First report of Longidorus leptocephalus Hooper, 1961 (Nematoda: Longidoridae) from Greece

Soil samples containing needle nematodes resembling L. leptocephalus were collected at a depth of 10 to 30 cm from the rhizosphere of grapevine in the area of Thessaloniki, northern Greece. Nematodes were extracted from soil by modified sieving and a decanting method (Brown and Boag, 1988). Extracted specimens were handpicked, heat killed, fixed in TAF (triethanolamine formalin), processed using glycerol by a slow evaporation method, and mounted on permanent slides (Hooper, 1986). The light micrographs and measurements of nematodes population, including the main diagnostic characteristics, were performed using a Leica DM6 compound microscope with a Leica DFC7000 T digital camera. All other abbreviations used were as defined in the study by Jairajpuri and Ahmad (1992).

For molecular analyses and in order to avoid mistakes in case of potential mixed populations in the same sample, single specimens from the sample were temporarily mounted in a drop of 1 M NaCl containing glass beads (to avoid nematode crushing/ damaging specimens) to ensure that specimens were conformed with the target population. All necessary morphological and morphometric data were recorded. This was followed by DNA extraction from single individuals as described by Archidona-Yuste et al. (2016). The D2-D3 segments were amplified using the D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') primers (De Ley et al., 1999). The internal transcribed spacer region 1 (ITS1) was amplified using forward primer 18S (5'-TTGATTACGTCCCTGCCCTTT-3') (Vrain et al., 1992) and reverse primer rDNA1 5.8S (5'-ACGAGCCGAGTGATCCACCG-3') (Cherry et al., 1997). Finally, the portion of the COI gene was amplified as described by Lazarova et al. (2006) using the primers COIF (5'-GATTTTTTGGKCATCCWGARG-3') and COIR (5'-CWACATAATAAGTATCATG-3').

All PCR assays were done according to the conditions described by Archidona-Yuste et al. (2016). Then, the amplified PCR products were purified using ExoSAP-IT (Affymetrix, USB products) and used for direct sequencing on a DNA multicapillary sequencer (Model 3130XL genetic analyzer; [Applied Biosystems Foster City, CA]), using the BigDye Terminator Sequencing Kit V.3.1 (Applied Biosystems, Foster City, CA), at the Stab Vida sequencing facilities (Caparica, Portugal). The newly obtained sequences were submitted to the GenBank database under the accession numbers indicated on the phylogenetic trees. Voucher specimens of the studied population were deposited in the nematode collection of the Institute for Sustainable Agriculture, IAS-CSIC, Córdoba, Spain.

D2-D3 expansion segments of 28S and ITS1 rDNA as well as COI mtDNA sequences of the recovered Longidorus population were obtained in this study. These newly generated sequences, and other sequences of Longidorus spp. retrieved from GenBank, were used for phylogenetic analyses. Outgroup taxa for each dataset were chosen following previously published studies (He et al., 2005; Archidona-Yuste et al., 2019; Bakhshi Amrei et al., 2022). Multiple sequence alignments of the three aforementioned loci were made using the FFT-NS-2 algorithm of MAFFT V.7.450 (Katoh et al., 2019). The alignments were manually visualized using BioEdit (Hall, 1999) and edited by Gblocks ver. 0.91b (Castresana, 2000) in the Castresana Laboratory server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) using 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 nonconserved positions: 8; minimum length of a block: 5; allowed gap positions: with half). Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The best-fit model of DNA evolution was obtained using JModelTest V.2.1.7 (Darriba et al., 2012) with the Akaike information criterion (AIC). The best-fit model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC were then used in MrBayes for the phylogenetic analyses. The general time-reversible model with invariable sites and a gamma-shaped distribution (GTR  +  I +  G) for the D2-D3 segments of 28S rDNA, GTR + G model for ITS1 rDNA, and the transitional model and a gamma-shaped distribution (TIM3 + G) for the partial COI gene and all three datasets were run with four chains for 4 ´ 10⁶ generations. A combined analysis of the two ribosomal genes was not undertaken due to some sequences not being available for all species. The Markov chains were sampled at intervals of 100 generations. After discarding burn-in samples of 30% and evaluating convergence, the remaining samples were retained for in-depth analyses. The topologies were used to generate a 50% majority-rule consensus tree. Posterior probabilities (PP) were given on appropriate clades. Trees from all analyses were visualized using FigTree software, version v.1.42 (Rambaut, 2014).

See Table 1.

Medium sized. Body habitus ventrally curved in a close C-shaped to single spiral when killed by gentle heat. Cuticle 2.9 ± 0.3 (2.6–3.2) mm thick at mid body. Lip region narrow and rounded, slightly offset by a depression from the rest of body, half body-width at the level of guiding ring. Amphidial pouches asymmetrically bilobed at base. Guiding ring single, at 2.6–2.9 times lip region diameter distance from anterior end. Odontostyle 1.3–1.7 times as long as odontophore; odontophore with slight basal swellings. Pharynx extending to a terminal pharyngeal bulb (87.7 ± 7.7 [80.0–103.0] μm long), with dorsal gland nucleus (DN) and ventrosublateral gland nuclei (SVN) separately located at 16.4 (11.9–19.8)% and 59.4 (52.9–67.1)% of distance from anterior end of pharyngeal bulb, respectively. Glandularium 77.0 (67.0–87.0) μm long. Cardia conoid-rounded. Vulva at about middle of the body. Vagina 13.3 (11.0–16.0) mm long, ovejector 25.3 (21.5–28.0) mm wide. Genital system amphidelphic, anterior and posterior genital branches almost equally developed, 237–568 and 260–554 mm long, respectively. Sperm cells not detected inside uterus or oviduct. Rectum 22.2 (19.0– 26.0) mm long. Tail conical, dorsally convex, ventrally concave to flat with bluntly rounded terminus.

Not found.

According to the polytomous key by Chen et al. (1997), and supplement by Loof and Chen (1999), the Greek population has the following codes (codes in parentheses are exceptions): A2(1) – B1 – C2 – D3 – E1 – F2(3) – G2– H4 – I1.

Up to our knowledge, the Greek population of L. leptocephalus collected from the rhizosphere of grapevine at Thessaloniki was the first report of this species from Greece and the second from the Mediterranean Basin. Morphology and morphometrics of this Greek population fit the type population (Hooper, 1961) and other populations from Europe (Lišková and Brown, 1995; Kumari and Decraemer, 2007; Širca et al., 2007). Body and odontostyle lengths of the Greek population of the species agreed with those by Flegg (1967) and Lišková and Brown (1995) identified as “small form” having a 60–70 μm long odontostyle. However, considering the great cryptic diversity within longidorids (Cai et al., 2020), an integrative taxonomical analysis should be carried out also on “large forms” characterized by having 70 to 80 μm long odontostyle to better confirm the species identification (Flegg, 1967; Hooper, 1973). The minor differences between Greek population and the type population of L. leptocephalus are as follows: slightly longer body (4.87 [4.25–5.53] mm vs 4.2 [3.6–4.6] mm), and wider c’ ratio range (1.4 [1.2– 1.5] vs 1.25). These small morphometric differences widen the range of morphometric data and may be due to geographical intraspecific variations.

Type population of Longidorus leptocephalus is morphologically and morphometrically close to type population of L. aetnaeus Roca et al., 1986, especially in the shape of the lip and tail regions, but differs in body (2.69–3.68 mm) and odontostyle lengths (72– 80 μm), position of the vulva (44–47), and presence/ absence of males. Furthermore, both species can be also separated by molecular markers, i.e., by differences in 28S and ITS1 rDNA and COI mtDNA sequences (see below).