Remarkable Diversity and Prevalence of Dagger Nematodes of the Genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) in Olives Revealed by Integrative Approaches

The genus Xiphinema includes a remarkable group of invertebrates of the phylum Nematoda comprising ectoparasitic animals of many wild and cultivated plants. Damage is caused by direct feeding on root cells and by vectoring nepoviruses that cause diseases on several crops. Precise identification of Xiphinema species is critical for launching appropriate control measures. We make available the first detailed information on the diversity and distribution of Xiphinema species infesting wild and cultivated olive in a wide-region in southern Spain that included 211 locations from which 453 sampling sites were analyzed. The present study identified thirty-two Xiphinema spp. in the rhizosphere of olive trees, ten species belonging to Xiphinema americanum-group, whereas twenty-two were attributed to Xiphinema non-americanum-group. These results increase our current knowledge on the biodiversity of Xiphinema species identified in olives and include the description of four new species (Xiphinema andalusiense sp. nov., Xiphinema celtiense sp. nov., Xiphinema iznajarense sp. nov., and Xiphinema mengibarense sp. nov.), and two new records for cultivate olives (X. cadavalense and X. conurum). We also found evidence of remarkable prevalence of Xiphinema spp. in olive trees, viz. 85.0% (385 out of 453 sampling sites), and they were widely distributed in both wild and cultivated olives, with 26 and 17 Xiphinema spp., respectively. Diversity indexes (Richness, Hill´s diversity, Hill´s reciprocal of D and Hill´s evenness) were significantly affected by olive type. We also developed a comparative morphological and morphometrical study together with molecular data from three nuclear ribosomal RNA genes (D2-D3 expansion segments of 28S, ITS1, and partial 18S). Molecular characterization and phylogenetic analyses allowed the delimitation and discrimination of four new species of the genus described herein and three known species. Phylogenetic analyses of Xiphinema spp. resulted in a general consensus of these species groups. This study is the most complete phylogenetic analysis for Xiphinema non-americanum-group species to date.


Introduction
Soil is most likely one of the more species-rich habitats of terrestrial ecosystems because over one quarter of all living species on Earth are inhabiting the soil [1,2]. One of the most diverse soil animals are nematodes although they are ubiquitous in all habitats that provide available organic carbon sources [3]. The phylum Nematoda includes species either free-living or parasites of animals or plants. Plant-parasitic nematodes (PPN) comprising about 15% of the total number of nematode species currently known, of which over 4,100 species have been identified as PPN [4,5]. The fact that new species of PPN are continually being described, combined with PPN gross morphology tends to be highly conserved, likewise the limitations of species concepts, results in an increase of the difficulty in the species identification [6][7][8][9][10][11][12][13]. However, accurate identification of PPN is essential for the selection of appropriate control measures against plant pathogenic species, as well as for a reliable method allowing distinction between species under quarantine or regulatory strategies and a better understanding of their implications in pest control and soil ecology [14]. Integrative taxonomy has been efficiently applied for the accurate diagnostic and identification over the wide range of PPN species [9,[11][12][13][15][16][17][18].
The most important nematodes economically include endoparasitic species such as the root-knot (Meloidogyne spp.) and cyst nematodes (Heterodera spp. and Globodera spp.), likewise the ectoparasitic nematodes belonging to the family Longidoridae Thorne, 1935 [19]. Dagger nematodes of the genus Xiphinema Cobb, 1913 [20] are one of the highest diversified group species of this family [21]. The phytopathological importance of this group of nematodes not only lies in its wide range of host and cosmopolitan distribution but some species of this genus are vectors of several important plant viruses (genus Nepovirus, family Comoviridae) that cause significant damage to a wide range of crops [21][22][23][24][25][26]. Considering the great morphological diversity, the genus Xiphinema was divided into two different species groups [14,22,27]: i) the Xiphinema americanum-group comprising a complex of about 60 species [22,28]; and ii) the Xiphinema non-americanum-group which comprises a complex of more than 215 species [14,17,18]. Species discrimination in Xiphinema is based mainly on classical diagnostic features; however, due to a high degree of intraspecific morphometric variability can lead to overlapping among Xiphinema species and increase the risk of species miss-identification [27][28][29].
Olive, in wild and cultivated forms, is widely distributed in the Mediterranean Basin, and particularly in southern Spain [13,[39][40][41]. Wild and cultivated olives are hosts and damaged by PPN, including dagger nematodes (Xiphinema spp.) [34,42]. However, little information is available about Xiphinema spp. associated with olive trees, except for the recent contributions of Archidona-Yuste et al. [13,18,28] reporting new species such as Xiphinema macrodora  [13,18,28]. Therefore, with the aim of deciphering the biodiversity of Xiphinema spp. infecting wild and cultivated olives in southern Spain, we surveyed a total of 211 localities at the eight provinces of Andalusia where both olive forms were present. This survey raised 385 populations of Xiphinema species, apparently morphologically related to other known Xiphinema spp. This prompted us to carry out an integrative taxonomic study to identify the species within this complex genus.
The general objectives of this research was to study the occurrence and abundance of Xiphinema species and to test the resemblance between morphological and molecular data within Xiphinema species, and the specific objectives were: i) to identify the 385 Spanish populations of Xiphinema spp. detected in wild and cultivate olives; ii) to carry out a molecular characterisation of these Xiphinema 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 Xiphinema 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 growing in Andalusia, southern Spain (Table 1, Fig 1). Soil samples were collected for nematode analysis with a shovel from four to five trees randomly selected in each sampling site. A total of 115 and 338 sampling sites from wild and cultivated olives, respectively, were arbitrarily chosen in the eight provinces of Andalusia where both olive subspecies 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 and analyzed as described by Archidona-Yuste et al. [13].
Nematodes were extracted from a 500-cm 3 sub-sample of soil by a modification of Cobb´s decanting and sieving method [43]. Since recovery nematode effectiveness is highest in Cobb´s decanting and sieving method [43,44], these data were used for prevalence and abundance data analyses. In some samples in which new taxa were detected and more specimens were required for suitable descriptions, additional soil samples were extracted by centrifugal-flotation [45]. The nematode sample processing was carried out as described by Archidona-Yuste et al. [13]. PPN from soil samples were identified to genus, and then we focussed on the species delineation of dagger nematodes of the genus Xiphinema.

Diversity indexes
Based on the Xiphinema spp. populations detected infesting soils from olives in Andalusia, conventional ecological and diversity indexes were performed in order to evaluate the distribution and changes in the diversity in wild and cultivated olives. In this regard, abundance and prevalence of each Xiphinema species identified were estimated. For each sampling site, abundance was calculated as the mean number of Xiphinema nematodes per 500 cm 3 of soil for all samples. The prevalence was computed by dividing the number of samples in which a Xiphinema species was detected by the total number of samples and expressed as a percentage.
Several diversity indexes including Hill´s diversity, Hill´s reciprocal of D (Simpson´s dominance index) and Hill´s evenness indexes [46] were calculated according to code indications

Morphological studies
Xiphinema specimens for light microscopy were killed by gentle heat, fixed and examined Xiphinema specimens as described by Archidona-Yuste et al. and Seinhorst [13,50]. The morphometric study and drawing of each nematode population was carried out as described in previous papers [13,14,22,27,51]. All abbreviations used are as defined in Jairajpuri & Ahmad [51]. In addition, a comparative morphological and morphometrical study of type specimens of some species were conducted with specimens kindly provided by Dr.  Siddiqi, 1964 [53]. Nematode populations of Xiphinema species already described were analysed morphologically and molecularly in this study and proposed as standard and reference populations for each species given until topotype material becomes available and molecularly characterized. Voucher specimens of these described species have been deposited in the nematode collection of Institute for Sustainable Agriculture, IAS-CSIC, Córdoba, Spain.

DNA extraction, PCR and sequencing
For molecular analyses, in order to avoid mistakes in the case of mixed populations, 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 to the unidentified populations of Xiphinema. Following morphological confirmation, the specimens were removed from the slides and DNA extracted. Detailed protocols for nematode DNA extraction, PCR and sequencing were applied as described by Castillo et al. [54]. The D2-D3 expansion segments of 28S rRNA, ITS1 region, and the portion of the 18S-rRNA were amplified using primers described in previous papers [13,[55][56][57][58]. PCR products were purified and sequenced as described by Archidona-Yuste et al. [13]. 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 Xiphinema spp. from GenBank were used for phylogenetic reconstruction. Outgroup taxa for each dataset were chosen according to previous published data [13,17,18]. Multiple alignments of the different genes were made using the Q-INS-i algorithm of MAFFT v. 7.205 [59], strategy FFT-NS-1 with default parameters. Sequence alignments were visualized using BioEdit [60] and edited by Gblocks v0.91b [61] in Castresana Lab server (http://molevol.cmima.csic.es/ castresana/Gblocks_server.html) using the less stringent option (Minimum number of sequences for a conserved or a flanking position: 50% of the number of sequences + 1; maximum number of contiguous non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half). Percentage similarity between sequences was calculated using a sequence identity matrix in 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. When position of both sequences has a gap they do not contribute as a difference. Phylogenetic analyses of the sequence data sets were performed based on Bayesian inference (BI) using MrBayes 3.1.2 [62]. The best fitted model of DNA evolution was obtained using jModelTest v. 2.1.7 [63] 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 GTR+I+G (namely, general time reversible of invariable sites and a gamma-shaped distribution) model for D2-D3 expansion segments of 28S and ITS1 rRNA, and TIM3+I+G (namely, transversional and a transitional of invariable sites and a 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 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 [64].

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 LSIDs (Life Science Identifiers) 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:CE945C7D-7B14-46DD-8A17-A93A05750590. 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, prevalence and diversity indexes of Xiphinema spp. in olive All Xiphinema spp. found in this study including specimens of sampling sites used in morphological and/or molecular analyses, are shown in Table 1. In addition, all positive Xiphinema spp. and sampling sites are presented in Fig 1. Overall, 32 Xiphinema spp. were detected in the rhizosphere of olive trees, ten species belonging to X. americanum-group, whereas 22 were attributed to X. non-americanum-group (Table 2). From all Xiphinema spp. identified in this study, 26 species were associated with wild olive, whereas seventeen Xiphinema species were associated with cultivated olive (Table 1; Fig 1). Eleven Xiphinema species occurred in both wild and cultivated olives (viz. X. adenohystherum Lamberti et al., 1992 [65], X. coxi europaeum Tarjan, 1964 [66], X. duriense Lamberti et al., 1993 [67], X. incertum Lamberti et al., 1983 [68], Xiphinema spp. were present in low to high densities (ca 33, from 1 to 414 nematodes per 500 cm 3 of soil) in both wild and cultivated olives, being observed in cultivated olives in higher densities than in wild olives ( Table 2 and S1 Table). Nematode abundance of X. americanumgroup species was significantly higher (P < 0.01) in cultivated than wild olives (Fig 2B), averaging ca 23 vs 43 nematodes per 500 cm 3 of soil for wild and cultivated olives, respectively. On the contrary, nematode density was similar (P > 0.05) in both olive types in the Xiphinema non-americanum-group (Fig 2C), being slightly higher in wild than cultivated olives. In general, Xiphinema spp. belonging to X. americanum-group showed higher densities than species identified within X. non-americanum-group (ca 38 vs 22 nematodes per 500 cm 3 of soil, respectively) ( Table 2 and S1 Table), which resulted in a higher abundance (P < 0.001) for X. americanum group than X. non-americanum-group species (Fig 2D). On the other hand, the Xiphinema species with the highest nematode density was X. pachtaicum (414 nematodes per 500 cm 3 of soil), which showed a higher average density in cultivated than wild olives (Table 2 and S1 Table). However, the subsequent species with high nematode density included X. italiae and X. nuragicum (350 and 218 nematodes per 500 cm 3 of soil, respectively), both belonging to X. non-americanum-group, showing lower average density in cultivated than in wild olives ( Table 2 and S1 Table).
The overall prevalence of Xiphinema spp. in olive was 85.0% (385 out of 453 sample sites) in Andalusia (Fig 1, Table 2 and S1 Table). However, Xiphinema spp. were more prevalent in wild olives (93.9%, 108 out of 115 sampling sites) than cultivated olives (81.7%, 276 out of 338 sampling sites) ( Table 2). In addition, the major differences between both olive types occurred in the Xiphinema non-americanum-group species, being more prevalent in wild than cultivated olives. Nevertheless, prevalence in X. americanum-group species was similar between both olive types ( Table 2). As indicated above for most of the Xiphinema spp. identified in this study, the prevalence was higher in wild than cultivated olive except for X. pachtaicum that was detected in both wild and cultivated olives in all provinces of Andalusia, and being the most prevalent Xiphinema species in our study (74.2%, 336 out of 453 sample sites) ( Table 2 and S1 Table). The subsequent species with a high prevalence was X. nuragicum (16.3%, 74 out of 453 sample sites) that was detected in both olive types in the most of the Andalusia provinces, at exception of Almería (Fig 1, Table 1 and S1 Table). Another prevalent Xiphinema species belonging also to X. non-americanum-group was X. italiae (10.2%, 46 out of 453 sample sites), that was found in both olive types in Almería, Cádiz, Huelva and Málaga provinces, but only in wild olive in Córdoba, Granada, Jaén and Seville provinces (Fig 1, Table 2 and S1 Table).
Several diversity indexes were estimated in our study (Richness, Hill´s diversity, Hill´s reciprocal of D (Simpson´s dominance index), and Hill´s evenness [46]), and tested for differences associated with presence of Xiphinema spp. in wild and cultivated olive (Fig 2). Overall, the number of Xiphinema spp. detected in each sampling site (Richness index) was significantly affected (P < 0.05) by olive type (Fig 2), showing higher values (P < 0.001) in wild than cultivated olives (Fig 2A). Similarly, Richness index in X. non-americanum-group species were significantly higher (P < 0.05) in wild than in cultivated olive ( Fig 2C), but the opposite occurred in the X. americanum-group species (Fig 2B). Overall, the Richness index was significantly higher (P < 0.001) in X. americanum-group than in X. non-americanum-group (Fig 2D). Diversity and dominance diversity indexes (Hill´s 1 and Hill´s 2, respectively) showed similar patterns for both olive types (Fig 2). Thus, significant differences (P < 0.05) for both diversity indexes were observed when Xiphinema species groups were considered separately (Fig 2B and  2C). On the other hand, the detection of a higher number of species belonging to X. non-americanum-group linked to the increased presence of prevalent species (viz. X. italiae, X. nuragicum or X. coxi europaeum) than X. americanum-group (Tables 1 and 2) resulted in significant differences (P < 0.01) among them when it was considered both olive types ( Fig 2D). Evenness diversity showed an inverse trend to that observed in diversity and dominance diversity indexes, with cultivated olives showing higher values (P < 0.01) than that of wild olives (Fig 2A  and 2C) according to the higher abundance and prevalence (P < 0.05) detected in cultivated than wild olives ( Table 2 and S1 Table). On the other hand, Evenness index in X. americanumgroup was significantly higher (P < 0.001) than that of X. non-americanum-group species (Fig 2D).
Etymology. The species epithet refers to the autonomous community from Spain, Andalusia, where the species was detected and moderately distributed.
Description of taxa. Female. Body cylindrical and habitus in specimens killed by gentle heat as open C-shape, more curved behind the vulva position, with increasing curvature towards the posterior extremity. Cuticle 3.5-4.0 μm thick at mid-body, but thicker at tail tip, 4.5-8.0 μm wide. Lateral hypodermical chords 18.0-29.0 μm wide at mid body or 29-57% of the corresponding maximum body diameter. Lip region hemispherical, rounded laterally and less so frontally, almost continuous or separated from the body contour by a slightly depression, 12.5-15.5 μm diam. and 5.0-7.5 μm high. Amphidial fovea aperture extending for ca 76-88% of lip region diam. and located at ca two-thirds of lip region height. Odontostyle long, 1.6-1.9 times longer than odontophore, and the latter with moderate-developed flanges 9.    [74]. In some specimens studied the tip of reserve odontostyle (vestigium) was ca 3.5 5 μm in size and directed anteriorly to the isthmus. Cardia conoid, 6.5-14.5 μm long. Prerectum variable in length, 372-783 μm long or 10-19 times anal body diam. Rectum 35.5-47.0 μm long ending in anus as a small rounded slit. Reproductive system didelphic-amphidelphic with branches equally developed and vulva slit-like situated located slightly anterior to mid body. Each branch composed of a 109-212 μm long reflexed ovary and a largely tubular oviduct with enlarged pars dilatata oviductus separated from uterus by a welldeveloped sphincter. Uterus tripartite, comprising a developed pars dilatata uteri continuing into a narrower, muscular tube-like portion including a Z-differentiation with weakly muscularised wall and containing 11-16 globular bodies of variable size, each one consisting of a large central portion, irregularly spherical surrounded by a variable number of refractive pieces, and petal shaped (Figs 3 and 4). Low numbers of small spiniform structures and crystalloid bodies along uterus, observed in fresh material in water. Abundant wrinkles observed in uterine wall along uterus, which may be confused as spiniform structures. No sperm was observed in the female genital tract. Ovejector well-developed 41.5-60.0 μm wide, and vagina perpendicular to body axis, 19.5-33.5 μm long or 27-52% of corresponding body diam. in lateral view. In some specimens studied, maturate eggs observed in the tubular part of uterus, 156-183 μm long and 35-43 μm wide. Tail short, varying from convex-conoid to conoid shape with digitate or subdigitate terminus, directed ventrally with respect to the body axis. Distinct terminal blind canal, and in most of specimens studied three caudal pores present on each side.
Male. No detected. 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 ( Table 3, Fig 5) [75,76]. Specifically, J1 were characterised by position of replacement odontostyle just posterior to functional odontostyle, its tip touching or very close to base of functional odontostyle; tail elongate conoid with a slightly dorsal depression at hyaline region and c' ratio ! 3.5 (Figs 3 and 4); and odontostyle length ca 66 μm. Tail morphology in second-juvenile stage similar to J1, becoming shorter and stouter than this developmental stage. However, tail morphology in third-and fourth-juvenile stages (except for undeveloped genital structures) similar to that of female, including almost conoid tail shape ending in a digitate terminus (Fig 4), becoming progressively shorter and stouter in each moult, and shorter distance from anterior end to guiding-ring in each moult.
Measurements, morphology and distribution. Morphometric variability is described in Tables 3 and 4 and morphological traits in Figs 3, 4 and 5. In addition to the type locality, Xiphinema andalusiense sp. nov. was collected from the rhizosphere of wild olive (Olea europaea subsp. silvestris (Miller) Lehr) of two localities belonging to Córdoba and Jaén provinces, being one of the new species described in this work which has a broader distribution in Andalusia, concretely on North of Andalusia (Table 1, Fig 1).
Xiphinema spp.  Uterus tripartite composed of pars dilatata uteri followed by a tubular part containing in the proximal part a well-developed Z-differentiation with weakly muscularised wall, comprising 12-19 small granular bodies similar in size (Figs 6, 7F and 7G). Small crystalloid bodies similar in size and lower in number, mixed with abundant wrinkles from uterine wall, which may be confused as spiniform structures, distributed over the entire length of the tube-like portion of uterus (Figs 6 and 7). In some specimens studied and in a proximal part of pars dilatata uteri spindle shaped sperm cells were observed. Ovejector well-developed 46.0-61.5 μm wide, and vagina perpendicular to body axis, 20.0-29.5 μm long or 27-42% of corresponding body diam. in lateral view. Vulva slit-like, situated slightly posterior the mid-body region. Tail short, always shorter than anal body diam., varying in shape from hemispherical to convex-conoid with rounded terminus, and bearing two or three caudal pores present on each side.
Male. Extremely rare, only one male specimen was found in type locality. Male genital tract diorchic with testes containing multiple rows of different stages of spermatogonia. Tail short, convex-conoid with a broadly rounded terminus and thickened outer cuticular layer. Spicules moderately long and slightly curved ventrally; lateral guiding pieces more or less straight or with curved proximal end. One pair of adanal and 4 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 ( Table 6, Fig 5) [75,76]. Specifically, J1 were characterised by position of replacement odontostyle just posterior to functional odontostyle, its tip touching or very close to base of functional odontostyle; tail conical elongate, ending in a knob-like expansion, more or less developed, separated from the anterior part of the tail by a depression more or less marked, but giving to the tail a very characteristic profile (Figs 6 and 7); c' ratio ! 4.0; and odontostyle length ca 75 μm. Tail morphology of second-stage juvenile similar to J1 expect to absence of knob-like expansion, and tail conoid and subdigitate with rounded terminus for third-stage juvenile. In J4 tail conoid with a short bulge rounded terminus (Fig 7). All juvenile developmental stages with tail becoming progressively shorter and stouter in each moult, and shorter distance from anterior end to guiding-ring in each moult (Table 6, Fig 7).
Measurements, morphology and distribution. Morphometric variability is described in Table 6 and morphological traits in Figs 5, 6 and 7. In addition to the type locality, Xiphinema celtiense sp. nov. was found in the rhizosphere soil of wild olive (Olea europaea subsp. silvestris (Miller) Lehr) in one additional locality belonging to Córdoba province. (Table 1, Fig 1).
Etymology. The species epithet refers to the type locality, Iznájar, where the species was detected.  [74]. In some specimens studied, vestigium (tip of reserve odontostyle), 2.5 μm long, observed in anterior region of slender part of pharynx. Cardia conoid and variable in length, 11.5-22.0 μm long. Prerectum reaching about 10-15% of nematode body from the anus to anterior part. Rectum 29.5-38.0 μm long ending in anus as a small rounded slit. Reproductive system didelphic-amphidelphic with branches about equally developed. Each branch composed of a short ovary (63.5-122.0 μm long), a reflexed oviduct with welldeveloped pars dilatata oviductus separated from uterus by a well-developed sphincter. Uteri tripartite, comprising a developed pars dilatata uteri continuing into a narrower, muscular tube-like portion, and a well-developed Z-differentiation with weakly muscularised wall and containing numerous small granular bodies. Uterine wall wrinkles present along uterus, being more numerous in the proximal part of pars dilatata uteri and ovejector (Fig 9E). Small spiniform structures and crystalloid bodies present, in low number, along uterus and observed when tubular part of uterus is wider and without wrinkles (Figs 8 and 9G and 9H). In some specimens studied and in a proximal part of pars dilatata uteri, spindle-shaped sperm cells were observed, being variable in length (3.0-6.5 μm long). Ovejector well-developed 35.5-56.0 μm wide, vagina perpendicular to body axis, 18.0-24.0 μm long in lateral view. Vulva slitlike, pre-equatorial. Tail conoid and short, dorso-ventrally convex, ending in a rounded and broadly terminus, bearing in four to five pairs of caudal pores on each side.
Male. Frequent but less abundant than female (ratio = 1: 2). Morphologically similar to female except for genital system and more curved posterior part of body. Male genital tract diorchic with testes containing multiple rows of different stages of spermatogonia. Tail short, convex-conoid with short bulge rounded terminus and thickened outer cuticular layer (Figs 8,  9N and 9O). Spicules moderately long and slightly curved ventrally; lateral guiding pieces more or less straight or with curved proximal end. One pair of adanal and 4-5 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  (Table 7, Fig 5) [75,76]. In particular, J1 were characterised by position of replacement odontostyle just posterior to functional odontostyle, its tip touching or very close to base   of functional odontostyle; tail blunty conoid elongate with a c´ratio ! 3.8 (Figs 8 and 9Q); and odontostyle length ca 63 μm. Tail morphology in second-stage juvenile similar to J1 expect for the presence a slightly depression at the level of the hyaline region in both sides. On the other hand, the tail was conoid and subdigitate with a rounded terminus for J3, while for fourthstage juvenile was conoid with rounded terminus and short bulge (Fig 9T). All juvenile developmental stages showed a tail becoming progressively shorter and stouter in each moult, and shorter distance from anterior end to guiding-ring in each moult (Table 7, Fig 9Q-9T). Measurements, morphology and distribution. Morphometric variability is described in Table 7 and morphological traits in Figs 5, 8 and 9. Xiphinema iznajarense sp. nov. was only found in type locality, Iznájar (Córdoba province), being extracted from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) (Table 1, Fig 1).
In addition, X. iznajarense sp. nov. is molecularly related to X. hispidum Roca & Bravo, 1994 [78] and X. adenohystherum, but it can be clearly differentiated by a combination of characters discussed below. From X. hispidum it can be differentiated by higher c ratio (119.  (Figs 8 and 9; [31,78]). And from X. adenohystherum it clearly differs in having the presence vs absence of Zdifferentiation containing numerous granular bodies, and presence vs absence of crystalloid bodies in the tubular portion of uterus (Figs 8 and 9; [17,65]).
Male. Functional, less abundant than females (ratio = 1: 2). Reproductive system diorchic with testes occupying 45-57% of body length, and spindle-shaped sperm. Spicules dorylaimoid, massive, well sclerotised, 57.5-66.0 μm long, ventrally curved with tubular lateral guiding pieces 13.5-18.0 μm long. One pair of adanal supplements located at 16.6 ± 1.2 (15.5-19.0) μm from cloacal opening and a series of four to five ventromedian supplements. Tail similar to that of female, dorsally more convex than female, and ending in a rounded terminus with short bulge.
Juveniles. All four juvenile stages were found and detected using body length, length of replacement and functional odontostyle ( Table 8, Figs 5 and 11) [75,76]. J1 were characterised by position of replacement odontostyle just posterior to functional odontostyle, its tip touching or very close to base of functional odontostyle; tail elongate, dorsally convex and ventrally concave with a slightly dorsal depression at hyaline region with a c' ratio ! 3.1 (Figs 10 and 11Q); odontostyle length ca 53 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages. Tail morphology in second and third juvenile stages similar to J1, becoming progressively shorter and stouter in each progressively moult. However, tail shape in fourthstage similar into that of female, broadly dorsally convex-conoid with slightly bulging rounded terminus (Fig 11Q and 11T).
Xiphinema cadavalense Bravo & Roca, 1995. The amphimictic population of Xiphinema collected from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) at Espiel (Córdoba province) corresponds fairly well with studied paratypes and original description of X. cadavalense. This population was characterised by a long body; lip region hemispherical, rounded both anteriorly and laterally and set off from body contour by slightly depression; long odontostyle and odontophore; reproductive system didelphic-amphidelphic with both branches about equally developed with a well-developed Z-differentiation with weakly muscularised wall and comprising 9-15 sclerotized bodies of variable size and petal shape, each one consisting of a large portion, irregularly spherical surrounded by a variable number of refractive pieces; spiniform structures and crystalloid bodies in very small size and low number present along the narrower and muscular tube-like of uterus; tail dorsally convex-conoid (dorsally convex and ventrally almost convex or slightly straight) ending in a terminal peg with blind canal (Fig 12 and Table 9). The observations on the general morphology nematode indicate that this Xiphinema population belongs to the X. non-americanum Group 5 in Loof and Luc [27], which agrees with the original description of X. cadavalense [52]. In addition, female morphometrics fit with those provided in the original description, except in having slightly longer body and odontostyle length (5.  [52]. These differences may be due to geographical intraspecific variability. Up to our knowledge, 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 of Loof & Luc [27], this Spanish population of X. cadavalense has the following specific alphanumeric codes (codes in parentheses are exceptions): A4-B2+3-C5a-D65-E56-F5-G4-H2-I3-J-K-L1.
Xiphinema conurum Siddiqi, 1964. The Spanish population of this species from the rhizosphere of olive was characterised by a lip region rounded offset from the rest of the body by a conspicuous depression, two equally developed female genital branches, vulva slightly anterior to mid-body, uterus with uterine differentiation, presence of Z-differentiation with small granular bodies plus small spines (in low number), female tail conical, ventral profile nearly straight, dorsal profile regularly curved with rounded terminus (Fig 13). The morphology and morphometric of this population agree closely with the original description and redescription of the species by Siddiqi [53] and Luc & Aubert [91], likewise recently examined specimens from Soukra, Tunisia by Guesmi-Mzoughi et al. [92]. Up to our knowledge, this is the first report of this species for Spain. D2-D3 sequence for X. conurum (KX244902) matched well, 99% similar with former sequences from Tunisia deposited in GenBank (KX062671-KX062673); and ITS1 (KX244934) was 95-96% similar with former sequences from Tunisia deposited in GenBank (KX062696-KX062697). And partial 18S (KX244947) was provided for the first time in this research, being 99% similar to several Xiphinema spp. such as X. nuragicum (GU725081) or X. israeliae Luc, Brown & Cohn, 1982 [93] (KJ802900), extending the molecular diversity of this species to newly studied area.
Xiphinema pseudocoxi Sturhan, 1984. The amphimictic population of Xiphinema collected from the rhizosphere of wild olive (Olea europaea subsp. silvestris (Miller) Lehr) at Alcaracejos (Córdoba province) agrees fairly well with original description of X. pseudocoxi. This population was characterised by a moderately long body in an open C-shaped after fixation; lip region distinct from the body contour by a depression, frontally rounded; female reproductive system didelphic-amphidelphic having both branches about equally developed; Z-differentiation with weakly muscularised wall formed by 6-10 globular bodies similar in size, and irregularly spherical surrounded by a variable number of refractive pieces; no spiniform structures and, crystalloid bodies nor sperm cells observed along uterus; female tail convex-conoid, varying slightly in shape, and ending in a terminal peg with a blind canal (Fig 14, Table 9). Based on the morphological character observations we confirm that this Xiphinema population belongs to the X. nonamericanum Group 5 in Loof and Luc [27], which is in agreement with the original description of X. pseudocoxi [87]. Additionally, female morphometrics fit with those provided in the original description and rather similar to data reported subsequently for other populations of Spain and Portugal, except for minor differences in nematode body and odontostyle length, which may be due to few specimens originally studied or geographical intraspecific variability [87,94,95]. This new Spanish population extends the species distribution in Europe, and confirms a wider distribution in the Iberian Peninsula, apart from other populations from Spain, Portugal, and original description in Germany. According to the polytomous key of Loof & Luc [27], the new Spanish population of X. pseudocoxi has the following specific alphanumeric codes (codes in parentheses are exceptions): A4-B2-C5a-D45-E4(5)-F4(5)-G2-H2-I3-J-K-L1.
Difficulties were experienced with alignment of the ITS1 sequences due to scarce similarity, thus, only related sequences were used. The alignment generated for the 45 sequences of ITS1, comprising several X. non-americanum-group species, was 1113 characters after discarding ambiguously aligned regions from the alignment. Two new accessions were used as outgroup, X. duriense (KX244935) and X. opisthohysterum (KX244938). The 50% majority rule consensus BI tree of X. non-americanum-group spp. showed two major clades (PP = 1.00) similar to those obtained for D2-D3 region (Fig 16). Clade I was formed by twelve Xiphinema species

Discussion
This study aimed to get knowledge and a better understanding on the occurrence, abundance and biodiversity of dagger nematodes of the genus Xiphinema associated with wild and cultivated olives in southern Spain, as well as their distribution and molecular phylogeny. This was conducted in an extensive and systematic nematological survey that included 211 locations and 453 sampling sites. We found 385 Spanish populations of Xiphinema spp. infesting olive soils. We described four new Xiphinema species, enlarging the diversity of Xiphinema species in the Iberian Peninsula which is in agreement with previous data obtained for the phylogeny and biogeography of the genus Xiphinema and Longidorus in the Euro-Mediterranean region [13,17,18,28,31,100,101]. To the date, to our knowledge, this work is the largest phylogenetic analysis of the genus Xiphinema based on nuclear rDNA markers.
The genus Xiphinema is one of the most diverse PPN associated with olive, with twenty species (viz. X. aequum Roca & Lamberti, 1988  italiae, X. israeliae, X. lusitanicum Sturhan, 1983 [105], X. macroacanthum Lamberti, Roca & Agostinelli, 1990 [106], X. macrodora, X. madeirense, X. nuragicum, X. oleae, X. opisthohysterum, X. pachtaicum, X. parapachydermum, X. plesiopachtaicum, X. rivesi, X. sahelense Dalmasso, 1969 [82], X. turcicum, X. vallense, X. vuittenezi and several unidentified species) reported in various countries of the Mediterranean Basin [18,28,33,34,42,80]. The present results increase the previous data about diversity of Xiphinema species detected in olive worldwide, including four new species from the X. non-americanum-group. All these species were new records for olive with the exception of X. pachtaicum, X. index, X. italiae, X. nuragicum and X. turcicum [34]. In addition to the remarkable prevalence of Xiphinema spp. observed in both olive types, our study showed a great species diversity, that was mainly associated with the X. non-americanum-group species (P < 0.05, Fig 2D), being widely distributed in Andalusia but in particular mainly associated with wild olive in Cádiz province, a more humid and ecologically diverse area than the rest of the Andalusian provinces. However, X. pachtaicum was present in the majority of the sampled localities in wild and cultivated olives showing the plasticity of this species for a wide diversity of ecological conditions (Fig 1). Overall, X. pachtaicum was detected in 74.2% of the total sampling sites, specifically 67 out of 115 and 268 out of 338 associated with wild and cultivated olive, respectively. As reported in previous studies, this species is widespread in the Mediterranean Basin [16,23,28,31,33,92,107,108], including olive [9,109,110]. In Spain, X. pachtaicum was also the most prevalent dagger nematode in vineyards and stone-fruit orchards [31,111]. The widespread distribution of X. pachtaicum may suggest also adaptability to a range of soil types, and reproduction sustained over a broad range of temperatures [112,113]. Nevertheless, these wider ecological requirements are difficult to explain regarding their low genetic diversity [108] and could be more associated with the presence of specific ovarial-intestine endosymbionts [114], but some of the other species from the X. americanum-group also possesses ovarial-intestine endosymbionts and were more restricted to some areas (viz. X. opisthohysterum, X. santos, X. incertum, X. madeirense, X. vallense, X. plesiopachtaicum and X. rivesi) [114]. Other species with a broad distribution were included in the X. non-americanum-group, i.e. X. italiae found in all provinces, X. nuragicum in 7 out of 8 provinces, and X. coxi europaeum in 5 out of 8 provinces. In this sense, the presence of a high number of frequent species belonging to X. non-americanum-group (i.e. X. italiae, X. nuragicum, X. coxi europaeum or to a lesser extent X. adenohystherum) explains the higher value observed in Hill´s 2 (Dominance diversity) index with respect to X. americanum-group (P < 0.01, Fig 2D).
Nematodes of the genus Xiphinema cause damage to olive by feeding on unmodified plant root cells and causing cell necrosis and galling in root apex [54,115]. However, some species are also capable to transmit pathogenic viruses to olive, specifically species belonging to the Nepovirus genus [24], such as X. diversicaudatum and X. vuittenezi [116]. Nevertheless, some dagger nematodes have been considered as major pathogens on olive trees in several countries including Chile or USA, where it was reported that Xiphinema spp. were responsible for 5 to 10% of loss production resulting in an estimated $39 million loss [117,118]. Although our results mainly revealed low densities of Xiphinema spp. in both olive types studied (Table 2 and S1 Table), in some sampling sites the densities were high, i.e. 414 or 350 nematodes per 500 cm 3 of soil for X. pachtaicum and X. italiae, respectively. In this regard, similar nematode densities of Xiphinema spp. have been reported to reduce plant-growth by feeding directly on olive roots, e.g. 65% in the case of X. elongatum [119], and in several plants including other crops [120,121] or ornamental plants [122]. On the other hand, total abundance of nematodes in each sampling site resulted significantly higher in X. americanum-group in comparison to X. non-americanum-group (P < 0.001, Fig 2D). We found a significant increase in the abundance in cultivated than in wild olive (P < 0.01, Fig 2B) for the X. americanum-group, mainly because of the prevalence and high average nematode density detected for X. pachtaicum on cultivated olive (Table 2 and S1 Table). Overall, these results could support the hypothesis that X. pachtaicum could be a real problem in olive orchards [123], although more studies would be required to clarify it. In general, Xiphinema spp. are difficult to culture under glasshouse conditions, and it is possible, that these nematodes are more pathogenic to olive in the field than is indicated by glasshouse test, since their population densities in such situations are likely to exceed those that can be attained in glasshouses [117].
Overall, nematode diversity decreases rapidly to agricultural management including plantparasitic nematodes [124]. Our results showed lower diversity indexes values, specifically for Richness diversity, in wild than in cultivated olives (P < 0.001, Fig 2A). These differences were emphasized when X. americanum-group and X. non-americanum-group species were analyzed separately (P < 0.05, Fig 2B and 2C). This fact showed the effect of agricultural management to a wide range of changes in physical, chemical and biological properties of the soil, and alterations in the autoregulation in nematode assemblages, when compared natural (wild olive) with agricultural ecosystems (cultivated olive). In this sense, several papers showed the effect of these parameters or agricultural practices in the olive nematode community [110,125,126]. However, according to the higher number of species identified from X. non-americanumgroup likewise the high prevalence of this group of nematodes associated to wild olive resulted in a higher value of Richness diversity in this type of olive in comparison to cultivated olive (P < 0.05, Table 2 and Fig 2C) in contrast to observed in X. americanum-group showing the possible plasticity of this species for a wide diversity of ecological requirements as discussed above. On the other hand, the distribution of the 385 Xiphinema populations collected in Andalusia did not revealed geographic associations to certain areas (Fig 1). Although agricultural activities may result in the widespread dissemination of Xiphinema species [112], the geographical distribution of Xiphinema species in wild and cultivated olives in southern Spain suggest a pattern linked to ecological factors. As previously reported by Archidona-Yuste et al. [13] for Longidorus species: "longidorids could have a lower dissemination level by human activities than other plant-parasitic 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 Xiphinema nematodes and further research is needed [112]. Some provinces as Cádiz, Córdoba and Jaén have showed a higher diversity than other with 17, 15 and 12 species, respectively. Some of these provinces as Cádiz showed more favorable environment for nematodes development due to the higher relative humidity and water content in the soil. By contrast, Longidorus spp. showed evidence of some geographic species associations in Andalusia [13]. Consequently, further research is needed in order to determine the influence of physico-chemical soil factors on the prevalence and distribution of Xiphinema spp. in southern Spain and other wider areas.
Sequences of nuclear ribosomal RNA genes, particularly D2-D3 and ITS1, are useful molecular markers for providing accurate species identification of Longidoridae [13, 16, 18, 28, 30,  127]. The majority of the identified species in the rhizosphere of olive matched former molecularly characterized species in other studies. In this sense, this study provides new molecular markers for partial 18S (X. cadavalense, X. pseudocoxi, and X. conurum) and for ITS1 (X. cadavalense, X. pseudocoxi, X. cohni, X. opisthohysterum and X. duriense). D2-D3 expansion region was more useful for establishing phylogenetic relationships among Xiphinema species than ITS1 or 18S. Phylogenetic analyses based on D2-D3, ITS1, and partial 18S using BI resulted in a consistent position for the newly described species of X. non-americanum-group species from Spain, which grouped in two separated clades, and mostly agree with the clustering obtained by other authors [17,18]. These species showed a good congruence between morphometric characters and phylogenetic positions as it is the case of X. andalusiense sp. nov., X. baetica, and X. cadavalense. In the case of X. andalusiense sp. nov. vs X. baetica, only lower a and c' ratios, the absence of spines in the uterus, the absence of males and different ribosomal genes could separate X. baetica from X. andalusiense sp. nov. These species probably evolved in the Iberian Peninsula as they occur only there. The Iberian Peninsula has been suggested as a possible center of recent speciation for PPN nematode genera such as Longidorus, Trichodorus or Rotylenchus species [33]. Xiphinema celtiense sp. nov., X. iznajarense sp. nov. and X. mengibarense sp. nov. could be clearly separated morphologically and molecularly from the other Xiphinema species. The majority of the species showed congruence in the phylogenetic relationships within D2-D3, ITS1, and partial 18S using the DNA from the same individual and these markers matched very well with the sequences deposited in the GenBank. This result is in contrast with the close related genus Longidorus found in a similar sampling scheme and localities in which the diversity of species was lower and all the species occupies two major positions in the phylogenetic clade [28].

Conclusions
In summary, this study provides new insights into the diversity of this genus associated with the olive in Mediterranean conditions with important differences related to the species within the X. americanum-group and the non-americanum group species. This research provides molecular markers for precise and unequivocal diagnosis of some species of Xiphinema in order to differentiate virus vector or quarantine species. Furthermore, it reflects that similar intensive and extensive integrative studies on Xiphinema species based on widest areas may help to elucidate the evolutionary origin of Xiphinema species. In this sense, further studies based on widespread species (i.e. X. pachtaicum) could also help to clarify if the main speciation occurred in Africa leading to many apomictic species in tropical and subtropical environments as hypothesised by Coomans [128], or in South America but in this case information is limited.