Unravelling the Biodiversity and Molecular Phylogeny of Needle Nematodes of the Genus Longidorus (Nematoda: Longidoridae) in Olive and a Description of Six New Species

The genus Longidorus includes a remarkable group of invertebrate animals of the phylum Nematoda comprising polyphagous root-ectoparasites of numerous plants including several agricultural crops and trees. Damage is caused by direct feeding on root cells as well as by transmitting nepoviruses that cause disease on those crops. Thus, correct identification of Longidorus species is essential to establish appropriate control measures. We provide the first detailed information on the diversity and distribution of Longidorus species infesting wild and cultivated olive soils in a wide-region in southern Spain that included 159 locations from which 449 sampling sites were analyzed. The present study doubles the known biodiversity of Longidorus species identified in olives by including six new species (Longidorus indalus sp. nov., Longidorus macrodorus sp. nov., Longidorus onubensis sp. nov., Longidorus silvestris sp. nov., Longidorus vallensis sp. nov., and Longidorus wicuolea sp. nov.), two new records for wild and cultivate olives (L. alvegus and L. vineacola), and two additional new records for wild olive (L. intermedius and L. lusitanicus). We also found evidence of some geographic species associations to western (viz. L. alvegus, L. intermedius, L. lusitanicus, L. onubensis sp. nov., L. vineacola, L. vinearum, L. wicuolea sp. nov.) and eastern distributions (viz. L. indalus sp. nov.), while only L. magnus was detected in both areas. We developed a comparative study by considering morphological and morphometrical features together with molecular data from nuclear ribosomal RNA genes (D2–D3 expansion segments of 28S, ITS1, and partial 18S). Results of molecular and phylogenetic analyses confirmed the morphological hypotheses and allowed the delimitation and discrimination of six new species of the genus described herein and four known species. Phylogenetic analyses of Longidorus spp. based on three molecular markers resulted in a general consensus of these species groups, since lineages were maintained for the majority of species. This study represents the most complete phylogenetic analysis for Longidorus species to date.


Introduction
The overall objective of this study was to test the congruence between morphological and molecular data within Longidorus species, and the specific objectives were: i) to identify and morphologically and morphometrically compare the 40 Spanish populations of Longidorus spp. detected in recent field samples from wild and cultivate olive-ecosystems; ii) to carry out a molecular characterisation of these Longidorus populations based on sequences of the D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene, the ITS1 of rRNA, and partial 18S rRNA sequences; and iii) to study the phylogenetic relationships of Longidorus spp.

Ethics Statement
No specific permits were required for the described fieldwork studies. Permission for sampling the olive orchards was granted by the landowner. The samples from wild olives were obtained in public areas, forests, and other natural areas studied and do not involve any species endangered or protected in Spain. The sites are not protected in any way.

Soil collection and nematode extraction
Nematodes were surveyed from 2012 to 2015 during the spring season in wild and cultivate olives groves in Andalusia, southern Spain (Table 1, Fig 1). Soil samples were collected for nematode analysis with a shovel from four to five trees in each sampling site. A total of 131 and 318 sampling sites from wild and cultivated olives, respectively, were arbitrarily chosen in the eight provinces of Andalusia where both olive types were present. The number of sampling sites was proportional to the area of wild and cultivated olive in each province ( Table 1, Fig 1). Soil samples were collected from a 5-to 50-cm depth, in the close vicinity of active plant roots, discarding the upper 5-cm of topsoil to ensure that roots from weeds or other herbaceous plants were not included. All soil samples from each site were thoroughly mixed to obtain a single representative sample before nematode extraction.
Nematodes were extracted from a 500-cm 3 sub-sample of soil using magnesium sulphate centrifugal-flotation and a modification of Cobb´s decanting and sieving methods [41,42]. The soil was washed thoroughly with tap water through a 710-μm mesh sieve, and the filtered water was collected in a beaker and thoroughly mixed with 4% kaolin (v/v). This mixture was centrifuged at 1,100×g for 4 min, and the supernatants discarded. Pellets were resuspended in 250 ml MgSO4 (δ = 1.16) and the new suspensions were centrifuged at 1,100×g for 3 min. The supernatants were sieved through a 5 μm mesh, and nematodes collected on the sieve were washed with tap water [42]. The nematode sample was poured into a counting dish (8 cm L × 8 cm W × 1.5 cm H) and the nematodes were identified and counted under a Leica MZ12, stereomicroscope (Leica Microsystems, Wetzler, Germany). PPN from soil samples were identified to genus, and then we focussed on the species delineation of needle nematodes of the genus Longidorus. Later on, abundance and prevalence of each Longidorus species was estimated. Abundance was calculated as the mean number of Longidorus nematodes per 500 cm 3 of soil for all samples. The prevalence was computed by dividing the number of samples in which the Longidorus species was detected by the total number of samples and expressed as a percentage.

Morphological studies
Longidorus specimens for light microscopy were killed by gentle heat, fixed in a solution of 4% formaldehyde + 1% propionic acid and processed to pure glycerine using Seinhorst's method [43]. Specimens were examined using a Zeiss III compound microscope with Nomarski differential interference contrast at up to 1,000x magnification. The morphometric study of each nematode population included classical diagnostic features in longidoridae (i.e. de Man body ratios, lip region and amphid shape, oral aperture-guiding ring, odontostyle and odontophore length) [44]. All measurements were expressed in micrometers (μm), unless otherwise indicated in text. For line drawing of the new species, light micrographs were imported to CorelDraw software version X6 (Corel Corporation, London, UK) and redrawn. All other abbreviations used are as defined in Jairajpuri & Ahmad [44]. In addition, a comparative morphological and morphometrical study of type specimens of some species were conducted with specimens kindly provided by Dr. A. Troccoli, from the nematode collection at the Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari

DNA extraction, PCR and sequencing
For molecular analyses, in order to avoid mistakes in the case of mixed populations in the same sample, two live nematodes from each sample were temporary mounted in a drop of 1M NaCl containing glass beads (to avoid nematode crushing/damaging specimens) to ensure specimens conformed in form to the unidentified populations of Longidorus. Morphometrics and photomicrographs recorded during this initial study were not used as part of the morphological study or analyses. Following morphological confirmation, the specimens were removed from the slides and DNA extracted. Nematode DNA was extracted from single individuals and PCR assays were conducted as described by Castillo et al. [48]. One nematode specimen of each sample was transferred to an Eppendorf tube containing 16 μl ddH 2 O, 2 μl 10x PCR buffer and 2 μl proteinase K (600 μg/ml) (Promega, Benelux, The Netherlands) and crushed during 2 min with a micro-homogeniser, Vibro Mixer (Zürich, Switzerland). The tubes were incubated at 65°C (1 h), then at 95°C (15 min), and finally at 80°C (15 min). One μl of extracted DNA was transferred to an Eppendorf tube containing: 2.5 μl 10X NH4 reaction buffer, 0.75 μl MgCl2 (50mM), 0.25 μl dNTPs mixture (10mM each), 0.75 μl of each primer (10mM), 0.2 μl BIOTAQ DNA Polymerase (BIOLINE, UK) and ddH 2 O to a final volume of 25 μl. The D2-D3 expansion segments of 28S rRNA was amplified using the D2A (5'-ACAAGTACCGTGAGG GAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') primers [49]. The ITS1 region was amplified using forward primer 18S (5´TTGATTACGTCCCTGCCCTTT-3´) [50] and reverse primer rDNA1 (5´-ACGAGCCGAGTGATCCACCG-3´) [51]. 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´) [52]. 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), quantified using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and used for direct sequencing in both directions using the primers referred to 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 GenBank database under accession numbers indicated on the phylogenetic trees and in Table 1.

Phylogenetic analysis
D2-D3 expansion segments of 28S rRNA, ITS1, and partial 18S rRNA sequences of different Longidorus spp. from GenBank were used for phylogenetic reconstruction. Outgroup taxa for each dataset were chosen according to previous published data [19,25,26,52,53]. The newly obtained and published sequences for each gene were aligned using MAFFT ver. 7 [54], strategy FFT-NS-1 with default parameters. Sequence alignments were manually edited using BioEdit [55]. Percentage similarity between sequences was calculated using the sequence identity matrix using BioEdit. For that, the score for each pair of sequences was compared directly and all gap or place-holding characters were treated as a gap [55]. When positions of both sequences have a gap they do not contribute [55]. Phylogenetic analyses of the sequence data sets were performed based on Bayesian inference (BI) using MRBAYES 3.1.2 [56]. The best fitted model of DNA evolution was obtained using JMODELTEST v. 2.1.7 [57] 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. BI analyses were performed under SYM+I+G (namely, symmetrical of invariable sites and a gamma-shaped distribution) model for D2-D3 expansion segments of 28S rRNA, TVM+I+G and TIM3+I+G (namely, transversional and a transitional of invariable sites and a gamma-shaped distribution) models for the two ITS1 region datasets, TVMef+I+G (namely, equal-frequency transversional of invariable sites and gamma-shaped distribution) model for the partial 18 S rDNA. These BI analyses were run separately per dataset using four chains for 2 × 10 6 , 1 and 1 × 10 6 , and 3 × 10 6 generations, respectively. The Markov chains were sampled at intervals of 100 generations. Two runs were performed 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) are given on appropriate clades. Trees were visualised using TreeView [58].

Nomenclatural Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), and hence the new names contained herein are available under that Code from the electronic edition. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSIDs) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn: lsid:zoobank.org:pub: C8230A9D-FD45-4AA4-9ABF-8445E8001CCC. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Results
Taxon Sampling, abundance and prevalence of Longidorus species All positive Longidorus spp.-sampling sites for this study, including specimens used in morphological and/or genetic analyses, are shown in Table 1 Fig 1). Except for L. alvegus, L. indalus sp. nov. and L. vineacola, that occurred in both olive types, all the remaining identified species where present only in either wild or cultivated olives.
Longidorus spp. were present in low to moderate densities (from 1 to 33 nematodes per 500 cm 3 of soil), and were moderately distributed in both wild and cultivated olives ( Table 2). The overall prevalence of Longidorus spp. in wild olives was 16.03% (21 out of 131 samples) whereas in cultivated olives was 5.97% (19 out of 318 samples) (Tables 1 and 2). Although wild and cultivated olives were present in all of the eight provinces of Andalusia, the genus Longidorus was not detected in Jaén and Málaga provinces, and in Granada only in cultivated olives (4 samples out of 39) ( Table 2, Fig 1). The three most prevalent Longidorus species, L. indalus sp. nov., L. oleae, and L. vineacola, were detected in both wild and cultivated olives, as well as L. alvegus, L. vallensis sp. nov. and L. wicuolea sp. nov. but with lower prevalence (Tables 1 and  2). Longidorus vineacola was rather moderately distributed among the studied zones having the highest overall prevalence in both wild and cultivated olives (Tables 1 and 2). However, some other Longidorus species showed a lower prevalence and were only detected either in wild (L. lusitanicus, L. silvestris sp. nov. and L. vinearum) or in cultivated olive (L. macrodorus sp. nov., L. magnus, L. onubensis sp. nov.) (Tables 1 and 2).
Male. Extremely rare, only one male specimen was found but not in type locality. Morphologically similar to female except for genital system, but with posterior region slightly curved ventrally. Male genital tract diorchic with testes opposed, containing multiple rows of different stages of spermatogonia. Tail conoid, dorsally conoid and ventrally concave with rounded terminus and thickened outer cuticular layer. Spicules very short, moderately developed and slightly curved ventrally; lateral guiding pieces more or less straight or with curved proximal end. Low number of supplements, one pair of adanal and 4 mid-ventral supplements.
Juveniles. Morphologically similar to adults, but smaller. All four juvenile stages were found, being distinguishable by relative lengths of body and functional and replacement odontostyle (Table 3, Figs 3 and 4; [66,67]). J1s were characterised by a bluntly conoid tail with a c' ratio 3.2, well curved dorsally with a dorsal depression at hyaline region level (Fig 3) odontostyle length ca 37 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages. However, morphology in second-, third-and fourth-stages (except for undeveloped genital structures) similar to that of female, including conoid tail shape, becoming progressively shorter and stouter in each moult and shorter distance from anterior end to guidingring in each moult.
Measurements, morphology and distribution. Morphometric variability is described in Tables 3 and 4 and morphological traits in Figs 2, 3 and 4. In addition to the type locality, Longidorus indalus sp. nov. was extracted from five cultivated olive samples causing enlarged swellings of root tips (Fig 3), and two wild olive samples of several localities distributed in Almería and Granada province, being one of the two species (together with L. magnus) located on Eastern Andalusia ( Table 1, Fig 1).
Relationships. According to the polytomous key by Chen et al. [64] and the supplement by Loof and Chen [65], and on the basis of sorting on matrix codes A (odontostyle length), B (lip region width), C (distance of guiding-ring from anterior body end), D (lip region shape), and E (shape of amphidial pouch), L. indalus sp. nov. is closely related to L. carpetanensis and L. unedoi from which it can be differentiated by a combination of these characters discussed below, but particularly in female and male tail shape (bluntly conoid vs conical, dorsally convex) (Fig 3, S1 Fig).  [19,68].
Etymology. The species name refers to the primarily distinguishing character of the long odontostyle (from Greek macros = long, and dorus = stylet).
Description of taxa. Female. Body very long and rather robust, sharply tapering towards anterior end, usually assuming a body straight or nearly so shape when heat relaxed. Cuticle very finely striated generally but mainly at the posterior extremity, 5.5 ± 0.7 (4.0-7.0) μm thick at mid-body but more thickened at tail tip where it is 13.5 ± 3.0 (8.5-17.0) μm thick, immediately anterior to anus. Lip region conoid-narrowed, anteriorly rounded, and continuous with body contour. Amphidial fovea pocket-shaped slightly symmetrically bilobed with lobes about equal length and extending about 2/3 part of distance between oral aperture and guiding-ring, openings obscure appearing as minute pores, not slit-like. Stylet guiding-ring single, located 5.3 ± 0.6 (4.1-6.0) times lip region diam. from anterior end. Lateral chord 26.2 ± (24.0-30.0) μm wide at mid-body or 20-27% of corresponding body diam. Odontostyle very long and robust straight or slightly arcuate, 3.9 ± 0.3 (3.4-4.0) times as long as distance between anterior end to guiding-ring, odontophore about 2/3 part of the odontostyle length, weakly developed with slightly enlarged at the base. Nerve ring encircling cylindrical part of pharynx at odontophore base, located 271. ). Cardia hemispherical, 18.7 ± 3.9 (14.5-25.0) μm long. Reproductive system with both genital branches equally developed, relatively short compared to body length, ranging between 622-1318 μm long, with reflexed ovaries very variable in length. Vulva in form of a transverse slit, located about mid-body, vagina perpendicular to body axis, extending to ca 2/ 3 corresponding body width, surrounded by well-developed muscles. Genital branches equally developed, 8.8 ± 1.6 (6.6-13.0), 8.8 ± 2.0 (6.5-14.0)% of body length, respectively. Uterus short, thick-walled, filled with sperm cells in most female specimens observed; well-developed sphincter between uterus and pars dilatata oviductus, usually containing numerous sperm cells too. Ovaries equally developed and very variable in length, 192-545 μm long, both of them with a single row of oocytes. Prerectum variable in length, 2170 ± 559.7 (1427-3045) μm long, and rectum 46.6 ± 7.9 (36.0-56.0) μm long, anus a small rounded slit. Tail short, bluntly conoid, rounded to almost hemispherical, bearing between three and four pairs of caudal pores.
Male. Common, as frequently as female. Morphologically similar to female except for genital system, posterior region being more strongly coiled with slightly longer tail. Male genital tract diorchic with testes opposed, containing multiple rows of different stages of spermatogonia. Spicules massive, robust, and curved ventrally; lateral guiding pieces more or less straight or with curved proximal end. Tail convex-conoid, dorsally conoid, ventrally being almost straight with broad blunt terminus and thickened outer cuticular layer. One pair of adanal supplements and 17-25 mid-ventral supplements.
Juveniles. Morphometrics obtained from juvenile specimens, and of the relative lengths of body, tail, and functional and replacement odontostyle, confirmed the presence of four juvenile stages ( Table 6, Figs 4 and 6; [66,67]). J1s were characterised by a bluntly rounded to cylindrical tail with a c' ratio 1.2 (Table 6), an odontostyle very long, ca 120 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages.
Measurements, morphology and distribution. Morphometric variability is described in Table 6, and morphological traits in Figs 4-7. Longidorus macrodorus sp. nov. was only found in the type locality in the rhizosphere of cultivated olive ( Table 1, Fig 1).
Longidorus Diagnosis. Longidorus onubensis sp. nov. is a gonochoristic species characterized by a long and rather body (7.4-9.5mm), assuming an open C-shaped when heat relaxed; lip region broadly rounded to truncate, continuous or separated from body contour by slight depression, 14.0-16.5 μm wide; guiding-ring located 31-44μm from anterior end; long odontostyle (103-121 μm); amphidial fovea pocket-shaped with lobes of about equal length; vulva almost equatorial; female tail very short, broadly conoid to hemispherical, and bearing two or three pairs of caudal pores; c' ratio (0.6-0.8); males frequent with long spicules (92-98 μm) and 14-16 ventromedian supplements; and specific D2-D3, ITS1 rRNA and partial 18S rRNA sequences (GenBank accession numbers KT308857-KT308858, KT308882-KT308883, and KT308897, respectively). According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], the new species has the following code (codes in parentheses are exceptions): Etymology. The species epithet refers to 'Onuba', the Roman name of the province of Huelva, where the type specimens were collected.
Description of taxa. Female. Body long and rather robust, slightly tapering towards anterior end, usually assuming an open C-shaped when heat relaxed, almost straight anteriorly and more curved behind the vulva to single spirals. Cuticle appearing smooth, 4.5 ± 0.8 (3.5-6.0) μm thick, 11.1 ± 2.3 (8.5-13.5) μm thick at tail tip, and marked by very fine superficial transverse striate mainly in tail region. Lip region broadly rounded frontally and more so laterally, separated from body contour by slight depression. However, lip region truncate, slightly concave anteriorly and continuous with body contour shape, observed in some female specimens. Amphidial fovea pocket-shaped symmetrically bilobed, with lobes of about equal length, occupying more of 2/3 part of distance between oral aperture and guiding-ring. Labial papillae prominent. Stylet guiding-ring single, located 2.5 ± 0.3 (2.1-3.0) times lip region diam. from anterior end. Lateral chord ca 25 μm wide at mid-body or one-fourth of corresponding body diam. Odontostyle moderate long and robust, usually straight, 1.9 ± 0.1 (1.6-2.29) times as long as odontophore; odontophore weakly developed, posterior slightly enlarged with rather weak basal swellings. Nerve ring encircling cylindrical part of pharynx, 11.3 ± 0.6 (10.  Male. Common, but less frequent (40%) than female. Morphologically similar to female except for genital system, but with posterior region slightly curved ventrally and longer tail. Male genital tract diorchic with testes opposed, containing multiple rows of different stages of spermatogonia. Tail rounded, dorsally convex conoid, ventrally slightly concave with broad blunt terminus and thickened outer cuticular layer. Spicules arcuate, robust, ca 2 times longer than tail length, lateral guiding pieces more or less straight or with curved proximal end. One pair of adanal supplements and 14-16 midventral supplements.
Juveniles. Morphologically similar to adults, but smaller. All four juvenile stages were found, being distinguishable by relative lengths of body and functional and replacement odontostyle (Table 7, Figs 4, 7 and 8; [66,67]). J1s were characterised by a conoid-rounded tail, curved dorsally and slightly concave ventrally with a dorsal-ventral depression at hyaline region level, c' ratio 1.5 (Table 7), an odontostyle length ca 58 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages.
Measurements, morphology and distribution. Morphometric variability is described in Table 7 and morphological traits in Figs 4, 7 and 8. Longidorus onubensis sp. nov. was only found in the type locality from the rhizosphere of cultivated olive ( Table 1, Fig 1).
Etymology. The species name refers to the habitat (silvestris, silvestre = sylvan, living in the wild forest), where the type specimens were collected.
Male. Extremely rare, only one male specimen was found. Morphologically similar to female except for genital system and posterior region slightly curved ventrally Tail convexconoid, ventrally slightly concave with broad blunt terminus and the thickened outer cuticular layer. Male genital tract diorchic with test opposed, containing multiple rows of different stages of spermatogonia. Spicules arcuate, robust, about 2 times longer than tail length, lateral guiding pieces more or less straight. One pair of adanal supplements preceded by a row of 10 ventromedian supplements.
Juveniles. Morphologically similar to adults in most respects except for size and development reproductive system. All juvenile developmental stages were detected and distinguished by relative lengths of body and functional and replacement odontostyle ( Table 8, Figs 4 and 10; [66,67]), and the genital primordium. J1s characterised by a conoid-rounded tail, slightly curved dorsally and dorsal-ventral depression at hyaline region level, subdigitate (Fig 10), with a c´ratio ca 2.5, odontostyle length ca 49 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages. However, morphology in second-, third-and fourthstages (except for undeveloped genital structures) similar to that of female, including broadly conoid to hemispherical tail shape with rounded terminus, which becoming progressively shorter and stouter in each moult and shorter distance from anterior end to guiding-ring in each moult (Fig 10).
Measurements, morphology and distribution. Morphometric variability is described in Table 8 and morphological traits in Figs 9 and 10. Longidorus silvestris sp. nov. was only found in type locality from the rhizosphere of wild olive (Table 1, Fig 1).
Male. Not found.
Juveniles. Morphometrics obtained from juvenile specimens, and of the relative lengths of body, tail, and functional and replacement odontostyle, confirmed the presence of four juvenile stages (Table 9, Figs 4 and 12; [66,67]). J1s were characterised by a conoid tail, dorso-ventrally curved with rounded terminus, and slightly depression at hyaline region level, c' ratio 2.3 (Table 9); an odontostyle length ca 53 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages.
Measurements, morphology and distribution. Morphometric variability is described in Table 9 and morphological traits in Figs 4, 11 and 12. In addition to the type locality, L. vallensis sp. nov. was found from one cultivated olive sample located in Córdoba province (Table 1, Fig 1).
Juveniles. Morphologically similar to adults, but smaller. All four juvenile stages were found, being distinguishable by relative lengths of body and functional and replacement odontostyle (Table 10, Figs 4 and 14; [66,67]). J1s were characterised by a conoid tail, dorso-ventrally curved with rounded terminus, and slightly depression at tip tail level, c' ratio 1.7 (Table 10); an odontostyle length ca 52 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages.
Measurements, morphology and distribution. Morphometric variability is described in Table 10 and morphological traits in Figs 4,13 and 14. In addition to the type locality, L. wicuolea sp. nov. was extracted from one wild olive sample located in Huelva province, being distributed only in Western Andalusia (Table 1, Fig 1).
Relationships. On the basis of body and odontostyle length, distance between guidingring from anterior body end, a, c and c´ratios, amphidial fovea, or female tail shape, L. wicuolea sp. nov. is very closely related to L. silvestris sp. nov. from which it can be differentiated by a combination of these characters, but particularly in lip region shape (separated from body contour by slight depression vs anteriorly rounded continuous), and J1 tail shape (conoid vs conoid-subdigitate) (Figs 9, 10, 13 and 14). In addition, according to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], and on the basis of sorting on matrix codes A (odontostyle length), B (lip region width), C (distance of guiding-ring from anterior body end), D (shape of anterior region), F (body length), H (tail shape) and I (presence/absence of males), L. wicuolea sp. nov. can be related with L. henanus Xu & Cheng, 1992 [79] and L. vallensis sp. nov. From L. henanus it differs mainly in having a longer body and tail length (6.1-8.  Table 5]. Intraspecific variation of D2-D3 segments detected between the two studied populations of L. wicuolea sp. nov. consisted of 6 nucleotides (99% similarity), and no indels. Similarly, the ITS1 (KT308887-KT308889) also showed a low intraspecific variability between the two studied populations with only 4 nucleotides (99% similarity). The closet ITS1 to that of L. wicuolea sp. nov. was L. silvestris sp. nov.

Morphology and morphometrics of known Longidorus species
Morphological and morphometrical data as well as molecular delineation (rDNA) of L. alvegus, L. intermedius, L. magnus, L. oleae and L. vineacola have been previously recorded within studies of dagger and needle nematodes infesting vineyards in southern Spain [19,28]. The new records of these species from wild and cultivated olive in Granada and Sevilla provinces presented here extend the geographical distribution of these species (S1 and S2 Tables) in southern Spain [19]. Consequently, only D2-D3 sequences had been reported here for these samples. For other known species studied, representing the first molecular characterization and new records for olive or for Spain (viz. L. lusitanicus and L. vinearum), a brief description and a morphometric comparison with previous records and paratypes is provided below (S1-S3 Figs, S1 and S2 Tables).
Longidorus lusitanicus Macara 1985. The gonochoristic population of Longidorus from wild olive at Sanlúcar de Barrameda (Cádiz province) agrees fairly well with studied paratypes and original description of L. lusitanicus. This population was characterised by a lip region expanded or distinctly offset by constriction, rounded laterally and almost flattened frontally; amphidial fovea pouch-shaped, distinctly asymmetrically bilobed; female tail conoid-rounded; and the same proportion of male specimens found (S1 Table, S2 Fig). Morphometrics were coincident with those provided in the original description, except for only minor differences in oral aperture-guiding ring distance, which may be due to few specimens originally studied, or geographical intraspecific variability [45]. This is the first report for Spain and confirms a wider distribution in the Iberian Peninsula, apart from original description in Portugal. According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], this species has the following code: A3 B34 C23 D4 E3 F234 G2 H1 I2.
Longidorus vinearum Bravo & Roca, 1995. The four gonochoristic populations of L. vinearum from wild olive at Santa María de Trassierra (Córdoba province) agree fairly well with studied paratypes and original description of L. vinearum. The four studied populations were characterised by a robust and long body, lip region anteriorly rounded and separated from body contour by a very slight depression; amphidial fovea pouch-shaped, distinctly asymmetrically bilobed; female tail short, bluntly rounded to hemispherical with rounded terminus; and the common presence of male specimens (S2 Table, S3 Fig). Morphometrics of female, male and J1 specimens were coincident with those provided in the original description and rather similar to data reported subsequently for other populations of Portugal, except for minor differences in a ratio and length of spicules, which may be due to few specimens originally studied or geographical intraspecific variability [46,73]. This is the first report for Spain and confirms a wider distribution in the Iberian Peninsula, apart from original description and other populations in Portugal. According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], this species has the following code: A45 B345 C34 D2 E3 F345 G12 H1 I2.
Phylogenetic relationships of the Longidorus spp.
Phylogenetic relationships among Longidorus species inferred from analyses of D2-D3 expansion segments of 28S, ITS1, and the partial 18S rDNA gene sequences using BI are given in Figs 15, 16 and 17, respectively. To facilitate discussion, clades that were well supported or are taxonomically well founded are labelled in roman numerals from I through VII (Fig 15).
Poorly supported lineages are not explicitly labelled. The 50% majority rule consensus 28S rRNA gene BI tree of Longidorus and Paralongidorus spp. based in a multiple edited alignment including 133 sequences and 748 total characters consisted of six moderate to highly supported major clades in the genus (Fig 15). Clade I is well-supported (PP = 100%) comprising 16

Discussion
The primary objective of this study was to unravel the biodiversity, distribution and molecular phylogeny of needle nematodes of the genus Longidorus associated with wild and cultivated olives in southern Spain. This was conducted in an extensive and systematic nematological survey that included 159 locations and 449 sampling sites. We found 40 Spanish populations of Longidorus spp. infesting olive soils. Our results demonstrate that the use of morphological studies together with rDNA molecular markers may decipher the specific biodiversity in this complex group of plant-parasitic nematodes. We described here six new Longidorus species, based on integrative taxonomy and the phylogenetic relationships of the genus Longidorus based on nuclear rDNA markers.
The comparative morphological and morphometrical study of the 40 Spanish populations of Longidorus spp. confirmed that diagnosis and identification of these species based solely on diagnostic morphometric features is quite complex since there is almost a continuous range of character measurements within populations as well as among species [8,19]. The present results (including new and known species) enlarge the biodiversity of Longidorus in the Iberian Peninsula and agree with previous data obtained for the phylogeny and biogeography of the genus Longidorus in the Euro-Mediterranean region [19,28,82,83], in which a dispersalist model was one of the primary explanations for the large groups of Longidorus species found in this region.
Considering the species richness of PPN associated with olive in different studies, the genus Longidorus is one of the most biodiverse with nine species (viz. L. africanus, L. belloi, L. closelongatus, L. cretensis, L. elongatus, L. macrosoma, L. oleae, L. pseudoelongatus, L. siddiqii, and L. vinearum) reported in several countries of the Mediterranean Basin such as Egypt, Greece, Jordan, Portugal and Spain [19,35,36,73,77,84,85,86,87,88,89]. Although all Longidorus spp. are obligate soil plant ecto-parasites of a wide range of wild and cultivated plants causing enlarged swellings of root tips, it is unlikely that these species could be detected in other wild and cultivated plants in the next future. The present results double the previous biodiversity of Longidorus species detected in olive worldwide, including six new species and two new records for wild and cultivated olives (L. alvegus and L. vineacola), as well as two additional new records for wild olives (L. intermedius and L. lusitanicus). The most recent major geological event having important effects for nematode biodiversity and distribution in Europe was the Quaternary glaciation which happened ca. 40,000 years ago. In Europe has been hypothesized that reduced species numbers in northern Europe is attributed to Quaternary glaciations, being the highly diverse nematofauna of the Mediterranean basin related to Miocene plate tectonics in that area [90]. Our study showed a great diversity in Southern Spain. However, because of no sampling North-South has been developed; more intensive studies are needed in northern areas in order to corroborate this hypothesis. The distribution of the 40 Longidorus populations collected in Andalusia showed that some of them revealed a certain geographic associations to western areas (viz. L. alvegus, L. intermedius, L. lusitanicus, L. onubensis sp. nov., L. vineacola, L. vinearum, L. wicuolea sp. nov.) and eastern regions (viz. L. indalus sp. nov.), while only L. magnus was detected in both areas (Fig 1). The present findings showed certain coincidences with the quantitative analysis of Longidorus spp. distribution carried out by Navas et al. [82], who recognized two main groups of species, the European-Atlantic and the Mediterranean. The widespread distribution of L. magnus may suggest a high ecological flexibility e.g. adaptability to a range of soil types, and reproduction sustained over a broad temperature range [91]. While other species seems to be better adapted to drier areas as it is the case for L. indalus sp. nov. in Eastern regions with markedly lower precipitation. Species showing a restricted distribution may be the result of isolation of populations in diverse biotopes which would result in reproductive isolation and hence the establishment of new species [91]. Also, although agricultural activities may result in the widespread dissemination of Longidorus species [91], the geographical distribution of Longidorus species in wild and cultivated olives in southern Spain suggest an established pattern related to ecological factors, on a geological timescale. These nematodes could have a lower dissemination by human activities than other plantparasitic nematodes (i.e. cyst-or root-lesion nematodes) because of their sensitivity to fast desiccation, large body size, and the absence of survival-resistance forms. Unfortunately, little is known about the ecological requirements of Longidorus nematodes and elucidation of speciation and species biodiversity has currently to be approached on the groupings of morphometric characters [91]. Consequently, further research is needed in order to determine the influence of physico-chemical soil factors on the incidence and distribution of these nematodes in southern Spain and other wider areas.
Sequences of nuclear ribosomal RNA genes, particularly D2-D3 and ITS1, have proven to be a powerful tool for providing accurate species identification of Longidoridae [22,25,92]. However D2-D3 expansion region was more useful for establishing phylogenetic relationships among Longidorus species than ITS1 or 18S. The great diversity detected in the ITS1 suggests that a variety of poorly understood factors are involved in the fast evolution of this region in nematodes. Thus, ITS1 appears better suited for differentiation of species than for phylogenetic relationships within Longidorus. Our findings also confirm that partial 18S sequence does not have enough resolution to distinguish species, because different species showed a low nucleotide differences amongst them. Phylogenetic analyses based on D2-D3, ITS1, and partial 18S using BI resulted in a congruent position for the newly sequenced species of Longidorus spp. from Spain, which grouped in a separate clade, except for L. vallensis sp. nov. (KT308861-KT308862) and L. indalus sp. nov. (KT308852-KT308854) in the D2-D3, partial 18S, and ITS1 trees, which grouped separately (Figs 15, 16 and 17). Longidorus vallensis sp. nov. clustered in all ribosomal markers with L. rubi. However these species showed several morphological differences that made it difficult to establish a correspondence between morphological characters and the phylogenetic trees inferred from the molecular data. The majority of the species showed congruence in the phylogenetic relationships within these ribosomal markers using the DNA from the same individual. However L. indalus sp. nov. phylogenetic position was not congruent amongst the different ribosomal markers used here. This could be a result of different mutation rates within the different ribosomal markers, or difficulties in sequence alignment in ITS1 sequences. The phylogenetic relationships inferred in this study based on the D2-D3 and ITS1 sequences mostly agree with the lineages obtained by other authors [19,25,28,93,94]. Most of the newly and known described species in this research (viz. L. lusitanicus, L. macrodorus sp. nov., L. magnus, L. oleae, L. onubensis, L. silvestris sp. nov., L. vineacola, L. vinearum, L. wicuolea sp. nov.) grouped genetically in the same clade. These species shared a long body and odontostyle and can be considered as the most evolved species in the genus [14]. These traits could be related to the feeding habits of these nematodes, since longer stylets are better adapted to penetrate major woody plants roots persisting during the hot-dry summer conditions prevalent in Southern Spain and with long body sizes to move quickly deeper in the soil to avoid dry conditions in summer.
To confirm the correlation of the results obtained by conventional morphological approaches and new molecular methods is important for the proper understanding of the evolution of the genus Longidorus. The close relationship of the morpho-species groups detected in this and previous studies in Spain was also supported by molecular data (most of the species described were in the same clade), an observation that points to the Iberian Peninsula as a possible center of recent speciation [19], as it was suggested for other genera such as Xiphinema [5,19,22,28], Trichodorus [95] or Rotylenchus species [11].

Conclusions
In summary, the present study establishes the importance of using integrative taxonomic identification highlighting the difficulty of a correct identification at species level within the genus Longidorus. This study also provides molecular markers for precise and unequivocal diagnosis of some species of Longidorus in order to differentiate virus vector or quarantine species. This and previous studies demonstrate that the genus Longidorus is clearly a complex group and much work remains to be done to elucidate species boundaries in this economically important group of PPN. Furthermore, similar intensive and extensive integrative studies on Longidorus species in several wider areas may help to elucidate if Longidorus species have originated in Southeast Africa and India, when these two areas were still united, and a later spread to Laurasia was followed by a main speciation of Longidorus in the Holarctic region, especially Europe, as hypothesised by Coomans [14]. This hypothesis is reinforced with the basal position of Asian species in D2-D3 region and partial 18S phylogenetic trees.