Integrative taxonomy of Xiphinema histriae and Xiphinema lapidosum from Spain

Abstract Three populations of Xiphinema non-americanum group species were detected in agricultural and natural ecosystems, during routine surveys for plant-parasitic nematodes in Spain. Based on morphological and molecular analyses, the species were identified as Xiphinema histriae and Xiphinema lapidosum, being this the first record and molecular characterization of both species in Spain. The morphometrics and morphology of the Spanish populations agree with those of the original description and paratype specimens and the present study provided a first description of the second to fourth juvenile stages of both species. A detailed study on the morphology in the Spanish populations of X. histriae, as well as in paratypes, showed a pseudo-Z-organ with weakly muscularized wall and containing numerous small dense granular bodies, which was different to the original description by Lamberti et al. (1993). This new finding suggests that X. histriae must be considered a member of the morphospecies Group 5 of X. non-americanum. Phylogenetic analysis based on D2 to D3 expansion segments of 28S gene, ITS1 and partial CoxI gene indicated that X. histriae and X. lapidosum are phylogenetically related with other Xiphinema non-americanum group spp. reported from Spain. Considering the pathological and economic importance of this group of nematodes, the combination of morphological characters, measurements, and molecular analysis is crucial for accurate identification of these species.

The genus Xiphinema Cobb, 1913 is a large and morphologically diverse group of plant-parasitic nematodes comprising more than 275 species (Archidona-Yuste et al., 2016a, 2016b. The economic importance of this group of nematodes is not only because of its extensive range of host plants and worldwide distribution, but for the transmission of several important plant viruses (genus Nepovirus, family Comoviridae) that cause direct damage to a wide variety of crops (Taylor and Brown, 1997;Decraemer and Robbins, 2007). Due to their economic importance, complex identification because of the sharing of a variety or morphological characters and existence of cryptic species, it is essential to identify species accurately and developing integrative taxonomy methods to control such plant pathogenic species (Archidona-Yuste et al., 2016a, 2016b. Species identification in this group is complex because of the sharing of a variety of morphological characters and the existence of cryptic species (Archidona-Yuste et al., 2016a, 2016b. According to the key for species of Xiphinema established by Loof and Luc (1990), the genus Xiphinema consists of X. americanum-group and X. non-americanum species. Later, non-americanum group was divided into eight morphospecies groups (Loof and 1990). Several authors have highlighted the great diversity of Xiphinema spp. detected in the Iberian Peninsula, in particular, around 40 species of the genus Xiphinema have been reported in Spain, mainly associated with woody, ornamental, and vegetable plant species (Gutiérrez-Gutiérrez et al., 2010, 2013, 2016Archidona-Yuste et al., 2016a, 2016b. Routine nematological surveys in agricultural and natural ecosystems in Spain yielded three populations of Xiphinema non-americanum group species, which were typologically different to previous reported species in Spain. Two populations of Xiphinema histriae were isolated from Quercus faginea Lam. and Pinus nigra Arnold, whereas one population of Xiphinema lapidosum was identified in association with Olea europaea subsp. europaea L. Lamberti et al. (1993aLamberti et al. ( , 1993b and Roca and Bravo (1993) described female and male stages of X. histriae and X. lapidosum, respectively, but in both species no juvenile stages were detected and described. The objectives of this study were: (i) to provide updated morphological descriptions of juvenile stages of X. histriae and X. lapidosum, (ii) to characterize the molecular data of both species using the D2 to D3 segments, ITS1 and partial CoxI gene sequences, and (iii) to determine the phylogenetic relationships of both species within the X. non-americanum group species.

Materials and methods
Nematode sampling, extraction, and morphological study Nematodes were surveyed from 2017 to 2018 during the spring season in natural ecosystems and olive growing area in Andalucia, southern Spain (Table 1). Soil samples were collected for nematode analysis with a shovel from four to five cores randomly selected in each sampling site. Nematodes were extracted from a 500-cm 3 sub-sample of soil by a modification of Cobb's decanting and sieving method (Flegg, 1967). Specimens were killed and fixed with hot formalin (4% with 1% glycerol), and processed in glycerin (Seinhorst, 1959) as modified by De Grisse (1969). The measurements and light micrographs of nematodes were performed using a Zeiss III compound microscope. A comparative morphological and morphometrical study of type specimens of X. histriae 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, Italy; and paratypes of X. lapidosum kindly provided by Dr Z.A. Handoo from USDA Nematode Collection, Beltsville, MD, USA (T-4406p; T4407p). Spanish nematode populations of both Xiphinema species in this study are 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.

Phylogenetic analysis
D2 to D3 segments, partial ITS1 rRNA, and partial CoxI sequences of different Xiphinema species belonging to the X. non-americanum group were obtained from GenBank and used for phylogenetic reconstruction. Outgroup taxa for each data set were chosen following previous published studies: Longidorus oleae (KT308871), Xiphinema americanum (KX263175); Longidorus caespiticola (KJ567469), Xiphinema duriense (KX244935), Xiphinema pachtaicum (HM921337); Scutellonema bradys (AY268114), Meloidogyne hapla (AY268113) (He et al., 2005;Holterman et al., 2006;Gutiérrez-Gutiérrez et al., 2013;Tzortzakakis et al., 2015;Archidona-Yuste et al., 2016a, 2016bSusulovska et al. 2018: Varela-Benavides et al., 2018. Multiple sequence alignments of the different genes were made using the Q-INS-i algorithm of MAFFT V.7.205 (Katoh and Standley, 2013), which accounts for secondary RNA structure. Sequence alignments were visualized and their percentage of similarity calculated using the sequences identity matrix using BioEdit (Hall, 1999) and manually edited by Gblocks ver. 0.91b (Castresana, 2000) in Castresana Laboratory server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) using options for a less stringent selection (minimum number of sequences for a conserved or a flanking position: 50% of the num-ber of sequences + 1; maximum number of contiguous non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half). Phylogenetic analyses of the sequence data sets were based on Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The best-fit model of DNA evolution was obtained using JMod-Note: a Newly sequences species in bold font; b (-) data not provided or sequence not used. elTest V.2.1.7 (Darriba et al., 2012) with the Akaike Information Criterion (AIC). The best-fit model, the base frequency, the proportion of invariable sites, the gamma distribution shape parameters, and substitution rates in the AIC were then given to MrBayes for the phylogenetic analyses. BI analyses were performed under the general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) for the D2 to D3 segments of 28S, rRNA ITS1 and partial CoxI gene. These BI analyses were run separately per data set using four chains for 2 × 10 6 generations for all of molecular markers. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each analysis.
After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) are given on appropriate clades. Trees from all analyses were visualized using FigTree software V.1.42 (http://tree.bio.ed.ac.uk/software/figtree/).

Systematics
Xiphinema histriae Lamberti et al. (1993aLamberti et al. ( , 1993b. (Figs. 1-3; Table 2). Figure 1. Light micrographs of Xiphinema histriae (Lamberti et al., 1993a(Lamberti et al., , 1993b. located at the beginning of basal bulb (9.0-12.7%) and two ventro-sublateral nuclei (SVN) are located near to the middle of bulb (51.9-57.5%); glandularium is 152.8 (129.5-170.5) μ m long; reproductive system didelphic-amphidelphic is with equally developed branches, and vulva slit-like and situated slightly posterior to mid-body; each branch comprises a reflexed ovary and a tubular oviduct with a developed pars dilatata oviductus separated from uterus by a sphincter; uteri tripartite with a long tubular part, consisting of a developed pars dilatata uteri link with a narrower, muscular tube-like portion containing crystalloid bodies distributed over the entire length, pseudo-Z-organ with weakly muscularized wall with numerous small dense granular bodies; ovejector is well developed, 22.2 (16.0-32.5) μ m wide, and vagina is 30.4 (20.0-40.0) μ m long or 47.4% (34.5-57.1%) of corresponding body width; prerectum is reaching around 8.9 to 11.5% of nematode body from the anus to anterior part; rectum is extending more or less than the body width at anus; and tail is short and hemispherical with a peg 6.0 to 9.5 μ m long.

Male
Very rare, only one male specimen was found in both Spanish populations. It is morphologically similar to female except for the genital system. Male genital tract is diorchic with testes with multiple rows of spermatogonia. Spicules are moderately long, curved ventrally, and lateral guiding pieces 21.0 μ m long. Tail is short and hemispherical with a peg 3.5 μ m long. One pair is of adanal supplements and seven of mid-ventral supplements.        and posteriorly and assuming a hook-shape upon fixation; cuticle appearing smooth, 5.0 (3.5-6.5) μ m thick at the middle body; lip region is flatly rounded, separated by a weak depression; odontostyle is robust, and odontophore is with well-developed basal flanges (10.5-14 μ m wide); guiding ring is double; pharynx is extending to a terminal pharyngeal bulb with three nuclei with one dorsal gland nucleus located at the beginning of pharyngeal bulb (DN = 8.5-10.5%), while two subventrolateral nuclei located at middle of bulb (SN12 = 56-60%); pharyngeal basal bulb 127 to 148 μ m long and 24.5 to 35 μ m diam; glandularium is 111.5 (106.5-115) μ m long; female reproductive system is didelphic, with two complete genital branches equally developed, each 541 (465-580) μ m long; the length of ovaries is variable, and a pars dilatata oviductus separated from the uterus by a conspicuous sphincter muscle, tripartite uterus consisting of a pars dilatata uteri followed by a tubular portion, a pseudo-Z-organ, a dilated part and an ovejector; pseudo-Z-organ well developed with a thick wall and longitudinal folding is easy to observe, comprising 15 to 20 sclerotized bodies of large size, but all of them of variable size; no spines or different structures are observed in the uterus; vulva is a transverse slit, vagina 33.0 (30.5-37.5) μ m wide and perpendicular to body-axis, ovejector well developed, 50.5 (40.5-58.5) μ m wide, extending inwards more than half of corresponding body diam; and tail short, convex dorsally and ending with bulge.

Male
Males are common but less frequent (50%) than female. They are morphologically similar to female except for the genital system; spicules are curved, lateral guiding pieces well sclerotized; tail is conoid with one pair of adanal supplements and five mid-ventral supplements (Table 3, Fig. 4).

Juveniles
Three juvenile stages (J2, J3, and J4) were found and they were basically similar to adults, except for their smaller size, shorter tails, and sexual characteristics (Table 3, Fig. 4). Tail becomes progressively wider and shorter after each moult.

Locality and habitat
The population was collected from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) at Aroche, Huelva province, Spain.

Juveniles
Three juvenile stages (J2, J3, and J4) were found and they were basically similar to adults, except for their smaller size, shorter tails, and sexual characteristics (Figs. 1, 2). The tails of juvenile stages become progressively wider after each moult. All of the stages are distinguishable by relative body lengths, functional, and replacement odontostyle (Robbins et al., 1996).

Locality and habitat
Spanish populations of Xiphinema histriae were collected in the rhizosphere of Portuguese oak (Quercus faginea Lam.) and black pine (Pinus nigra Arnold) at Arroyo Frío and Nava de San Pedro, Cazorla, Jaén Province, Spain.

Remarks
The two amphimictic populations of X. histriae agree fairly with studied paratypes (Fig. 3) and original description of X. histriae by Lamberti et al. (1993aLamberti et al. ( , 1993b. According to the polytomous key (Loof and Luc, 1990), these populations belong to the X. non-americanum Group 5 and has the following specific α-numeric codes: . No juvenile stages were described in the original description. This is the first time that J2 to J4 juvenile stages were detected and described, being similar to adults, except in body length, tail morphology, and sexual characteristics. Additionally, females of the Spanish populations of X. histriae, a pseudo-Z-organ with weakly muscularized wall, containing numerous small dense granular bodies was observed, which differ from the original description by Lamberti et al. (1993aLamberti et al. ( , 1993b. This pseudo-Z-organ was also confirmed in detailed examination of paratypes (Fig. 3). Therefore, X. histriae should be placed in morphospecies Group 5. To our knowledge, this is the first report of this species in Spain. Roca and Bravo (1993). (Figs. 4-6; Table 3).

Description Female
The female body of Xiphinema lapidosum is as follows: body is cylindrical, slightly tapering anteriorly   Table 3. Morphometrics of Xiphinema lapidosum (Roca and Bravo, 1993) from cultivated olive at Aroche (Huelva, Spain). All measurements are in µm and in the form: mean ± s.d. (range) a . Roca and Bravo (1993)   D3 and CoxI sequences from X. lapidosum obtained in this study (MK801306-MK801307, MK796913-MK796915). Phylogenetic relationships among Xiphinema non-americanum group species inferred from analyses of D2 to D3 expansion segments of 28S, ITS1, and the partial CoxI gene sequences using BI are given in Figures 7 to 9, respectively. Poorly supported clusters were not explicitly labelled. The 50% majority rule consensus 28S rRNA gene BI tree of X. non-americanum group spp. based in a multiple edited alignment including 70 sequences and 771 total characters showed two clearly separated (PP = 1.00) major clades (Fig. 7). Clade I grouped species from all morphospecies groups, including the new accessions obtained in this study of X. histriae and X. lapidosum. Clade II was not well supported (PP = 0.84) and was mostly composed by species from the morphospecies Group 5, except for X. tica, X. bakeri, and X. index which belong to Groups 4, 7, and 8, respectively. Xiphinema histriae (MK801302- MK801305) occupies a superior position within this major clade I clustering with X. hispanum, X. celtiense, and X. cohni in a well-supported subclade (PP = 0.97). On the contrary, X. lapidosum (MK801306-MK801307) occupied a basal position and seemed to be related with X. lupini, X. turcicum, and X. oleae since all of them formed a well-supported subclade (PP = 0.99). The low similarity and small coverage between the ITS1 region from X. lapidosum and the rest of the ITS1 sequences available in GenBank made it impossible to perform a phylogenetic analysis for this region. For X. histriae, only ITS1-related sequences were used, the edited alignment generated for the 29 sequences of ITS1 was of 1,104 characters after discarding ambiguously aligned regions. This ITS phylogenetic tree (Fig. 8) showed two major clades (PP = 1.00), similar to those obtained for D2 to D3 region. Xiphinema histriae (MK801298-MK801299) appeared in the basal major clade but their phylogenetic position was not well resolved for this marker (Fig. 8). The CoxI region using a multiple alignment of 52 sequences and 390 characters after editing was used to obtain the 50% majority rule BI tree (Fig.  9). The position of X. histriae (MK796911-MK796912) was not well-defined, but clustering with X. hispanum, X. hispidum, X. cohni, and X. celtiense. By contrast, the relationship among X. lapidosum (MK796913-MK796915) and X. lupini was maintained.

Discussion
This study aimed to provide and to characterize morphometrically and molecularly two Xiphinema species belonging to Xiphinema non-americanum Group 5 from Spain, and to carry out an updated phylogenetic study of both species within the X. non-americanum group species. To date, this is the first record of the occurrence of X. histriae and X. lapidosum in Spain and the first time that describes the molecular characterization and the juvenile stages of both species.
Xiphinema histriae was originally described from Italy associated with grapevine (Lamberti et al., 1993a(Lamberti et al., , 1993b, and later on, reported from the rhizosphere of wild growing grape (Vitis vinifera ssp. silvestris) in Austria (Tiefenbrunner and Tiefenbrunner, 2004). Based on the detailed study of paratypes and both Spanish populations described here, we detected that this species is characterized by having a pseudo-Z-organ with weakly muscularized wall with numerous small dense granular bodies against that initially described by Lamberti et al. (1993aLamberti et al. ( , 1993b. Therefore, X. histriae must be transferred to morphospecies Group 5 (Loof and Luc, 1990). This study illustrates the importance of paratypes deposited in different official collections and reference nematology laboratories of nematodes, which are provided as a useful tool in the accurate identification and revision of nematodes species. On the other hand, X. lapidosum was, first, described from the rhizosphere of broad bean and pea in the south of Portugal (Roca and Bravo, 1993) and now it is reported from cultivated olive at Huelva, southwestern Spain. These data suggest that X. histriae may have a wider distribution than that described until now (including agricultural and natural ecosystems), and X. lapidosum may be an Iberian endemism, also associated with cultivated hosts.
The use of different ribosomal and mitochondrial markers in this study, D2 to D3, ITS1, and partial CoxI, provides a precise and unequivocal tool for the identification of X. histriae and X. lapidosum. Phylogenetic analyses based on D2 to D3, ITS1, and CoxI gene using BI resulted in a consistent position for X. histriae and X. lapidosum. Xiphinema histriae clustered with Xiphinema species from morphospecies Group 5, such as X. hispanum, X. cohni, X. celtiense, and X. hispidum, while X. lapidosum seems to be related with X. lupini because of both species clustered together in all the analyses carried out in this study. The present study on the phylogeny based on D2 to D3 segments supported a very weak correlation in the phylogenetic relationships among the different morphospecies groups within Xiphinema, a finding already reported by several authors namely, Gutiérrez-Gutiérrez et al., 2013;De Luca et al., 2014;Tzortzakakis et al., 2014Tzortzakakis et al., , 2015Archidona-Yuste et al., 2016a, 2016b, 2016c.
In summary, this study highlighted the diagnosis of Xiphinema non-americanum group species because a large number of species and the lack of good diagnostic characteristics among the X. non-americanum group (Loof and Luc, 1990;Loof et al., 1996). For this reason, we recommend the use of integrative taxonomy that are crucial for accurately identify species and better understanding of the present geographical distribution and host range of X. non-americanum group species. In this case, we provide new morphological and molecular data for the precise identification of these species, the first reports of these species in Spain, new hosts, and their phylogenetic position in the Xiphinema genus.