Remarkable Cryptic Diversity of Paratylenchus spp. (Nematoda: Tylenchulidae) in Spain

Simple Summary Paratylenchus spp. are vermiform organisms distributed throughout the world that can parasitize a wide variety of cultivated and wild plants. Some species are considered pathogenic in crops; therefore, correct identification is essential to design management strategies. However, the conserved morphology, similarity of morphometric characters and other factors as co-occurrence of more than one species of Paratylenchus in the same soil sample hinder identification to species level. In consequence, this identification should be carried out jointly with morphological, morphometrical and molecular data. The present research aims to provide morphological and molecular characterization of some Paratylenchus species found in Spain and the description of several new species. Abstract In previous studies, fifteen species of Paratylenchus, commonly known as pin nematodes, have been reported in Spain. These plant-parasitic nematodes are ectoparasites with a wide host range and global distribution. In this research, 27 populations from twelve Paratylenchus species from 18 municipalities in Spain were studied using morphological, morphometrical and molecular data. This integrative taxonomic approach allowed the identification of twelve species, four of them were considered new undescribed species and eight were already known described. The new species described here are P. caravaquenus sp. nov., P. indalus sp. nov., P. pedrami sp. nov. and P. zurgenerus sp. nov. As for the already known described species, five were considered as first reports for the country, specifically P. enigmaticus, P. hamatus, P. holdemani, P. israelensis, and P. veruculatus, while P. baldaccii, P. goodeyi and P. tenuicaudatus had already been recorded in Spain. This study provides detail morphological and molecular data, including the D2-D3 expansion segments of 28S rRNA, ITS rRNA, and partial mitochondrial COI regions for the identification of different Paratylenchus species found in Spain. These results confirm the extraordinary cryptic diversity in Spain and with examples of morphostatic speciation within the genus Paratylenchus.


Introduction
Pin nematodes of the genus Paratylenchus Micoletzky, 1922 [1] are one of the smallest plant-parasitic nematodes; their body length varies from 160 to 600 µm [2]. Stylet length is the organ which drives the feeding habit and morphology of adult females. Some species

PR_011
Paratylenchus israelensis (Raski, 1973) Siddiqi, 1986 Córdoba ( A total of 231 individuals including 214 females and 17 males were used for morphological and morphometrical analyses. Specimens for study using light microscopy (LM) and morphometrical studies were killed and fixed in an aqueous cold solution of 4% formaldehyde + 1% glycerol, dehydrated using alcohol-saturated chamber and processed to pure glycerine using Seinhorst's method [54] as modified by De Grisse [55]. Light micrographs were taken using fresh nematodes and measurements of each nematode population including important diagnostic characteristics (i.e., de Man indices, body length, stylet length, lip region, tail shape) [56] were performed using a Leica DM6 compound microscope with a Leica DFC7000 T digital camera using fixed and embedded nematodes in glycerin. Nematodes were identified at the species level using an integrative approach combining molecular and morphological techniques to achieve efficient and accurate identification [21,24,28]. For each nematode population, key diagnostic characters were determined, including body length, stylet length, a ratio (body length/maximum body width), b ratio (body length/total pharynx length), c ratio (body length/tail length), c' ratio (tail length/body width at anus), V ratio (distance from anterior end to vulva/body length × 100), and o ratio (distance from stylet base to dorsal pharyngeal opening/stylet length 100) [21,24,28], and the sequencing of specific DNA fragments (described below) confirmed the identity of the nematode species for each population.

Nematode Molecular Characterization
For molecular analyses, and in order to avoid mistakes in case of mixed populations in the same sample (being common in several soil samples), single specimens from the sample were temporarily mounted in a drop of 1 M NaCl containing glass beads (to avoid nematode crushing/damaging specimens) to ensure homogenous morphology with specimens conformed with the unidentified population. All necessary morphological and morphometrical data by taking pictures and measurements using the above camera-equipped microscope were recorded. This was followed by DNA extraction from single individuals as described by Palomares-Rius et al. [57], and more importantly, for all the 27 studied isolates, all the three molecular markers of each Paratylenchus isolate belong to the same single extracted individual in each PCR tube without any exception. The D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A (5 -ACAAGTACCGTGAGGGAAAGTTG-3 ) and D3B (5 -TCGGAAGGAACCAGCTACTA-3 ) primers [58]. The Internal Transcribed Spacer region (ITS) was amplified by using forward primer TW81 (5 -GTTTCCGTAGGTGAACCTGC-3 ) and reverse primer AB28 (5 -ATATGCTTAAGTTCAGCGGGT-3 ) [59]. The COI gene was amplified using the primers JB3 (5 -TTTTTTGGGCATCCTGAGGTTTAT-3 ) and JB5 (5 -AGCACCTAAACTTAAAACAT AATGAAAATG-3 ) [60]. The PCR cycling conditions for the 28S rRNA and ITS regions were as follows: 95 • C for 15 min, followed by 35 cycles of 94 • C for 30 s, an annealing temperature of 55 • C for 45 s, and 72 • C for 1 min, and 1 final cycle of 72 • C for 10 min. The PCR cycling for COI primers was as follows: 95 • C for 15 min, 39 cycles at 94 • C for 30 s, 53 • C for 30 s, and 68 • C for 1 min, followed by a final extension at 72 • C for 7 min. PCR volumes were adapted to 25 µL for each reaction, and primer concentrations were as described in De Ley et al. [58], Subbotin et al. [59] and Bowles et al. [60]. We used 5× HOT FIREpol Blend Master Mix (Solis Biodyne, Tartu, Estonia) in all PCR reactions. The PCR products were purified after amplification using ExoSAP-IT (Affimetrix, USB products, Kandel, Germany) and used for direct sequencing in both directions with the corresponding primers. The resulting products were purified and 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 (ITS, COI and D2-D3 expansion segments of 28S rRNA) were analyzed using DNASTAR LASERGENE SeqMan v. 7.1.0. Basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) was used to confirm the species identity of the DNA sequences obtained in this study [61]. The newly obtained sequences were deposited in the GenBank database under accession numbers indicated on the phylogenetic trees and in Table 1.

Phylogenetic Analyses
D2-D3 expansion segments of 28S rRNA, ITS rRNA, and COI mtDNA sequences of the 27 Paratylenchus isolates were obtained in this study. These sequences and other sequences from species of Paratylenchus from GenBank were used for phylogenetic analyses. Selection of outgroup taxa for each dataset were based on previously published studies [24,28,29]. Multiple sequence alignments of the different genes were completed using the FFT-NS-2 algorithm of MAFFT V.7.450 [62]. BioEdit program V. 7.2.5 [63] was used for sequence alignments visualization and edited by Gblocks ver. 0.91b [64] in Castresana Laboratory server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html (accessed on 27 March 2021)) using options for a less stringent selection (minimum number of sequences for a conserved or a flanking position: 50% of the number of sequences +1; maximum number of contiguous non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half). Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes 3.1.2 [65]. The best-fit model of DNA evolution was achieved using JModelTest V.2.1.7 [66] with the Akaike Information Criterion (AIC). 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 and the partial ITS rRNA and the general time-reversible model with a gamma-shaped distribution (GTR + G) for COI gene, were run with four chains for 4, 4, and 10 × 10 6 generations, respectively. A combined analysis of the three ribosomal genes was not undertaken due to several sequences not being available for all species. The sampling for Markov chains was carried out 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 [67] was used for visualizing trees from all analyses.

Results
Twelve species were identified from 27 isolates of Paratylenchus spp. from 23 soil samples (codified as PR_014, PR_115, PR_118 and PR_193 contain more than one Paratylenchus species) in 18 municipalities in Spain. These populations were morphologically studied in detail and molecular markers for their identification were provided (Table 1). From these, 4 were considered new undescribed species and 8 were already known described species (Table 1 Female: Body slender, ventrally arcuate to form an open, C-shaped body habitus when heat relaxed; cuticle finely annulated; lateral field equidistant with four distinct lines; lip region conoid rounded, with anterior end flattened, continuous with the rest of the body, small submedian lobes or almost indistinguishable in some specimens. Labial framework sclerotization weak; pharyngeal region typical paratylenchoid type. Stylet rigid, straight; stylet knobs rounded; dorsal pharyngeal gland opening 5.5-8.0 µm behind stylet knobs. Median pharyngeal bulb slender elongate, bearing distinct large valves; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, pharyngeal-intestinal valve rounded; excretory pore situated at the level or anterior to pharyngeal basal bulb. Hemizonid 1-2 annuli long, situated immediately anterior to excretory pore. Body slightly narrower posterior to vulva; ovary outstretched, well developed; spermatheca and crustaformeria well developed; spermatheca rounded; vulva a transverse slit occupying half of the corresponding body width. Vulval lips prominent, the anterior lip is protruding further than the posterior lip; advulval flaps present, but not prominent in fresh specimens. Anus difficult to distinguish in some specimens; tail slender, conoid, finely annulated, and gradually tapers to form a rounded or subacute terminus in some individuals.
Male: Body slender than female, tapering towards both ends, posterior region ventrally arcuate when heat relaxed. Cuticle apparently smooth with fine annulations; labial region similar to that of female but narrower and slightly truncated, continuous with body, sclerotization in labial region weak; stylet lacking. Pharynx rudimentary and non-functional, procorpus, metacorpus, and basal bulb inconspicuous; excretory pore located 73.0 µm away from anterior end. Testis outstretched, with small spermatozoa; spicule slender, slightly curved towards end; gubernaculum curved; bursa absent. Tail elongate-conoid, tapering gradually to a finely pointed tip.
Juveniles: It was the most abundant developmental stage at the end of summer in the type locality, most probably J4. They were similar in morphology to the adult females. However, they are characterized by the presence of weak stylet; underdeveloped pharynx components; underdeveloped genital primordium; indistinct anus; and posterior body with a rounded terminus.

Diagnosis and Relationships
The new species can be characterized by the presence of 4 lateral lines in lateral field, advulval flaps present, and a moderate female stylet length of 29.8 (26.5-32.0) µm. Lip region conoid-rounded, with the anterior end flattened, continuous with the rest of the body. Excretory pore situated at the level or anterior to the pharyngeal basal bulb. Spermatheca rounded. Tail elongate-conoid gradually tapering to form a rounded terminus. According to species grouping by Ghaderi et al. [36] belongs to group 3 characterized by stylet length less than 40 µm, four lateral lines and advulval flaps present.
Animals 2021, 11,1161 10 of 55 lobes in P. nawadus [27]. Unfortunately, no data for ITS or COI from P. nawadus are available in the GenBank. From our P. baldaccii population (PR_152), P. caravaquenus sp. nov. is 79%, 79%, and 84% similar in D2-D3 region, ITS and COI sequences, respectively, clearly separating both species.      Morphologically and morphometrically, the new species is close to P. baldaccii, P. salubris , P. coronatus Colbran, 1975 and P. mimulus . Paratylenchus caravaquenus sp. nov. differs from P. baldaccii in having males without stylet vs. males with stylet, other morphometrical characters are in the same range. From a molecular point of view, this species differs from other populations identified as P. baldaccii in all the molecular markers studied (D2-D3, ITS and COI). Paratylenchus caravaquenus sp. nov. differs from P. salubris in tail shape acute to finely rounded vs. usually bluntly rounded

Remarks
This species has been found in only one forest close to an almond field. The population presented moderate numbers of individuals in soil (224 individuals/500 cm 3 of soil), but the majority of them were J4 individuals.

Etymology
The species epithet, caravaquenus, refers to the name of the type locality (Caravaca).

Type Material
Holotype female, 17 paratypes females and 6 male paratypes (slide numbers PI_AR-01 to PI_AR-11) were deposited in the Nematode Collection of the Institute for Sustainable Agriculture, CSIC, Córdoba, Spain, and two females deposited at the USDA Nematode Collection (slide T-7479p). Female: Body slender, ventrally arcuate to form an open C-shaped when heat relaxed. Cuticle finely annulated; lateral field equidistant with four distinct lines. Lip region rounded, with anterior end flattened, continuous with the rest of the body, presence of small submedian lobes; labial framework sclerotization weak. Pharyngeal region typical paratylenchoid type; stylet rigid, straight; stylet knobs rounded; dorsal pharyngeal gland opening 5.5-6.5 µm behind stylet knobs. Median pharyngeal bulb slender elongate, bearing distinct large valves; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, pharyngeal-intestinal valve rounded; excretory pore situated anterior to pharyngeal basal bulb. Hemizonid 1-2 annuli long situated immediately anterior to excretory pore; body slightly narrower posterior to vulva; ovary outstretched, well developed. Spermatheca and crustaformeria well developed; spermatheca rounded; vulva a transverse slit occupying half of the corresponding body width; vulval lips prominent, the anterior lip is protruding further than the posterior lip; advulval flaps present, but not prominent in fresh specimens. Anus difficult to distinguish. Tail slender, conoid, finely annulated, and gradually tapers to form a rounded terminus or pointed in some individuals ( Figure 5).       Lip width 6.0 6.2 ± 0.3 (5.5-6.5) 6.5 ± 0.4 (6.0-7.0) 6.5, 7.0 6.5 ± 0.5 (6.0-7.0) Median bulb length 21.0 20.7 ± 2.0 -28.0 - Male: Not found. Juveniles: It is the most abundant developmental stage at the end of summer in the type locality and other localities. They were similar in morphology to females. However, they are characterized by the presence of a weak stylet; underdeveloped pharynx components; underdeveloped genital primordium; indistinct anus; and posterior body with a rounded or pointed terminus.

Diagnosis and Relationships
The new species can be characterized by the presence of 4 lateral lines, advulval flaps, and a stylet length of 28.3 (26.0-29.5) µm. Lip region rounded, with the anterior end flattened, continuous with the rest of the body. Excretory pore situated at the level or anterior to the pharyngeal basal bulb. Spermatheca rounded. Tail conoid gradually tapering to form a rounded terminus.
Morphologically, the new species is close to P. projectus, P. neoprojectus Wu & Hawn, 1975 and P. enigmaticus. From these species no important and clear morphological and morphometrical differences can be detected. However, P. projectus and P. neoprojectus have important differences molecularly to P. indalus sp. nov. Molecular differences using population paratypes from P. projectus and P. neoprojectus must be necessary in order to separate these three closely related morphologically species. Paratylenchus projectus differs from P. neoprojectus by having a trapezoid-shaped lip region, more anterior position of excretory pore and often digitate tail terminus, but these characters could have some variation [36]. These species are also similar to P. nanus and P. neoamblycephalus from which differ by having empty spermatheca and absence of males [36]. Molecularly, this species is closely related but different to paratypes from P. enigmaticus. This is a case of cryptic speciation among these three species comprising a complex group of pin nematodes.

Molecular Characterization
Ten D2-D3 of 28S rRNA (MW798273-MW798282), ten ITS (MW798319-MW798328), and four COI gene sequences (MW797005-MW797008) from the four different populations were generated herein for this species. All sequences showed no intraspecific variation, except for the ITS sequences, where one variable position was found. Paratylenchus indalus sp. nov. was closely related with P. enigmaticus, showing similarity values of 96% (differing by 25 nucleotides and 1 indel) for the D2-D3 region with several accession from P. enigmaticus, such as MN535546 or MW282761. For the ITS region, the similarity values were from 94 to 96% (differing by 24 to 39 nucleotides and from 10 to 15 indels) with the P. enigmaticus accessions MW282773 and MN535549, respectively. Finally, the similarity found for the COI gene sequences was 97% (differing by 11 nucleotides) with P. enigmaticus accessions (MW421686, MN782403 and MW316640). Paratylenchus indalus sp. nov. is not so closely molecularly related to P. projectus and P. neoprojectus as for P. enigmaticus, with a similarity of 92-93%, 89% and 87% for D2-D3 of 28S rRNA, ITS region and COI, respectively.

Remarks
This species has been found in four almond orchards with undetermined rootstock in four different localities in Almería province (South-eastern Spain). The population presented moderate to high numbers of individuals in soil (from 68 to 2268 individuals/500 cm 3 of soil), being the majority of them J4 at the end of summer.

Type Habitat and Locality
Paratylenchus indalus sp. nov. was found in the rhizosphere of almond at Santa María de Nieva, Almería province (coordinates 37 • 35 26.8 N; 2 • 02 03.5 W), and also has been found in four localities in Almería province (Table 1).

Etymology
The species epithet, indalus, is derived from the name 'indalo' a prehistoric symbol found in a cave of Almería, the province of the locality where the type specimens were collected.

Type Material
Holotype female, and 11 paratypes females (slide numbers PR_104-01 to PR_104-06) were deposited in the Nematode Collection of the Institute for Sustainable Agriculture, CSIC, Córdoba, Spain, and two females deposited at the USDA Nematode Collection (slide T-7480p). Female: Body slender, ventrally arcuate to form an open C-shaped when heat relaxed. Cuticle finely annulated; lateral field equidistant with four distinct lines. Lip region rounded, with anterior end flattened, continuous with the rest of the body, presence of small submedian lobes. Labial framework sclerotization weak; pharyngeal region typical paratylenchoid type. Stylet rigid, straight; stylet knobs rounded; dorsal pharyngeal gland opening 3.5-5.0 µm behind stylet knobs. Median pharyngeal bulb slender elongate, bearing distinct large valves; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, pharyngeal-intestinal valve rounded; excretory pore situated at the level or anterior to pharyngeal basal bulb. Hemizonid 1-2 annuli long situated immediately anterior to excretory pore; body slightly narrower posterior to vulva; ovary outstretched, well developed. Spermatheca and crustaformeria well developed; spermatheca rounded; vulva a transverse slit occupying half of the corresponding body width; advulval lips prominent, the anterior lip is protruding further than the posterior lip; vulval flaps present, but not prominent in fixed specimens. Anus difficult to distinguish. Tail slender, conoid, finely annulated, and gradually tapering to form a rounded terminus or pointed in some individuals ( Figure 8).
Male: Body slender than female, tapering towards both ends, cuticle apparently smooth with fine annulations; labial region similar to that of female but narrower and slightly truncated, continuous with body, sclerotization in labial region weak. Stylet lacking; pharynx rudimentary, procorpus, metacorpus, and basal bulb inconspicuous, and nonfunctional. Excretory pore located 65.5 µm from anterior end. Testis outstretched, with small spermatozoa; spicule slender, slightly curved towards end; gubernaculum curved; bursa absent; tail short and rounded.
Lip width 4. Juveniles: It is the most abundant developmental stage at the end of summer in the type locality and other localities. They were similar in morphology to adult females. However, they are characterized by the presence of weak stylet; underdeveloped pharynx components; underdeveloped genital primordium; indistinct anus; and posterior body with a rounded or pointed terminus.

Diagnosis and Relationships
The new species can be characterized by the presence of 4 lateral lines, advulval flaps, and a stylet length of 28.3 (26.0-29.5) µm. Lip region rounded, with the anterior end flattened, continuous with the rest of the body. Excretory pore situated at the level or anterior to the pharyngeal basal bulb. Spermatheca rounded. Tail conoid gradually tapering to form a rounded terminus. Males with a short and rounded tail. According to species grouping by Ghaderi et al. [36] belongs to group 3 characterized by stylet length less than 40 µm, four lateral lines and advulval flaps present.

Remarks
This species has been found in two almond orchards with undetermined rootstock in two different localities in Córdoba province (Southern Spain). The population presented moderate numbers of individuals in soil (200 and 216 individuals/500 cm 3 of soil), being the majority of them J4 at the end of summer.
Paratylenchus pedrami sp. nov. was found in the rhizosphere of almond at Córdoba, Córdoba province (coordinates 37 • 49 39.9" N; 4 • 53 22.0" W), and also has been found in other place at the same locality (        Female: Body slender, ventrally arcuate to form a C-shaped when heat relaxed. Cuticle finely annulated; lateral field equidistant with four distinct lines. Lip region rounded, with anterior end flattened, continuous with the rest of the body, absence of submedian lobes. Labial framework sclerotization relatively strong; pharyngeal region typical paratylenchoid type. Stylet delicate, straight; stylet knobs rounded; dorsal pharyngeal gland opening 3.5-5.0 µm behind stylet knobs. Median pharyngeal bulb slender elongate, bearing distinct large valves; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, pharyngeal-intestinal valve rounded. Excretory pore situated at the level or anterior to pharyngeal basal bulb; hemizonid 1-2 annuli long situated immediately anterior to excretory pore; body slightly narrower posterior to vulva. Ovary outstretched, well developed; spermatheca and crustaformeria well developed; spermatheca rounded; vulva a transverse slit occupying half of the corresponding body width; vulval lips prominent, the anterior lip is protruding further than the posterior lip; vulval flaps present, but not prominent in fresh specimens. Anus difficult to distinguish (more distinguishable in alive specimens). Tail slender, conoid, finely annulated, and gradually tapering to form a rounded terminus or pointed in some individuals ( Figure 11).

Molecular Characterization
Four D2-D3 of 28S rRNA (MW798286-MW798289), and four ITS sequences (MW798331-MW798334) were obtained for this species. In both ribosomal genes, no intraspecific variability was detected between three of the four individuals sequenced, whereas in the fourth one, 11 different nucleotides were detected for the D2-D3 (MW798289) and 24 nucleotides and 4 indels for the ITS region (MW798334). Two identical COI gene sequences (MW797010-MW797011) were generated for P. zurgenerus sp. nov. The D2-D3 sequences were found to be 99% similar (differing from 1 to 11 nucleotides) and in the same phylogenetic clade of Paratylenchus sp.7 (KF242242) from California (USA) that should be consider as conspecific to P. zurgenerus sp. nov., however, only molecular and no morphological data are available for this species [23]. The closest Paratylenchus spp. for the rest of the molecular markers studied was P. microdorus (MW413599-MW413600) being 87% similar for the ITS region (differing by 38 to 41 nucleotides and from 28 to 31 indels) and 91% for COI sequences (MW421666-MW421667) (differing by 28 nucleotides and no indels).

Remarks
This species has been found in one almond orchard with undetermined rootstock in one locality in Granada province (Southern Spain). The population presented moderatehigh numbers of individuals in soil (1470 individuals/500 cm 3 of soil), being the majority of them J4 at the end of summer. Probably this species could be the same previously described by Gomez-Barcina et al. [45] as P. microdorus as its measurements are identical and in geographically related areas in Southern Spain, but further studies are needed for confirming this hypothesis.

Etymology
The species epithet, zurgenerus, refers to the name of the gentilice of inhabitants of the type locality (Zurgena).

Type Material
Holotype female, and 17 paratypes females (slide numbers PR_111-01 to PR_111-09) were deposited in the Nematode Collection of the Institute for Sustainable Agriculture, CSIC, Córdoba, Spain, and two females deposited at the USDA Nematode Collection (slide T-7482p).

Morphometrics and Remarks of Known Paratylenchus Spanish Populations
Morphometrical data as well as molecular characterization of other already known Paratylenchus populations detected in the present study were compared with original and previous reported populations, and these species included P. baldaccii, P. enigmaticus, P. goodeyi, P. hamatus, P. holdemani, P. israelensis, P. tenuicaudatus, and P. veruculatus.
Paratylenchus enigmaticus Munawar, Yevtushenko, Palomares-Rius & Castillo, 2021. This species has been recently described from Canada [21]; and it has also been reported in Belgium [22,24]. In the present study, the P. enigmaticus population from Spain matches with the original species description, except for minor differences in body length and a ratio; the Spanish population is slightly shorter than the original one (324-383 µm vs. 343-431 µm) and with smaller a ratio (17.6-21.6 vs. 21.7-28.7) (Table 6, Figure 12). This population is closely related morphometrically to the T1-T5 populations found in Belgium [22]. This species has not been reported before in Spain and constitutes the first record for the country.
Gubernaculum ----5.1 ± 0.25 (5.0-5.5) -* Abbreviations: a = body length/greatest body diameter; b = body length/distance from anterior end to pharyngo-intestin junction; DGO = distance between stylet base and orifice of dorsal pharyngeal gland; c = body length/tail length; c' = t length/tail diameter at anus or cloaca; G1 = anterior genital branch length expressed as percentage (%) of the body leng L = overall body length; m = length of conus as percentage of total stylet length; MB = distance between anterior end body and center of median pharyngeal bulb expressed as percentage (%) of the pharynx length; n = number of specime on which measurements are based; O = DGO as percentage of stylet length; T = distance from cloacal aperture to anter end of testis expressed as percentage (%) of the body length; V = distance from body anterior end to vulva expressed percentage (%) of the body length.   Molecular characterization: Molecular markers agree with the identification of this species. One D2-D3 of 28S rRNA (MW798292), one ITS (MW798337) and one COI gene sequences (MW797013) were generated for this species, and all of them were found to be identical to several accessions from P. enigmaticus deposited in GenBank such as, MN535546 for D2-D3 of 28S, MW319816 and MN535551 for the ITS region, and MW421686 for the COI gene sequences.
The Spanish population from Córdoba characterized in this study coincides mainly with the original description of the species (Table 6, Figure 13) at exception of the position of the excretory pore (82.0-102.0 μm vs. 64 μm). This species matches well with other populations, as the Spanish population described by Castillo et al. [48], at exception of a shorter body [396-427 μm vs. 410-450 μm] and smaller c' ratio (3. 2-3.8 vs. 4.1-4.7).
Molecular characterization: Molecular markers of this population agree with those provided for this species by Singh et al. [24]. Two D2-D3 of 28S rRNA (MW798293-MW798294), two ITS (MW798338-MW798339) and two COI gene sequences (MW797014-MW797015) were generated in this study without intraspecific sequence variations for this population. The D2-D3 of 28S rRNA sequences were 99% similar (differing by 9 nucleotides) with P. goodeyi from Belgium (MW413631-MW413633). The ITS sequences were 96% similar (differing by 27 nucleotides and 11 indels) with the P. goodeyi sequence MW423594 and finally, the COI gene sequences showed 95% similarity (differing by 19 nucleotides) with the accessions from P. goodeyi deposited in GenBank (MW421648-MW421649). Molecular characterization: Molecular markers of this population agree with those provided for this species by Singh et al. [24]. Two D2-D3 of 28S rRNA (MW798293-MW798294), two ITS (MW798338-MW798339) and two COI gene sequences (MW797014-MW797015) were generated in this study without intraspecific sequence variations for this population. The D2-D3 of 28S rRNA sequences were 99% similar (differing by 9 nucleotides) with P. goodeyi from Belgium (MW413631-MW413633). The ITS sequences were 96% similar (differing by 27 nucleotides and 11 indels) with the P. goodeyi sequence MW423594 and finally, the COI gene sequences showed 95% similarity (differing by 19 nucleotides) with the accessions from P. goodeyi deposited in GenBank (MW421648-MW421649).
Paratylenchus hamatus Thorne & Allen, 1950 and Paratylenchus baldaccii . These species are closely related morphologically to other species such as P. tenuicaudatus. Van den Berg et al. [23] included P. tenuicaudatus within the P. hamatus sensu stricto, P. baldaccii and two other putative species within the P. hamatus "species complex". Only one character has been pointed by different authors to separate P. hamatus from P. baldaccii, viz. slenderer and sharply conoid female tail tip and male tail tips in P. hamatus vs. P. baldaccii which was described as finely rounded to almost acute [75,77]. Paratylenchus baldaccii is described morphologically in this study (Table 6, Figure 14) with molecular markers provided too (see description below) and different to other molecular species descriptions within the P. hamatus "species complex" [23]. Our population of P. baldaccii matches with the original description of the species, but in our samples it is really very difficult to separate P. baldaccii and P. hamatus only based on morphological traits. However, topotypes of P. baldaccii should be necessary in order to assign a definitive molecular marker association between morphology and molecular differences in this complex species group. Specimens of P. hamatus from fig orchards at the type locality (Planada, CA, USA) were molecularly characterized by Van den Berg et al. [23]. Paratylenchus hamatus has not been described before in Spain and constitute a first record for the country.
Paratylenchus hamatus has a worldwide distribution and has been reported in many countries including Australia, Belgium, Canada, Pakistan, etc. [36]. This species can cause damage in different crops as figs and several vegetables [36]. Different populations of this species have been found in our study in the rhizosphere of peach/rootstock peach × almond [GxN] (sample codes PR_44 and PR_207) and almond/rootstock almond orchards (sample codes PR_115 and PR_187). Nematode soil population levels were high or very high (42400, 4212, 2042 and 8250 individuals per 500 cm 3 of soil) in peach orchards sample codes PR_44, PR_115, PR_187 and PR_207, respectively. However, peach trees did not show any apparent growth reduction or symptomatology associated with these high levels of nematodes in the soil. Morphologically, the Spanish populations studied herein ( Figure 15, Table 7) were in the range of the described populations and topotypes from P. hamatus [23].
Paratylenchus baldaccii has been described in grapevine in Sicily (Italy), Bari (Italy) and South of France [77] and later in Italy [75] and Spain [44]. In our sampling has been recorded in one locality in a peach orchard at a density of 200 individuals per 500 cc of soil.
Paratylenchus holdemani .  This species has been described from Santa Ana, El Salvador [77] and also reported in Czech Republic [33] and Belgium [24]. It resembles morphologically P. hamatus and P. baldaccii but differs from them in having a shorter female stylet [36]. The morphology and morphometry of the Spanish population from Martos, Jaén province (Southern Spain) (Table 6, Figure 16) agrees with the original species description and other populations described, as well as molecularly in various molecular markers to populations sequenced for this species [24]. Minor differences were found in longer female body (345-441 µm vs. 290-350 µm), higher a ratio (23.5-27.2 vs. 19-24) and longer stylet (24-29 µm vs. 21-23 µm) in comparison to paratypes. However, later species descriptions increased the overlapping ranges for some important characters as longer body (285-475 µm), longer stylet (19.0-26.1 µm) [24]. In our case the morphometrics and molecular data were coincident with Singh et al. [24] and increase the morphological traits range of the species even further than the original description of the species. The presence of males is also reported in the Spanish population. The integrative taxonomical identification of this population confirms the morphometrical plasticity of this species. Only one population has been found in an almond orchard in Martos, Jaén province with 4735 individuals per 500 cm 3 of soil. This species has not been described before in Spain and constitutes a first report for the country and expand their distribution in Europe.    Paratylenchus israelensis (Raski, 1973) Siddiqi, 1986 This species was described in Shiller, Israel [79]. It is characterized by a strong sclerotization of the lip region. This species is similar to P. sheri, from which differs in its longer more robust stylet, stronger sclerotization of the lip region and different outline of lateral field in cross section [36]. The Spanish populations from two localities in Córdoba province (Southern Spain) fit the original description of P. israelensis and no differences were found (Table 6, Figure 17). Soil populations from almond orchards in Córdoba (sample code PR_011) and in Valenzuela (sample code PR_079) showed 368 and 320 individuals/500 cm 3 of soil, respectively. No symptoms of decline were apparently detected in the trees. This species has not been described before in Spain and constitute a first record for the country.

Measurements and Ratios
Molecular characterization: Five D2-D3 of 28S rRNA (MW798301-MW798305), four ITS (MW798343-MW798346) and two COI (MW797019-MW797020) gene sequences were generated for the first time from this species without intraspecific sequence variations, except for the ITS sequences (differing by 1 nucleotide and 2 indels). The closest Paratylenchus

Molecular characterization:
One sequence from all regions were generated in this study, MW798300, MW798342 and MW797018 (D2-D3 of 28S rRNA, ITS and COI gene, respectively) being all of them identical to several accessions from P. holdemani deposited in GenBank, such as MW413642 for the D2-D3 of 28S, MW413596 for the ITS region and MW421652 for the COI gene [24].
Paratylenchus israelensis (Raski, 1973) Siddiqi, 1986. This species was described in Shiller, Israel [79]. It is characterized by a strong sclerotization of the lip region. This species is similar to P. sheri, from which differs in its longer more robust stylet, stronger sclerotization of the lip region and different outline of lateral field in cross section [36]. The Spanish populations from two localities in Córdoba province (Southern Spain) fit the original description of P. israelensis and no differences were found (Table 6, Figure 17). Soil populations from almond orchards in Córdoba (sample code PR_011) and in Valenzuela (sample code PR_079) showed 368 and 320 individuals/500 cm 3 of soil, respectively. No symptoms of decline were apparently detected in the trees. This species has not been described before in Spain and constitute a first record for the country. MW413606-MW413609, and finally sequences from COI gene regions showed a similarity of 90% (37 nucleotides) with the accession (MW421675-MW421682). However, morphologically and morphometrically P. israelensis and P. neoamblycephalus can be clearly separated by: labial framework (with strong sclerotization vs. light sclerotization), lip region shape (conical with protruding submedian lobes surrounding the oral aperture vs. conical-truncate with submedian lobes indistinct), and stylet length (24-26 μm vs. 26-34 μm) [36].  Molecular characterization: Five D2-D3 of 28S rRNA (MW798301-MW798305), four ITS (MW798343-MW798346) and two COI (MW797019-MW797020) gene sequences were generated for the first time from this species without intraspecific sequence variations, except for the ITS sequences (differing by 1 nucleotide and 2 indels). The closest Paratylenchus spp. was P. neoamblycephalus described in Singh et al. [24] with 99% similarity for the D2-D3 of 28S rRNA (differing by 7 nucleotides) to MW413660-MW413663, for the ITS sequences the similarity was 95% (differing by 45 nucleotides and 13 indels) with MW413606-MW413609, and finally sequences from COI gene regions showed a similarity of 90% (37 nucleotides) with the accession (MW421675-MW421682). However, morphologically and morphometrically P. israelensis and P. neoamblycephalus can be clearly separated by: labial framework (with strong sclerotization vs. light sclerotization), lip region shape (conical with protruding submedian lobes surrounding the oral aperture vs. conical-truncate with submedian lobes indistinct), and stylet length (24-26 µm vs. 26-34 µm) [36].
Paratylenchus tenuicaudatus Wu, 1961. Described from Ontario, Canada in soil around roots of T. pratense, T. repens L., Acer saccharum Marsh, M. sativa, and grass sod. It has also been reported in several localities of USA [77,80] and Iran [81]. The Iranian population of P. tenuicaudatus was also morphologically and molecularly similar to Paratylenchus sp. 1 from USA [23], suggesting that Paratylenchus sp. 1 from USA is conspecific with P. tenuicaudatus as mentioned by Esmaeili et al. [81].
The four Spanish populations of P. tenuicaudatus detected in our study agree with original description and others populations with molecular data available (Table 8, Figure 18). Only minor differences were found in all populations measured in a slightly shorter female body in comparison to the original description (354-392 µm, 307-407 µm, 292-389 µm, and 394-414 µm vs. 381-600 µm), but with a good size matching with the Iranian population (305-365 µm); slightly posterior positon of the vulva in the Caravaca population (sample code PR_124) (82.1-84.7% vs. 77.6-81.6%) and longer female stylet in Sástago population (sample code PR_208) (32.0-33.5 µm vs. 25.1-31.5 µm). Molecularly the Spanish populations are identical to the populations with molecular data available from Iran [81] and USA population (identified as Paratylenchus sp. 1) [23]. These four populations have been found in the rhizosphere of almond in Caravaca, Murcia province (sample code PR_124), peach in Calasparra, Murcia province (sample code PR_129), peach in Sollana, Valencia province (sample code PR_168) and apricot in Sástago, Zaragoza province (sample code PR_208) with 212, 10149, 15.050 and 12.950 individuals/500 cm 3 of soil, respectively. This species has been previously reported in Navarra forests (North Spain) by Hernández et al. [52], but no detailed morphology and measurements were provided.
Molecular characterization: Four D2-D3 of 28S rRNA (MW798306-MW798309), three ITS (MW798347-MW798349), and three COI gene sequences (MW797021-MW797023) were obtained from this species without intraspecific sequence variations. COI sequences were generated the first time in this study. D2-D3 of 28S rRNA sequences were shown to be 99% similar to the accession KU291239 (differing by 4 nucleotides) and ITS sequences were identical to the accession KF242260 [24,81].
The five Spanish populations of P. veruculatus agree with the original species description and other populations [24,75,83,84] tively. This species has not been reported before in Spain and constitute a first record for the country.      Molecular characterization: Six D2-D3 of 28S rRNA (MW798310-MW798315) with a intraspecific sequence variation of 2% (differing from 0 to 13 nucleotides and 1 indel), five ITS (MW798350-MW798354) (96% similarity; 27 nucleotides and 6 indels), and finally, six COI gene sequences (MW797024-MW797029) with a intraspecific sequence variation of 5% (differing from 0 to 19 nucleotides). The D2-D3 of 28S rRNA sequences matched well with other accession of P. veruculatus deposited in GenBank showing similarity values from 98 to 99% (differing from 1 to 10 nucleotides and 1 indel) with MW413687, and from 94 to 95% for the COI gene sequences (differing from 19 to 21 nucleotides) with MW421717 [24]. ITS sequences (MW798350-MW798354) from P. veruculatus were generated for the first time in this study.
The ITS rRNA gene alignment (778 bp long) included 84 sequences of 48 Paratylenchus species and three outgroup species [Hemicriconemoides californianus (KF856557), Hemicriconemoides alexis (KF856562) and Hemicycliophora poranga (KF430598)]. Thirty-seven new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the ITS alignment is given in Figure 21. The tree contained one highly supported major clade (PP = 1.00) and other clade not supported (but highly supported if excluding P. idalimus (KF242275)). These clades were partially coincident with previous studies with in some case similar or different clade support [24,28].
The COI gene alignment (340 bp long) included 161 sequences of 34 Paratylenchus species and three outgroup species [Hemicriconemoides californianus (KM516192), Hemicycliophora floridensis (MG019867) and Hemicycliophora poranga (MG019892)]. Seventy-one new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the COI sequence alignment is given in Figure 22. The tree contained one highly supported major clade (PP = 1.00) and one moderately supported clade (PP = 0.75). These clades were partially coincident with other studies with in some case similar or different clade support [24].
Paratylenchus zurgenerus sp. nov. was closely related to Paratylenchus sp. 7 SAS-2014 (KF242242) in D2-D3 tree (most probably is conspecific with P. zurgenerus sp. nov.) and to P. microdorus (MW413654) and an undescribed species Paratylenchus sp. BE11 (MW413672), and in ITS tree also related to P. microdorus (MW413597) and an undescribed species Paratylenchus sp. BE11 (MW413617) (Figures 20 and 21). COI marker did not show a clear relationship with other species, but this species was clearly separated from the other species in a unique clade ( Figure 22).  P. holdemani, P. enigmaticus, P. veruculatus, and P. goodeyi . Species with new molecular information but already described in the literature include P. israelensis and P. baldaccii. Paratylenchus israelensis is closely related to P. neoamblycephalus C Belgium (MW413662) in a moderately supported clade (PP = 0.84) in the D2-D3 domains of the 28S rRNA, in a high-supported clade (PP = 1.00) in the ITS region (MW413609), and a low-supported clade (PP < 0.70) in the COI tree (MW421675-MW421681) (Figures 20-22). Paratylenchus baldaccii is closely related phylogenetically with P. pedrami sp. nov. as described before (Figures 20-22).

Figure 21.
Phylogenetic relationships within the genus Paratylenchus. Bayesian 50% majority rule consensus tree as inferred from ITS rRNA sequence alignment under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Posterior probabilities of more than 0.70 Figure 21. Phylogenetic relationships within the genus Paratylenchus. Bayesian 50% majority rule consensus tree as inferred from ITS rRNA sequence alignment under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Posterior probabilities of more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. The scale bar indicates expected changes per site.
Species identification with molecular markers deposited in GenBank was completely congruent with their phylogenetic position as it the case for P. hamatus, P. tenuicaudatus, P. holdemani, P. enigmaticus, P. veruculatus, and P. goodeyi (Figures 20-22). Species with new molecular information but already described in the literature include P. israelensis and P. baldaccii. Paratylenchus israelensis is closely related to P. neoamblycephalus C Belgium (MW413662) in a moderately supported clade (PP = 0.84) in the D2-D3 domains of the 28S rRNA, in a high-supported clade (PP = 1.00) in the ITS region (MW413609), and a low-supported clade (PP < 0.70) in the COI tree (MW421675-MW421681) (Figures 20-22). Paratylenchus baldaccii is closely related phylogenetically with P. pedrami sp. nov. as described before (Figures 20-22).

Discussion
This research is the major study of pin nematodes of the genus Paratylenchus carried out in Spain, increasing their biodiversity, confirming the great cryptic morphology among several species, and expanding the number of species of this genus with molecular information for their unequivocal identification. Herein, we provide detailed morphological and molecular data on 27 studied populations from Spain and also discuss and confirm the existence of cryptic species as suggested in other plant-parasitic nematodes [85,86]. More specifically, four new species were described and additionally of them, five out of eight species identified are considered first reports in Spain (P. enigmaticus, P. hamatus, P. holdemani, P. israelensis, and P. veruculatus). This study also confirms that we have only found just a minor part of the species was already reported in Spain as P. baldaccii, P. goodeyi and P. tenuicaudatus [44,48,52], indicating that the biodiversity of this group is far to be adequately explored in Spain and many of the data need to be revisited and complemented with molecular tools for their accurate identification by integrative taxonomy. However, our data in the present research were mainly based on agricultural systems and some of these species previously reported in Spain may not be coincident with our present species found mainly in Prunus plantations. Some of our data, even for new species, reinforces the idea that some of these species might be pathogenic in some periods of the year or feed with other plant hosts different from the main crop observed at the time of the soil sampling. This idea is reinforced because the majority of the species described here are in an arrested juvenile stage at the moment of sampling. Additionally, many ecological requirements of these nematodes are fairly deciphered (degree of soil humidity, temperature, etc.), and further studies are required for clarifying these aspects.
This study gave molecular markers for the first time for several Paratylenchus species for their accurate identification in an integrative taxonomic approach (including molecular and morphological traits). This is even more important where the presence of cryptic speciation is clearly detected in this genus (as for example, P. aquaticus, P. straeleni or P. hamatus [23,24]), with an excellent new example described in this study (i.e., P. indalus sp. nov., P. projectus, P. neoprojectus and P. enigmaticus). Several authors studied and give molecular markers in an integrative approach for this genus [21,23,24,[26][27][28][29]. Singh et al. [24] gives a DNA-based species delimitation study using different calculation approaches and the markers most congruent between species separation by morphology and molecular identification was COI and D2-D3 region of 28S rRNA. These same authors recommend the use of multilocus approaches (D2-D3 region of 28S rRNA and COI) for a posterior double-check for contamination, sequencing errors of mitochondria-specific pitfalls [87]. Our data agrees with this result, but the phylogeny with COI marker was not completely congruent with the ribosomal molecular markers, and also some clades were low-supported. Phylogenetic analyses based on D2-D3, ITS, and partial COI gene using BI resulted in a consistent position for the newly described species of Paratylenchus species from Spain, and mostly agree with the clustering obtained by other authors [24,28]. Although the position of some species varies, probably due to the large number of species and additional molecular diversity included in this study. The molecular markers for this genus match with our identified species as mentioned in results, giving evidence that they could help in the identification process for the majority of our species. In our study, the majority of the species showed no or low intraspecific molecular variability for ribosomal and mitochondrial regions irrespective of the geographic origin of the population, with illustrative examples in P. indalus sp. nov., P. goodeyi or P. hamatus. The highest intraspecific variability for COI and ITS regions (95%, 96%, respectively) was found in P. veruculatus. Singh et al. [24] detected also important COI gene sequence variations within some species such as P. enigmaticus, P. microdorus and P. veruculatus, despite these sequences were originated from the same population. Usually, the most variable marker for plant-parasitic nematode species separation is COI, followed by ITS, D2-D3 region of 28S rRNA and partial 18S rRNA [85,88]. However, in this study we found a different molecular variability among the different molecular markers between our four populations of P. indalus sp. nov. and molecular markers for P. enigmaticus in Canada [21] and Belgium [24]. In this case, the variability is higher or similar for the D2-D3 region of 28S rRNA marker than for ITS region and COI (96% vs. 94-96% and 97% similarity for D2-D3 region of 28S rRNA, ITS region and COI, respectively when compared both species). This point is reinforced because many sequences were obtained for P. indalus sp. nov. (ten D2-D3 of 28S rRNA, thirteen ITS, and four COI gene sequences) from the four different populations and all populations showed no intraspecific variation, except for the ITS sequences, where one variable position was found. This result is difficult to explain, and reinforces the idea that more than one marker is necessary for the molecularly species identification in this group of nematodes. In this case, the low molecular differences for COI marker needs to be explored in further studies with different mitochondrial markers or different primers for this gene.
Interestingly, the morphology in some complex species did not match the phylogenetic results, for example, the "P. hamatus complex" of species has some species closely related phylogenetically, but other far related such as P. pedrami sp. nov. and P. baldaccii, but these later related morphologically. Clear morphological characters did not match our phylogenies, for example the long stylet length (>40 µm) for some species did not correspond with a unique clade (P. straeleni, P. goodeyi, P. idalimus.) as this character seems evolved independently several times in the different phylogenetic trees. Two clades of species seem to evolved from an exclusive long stylet ancestor, for example clade II (P. idalimus, P. sinensis and others) or the subclade (P. colinus, P. audriellus, P. aculentus, P. paralatescens, P. nanjingensis and P. peraticus) in the D2-D3 region of 28S rRNA tree, but not clearly supported in the other trees in this study or only partially in specific subclades (ITS region and COI). In this sense, the use of fresh material is essential to observe several characters in these nematodes as the presence/absence of stylet in male and juveniles, advulval flaps, lateral field incisures, and other characters difficult to visualize in the glycerin mounted specimens.
The distribution of many of these Paratylenchus species using an integrative approach showed that these species are more widely spread among continents that suspected. Cases as P. hamatus, P. tenuicaudatus, P. enigmaticus, . . . ) shows us that these nematodes have a potential to be distributed on soil remains even with the absence of plants because of their resistant strategies not properly studied. As commented before, the majority of our samples are based on cultivated soils, but probably a higher diversity could be present in samples from wild environments.

Conclusions
This study describes and provides unequivocal molecular markers for the identification of different Paratylenchus species found in Spain. This is particularly important in this group of nematodes as only a few morphological characters can be used in their identification. As pointed in this and other studies, molecular markers could help in their identification, even when morphological characters might be variable and not overlapping ranges can be found. In summary, the present study confirmed the cryptic diversity of Paratylenchus species in Spain and comprises a good example of morphostatic speciation of pin nematodes in Spain. However, this genus is started recently to be studied by integrative taxonomy and increasing numbers of examples of cryptic diversity is expected to be found in the future.  Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.