Molecular phylogenetic analysis and comparative morphology reveals the diversity and distribution of needle nematodes of the genus Longidorus (Dorylaimida: Longidoridae) from Spain

The genus Longidorus constitutes a large group of approximately 170 species of plant-ectoparasitic nematodes that are polyphagous and distributed almost worldwide. Some of the species of this genus are vectors of plant viruses. Species discrimination in Longidorus is difficult because the morphology is very conservative, and morphometric characters often overlap, leading to potential misidentification. Integrative taxonomy, based on the combination of molecular analyses with morphology, is a useful and necessary approach in Longidorus species identification. In Spain from 2014 to 2017, we conducted nematode surveys among cultivated and wild plants, from which we identified 13 populations of Longidorus , two of which appeared to represent new species and are described herein as L. iliturgiensis sp. nov. and L. pacensis sp. nov., and 11 populations belonging to eight known species: L. africanus , L. baeticus , L. carpetanensis L. carpetanensis and L. pini for the first time. Additionally, we describe the males of L. pini and the juveniles of L. cf. olegi .


Introduction
Nematodes include some of the most abundant metazoans on earth with a global distribution and an estimated ~100,000 species (Boucher & Lambshead, 1995;Blaxter et al., 1998;Coomans, 2000). In addition, among soil fauna, nematodes are the most common and diverse multicellular animals, being found in many environments and representing one of the most ubiquitous animal phyla in the soil (Ferris et al., 2001). Nematodes occupy all trophic levels within the soil food web, which leads them to play a central role in numerous soil functions (Ferris et al., 2001).
Due to the highly conserved morphology, with similar anatomical characteristics and high inter-and intraspecific morphometric variability, species delimitation in Longidorus is a very complex and time-consuming task (Coomans et al., 2001;Archidona-Yuste et al., 2016a). Accurate identification of needle nematodes is essential to establish an unequivocal diagnosis to discriminate virus vector species, select appropriate management strategies for preventing their spread and establish efficient control measures. Recent studies using an integrative approach for identification highlighted the difficulty of correct identification at the species level within the genus Longidorus, as well as cryptic species separation (Subbotin et al., 2015; Archidona-Yuste et al., 2016a; Peraza-Padilla et al., 2017;Xu et al., 2017Xu et al., , 2018. These studies provide molecular markers based on ribosomal RNA (rRNA) (D2-D3 expansion domains of 28S rRNA, ITS and 18S rRNA) and mitochondrial DNA (mtDNA) genes (cytochrome c oxidase subunit I or CoxI) for precise and unequivocal diagnoses of some species of Longidorus, demonstrating that this genus is a complex and taxonomically important group of plantparasitic nematodes.
Approximately forty species of Longidorus have been reported from the Iberian Peninsula, including the recent descriptions of fifteen new species (Peña Santiago et al., 2003;Gutiérrez-Gutiérrez et al., 2013;Archidona-Yuste et al., 2016a). A survey of nematodes from cultivated and wild plants in Spain revealed the presence of thirteen unidentified populations of needle nematodes belonging to the genus Longidorus. Preliminary morphological observations indicated that two of these populations appeared to be morphologically different from all other species described in the genus, while the other populations were assigned to eight known species of Longidorus. Detailed observations using light microscopy and molecular characterisation indicated that these two populations should be assigned to two new species. In the present study, we describe the two new species, Longidorus iliturgiensis sp. nov. and Longidorus pacensis sp. nov. and present phylogenetic data that confirm their species status.
The objectives of this study were (1) to morphologically and morphometrically characterise the two new species belonging to the genus Longidorus and to compare them with previous records; (2) to characterise molecularly the sampled Longidorus spp. populations using the D2-D3 expansion domains of the 28S rRNA gene, ITS1, partial 18S rRNA gene, and the partial mitochondrial CoxI gene sequences; and (3) to study the phylogenetic relationships of the identified Longidorus species with available sequenced species.

Nematode populations and morphological studies
Nematode surveys were conducted from the spring of 2014 to 2017 in the soil around cultivated and wild plants in Spain (supplementary fig. S1). Soil samples were collected with a shovel from the upper 50 cm of soil surrounding four or five plants arbitrarily chosen in each locality. Nematodes were extracted from 500 cm3 of soil by centrifugal flotation (Coolen, 1979) and a modification of Cobb's decanting and sieving (Flegg, 1967) methods. In some cases, additional soil samples were collected from the same locality for additional specimens for morphological and/or molecular identification.
Specimens for light microscopy were killed by hot fixative using a solution of 4% formaldehyde + 1% propionic acid and embedded in pure glycerine using Seinhorst's (1966) method. Specimens were examined using a Zeiss III compound microscope with Nomarski differential interference contrast at powers up to 1,000x magnification. The morphometric study of each nematode population included morphology-based diagnostic features in Longidoridae (i.e., de Man body ratios (Jairajpuri & Ahmad, 1992)), lip region and amphid shape, oral aperture-guiding-ring, odontostyle length and female tail shape) (Jairajpuri & Ahmad, 1992). For line drawings of the new species, light micrographs were imported to CorelDraw ver. X7 and redrawn. In addition, a comparative morphological study on the type specimens of one species was conducted with specimens kindly provided by Dr. A. Navas from the Nematode Collection of the Spanish National Museum of Natural Sciences-CSIC, Madrid, Spain (viz. L. pini .
Topotype specimens of L. carpetanensis  were used for morphological and molecular studies after verifying that their morphology was congruent with Downloaded from Brill.com03/10/2020 02:14:16AM via free access that of the original description. Nematode populations of known Longidorus species were analysed morphologically and molecularly in this study and proposed as standard and reference populations for each species until topotype specimens become available and were molecularly characterised.

DNA extraction, PCR and sequencing
To avoid mistakes caused by mixed populations in the same sample, two live nematodes from each sample were temporarily mounted in a drop of 1 M NaCl containing glass beads and were measured and identified to ensure specimens belong to the unidentified populations of Longidorus. Morphometrics and photomicrographs recorded during this initial study were not used as part of the morphological study. Following morphological confirmation of the species, the specimens were removed from the slides, and DNA was extracted. Nematode DNA was extracted from single individuals as described by Subbotin et al. (2000). The D2-D3 expansion domains of 28S rRNA were amplified using the D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and D3B (5´-TCGGAAGGAACCAGCTACTA-3´) primers (Nunn, 1992). The ITS1 region was amplified using forward primer 18S rRNA (5´-TTGATTACGTCCCTGCCCTTT-3´) (Vrain et al., 1992) and reverse primer rDNA1 (5´-ACGAGCCGAGTGATCCACCG-3´) (Cherry et al., 1997). Finally, the portion of the 18S rRNA was amplified using primers 988F (5´-CTCAAAGATTAAGCCATGC-3´), 1912R (5´-TTTACGGTCAGAACTAGGG-3´), 1813F (5´-CTGCGTGAGAGGTGAAAT-3´) and 2646R (5´-GCTACCTTGTTACGACTTTT-3´) (Holterman et al., 2006). Finally, the portion of the CoxI gene was amplified as described by Lazarova et al. (2006) using the primers COIF (5′-GATTTTTTGGKCATCCWGARG-3′) and COIR (5′-131 CWACATAATAAGTATCATG-3′). PCR cycle conditions were one cycle of 94 °C for 2 min., followed by 35 cycles of 94 °C for 30 s., annealing temperature of 55 °C for 45 s; 72 °C for 3 min; and finally one cycle of 72 °C for 10 min. PCR products were purified after amplification using ExoSAP-IT (Affmetrix, USB products, High Wycombe, UK), quantified using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and used for direct sequencing in both directions using the primers noted above. The resulting products were purified and run on a DNA multicapillary sequencer (Model 3130XL genetic analyser; Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator Sequencing Kit v.3.1 (Applied Biosystems, Foster City, CA, USA) at the Stab Vida sequencing facilities (Caparica, Portugal). The newly obtained sequences were submitted to the Gen-Bank database under accession numbers indicated in table 1 and on the phylogenetic trees.

Phylogenetic analysis
D2-D3 expansion domains of 28S rRNA, ITS1, partial 18S rDNA and the mtDNA gene and CoxI sequences of different Longidorus spp. from GenBank were used for phylogenetic reconstruction. Outgroup taxa for each dataset were chosen based on previous published studies (He et al., 2005;Holterman et al., 2006;Gutiérrez-Gutiérrez et al., 2013;Subbotin et al., 2015;Archidona-Yuste et al., 2016a;Xu et al., 2017). Multiple sequence alignments of the different genes were made using the Q-INS-i algorithm of MAFFT v. 7.205 (Katoh & Standley, 2013) which accounts for secondary RNA structure. Sequence alignments were visualised using BioEdit (Hall, 1999) and edited by Gblocks ver. 0.91b (Castresana, 2000) in Castresana Lab 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 no conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half).
Phylogenetic analyses of the sequence data sets were based on Bayesian inference (BI) using MRBAYES 3.1.2 (Ronquist & 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 Akaike-supported 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 phylogenetic analyses. A symmetrical model with invariable sites and a gamma-shaped distribution (SYM + I + G) for the D2-D3 expansion domains of 28S rRNA, a 3-parameter model with invariable sites and a gamma-shaped distribution (TPM3 μf + I + G) for the ITS1 region, a transitional model with invariable sites and a gamma correction (TIM2 + I + G) for the partial 18S rRNA, and a general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) model for the partial CoxI gene were run with four chains for 2 × 106 generations. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each analysis. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further 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 visualised using FigTree software version v.1.42 (http:// tree.bio.ed.ac.uk/software/figtree/).

Species identification and delimitation
According to the polytomous key by  and the supplement by Loof & Chen (1999), matrix codes A (odontostyle length), B (lip region width), C (distance of guiding-ring from anterior body end), D (lip region shape), E (shape of amphidial fovea), F (body length), G (index "a"), H (tail shape), and I (presence/ absence of males) were used for species identification and delimitation. Additionally, classical diagnostic features in Longidoridae (i.e., de Man body ratios (Jairajpuri & Ahmad, 1992)), lip region and amphid shape, distance from oral aperture to guiding-ring, odontostyle length and female tail shape (Jairajpuri & Ahmad, 1992) were used for species delimitation and presented in species descriptions. Molecular data were also considered to differentiate species within the genus. Based on an integrative taxonomy, we described two new species: L. iliturgiensis sp. nov. and L. pacensis sp. nov. We found additional species for which morphological and morphometric data as well as molecular data were provided for L. africanus Merny, 1966, L. carpetanensis, L. nevesi Macara, 1985, L. cf. olegi Kankina & Metlitskaya, 1983. For other previously studied species, such as L. baeticus Gutiérrez-Gutiérrez, Cantalapiedra-Navarrete, Montes-Borrego, Palomares-Rius & Castillo, 2013, L. fasciatus, and L. vallensis Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, 2016a, only the D2-D3 expansion domains of 28S rRNA or ITS1 sequences have been provided. Paratype materials of L. pini were used for measurements and morphological studies to compare with our specimens. For L. africanus, L. carpetanensis, L. nevesi, L. cf. olegi, and L. pini, a brief description and comparison with previous records are provided in the Appendix because these species records represent the first molecular characterisation and/or new records in Spain. The high variability of the ITS1region makes it difficult to determine similarity values among the different Longidorus spp., and they also show low coverage values in the sequence pairwise BLAST comparisons. Intrapopulation variability of this region was low for L. nevesi (MH429997-MH429998), with 21 variable nucleotides, L. carpetanensis (MH429991-MH429993), from 1 to 20 variable nucleotides, L. cf. olegi (MH429999-MH430000), with 11 nucleotides, and finally only one different position for L. iliturgiensis sp. nov., and no variability for L. pacensis sp. nov. However, L. fasciatus (MH429994-MH429996) showed a much higher intraspecific variability for the ITS1 region, being 96-97% similar (from 31 to 51 variable nucleotides) among the 3 studied populations in this study and 88-89% similar with L. fasciatus (JX445097, Gutierrez-Gutierrez et al., 2013) (31-133 variable nucleotides).
For the 18S rRNA, ten new sequences were obtained in this study, and all of them showed very high similarity values with the other accessions from Longidorus spp. deposited in GenBank, being 98-99% similar. Finally, six new CoxI sequences were deposited in GenBank in this study. This gene showed an interspecific variability very similar among all Longidorus spp. deposited in GenBank (similarity values ranging from 77 to 80% among all the accessions with CoxI sequences available).

Phylogenetic relationships
Phylogenetic relationships among Longidorus species inferred from analyses of D2-D3 expansion domains of 28S rRNA, ITS1, the partial 18S rRNA and the partial CoxI mtDNA gene sequences using BI are shown in figs.   To facilitate discussion, in trees of nuclear markers, clades that were well supported or taxonomically well founded were labelled in Roman numerals from I to VI. Poorly supported lineages were not explicitly labelled. These trees showed a very similar topology, which consisted of a major clade that clustered the majority of Longidorus spp. (approximately 75% of species with molecular data available). Clade I from the 50% majority rule consensus 28S rRNA gene BI tree (PP = 1.00) comprised 19 species, all of them reported in the Iberian Peninsula with a characteristic tail (hemispherical convex-conoid tail shape), and included L. pacensis sp. nov. ( fig. 1). Longidorus pacensis sp. nov. was phylogenetically related to L. baeticus and L. macrodorus in a well-supported clade ( fig. 1). Clade II (PP = 1.00) comprised 12 species, mostly central European ( fig. 1). Clade III (PP = 1.00) comprised 8 species with a similar distance from the anterior end to the guiding-ring and amphids with bilobed basal lobes including two newly sequenced species from Spain, viz. L. carpetanensis and L. pini ( fig. 1). Clade IV (PP = 1.00) comprised five species from Crete and Iran, including nematodes with similar sizes and males absent or rare. Clade V (PP = 1.00) comprised seven species from Western Europe with a lip region continuous with body contour and short hemispherical tail (c' < 1). Finally, Clade VI (PP = 1.00) comprised five species from Asia characterised by a long distance between the guiding-ring and anterior end ( fig. 1). The second new species described here, L. iliturgiensis sp. nov., clustered in a clade with L. alvegus tree ( fig. 1). The 50% majority rule consensus ITS1 BI tree showed three major clades (I to III), although Clade II was moderately supported. Clade I (PP = 1.00) comprised 43 species with different geographical origins and morphologies and included the two new and five known species studied here, clustering with the same species as in the 28S rRNA gene tree but with lower supports ( The 50% majority rule consensus 18S rRNA gene BI tree contains three major clades. Clade I (PP = 0.99) included the majority of the species of the genus, comprising the new and known species studied here. Longidorus iliturgiensis sp. nov. clustered with L. leptocephalus in a not well-supported clade ( fig. 3). Unfortunately, no data were available for the partial 18S rRNA of L. alvegus. In addition to the 28S rRNA gene and ITS1 trees, L. pacensis sp. nov. clustered with some species described from the Iberian Peninsula; however, these relationships were not well supported. Finally, L. carpetanensis and L. pini seem to be phylogenetically related since they appear together within a well-supported clade (PP = 1.00) ( fig. 3). In addition, Clade II (PP = 0.90) included nine Asian species (fig. 3). The principal differences among the three rRNA trees were the support for some clades in partial 18S rRNA and ITS1.

Discussion
The primary objective of this study was to identify and molecularly characterise species belonging to the genus Longidorus associated with different plant hosts and environments in Spain. Our results demonstrate that the use of morphological-morphometrical studies integrated with rRNA and mtDNA molecular markers deciphered the important diversity in this difficult group of nematodes. We described two new species and broadened the knowledge on the distribution and molecular and biological data of eight known species of the genus Longidorus based on integrative taxonomy and the phylogenetic relationships among the new and known species of the genus Longidorus (see Appendix).
The results (including new and known species) increased data on the biodiversity of Longidorus in the Iberian Peninsula and agree with previous results obtained for the phylogeny and biogeography of the genus Longidorus in the Euro-Mediterranean region (Navas et al., 1993;Archidona-Yuste et al., 2016a). The diversity of this genus in Spain is remarkable, with approximately 40 species reported including the species described in this article Andrés et al., 1991;Gutiérrez-Gutiérrez et al., 2011;Gutiérrez-Gutiérrez et al., 2013; Archidona-Yuste et al., 2016a). This study, in addition to the description of two new species, provides three first reports for Spain (L. africanus, L. nevesi and L. cf. olegi), and new geographic records for other species, such as L. baeticus, L. vallensis, L. carpetanensis and L. pini. The species distribution knowledge is important to understand nematode expansion, phytopathological risks and how some species are native to the Iberian Peninsula (i.e., L. baeticus, L. macrodorus, L. pacensis sp. nov.) while others are distributed in the Mediterranean region (i.e., L. iuglandis and L. fasciatus). Additionally, the biological knowledge of L. pini and L. cf. olegi is extended by presenting data for males in the case of L. pini and describing juveniles for L. cf. olegi. Males and juveniles are important for the diagnosis of Longidorus (Robbins et al., 1996;Peneva et al., 2013).
Sequences of nuclear ribosomal RNA genes, particularly D2-D3 expansion domains of the 28S rRNA gene, ITS1 region, and the mtDNA gene CoxI, have proven to be a powerful tool for providing accurate species identification of Longidoridae (Palomares-Rius et al., 2017). However, the low nucleotide variability found in partial 18S rRNA makes it difficult to identify individuals to the species level. New molecular markers were provided for L. nevesi, L. cf. olegi, L. carpetanensis and L. pini, in addition to the newly described species (L. iliturgiensis sp. nov. and L. pacensis sp. nov.). For other species, we increased their molecular diversity (L. africanus, L. baeticus, L. fasciatus, and L. vallensis). This latter point is important for the use of barcoding techniques for species identification in this genus (Palomares-Rius et al., 2017).
Phylogenetic analyses based on three rDNA molecular markers (D2-D3 expansion domains of 28S rRNA gene, ITS1 region and the partial 18S rRNA) resulted in a general consensus of species phylogenetic positions for the majority of species and were generally congruent with those given by previous phylogenetic analysis (Subbotin et al., 2014; Archidona-Yuste et al., 2016a;Esmaeili et al., 2016;Palomares-Rius et al., 2017). Only one previous work on the phylogeny of the CoxI gene in the family Longidoridae has been published to date (Palomares-Rius et al., 2017). However, this recent work, including all the genera with molecular data available within the family, did not resolve the phylogenetic relationships among the major different groups of Longidorus species, so it was difficult to compare both results. Base saturation (third nucleotide position in each codon), the short fragment used in this study, different mutation rates in the mitochondrial genome and the wide evolutionary differences within these studied groups could complicate the phylogeny for the CoxI marker (Palomares-Rius et al., 2017). As mentioned before, the phylogenetic position of all species sequenced in this study was coincident; L. pacensis sp. nov., L. nevesi, L. cf. olegi, L. baeticus and L. fasciatus were grouped in a clade with other species from the Iberian Peninsula in all analysed markers, although the relationships within this clade varied depending on which marker was used. Longidorus iliturgiensis sp. nov. clustered with L. alvegus in D2-D3 expansion region domains of 28S rRNA and ITS1 phylogenetic trees; however, it was not possible to study this relationship for the partial 18S rRNA because there were no 18S rRNA sequences from L. alvegus in GenBank. Longidorus carpetanensis and L. pini seem to be strongly phylogenetically related using all nuclear markers studied and only when mtDNA was analysed did these species appear separately, probably because mtDNA evolves faster than ribosomal DNA (Lazarova et al., 2006;Kumari et al., 2010;Palomares-Rius et al., 2017). Some clades were strongly supported and clustered species of a geographical area, as was the case for Asian species. This clade usually occupies basal positions showing their ancestral position in the genus, suggesting this area as the possible origin of the genus Longidorus, but this hypothesis needs to be confirmed with additional studies.
In summary, the present study updates the biodiversity within the genus Longidorus, showing the plasticity of these nematodes and the importance of describing new species by integrating morphometrical and molecular approaches. The description of the two new species also expands the distribution of these nematodes in the Iberian Peninsula, specifically in Spain. New reports and molecular data of these nematodes with descriptions of juveniles in some species provide new data for the identification, biology and ecology of these plant-parasitic nematodes in the field. lent technical assistance and four anonymous reviewers and editors for their valuable suggestions to improve the manuscript. Our research was supported by grants P12-AGR-1486 from 'Consejería de Economía, Innovación y Ciencia' of Junta Type locality. Andújar, Jaen province, Spain: 38° 9'7.45"N, 4°0'54.35"W; 267 m above sea level (a.s.l.).
Etymology. The species name is derived from "iliturgi" the Roman name of Andújar, where the type specimens were found.
Male. Almost as frequent as female. Morphologically similar to female except for genital system and posterior region slightly curved ventrally. Male genital tract diorchic with testes opposed, containing multiple rows of spermatogonia in the germinal zone. Tail conoid, dorsally conoid and ventrally concave with acute terminus and thickened outer cuticular layer. Spicules very short, moderately developed and slightly curved ventrally, approximately 0.7-0.9 times shorter than tail length; lateral guiding pieces straight with curved proximal end. Moderate number of supplements, one pair of adanal and from 6 to 10 mid-ventral supplements.
Juveniles. All four juvenile stages (first-, second-, third-and fourth-stage) were identified using morphological characters such as body length, length of replacement and functional odontostyle (Robbins et al., 1996). Juveniles were similar to adults apart from developed reproductive system, shorter body length, tail shape and presence of replacement odontostyle (figs. 5, 6-7). Tail becomes progressively shorter and stouter in each moult (figs. 5, 6-7 and table 2). As for other longidorids, first-juvenile stage was characterised by the replacement odontostyle tip close to base of functional odontostyle and located at level of odonto phore. In J2-J4, replacement odontostyle located at some distance from odontophore base. J1s were characterised by a bluntly rounded to cylindrical tail with a c' ratio >3.0 (figs. 5-6 and table 2).
Remarks. According to the updated polytomous key by  and the supplement by Loof & Chen (1999), the specific matrix code for this species was A2(1)-B1-C2-D4-E2-F23-G3-H6 (5)   Etymology. The species name is derived from the Latin word "Pacensis" the Roman name of Badajoz, where the type specimens were found.
Male. Less frequent than female (1:2 ratio). Morphologically similar to female except for genital system and posterior region considerably curved ventrally. Tail convex conoid, ventrally concave with broad blunt terminus, a deep depression posterior to anus and the thickened outer cuticular layer. Male genital tract diorchic with testis opposed, containing multiple rows of spermatogonia in the germinal zone. Spicules arcuate, robust, 1.5-2.1 times longer than tail length, lateral guiding pieces more or less straight. One pair of adanal supplements preceded by a row of 14-19 ventromedian supplements.

Paratype
10.1 ± 1.3 (8.5-11.5) 11.5 Remarks. Longidorus carpetanensis was originally described from around roots of common broom (Cytisus scoparius L.), in Navalmoral, Avila Province, Spain. Subsequently, Bravo & Lemos (1997) reported it in the rhizosphere of cereals and peach trees from Constância and Abrantes, province of Ribatejo, Portugal. A Longidorus population resembling this species was detected in the rhizosphere of common oak, at Puebla de Sanabria, Zamora Province, Spain, which prompted us to study this population and to characterise molecularly the topotype specimens in order to confirm its identification.
The population of Puebla de Sanabria agrees closely with original description  in morphology and morphometry ( fig. 11  and table 4). This study expands its distribution to another province in north-western Spain. According to the polytomous key by , the supplement by Loof & Chen (1999), and additional codes (Peneva et al., 2013;Archidona et al., 2016), this species has the following code: A12-B12-C2-D4-E3-F2-G2-H56-I2-J1-K?. Macara, 1985 (fig. 12, table 5) Remarks. The Spanish population of this species is characterised by a long body, ventrally curved in an open C when killed by heat, lip region conoid and continuous with body contour. Amphidial fovea bilobed, slightly asymmetrical, odontostyle long and robust, approximately 2 times longer than odontophore. Female tail short, bluntly conoid with rounded terminus and c' < 1.0. Males frequent with robust spicules, ventrally arcuate approximately 100 μm long. The morphology and morphometrics of this population closely agree with the original description (Macara, 1985). Apart from the original description, this species has been reported also from forests in Portugal (Macara, 1994;Bravo & Lemos, 1997), and to our knowledge, this is the first report from Spain. This new report confirms the hypothesis that this species is an Iberian endemic (Gutiérrez-Gutiérrez et al., 2016). According to the polytomous key by , the supplement by Loof & Chen, (1999), and additional codes (Peneva et al., 2013;Archidona et al., 2016), this species has the following code: A56-B34-C34-D1-E3-F35-G12-H1-I2 -J1-K3. Kankina & Metlitskaya, 1983 (figs. 13 and 14, table 6) Remarks. This species was described by Kankina & Metlitskaya (1983) from the rhizosphere of raspberry at the experimental farm of the Rossoshanski fruit-berry experimental station in the    Kankina & Metlitskaya, 1983 Rossoshanski region of Voronezh Province (Russia), based only on two females and three males; there is no other report on it. The Spanish population of Longidorus collected from the rhizosphere of Portuguese oak (Quercus faginea Lam.), at Arroyo Frío, Jaén province, Spain showed morphological and morphometric characteristics resembling this species, which prompted us to study this population, characterising morphologically females and males, as well as providing descriptions for the first time of first-, second-, third-and fourth-stage juveniles, including a new molecular characterisation by D2-D3, ITS1, and partial 18S sequences (supplementary fig. S1 and table 6). Given the low number of specimens used in the original description of this species, a detailed characterisation of both morphology and morphometrics was provided in the present study. However, the taxonomic assignment is here given as L. cf. olegi because of the few specimens used in the original species description. For this reason, we consider this species as L. cf. olegi until topotypes of this species can clarify the similarity, or not, with the Spanish population.

Longidorus cf. olegi
The morphology and morphometrics of the Spanish population from Portuguese oak at Arroyo Frío, Jaén Province, agree closely with those of the original description by Kankina & Metlitskaya (1983) (table 6) 2) μm). These differences further expand the intraspecific variation in this species that can be a consequence of the few specimens measured in the original description by Kankina & Metlitskaya (1983). The population examined by us represents the first report of the species in a country outside of Russia. According to the polytomous key by , the supplement by Loof & Chen (1999), and additional codes (Peneva et al., 2013;Archidona et al., 2016), this species has the following code (codes in parentheses are exceptions): A4(3)-B2(3)-C4-D1-E2-F4-G2-H1-I2-J1-K6.
Male. Very rare, only two male specimens were found (1:7 ratio). Morphologically similar to female except for genital system and posterior region slightly curved ventrally. Tail convex conoid, ventrally slightly concave with broad blunt terminus and thickened outer cuticular layer. Male genital tract diorchic with testis opposed, containing multiple rows of spermatogonia in the germinal zone. Spicules arcuate, robust, 1.6-1.8 times longer than tail length, lateral guiding pieces more or less straight. One pair of adanal supplements preceded by a row of 13-14 ventromedian supplements.
Juveniles. All four juvenile stages (J1-J4) distinctly separated by differences in body length, odontostyle and replacement odontostyle length (Robbins et al., 1996) were detected (figs. 13 and 14). Morphologically, juveniles resemble adults except for the smaller size and undeveloped reproductive system. J1s were characterised by a subdigitate tail with a rounded tip and a c' ratio 2.4-2.6 (  . A Longidorus population resembling this species was detected in the rhizosphere of Pyrenean oak, at Nava de Francia, Salamanca Province, Spain, which prompted us to study this population in order to characterise it molecularly and compare it with paratype specimens, thus supporting identification. The population from Nava de Francia was characterised by a medium body size, lip region offset and slightly expanded, amphidial fovea symmetrically bilobed, and female tail conical dorsally convex, ventrally concave. One male specimen was found, representing the first report for this species and showing a similar morphology than that of females, except for the genital system and posterior region being slightly curved ventrally. Spicules arcuate, robust, 0.7 times shorter than tail length, lateral guiding pieces more or less straight. One pair of Table 6 Morphometrics of Longidorus cf. olegi Kankina and Metlitskaya, 1983 from Portuguese oak at Arroyo Frío (Jaén, Spain). Measurements are in micrometres (μm) and in the form: mean standard deviation (range) Arroyo Frío (Jaén, Spain) Paratypes (Kankina and Metlitskaya, 1983) Characters/ratios adanal supplements preceded by a row of 7 ventromedian supplements ( fig. 15). The population of Nava de Francia agree with the original description and examined paratypes  in morphology and morphometry ( fig. 15 and table 5), except for a slightly longer body (5.7-6.0 mm vs. 4.0-5.7 mm), longer odontostyle (79.0-80.0 μm vs. 65-74 μm), and shorter female tail length (60.0-66.5 μm vs. 57.0-75.0 μm). These differences should be regarded as geographical intraspecific variation. In addition, this study expands its distribution to another province in north-western Spain. According to the polytomous key by , the supplement by Loof & Chen (1999), and additional codes (Peneva et al., 2013;Archidona et al., 2016), this species has the following code (codes in parentheses are exceptions): A2-B1-C2-D3-E2-F3-G3-H6-I12-J?-K?.