Integrative Taxonomy Reveals Hidden Cryptic Diversity within Pin Nematodes of the Genus Paratylenchus (Nematoda: Tylenchulidae)

This study delves into the diagnosis of pin nematodes (Paratylenchus spp.) in Spain based on integrative taxonomical approaches using 24 isolates from diverse natural and cultivated environments. Eighteen species were identified using females, males (when available) and juveniles with detailed morphology-morphometry and molecular markers (D2-D3, ITS and COI). Molecular markers were obtained from the same individuals used for morphological and morphometric analyses. The cryptic diversity using an integrative taxonomical approach of the Paratylenchus straeleni-species complex was studied, consisting of an outstanding example of the cryptic diversity within Paratylenchus and including the description of a new species, Paratylenchus parastraeleni sp. nov. Additionally, 17 already known species were identified comprising P. amundseni, P. aciculus, P. baldaccii, P. enigmaticus, P. goodeyi, P. holdemani, P. macrodorus, P. neoamblycephalus, P. pandatus, P. pedrami, P. recisus, P. sheri, P. tateae, P. variabilis, P. veruculatus, P. verus, and P. vitecus. Eight of these species need to be considered as first reports for Spain in this work (viz. P. amundseni, P. aciculus, P. neoamblycephalus, P. pandatus, P. recisus, P. variabilis, P. verus and P. vitecus). Thirty-nine species of Paratylenchus have been reported in Spain from cultivated and natural ecosystems. Although we are aware that nematological efforts on Paratylenchus species in Southern Spain have been higher than that carried out in central and northern part of the country, the present distribution of the genus in Spain, with about 90% of species (35 out of 39 species, and 24 of them confirmed by integrative taxonomy) only reported in Southern Spain, suggest that this part of the country can be considered as a potential hotspot of biodiversity.

The taxonomic consideration for several genera of pin nematodes sensu lato historically included in this group comprise Gracilacus, Paratylenchoides, Gracilpaurus, Cacopaurus, has been recently discussed by Singh et al. [3] concluding that all these genera were confirmed as synonyms with Paratylenchus since no clear separations were detected under phylogenetic relationships of ribosomal and mitochondrial genes [3]. Stylet drives the feeding habit approaches, with the final aim to disentangle the real biodiversity of these nematodes in cultivated and natural environments in Spain. The first one dealt with pin nematodes associated with cultivated Prunus spp. in Spain, including almond, apricot, cherry, nectarine and peach [4]. This study tries to understand the biodiversity of Paratylenchus spp. in some almond samples and additional new natural environments as well as re-analyzing some previous studies carried out by our laboratory 30 years ago based on morphology and morphometry only [9,17,20], but now using more accurate and precise integrative taxonomical approaches.
In the genus Paratylenchus, species display a particular resting-stage which accumulates in soil under adverse environmental conditions (viz. drought conditions) [4]. This state is non-feeding, molting to adults after stimulation by host-plant roots, and may provide some useful data for species identification [25,26]. Usually the resting stage is fourth-stage juvenile (J4), but third-stage (J3) appears in other species, recognized by granular body contents and presence/absence of stylet [2,26]. In P. straeleni, all juveniles had a well-developed stylet and pharynx, while the body of J4 contained numerous dark granules and this is considered the resting stage [26]. However, in close-related species such as P. steineri, stylet and pharynx are well-developed in second-and third-stage juveniles (J2 and J3), but J4 had no stylet and pharynx is much reduced. Morphological changes in stylet morphology in juveniles of some Paratylenchus species need to be studied with regard to adult state. In this research we study the stylet morphology of quiescent juvenile stages (J4) based on an integrative taxonomical approach [4].
The main objectives of this study were to (i) conduct identification with morphological and morphometrical approaches of some Paratylenchus species collected in several nematode surveys on almond and natural environments in Spain; (ii) provide molecular characterization of several species using ribosomal (D2-D3 expansion segments of 28S rRNA, Internal Transcribed Spacer region (ITS) rRNA) and the mitochondrial region cytochrome c oxidase subunit 1 (COI); (iii) study phylogenetic relationships within Paratylenchus spp. using the obtained molecular markers.
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 smooth lines. Lip region rounded, truncate, submedian lobes almost indistinct; with very slight sclerotization. Stylet flexible, 11.3-14.6% of body length. Conus of stylet 2.4-3.5 times longer than shaft, 73-80% of total stylet length. Stylet knobs small, 2.5-3.0 µm across, laterally directed. Procorpus cylindrical, about 50 µm long. Excretory pore situated at distal end of basal pharyngeal bulb. Hemizonid conspicuous, located two annuli anterior to excretory pore. Valvular apparatus in metacorpus 6.0-7.0 µm long, at 58-70% of pharynx length from anterior end. Basal pharyngeal bulb pyriform. Ovary outstretched, spermatheca almost spherical, 21 (19)(20)(21)(22)(23)(24)(25)(26)(27)(28) µm wide, filled with rounded sperm 1.0-1.5 µm in diameter. Lateral vulval membranes, 5.5-6.0 µm long. Tail elongate-conoid gradually tapering to form a rounded terminus, 0.5-0.8 times as long as vulva-anus distance.    * 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.
Male: Less common than females (ratio ca. 1:4). Male body is slender than female body, 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 81.5 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.

Diagnosis and Relationships
The new species can be characterized by the presence of four lateral lines in lateral field, advulval flaps present, and a moderately long female stylet of 53.5 (52.0-56.0) µm. Lip region rounded, truncate, submedian lobes almost indistinct; with very slight sclerotization, continuous with the rest of the body. Spermatheca spherical. Tail elongate-conoid gradually tapering to form a rounded terminus. According to species grouping by Ghaderi et al. [2] belongs to group 10 characterized by stylet length more than 40 µm, four lateral lines and advulval flaps present.
Morphologically and morphometrically, the new species is very close to P. straeleni, and can be also similar to P. goodeyi and P. ivorensis Luc and de Guiran, 1962. In fact, the description of the Spanish population agrees well with original description by De Coninck [27], and other populations from The Netherlands, Poland, Italy, Czech Republic, Iran, USA, Turkey and Belgium [3,6,10,26,[28][29][30], and no major differences in morphology or morphometry can be detected. Consequently, based on the molecular markers, this is an extraordinary example of cryptic species within the P. straeleni-complex species, and this can help to clarify the identity of other populations with similar morphology and morphometry. From P. goodeyi can be differentiated by lip region shape (conoid-rounded to truncate vs. conoid) [2], and from P. ivorensis in a posterior position of vulva (80.2-83.5 vs. 73-77).

Molecular Characterization
Seven D2-D3 of 28S rRNA (MZ265064-MZ265070), four ITS (MZ265004-MZ265007) and four COI gene sequences (MZ262208-MZ262211) were generated for this new species without intraspecific sequence variations, except for the ITS where only one variable position was detected. The closest species to P. parastraeleni sp. nov. was P. straeleni, being 95% similar for the D2-D3 region (MZ265064-MZ265070) (differing from 32 to 38 nucleotides and no indels) to several accessions deposited in GenBank. For the COI gene sequences (MZ262208-MZ262211), the similarity values were 93 and 94% (differing from 21 to 26 nucleotides and no indels) from P. straeleni sequences deposited in GenBank; finally, the similarity for the ITS region was 86-88% (differing from 89 to 111 nucleotides and 35 to 43 indels) from P. straeleni sequences deposited in GenBank. All molecular markers studied clearly separate both species. Due to the presence of more than one species in the same soil sample, J4 individual identification for morphological-morphometrical analysis was based on a molecular barcoding using the 28S rRNA markers, and nematodes with identical sequences as adults were considered as the same species, in this case, P. parastraeleni sp. nov.

Etymology
The species epithet, parastraeleni, refers to Gr. prep. para, alongside of and resembling, because of its close resemblance to Paratylenchus straeleni.

Type Material
Holotype female, 17 paratypes females, 5 fourth-stage juveniles and 4 male paratypes (slide numbers CAZ_05-01 to CAZ_05-12) were deposited in the Nematode Collection of the Institute for Sustainable Agriculture, CSIC, Córdoba, Spain, and four females deposited at the USDA Nematode Collection (slides T-7511p and T-7512p). Brown, 1959 ( Figure 4, Table 3).   According to species grouping by Ghaderi et al. [2] this species belongs to group 9 characterized by stylet length more than 40 µm, three lateral lines and advulval flap absent. The Spanish population from Coto Ríos, Jaén province, was characterized by long flexible stylet 67.5-75.0 µm, lip region rounded and continuous with body contour, female tail subacute to finely rounded, and spermatheca ellipsoid and filled with sperm, which indicates that males are required for reproduction but their numbers are lower than females. J4 not found. Morphometrics of the Spanish population agree well with original description as well as other populations with small differences in stylet length (67.5-75.0 µm vs. 61.0-69.0 µm), which may be due to geographical intraspecific variability [2]. This species was described from Canada and has been reported in USA, several European countries, including the recent integrative identification from Belgium [3], and this study comprises the first report from Spain. Although ribosomal markers (D2-D3 and ITS) between the Spanish population of P. aciculus and the Belgian population of P. aculentus are quite similar (see below), these species can be separated by COI (see below), and by clear differences in stylet length (67.5-75.0 µm vs. 52.4-61.2 µm), advulval flap (absent vs. small advulval flap present), and spermatheca shape (ellipsoid vs. rounded) [3].

Molecular Characterization
Five D2-D3 of 28S rRNA (MZ265071-MZ265075), four ITS sequences (MZ265008-MZ265011), and three COI sequences (MZ262212-MZ262214) were obtained for this species. In both ribosomal genes, no intraspecific variability was detected, however, one variable position was found between the three COI sequences included in this study (MZ262212-MZ262214). Ribosomal genes (MZ265071-MZ265075, MZ265008-MZ265011) showed a high similarity with P. aculentus, being 99% (2 out of 698 bp difference) and 98% (11-12 out of 742 bp difference) similar for the D2-D3 (MW413626-MW413628) and ITS region (MW413588-MW413589), respectively. However, the separation of both species is possible using the COI gene (MZ262212-MZ262214), since for this marker the similarity found was 89% (differing by 40-41 nucleotides and no indels) with the accessions belonging to P. aculentus (MW421639-MW421641).  Table 4). According to species grouping by Ghaderi et al. [2] this species belongs to group 3 characterized by stylet length less than 40 µm, four lateral lines and advulval flaps present. The Spanish population from La Iruela, Jaén province, was characterized by a conoid-truncate lip region with submedian lobes indistinct, a female tail finely rounded to acute, and a rounded spermatheca filled with sperm, which indicates that males are required for reproduction but their numbers are lower than females. J4 bearing a delicate stylet. Some morphometric differences with original description include slightly larger body length (335-450 µm vs. 320-370 µm), slightly shorter stylet length (16.0-18.0 µm vs. 17.0-19.0 µm), and slightly posterior position of vulva (78.6-82.8 vs. 76.0-80.0), which may be considered as intraspecific variability. This species is very close morphologically and morphometrically to P. tateae, from which they can be separated by lip region (conoidtruncate and submedian lobes indistinct vs. conoid narrow, with anterior end flattened and protuberant submedian lips) ( Figure 5), as well as by molecular markers (see below). This species has only been reported from original description in the rhizosphere of grasses (Leymus mollis (Trin.) Pilg.) at Adak Island, Alaska (USA) [32], and this consists of the first report from Spain and the second written record.
Paratylenchus baldaccii, P. enigmaticus, P. holdemani, P. neoamblycephalus, P. pedrami, and P. veruculatus have been previously recorded within recent studies of pin nematodes in Spain [4,15], and morphological and morphometrical data of them were coincident with previous reports. Consequently, only some morphometric data or D2-D3 sequences had been reported here for these nematode samples. Paratylenchus baldaccii was identified in grasses at Arroyo Frío, Jaén province, in the same sample that we previously identified a population of P. vandenbrandei [17]. These data suggest that most probably the previous record of P. vandenbrandei [17] needs to be considered as P. baldaccii, as well as other reports [16,33], but additional studies need to be carried out to confirm these potential misidentifications on the basis of application of integrative taxonomy. Paratylenchus baldaccii has been reported in several localities at south and southeastern Spain, including Jaén, Granada and Murcia provinces [4,15,22,34]. Paratylenchus enigmaticus was detected in the rhizosphere of grasses at campus Alameda del Obispo, Córdoba; this report confirms a wider distribution than previously estimated, since it was detected only in the rhizosphere of cherry at Northeastern of Spain at La Almunia, Zaragoza province [4]. Paratylenchus holdemani has been recently reported in the rhizosphere of almond at Martos, Jaén province [4]. This new report under a natural environment (wild olive) at St. Maria de Trasierra, Córdoba province, also suggests that this species can be common in Andalucia (Southern part of the Iberian Peninsula). Finally, P. neoamblycephalus was confirmed by molecular and morphometrical data under a natural environment (Portuguese oak forest). Unfortunately, only a mature female was detected (Table 5), but morphometrics agree with original description [35] and recent data by Singh et al. [3]. Consequently, up to our knowledge, this is the first report of this species for Spain. Finally, the new findings of P. pedrami and P. veruculatus from natural environments (wild olive) at Córdoba province confirms also that these species are widely distributed in Spain [4].

Molecular Characterization
Several populations of species already molecularly characterized in previous works, such as P. baldaccii, P. enigmaticus, P. holdemani, P. neoamblycephalus, P. pedrami, and P. veruculatus have been sequenced herein. All sequences obtained for these species matched well with the accessions from the same species deposited in GenBank, showing similarity values from 99 to 100% [3,4]. Oostenbrink, 1953 ( Figure 6, Table 6).    20     This species has been detected in several samples of almond and natural environment (wild olive) in several localities of Córdoba and Jaén provinces (Table 1). Morphology and morphometrics of adult females are coincident with the original description and recent studies [3,4]. However, in none of the previous studies on this species J4 were studied under an integrative taxonomic point of view. In all of our populations, irrespective of cultivated almond fields or natural environments, all the J4 of this species were characterized by bearing a short rigid and straight stylet (15.0-18.5 µm), lip region-truncate with labial framework sclerotization strong; with numerous dark granules into the body ( Figure 6, Table 6), and considered the resting-stage [26]. In the original description of P. goodeyi it is mentioned that "J3 and J4 from soil samples, which probably belonged to this species, on account of the typical shape of the lip region, all had a short spear below 20 µm" [36]. However, this is the first report documenting, by morphometric and molecular markers (see below), a clear stylet and lip region metamorphosis between J4 and adult female, from short rigid stylet and conoid-truncate lip region with strong labial sclerotization moving to a long and slender flexible stylet and a conoid-rounded lip region without labial sclerotization ( Figure 6). These data suggest, that apart from the reserve dark granules for resting during adverse environmental conditions (such as the hard drought during the summer season in Mediterranean climates), J4 of P. goodeyi is ready for feeding on susceptible roots during the beginning of the next season. Except for the stylet and lip region, J4 showed similar morphology to adult females with a posterior body rounded terminus. The present reports extend the geographical distribution of this species in Spain which has been already reported in several provinces including Navarra [12], Jaén [20,21], Barcelona [19], and Córdoba [4].
This species was described from vegetables from Poland [39] and has been reported from the Netherlands, Germany and Belgium [34], and New Caledonia [40]. This is the second report from Spain, the first being from natural environments in Almeria province [18].

Molecular Characterization
Six D2-D3 sequences of 28S rRNA (MZ265108-MZ265113), five ITS (MZ265034-MZ265038), and six COI gene sequences (MZ262239-MZ262244) were generated for P. macrodorus without intraspecific sequence variations for ribosomal genes, and J4 and adult female sequences were identical, confirming the identity of these juvenile individuals as P. macrodorus. Paratylenchus macrodorus showed high molecular similarity with P. pandatus and P. wuae, being 99% similar for the D2-D3 of 28S rRNA (varying from 3 to 7 nucleotides and no indels). For the ITS region, similarity values found for P. macrodorus ranging from 96% (34 nucleotides and 13 indels) to 98% (13 nucleotides and 2 indels) to P. wuae (KM061783) and P. pandatus (MZ265041-MZ265042), respectively. Similarity values detected in the COI gene were lower than in the ribosomal genes, being 96% (14 nucleotides and no indels) to P. wuae and 94% (24 nucleotides and no indels) to P. pandatus. However, morphologically and morphometrically P. macrodorus, P. pandatus and P. wuae can be clearly separated (see above).  Table 8). According to species grouping by Ghaderi et al. [2] this species belongs to group 10 characterized by stylet length more than 40 µm, four lateral lines and advulval flaps present. The Spanish population from Caravaca, Murcia province, was characterized by moderately long flexible stylet 57.0-68.5 µm, lip region rounded, continuous with body contour, with distinct submedian lobes, spermatheca elongate and filled with sperm, which indicates that males are required for reproduction but were not detected, female tail tapering gradually to rounded terminus. J4 was similar to female, except for absent stylet (stages confirmed belonging to this species by molecular markers). Morphometrics of the Spanish populations agree well with the original description, as well as Vietnam population with small differences in stylet length (57.0-68.5 µm vs. 63.0-70.0 µm), V ratio (74.5-77.7 vs. 70.0-76.0), and shape of tail terminus (finely rounded in Spanish and Vietnam populations while almost acute in original description), which may be due to geographical intraspecific variability [2]. This species was described from grapefruit in Nigeria [37] and has been reported from Vietnam [41] and Ethiopia [42], and this study comprises the first report from Spain. This species is closely related molecularly to P. macrodorus, but they have important morphological differences such as the presence vs. absence of advulval flaps and J4 without stylet vs. J4 with stylet.

Molecular Characterization
Two identical D2-D3 of 28S rRNA (MZ265116-MZ265117), two identical ITS sequences (MZ265041-MZ265042) and five identical COI gene sequences (MZ262247-MZ262251) were obtained from P. pandatus in the present study. Sequences obtained from J4 and females for all genes were identical, confirming that are the same species. Paratylenchus pandatus showed high molecular similarity with P. macrodorus and P. wuae, being 99% similar for the D2-D3 of 28S rRNA (varying from 7 to 8 nucleotides and no indels). For the ITS region, the similarity values were from 97% to 98% (differing by 13-15 nucleotides and from 2 to 6 indels) with P. macrodorus and P. wuae, respectively. Similarity values detected in the COI gene were lower than in the ribosomal genes, being 93% (23 nucleotides and no indels) to P. wuae and 94% (24 nucleotides and no indels) to P. macrodorus. Siddiqi, 1996 ( Figure 9, Table 9).     According to species grouping by Ghaderi et al. [2] this species belongs to group 3 characterized by stylet length less than 40 µm, four lateral lines and advulval flaps present. The Spanish population from Arroyo Frío, Jaén province, was characterized by a short stylet 14.5-16.0 µm with rounded basal knobs, lip region rounded to truncate, continuous with body contour, indistinct submedian lobes, spermatheca rounded and filled with sperm, which indicates that males are required for reproduction but were not detected, female tail ventrally arcuate, tapering gradually to rounded terminus. J4 was similar to female, except for absent stylet (stage confirmed belonging to this species by molecular markers). Morphometrics of the Spanish population agree well with original description from Colombia [43], with small differences in stylet length (14. , which may be due to geographical intraspecific variability. This species was described from Llanos Oriental in Colombia [43] and this study comprises the first report from Spain. This species is morphologically close to P. microdorus, from which can be differentiated by vulva-anus distance with regard to tail length and tail terminus, and probably has been misidentified in some previous records with P. microdorus, therefore additional studies need to clarify the real biodiversity in the P. microdorus-species complex in Spain by applying integrative taxonomy.
This species was described from Digne, France [44], and has been reported in Spain [17,18] and Italy [28]. This population was from the same locality as that reported by Gomez-Barcina et al. [17], which was confirmed by Prof. Raski [17]. The species was recently synonymized with P. israelensis by Ghaderi et al. [2] based on similar morphology, including strong labial sclerotization. However, the present results together with the recent integrative taxonomical diagnosis of P. israelensis [4] demonstrated that both species are closely related morphologically and molecularly (see below) and need to be considered as nominal valid species. This species has also been reported in Iran [45]; however, the single D2-D3 sequence provided for this Iranian population was 99.7% similar to P. tateae from Spain and Canada (see below) and needs to be revised by the authors.

Remarks on Paratylenchus variabilis
The Spanish population from Córdoba, Córdoba province, was characterized by a rounded lip region with indistinct submedian lobes, continuous with the rest of the body, short stylet 14.0-16.0 µm long, spermatheca oval and filled with sperm, which indicates that males are required for reproduction but were not found, and female tail narrows gradually to a bluntly rounded terminus. J4 with similar morphology to that of adult females, except sexual characters and shorter body length and stylet. This species was described from California and Utah [46] and has been reported in Israel and Iran [30], and this study comprises the first report from Spain. This species is morphologically close to P. microdorus, from which can be differentiated by the shape of female tail terminus, and probably has been misidentified in previous records with P. microdorus, therefore, additional studies need to clarify the real biodiversity in the P. microdorus-species complex in Spain by applying integrative taxonomy. In addition, P. variabilis is morphologically and morphometrically almost indistinguishable from P. zurgenerus [4], from which it can be separated by molecular markers (see below), and both can be considered as cryptic species.
2.1.11. Remarks on Paratylenchus verus (Brzeski, 1995) Brzeski, 1998 ( Figure 13, Table 12).   According to species grouping by Ghaderi et al. [2] this species belongs to group 10 characterized by stylet length more than 40 µm, four lateral lines and advulval flaps present. The Spanish population from Sta. Maria de Trasierra, Córdoba province, was characterized by a rounded lip region with distinct submedian lobes, continuous with the rest of the body, long flexible stylet 79.0-97.0 µm long, excretory pore opposite to median bulb, spermatheca oval and filled with sperm, which indicates that males are required for reproduction but were not found, and female tail narrows gradually to a rounded terminus. J4 with similar morphology to that of adult females, except sexual characters and shorter body length and stylet. This species was described from Texcoco, Mexico [28], and this study comprises the first report from Spain. Several females of the Spanish population had conspicuous infections of Pasteuria sp. on cuticle, especially on anterior and posterior ends ( Figure 13).

Molecular Characterization
Four D2-D3 of 28S rRNA (MZ265130-MZ265133), five ITS (MZ265054-MZ265058) and four COI (MZ262268-MZ262271) gene sequences were generated for the first time from this species, including J4 and adult females, without intraspecific sequence variations, except for the ITS sequences with 98-100% similarity (differing from 2 to 11 nucleotides and 0 to 2 indels). The closest Paratylenchus spp. was P. idalimus being 96% similar (22 nucleotides and no indels) for the D2-D3 of 28S rRNA, 90% similar for the ITS region (differing by 69-75 nucleotides and from 21 to 23 indels) and, finally, 93% for COI sequences (MW411839) (differing by 26 nucleotides and no indels).  Table 13). According to species grouping by Ghaderi et al. [2] this species belongs to group 11 characterized by stylet length more than 40 µm, four lateral lines in and advulval flaps absent. The Spanish population from Córdoba, Córdoba province, was characterized by a conoid-rounded lip region with distinct submedian lobes, continuous with the rest of the body, long flexible stylet 62.0-70.0 µm long, spermatheca elongate and filled with sperm, which indicates that males are required for reproduction but not found, and female tail finely rounded. J4 with similar morphology to that of adult females, except sexual characters and shorter body length and stylet. Morphometrics of the Spanish population agree well with original description with small differences in stylet length ( ). This species was described from Manipur, India [47], and this study comprises the first report from Spain.

Molecular Characterization
Six D2-D3 of 28S rRNA (MZ265136-MZ265141) and four ITS (MZ265059-MZ265062) with one and two variable positions, respectively, and three identical COI gene sequences (MZ262272-MZ262274) were generated for this species, including sequences from J4 and adult females. The closest Paratylenchus spp. was P. teres with 97% similarity for the D2-D3 of 28S rRNA (differing by 25 nucleotides) to MN088376. Unfortunately, no data for ITS or COI from P. teres are available in the GenBank.

Distribution of Paratylenchus spp. in Spain
In the exhaustive review of the geographical distribution of Paratylenchus species in cultivated and natural environments in Spain, we detected that pin nematodes exhibited a wide distribution across an extensive variety of herbaceous and woody hosts, including 39 species (Figure 15). It should be noted that the highest diversity seems to be associated with southern Spain (Andalucia), with 35 out of 39 species in the country ( Figure 15). Although the data suggest that the nematode survey efforts were higher in southern than in central and northern parts of the country, the biodiversity of Paratylenchus in Andalucia is really remarkable (Figure 15). In any case, the Paratylenchus species distribution observed herein revealed that this genus is adapted to a wide variety of host plants and heterogeneous environmental conditions (climatic, edaphic) from all over the country (ca. 1000 km across north-south, and ca. 600 km across east-west).

Phylogenetic Analyses of Paratylenchus spp.
The D2-D3 domains of the 28S rRNA gene alignment (702 bp long) included 148 sequences of 64 Paratylenchus species and three outgroup species (Basiria gracillis (DQ328717), Aglenchus agricola (AY780979), and Coslenchus costatus (DQ328719)). Seventy-eight new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the D2-D3 alignment is given in Figure 16. The tree contained two moderately supported clades (PP = 0.94, PP = 0.84). These clades are mainly coincident with other recent studies on Paratylenchus spp. [3,4]. The new species, P. parastraeleni sp. nov., clustered with several accessions of P. straeleni from Belgium, Iran, South Africa, and Turkey, but clearly separated into two different subclades (PP = 1.00) (Figure 16). Newly sequenced species clustered in separated clusters and subclusters, viz. P. variabilis, P. amundseni, P. recisus, P. verus, P. macrodorus, P. pandatus, P. vitecus and P. aciculus, but with mixed stylet patterns (long and flexible stylet > 40 µm with conus representing about more than 70% of the total stylet and short and rigid stylet < 40 µm with conus about 50% of the total stylet) within the main clusters, except for a basal clade moderately supported (PP = 0.84) comprising 14 species with stylet > 40 µm, including the four species newly sequenced herein (P. aciculus, P. macrodorus, P. pandatus, and P. vitecus) (Figure 16). The ITS rRNA gene alignment (836 bp long) included 117 sequences of 55 Paratylenchus species and three outgroup species (Hemicycliophora lutosa (GQ406237), H. wyei (KC329575) and H. poranga (KF430598)). Fifty-nine new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the ITS alignment is given in Figure 17. The tree contained two highly supported major clades I and II (PP = 0.99 and PP = 1.00, respectively) and several subclades ( Figure 17). Clade I includes mostly species with short stylet (<40 µm), but also species with long stylet (>40 µm), including all isolates of P. goodeyi, the new species P. parastraeleni sp. nov., P. straeleni, P. verus and P. idalimus (Figure 17). Clade II mostly includes species with long stylet (>40 µm), but also species with short stylet (<40 µm), including P. baldaccii, P. pedrami, P. jasminae, P. minor, and P. rostrocaudatus (Figure 17). These clades were partially coincident with previous studies with, in some cases, similar or different clade support [3,4]. The COI gene alignment (384 bp long) included 245 sequences of 51 Paratylenchus species and three outgroup species (Hemicriconemoides californianus (KM516192), Hemicycliophora floridensis (MG019867) and H. poranga (MG019892)). Sixty-seven new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the COI sequence alignment is given in Figure 18. The tree contained four major clades, but only one basal clade (IV) was well supported (PP = 1.00), including two unidentified Paratylenchus species and P. verus and P. idalimus, and all others (clades I, II, and III) low supported (PP < 0.70 to 0.89). The P. straeleni-complex clustered in a well-supported subclade (PP = 1.00) within clade II, and the new species, P. parastraeleni sp. nov., was clearly separated from all other isolates of P. straeleni from Belgium, Canada, Ireland, and USA ( Figure 18). Similar as in ribosomal markers, stylet length patterns (> or <40 µm) were mixed in clusters II and III, whereas cluster I comprises species with short stylets and clade IV species with long stylets (Figure 18). These clades were partially coincident with other studies with, in some cases, similar or different clade support [3,4].

Discussion
This research comprises the second part focused on the integrative taxonomical identification of pin nematodes of the genus Paratylenchus in Spain. These results increase the number of species with morphological and molecular data for their unequivocal identification, as well as confirming the huge biodiversity of this group including the description of a new species viz. P. parastraeleni sp. nov., within the P. straeleni-complex.
Eighteen Paratylenchus spp. from nine different localities, including almond and natural environment soil samples, were identified. All of them except one, were already known (P. amundseni, P. aciculus, P. baldaccii, P. enigmaticus, P. goodeyi, P. holdemani, P. macrodorus, P. neoamblycephalus, P. pandatus, P. pedrami, P. recisus, P. sheri, P. tateae, P. variabilis, P. veruculatus, P. verus, and P. vitecus), and eight considered as first reports for Spain in this work (viz. P. amundseni, P. aciculus, P. neoamblycephalus, P. pandatus, P. recisus, P. variabilis, P. verus and P. vitecus). Finally, one of the 18 species detected was identified as a new species, P. parastraeleni sp. nov., which confirmed the cryptic diversity within the P. straeleni-species complex group by applying integrative taxonomical approaches verifying an outstanding example of the cryptic diversity. Overall, the results of this and previous studies reported a total of 39 species of Paratylenchus in Spain, widespread in cultivated and natural ecosystems.
In Paratylenchus spp. with longer stylet (>40 µm) most juveniles bear elongate flexible stylet (formerly belonging to the genus Gracilacus), but some species are found to have what appears to be fourth-stage juveniles with very length reduced and rigid stylets, a characteristic most frequently found in species of Paratylenchus sensu stricto with female stylets of 40 µm or less [36]. Since many soil samples from natural environments comprise mixed species (even four different species), it is very difficult to associate specimens of one developmental stage with the appropriate adult state [3,4]. However, applying integrative taxonomical approaches (molecular barcoding of juvenile and adult individuals) we can accurately study juvenile and adult forms in each soil sample. For the first time, morphological and molecular data (D2-D3, ITS and COI for the same individual) of J4 for the majority of the species detected in this study were provided herein, allowing the first report for authenticating a clear example of stylet and lip region metamorphosis between J4 and adult female. Within several isolates of P. goodeyi studied here, we verified that short rigid stylet and conoid-truncate lip region with strong labial sclerotization in J4 moved to a long and slender flexible stylet and a conoid-rounded lip region without labial sclerotization in adult females. Apart from the unequivocal identification of juvenile stages of each species, the integrative taxonomical identification of J4 allows to document some important biological aspects for some species, as well as a useful tool for the species identification in periods when the resting-stage accumulates predominantly in soil under adverse environmental conditions (viz. drought conditions) [3,4].
Although we are aware that nematological efforts on Paratylenchus species in Southern Spain have been higher than that carried out in central and northern parts of the country, the present distribution of the genus in Spain, with about 90% of species (35 out of 39 species, and 24 of them confirmed by integrative taxonomy) only reported in Southern Spain, suggest that this part of the country can be considered as a potential hotspot of biodiversity. Nevertheless, further research is needed to definitely confirm this hypothesis. This study also ratifies the previous proposed hypothesis [4] that we have only deciphered just a small part of the species diversity of pin nematodes reported in Spain, indicating that the biodiversity of this group is far from being adequately explored all over the world [3,4]. The present data also suggest that species richness was higher in natural environments than in cultivated areas, since the number of Paratylenchus species detected within the same sample in natural environments included four different species (viz. P. holdemani, P. macrodorus, P. pedrami, and P. veruculatus in wild olive sample code as AR_102), and more than 60% of soil samples from natural environments exhibited at least two Paratylenchus species, whereas in the majority of samples from cultivated areas only one or maximum two mixed species were frequently detected in the same sample [3,4,8]. Nevertheless, this hypothesis needs to be contrasted with further investigations.
Paratylenchus microdorus has been extensively reported in Spain in cultivated and natural environments [15][16][17][18]. The present results, comprising integrative studies on some geographical areas with previous records of P. microdorus, suggest that probably this species was misidentified in previous records. In this case, P. recisus, P. variabilis, P. veruculatus, and P. zurgenerus, are close morphologically to P. microdorus, but molecularly well separated. This study suggests that previous records of P. microdorus could be misidentified, since only detailed traits can separate these species (short differences in stylet length, shape of tail terminus, vulva anus distance with regard to tail length), and therefore additional studies are needed to clarify the real biodiversity in the P. microdorus-species complex in Spain by applying integrative taxonomy. Probably, this potential misidentification can also be referred to the numerous records of P. microdorus in other countries such as Bulgaria, Germany, Hungary, Poland, and Romania [2], which need further investigations. Molecularly, P. microdorus-species complex was separated in two subgroups, one comprising P. microdorus, P. recisus and P. zurgenerus, and another very separate subclade including P. variabilis and P. veruculatus, being consistent for ribosomal and mitochondrial genes.
The genus Paratylenchoides was proposed by Raski [44] to accommodate two Paratylenchus species populations from France and Israel with heavy sclerotization in the lip region and narrow lip region dorso-ventrally. This action was partially followed by Siddiqi [48] proposing a subgeneric rank within the genus Paratylenchus. However, Raski and Luc [49] considered that differences between Paratylenchoides and Paratylenchus were minor and cannot be considered important to separate both taxa, synonymizing Paratylenchoides with Paratylenchus. The present results confirm that, molecularly, P. sheri and P. israelensis (formerly Paratylenchoides species) clustered together in two separate subclades in D2-D3, ITS and COI trees, but always together with other Paratylenchus species with long and short stylets, such as P. neoamblycephalus, P. veruculatus or P. parastraeleni sp. nov. and P. goodeyi and cannot be considered a separate genus as it is the case for Gracilacus already discussed [3,4,6].
The results obtained in the present study, reinforce the idea that for accurate identification of Paratylenchus spp. it is essential to carry out an integrative identification, including morphological, morphometrical and molecular analysis, the latter of which should be based on multilocus approaches (D2-D3 region of 28S rRNA and COI) [3,4,6]. In our case several species demonstrate low differences in ribosomal markers (98-99%) among species, but clear differences on COI and are also clearly different morphologically. This situation has been observed among P. sheri-P. israelensis-P. neoamblycephalus, P. macrodorus-P. pandatus-P. wuae or between P. aciculus-P. aculentus. This is because mitochondrial DNA display a high mutation rate and maternal inheritance, which also enables better discrimination of closely related species [50,51]. On the other hand, several species showed some molecular intraspecific variability in the three regions studied herein (0.5-4%) but with identical morphology and morphometry, such as P. goodeyi, P. enigmaticus.
Phylogenetic analyses based on D2-D3, ITS, and COI gene using BI mostly agree with the clustering obtained by other authors [3,4,6]. Ribosomal and mitochondrial phylogenies did not separate the long stylet length (>40 µm) with the short stylet length (>40 µm) supporting the synonymy of Gracilacus and suggesting that stylet length in Paratylenchus has evolved independently several times during the evolution of this genus [3,4,6].  (Table 1). Samples were collected using a shovel and considering the upper 5-40 cm depth of soil. Nematodes were extracted from a 500-cm 3 subsample of soil by centrifugal flotation [52].

Nematode Sampling and Morphological Identification
A total of 232 individuals including 160 females, 5 males and 67 juveniles were used for morphological and morphometrical analyses. Specimens for study using light microscopy (LM) and morphometrical studies 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 [53] as modified by De Grisse [54]. The developmental stage of the juveniles was determined according to the body length and the degree of development of genital cells [26]. 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) [55], 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 [3,4,8]. 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) [3,4,8], and the sequencing of specific DNA fragments (described below) confirmed the identity of the nematode species for each population. Specimens for SEM observations were processed using Wergin's method [56], coated with gold and observed with a JEOL 50A scanning electron microscope at 10 kV of accelerating voltage.
Nematode populations of Paratylenchus species already described were analyzed morphologically and molecularly in this study and proposed as standard and reference populations for each species given until topotype material becomes available and molecularly characterized. Voucher specimens of these described species have been deposited in the nematode collection of Institute for Sustainable Agriculture, IAS-CSIC, Córdoba, Spain.

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 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. Then DNA extraction from single individuals was performed as described by Palomares-Rius et al. [57], and more importantly, for all the 24 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. In addition, male and juveniles conspecificity was proven by single DNA extraction of male or juveniles for each species.
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 one 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 using ExoSAP-IT (Affimetrix, USB products, Kandel, Germany) and used for direct sequencing in both directions with the corresponding primers. The resulting 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 (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) 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 24 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 [3,4,7,62]. Multiple sequence alignments of the different genes were completed using the FFT-NS-2 algorithm of MAFFT V.7.450 [63]. BioEdit program V. 7.2.5 [64] was used for sequence alignments visualization and edited by Gblocks ver. 0.91b [65] in Castresana Laboratory server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html accessed on 13 May 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 [66]. The best-fit model of DNA evolution was achieved using JModelTest V.2.1.7 [67] 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 some 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 [68] was used for visualizing trees from all analyses.

Conclusions
This study reveals the existence of a huge cryptic biodiversity within the genus Paratylenchus, increasing and expanding the diversity of this group in Spain. For the first time, morphological and molecular data (D2-D3, ITS and COI for the same individual) of J4 allowed to authenticate an example of stylet and lip region metamorphosis between J4 and adult females in P. goodeyi (from short rigid stylet and conoid-truncate lip region with strong labial sclerotization in J4 to a long and slender flexible stylet and a conoid-rounded lip region without labial sclerotization in adult females). This study also ratifies the previous proposed hypothesis that we have only deciphered just a small part of the species diversity within pin nematodes reported in Spain and most probably all over the world. Our data also suggest that P. microdorus comprise a complex of species morphologically very close, but molecularly well separated, and therefore additional studies are needed to clarify the real biodiversity within the P. microdorus-species complex in Spain and all over the world by applying integrative taxonomy. Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.