DNA Barcoding and Morphometry Reveal Further Cryptic Bio-Diversity within the Pin Nematode Genus Paratylenchus (Nematoda: Tylenchulidae)

Paratylenchus species are obligate ectoparasitic nematodes on cultivated and wild herbaceous and woody plants occupying numerous soil categories. Several species may cause damage to several crops (viz. P. dianthus, P. enigmaticus, P. microdorus, P. hamatus and P. epacris on carnation, lettuce, rose and walnut, respectively). This investigation proves and emphasizes the relevance of applying integrative taxonomy for the accurate detection of Paratylenchus species in mountainous wild environments in the Malaga province, Southern Spain. This research analyzed 45 soil samples of maritimus pine and one of green heather in southern Spain and identified fourteen Paratylenchus species, two of them are described herein as new species (P. paraaonli sp. nov., P. plesiostraeleni sp. nov.), six of them were first reports for Spain (P. canchicus, P. nainianus, P. neonanus, P. salubris, Paratylenchus sp. 2 SAS, and P. wuae), and six species (P. caravaquenus, P. microdorus, P. nanus, P. neoamblycephalus, P. sheri, and P. variabilis) have been already reported in Spain. Accordingly, these data increase the biodiversity of pin nematodes in Spain comprising a total of 47 species (33.1% out of 142 total species of this genus). Phylogenetic analyses based on ribosomal and mitochondrial markers (D2-D3, ITS, and partial COI) resulted in a consistent position for the newly described Paratylenchus species in this study (P. plesiostraeleni sp. nov., P. paraaonli sp. nov.). Paratylenchus plesiostraeleni sp. nov. grouped in a separated subclade as unequivocal species from the P. straeleni-complex species (including P. straeleni and P. parastraeleni), and P. paraaonli sp. nov. clustered with P. vitecus, but clearly separate from this species. This study indicates that Paratylenchus species diversity in natural environments may be higher than expected, and this study may help in accurate identifications.

Male: not found, but the spermatheca was detected filled with sperm in several specimens, suggesting that males are essential for reproduction but were not detected in this survey.
Juveniles: J3 and J4 were detected with similar morphology to adult females ( Figure 3). J3 bearing flexible stylet 46.3 (43.0-49.0) µm-long, and a functional pharynx, well developed. However, in J4, stylet is absent, and pharynx is not functional with numerous granular body content (Figure 3), representing the resting stage.

Diagnosis and Relationships
Paratylenchus paraaonli sp. nov. can be delineated by lateral field with four lines, withadvulval flap membranes, and a discreetly long and flexible female stylet of 72.2 (67.0-79.0) µm. Lip region continuous with the rest of the body, conoid-truncate, with submedian lobes small; with scanty sclerotization. Spermatheca elongated, oval. Elongate-conoid tail with finely to broadly rounded terminus. It belongs to group 10 by Ghaderi et al. [2], characterized by a long stylet (>40 µm), four lateral lines and present advulval flaps.
The morphology and morphometry, P. paraaonli sp. nov. is almost identical to P. aonli and is also similar to P. brasiliensis and P. marylandicus. Although general morphology and many matrix codes of P. paraaonli sp. nov. by the polytomous key by Palomares-Rius et al. [8] are quite similar to P. aonli, both species can be separated by the length of stylet  (10.4-14.0 vs. 14-20) [12]. Interestingly, P. aonli has been already reported in Navarra, northern Spain, and could be of interest to confirm this identification by integrative taxonomical approaches [13]. It differs from P.

Type Habitat and Locality
Paratylenchus paraaonli sp. nov. was detected in the rhizosphere of maritimus pine (Pinus pinaster Ait.), coordinates 36 • 28'55.1" N, 5 • 4 37.1" W; in the municipal district of Casares, Malaga province, on the Bermeja-Crestellina Mountain, southeastern Spain. An additional sample from the same host plant and locality are stated in Table 1. The species name, paraaonli, refers to Gr. prep. para, alongside of and resembling, N.L. masc. n. aonli, since it is very close to Paratylenchus aonli.
Paratylenchus paraaonli sp. nov. was detected in the rhizosphere of maritimus pine (Pinus pinaster Ait.), coordinates 36°28'55.1″ N, 5°4′37.1″ W; in the municipal district of Casares, Malaga province, on the Bermeja-Crestellina Mountain, southeastern Spain. An additional sample from the same host plant and locality are stated in Table 1.

Etymology
The species name, paraaonli, refers to Gr. prep. para, alongside of and resembling, N.L. masc. n. aonli, since it is very close to Paratylenchus aonli.
Male: not found, but sperm was detected filling the spermatheca in several specimens, suggesting that males are essential for reproduction but were not detected in this survey.
Juveniles: J3 and J4 were detected with similar body morphology to adult females ( Figure 6). J3 bearing flexible stylet 37.7 (36.0-39.0) µm long, and pharynx well developed, functional. However, in J4, stylet is absent, and pharynx is not functional with a granular body content (Figure 6), representing the resting stage.

Diagnosis and Relationships
Paratylenchus plesiostraeleni sp. nov. is characterized by lateral field with four lines, advulval flap membranes well developed, and a moderately long flexible stylet of 48.7 (43.5-51.0) µm. Lip region continuous with the rest of the body, conoid-rounded, with submedian lobes almost indistinct; and very slight sclerotization; with very slight sclerotization. Spermatheca rounded to spherical. Conoid tail progressively narrowing to form a terminus subacute to finely rounded, corresponding to 0.5-0.8 times as long as the vulva-anus distance. It belongs to group 10 by Ghaderi et al. [2], characterized by a long stylet (>40 µm), lateral field with four lines, and advulval flap membranes.
Morphologically and morphometrically, P. plesiostraeleni sp. nov. is almost identical to P. parastraeleni and P. straeleni, and can be also analogous to P. goodeyi. Although general morphology and many matrix codes of P. plesiostraeleni sp. nov. by the polytomous key by Palomares-Rius et al. [8] are almost identical to P. parastraeleni and P. straeleni, both species can be only separated by lip region shape in the lateral view (conoid-rounded, E12 code vs. truncate, anteriorly flattened E4 code, conoid E1 code, respectively) [2,5,8], for all the other characters and matrix codes of all three species are within the same range. Additionally, no considerable differences in morphology and morphometrics can be distinguished among the new species and several P. straeleni populations reported from Belgium, Czech Republic, Iran, Italy, Poland, The Netherlands, Turkey, and USA [3,9,[19][20][21][22][23]. Consequently, considering the specific molecular markers (D2-D3, ITS and COI) this species could be separated as a new species, this being a valuable illustration of cryptic species within the P. straeleni-complex species, and the new species identification can support to delineate the identity of morphometrically related species. P. goodeyi can be also separated by lip region shape (conoid-rounded vs. conoid) and c' ratio

Type Habitat and Locality
Paratylenchus plesiostraeleni sp. nov. was detected in the rhizosphere of maritimus pine (Pinus pinaster Ait.), coordinates 36 • 40 59.2" N, 4 • 55 13.3" W; in the municipal district of Tolox, Malaga province, on the Nieves Mountain, southeastern Spain. Additional specimens of this species were detected in two samples from the same host-plant and two different localities from Bermeja-Crestellina Mountain and Tejeda-Almijara Mountain reported in Table 1.

Etymology
The species epithet, plesiostraeleni, is related to a compound name from the Greek word plesios = near, and straeleni, the morphologically closest species of the genus Paratylenchus.

Type Material
Holotype female, 16 paratypes females, 3 third-stage juveniles and 3 fourth-stage juveniles paratypes (slide numbers CMPp4-01, CMPp4-02-CMPp4-10) were maintained in the Nematode Collection of the Institute for Sustainable Agriculture, CSIC, Córdoba, Spain; two females at Istituto per la Protezione delle Piante (IPP) of Consiglio Nazionale delle Ricerche (C.N.R.), Sezione di Bari, Bari, Italy (CMPp4-11); and two females deposited at the USDA Nematode Collection (slide T-7737t). The species epithet, plesiostraeleni, is related to a compound name from the Greek word plesios = near, and straeleni, the morphologically closest species of the genus Paratylenchus.

Type Material
Holotype female, 16 paratypes females, 3 third-stage juveniles and 3 fourth-stage juveniles paratypes (slide numbers CMPp4-01, CMPp4-02-CMPp4-10) were maintained in the Nematode Collection of the Institute for Sustainable Agriculture, CSIC, Córdoba, Spain; two females at Istituto per la Protezione delle Piante (IPP) of Consiglio Nazionale delle Ricerche (C.N.R.), Sezione di Bari, Bari, Italy (CMPp4-11); and two females deposited at the USDA Nematode Collection (slide T-7737t).     Table 4). The Spanish population of P. canchicus is characterized by a conoid-rounded lip region, moderate-short stylet, with four lines on the lateral field and advulval flap present, belonging to Group 3 by Ghaderi et al. [2]. Morphology and morphometry of this population is close to original description from Uttar Pradesh, India [24], from which only minor differences were detected in body length (295.8 (281-304) vs. 360-420 μm), which can be associated with the low number of specimens detected and measured in the Spanish population vs. original one (4 vs. 10). This species is morphologically and morphometrically quite close to P. alleni [2,25]. However, the available morphological and molecular   Table 4). The Spanish population of P. canchicus is characterized by a conoid-rounded lip region, moderate-short stylet, with four lines on the lateral field and advulval flap present, belonging to Group 3 by Ghaderi et al. [2]. Morphology and morphometry of this population is close to original description from Uttar Pradesh, India [24], from which only minor differences were detected in body length (295.8 (281-304) vs. 360-420 µm), which can be associated with the low number of specimens detected and measured in the Spanish population vs. original one (4 vs. 10). This species is morphologically and morphometrically quite close to P. alleni [2,25]. However, the available morphological and molecular data on an Iranian population of P. alleni (MN168893, annotated in NCBI as Nematoda sp. Dezful, see below) [26] suggest that both species can be a complex of cryptic species; nonetheless, topotype specimens of both species need to be identified by integrative taxonomy to confirm this hypothesis. Thus, these reports are recommended as accepted and referral populations for each species until the topotype material of P. alleni and P. canchicus becomes available and molecularly characterized. This is the first record for P. canchicus in Spain, and represents the second world record after the original description in India [24]. According to the polytomous key of Palomares-Rius et al. [8], codes for the Spanish population of P.  (2), and all of them are identical or within the range for original population [24].

Molecular Characterization
Two new populations of P. caravaquenus and eight populations of P. sheri were also detected in this study. Since both species were recently morphometrical and molecularly characterized [4,5], only sequences from D2-D3 of 28S rRNA were provided, confirming their accurate identification (Table 1), and avoiding data repetition.
Two D2-D3 of 28S rRNA (ON873212-ON873213), and two COI sequences (ON873964-ON873965) were obtained for the first time for P. canchicus in this study. In ribosomal genes, no intraspecific variability was detected; however, some molecular

Molecular Characterization
Two new populations of P. caravaquenus and eight populations of P. sheri were also detected in this study. Since both species were recently morphometrical and molecularly characterized [4,5], only sequences from D2-D3 of 28S rRNA were provided, confirming their accurate identification (Table 1), and avoiding data repetition.
Finally, only one D2-D3 of 28S rRNA sequence (ON873216) was obtained for P. nanus in this study. This sequence showed a high similarity of 99.9% (1 bp, 0 indels difference) with P. nanus from Belgium and California, USA (KF242191-KF242195, MW413657-MW413659) [3,9].  Table 5). Two populations of P. nainianus were detected in this study, one from maritime pine and another from green heather, both in the same locality (Table 1, Figure 9). The Spanish populations of P. nainianus are characterized by a conoid-truncate lip region, moderateshort stylet, distance of the base of median valve to base of stylet knobs 63-72% of the stylet length, female tail terminus rounded, with four lines at the lateral field and advulval flap present, belonging to Group 3 by Ghaderi et al. [2]. This species can be separated from P. arculatus, which is already reported in Spain [34] by prominent submedian lobes forming a disc-like structure [2]. Brzeski et al. [34] proposed the synonymization of P. nainianus with P. arculatus; however, molecular data on the latter species are lacking in order to confirm the synonymization of both species. The morphology and morphometry of these populations are close to the original description from Uttar Pradesh, India [35] and Iran [36]. According to the polytomous key of Palomares-Rius et al. [8], codes for the Spanish populations of P. nainianus are (codes in parentheses are exceptions): A2, B3, C3, D1, E4, F2, G1, H1, I1, J1, K?, L?, M1, N2(3), O2(1), P?, Q2, R3(2), S1, T?, U3, V1, W1, X1(2), all of them are identical or within the same range than type population [35].
The Spanish population of P. neonanus is characterized by a moderately long stylet (Table 5, Figure 10), lip region conoid-truncate and continuous with body contour, four lateral lines, excretory pore located at isthmus level, advulval flap and post-vulval uterine sac present, spermatheca rounded and filled with sperm, and female tail terminus rounded. A single male was detected for the first time in this species, confirmed by molecular markers (D2-D3 and COI), characterized by a narrower body than a female (Table 5), lessening towards both ends, cuticle finely annulated, with a smooth appearance; lip region analogous to female but slenderer and somewhat truncated, continuous with body, lip region with weak sclerotization, without stylet; pharynx undeveloped and not functional, procorpus, metacorpus, and basal bulb indistinct, outstretched testis, with small sperm, spicule delicate, somewhat bent towards end; gubernaculum slightly curved, without bursa, and short tail, conoid-rounded. According to the polytomous key of Palomares-Rius et al.  (2), all of them identical or within the same range as type population [37], except for small differences in the c and c' ratio [37]. The presence of sperm in spermatheca and the first report of the male confirms the amphimictical reproduction of this species.   (2), all of them identical or within the same range as type population [37], except for small differences in the c and c' ratio [37]. The presence of sperm in spermatheca and the first report of the male confirms the amphimictical reproduction of this species.   38.9 ± 6.5 ---4.5 * Abbreviations: a = body length/greatest body diameter; b = body length/distance from anterior end to pharyngo-intestinal junction; DGO = distance between stylet base and orifice of dorsal pharyngeal gland; c = body length/tail length; c' = tail length/tail diameter at anus or cloaca; G1 = anterior genital branch length expressed as percentage (%) of the body length; L = overall body length; m = length of conus as percentage of total stylet length; MB = distance between anterior end of body and center of median pharyngeal bulb expressed as percentage (%) of the pharynx length; n = number of specimens on which measurements are based; O = DGO as percentage of stylet length; T = distance from cloacal aperture to anterior end of testis expressed as percentage (%) of the body length; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

Discussion
This study intends to decipher the biodiversity of pin nematodes in mountainous natural environments in Southern Spain and complements other studies mainly related to cultivated and wild areas, demonstrating the existence of the cryptic diversity of this group of nematodes [4,5]. We found 27 Spanish populations of Paratylenchus spp. in the rhizosphere of maritimus pine and green heather, from which we identified fourteen species, two of them are described herein as new species (P. paraaonli sp. nov., P. plesiostraeleni sp. nov.), six of them were first reports for Spain (P. canchicus, P. nainianus, P. neonanus, P. salubris, Paratylenchus sp. 2 SAS, and P. wuae), and six species (P. caravaquenus, P. microdorus, P. nanus, P. neoamblycephalus, P. sheri, and P. variabilis) have been already reported in our country and characterized under integrative taxonomical approaches [4,5]. Consequently, these data increase the biodiversity of pin nematodes in Spain comprising a total of 47 species (33.1% out of 142 total species), from which only 8 species have not been molecularly characterized in Spain (viz. P. aonli, P. arculatus, P. ciccaronei, P. mirus, P. projectus, P. steineri, P. straeleni, and P. vandenbrandei), and need to be completed in order to clarify if these morphological identifications harbor new cryptic diversity. Interestingly, some species expand their distribution geographically (i.e., P. caravaquenus and P. sheri), considering only species with available molecular data and identified using an integrative approach in Spain [4,5,7]. Some of these species shared cultivated and wild habitats, indicating that the ecological requirements are different, and can be due to the importation of nematode individuals with soil movement between regions/countries or by other means to the cultivated areas. Surprisingly, some species with molecular data available and identified using an integrative taxonomy are detected in different continents under wild habitats (USA-Spain, Paratylenchus 2 SAS) [9] or under cultivated (Canada) [17] vs. wild environments in two locations in Spanish forests for P. wuae. This could raise the point of a possible introduction of P. wuae from wild environments to cultivated environments in other countries and their adaptation or this species occupied a former wider distribution in the planet. In any case, upon the present results, new studies on wild environments in Spain are needed to unravel the actual biodiversity of these nematodes and corroborate if this area is a hotspot of biodiversity as previously suggested [4,5]. Cryptic speciation has frequently been described within pin nematodes, subsequently these data enhanced the hypothesis that the genus Paratylenchus may be a hyperdiverse group of nematodes [3][4][5][6][7]9], although further surveys are needed to validate this point.
The specific identification of Paratylenchus spp. is also problematic by the presence of several Paratylenchus species within the same soil sample, particularly in wild and cultivated environments [4,5]. In this research we recognized the presence of up to three Paratylenchus species (viz. P. paraaonli sp. nov., P. plesiostraeleni sp. nov., P. neonanus) within the same sample in several cases (Table 1), corroborating the need for developing molecular markers to support this laborious task. Additionally, these nematodes showed a great phenotypic plasticity with limited species-specific diagnostic characters. Recent studies confirmed the prerequisite of using ribosomal and mitochondrial markers for an accurate identification under integrative taxonomical approaches [3][4][5][6][7][8][9]42]. Morphological studies integrated with ribosomal and mitochondrial markers (D2-D3 expansion domains of the 28S rRNA gene, ITS rRNA gen, and mtDNA gene COI) are imperative tools for precise identification of Paratylenchus spp. and deciphering the cryptic diversity of pin nematodes in a complex scenario such as natural environments and give unequivocal molecular markers associated with a specific morphology-morphometry for species identification. Our data support also that P. straeleni-complex species with three recognized species (P. straeleni, P. parastraeleni and P. plesiostraeleni sp. nov.) and several putative undescribed species comprise a prototypical case of morphostatic speciation (that is, genetic modifications not reproduced in morphoanatomy) [3][4][5][6]9], since independent methods based on molecular analyses by means of ribosomal and mitochondrial sequence data clearly separate the P. straeleni-complex species.
Phylogenetic analyses constructed on D2-D3, ITS, and partial COI using BI give rise to a consistent position for the new Paratylenchus species from Spain described herein (P. plesiostraeleni sp. nov., P. paraaonli sp. nov.), which were grouped in a separated subclade as a valid species from the P. straeleni-complex species (including P. straeleni and P. parastraeleni), and P. paraaonli sp. nov. clustered with P. vitecus, but clearly separate from this species. Ribosomal and mitochondrial phylogenies essentially agree with the clustering achieved by other nematologists [3][4][5]9]. As indicated in phylogenetic results and in previous reports by several authors, ribosomal and mitochondrial phylogenies confirm that flexible and long stylet length species (> 40 µm, initially belonging to 'Gracilacus' or 'Cacopaurus') and rigid short stylet length species (< 40 µm, initially belonging to 'Paratylenchus s.s.') cannot be separated in consistent clades, suggesting several convergent evolution events for this trait [3][4][5][6]9].
Finally, the present results emphasized former results on the noteworthy biodiversity of several genera of plant-parasitic nematodes in southern Spain, such as species within the family Longidoridae (including virus vector nematodes of the genera Xiphinema and Longidorus) or pin nematodes of the genus Paratylenchus [4,5,49], and warranty supplementary sampling efforts to elucidate the actual biodiversity in Spain.

Sampling Sites and Nematode Morphological Identification
Fifty-six soil samples were gathered primarily from the rhizosphere of maritimus pine (Pinus pinaster Ait.) forests and one single sample from green heather (Erica scoparia L.) in three mountains (including Bermeja-Crestellina, Nieves and Tejeda-Almijara Mountains, located at western, central and eastern part of Malaga province) belonging to five municipalities (Casares, Tolox, Igualeja, Canillas de Albaida, and Carratraca) in the Malaga province, Southern Spain (Table 1). Samples were taken using a shovel and considering the upper 5-40 cm depth of soil. Nematodes were analyzed from a 500-cm 3 subsample of soil by centrifugal flotation [50].
Morphological and morphometrical analyses included a total of 137 specimens, comprising 124 females, 1 male and 12 juveniles. Individuals for light microscopy (LM) analysis were killed and fixed in an aqueous solution of 4% formaldehyde + 1% glycerol, dehydrated using alcohol-saturated chamber and processed to pure glycerine using Seinhorst's method [51] according to De Grisse [52]. The life-stage of the juveniles for the undescribed species was identified considering the body length and the grade of progress of genital cells [22]. Light micrographs were taken using fresh nematodes and measurements of each nematode population, including significant diagnostic characteristics (i.e., de Man indices, body length, stylet length, lip region, tail shape) [53], were completed by means of a Leica DM6 compound microscope with a Leica DFC7000 T digital camera (Wetzlar, Germany) and comprising fixed and mounted nematodes in glycerin. Nematodes were identified at specific levels applying an integrative taxonomy merging morphological techniques (including the recent web-assisted polytomous key of Palomares-Rius et al. [8]) and molecular analyses to achieve an efficient and accurate identification [3][4][5]. Within each nematode population, significant diagnostic traits were evaluated, comprising body length, stylet length, a ratio (body length/maximum body diameter), b ratio (body length/total pharyngeal length), c ratio (body length/tail length), c' ratio (tail length/body width at anus level), V ratio ((distance from anterior end to vulva level/body length) × 100), and o ratio ((distance from stylet base to dorsal pharyngeal orifice/stylet length) × 100) [3][4][5], and the sequencing of specific molecular markers (listed below) corroborated the distinctiveness of the nematode species for individual populations.
Nematode populations of Paratylenchus species previously described and molecularly analyzed in this study for the first time were recommended as accepted and referral populations until topotype material for separate species becomes available and molecularly characterized. Voucher individuals of these defined species have been maintained in the nematode collection of Institute for Sustainable Agriculture, IAS-CSIC, Córdoba, Spain.

DNA Extraction, PCR and Sequencing
DNA extraction was always based on single nematode specimens as defined by Palomares-Rius et al. [54], and more decisive, for all the 27 considered populations, all the three molecular markers of each Paratylenchus population are coming from the same single DNA extracted nematode in individually PCR tube without any exemption. Furthermore, assignation of male and juvenile stages to one species always was proven by single DNA extraction of these individuals. Additionally, for avoiding mistakes, in the case of mixed Paratylenchus populations within the same soil sample (being common in this study), single nematodes were provisionally deposited in a drop of 1 M NaCl containing glass beads (to avoid nematode crushing/damaging specimens) to ensure specimens were coincident with the unidentified population. This saline solution did not affect the morphology of nematodes.
The D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A (5 -ACAAGTACCGTGAGGGAAAGTTG-3 ) and D3B (5 -TCGGAAGGAACCAGCTACTA-3 ) primers [55]. The Internal Transcribed Spacer region (ITS) was amplified using forward primer TW81 (5 -GTTTCCGTAGGTGAACCTGC -3 ) and reverse primer AB28 (5 -ATATGCTTAAGTTCAGCGGGT -3 ) [56]. The COI gene was amplified using the primers JB3 (5 -TTTTTTGGGCATCCTGAGGTTTAT-3 ) and JB5 (5 -AGCACCTAAACTTAAAACA TAATGAAAATG-3 ) [57]. The PCR cycling conditions for all three molecular markers were as described in Clavero-Camacho et al. [5], De Ley et al. [55], Subbotin et al. [56] and Bowles et al. [57]. In all PCR reactions, we used 5× HOT FIREpol Blend Master Mix (Solis Biodyne, Tartu, Estonia). ExoSAP-IT (Affimetrix, USB products, Kandel, Germany) was used to purify the PCR products and used for direct sequencing in both directions with the corresponding primers. The subsequent products were run in a DNA multicapillary sequencer (Model 3130XL Genetic Analyzer; Applied Biosystems, Foster City, CA, USA), using the BigDye Terminator Sequencing Kit v.3.1 (Applied Bio-systems) at the Stab Vida sequencing facility (Caparica, Portugal). The sequence chromatograms of the 3 markers (D2-D3 expansion segments of 28S rRNA, ITS rRNA, and COI) were analyzed using DNASTAR LASERGENE SeqMan v. 7.1.0. Basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (NCBI) was used to confirm the species identity of the DNA sequences obtained in this study [58]. The newly obtained sequences were sent to the GenBank database under accession numbers shown on the phylogenetic trees and in Table 1.

Phylogenetic Analyses
In this study, D2-D3 expansion segments of 28S rRNA, ITS rRNA, and COI mtDNA fragments of the 27 Paratylenchus populations were sequenced. Obtained sequences and other from species of Paratylenchus from NCBI were employed for phylogenetic analyses. For each dataset, the outgroup taxa selection was constructed according to previously published phylogenies and considering the molecular diversity of each dataset [3,5,29,42,59]. FFT-NS-2 algorithm of MAFFT V.7.450 [60] was used for multiple sequence alignments of the different genes. BioEdit program V. 7.2.5 [61] was used for sequence alignments visual-ization. Alignments were manually edited and trimmed of the poorly aligned positions, using a light filtering strategy (up to 20% of alignment positions), which has little impact on tree accuracy and may save some computation time, as suggested by Tan et al. [62]. Methods for automated filtering of multiple sequence alignments frequently worsen singlegene phylogenetic inference [62]. Bayesian inference (BI) applying MrBayes 3.1.2 [63] was used for phylogenetic analyses of the sequence datasets. JModelTest V.2.1.7 [64] with the Akaike information criterion (AIC) was used to get the best-fit model of DNA evolution. The best-fit model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC were then used in MrBayes for the phylogenetic analyses. The general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) for the D2-D3 segments of 28S rRNA, the partial ITS rRNA, and COI gene, were run with four chains for 4, 4, and 10 × 10 6 generations, respectively. A joint analysis of the two ribosomal genes was not performed due to some sequences not being accessible for all species. The Markov chains were sampled at intervals of 100 generations. For each analysis, two runs were conducted. After discarding burn-in samples of 30% and evaluating convergence, the remaining samples were retained for more in-depth analyses. The topologies were used to generate a 50% majority-rule consensus tree. On each appropriate clade, posterior probabilities (PP) were given. FigTree software version v.1.42 [65] was used for visualizing trees from all analyses.

Conclusions
This research proves and emphasizes the importance of using integrative taxonomy for the accurate identification of Paratylenchus species in complex scenarios such as wild environments. Our results also establish the presence of further cryptic biodiversity within the P. straeleni-complex species, augmenting and increasing the diversity of these plantparasitic nematodes in Spain. This study delivers ribosomal and mitochondrial markers for accurate and unambiguous diagnosis of P. straeleni-complex and advises that other reports of P. straeleni in Spain and all over the world need to be confirmed with molecular markers. In addition, these data also indicate that species diversity in natural environments may be higher than that in cultivated areas, since two new Paratylenchus species to science and six first reports were detected with respect to previous studies, two of them new species for science (P. paraaonli sp. nov., P. plesiostraeleni sp. nov.), and six species are considered as first reports for Spain in this study (viz. P. canchicus, P. nainianus, P. neonanus, P. salubris, Paratylenchus sp. 2 SAS, and P. wuae). Then, our data endorse the anticipated hypothesis that until now we have only elucidated to barely a minimal part of the biodiversity within Paratylenchus described in Spain in wild habitats and possibly worldwide.  Data Availability Statement: The datasets generated during and/or analyzed during the current study are available NCBI and from the corresponding author on reasonable request.