945 | Unknown |
| Prodorylaimus uliginosus | AY593034 | Unknown |
| Pungentus engadinensis | MH346473 | Semnan province, Iran, fruit trees |
| Pungentus engadinensis | MH346474 | Mazandaran province, Iran, forest trees |
| Pungentus silvestris | AY593052 | Unknown |
| Pungentus silvestris | AY593054 | Unknown |
| Pungentus silvestris | AY593053 | Unknown |
| Pungentus azarbaijanensis | MH346476 | West Azarbaijan province, Iran, grasses |
| Pungentus azarbaijanensis | MH346477 | West Azarbaijan province, Iran, grasses |
| Pungentus engadinensis | AY593050 | Unknown |
| Pungentus monohystera | MF325344 | Germany, lime tree |
| Pungentus monohystera | MF325343 | Germany, lime tree |
| Rhyssocolpus vinciguerrae | KP204547 | Gilan province, Iran, forest trees |
| Thonus minutus | AY593047 | Unknown |
| Thonus minutus | AY593048 | Unknown |
| Thonus circulifer | AY593039 | Unknown |
| Thonus sp. | AY593041 | Unknown |
| Thonus sp. | AY593040 | Unknown |
| Thonus circulifer | AY593038 | Unknown |
| Tylencholaimellus sp. | AY593055 | Unknown |
Figure 1:
Line drawings of Longidorella (Saevadorella) caspica n. sp. A-C, E-G: Female; D: Male. A: Pharynx; B: Anterior body region; C: Anterior end; D: Posterior body region; E and G: Tail and posterior body region, F: Posterior genital tract.
Figure 2:
Photomicrographs of Longidorella (Saevadorella) caspica n. sp. A-D, F-I: Female; E: Male. A-D: Anterior and cephalic region; E and F: Entire body; G: Vulva in ventral view; H: Mature egg inside female reproductive system; I: Sperm inside the uterus. (Scale bars = 10 μm, except E and F = 100 μm).
Figure 3:
Photomicrographs of Longidorella (Saevadorella) caspica n. sp. A and B, F and G: Male; C-E: Female. A and B: Posterior body region (Arrows showing supplements). C. Pharyngeal bulb. F-G: Posterior body region with spicules. (All scale bars = 10 μm).
Measurements of the new species are given in Table 2.
Morphometrics of Longidorella (Saevadorella) caspica n. sp.
| Holotype | Paratype | ||
|---|---|---|---|
| Characters | Female | Females | Males |
| n | 1 | 3 | 3 |
| L | 852 | 832 ± 38.5 (788-874) | 811 ± 74 (725-856) |
| a | 19.8 | 19 ± 2 (16.8-21.3) | 22.0 ± 1.5 (20.7-23.8) |
| b | 3.8 | 3.7 ± 0.2 (3.5-3.8) | 3.8 ± 0.2 (3.6-4.0) |
| c | 25.8 | 27.2 ± 7 (20.7-37.0) | 25.2 ± 2.2 (23.0-27.5) |
| c′ | 1.3 | 1.3 ± 0.3 (0.9-1.5) | 1.3 ± 0.1 (1.2-1.4) |
| V | 59.2 | 55.7 ± 3.0 (52.5-59.0) | – |
| Anterior end-vulva | 504 | 463.5 ± 32.5 (425-504) | – |
| Cephalic region diam. | 12 | 11.5 ± 1.0 (10-12.5) | 12.5 ± 0.5 (12-13) |
| Cephalic region height | 5 | 5.0 ± 0.0 (5-5) | 4.7 ± 0.6 (4-5) |
| Odontostyle length | 33 | 33.0 ± 0.5 (32-33) | 35 ± 1 (34-36) |
| Odontophore length | 38 | 35.5 ± 2.5 (33-38) | 33.3 ± 3.0 (30-36) |
| Stylet total length | 71 | 68.5 ± 2.0 (66-71) | 68.5 ± 2.5 (66-71) |
| Guiding ring from ant. end | 2.8 | 18 ± 1 (18-20) | 18.0 ± 0.5 (18.0-18.5) |
| Neck length | 18 | 229 ± 2 (227-231) | 212 ± 8 (203-220) |
| Pharyngeal expansion length | 92 | 93 ± 1 (92-94) | 85.5 ± 5.5 (80-91) |
| Diam. at guiding ring level | 16 | 18.5 ± 0.0 (18.5-18.5) | 18.5 ± 0.0 (18.5-18.5) |
| -at neck base | 40 | 41.0 ± 3.5 (38-46) | 36.5 ± 2.5 (34-39) |
| -at mid-body | 43 | 44.0 ± 2.5 (41-47) | 37.0 ± 2.5 (35-40) |
| -at anus | 25 | 25.0 ± 0.5 (24-25) | 25 ± 1 (24-26) |
| Prerectum | 56 | 57.0 ± 7.5 (47-65) | 81 ± 20 (67-95) |
| Rectum | 22 | 23.5 ± 3.5 (19-27) | 38.0 ± 1.5 (37-39) |
| Tail length | 33 | 34.5 ± 2.5 (33-38) | 32.5 ± 4.0 (29-37) |
| Spicules length | – | – | 33.0 ± 1.5 (32-35) |
Note: All measurements are in μm and in the form Mean ± SD (range).
Female: Body fusiform, tapering gradually towards both ends, very slightly curved ventrad. Cuticle with two layers, 3 to 4 μm thick at anterior body region and mid body, 4 to 5 μm thick at the anterior lip of anus, with very delicate transverse striae visible at the dorsal side of the tail. Cephalic region separated from the rest of the body by a deep constriction. Labial papillae protruding, large and distinct. Amphidial fovea cup-shaped, large, their opening ca. 75% of cephalic region width wide, at the level of constriction. Odontostyle long and thin, 2.5 to 3.0 times longer than cephalic region width. Odontophore rod-like, its base simple, muscles at its base slightly swollen, but not flanged or sclerotized, approximately equal in size with odontostyle or slightly longer. Guiding ring single. Pharynx dorylaimoid, the anterior part narrower, enlarging gradually to the pharyngeal bulb. Location of pharyngeal glands’ nuclei follow: DN = 65 to 68, S1N = 32 to 40, S2N = 76 to 79. Cardia hemispheroid, 10-17 × 9-12 μm. Intestine simple with no specific features. Reproductive system didelphic-amphidelphic, genital branches 150 to 160 μm long, each branch composed of an ovary 90 to 120 μm long, oviduct and sphincter, a tubular uterus 55 to 60 μm long usually containing sperm, vagina 30 to 37% of body width, composed of pars proximalis vaginae 8-10 × 7-10 μm in size, pars refringens vaginae with two weakly sclerotized pieces triangular to trapezoid with 4 μm height and 3 to 5 μm width, pars distalis vaginae 2 to 4 μm long, and vulva a small rounded pore. Prerectum twice the anal body diameter and rectum equal to the anal body width in length. Tail conical with finely rounded tip, ventrally nearly straight, dorsally convex with a very weak dorsal concavity at the end, appearing slightly sub-digitate.
Male: Similar to females in general morphology except for the posterior body end more ventrally bent. Spicules dorylaimoid, almost slender, about five times longer than wide, their head (capitulum) narrow, a well-developed hump and deep hollow (sensu Peña-Santiago et al., 2014) lacking. Lateral guiding pieces cylindroid, 8 to 10 μm long. The copulatory supplements composed of a cloacal pair at 7 to 10 μm anterior to cloacal aperture, and a series of five to seven ventromedian supplements ending at 48 to 55 μm from the cloacal pair. Tail similar to that in female.
Rhizosphere of grasses, Mazandaran province, north Iran. GPS coordinates: 36°38ʹ6.225ʺN, 51°33ʹ52.236ʺE.
Holotype female, paratype females and males were deposited in Nematology Collection of Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran (slide accession codes: TM5100-TM5103). The ZooBank Life Science Identifier (LSID) for this publication is as follows: urn:lsid:zoobank.org:pub:1915B3CE-4B79-480B-8117-E926CA3CD090.
The specific epithet was derived from Caspian Sea, from where the new species was recovered in its vicinity.
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 μm long females having 92 to 94 μm long odontostyle in females and 80 to 91 μm 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.
The new species was morphologically compared with seven nominal species under the subgenus viz. Longidorella (Saevadorella) arenicola (Vinciguerra and Zullini, 1980), L. (S.) cuspidata (Andrássy, 1964; Jairajpuri and Hooper, 1969), L. (S.) magna (Loof, 1971), L. (S.) perplexa (Siddiqi, 2007), L. (S.) saadi (Siddiqi, 2007), L. (S.) saeva (Siddiqi, 1982) and L. (S.) tharensis (Nasira et al., 2010) as follow.
From Longidorella (Saevadorella) arenicola by a longer body of female (788-874 vs 630-750 μm), shorter odontostyle (32-33 vs 40-42 μm), greater b (3.5-3.8 vs 2.7-3.3) value, pore-like vulval opening (vs transverse slit), less ventromedian male copulatory supplements (five to seven vs nine) with the last supplement distantly placed to the spicules’ head (vs close).
From L. (S.) cuspidata by a longer body of female (788-874 vs 510-760 μm), greater a (16.8-21.3 vs 12.5-14.7), b (3.5-3.8 vs 2.6-3.0), and c (20.7-37.0 vs 16.5-20.0) ratios, vulva pore-like (vs transverse, after its original drawing) and tail ventrally slightly convex (vs concave).
From L. (S.) magna by a shorter body of female (788-874 vs 1,050-1,100 μm), longer odontophore (33-38 vs 20-24 μm) and shorter tail (33-38 vs 47-50 μm).
From L. (S.) perplexa by a longer body of female (788-874 vs 580-680 μm), greater b (3.5-3.8 vs 2.5-3.0), anteriorly located vulva (V = 52-59 vs 58-67), shorter odontostyle (32-33 vs 38-46 μm), shorter odontophore (33-38 vs 39-44 μm), pore-like vulval opening (vs transverse slit), longer spicules (32-35 vs 31-32 μm), and five to seven male copulatory supplements (vs three).
From L. (S.) saadi by a shorter odontostyle (32-33 vs 40-50 μm), shorter odontophore (33-38 vs 40-44 μm), less c′ (0.9-1.5 vs 1.4-1.7) value, different range of V (52.5-59.0 vs 54-66), longer prerectum (47-65 vs 20-30 μm) and shorter spicules (32-35 vs 40 μm).
From L. (S.) saeva by shorter odontostyle (32-33 vs 39-42 μm), shorter odontophore (33-38 vs 42-48 μm), pore-like vulval opening (vs transverse), smaller c′ (0.9-1.5 vs 1.2-2.0), shorter spicules (32-35 vs 38-40 μm) and number of copulatory supplements of male (five to seven vs two).
From L. (S.) tharensis by cephalic region separated from the rest body by a deep constriction (vs slightly offset), shorter odontostyle (32-33 vs 35-38 μm), longer neck (227-231 vs 197-223 μm), longer pharyngeal bulb (92-94 vs 67-68 μm), pore-like vs transverse vulval slit and longer tail of female (33-38 vs 20.2-26.4 μm).
Based on similar general morphology, the new species was further compared with two species under the subgenus Longidorella (Enchodorella) (Siddiqi, 2007) viz. L. (E.) deliblatica (Krnjaic, 1971) and L. (E.) murithi (Altherr, 1950) as follow.
From L. (E.) deliblatica by a longer body (788-874 vs 602-700 μm), slightly longer odontostyle (32-33 vs 26.8-31.4 μm) and odontophore (33-38 vs 22.4-30.0 μm), narrower tail tip (vs wider) and almost slender spiclues (vs with deep hollow).
From L. (E.) murithi by a cephalic region separated from the rest body by a sharp constriction (vs not), shorter odontostyle (32-33 vs 40-44 μm) and tail tip not remarkably narrowing (Andrássy, 2009).
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.
Figure 4:
Bayesian 50% majority rule consensus tree inferred from the 28S rDNA D2-D3 sequences of Longidorella (Saevadorella) caspica n. sp. under the GTR + G + I model. Bayesian posterior probabilities (BPP) and maximum likelihood bootstrap (ML BS) values > 0.50 are given for appropriate clades in the form: BPP/ML BS. The newly generated species is in bold font.
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.