**The Genus** *Pratylenchus* **(Nematoda: Pratylenchidae) in Israel: From Taxonomy to Control Practices**

**Patricia Bucki 1, Xue Qing 1,2, Pablo Castillo 3, Abraham Gamliel 4, Svetlana Dobrinin 5, Tamar Alon <sup>5</sup> and Sigal Braun Miyara 1,\***


Received: 11 October 2020; Accepted: 29 October 2020; Published: 2 November 2020

**Abstract:** Due to Israel's successful agricultural production and diverse climatic conditions, plant-parasitic nematodes are flourishing. The occurrence of new, previously unidentified species in Israel or of suggested new species worldwide is a consequence of the continuous withdrawal of efficient nematicides. Among plant-parasitic nematodes, migratory endoparasitic species of the genus *Pratylenchus* are widely distributed in vegetable and crop fields in Israel and are associated with major reductions in quality and yield. This review focuses on the occurrence, distribution, diagnosis, pathogenicity, and phylogeny of all *Pratylenchus* species recorded over the last few decades on different crops grown throughout Israel—covering early information from nematologists to recent reports involving the use of molecular phylogenetic methodologies. We explore the accepted distinction between *Pratylenchus thornei* and *Pratylenchus mediterraneus* isolated from Israel's northern Negev region, and address the confusion concerning the findings related to these *Pratylenchus* species. Our recent sampling from the northern Negev revealed the occurrence of both *P. thornei* and *P. mediterraneus* on the basis of molecular identification, indicating *P. mediterraneus* as a sister species of *P. thornei* and their potential occurrence in a mixed infection. Finally, the efficiencies of common control measures taken to reduce *Pratylenchus*' devastating damage in protected crops and field crops is discussed.

**Keywords:** *Pratylenchus*; root lesion nematode; pathogenicity; distribution; molecular phylogeny; taxonomy; control management practices

#### **1. Introduction**

Root-lesion nematodes of the genus *Pratylenchus* are migratory endoparasites belonging to the family Pratylenchidae (Nematoda, Tylenchina), with around 100 species recognized today [1–3]. *Pratylenchus* species can cause yield losses of up to 85% of expected production [4], and even higher losses when nematodes interact synergistically with certain soilborne plant pathogens [5]. Hence, *Pratylenchus* species are highly relevant to agriculture.

Israel is a small semiarid country located in western Asia, only 22,000 km2 in size. Despite the fact that the geography of the country is not naturally conducive to agriculture, advanced irrigation, cultivation mastery, use of elite varieties, and the introduction of state-of-the-art agricultural technologies contribute, in practice, to intensive and efficient farming. On the other hand, this success in agricultural productivity along with a diversity of climatic conditions have led to the proliferation of devastating plant-parasitic nematodes. Among them, *Pratylenchus* species are widely distributed in vegetable and crop fields in Israel and are associated with a major reduction in quality and yield. The genus *Pratylenchus* was first reported from Israel in 1957 [6]. Since then, several studies related to this nematode have been published [7–14]. However, these studies are largely scattered. Some of them are published in less accessible local journals, such as master's or PhD theses, or in scientific reports written in Hebrew. In this review, we collected all available information on *Pratylenchus* in Israel, spanning the last few decades, from local Hebrew journals to international peer-reviewed ones, revealing that *Pratylenchus* species are major pests in many crops throughout the country. We provide a comprehensive summary of the occurrence, taxonomy, distribution, diagnosis, and pathogenicity of *Pratylenchus* in Israel, along with an overview of the status and perspectives for *Pratylenchus* research in this country.

#### **2. Overview of Israeli Agriculture**

Agriculture is an important sector for the Israeli economy, representing around 2.5% of Israel's GDP and about 3.5% of its exports. Agricultural production is especially significant in certain areas, such as the Arava, Jordan Rift Valley, and northern Negev, where it provides almost the sole means of livelihood for the population. Although some of these regions are characterized by semiarid land with varied climatic [15,16], topographical, and soil conditions, determination and farming ingenuity have produced maximum yields and crop quality [17]. Among the most common agricultural sectors, vegetable growing has become a specialized skill of Israeli farmers, on the basis of selecting suitable hybrid varieties, fertilizers, irrigation methods, greenhouse covers designed for specific crops, innovative growing tools, and plant protection management. Moreover, vegetable growing exploits the sunshine and high temperatures, providing high-quality vegetables during the competitors' off seasons in other countries. As a result, vegetables account for about 17% of Israel's total crop output value. About two-thirds of Israel's field crops are grown on non-irrigated land. These rain-fed crops include wheat for grains, silage and hay, legumes for seeds, and sunflower for oil. The remaining field crops are summer crops, including cotton, chickpeas, green peas, beans, corn, groundnuts, and watermelon for seeds, most of which are irrigated. Fruit trees mainly include deciduous fruit orchards that are among the main crops in northern Israel, including grapevine, fig, almond, apple, pear, stone fruit, pomegranate, and persimmon, as well as subtropical varieties (citrus, avocado, mango, olive, litchi) and small fruit (various berries).

#### **3. Occurrence of** *Pratylenchus* **Species in Israel**

The most comprehensive survey of Israel's soil nematodes was performed by Cohn et al. [7], wherein 320 soil samples were taken from natural agro-ecosystems, providing a backbone for soil nematode diversity and distribution in Israel (Table 1, Cohn et al. [7]). This survey suggested that in cultivated crops grown throughout Israel, *Pratylenchus* species were among the three most prevalent plant-parasitic nematodes infecting vegetables (49% of the samples, see below), cereal and pasture grasses (68%), pasture legumes (48%), and deciduous fruit trees (47%). Less commonly, *Pratylenchus* species were found in natural vegetation fruit trees (35%) and forest trees (30%), and in cultivated crops of subtropical and tropical fruit trees (20%), grapevines (29%) and lawns (27%) (Table 1). Geographically, *Pratylenchus* was most prevalent in the Negev, located in southern region of the country (54%), while its abundance in the rest of the geographical locations ranged between 33 and 49% [7].


**Table 1.** Percentage occurrence of plant-parasitic nematode genera in the rhizosphere of plant groups (genera occurring in 20% or more of samples) covering natural vegetation and cultivated crops in Israel [7].

#### **4. Taxonomy and Diversity of** *Pratylenchus* **Species in Israel**

The first species of *Pratylenchus* were identified by Minz in 1957 [6]: *Pratylenchus brachyurus* (Godfrey, 1929), *Pratylenchus neglectus* (=*P. minyus*) (Rensch, 1924), *Pratylenchus penetrans* (Cobb, 1917), and *Pratylenchus scribneri* (Steiner in Sherbakoff & Stanley, 1943). Later, Cohn et al. [7] added three more species: *Pratylenchus pratensis* (de Man, 1880), *Pratylenchus thornei* (Sher and Allen, 1953), and *Pratylenchus vulnus* (Allen & Jensen, 1951), and Corbett (1983) described a new species, *Pratylenchus mediterraneus*. Most recently, Qing et al. [18] added another new species, *Pratylenchus capsici*.

To date, nine species of *Pratylenchus* have been reported throughout the country (Figure 1). Most of these species have only been identified by morphological characteristics, but three of them have been recently confirmed by molecular data. Their distribution and associated plant hosts are detailed below.

**Figure 1.** Map of the known distribution of *Pratylenchus* species recorded in Israel's farming regions. Only recorded infested regions are indicated for each *Pratylenchus* species.

#### *4.1. Pratylenchus mediterraneus Corbett, 1983*

Orion et al. [19] and Krikun and Orion [9] observed an unusual population of *P. thornei* parasitizing potatoes in the northern Negev. After a detailed morphological and morphometric study, Corbett and Clark [20] designated this population as a new species, *P. mediterraneus*. Although the validity of *P. mediterraneus* designation was questioned [21], it is generally accepted as a valid species [1,2,22,23], being further supported by a variety of molecular evidence, such as restriction fragment length polymorphism (RFLP) analysis of ribosomal (r)DNA fragments [24–26], sequences of rDNA D3 expansions [27], and sequences of 18S and 28S rDNA [18]. Morphologically, *P. mediterraneus* is closely related to *P. thornei* in labial region shape en face pattern, and only differs in having a shorter stylet, sexual reproduction, and males being common [28]. Therefore, the identities of several *P. thornei* populations reported from various Middle Eastern countries [29,30] are suspected to be *P. mediterraneus*. The matrix code for *P. mediterraneus* is A2, B2, C2, D2, E2, F3, G2, H1, I3, J1, K1 ([23]; Supplementary Material Table S1; Supplementary Material File S2).

*Pratylenchus mediterraneus* was originally found in the northern Negev region of Israel [14,19]. Later, this species was recorded on chickpea in Turkey [31,32]; chickpea and lentil in Syria [33,34]; legumes in Algeria, Tunisia, and Morocco [33,35]; and chrysanthemum in Korea [36]. In Israel, *P. mediterraneus* primarily parasitizes legumes and cereals, which are the prevalent crops in the northern Negev, but carrot and potato can also be hosts [37]. Hosts reported by the Plant Protection and Inspection Services (PPIS) of the Israeli Ministry of Agriculture and Rural Development currently include alfalfa, barley, beans, broad beans, cabbage, carrot, chickpea, clover, coriander, lovage, sweet potato, vetch, and wheat [38].

#### *4.2. Pratylenchus thornei Sher and Allen, 1953*

In Israel, *P. thornei* has been reported on potato [13], cereals such as wheat and barley [8,10,19], carrots [37], legumes such as *Vicia sativa*, alfalfa and trifolium [39], watermelon [19], and cabbage [38], all in the northern Negev. However, most of these are likely to be *P. mediterraneus* [13,14,37]. Given the similarity between these two species, and the fact that *P. thornei* can occur in a wide range of soil types and is commonly found in mixed populations [39,40], the existence of *P. thornei* as part of a mixed population alongside *P. mediterraneus* in these studies is suspected. Notably, 28S rDNA-based

molecular identification in recent samplings (2018 and 2019) has suggested the wide distribution of *P. thornei* in barley fields in Gevim, Alumim, and Nir-Oz located in the northern Negev (Table 2, Figure 1), and wheat fields in the Khavat Shif'a region and Avuka (Bet Shean Valley) located in the north of Israel [41].



This confirms the presence of *P. thornei* but fails to support the co-existence of *P. mediterraneus* and *P. thornei* within the same field populations. Diagnostic parameters described a labial region with three annuli, not offset from the body, an outer margin of sclerotized labial framework extending conspicuously around two annuli into the body, and one annulus into the labial region; lateral fields with four lines—the outer ones straight or weakly crenate; medium-length stylet (17–19 μm), a spermatheca that is difficult to see and does not contain spermatozoa; and males being very rare. The matrix code for *P. thornei* is A2, B2, C3, D1, E2, F2, G3, H1, I3, J1, K1. According to Castillo and Vovlas [23], it can be distinguished from the closely related species *P. penetrans* and *P. mediterraneus* by labial region shape, stylet length, the low proportion of males, and spermatheca and tail shapes.

#### *4.3. Pratylenchus neglectus (Rensch, 1924) Filipjev and S. Stekhoven, 1941*

In Israel, *P. neglectus* was first recorded by Minz [6] in association with fig tree roots. It is also known as the California meadow nematode, and has been reported by the Israeli Society of Plant Pathology (ISPP) on cotton crops and fig trees [38]. *Pratylenchus neglectus* is characterized by a labial region with two annuli, the second annulus wider than the first, anteriorly indented stylet knobs, a post-vulval uterine sac that is less than or equal to the body diameter, a variably shaped tail that is usually conoid with a little curvature of the ventral surface, and a tail terminus without annulation that is usually rounded but may be obliquely truncate or slightly digitate [23]. The matrix code for *P. neglectus* is A1, B2, C3, D1, E2, F1, G3, H1, I1, J1, K1.

#### *4.4. Pratylenchus vulnus Allen and Jensen, 1951*

*Pratylenchus vulnus* was first recorded by Cohn et al. [7] It is reported to be the most frequently encountered nematode associated with several pome and stone fruit trees, e.g., cherry, pear, plum, olive, apricot, nectarine, mango, persimmon, almond, citrus, fig, peach, and avocado, as well as some ornamentals including roses [38]. It is frequently found in rose nurseries, as well as in loquat, stone fruit, and apple trees in the north of Israel, very often in dense populations [42].

*Pratylenchus vulnus* is characterized by a labial region that is almost continuous with the body contour, with three or four annuli, a pharynx overlapping the intestine ventrally in a long lobe, an oblong spermatheca, a post-vulval uterine sac that is around two vulval body diameters long with a rudimentary ovary, and a tapering tail with a narrowly rounded subacute smooth tip; males are common. The matrix code for *P. vulnus* is A2, B2, C2, D3, E2, F6, G3, H3, I2, J1, K1.

#### *4.5. Pratylenchus pratensis (de Man, 1880) Filipjev, 1936*

*Pratylenchus pratensis* was first recorded by Cohn et al. [7], being described as *Anguillulina pratensis*. *Pratylenchus pratensis* has been found on Chinese cabbage, turnip, cauliflower, kohlrabi, white cabbage, radish, and cabbage by the ISPP [38]. This nematode species is characterized by a finely annulated cuticle, a labial region with three annuli, an oval to rectangular spermatheca, a post-vulval uterine sac length similar to the body diameter, and a tail with 20–28 annuli that are annulated to the terminus [23]. The matrix code for *P. pratensis* is A2, B2, C2, D4, E2, F3, G3, H2, I1, J1, K1. This species can be differentiated from closely related species by stylet length, the position of the vulva, shape of the spermatheca, shape of the tail, tail annuli, tail tip, and the presence of males.

#### *4.6. Pratylenchus capsici Qing, Bert, Gamliel, Bucki, Duvrinin, Alon, Braun-Miyara, 2019*

*Pratylenchus capsici* is an endemic Israeli species that has been recently identified from the roots of pepper [18], currently its only known host, with substantial damage observed. With the type population recovered from Tsofar farm, this species is widely spread across the pepper-growing region in the Arava Rift Valley of Israel. *Pratylenchus capsici* has been shown to be a cryptic species of *Pratylenchus oleae*, as they are almost indistinguishable morphologically. In fact, in the tabular key for *Pratylenchus* species identification proposed by Castillo and Vovlas [23], 10 out of 11 traits were identical for the two species. However, *P. capsici* differs from *P. oleae* in several molecular markers, as well as by several minor morphological differences, including the presence of males in the former, a functional spermatheca (vs. nonfunctional and empty in the latter), a larger body (559–642 for *P. capsici* vs. 412–511 μm for *P. oleae*), and a shorter stylet (14–15 vs. 15–17 μm, respectively) [18]. The matrix code for *P. capsici* is A2, B2, C2, D2, E1–3, F4–5, G2–3, H2, I1–2, J1, K2.

#### *4.7. Pratylenchus penetrans (Cobb, 1917) Filipjev and Stekhoven, 1941*

*Pratylenchus penetrans* was first recorded by Minz [6] in soil from a banana plantation. The ISPP has reported this species on lily, olive, nectarine, buttercup, apple, ruscus, strawberry, and peach [38]. It is also associated with grasses, cereals, and potatoes [42]. It was associated with the decline in pepper plants in the last decade in the Arava in a study carried out from 2004–2007, aimed at elucidating the causal agent of pepper collapse in that region [43]. Later on, *P. penetrans* continued to be identified in other studies as well [44]. Notably, during our intensive sampling of the Arava Rift Valley, *P. capsici* was the only root-lesion nematode associated with pepper. Given that *P. capsici* is morphologically similar to *P. penetrans* and species identification in these studies relied solely on morphology, here we consider that *P. penetrans* reported from the Arava might be *P. capsici*. Further morphology and molecular analyses are needed to confirm the distribution and host range of the former species. *Pratylenchus penetrans* is characterized by a labial region that is slightly offset, low, and flat in front with rounded outer margins, with three annuli; a pharynx overlapping the intestine ventrally; a lobe of around 1.5 body diameters in length; a short, undifferentiated post-vulval uterine sac, and a tail that is generally rounded with a smooth tip. The matrix code for *P. penetrans* is A2, B2, C3, D2, E3, F4, G2, H1, I3, J1, K1. It can be distinguished from closely related species by body and stylet length, number of lip annuli, labial framework, position of the vulva, and shape of the spermatheca and tail terminus [23].

#### *4.8. Pratylenchus scribneri Steiner in Sherbako*ff *and Stanley, 1943*

*Pratylenchus scribneri* was first recorded by Minz [6] in soils of banana, fig, plum, and quince trees. It has also been found on strawberry by the ISPP [38]. According to ISPP nematologists, *Pratylenchus* occurrence in banana plantations throughout Israel is very sparse [42].

This species is characterized by a labial region with two annuli that is slightly offset from the body, a stout stylet with rounded knobs, a pharyngeal overlap of medium length, an oblong spermatheca, and a slightly tapering tail with a smooth terminus. The matrix code for *P. scribneri* is A1, B2, C2, D3, E2, F4, G3, H1, I2, J1–3, K1.

#### *4.9. Pratylenchus brachyurus (Godfrey, 1929) Filipjev and Stekhoven, 1941*

*Pratylenchus brachyurus* was first recorded by Minz [6] and was found associated with other nematodes in soil from Cavendish banana. This species has also been reported on citrus [38].

This species is characterized by a labial region with two annuli, the anterior one showing an angular contour; a stylet with stout, rounded basal knobs; a vulva that is 82–89% of the body length; a post-vulval uterine sac that is less than one body diameter long; an inconspicuous nonfunctional spermatheca; and a tail that is broadly conoid, smooth, and broadly rounded, and truncate or spatulate at the tip. Males are rare. The matrix code for *P. brachyurus* is A1, B2, C4, D1, E4, F3, G3, H1, I4, J2–3, K1.

#### **5. Biology and Pathogenicity of** *Pratylenchus* **Species**

*Pratylenchus* species are polyphagous, migratory root endoparasites, developing and reproducing in the soil or roots. Their life cycle is simple and direct. The female lays its eggs singly or in small groups in the host root or in the soil near the root surface. Although little information is available about the true length of the *Pratylenchus* life cycle, on the basis of laboratory observations, research has estimated it to last from 45 to 65 days [45]. Symptoms caused by *Pratylenchus* are variable and depend on the host; they can include stunted and inefficient plant growth with reduced numbers of tillers and yellowed leaves.

Pathogenicity studies indicate that *Pratylenchus* species are very well adapted to parasitism, as extremely high populations in the soil do not kill their host plants. Nevertheless, damage thresholds range from 0.05 to 30 nematodes/cm<sup>3</sup> of soil. Apart from direct damage to the roots, *Pratylenchus* species may also predispose plants to other pathogens (e.g., *Verticillium* and *Fusarium*), thereby increasing the damage extent [46,47]. Consequently, elimination of the nematode or reduction of its population causes a marked reduction in the incidence of fungi and an increase in crop yield. In Israel, the synergistic relationship between *P. thornei* and the fungus *Verticillium dahliae* caused a significant increase in the populations of both pathogens and in their damage to potato crops in the northern Negev [48].

Among the nine species recorded in Israel, *P. mediterraneus*, *P. thornei*, and *P. capsici* have been relatively more studied, and their biology and pathogenicity are discussed below.

*Pratylenchus mediterraneus* parasitism occurs mainly in the winter, but the nematode can survive for 7–8 months in a state of anhydrobiosis during the hot and dry season [8,49]. It is reactivated by the subsequent winter rains. In a field observation conducted by Orion et al. [10] from 1974 to 1983, the highest population of *P. mediterraneus* (as *P. thornei* in their paper) was recorded in the drought of 1978 and partial drought of 1982, and the lowest population in the unusually wet years of 1980 and 1983. Moreover, nematode populations with auxiliary irrigation treatments were extremely low. These data suggest that low moisture level—the natural condition in the northern Negev region—is a major ecological factor required for *P. mediterraneus* to build up its population, supporting the notion that *P. mediterraneus* is native to the semiarid zones of the Middle East [8,19] or, more specifically, the eastern Mediterranean region [50]. During the long hot season (April–November), the nematode population level remains stable due to anhydrobiosis [8]. In this state, the nematode can withstand conditions of 0% relative humidity, and desiccated nematodes can withstand temperatures of up to 40 ◦C. This characteristic enables their survival and facilitates their field or regional transmission in

the northern Negev, where soil temperatures typically reach 40 ◦C in the hot season. This species is also likely to require the higher temperatures found in the Mediterranean region for its development, but this needs to be further studied.

In contrast to *P. mediterraneus*, the optimal temperature for *P. thornei*reproduction seems to be lower, ranging between 20 and 25◦ C [51,52], suggesting that the northern Negev may not be a suitable area for its survival. However, our molecular- and morphological-based analyses suggested that *P. thornei* is present not only in the mild northern Israel (Mesilot, Avuka, Shif'a), but also in the hot and dry region of the northern Negev [41]. In comparison, *P. mediterraneus* was only recovered from the northern Negev, suggesting that *P. thornei* may be able to adapt to a wider range of environmental conditions than *P. mediterraneus,* with the latter being more specialized for the hot and dry northern Negev.

The pathogenic effect of *P. mediterraneus* is limited to the early plant stages, resulting in reduced foliage and root growth of cereals and legumes, and thus influencing final plant density at harvest [12,14]. *Pratylenchus mediterraneus* was shown to be most concentrated in the root-tip region of hosts *Vicia sativa* and *Trifolium alexandrinum*. A histopathological study using scanning electron microscopy (SEM) showed nematodes penetrating the root epidermis and the cortical parenchyma through a clean-cut hole, probably a result of enzymatic activity and mechanical force [53]. When passing through parenchyma cells, *P. mediterraneus* can consume the cell contents, and these cells are thus void of cytoplasmic structures compared to the prominent nuclei and cytoplasmic structures in adjacent intact cells [12]. Typical symptoms caused by *P. mediterraneus* on common vetch were lesions produced along roots. These lesions lacked root hairs, with necrotic epidermal cells consisting of many holes, leading to severely deformed roots. Similar to *P. penetrans* [54], Orion and co-workers [12,37] speculated that *P. mediterraneus* can infect root tips as ectoparasites as well. Further SEM analysis showed the collapse of the parenchyma cells in the root lesion as the result of nematode feeding activity. The observed destruction was limited to the root cortex with an intact central cylinder, while nematode egg deposition was observed in cavities of the root cortex. These findings were similar to observations of *P. vulnus* in sour orange [55], *P. penetrans* in broad beans [56], and *Pratylenchus zeae* in maize [57].

*Pratylenchus penetrans* and *P. crenatus* Loof, 1960 have been reported worldwide as the major causal agent of carrots and Kuroda-type carrots [58–61]. In an investigation of carrot nematodes in Shoval, located in the northern Negev, we failed to detect these species. Instead, the field was infested with *P. thornei*, resulting in significant quality loss due to forking of carrot taproots [41]. However, whether *P. thornei* is the causal agent of these symptoms still needs to be confirmed, as continuous sampling from carrots demonstrated that the forking symptoms were not necessarily related to nematode occurrence [41].

*Pratylenchus capsici* is an endemic Israeli species that is widely distributed in the Arava Rift Valley, causing significant yield reduction of pepper (Figure 2).

**Figure 2.** Symptoms caused by *Pratylenchus capsici*. (**A**) Pepper plant decline in the Arava Rift Valley characterized by stunted growth and wilting. (**B**) Heavily infected roots, with pronounced lesions along primary and secondary roots. (**C**) Photograph of developed root lesion taken under a dissecting microscope.

The emergence of this species was surprising, as this remote region is isolated from the country's other agricultural areas. Moreover, until 1995, the entire region was free of reported nematodes, mainly due to intensive soil fumigation with methyl bromide [62]. Since the phase-out of this fumigant, certain species of *Meloidogyne* and *Pratylenchus* have become established in the soil, causing substantial damage to vegetable crops. Further biogeographical analysis suggested that a *P. capsici* population in weeds (*Chenopodium album* and *Sonchus oleraceus*) was an important source for *P. capsici* dispersal, either as the original nematode source or in maintaining the population between growing seasons (Figure 3).

**Figure 3.** Weed distribution and function as a reservoir for *Pratylenchus capsici* during and in between growing seasons. (**A**) Weeds emerging early after pepper seedling planting, and (**B**) throughout the pepper-growing season. (**C**) Lesions caused by *P. capsici* on *Chenopodium album* growing alongside the pepper plants.

Similar findings were observed for *P. penetrans* [63], *P. brachyurus* [64,65], *Pratylenchus co*ff*eae* [66], *P. zeae* [67], *P. scribneri* and *P. vulnus* [68], and *P. thornei* and *P. neglectus* [69].

*Pratylenchus capsici* has been shown to survive through the seasons with no host from April to July. During this period, nematode activation is prevented by the high temperature and low moisture in the soil. Extensive nematode extraction from roots and soils yielded a high number of nematodes in the former and low numbers in the latter, supporting its exclusive endoparasitic life strategy. Therefore, these observations raise the question of whether *P. capsici* is ever anhydrobiotic, and if so, whether it goes through anhydrobiosis in the roots or in the soil. Similarly, *P. capsici*'s capacity to migrate to lower soil levels during the off seasons is not known. Further study is needed to clarify this question.

#### **6. Phylogeny and Evolution of** *Pratylenchus* **Species Occurring in Israel**

To date, nine species of *Pratylenchus* have been reported from Israel, with molecular data available for only three of them (*P. thornei*, *P. mediterraneus*, and *P. capsici*) (Figure 4). The concatenated phylogeny based on 18S and 28S rDNA and internal transcribed spacer (ITS) suggests that *P. thornei* and *P. mediterraneus* form a well-supported (posterior probability (PP) = 1, bootstrap (BS) = 100) monophyletic group, concurring with previous studies [18,24].

Orion [50] suspected that *P. mediterraneus* is a native or at least old inhabitant of the semiarid region of the Eastern Mediterranean. Given the similarities in morphology and morphometric features, the overlapping geographical area (Mediterranean region), the same hosts (mostly cereal and legumes), and the anhydrobiotic survival properties, *P. thornei* and *P. mediterraneus* could be derived from recent speciation events, with insufficient time to attain complete morphological differentiation.

Similarly, *P. capsici* is sister to *P. oleae* in concatenated phylogeny (Figure 4, PP = 1, BS = 100), as well as in a previous study [18]. *Pratylenchus oleae* was found in the Mediterranean region, parasitizing both wild and cultivated olive trees in Spain and Tunisia, with the presence of the nematode in wild olive not showing any clear symptoms in the aboveground plant or roots [3]. Interestingly, *P. capsici* was found in both pepper and weeds, markedly damaging the pepper but causing very mild symptoms on the weeds. Later, population genetic analysis revealed that *P. capsici* is likely to have been native to wild grass and transmitted to pepper by a recent expansion [18]. The adjacent distribution, similar morphology and presumably similar transmission background give rise to the idea that the two closely related species, *P. capsici* and *P. oleae*, may be native to the Mediterranean region.

**Figure 4.** Bayesian 50% majority rule consensus tree inferred on concatenated sequences of 28S; asterisks indicate species that were only identified by morphology. The dataset was aligned by MAFFT v. 7.205 [70] using the G-INS-i algorithm. The phylogeny was reconstructed by maximum likelihood (ML) and Bayesian inference (BI) using RAxML v.8.1.11 [71] and MrBayes 3.2.3. [72]. Branch support is indicated in the following order: posterior probability (PP) value from BI analysis/bootstrap (BS) value from ML analysis. Red marked species indicate local Israeli isolates.

#### **7. Control and Management Practices**

Plant growth and yield losses in any nematode–plant interactions depend primarily on soil nematode densities at planting. In the last few decades, intensive studies in Israel have been dedicated to the development of systems-based approaches to reducing soilborne pathogen densities at planting in different climatic regions [73–77]. These studies have shown that soil fumigants with nematicidal properties can reduce nematode infestation level but fail to eradicate the soil nematode, whereas a combination of fumigants with solarization can enhance the killing of soilborne pathogens [73,78,79], emphasizing the importance of using an appropriate combined application of pesticides and solarization.

#### *7.1. The Use of Soil Fumigants*

Three commercial soil fumigants are registered and commonly used in Israel: (i) 1,3 dichloropropene (1,3-D), a liquid fumigant (boiling point 104–112 ◦C) that is considered to be highly effective against nematodes and has been adopted as an alternative to methyl bromide [80]; 1,3,-D is registered for use in the control of all plant-parasitic nematodes and bacterial plant diseases, insects, and weeds. In practice, nematodes are the main target of 1,3-D use on most crops; 1,3,-D is labeled as a pre-planting soil treatment, and its effectiveness is dependent on environmental factors such as length of the growing season, moisture, temperature and soil type. (ii) Metam sodium (sodium N-methyldithiocarbamate, metam-Na) is widely used to control soilborne plant pathogens, mainly fungi and weeds, while its efficacy in the control of plant-parasitic nematodes is

limited [81,82]; because metam sodium undergoes rapid decomposition in moist soils to the active compound methyl isothiocyanate [83], soil fumigation of vegetable crops with metam sodium or metam potassium results in inconsistent control, particularly against root-knot nematodes, while intensive experience indicates its efficiency toward migratory plant-parasitic nematodes but no effect on root-knot nematodes. (iii) Dimethyl disulfide (DMDS), which was registered in the last decade and is effective at controlling both sedentary and migratory nematodes, as well as weeds and soilborne fungal pathogens. Unfortunately, the performance of these three fumigants is inferior to that of methyl bromide. In Israel, the prevalent treatment for nematode management in vegetables is targeted to reducing nematode population density primarily through soil fumigation with 1,3-D or DMDS. However, these fumigants do not provide adequate protection of crop health throughout the entire growing season. Therefore, an integrated approach is needed to achieve successful management of lesion nematodes.

#### *7.2. Common Control* Methods in Used to Manage Plant Parasitic Nematodes

Currently recommended soil disinfestation approaches against soilborne plant-parasitic nematodes in conventional farming—mainly *Pratylenchus* species and *Meloidogyne* species root-knot nematodes—include the following steps [84]: (i) destruction of the plant roots at the end of the crop season before plant removal (Figure 5A); (ii) plant and root removal followed by tillage, although this latter recommendation is not always followed (Figure 5B); (iii) soil disinfection approaches using effective soil fumigants combined with soil solarization for a minimal period of 4 weeks during the summer (Figure 5C). At this time, nets above protected houses are removed to increase soil solarization efficiency, and shade nets are then reinstalled at seedling planting time (Figure 5D).

**Figure 5.** Integrated nematode management. Protocol used in practice to control migratory or sedentary plant-parasitic nematodes. (**A**) Destruction of previous crop's roots before removal to reduce primary inoculum. (**B**) Root removal, tilling, and soil preparation for fumigation and solarization requirements. (**C**) Soil-disinfection approaches using different soil fumigants in combination with soil solarization for at least 4 weeks during the summer. (**D**) Planting of seedlings and reinstallation of shade net.

A combination of solarization with organic material (biosolarization) can reduce nematode densities but not achieve full eradication [85]. Similarly, Oka and Pivonia [86] explored the possibility of using ammonia for controlling soilborne diseases under variable environmental conditions in the Arava region of Israel. Given that soil pH may be the most important factor affecting the nematicidal activity of ammonia, where alkaline soils support better activity [87], as well as the fact that neutral to weakly alkaline sandy soils are common in Israel, the use of ammonia for nematode control is promising [86]. As expected, the use of NH4OH (at 500 and 1000 kg N/ha) increased tomato yield and reduced the galling index (at 1000 kg N/ha). However, despite its positive control effect, a high percentage of ammonia may be deleterious to the environment. This needs to be further evaluated under different soil conditions, nematicidal activities, and application methods. Another approach to exploiting ammonia for nematode control is the application of ammonia generators such as chicken

manure, soy bean meal, and other organic materials [88]. Further studies by Oka et al. [89] demonstrated that application of ammonium sulfate, chicken litter and chitin, or neem (*Azadirachta indica*) extract alone failed to reduce the root galling index of tomato plants, but application of the amendments in combination with the neem extract reduced root galling significantly. Soil analysis indicated that the neem extract inhibits the nitrification of the ammonium released from the amendments and extends the persistence of the ammonium concentrations in the soil. In addition, biosolarization using chicken compost resulted in effective control of root-knot nematodes in a lettuce crop [88].

Field crops that are not under intensive production pose a challenge for nematode management. Orion et al. [10] found that leaving the soil fallow for 2 years reduced the *P. mediterraneus* population by 90% and increased wheat grain yields by 40–90%. By monitoring a 30-year rotation trial over several seasons of wheat-cropping systems, researchers found that the use of legumes (vetch, lentil) can increase *P. thornei* populations, whereas sunflower or safflower followed by a fallow period provided the best reduction of *P. thornei* [90]. Alternatively, soil treatment with metam sodium controlled *P. mediterraneus* by 90% and increased yield by 50–70% [91]. The biannual fallowing system was the most desirable environmentally, but it occupied 50% of the land, which in practice is problematic because cultivated land is quite limited in Israel. Since metam sodium treatment is less feasible in dryland agriculture, several alternative control methods were evaluated. Those studies suggested that nitrogen fertilizer does not affect *P. mediterraneus* populations in either dry farming or as a supplement in irrigation treatments [10]. Use of the nematicide formulation of furathiocarb, a systemic soil insecticide, as a seed dressing could reduce *P. mediterraneus* population level and increase yield, while the best nematode killing was achieved by soil application [11,14].

#### *7.3. Resistance to Root-Lesion Nematodes*

The wide host range of *Pratylenchus* species, and the restrictions, cost, and inefficiency of chemical nematicides have raised the importance of developing resistant cultivars as a control measure [92,93]. Unfortunately, only a few studies have considered the effects of resistance on *Pratylenchus* biology. Talavera and Van Stone [94] demonstrated that *P. thornei* is able to penetrate resistant cultivars. Farsi [95] observed equal root penetration by *P. neglectus* in both resistant and susceptible wheat lines. Other studies in various plant hosts have shown that, in other *Pratylenchus* species, resistance is associated with reduced motility and reproduction [96]. While the major studies of resistance have focused on wheat varieties [5], vegetable crops have been less investigated. The use of resistant cultivars is advantageous in integrated control programs because an accurate assessment of nematode infestations and infections is critical for the evaluation of plant resistance and tolerance to *Pratylenchus* species.

#### **8. Challenges and Perspectives for** *Pratylenchus* **Research in Israel**

In the last decade, several studies have been implemented toward the development of an integrated nematode management system that includes available and efficient means. Like elsewhere, most soil fumigants and nematicides belonging to containing organophosphates and carbamates have been withdrawn from the market or have strict use restrictions, mainly for environmental and safety reasons [97]. In general, there appears to be little prospect for the management of nematodes in many susceptible crops without repeated application of nematicides, which is economically justified in only a few cases. Alternatively, a number of products and formulations of fumigant–nematicides are available for use [98]. However, the effectiveness of traditional fumigants and nematicides with broad biocidal activity is declining, and the development of new classes of nematicides with novel activity and specific pest targets is perhaps an idealistic pipe dream. Recent research carried out in Israel has shown that the incorporation of nematicidal fluensulfone into the soil can reduce the populations of several migratory nematodes under laboratory conditions [44]. An additional new nematicidal compound, fluopyram, has been evaluated in vitro against root-knot nematodes [99], but its effect on migratory nematodes has not yet been confirmed in the field.

#### *8.1. Taxonomy and Diagnosis of Pratylenchus Species*

Given the wide distribution and severe damage caused by *Pratylenchus*, its taxonomy and diagnosis are crucial for *Pratylenchus* research and agricultural production in Israel. Despite its importance, the morphological diagnosis is greatly hampered by phenotypic plasticity, interspecific similarities, and a lack of molecular taxonomy specialists. Today, routine plant-parasitic nematode identification is conducted by the PPIS of the Israeli Ministry of Agriculture and Rural Development using only diagnostic morphological characteristics. The information provided to farmers, agronomists, nurseries, and inspectors consists mostly of identification at the genus level and the density of the nematode population found in the soil or root samples. Similarly, identification of *Pratylenchus* species is limited in most instances to the genus level, while species identification relies on the host from which they were recovered. Thus, molecular barcoding is a powerful, efficient, and reliable tool to simplify and standardize nematode identification, but such a method is not yet fully established for routine identification of *Pratylenchus* species, especially for basic research stations and production departments. Further effort is needed to expand *Pratylenchus* diagnostic techniques and improve farms' awareness of them.

#### *8.2. Control*/*Management of Pratylenchus Species*

Extensive research is being performed on alternative chemical and nonchemical methods for controlling nematode diseases. However, these methods are generally less effective than soil fumigation in reducing soil nematode densities, and many have not proven consistent enough when used in intensive crop farming. Long-term field trials comparing the nematicide efficacies of several soil disinfestation methods would provide valuable information for nematode management. New nematicides are continually being introduced to the market although their efficiency against *Pratylenchus* species is not always known, and if it is, their label should refer to specific hosts, soils, and environmental conditions. Thus, the participation of professional nematologists is crucial in laboratory and field experiments evaluating nematicides. Symptoms caused by *Pratylenchus* species are frequently overlooked and a lack of nematological knowledge might lead to erroneous interpretations. Moreover, the migratory endoparasitic lifestyle, which might support the association of additional plant pathogens, should be studied for each plant–*Pratylenchus* interaction. In such cases, control strategies need to target both the nematode and the associated pathogen. A study of the etiology underlying nematode survival between seasons under extreme conditions is required to address important questions regarding the occurrence of anhydrobiosis, migration ability to lower soil levels, and factors required for these nematodes' recovery. Exploration of these aspects is expected to contribute to the development of efficient integrated control management of *Pratylenchus*.

#### **9. Conclusions**

Delimitation of the various *Pratylenchus* species is considered to be very complicated, especially because of the small number of diagnostic features available at the species level and the intraspecific variability of some of these characteristics [23]. Nevertheless, due to the difficulty in separating species, the number of new proposed species of *Pratylenchus* has increased almost linearly, with a slope of 1.1 species per year between 1940 and 2006 [23]. Although morphology continues to be the basis for identification of *Pratylenchus* species, new technologies based on biochemical and molecular analyses are becoming increasingly important for nematode systematics and practical diagnoses [27,100–102]. New species are continuously being described through extensive morphological and molecular studies of the 28S D2-D3 expansion domains and ITS. The highest biodiversity of the genus is found in Asia, where 40 species have been reported, followed by Europe with 32, North America with 27, Central and South America with 22, Africa with 16, Oceania with 12, and Antarctica with a single species. The most widely distributed and common species are *P. neglectus*, *P. penetrans*, *P. thornei*, and *P. vulnus*, which have been reported on every continent with the exception of Antarctica. Thirty-seven species (54% of the 68

nominal species) in the genus have only been reported from a single continent, while the remaining 31 species (46%) have been reported from two or more continents. Nevertheless, despite the global distribution of the genus, some 32 of the described species have thus far only been recorded from their type locality. Along these lines, it will be interesting to determine whether, similar to *P. mediterraneus*, which was first found in Israel and later in other Middle Eastern countries, the occurrence of *P. capsici* will be identified in neighboring countries as well.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2223-7747/9/11/1475/s1, Table S1: Morphometrics of *Pratylenchus* species reported from Israel. Reference [103] is cited in Table S1; Material File S2: Matrix Key Codes for the identification of *Pratylenchus* spp. according to Castillo and Vovlas.

**Author Contributions:** Conceptualization, S.B.M., X.Q. and P.C.; Methodology, P.B.; Validation, P.B., T.A. and S.D.; Investigation, S.B.M., P.B.; Resources, P.C., S.B.M.; Data Curation, S.B.M.; Writing—Original Draft Preparation, S.B.M., P.C., P.B., X.Q.; Writing—Review and Editing, S.B.M., P.C., A.G.; Visualization, P.C., S.B.M., X.Q.; Supervision, S.B.M., X.Q.; Project Administration, P.B.; Funding Acquisition, S.B.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by The Chief Scientist Ministry of Agriculture and Rural Development grant number 131-4544.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article Pratylenchus penetrans* **Parasitizing Potato Crops: Morphometric and Genetic Variability of Portuguese Isolates**

**Diogo Gil, Joana M.S. Cardoso , Isabel Abrantes and Ivânia Esteves \***

Centre of Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal; diogo.gil12@gmail.com (D.G.); joana.cardoso@uc.pt (J.M.S.C.); isabel.abrantes@uc.pt (I.A.)

**\*** Correspondence: iesteves@uc.pt

**Abstract:** The root lesion *Pratylenchus penetrans* is an economically important pest affecting a wide range of plants. The morphometry of five *P. penetrans* isolates, parasitizing potato roots in Portugal, was compared and variability within and between isolates was observed. Of the 15 characters assessed, vulva position (V%) in females and the stylet length in both females/males showed the lowest coefficient of intra and inter-isolate variability. Moreover, DNA sequencing of the internal transcribed spacers (ITS) genomic region and cytochrome c oxidase subunit 1 (COI) gene was performed, in order to evaluate the intraspecific genetic variability of this species. ITS revealed higher isolate genetic diversity than the COI gene, with 15 and 7 different haplotypes from the 15 ITS and 14 COI sequences, respectively. Intra- and inter-isolate genetic diversity was found considering both genomic regions. The differentiation of these isolates was not related with their geographical origin. In spite of the high intraspecific variability, phylogenetic analyses revealed that both ITS region and COI gene separate *P. penetrans* from other related species. Our findings contribute to increasing the understanding of *P. penetrans* variability.

**Keywords:** COI; cloning; ITS; morphometrics; plant-parasitic nematode; potato; molecular diversity

#### **1. Introduction**

The root lesion nematode (RLN) *Pratylenchus penetrans* (Cobb, 1917) Filipjev and Schuurmans Stekhoven, 1941 is an important migratory endoparasite, often reported as a limiting factor of several herbaceous and fruit crops [1–3]. On potato (*Solanum tuberosum* L.), the nematode causes necrotic lesions on tubers and roots due to migration and feeding, and its presence increases the severity of the "potato early dying" disease caused by *Verticillium dahliae* Kleb. [3,4]. Damage of roots by *P. penetrans* diminishes water and uptake of nutrients, resulting in poor plant growth and consequent crop losses. In Europe and North America, *P. penetrans* has been considered a damaging species associated to potato crop [5–11]. In Portugal, *P. crenatus* Loof, 1960, *P. neglectus* Filipjev and S. Stekhoven, 1941, *P. penetrans* and *P. thornei* Sher and Allen, 1953 have been found parasitizing potato, coexisting frequently in soil with other plant–parasitic nematodes [12]. The correct identification and characterization of *Pratylenchus* species are thus important, for example, to inform and advise farmers on the application of suitable pest management strategies. *Pratylenchus* species can be identified by means of morphological and morphometrical characters but requires specialized expertise since a considerable number of species share many morphological features and most specific differences can only be observed using high magnifications [3,13]. In addition, intraspecific morphological variability has been demonstrated in *P. penetrans* isolates in populations from different geographical locations [14]. To overcome the issues of overlapping morphological and morphometrical characters, identification of RLN should be complemented with the molecular analysis for accurate diagnosis of this group of nematodes [15]. Molecular methods based on restriction fragment length polymorphism (RFLP) analysis of the ribosomal ribonucleic acid (rRNA) genes [16]

**Citation:** Gil, D.; Cardoso, J.M.S.; Abrantes, I.; Esteves, I. *Pratylenchus penetrans* Parasitizing Potato Crops: Morphometric and Genetic Variability of Portuguese Isolates. *Plants* **2021**, *10*, 603. https:// doi.org/10.3390/plants10030603

Academic Editor: Pablo Castillo

Received: 2 March 2021 Accepted: 19 March 2021 Published: 23 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and sequencing of different fragments of the rDNA cluster, including internal transcribed spacers (ITS) [17,18], 18S [19,20] and 26S [19,21,22] have been used for diagnostics of RLN species [15]. Moreover, sequencing of the mitochondrial DNA (mtDNA), cytochrome c oxidase subunit 1 (COI) gene [15,23–27] and the nuclear hsp90 gene [23,24,27–29] have also been largely used in the molecular characterization of RLN species. *Pratylenchus fallax* Seinhorst, 1968, and *P. convallariae* Seinhorst, 1959, were shown to be closely related to *P. penetrans* (96–97% similarity) after sequence analysis of the D2–D3 region of the 28S rRNA gene [22,30]. Phylogenetic analyses of sequences of D2-D3 of 28S rDNA or partial 18S rDNA conducted by Subbotin et al. [19] grouped *P. penetrans* with *P. arlingtoni* Carta and Skantar, 2001, *P. convallariae*, *P. dunensis* de la Pena, van Aelst, Karssen and Moens, 2006, *P. fallax* and *P. pinguicaudatus* Corbett, 1969, in clade IV, forming the Penetrans group. Later, Palomares-Rius et al. [23] added *P. brachyurus* Filipjev and Schuurmans Stekhoven, 1941, and *P. oleae* n. sp. into this clade. Using a combination of phylogenetic data with molecular species delineation analysis, population genetics, morphometric information and sequences, Janssen et al. [15] reconstructed a multi-gene phylogeny of the Penetrans group using the ITS, D2-D3 of the 28S rDNA regions from nuclear rDNA and the COI gene from mtDNA. The authors were able to confirm the taxonomic status of *P. penetrans*, *P. fallax* and *P. convallariae*, clarifying the boundaries within the Penetrans group. In the same study, *P. fallax* populations demonstrated low intraspecific variability whereas *P. penetrans* showed diverse haplotypes, with extremely variable intraspecific variability. Nonetheless, identical *P. penetrans* haplotypes were found to be geographically widespread, suggesting that *P. penetrans* could have spread anthropogenically through agricultural development and crop exchange [15]. Despite *P. penetrans* has already been detected in Portuguese potato crops, little information is still available on the morphometric and molecular variability of these *P. penetrans* isolates. The knowledge acquired in this study can be valuable in help defining effective strategies for RLN management in this crop. Therefore, the objectives of this research were to evaluate the morphometric variability of *P. penetrans* Portuguese isolates and to assess their genetic diversity, geographical and host relations. Information on intraand interspecific variation of *P. penetrans* parasitizing potato increase the awareness of the genetic diversity of this species, and relationships with other *P. penetrans* isolates in other countries and hosts.

#### **2. Results**

#### *2.1. Morphology of P. penetrans Portuguese Isolates*

#### 2.1.1. Female

Body moderately slender, almost straight when heat relaxed, with body length 672.5 (522.1–869.5) μm long (Figure 1A and Table 1). Lip region slightly offset from body, body annules distinct, lip with three annules low flat anteriorly with rounded margins. Stylet stout 16.5 (15.2–17.9) μm long, with knobs varying from rounded to cupped anteriorly (Figure 1C–E). Lateral fields with four straight lines (Figure 1F). Pharyngeal glands overlapping intestine ventrally and slightly laterally. Excretory pore at from anterior extremity located opposite to pharyngo-intestinal junction. Spermatheca rounded, filled with sperm. Post uterine sac 1–1.5 times longer than vulval body diameter. Vulva located at 81.1 (75.7–83.9) % of body length (Figure 1G and Table 1). Tail cylindrical, 30.34 (19.0–41.5) μm long with smooth tip (Figure 1H,I and Table 1).

**Figure 1.** *Pratylenchus penetrans* specimens from Portuguese isolates: **A**—Entire female body, **B** entire male body; **C**–**E**—anterior region; **F**—lateral field; **G**—female posterior region showing vulva; **H**,**I**—female tail variability; **J**—male posterior region; **K**—male tail (ventral side). Scale bars **A**,**B**: 100 μm; **C**–**E**, **J**: 20 μm; **F**–**I**, **K**:10 μm.

**Table 1.** Morphometric characters of *Pratylenchus penetrans* females from Portugal. All measurements are in μm. Data are means of 10 nematodes ± standard deviation (range). In each row means followed by the same letters do not differ significantly at *p* > 0.05, according to the Fisher Least Significant Difference test.



**Table 1.** *Cont.*

\* L—body length; V—position of vulva from the anterior end expressed as the percentage of body length; a—body length/maximum body width; b'—body length/distance from the anterior end to the base of esophageal glands; c—body length/tail length; c'—tail length/tail diameter at the anus.

#### 2.1.2. Male

Males were common in all the isolates, morphologically similar to females for all nonsexual characters but smaller, with body length 555.64 (470.5–670.1) μm long (Figure 1B and Table 2). Lateral field with four lines ending on bursa, spicules slender, gubernaculum ventrally curved. Bursa irregularly crenate along margin, enveloping the tail tip (Figure 1J,K).

**Table 2.** Morphometric characters of *Pratylenchus penetrans* males from Portugal. All measurements are in μm. Data are means of 10 nematodes ± standard deviation (range). In each row, means followed by the same letters do not differ significantly at *p* > 0.05, according to the Fisher Least Significant Difference test.


\* L—body length; V—position of vulva from anterior end expressed as percentage of body length; a—body length/maximum body width; b'—body length/distance from anterior end to base of esophageal glands; c—body length/tail length; c'—tail length/tail diameter at anus.

#### *2.2. Morphometry of P. penetrans Portuguese Isolates*

The morphometric measurements of *P. penetrans* isolates of Portugal were, in average, within the range described by Loof after [31], except for the c ratio in both PpA34L3 females and males, overall body length of PpA24L1 and PpA44L2 males and spicule length of PpA34L3 and PpA44L2 (Tables 1 and 2). Morphometric comparisons using ANOVA revealed a significant degree of intra- and inter-isolate variability on most studied characters. Nine out of fifteen morphometric characters studied in *P. penetrans* females, varied significantly among isolates (*p* < 0.05) (Table 1). Inter-isolate variability was high for the overall body length, anterior end to excretory pore, anterior end to vulva, body width at anus, vulva–anus distance, tail length and ratios b', c and c', whereas the stylet length, distance of anterior end to median bulb, anterior end to pharyngeal gland lobe, maximum body width, V% and ratio a did not vary significantly among isolates (*p* > 0.05). The stylet length and V% had the lowest CV intra and inter-isolates of females and the highest values of CV were found in tail length, vulva–anus distance and b' ratio (Table 3). In males, inter-isolate variability was found in nine out of thirteen morphometric characters: overall body length, anterior end to median bulb, anterior end to excretory pore, spicule length, tail and a, b', c and c' ratios (*p* < 0.05). The stylet length, distance from the anterior end to the tip of esophageal glands, body width at the anus and maximum body width were similar among isolates (*p* > 0.05) (Table 2). The stylet was the least variable character, whereas tail, c' ratio and spicule length were the most variable among isolates, supporting the results given by the ANOVA (Table 4).


**Table 3.** Intra- and inter-isolate coefficient of variability (%) of *Pratylenchus penetrans* females from Portugal.

\* L—body length; V—position of vulva from anterior end expressed as percentage of body length; a—body length/maximum body width; b'—body length/distance from anterior end to base of esophageal glands; c—body length/tail length; c'—tail length/tail diameter at the anus.

**Table 4.** Intra- and inter-isolate coefficient of variability (%) of *Pratylenchus penetrans* males from Portugal.


\* L—body length; V—position of vulva from anterior end expressed as percentage of body length; a—body length/maximum body width; b'—body length/distance from anterior end to base of esophageal glands; c—body length/tail length; c'—tail length/tail diameter at the anus.

#### *2.3. Genetic Diversity of P. penetrans Portuguese Isolates*

ITS sequences of three clones from each isolate were determined and submitted in GenBank database under the accession numbers MW633839-MW633853. For the COI gene, sequences of two clones from isolate PpA21L2 and three clones from the other isolates were determined and also submitted under the accession numbers MW660605-MW660618. A BLAST search against NCBI database of the determined ITS and COI sequences confirmed the species identity, with sequences homologies ranging from 94.7% to 98.4%, and 99.2% to 100.00%, to other *P. penetrans* ITS and COI sequences, respectively, present in the database. The length variation on ITS region of all clones (671–683 bp) and the sequence analysis revealed high variability, not only between isolates but also within isolates, with a high number of polymorphic (S), mutation (Eta) sites and average number of nucleotide differences (k) then the ones found for COI region. All 15 ITS sequences and 14 COI sequences corresponded, respectively, to 15 and 7 different haplotypes (Table 5). Intra-isolate nucleotide diversity (Pi) for the ITS region was lower for the PpA34L3 isolate (Pi = 0.00997) and higher for the PpA44L2 isolate (Pi = 0.06115). For the COI gene, a low number of polymorphic and mutation sites were found considering each isolate or even considering all isolates. The COI intra-isolate Pi was lower for PpA44L2 isolate (Pi = 0.00000), with all three clones being identical, and higher for PpA21L2 isolate (Pi = 0.00509). Considering all isolates, a higher Pi was found for the ITS region (Pi = 0.03350) than for the COI gene (Pi = 0.00587) (Table 5).


**Table 5.** Genetic diversity of cloned ITS and COI regions of five *Pratylenchus penetrans* isolates from Portugal.

\* S—number of polymorphic sites; Eta—total number of mutations; Hd—haplotype diversity; Pi—nucleotide diversity; k—average number of nucleotide differences.

#### *2.4. Phylogenetic and Molecular Evolution Relationships*

Phylogenetic analysis was performed with the alignment of the 15 sequences obtained in this study and other ITS sequences from *P. penetrans*, *P. fallax*, *P. pinguicaudatus* and *P. thornei* present in the GenBank database. Results showed that *P. penetrans* isolates from Portugal clearly group up with other *P. penetrans* isolates but ITS sequences from the same isolates do not group together, reflecting the high intra- and inter-isolate estimated ITS divergence. Additionally, no grouping of isolates belonging to the same country or originated from the same host was found (Figure 2). On the other hand, phylogenetic analysis based on COI sequences revealed lower divergence between sequences from the same isolate and also from different isolates, comparing to the ITS region phylogenetic analysis. All Portuguese *P. penetrans* COI sequences grouped together and with other *P. penetrans* isolates, revealing a closer relationship with one Dutch isolate from apple (KY816941), one African isolate from onion (KY817013) and five American isolates from potato (MK877988; MK877989; MK877990; MK877991 and MK877992) (Figure 3). The differences between the *P. pinguicaudatus*, *P. fallax* and *P. thornei*, included in the phylogenetic analysis, were visible on both trees, as they did not group together (Figures 2 and 3).

**Figure 2.** Neighbor-joining phylogenetic tree based on ITS nucleotide sequences of *Pratylenchus penetrans*, *P. pinguicaudatus* and *P. fallax*. ITS sequence from *P. thornei* was used as an outgroup. Bootstrap values are shown next to the branches and values with less than 50% confidence were not shown. Scale bar represents nucleotide substitutions per site.

**Figure 3.** Neighbor-joining phylogenetic tree based on COI nucleotide sequences of *Pratylenchus penetrans*, *P. pinguicaudatus* and *P. fallax*. COI sequence from *P. thornei* was used as the outgroup. Bootstrap values are shown next to the branches and values with less than 50% confidence are not shown. Scale bar represents nucleotide substitutions per site.

The estimate of evolutionary divergence between sequences of *P. penetrans* Portuguese isolates showed that ITS region diverges by at least 0.01513 base substitutions per site (±0.00487), considering different isolates and that value decreases for 0.00149 (±0.00149), considering intra-isolate divergence (isolate PpA34L3). However, there were ITS sequences from clones from the same isolate with an estimated divergence higher than from distinct isolates. The higher value of base substitutions per site, 0.08121 (±0.01253), was found between the PpA44L2 isolate, clone 1, and PpA24L1 isolate, clone 2 (Table S1). On the other hand, the COI gene revealed much lower nucleotide divergence with a minimum of 0.00000 base substitutions per site (±0.00000), considering both, intra and inter-isolate divergence. A maximum of 0.01821 (±0.00667) base substitutions per base on the COI gene was found between PpA21L2 isolate, clone 1 and PpA44L4 isolate, clone 3 (Table S2).

From neutrality tests, estimated Tajima's D values, using the total number of mutations, were −1.54235 (*p* > 0.05) and −1.03620 (*p* > 0.05) for ITS and COI respectively, indicating that the changes were not significant and all sequences underwent neutral selection. Additionally, the mismatch distribution of both ITS and COI sequences revealed to be a multimodal distribution, with several peaks of pairwise differences, excluding the possibility of abrupt selection events (Figure 4).

0.050

**Figure 4.** Mismatch distribution of ITS (**A**) and COI (**B**) sequences of five Portuguese isolates of *Pratylenchus penetrans*.

The possible correlation of genetic distance and geographic distance of the five *P. penetrans* isolates were also investigated, considering both ITS and COI gene, and there were no significant correlation between this two variables with a Kendall tau of 0.02458 (*p* > 0.05) for ITS region and a Kendall tau of 0.08254 (*p* > 0.05) for the COI gene, showing that geographical distance is not the main factor leading to *P. penetrans* isolates differentiation.

#### **3. Discussion**

In this study, *P. penetrans* isolates from potato in different geographic locations of Portugal were characterized for the first time, using both morphometric and molecular analyses. The comparative morphometrical analyses revealed the presence of substantial inter and intra variability between isolates, although differences fall within the range of the morphometrical variability described previously in *P. penetrans* [3,31]. The body size of these isolates appears to be larger than that described by Rusinque et al. in *P. penetrans* parasitizing amaryllis (*Hippeastrum* × *hybridum*), in Portugal [32]. Spicule size of males and overall body length of the Portuguese isolates were also greater than those observed by Mokrini et al. in populations associated to maize (*Zea mays* L.) in Morocco [33]. Variations in morphometric characters can be caused from differences in fixation methods or changes in environmental conditions [34]. The morphometric characters of Portuguese isolates were recorded on fresh mounted nematodes (not glycerin-infiltrated specimens) and compared with type specimens in permanent mounts, and therefore affected by "shrinkage" due to the fixation process. Environmental conditions, like the host plant, influence morphometric characters such as body length, width, esophagus length, stylet length, V value, a and b ratios and qualitative characters such as tail terminus, growth of ovary and shape of the median bulb [14]. Townshend [35] reported that morphometric variations existed between populations of *P. penetrans* associated with strawberry (*Fragaria* × *ananassa*) and those associated with celery (*Apium graveolens* L.) in Ontario, Canada. Furthermore, variations in size were also found between *P. penetrans* populations recovered from strawberry collected at different geographical areas [35]. In our study, intra- and inter-isolate variability was found in most of the morphometric characters that were analyzed in females and males. However, the results obtained with ANOVA and the analysis of the CV allowed one to verify that the characters V and stylet length proved to be stable among isolates and between replicates within the same isolate. As previously noted by Roman and Hirschmann [13] and Tarte and Mai [14], the stability of these characters confirms its usefulness for discriminating this species. All other morphometrical characters, including those commonly used in nematode taxonomy (body length, body width, anterior end to esophageal glands and a, b', c and c' ratios), have shown relatively high coefficients of variation.

The ITS and COI genomic regions from the five Portuguese *P. penetrans* isolates were selected for sequencing to evaluate the intraspecific genetic diversity of this species. From the two regions, the ITS region revealed higher genetic diversity than the COI gene with 15 and 6 different haplotypes from the 15 ITS and 14 COI sequences, respectively. Besides, inter-isolate genetic diversity also intra-isolate genetic diversity was found in all isolates with exception for one isolate in the COI gene. Sequence comparisons performed by De Luca et al. [17] revealed high intraspecific variability in ITS sequences of several *Pratylenchus* species, including *P. penetrans*. Sequence analyses showed high sequence variability not only between populations or isolates but also within individuals. The same study concluded that ITS sequences allow a clear separation of the *Pratylenchus* species, despite the high intraspecific variability. Janssen et al. [15] also reported intraspecific variability of *P. penetrans* isolates based on sequence analysis and phylogenetic reconstruction of the ITS, D2-D3 regions of 28S rDNA and the COI gene. Furthermore, the phylogenetic analyses based on the sequences of the ITS and D2-D3 regions also confirmed high sequence variability among populations of *P. penetrans* [29].

Despite the high intraspecific diversity found for *P. penetrans* in our studies, phylogenetic analyses revealed that both ITS and COI genomic regions separate *P. penetrans* from other related species, such as *P. pinguicaudatus*, *P. fallax* and *P. thornei*, which is also in accordance with that previously reported [15,17,29]. Additionally, no grouping of isolates belonging to the same country or originated from the same host was found in phylogenetic analyses of both ITS and COI genomic regions. This is in agreement with the no correlation of genetic and geographic distance found among the Portuguese isolates, being the same COI haplotypes from isolates sampled in fields that are more than 90 km away, suggesting that geographical distance is not the main factor leading to the differentiation of isolates. Janssen et al. [15] referred that although the large intraspecific variability recovered in *P. penetrans*, identical haplotypes were found to be geographically widespread and this could be a result of the anthropogenic spread of *P. penetrans* through agriculture development and crop exchange. Our findings contribute to increase the understanding of *P. penetrans* variability.

#### **4. Materials and Methods**

#### *4.1. Pratylenchus penetrans Isolates*

Five *P. penetrans* isolates, obtained previously from potato roots sampled in the north and centre regions of mainland Portugal [12], were used in this study. The isolates were originated from a gravid female and propagated on carrot discs [36]. Isolates PpA21L2, PpA4L1 and PpA34L3 are from potato fields in different geographical locations, whereas PpA44L2 and PpA44L4 shared the same sampling geographic origin (Table 6).

#### *4.2. Morphometrical Analyses*

Twenty individual adults (10 females and 10 males), from each isolate, were mounted into a drop of water and used for the morphometric analyses. Before covering and sealing slides with the coverslips, nematodes were immobilized by gently heating the slide underneath, just enough to stop movement. Nematode measurements were made directly using a DM2500 microscope equipped with a ICC50HD digital camera (Leica Microsystems, Wetzlar, Germany) and LAS 4.8.0 software (Leica) and results compared with previous descriptions for this species [31]. Microscopic observations were made in nematodes without using a fixation method since the software used for nematode measurements allows the capture of the image and simultaneous measurement of specimens, without the need of a preservation method. All measurements were expressed in micrometers (μm). To assess the morphometric variation of the isolates, data was subjected to a one-way analysis of variance (ANOVA) using Statistica® V.7 (StatSoft, Tulsa, Germany), after ensuring that the assumptions of normality and constant variance were met, as checked by using the Shapiro–Wilk and Levene's tests, respectively. Logarithmic and square root transformations were applied to data whenever needed. Following ANOVA, to test differences between

isolates Fisher Least Significant Difference test at the 95% confidence level was applied. The coefficients of variability (CV) were calculated to determine which characters were most stable and more variable among isolates.



#### *4.3. DNA Extraction, PCR, Cloning and Sequencing*

Nematode DNA was extracted from 50 to 100 mix developmental stages of *P. penetrans* PpA21L2, PpA24L1, PpA34L3, PpA44L2 and PpA44L4 isolates (Table 6) using the DNeasy®® Blood and Tissue Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.

Two genomic regions were selected to evaluate the intraspecific genetic diversity of this species, the internal transcribed spacers (ITS) rDNA region containing partial 18S and 28S and complete ITS1, 5.8S and ITS2 sequences and partial cytochrome c oxidase subunit I (COI) gene.

PCR amplifications were carried out using 20–50 ng extracted DNA and 2 U of BioTaq DNA polymerase (Meridian Bioscience, Memphis, TN, USA) in the 1× reaction buffer, 0.2 mM each dNTPs, 1.25 mM MgCl2 and 2.0 μM of each primer, PRATTW81 (5 GTAGGTGAACCTGCTGCTG3 ) and AB28 (5 ATATGCTTAAGTTCAGCGGGT3 ) for ITS region [16] and JB3 (5 TTTTTTGGGCATCCTGAGGTTTAT3 ) and JB4.5 (5 TAAAGAAA GAACATAATGAAAATG3 ) for the COI gene [37]. Reactions were carried out in a Thermal Cycler (Bio-Rad, California, USA) with an initial denaturation step of 95 ◦C for 3 min followed by 35 reaction cycles of 94 ◦C for 30 s, annealing for 30 s at 60 ◦C and 54 ◦C for ITS region and COI region, respectively, extension at 72 ◦C for 30 s and a final extension at 72 ◦C for 7 min. The PCR products were purified using the NucleoSpin®® Gel and PCR Clean-up kit (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions and cloned. Purified ITS and COI amplified products were ligated into pGEM®®-T Easy Vector (Promega, Madison, USA using 50 ng vector in a 10 μL reaction with 3 U T4 DNA Ligase (Promega) and 36 ng purified ITS or 22 ng of COI products in the 1× Rapid Ligation Buffer (Promega). Ligation reactions were incubated for 1 h at room temperature. Then, 2 μL of the ligation product was used to transform *Escherichia coli* JM109 high efficiency competent cells (Promega) following the manufacturer's instructions. Plasmid DNA was extracted from *E. coli* cells using the Nzymini Prep kit (Nzytech, Lisbon, Portugal and three selected positive clones for each genomic region and each *P. penetrans* isolate were fully sequenced in both strands in an Automatic Sequencer 3730xl under BigDyeTM terminator cycling conditions at Macrogen Company (Madrid, Spain).

#### *4.4. Sequence Analysis*

Sequence analysis and alignments were achieved using BioEdit [38]. The region containing primers sequence was removed from all sequence analyses. Homologous sequences in the databases were searched using the Basic Local Alignment Search Tool [39]. Sequence statistics such as number of polymorphic (S) and mutation (Eta) sites, nucleotide diversity (Pi), haplotype diversity (Hd), average number of nucleotide differences (k) and mismatch distributions were estimated using DnaSP 6.12.03 software [40]. Intra-isolate sequence analyses were performed from the alignments obtained with sequences of each isolate and overall sequence diversity with the alignment obtained with all sequences of the five isolates.

#### *4.5. Phylogenetic and Molecular Evolutionary Analyses*

Phylogenetic and molecular evolutionary analyses were conducted in MEGA v10.1.8 software [41]. Phylogenetic trees were constructed by the neighbor-joining method [42] with 1000 replications of bootstrap, with the evolutionary distances computed using the maximum composite likelihood [43] model and ambiguous positions removed for each sequence pair (pairwise deletion option), using the ITS and COI nucleotide sequence alignments of the five isolates used in this study and homologous sequences retrieved from the GenBank database (Table 7). Genetic distance between sequences from the five Portuguese isolates were accomplished by pairwise distance using the maximum composite likelihood model with pairwise deletion option and standard error estimate by a bootstrap procedure (1000 replicates), using the alignments of ITS and COI nucleotide sequences determined in this study. Additionally, Tajima's D neutrality tests [44], which distinguish between a DNA sequence evolving randomly (or neutrally) and one evolving under a non-random process, and mismatch distribution of ITS and COI sequences of Portuguese *P. penetrans* isolates were performed in DnaSP v6.12.03 software.




**Table 7.** *Cont.*

The correlation between genetic and geographic distance of Portuguese *P. penetrans* isolates was also evaluated computing the determined pairwise distance versus the distance between the sampling locations of each of the five isolates. Geographic distance between isolates was calculated using the script available at https://www.movable-type.co.uk/ scripts/latlong.html (accessed on 15 January 2021) with the GPS coordinates of each isolate sampling location (Table 6 and Table S3). The significance of genetic and geographic distance correlation was calculated using Kendall tau rank correlation in Free Statistics Software v1.2.1 [45].

#### **5. Conclusions**

In conclusion, morphometric and genetic diversity were found among *P. penetrans* isolates and this variability was not only a result of the diversity found between isolates but also due to the diversity within each isolate. The information gathered highlights the importance of the knowledge about this relevant plant–parasitic nematode in potato crops, and can be used further in larger genetic studies, focusing this nematode species. Future research should also be conducted to evaluate whether the differences in pathogenicity among *P. penetrans* isolates are related to the observed morphometric and molecular variability.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2223-7 747/10/3/603/s1, Table S1: Data on genetic distance among the 15 internal transcribed spacers (ITS) sequences from the five Portuguese *Pratylenchus penetrans* isolates; Table S2: Data on genetic distance among the 14 cytochrome c oxidase subunit 1 (COI) gene sequences from the five Portuguese *Pratylenchus penetrans* isolates; Table S3: Data on geographic distance among the five Portuguese *Pratylenchus penetrans* isolates. Geographic distance (Km) estimated by the distance between the GPS coordinates of each of the five isolates sampling locations.

**Author Contributions:** Conceptualization, J.M.S.C., I.A. and I.E.; methodology, D.G., J.M.S.C., I.A. and I.E.; software, D.G., J.M.S.C. and I.E.; validation, J.M.S.C., I.A. and I.E.; formal analysis, D.G., J.M.S.C., I.E.; investigation, D.G., J.M.S.C., I.A. and I.E.; resources J.M.S.C., I.A. and I.E.; data curation, D.G., J.M.S.C., I.A. and I.E.; writing—original draft preparation, D.G.; writing—review and editing, J.M.S.C., I.E. and I.A.; visualization, D.G., J.M.S.C., I.A. and I.E.; supervision, I.E., I.A., J.M.S.C.; project administration, I.E.; funding acquisition, I.A., I.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was carried out at the R&D Unit Centre for Functional Ecology-Science for People and the Planet (CFE) with references UIDB/04004/2020, funded by "Fundação para Ciência e a Tecnologia" (FCT)/MCTES through National funds and at the European Regional Development Fund (FEDER), grants: PTDC/AGR-PRO/2589/2014, PTDC/ASP-PLA/31946/2017, POCI-01-0145- FEDER-031946, funded by FCT/FEDER; CENTRO-01-0145-FEDER-000007, funded by the Comissão de Coordenação da Região Centro (CCDR-C)/FEDER; and from Instituto do Ambiente, Tecnologia e Vida (IATV).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in Plants 2021, 10, 603. https://doi.org/10.3390/plants10030603. DNA sequence information were deposited in Gen-Bank database under the accession numbers: MW633839-MW633853 (ITS sequences) e MW660605- MW660618 (COI sequences).

**Acknowledgments:** Ivânia Esteves acknowledges FCT through project CEECIND/02082/2017.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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

**Ilenia Clavero-Camacho 1, Juan Emilio Palomares-Rius 1, Carolina Cantalapiedra-Navarrete 1, Guillermo León-Ropero 1, Jorge Martín-Barbarroja 1, Antonio Archidona-Yuste 2,3 and Pablo Castillo 1,\***


**Abstract:** 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.

**Keywords:** cytochrome c oxidase subunit 1; ITS rRNA; D2-D3 of 28S rRNA; molecular; morphology; phylogeny; rRNA; taxonomy

#### **1. Introduction**

Pin nematodes of the genus *Paratylenchus* Micoletzky, 1922 [1] are obligate plant-ectoparasitic nematodes of small body length (<600 μm) with variable stylet length (10–120 μm), widely dispersed in different natural environments and crops, and distributed worldwide [2–4].

The taxonomic consideration for several genera of pin nematodes *sensu lato* historically included in this group comprise *Gracilacus*, *Paratylenchoide*s, *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

**Citation:** Clavero-Camacho, I.; Palomares-Rius, J.E.; Cantalapiedra-Navarrete, C.; León-Ropero, G.; Martín-Barbarroja, J.; Archidona-Yuste, A.; Castillo, P. Integrative Taxonomy Reveals Hidden Cryptic Diversity within Pin Nematodes of the Genus *Paratylenchus* (Nematoda: Tylenchulidae). *Plants* **2021**, *10*, 1454. https://doi.org/10.3390/plants10071454

Academic Editors: Carla Maleita, Isabel Abrantes and Ivânia Esteves

Received: 13 June 2021 Accepted: 11 July 2021 Published: 15 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and many species have a long stylet (>40 μm), becoming swollen and feeding from deeper layers in the root cortex as sedentary ectoparasites. Two stylet pattern shapes can be found in this genus: (a) long and flexible stylet > 40 μm with conus representing about more than 70% of the total stylet (m ratio), and juveniles with well-developed stylet, initially included in the genus *Gracilacus* Raski [5]; (b) short and rigid stylet < 40 μm with conus about 50% of the total stylet, and juveniles without well-developed stylet, initially included in the genus *Paratylenchus.* Nevertheless, these differences were not sufficiently supported by molecular analyses to separate and maintain both genera [3,4,6]. The genus *Paratylenchus* is a wide diverse group with about 130 nominal species, from which about 54 of them are molecularly characterized [2–4,6–8]. Consequently, about half of the nominal species of this genus are not yet linked to molecular data, and there is a need for completing that information. The conserved morphology that characterizes *Paratylenchus* species led to the development of molecular methods using different fragments of nuclear ribosomal and mitochondrial DNA gene sequences to be used in DNA barcoding [3,4,6–8]. Use of molecular markers in species identification of pin nematodes over the last years has indicated that many widespread species actually comprise multiple genetically divergent and morphologically similar cryptic species [3,4,6–8]. An emblematic example of these species complexes comprises the *Paratylenchus straeleni*-species complex, which Singh et al. [3] distinguished among 4–9 putative species within this complex considering all the available ribosomal and mitochondrial sequences. In 1988, Castillo and Gomez Barcina [9] identified a population of *P. straeleni* (De Coninck, 1931) Oostenbrink, 1960 from a natural environment (Portuguese oak forest, *Quercus faginea* Lam.) at southern Spain based on morphological and morphometric traits. This raises the possibility that this population was potentially misidentified and included under the common and widely distributed species *P. straeleni*. Consequently, this is an excellent opportunity which prompted us to apply integrative taxonomical approaches to unravel the cryptic diversity of this species complex. This study allowed us to verify if this species identification was correct or to prove if close morphology and morphometry with original description comprise some genetic diversity with recent molecularly studied *P. straeleni* populations from Belgium, USA, and Turkey [3,6,10,11].

Thirty species of *Paratylenchus* have been reported in Spain from cultivated and natural ecosystems including *P. aonli* Misra and Edward, 1971 [12], *P. arculatus* Luc and de Guiran, 1962 [13,14], *P. baldaccii* Raski, 1975 [4,15], *P. caravaquenus* Clavero Camacho, Cantalapiedra-Navarrete, Archidona-Yuste, Castillo and Palomares-Rius, 2021 [4], *P. ciccaronei* Raski, 1975 [16–18], *P. enatus* (Raski, 1976) Siddiqi, 1986 [19], *P. enigmaticus* Munawar, Yevtushenko, Palomares-Rius and Castillo, 2021 [4], *P. goodeyi* Oostenbrink, 1953 [20], *P. hamatus* Thorne and Allen, 1950 [4], *P. holdemani* Raski, 1975 [4], *P. indalus* Clavero Camacho, Cantalapiedra-Navarrete, Archidona-Yuste, Castillo and Palomares-Rius, 2021 [4], *P. israelensis* (Raski, 1973) Siddiqi, 1986 [4], *P. macrodorus* Brzeski, 1963 [18], *P. microdorus* Andrássy, 1959 [15–18], *P. minusculus* Tarjan, 1960 [21], *P. mirus* (Raski, 1962) Siddiqi and Goodey, 1964 [12], *P. nanus* Cobb, 1923 [18,22], *P. pedrami* Clavero-Camacho, Cantalapiedra-Navarrete, Archidona-Yuste, Castillo and Palomares-Rius, 2021 [4], *P. peraticus* (Raski, 1962) Siddiqi and Goodey, 1964 [20], *P. projectus* Jenkins, 1956 [19,23], *P. sheri* (Raski, 1973) Siddiqi, 1986 [16–18,22], *P. similis* Khan, Prasad and Mathur, 1967 [18,22], *P. steineri* Golden, 1961 [18,20], *P. straeleni* (De Coninck, 1931) Oostenbrink, 1960 [9], *P. tateae* Wu and Townshend (1973) [4], *P. tenuicaudatus* Wu, 1961 [12], *P. teres* (Raski, 1976) Siddiqi, 1986 [24], *P. vandenbrandei* de Grisse, 1962 [16,17], *P. veruculatus* Wu, 1962 [4], and *P. zurgenerus* Clavero-Camacho, Cantalapiedra-Navarrete, Archidona-Yuste, Castillo and Palomares-Rius, 2021 [4]. However, for the majority of these studies, except that of Clavero-Camacho et al. [4], no molecular analyses were carried out for their identification, and the cryptic biodiversity of these nematodes could be underexplored, including some species identifications for Spanish populations performed by our group some years ago. For this reason, the identification and reliable estimation of pin nematode diversity in Spain is needed. This paper is the second in a series deciphering the cryptic diversity of pin nematodes in Spain using integrative taxonomical

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.

#### **2. Results**

Eighteen species were identified from 24 isolates of *Paratylenchus* spp. from 15 soil samples in nine municipalities in Spain (Table 1). In these populations, females, males (when available) and juveniles were morphologically and morphometrically studied in detail and molecular markers for their identification were provided (Table 1). From these, one isolate was considered a new undescribed species and 17 were already known described species (Table 1). The new species include an isolate from the *P. straeleni*-complex and was described herein as *Paratylenchus parastraeleni* sp. nov. The already known species included *P. amundseni* Bernard, 1982, *P. aciculus* Brown, 1959, *P. baldaccii* (Oostenbrink, 1953) Raski, 1962, *P. enigmaticus* Munawar et al., 2021, *P. goodeyi* Oostenbrink, 1953, *P. holdemani* Raski, 1975, *P. macrodorus* Brzeski, 1963, *P. neoamblycephalus* Geraert, 1965, *P. pandatus* (Raski, 1976) Siddiqi, 1986, *P. pedrami* Clavero-Camacho et al., 2021, *P. recisus* Siddiqi, 1996, *P. sheri* (Raski, 1973) Siddiqi, 1986, *P. tateae* Wu and Townshend (1973), *P. variabilis* Raski, 1975, *P. veruculatus* Wu, 1962, *P. verus* (Brzeski, 1995) Brzeski, 1998, and *P. vitecus* (Pramodini et al., 2006) Ghaderi et al., 2014. Eight of these species need to be considered as first reports for Spain in this research (*viz*. *P. amundseni*, *P. aciculus*, *P. neoamblycephalus*, *P. pandatus*, *P. recisus, P. variabilis, P. verus* and *P. vitecus*) and measurements from females, males (if available) and juveniles, as well as molecular markers were provided for their unequivocal identification.


**Table 1.** Isolates sampled and sequenced for *Paratylenchus* spp. from several localities in Spain used in this study.

#### *2.1. Systematics*

2.1.1. Description of *Paratylenchus parastraeleni* sp. nov.

#### (Figures 1–3, Table 2) http://zoobank.org/urn:lsid:zoobank.org:act:61B40ACF-177F-4D92-A16F-0A3CAF78FD4A (accessed on 8 July 2021).

*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 (19–28) 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.

**Figure 1.** Line drawings of *Paratylenchus parastraeleni* sp. nov. (**A**): Female pharyngeal region; (**B**,**C**): Female posterior region; (**D**): Entire female; (**E**): Male posterior region.

**Figure 2.** Light photomicrographs of *Paratylenchus parastraeleni* sp. nov. female and male. (**A**,**C**) Entire female with vulva arrowed; (**B**) detail of lateral fields; (**D**,**F**) detail of female stylet region; (**E**) female pharyngeal region; (**G**–**J**) female posterior region with vulva and anus (arrowed) and detail of vulva showing advulval flap (arrowed); (**K**) male pharyngeal region showing absence of stylet; (**L**,**M**) male posterior region showing spicules (arrowed). Scale bars (**A**–**M** = 20 μm). (Abbreviations: a = anus; avf = advulval flap; ep = excretory pore; lf = lateral field; sp = spicules; spm = spermatheca; V = vulva).

**Figure 3.** Light photomicrographs of *Paratylenchus parastraeleni* sp. nov. fourth-stage juveniles. (**A**,**B**) Entire fourth-stage juveniles showing stylet (arrowed); (**C**,**D**) fourth-stage juvenile pharyngeal region showing stylet; (**E**) fourth-stage juvenile anterior and posterior region showing stylet and initial vestigium of vagina (arrowed); (**F**) fourth-stage juvenile tail. Scale bars (**A**–**F** = 20 μm). (Abbreviations: ep = excretory pore; st = stylet; vv = vaginal vestigium).


**Table 2.** Morphometrics of *Paratylenchus parastraeleni* sp. nov. paratype females, males and fourth-stage juveniles. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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.

*Juveniles*: J4 similar in morphology to adult females (Figures 2 and 3), bearing flexible stylet 45.8 (43.0–48.0) μm-long. Pharynx well developed, functional. Genital primordium underdeveloped, primordium of vagina discernible, anus indistinct, posterior region similar to female but slightly more rounded terminus.

#### 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 [3,6,10,26,28–30], 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.

#### Type Habitat and Locality

*Paratylenchus parastraeleni* sp. nov. was found in the rhizosphere of a *Quercus faginea* Lam., forest (coordinates 37◦58 33.0" N 2◦54 18.8" W); the municipal district of Arroyo Frío, Jaén province, Spain.

#### 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).

2.1.2. Remarks of *Paratylenchus aciculus* Brown, 1959

(Figure 4, Table 3).

**Figure 4.** Light photomicrographs of *Paratylenchus aciculus* Brown, 1959. (**A**,**B**) Entire female with vulva arrowed; (**C**) female pharyngeal region; (**D**,**E**) female lip region; (**F**) detail of lateral field; (**G**,**H**) female posterior region with vulva and anus (arrowed). Scale bars (**A**–**H** = 20 μm). (Abbreviations: a = anus; ep = excretory pore; lf = lateral field; st = stylet; V = vulva).


**Table 3.** Morphometrics of *Paratylenchus aciculus* Brown, 1959 from Coto Ríos, Jaén, Spain, type population, and *P. aculentus* from Belgium. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

> 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).

#### 2.1.3. Remarks of *Paratylenchus amundseni* Bernard, 1982

#### (Figure 5, 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.

#### Molecular Characterization

Three D2-D3 of 28S rRNA (MZ265076-MZ265078), three ITS (MZ265012-MZ265014), and five COI gene sequences (MZ262215-MZ262219) were generated herein for this species, including J4 and female adult sequences. All sequences showed no intraspecific variation. *Paratylenchus amundseni* was molecularly closely related with *P. tateae*, showing similarity values of 98% (differing from 11 to 14 nucleotides and no indels) for D2-D3 region. However, for the ITS region, the similarity value was 95% (differing by 31 to 43 nucleotides and 7 to 11 indels) with *P. tateae* accessions (MW282766-MW282771) from Spain and Canada [8]. Finally, the similarity found for COI gene sequences was 90% (differing by 34–36 nu-

cleotides) with the COI accessions of *P. tateae* (MZ262262-MZ262264) from Spain, newly obtained in the present study.

**Figure 5.** Light photomicrographs of *Paratylenchus amundseni* Bernard, 1982. (**A**) Entire female with vulva arrowed; (**B**) female pharyngeal region; (**C**) female lip region; (**D**) detail of lateral field; (**E**–**H**) female posterior region with vulva, anus, and advulval flap (arrowed); (**I**) entire fourth-stage juvenile with stylet (arrowed). Scale bars (**A**–**I** = 20 μm). (Abbreviations: a = anus; avf = advulval flap; eg = egg; lf = lateral field; st = stylet; V = vulva).


**Table 4.** Morphometrics of *Paratylenchus amundseni* Bernard, 1982 from La Iruela, Jaén, Spain. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

2.1.4. Remarks on *Paratylenchus baldaccii* (Oostenbrink, 1953) Raski, 1962, *Paratylenchus enigmaticus* Munawar, Yevtushenko, Palomares-Rius and Castillo, 2021, *Paratylenchus holdemani* Raski, 1975, *Paratylenchus neoamblycephalus* Geraert, 1965, *Paratylenchus pedrami* Clavero-Camacho, Cantalapiedra-Navarrete, Archidona-Yuste, Castillo and Palomares-Rius, 2021, and *Paratylenchus veruculatus* Wu, 1962

(Table 5).

*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].

2.1.5. Remarks on *Paratylenchus goodeyi* Oostenbrink, 1953

(Figure 6, Table 6).


**Table 5.** Morphometrics of *Paratylenchus baldaccii* (Oostenbrink, 1953) Raski, 1962 and *Paratylenchus enigmaticus* Munawar, Yevtushenko, Palomares-Rius and Castillo, 2021, *Paratylenchus holdemani* Raski, 1975, and *Paratylenchus neoamblycephalus* Geraert, 1965 from several localities in Spain. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

**Figure 6.** Light photomicrographs of *Paratylenchus goodeyi* Oostenbrink, 1953. (**A**) Entire female with vulva arrowed; (**B**–**D**) female lip region with stylet arrowed; (**E**) detail of vulval region showing spermatheca arrowed; (**F**) entire fourthstage juvenile with short stylet arrowed; (**G**–**L**) fourth-stage juvenile lip regions showing labial sclerotization and short stylet (arrowed). Scale bars (**A**–**L** = 20 μm). (Abbreviations: dgo = pharyngeal dorsal gland orifice; hls = heavy labial sclerotization; spm = spermatheca; st = stylet; V = vulva).




**Table 6.** *Cont.*

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].

#### Molecular Characterization

Twenty-two D2-D3 sequences of 28S rRNA (MZ265084-MZ265105), 14 ITS (MZ265020- MZ265033), and 12 COI gene sequences (MZ262227-MZ262238) of *P. goodeyi* were generated in this study, with an intraspecific sequence variation from 0 to 9 nucleotides for D2-D3 of 28S rRNA (MZ265084-MZ265105), 0 to 17 nucleotides for ITS region (MZ265020-MZ265033), and finally, 0 to 29 nucleotides for COI gene (MZ262227-MZ262238). Some intraspecific sequence variations were detected when comparing with the accessions of *P. goodeyi* deposited in GenBank, showing similarity values of 99% for the D2-D3 of 28S rRNA, from 96 to 99% for the ITS region and finally, from 96 to 98% for the COI gene [3,4]. Some accessions from the different populations, belonging to J4, and all of them, matched well, from 99 to 100% similarity, with the sequences obtained for adult females of the same population.

#### 2.1.6. Remarks on *Paratylenchus macrodorus* Brzeski, 1963

#### (Figure 7, Table 7).

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 and advulval flaps absent. The Spanish population from Santa Mª de Trasierra, Córdoba province, was characterized by long flexible stylet 70.0–84.0 μm, lip region continuous with body contour, tapering slightly to a blunt anterior end, submedian lobes fairly distinct, female tail tapering gradually to finely rounded terminus. Males without stylet, and J4 similar to female, except for shorter stylet (both stages confirmed belonging to this species by molecular markers). Morphometrics of the Spanish population agree well with original description as well as other populations with small differences in stylet length (70.0–84.0 μm vs. 75.0–92.0 μm), which may be due to geographical intraspecific variability [2]. Molecularly *P. macrodorus* is close to *P. pandatus* and *P. wuae* (using D2-D3 region of 28S rRNA) from which can be morphological and morphometrically separated by submedian lobes (fairly distinct vs. clearly distinct, pronounced submedian lobes, respectively), body length (317–410 vs. 290–339, 300–360 μm, respectively), c and c' ratios (7.4–11.1 vs. 9.2–16.6, 10.5–11.3, and 3.5–4.9 vs. 2.2–3.0, 3.4–3.8, respectively), and J4 stylet (present vs. absent, absent, respectively) [2,37,38]. 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].

**Figure 7.** Light photomicrographs of *Paratylenchus macrodorus* Brzeski, 1963. (**A**,**B**) Entire female with stylet and vulva arrowed; (**C**) female pharyngeal region; (**D**,**E**) female lip region; (**F**) female posterior region with vulva and anus (arrowed); (**G**) detail of vulva (arrowed); (**H**) female tail region with anus arrowed; (**I**) male pharyngeal region showing absence of stylet; (**J**) male posterior region showing spicules (arrowed). Scale bars (**A**–**J** = 20 μm). (Abbreviations: a = anus; ep = excretory pore; sp = spicules; st = stylet; V = vulva).


**Table 7.** Morphometrics of *Paratylenchus macrodorus* Brzeski, 1963 from Santa María de Trasierra, Córdoba, Spain. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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.

#### 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).

#### 2.1.7. Remarks on *Paratylenchus pandatus* (Raski, 1976) Siddiqi, 1986

#### (Figure 8, 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.*

2.1.8. Remarks on *Paratylenchus recisus* Siddiqi, 1996

(Figure 9, Table 9).

**Figure 8.** Light photomicrographs of *Paratylenchus pandatus* (Raski, 1976) Siddiqi, 1986. (**A**,**B**) entire females with vulva arrowed; (**C**,**D**) female pharyngeal region; (**E**,**F**) female posterior region showing vulva and anus (arrowed); (**G**) detail of lateral field (arrowed) at mid-body; (**H**) detail of spermatheca and sperm (arrowed); (**I**) detail of lateral field and advulval flap (arrowed); (**J**) entire fourth-stage juvenile, stylet absence arrowed; (**K**) fourth-stage juvenile lip region showing stylet absence (arrowed); (**L**) fourth-stage juvenile tail. Scale bars (**A**–**L** = 20 μm). (Abbreviations: a = anus; avf = advulval flap; dgo = pharyngeal dorsal gland orifice; ep = excretory pore; lf = laterl field; spm = spermatheca; spr = sperm; V = vulva).


**Table 8.** Morphometrics of *Paratylenchus pandatus* (Raski, 1976) Siddiqi, 1986 from Caravaca, Murcia, Spain. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.


**Table 9.** Morphometrics of *Paratylenchus recisus* Siddiqi, 1996 from Arroyo Frío, Jaén, Spain. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

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 (14.5–16.0 μm vs. 15.0–17.0 μm), c' ratio (2.8–3.5 vs. 2.7–3.3), vulva–anus distance (1.5–1.8 times tail length), and tail length (28.0–34.0 μm vs. 18.0–29.0 μm), 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.

**Figure 9.** Light photomicrographs of *Paratylenchus recisus* Wu, 1974. (**A**) Entire female with stylet and vulva arrowed; (**B**) female pharyngeal region; (**C**–**E**) female posterior region with vulva, advulval flap, spermatheca and anus (arrowed); (**F**) detail of lateral field at mid-body (arrowed); (**G**) fourth-stage juvenile pharyngeal region showing absence of stylet (arrowed) and undeveloped pharynx; (**H**) fourth-stage juvenile posterior region showing vaginal vestigium (arrowed). Scale bars (**A**–**H** = 20 μm). (Abbreviations: a = anus; avf = advulval flap; dgo = pharyngeal dorsal gland orifice; ep = excretory pore; lf = lateral field; spm = spermatheca; st = stylet; vv = vaginal vestigium; V = vulva).

Molecular Characterization

Two D2-D3 of 28S rRNA (MZ265119-MZ265120), one ITS (MZ265043), and one COI gene sequence (MZ262252) were generated herein without intraspecific sequence variations. The closest *Paratylenchus* sequences to *P. recisus* were those of *P. microdorus* with 97, 93 and 91% similarity for the D2-D3 of 28S rRNA, ITS region and COI gene (MW421666- MW421667), respectively.

2.1.9. Remarks on *Paratylenchus sheri* (Raski, 1973) Siddiqi, 1986

(Figures 10 and 11, Table 10).

**Figure 10.** Light and SEM photomicrographs of *Paratylenchus sheri* (Raski, 1973) Siddiqi, 1986. (**A**) Entire female with stylet and vulva arrowed; (**B**,**C**) female pharyngeal region showing heavy lip sclerotization (arrowed); (**D**–**F**) female lip region showing heavy lip sclerotization (arrowed); (**G**–**I**) detail of lip region at SEM showing smooth lip region and labial disc (arrowed); (**J**,**K**) female posterior region with vulva and anus (arrowed); (**L**) detail of lateral field at mid body (arrowed); (**M**) detail of vulva showing advulval flap (arrowed). Scale bars (**A**–**F** = 20 μm; **G**–**I** = 5 μm; **J**–**M** = 20 μm). (Abbreviations: a = anus; avf = advulval flap; dgo = pharyngeal dorsal gland orifice; ep = excretory pore; hls = heavy lip sclerotization; ld = labial disc; slr = smooth lip region; st = stylet; V = vulva).

**Figure 11.** Light photomicrographs of *Paratylenchus sheri* (Raski, 1973) Siddiqi, 1986. (**A**,**B**) Entire fourth-stage juvenile with stylet and vaginal vestigium arrowed; (**C**–**G**) fourth-stage juvenile lip region showing heavy lip sclerotization (arrowed); (**H**) fourth-stage juvenile with vaginal vestigium arrowed. Scale bars (**A**–**H** = 20 μm). (Abbreviations: dgo = pharyngeal dorsal gland orifice; hls = heavy lip sclerotization; st = stylet; vv = vaginal vestigium).


**Table 10.** Morphometrics of *Paratylenchus sheri* (Raski, 1973) Siddiqi, 1986 from Arroyo Frío and Coto Ríos, Jaén, Spain. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

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 conoidtruncate lip region, with an unstriated depression from body contour (4–5.5.0 μm wide) and strong sclerotization. Small projecting oral lips present. SEM face view (Figure 10) shows an unstriated, dorso-ventrally flattened lip region. Stylet robust, occupying 19 (15–23) annuli and 22.5–25.0 μm long. Stylet knobs weakly backwardly directed, 4.8 (4.0–5.5) μm across. Lateral field with four incisures with smooth margins (central two are very faint), 3.2 (2.5–4.0) μm wide. Orifice of dorsal pharyngeal gland 5.9 (4.5–6.5) μm from stylet base. Metacorpus with well-developed valvular apparatus 5.6 (4.5–7.5) μm long, its posterior margin situated at 70 (63–80) μm from anterior end. Excretory pore located near anterior end of basal bulb, immediately posterior to hemizonid. Cardia well developed, 2.5–3.5 μm wide. Distinct cuticular vulval flap, 6 (5.0–7.0) μm long. Large round spermatheca 13.5 (11.5–15.5) μm wide, filled with sperms 1–2 μm wide, which indicates that males are required for reproduction but their numbers are lower than females. Tail almost straight to slight ventrally curved, with rounded terminus, 0.8 (0.6–1.3) times vulva–anus distance or 3.8 (3.1–4.7) times anal body diameter. J4 with similar morphology to that of adult females, except sexual characters and shorter body length and stylet.

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.

#### Molecular Characterization

Six D2-D3 of 28S rRNA (MZ265121-MZ265126) with an intraspecific sequence variation of 0.5% (differing from 0 to 2 nucleotides), seven ITS (MZ265044-MZ265050) (99% similarity; nine nucleotides and no indels), and finally, nine COI gene sequences (MZ262253- MZ262261), with an intraspecific sequence variation of 5% (differing from 0 to 23 nucleotides), were generated. Two J4 from the Arroyo Frío population were sequenced, including D2–D3 of 28S (MZ265121-MZ265122), ITS region (MZ265044-MZ265045) and COI gene (MZ262253-MZ262254) being identical to the adult female sequences from this population. The D2-D3 of 28S rRNA sequences (MZ265121-MZ265126) showed high similarity with accession from *P. israelensis* (MW798301-MX798305) and *P. neoamblycephalus* (MW413660- MW413663) being 99% similar between them (differing from 2 to 7 nucleotides). For the ITS (MZ265044-MZ265050), the similarity detected was 98% (differing by 17–23 nucleotides and 6–8 indels) with *P. israelensis* (MW798343) and 95% (differing by 44–50 nucleotides and 20 indels) with *P. neoamblycephalus* (MW413607). Finally, for the COI gene sequences (MZ262253-MZ262261), *P. sheri* showed similarity values of 92–94% (differing from 21 to 29 nucleotides) with *P. israelensis* (MW797019-MW797020) and 89–91% (differing from 34 to 38 nucleotides) with *P. neoamblycephalus* (MW421677-MW421682). D2–D3 of 28S sequences from *P. sheri* obtained herein showed similarity values of 91% with the accession MN088374 of *P. sheri* from Iran, thus reinforcing the idea that this sequence belongs to *P. tateae* instead of *P. sheri,* as already suggested by Munawar et al. [8].

#### 2.1.10. Remarks on *Paratylenchus variabilis* Raski, 1975

(Figure 12, Table 11).

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 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.

**Figure 12.** Light photomicrographs of *Paratylenchus variabilis* Raski, 1975. (**A**,**B**) Entire female with stylet and vulva arrowed; (**C**,**D**) female pharyngeal region; (**E**–**G**) female lip region; (**H**–**J**) female posterior region with vulva, spermatheca, advulval flap and anus (arrowed); (**K**–**L**) fourth-stage juvenile with stylet and vaginal vestigium arrowed. Scale bars (**A**–**L** = 20 μm). (Abbreviations: a = anus; avf = advulval flap; dgo = pharyngeal dorsal gland orifice; spm = spermatheca; st = stylet; vv = vaginal vestigium; V = vulva).


**Table 11.** Morphometrics of *Paratylenchus variabilis* Raski, 1975 from Córdoba, Córdoba, Spain. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

Molecular Characterization

Three D2-D3 of 28S rRNA (MZ265127-MZ265129), three ITS (MZ265051-MZ265053) and three COI gene sequences (MZ262265-MZ262267) were generated in this study from two adult females and one J4 specimen without intraspecific sequence variations. *Paratylenchus variabilis* was closely related with *P. nanus*, showing similarity values of 96% (differing by 31 nucleotides and 1 indel) for the D2-D3 region with several accessions of *P. nanus* (MW413657-MW413659, MW234449-MW234450). However, for the ITS region the similarity was lower, with values about 87% with accessions belonging to several *Paratylenchus* spp., such as, *P. veruculatus*, *P. goodeyi* and *P. nanus*. Finally, the closest species for the COI gene sequences was *P. goodeyi* (MW421648-MW421649), being 95% similar between them (19 nucleotides and no indels). Finally, *P. variabilis* can also be clearly separated molecularly from *P. zurgenerus* by D2-D3 and ITS, 88.9%, 78.3% similarity (differing in 79 bp, 166 and 20, 66 indels), respectively; low similarity was detected among COI sequences of both species.

2.1.11. Remarks on *Paratylenchus verus* (Brzeski, 1995) Brzeski, 1998

(Figure 13, Table 12).

**Figure 13.** Light photomicrographs of *Paratylenchus verus* (Brzeski, 1995) Brzeski, 1998. (**A**,**B**) Entire female with stylet and vulva arrowed; (**C**–**F**) female lip region with stylet, excretory pore and *Pasteuria* endospore arrowed; (**G**) detail of lateral field at mid-body and *Pasteuria* endospore arrowed; (**H**) female posterior region with vulva and *Pasteuria* endospore arrowed; (**I**,**J**) entire fourth-stage juvenile with stylet arrowed; (**K**–**N**) fourth-stage juvenile lip region with stylet and excretory pore arrowed; (**O**) fourth-stage juvenile posterior region with vaginal vestigium arrowed; (**P**,**Q**) fourthstage juvenile posterior region with anus arrowed. Scale bars (**A**–**Q** = 20 μm). (Abbreviations: a = anus; ep = excretory pore; lf = lateral field; pe = *Pasteuria* endospore; st = stylet; vv = vaginal vestigium; V = vulva).


**Table 12.** Morphometrics of *Paratylenchus verus* (Brzeski, 1995) Brzeski, 1998 from Santa María de Trasierra, Córdoba, Spain. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

> 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).

#### 2.1.12. Remarks on *Paratylenchus vitecus* (Pramodini et al., 2006) Ghaderi et al., 2014

(Figure 14, 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 (62.0–70.0 μm vs. 42.0–65.0 μm), V ratio (68.1–75.4 vs. 72.0–77.0), which may be due to geographical intraspecific variability [2]. Molecularly, *P. vitecus* is close to *P. teres* (see below), however, it can be morphologically separated by clear differences in stylet length (42.0–65.0 μm, 62.0–70.0 μm vs. 69.0–83.0 μm, 67.0–96.0 μm) and c' ratio (2.9, 2.7–3.5 vs. 4.2, 3.1–3.9). 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.

**Figure 14.** Light photomicrographs of *Paratylenchus vitecus* (Pramodini et al., 2006) Ghaderi et al., 2014. (**A**) Entire female with stylet and vulva arrowed; (**B**) female pharyngeal region with stylet and excretory pore arrowed; (**C**–**E**) female lip region; (**F**) entire fourth-stage juvenile with stylet arrowed; (**G**) detail of lateral field at mid-body (arrowed). Scale bars (**A**–**G** = 20 μm). (Abbreviations: ep = excretory pore; lf = lateral field; st = stylet; V = vulva).


**Table 13.** Morphometrics of *Paratylenchus vitecus* (Pramodini et al., 2006) Ghaderi et al., 2014 from Córdoba, Córdoba, Spain. All measurements are in μm and in the form: mean ± s.d. (range).

\* 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; V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

#### *2.2. 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).

**Figure 15.** Spain map distribution of *Paratylenchus* species across all of the country. Species list with asterisk (\*) indicated species identified by integrative taxonomy and including molecular analyses confirmation.

#### *2.3. 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).

**Figure 16.** Phylogenetic relationships within the genus *Paratylenchus*. Bayesian 50% majority rule consensus tree as inferred from D2-D3 expansion domains of the 28S 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. \*\*\* Red font names refer to the previous consideration in NCBI.

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].

**Figure 17.** 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.

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].

**Figure 18.** Phylogenetic relationships within the genus *Paratylenchus*. Bayesian 50% majority rule consensus tree as inferred from cytochrome c oxidase subunit 1 (COI) sequence alignment under the general time-reversible model of sequence evolution with a gamma-shaped distribution (GTR + 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.

#### **3. 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 [15–18]. 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].

#### **4. Materials and Methods**

#### *4.1. Nematode Sampling and Morphological Identification*

Fifteen soil samples were collected mainly from the rhizosphere of herbaceous and woody plants including 5 samples from almond with different rootstocks (*Prunus* spp.), 2 samples from Portuguese oak (*Quercus faginea* Lam.), 3 samples from Aleppo pine (*Pinus halepensis* Mill.), 3 samples from wild olive (*Olea europaea* sbsp. *silvestris* (Mill.) Lehr), and 2 samples from grasses, in 10 localities in Spain (Table 1). Samples were collected using a shovel and considering the upper 5–40 cm depth of soil. Nematodes were extracted from a 500-cm3 subsample of soil by centrifugal flotation [52].

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.

#### *4.2. 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.

#### *4.3. 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 <sup>10</sup> × 106 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.

#### **5. 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.

**Author Contributions:** Conceptualization, J.E.P.-R., I.C.-C., C.C.-N., A.A.-Y. and P.C., methodology, I.C.-C., C.C.-N., G.L.-R., J.M.-B., J.E.P.-R. and P.C., software, I.C.-C., C.C.-N., A.A.-Y., G.L.-R., J.E.P.-R. and P.C. analysis, I.C.-C., C.C.-N., A.A.-Y., G.L.-R., J.E.P.-R. and P.C., resources, P.C. and J.E.P.-R., writing, I.C.-C., C.C.-N., A.A.-Y., J.E.P.-R. and P.C. All authors contributed to the final discussion data and have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by grant RTI2018-095925-A-100 from Ministerio de Ciencia, Innovación y Universidades, Spain.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

**Acknowledgments:** This research is part of the PhD project of the first author. The first author is a recipient of a contract from Ministry of Science and Innovation for Predoctoral Researchers in Spain, PRE2019-090206.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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