Figures
Abstract
The genus Longidorus includes a remarkable group of invertebrate animals of the phylum Nematoda comprising polyphagous root-ectoparasites of numerous plants including several agricultural crops and trees. Damage is caused by direct feeding on root cells as well as by transmitting nepoviruses that cause disease on those crops. Thus, correct identification of Longidorus species is essential to establish appropriate control measures. We provide the first detailed information on the diversity and distribution of Longidorus species infesting wild and cultivated olive soils in a wide-region in southern Spain that included 159 locations from which 449 sampling sites were analyzed. The present study doubles the known biodiversity of Longidorus species identified in olives by including six new species (Longidorus indalus sp. nov., Longidorus macrodorus sp. nov., Longidorus onubensis sp. nov., Longidorus silvestris sp. nov., Longidorus vallensis sp. nov., and Longidorus wicuolea sp. nov.), two new records for wild and cultivate olives (L. alvegus and L. vineacola), and two additional new records for wild olive (L. intermedius and L. lusitanicus). We also found evidence of some geographic species associations to western (viz. L. alvegus, L. intermedius, L. lusitanicus, L. onubensis sp. nov., L. vineacola, L. vinearum, L. wicuolea sp. nov.) and eastern distributions (viz. L. indalus sp. nov.), while only L. magnus was detected in both areas. We developed a comparative study by considering morphological and morphometrical features together with molecular data from nuclear ribosomal RNA genes (D2–D3 expansion segments of 28S, ITS1, and partial 18S). Results of molecular and phylogenetic analyses confirmed the morphological hypotheses and allowed the delimitation and discrimination of six new species of the genus described herein and four known species. Phylogenetic analyses of Longidorus spp. based on three molecular markers resulted in a general consensus of these species groups, since lineages were maintained for the majority of species. This study represents the most complete phylogenetic analysis for Longidorus species to date.
Citation: Archidona-Yuste A, Navas-Cortés JA, Cantalapiedra-Navarrete C, Palomares-Rius JE, Castillo P (2016) Unravelling the Biodiversity and Molecular Phylogeny of Needle Nematodes of the Genus Longidorus (Nematoda: Longidoridae) in Olive and a Description of Six New Species. PLoS ONE 11(1): e0147689. https://doi.org/10.1371/journal.pone.0147689
Editor: Chao-Dong Zhu, Institute of Zoology, CHINA
Received: September 11, 2015; Accepted: January 7, 2016; Published: January 25, 2016
Copyright: © 2016 Archidona-Yuste et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All genetic sequences are available from GenBank (accession numbers KT308852-KT308903). All relevant data are within the paper and its Supporting Information files.
Funding: Financial support was received by Projects AGL-2012-37521 from ‘Ministerio de Economía y Competitividad’ of Spain, Project P12-AGR-1486 from ‘Consejería de Economía, Innovación y Ciencia’ of Junta de Andalucía, and FEDER financial support from the European Union is gratefully acknowledged. The grant 219262 ArimNET_ERANET FP7 2012–2015 Project PESTOLIVE ‘Contribution of olive history for the management of soilborne parasites in the Mediterranean basin’ from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), also provided partial financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The phylum Nematoda comprises the most species-rich metazoans on earth with a global distribution and estimated realistic number of species of ca. 105 [1, 2, 3]. Soil nematode gross morphology tends to be highly conserved, making species identification a very difficult task [3, 4]. Accurate diagnostic studies of plant-parasitic nematode (PPN) species are important because of their implications in pest control and soil ecology [5]. With most nematode species likely remaining undescribed, efforts to catalogue and explain biodiversity need to be prioritised [6]. However species concept ranges among typological species (a community of specimens described by characteristic features of its type specimen), biological species (populations which successfully interbreed with each other), and phylogenetic species (phylogenetic lineages). All of these concepts have limitations, including the popular biological species concept which is restricted to sexual, outcrossing populations and excludes parthenogenetic organisms [7, 8]. Species delimitation in nematodes typically uses a phenotypic view of the animal, based in relatively few anatomical and morphological characters, such as lip region and female tail shape, pharyngeal glands, stylet shape and length, type of female reproductive system, etc. Additionally, many nematodes have complex life-cycles and it can be difficult to demonstrate the validity of a species by means of intercrossing of individuals and production of viable progeny. For these reasons the possibility of undescribed or misdescribed species is very high, as demonstrated by several authors [8, 9, 10, 11].
The family Longidoridae Thorne, 1935 [12] includes a wide and diverse group of migratory ectoparasitic nematode species, where the needle nematodes of the genus Longidorus Micoletzky, 1922 [13] is one of the most evolved group species of this family [14]. This genus includes a number of long to very long body (2–12 mm) specimens with long stylet (80–260 μm). They are polyphagous species of many plants including various agricultural crops, and cause damage by direct feeding on root cells as well as by transmitting nepoviruses (nepoviruses are spherical, single-stranded RNA of positive-sense) [15, 16, 17]. Some Longidorus spp. are cosmopolitan whilst others have a limited geographic distribution [14]. The genus Longidorus is a diverse group with about 160 nominal species [18, 19], but only 11 species (6.9%) (L. apulus, L. arthensis, L. attenuatus, L. caespiticola, L. diadecturus, L. elongatus, L. fasciatus, L. leptocephalus, L. macrosoma, L. martini, and L. profundorum) have been reported as virus vector, but transmitting seven out of the 38 known nepoviruses [15, 20]. Nepoviruses vectored by Longidorus species damage vegetable and fruit crops including: Artichoke Italian latent virus, Cherry rosette disease virus, Tomato black ring virus, Raspberry ringspot virus, Arabis mosaic virus, Peach rosette mosaic virus, and Mulberry ringspot virus [15, 20]. Therefore, correct identification of Longidorus species is essential to establish appropriate control measures. Species discrimination in Longidorus has classically been based mainly on morphology and morphometrics of diagnostic features. However morphologically based species characterization is complicated by a high degree of intraspecific variability within morphometrics, as well as slight interspecific differences that lead to substantial overlapping among Longidorus species and increase the risk of species miss-identification [10, 19]. As a result, taxonomic difficulties often arise from under- or over-estimation of intraspecific variability of certain morphological characters currently being used for species diagnosis.
Integrative taxonomy assembles and assimilates all available data and information to frame species limits (phenotypic, genotypic and phylogenetic) [7, 8]. Although this approach is more complex and has a higher cost than traditional taxonomy, its application reduce the degree of subjectivity that is common in traditional alpha taxonomic practices, as has been recently reported in studies showing the potential for these methods in the discovery of new and cryptic species in taxa poorly known or composed of morphologically conserved species [6, 8, 10, 19, 21, 22].
Recently, 68 Longidorus species (about 42% of total species) have been characterized molecularly, constituting a useful tool for molecular-based species identification. Molecular approaches using multiple regions of the ribosomal DNA (rDNA) genes sequences including (28S, 18S, and 5.8S genes and internal transcribed spacers (ITS1 and ITS2)), have been investigated to better understand the taxonomic relationships within the genus Longidorus [19, 21, 23, 24, 25, 26, 27, 28]. These molecular markers have been shown to be useful diagnostic tools in the characterization and phylogenetic relationships within Longidoridae, particularly in cases where morphological characters may lead to ambiguous interpretation, such as species in the Xiphinema americanum group [19, 21, 23, 24, 25, 26, 27, 28]. D2–D3 expansion segments of 28S rRNA and ITS1 rRNA have proven to be a powerful tool for providing accurate and molecular species identification in Longidoridae compared to partial 18S, since both molecular markers showed more species variability (nucleotides and indels) than partial 18S, which in some cases did not show enough resolution to distinguish species [19, 24, 25, 28, 29].
Longidorus species identification remains quite challenging when dealing with species that closely resemble one another and which co-occur in a region, as is often the case in the Iberian Peninsula. Furthermore, soil samples often contain mixed populations with more than one species in the same sample. In this study we focus mostly on the Longidorus species that occur throughout wild and cultivate olives at southern Spain. Morphological and morphometric evaluation as well as molecular sequencing of each Longidorus population were used simultaneously for species delineation and grouping specimens into species.
Olive, the emblematic tree of the Mediterranean Basin, is found in two forms, namely wild (Olea europaea subsp. europaea var. sylvestris) and cultivated (Olea europaea subsp. europaea var. europaea) [30]. Wild olives occur throughout many Mediterranean environments, characterized by semi-arid climatic conditions with different altitudes, plant communities and soils, including those with extreme dry conditions [30]. Cultivated olive is extensively grown in the Mediterranean Basin, as well as the subtropical regions of Australia, southern Africa, and North and South America [31]. Olive is the most cultivated non-tropical fruit trees and is among the most ancient crops in the Mediterranean Basin [31]. Approximately 10.5 million ha of cultivate olive are growing in the world, of which about 85% are in Mediterranean countries, including North Africa, and about 25% of them in Spain [32]. In Andalusia, southern Spain, cultivated olive trees cover more than 1.6 million ha accounting for 19% of the total surface area in an impressive monoculture [33, 34].
Both wild and cultivated olive trees serve as hosts to a large number of plant-parasitic nematodes, of which root-knot nematodes (Meloidogyne spp.), root-lesion nematodes (Pratylenchus spp.), spiral nematodes (Helicotylenchus spp.), and needle and dagger nematodes (Longidorus spp., Xiphinema spp.) are widely distributed and damage this crop [35, 36]. However, little information is available about needle nematodes associated with olive trees, except for the recent contribution of Palomares-Rius et al. [37] reporting Longidorus magnus Lamberti, Bleve-Zacheo and Arias 1982 [38] and Longidorus sp. According to Gutiérrez-Gutiérrez et al. [19] and other authors, 30 species of the genus Longidorus have been reported in Spain, mainly associated with fruit, forest, ornamental and vegetable plant species [19, 28, 39, 40].
With the aim of deciphering the biodiversity of Longidorus spp. infecting wild and cultivated olives in southern Spain, we sampled a total of 159 nine localities at the eight provinces of Andalusia where both olive types were present. In this survey we detected 40 populations of Longidorus species characterized by moderate to large body and stylet length, apparently morphologically related to other known Longidorus spp. This prompted us to carry out an integrative taxonomic study to assess the power of this approach for species identification within this complex genus.
The overall objective of this study was to test the congruence between morphological and molecular data within Longidorus species, and the specific objectives were: i) to identify and morphologically and morphometrically compare the 40 Spanish populations of Longidorus spp. detected in recent field samples from wild and cultivate olive-ecosystems; ii) to carry out a molecular characterisation of these Longidorus populations based on sequences of the D2–D3 expansion segments of the 28S nuclear ribosomal RNA gene, the ITS1 of rRNA, and partial 18S rRNA sequences; and iii) to study the phylogenetic relationships of Longidorus spp.
Material and Methods
Ethics Statement
No specific permits were required for the described fieldwork studies. Permission for sampling the olive orchards was granted by the landowner. The samples from wild olives were obtained in public areas, forests, and other natural areas studied and do not involve any species endangered or protected in Spain. The sites are not protected in any way.
Soil collection and nematode extraction
Nematodes were surveyed from 2012 to 2015 during the spring season in wild and cultivate olives groves in Andalusia, southern Spain (Table 1, Fig 1). Soil samples were collected for nematode analysis with a shovel from four to five trees in each sampling site. A total of 131 and 318 sampling sites from wild and cultivated olives, respectively, were arbitrarily chosen in the eight provinces of Andalusia where both olive types were present. The number of sampling sites was proportional to the area of wild and cultivated olive in each province (Table 1, Fig 1). Soil samples were collected from a 5- to 50-cm depth, in the close vicinity of active plant roots, discarding the upper 5-cm of topsoil to ensure that roots from weeds or other herbaceous plants were not included. All soil samples from each site were thoroughly mixed to obtain a single representative sample before nematode extraction.
This map may be similar but not identical to other published maps of Andalusia and is therefore for illustrative purposes only on the sampling sites.
Nematodes were extracted from a 500-cm3 sub-sample of soil using magnesium sulphate centrifugal-flotation and a modification of Cobb´s decanting and sieving methods [41, 42]. The soil was washed thoroughly with tap water through a 710-μm mesh sieve, and the filtered water was collected in a beaker and thoroughly mixed with 4% kaolin (v/v). This mixture was centrifuged at 1,100×g for 4 min, and the supernatants discarded. Pellets were resuspended in 250 ml MgSO4 (δ = 1.16) and the new suspensions were centrifuged at 1,100×g for 3 min. The supernatants were sieved through a 5 μm mesh, and nematodes collected on the sieve were washed with tap water [42]. The nematode sample was poured into a counting dish (8 cm L × 8 cm W × 1.5 cm H) and the nematodes were identified and counted under a Leica MZ12, stereomicroscope (Leica Microsystems, Wetzler, Germany). PPN from soil samples were identified to genus, and then we focussed on the species delineation of needle nematodes of the genus Longidorus. Later on, abundance and prevalence of each Longidorus species was estimated. Abundance was calculated as the mean number of Longidorus nematodes per 500 cm3 of soil for all samples. The prevalence was computed by dividing the number of samples in which the Longidorus species was detected by the total number of samples and expressed as a percentage.
Morphological studies
Longidorus specimens for light microscopy were killed by gentle heat, fixed in a solution of 4% formaldehyde + 1% propionic acid and processed to pure glycerine using Seinhorst’s method [43]. Specimens were examined using a Zeiss III compound microscope with Nomarski differential interference contrast at up to 1,000x magnification. The morphometric study of each nematode population included classical diagnostic features in longidoridae (i.e. de Man body ratios, lip region and amphid shape, oral aperture-guiding ring, odontostyle and odontophore length) [44]. All measurements were expressed in micrometers (μm), unless otherwise indicated in text. For line drawing of the new species, light micrographs were imported to CorelDraw software version X6 (Corel Corporation, London, UK) and redrawn. All other abbreviations used are as defined in Jairajpuri & Ahmad [44]. In addition, a comparative morphological and morphometrical study of type specimens of some species were conducted with specimens kindly provided by Dr. A. Troccoli, from the nematode collection at the Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (viz. Longidorus lusitanicus Macara 1985 [45], Longidorus vinearum Bravo & Roca, 1995 [46]), and Dr. A. Navas from the Nematode Collection of the Spanish National Museum of Natural Sciences-CSIC, Madrid, Spain (viz. Longidorus carpetanensis Arias, Andrés & Navas, 1986 [47] and Longidorus unedoi Arias, Andrés & Navas, 1986 [47]).
DNA extraction, PCR and sequencing
For molecular analyses, in order to avoid mistakes in the case of mixed populations in the same sample, two live nematodes from each sample were temporary mounted in a drop of 1M NaCl containing glass beads (to avoid nematode crushing/damaging specimens) to ensure specimens conformed in form to the unidentified populations of Longidorus. Morphometrics and photomicrographs recorded during this initial study were not used as part of the morphological study or analyses. Following morphological confirmation, the specimens were removed from the slides and DNA extracted. Nematode DNA was extracted from single individuals and PCR assays were conducted as described by Castillo et al. [48]. One nematode specimen of each sample was transferred to an Eppendorf tube containing 16 μl ddH2O, 2 μl 10x PCR buffer and 2 μl proteinase K (600 μg/ml) (Promega, Benelux, The Netherlands) and crushed during 2 min with a micro-homogeniser, Vibro Mixer (Zürich, Switzerland). The tubes were incubated at 65°C (1 h), then at 95°C (15 min), and finally at 80°C (15 min). One μl of extracted DNA was transferred to an Eppendorf tube containing: 2.5 μl 10X NH4 reaction buffer, 0.75 μl MgCl2 (50mM), 0.25 μl dNTPs mixture (10mM each), 0.75 μl of each primer (10mM), 0.2 μl BIOTAQ DNA Polymerase (BIOLINE, UK) and ddH2O to a final volume of 25 μl. The D2–D3 expansion segments of 28S rRNA was amplified using the D2A (5’-ACAAGTACCGTGAGGGAAAGTTG-3’) and D3B (5’-TCGGAAGGAACCAGCTACTA-3’) primers [49]. The ITS1 region was amplified using forward primer 18S (5´TTGATTACGTCCCTGCCCTTT-3´) [50] and reverse primer rDNA1 (5´-ACGAGCCGAGTGATCCACCG-3´) [51]. Finally, the portion of the 18S-rRNA was amplified using primers 988F (5´-CTCAAAGATTAAGCCATGC-3´), 1912R (5´TTTACGGTCAGAACTAGGG-3´), 1813F (5´- CTGCGTGAGAGGTGAAAT-3´) and 2646R (5´-GCTACCTTGTTACGACTTTT-3´) [52].
PCR cycle conditions were: one cycle of 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, annealing temperature of 55°C for 45 s, 72°C for 3 min, and finally one cycle of 72°C for 10 min. PCR products were purified after amplification using ExoSAP-IT (Affmetrix, USB products), quantified using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and used for direct sequencing in both directions using the primers referred to above. The resulting products were purified and run on a DNA multicapillary sequencer (Model 3130XL genetic analyser; Applied Biosystems, Foster City, CA, USA), using the BigDye Terminator Sequencing Kit v.3.1 (Applied Biosystems, Foster City, CA, USA), at the Stab Vida sequencing facilities (Caparica, Portugal). The newly obtained sequences were submitted to the GenBank database under accession numbers indicated on the phylogenetic trees and in Table 1.
Phylogenetic analysis
D2–D3 expansion segments of 28S rRNA, ITS1, and partial 18S rRNA sequences of different Longidorus spp. from GenBank were used for phylogenetic reconstruction. Outgroup taxa for each dataset were chosen according to previous published data [19, 25, 26, 52, 53]. The newly obtained and published sequences for each gene were aligned using MAFFT ver. 7 [54], strategy FFT-NS-1 with default parameters. Sequence alignments were manually edited using BioEdit [55]. Percentage similarity between sequences was calculated using the sequence identity matrix using BioEdit. For that, the score for each pair of sequences was compared directly and all gap or place-holding characters were treated as a gap [55]. When positions of both sequences have a gap they do not contribute [55]. Phylogenetic analyses of the sequence data sets were performed based on Bayesian inference (BI) using MRBAYES 3.1.2 [56]. The best fitted model of DNA evolution was obtained using JMODELTEST v. 2.1.7 [57] with the Akaike Information Criterion (AIC). The Akaike-supported 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 phylogenetic analyses. BI analyses were performed under SYM+I+G (namely, symmetrical of invariable sites and a gamma-shaped distribution) model for D2–D3 expansion segments of 28S rRNA, TVM+I+G and TIM3+I+G (namely, transversional and a transitional of invariable sites and a gamma-shaped distribution) models for the two ITS1 region datasets, TVMef+I+G (namely, equal-frequency transversional of invariable sites and gamma-shaped distribution) model for the partial 18 S rDNA. These BI analyses were run separately per dataset using four chains for 2 × 106, 1 and 1 × 106, and 3 × 106 generations, respectively. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) are given on appropriate clades. Trees were visualised using TreeView [58].
Nomenclatural Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), and hence the new names contained herein are available under that Code from the electronic edition. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSIDs) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn: lsid:zoobank.org:pub: C8230A9D‐FD45‐4AA4‐9ABF‐8445E8001CCC. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.
Results
Taxon Sampling, abundance and prevalence of Longidorus species
All positive Longidorus spp.-sampling sites for this study, including specimens used in morphological and/or genetic analyses, are shown in Table 1 and Fig 1. Ten Longidorus species were associated with wild olive (viz. Longidorus alvegus Roca, Pereira and Lamberti 1989 [59], Longidorus indalus sp. nov., Longidorus intermedius Kozlowska & Seinhorst, 1979 [60], L. lusitanicus, Longidorus oleae Gutiérrez-Gutiérrez, Cantalapiedra-Navarrete, Montes-Borrego, Palomares-Rius & Castillo, 2013 [19], Longidorus silvestris sp. nov., Longidorus vallensis sp. nov., Longidorus vineacola Sturhan & Weischer, 1964 [61], L. vinearum, and Longidorus wicuolea sp. nov.), whereas nine Longidorus species (viz. L. alvegus, Longidorus indalus sp. nov., Longidorus macrodorus sp. nov., L. magnus, L. oleae, Longidorus onubensis sp. nov., Longidorus vallensis sp. nov., L. vineacola, and Longidorus wicuolea sp. nov.) were associated with cultivated olive in Andalusia (Table 1; Fig 1). Except for L. alvegus, L. indalus sp. nov. and L. vineacola, that occurred in both olive types, all the remaining identified species where present only in either wild or cultivated olives.
Longidorus spp. were present in low to moderate densities (from 1 to 33 nematodes per 500 cm3 of soil), and were moderately distributed in both wild and cultivated olives (Table 2). The overall prevalence of Longidorus spp. in wild olives was 16.03% (21 out of 131 samples) whereas in cultivated olives was 5.97% (19 out of 318 samples) (Tables 1 and 2). Although wild and cultivated olives were present in all of the eight provinces of Andalusia, the genus Longidorus was not detected in Jaén and Málaga provinces, and in Granada only in cultivated olives (4 samples out of 39) (Table 2, Fig 1). The three most prevalent Longidorus species, L. indalus sp. nov., L. oleae, and L. vineacola, were detected in both wild and cultivated olives, as well as L. alvegus, L. vallensis sp. nov. and L. wicuolea sp. nov. but with lower prevalence (Tables 1 and 2). Longidorus vineacola was rather moderately distributed among the studied zones having the highest overall prevalence in both wild and cultivated olives (Tables 1 and 2). However, some other Longidorus species showed a lower prevalence and were only detected either in wild (L. lusitanicus, L. silvestris sp. nov. and L. vinearum) or in cultivated olive (L. macrodorus sp. nov., L. magnus, L. onubensis sp. nov.) (Tables 1 and 2).
Taxonomic treatment
Nematoda Linnaeus, 1758 [62]
Dorylaimida Pearse, 1942 [63]
Longidoridae Thorne, 1935 [12]
Longidorinae Thorne, 1935 [12]
Longidorus Micoletzky 1922 [13]
Longidorus indalus Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, sp. nov. urn:lsid:zoobank.org:act:CE07DF59-E705-43D1-9CFF-1A8D793FA58D Figs 2–4.
A) Pharyngeal region. B, C) Details of lip region. D) Vulval region. E-G) Female tails. H) Male tail. I) First-stage juvenile tail (J1).
A) Olive apical galled roots infected by the nematode. B–E) Female anterior regions. F) Detail of odontostyle and odontophore. G) Vulval region. H-K) Female tails. L, M) Male tail with detail of spicules. N-Q) First-, second-, third-, and fourth-stage juvenile (J1–J4) tails, respectively. Abbreviations: a = anus; gr = guiding-ring; odt = odontostyle; odp = odontophore; lp = lateral accessory piece; spl = ventromedian supplements; v = vulva. Scale bars B, C, F = 10 μm; D, E, G-Q = 20 μm.
A) Longidorus indalus sp. nov. B) Longidorus macrodorus sp. nov. C) Longidorus onubensis sp. nov. D) Longidorus silvestris sp. nov. E) Longidorus vallensis sp. nov. F) Longidorus wicuolea sp. nov.
Holotype.
Adult female, collected from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) (37°08'47.5"N, 002°43'31.7"W), at Las Tres Villas, Almería province, Spain; collected by G. Leon Ropero, April 11, 2013; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number ST41-21).
Paratypes.
Female, male and juvenile paratypes extracted from soil samples collected from the same locality as the holotype; mounted in pure glycerine and deposited in the following nematode collections: Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers ST41-01-ST41-17); two females at Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (ST41-20); two females at Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RIT837); and four females at USDA Nematode Collection, Beltsville, MD, USA (T-6629p); collected by G. Leon Ropero, April 11, 2013.
Diagnosis.
Longidorus indalus sp. nov. is characterized by a moderate long body (4.1–6.0 mm), assuming an open C-shaped when heat relaxed; lip region expanded distinctly set off from body contour, 8.5–10.0 μm wide and 3.0–4.5 μm high; guiding-ring located 19.0–27.5 μm from anterior end; relatively short odontostyle (53.5–60.5 μm); amphidial fovea pocket-shaped, slightly asymmetrically bilobed; vulva almost equatorial; female tail long, conoid, and bearing three pairs of caudal pores; c’ ratio (1.8–2.9); males extremely rare, only one male was found, with very short spicules (34.5 μm) and 5 ventromedian supplements; and specific D2–D3, ITS1 rRNA and partial 18S rRNA sequences (GenBank accession numbers KT308852-KT308854, KT308878-KT308879, and KT308894-KT308895, respectively). According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], the new species has the following code (codes in parentheses are exceptions): A1-B1-C2-D4-E2-F23-G3-H56-I12.
Etymology.
The species name is derived from the name ‘indalo’ a prehistoric symbol found in a cave of Almería, the province of the locality where the type specimens were collected.
Description of taxa.
Female. Body somewhat helicoid to arcuate, cylindrical, relatively long and thin, slightly tapering towards at both ends. When heat relaxed, body ventrally curved in open C-shaped. Cuticle thin appearing smooth under low magnifications, 1.9 ± 0.4 (1.5–2.5) μm thick at mid body, but slightly thicker (3.1 ± 0.8 (2.0–4.5) μm) and marked by very fine superficial transverse striate mainly in tail region, as shown by higher magnifications. Lip region expanded distinctly set off from body contour, anteriorly flattened, 9.2 ± 0.5 (8.5–10.0) μm wide and 3.9 ± 0.4 (3.0–4.5) μm high. Amphidial fovea pocket-shaped, slightly asymmetrically bilobed with lobes occupying about 1/3 part of distance between oral aperture and guiding-ring. Stylet guiding-ring single, located 2.8 ± 0.2 (2.5–3.2) times lip region diam. from anterior end. Odontostyle typical of genus, 1.5 ± 0.2 (1.1–1.9) times as long as odontophore, straight or slightly arcuate; odontophore weakly developed, with rather weak basal swellings. Nerve ring surrounding odontophore base at 94.3 ± 4.9 (85.5–107.0) μm from anterior end. Anterior slender part of pharynx usually coiled in its posterior region. Basal bulb short and cylindrical, 92.3 ± 9.6 (72.0–103.5) μm long and 15.6 ± 1.8 (12.5–19.5) μm in diam. Glandularium 83.3 ± 8.7 (63.5–96.0) μm long. Dorsal pharyngeal gland nucleus (DN) and ventrosublateral nuclei (SVN) located at 33.5 ± 4.0 (27.3–39.5)% and 57.0 ± 4.4 (48.9–63.7)% of distance from anterior end of pharyngeal bulb, respectively. Nucleolus of DN larger than nucleoli of two SVN (4.0–4.5 vs 3.0–3.5 μm). Cardia conoid-rounded, 8.2 ± 0.2 (5.5–10.5) μm long. Lateral chord ca 9.6 μm wide at mid-body or ca 28% of corresponding body diam. Reproductive system with both genital branches equally developed, each branch 314–800 μm long, with reflexed ovaries very variable in length (85.5–161 μm long). Vulva in form of a transverse slit, located slightly anterior of the middle of the body, vagina perpendicular to body axis, 13.7 ± 3.2 (8.5–16.5) μm long or 24–47% of corresponding body width, surrounded by well-developed muscles. Genital branches equally developed, 9.7 ± 2.4 (6.8–13.9), 9.9 ± 2.2 (6.7–13.9)% of body length, respectively. Uteri highly variable in length (250–594 μm long), without sperm cells in all female specimens examined; sphincter well-developed, between uterus and oviduct. Eggs mature observed in some gravid female specimens along uterus from one gonoduct, 228.3 ± 8.0 (220.0–236.0) μm long and 32.2 ± 2.0 (30.0–34.0) μm wide. Anterior and posterior oviduct of similar size. Prerectum very variable in length, 673.1 ± 120.7 (489.0–861.0) μm long, and rectum 17.9 ± 3.2 (8.5–16.5) μm long ending in anus as a small rounded slit. Tail long, bluntly conoid, with rounded terminus, bearing three pairs of caudal pores.
Male.
Extremely rare, only one male specimen was found but not in type locality. Morphologically similar to female except for genital system, but with posterior region slightly curved ventrally. Male genital tract diorchic with testes opposed, containing multiple rows of different stages of spermatogonia. Tail conoid, dorsally conoid and ventrally concave with rounded terminus and thickened outer cuticular layer. Spicules very short, moderately developed and slightly curved ventrally; lateral guiding pieces more or less straight or with curved proximal end. Low number of supplements, one pair of adanal and 4 mid-ventral supplements.
Juveniles.
Morphologically similar to adults, but smaller. All four juvenile stages were found, being distinguishable by relative lengths of body and functional and replacement odontostyle (Table 3, Figs 3 and 4; [66, 67]). J1s were characterised by a bluntly conoid tail with a c’ ratio ≥ 3.2, well curved dorsally with a dorsal depression at hyaline region level (Fig 3) odontostyle length ca 37 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages. However, morphology in second-, third- and fourth-stages (except for undeveloped genital structures) similar to that of female, including conoid tail shape, becoming progressively shorter and stouter in each moult and shorter distance from anterior end to guiding-ring in each moult.
Measurements, morphology and distribution.
Morphometric variability is described in Tables 3 and 4 and morphological traits in Figs 2, 3 and 4. In addition to the type locality, Longidorus indalus sp. nov. was extracted from five cultivated olive samples causing enlarged swellings of root tips (Fig 3), and two wild olive samples of several localities distributed in Almería and Granada province, being one of the two species (together with L. magnus) located on Eastern Andalusia (Table 1, Fig 1).
Relationships.
According to the polytomous key by Chen et al. [64] and the supplement by Loof and Chen [65], and on the basis of sorting on matrix codes A (odontostyle length), B (lip region width), C (distance of guiding-ring from anterior body end), D (lip region shape), and E (shape of amphidial pouch), L. indalus sp. nov. is closely related to L. carpetanensis and L. unedoi from which it can be differentiated by a combination of these characters discussed below, but particularly in female and male tail shape (bluntly conoid vs conical, dorsally convex) (Fig 3, S1 Fig). Longidorus indalus sp. nov. differs from L. carpetanensis by a longer body length (4.1–5.7 vs 3.5–4.4 mm), higher a ratio (115.0–178.2 vs 96.0–118.0), slightly higher c and c´ ratio (81.0–122.8 vs 77.0–96.0, 1.8–2.9 vs 1.6–2.2, respectively), and a lower frequency of males (extremely rare vs frequent) [47]. On the other hand, L. indalus sp. nov. differs from L. unedoi in shaving lower c and V ratio (81.0–122.8 vs 122.0–156.0, 42.0–52.0 vs 52.0–58.0; respectively), and slightly higher c´ ratio (1.4–2.0 vs 1.8–2.9) [47]. Finally, L. indalus sp. nov. is molecularly related to L. rubi [68] from which it can be mainly differentiated morphologically in having a smaller odontostyle and spicules length (53.5–60.5 vs 72.0–90.0, 35.0 vs 40.0–45.0 μm; respectively), and lower number of ventromedian supplements in male tail (5 vs 11–12) [19, 68].
Molecular divergence of the new species.
D2–D3 region of L. indalus sp. nov. (KT308852-KT308854) was 91% similar to several Longidorus species such as L. closelongatus (KJ808866), L. pseudoelongatus (KJ802873) and L. rubi (JX4455116) (Table 5). Longidorus indalus sp. nov. showed a high homogeneity for the D2–D3 region (99% similarity, 3 nucleotides) in the eight sampled populations. However, this homogeneity was lower for the ITS1 sequences (KT308878-KT308879) (98% similar, 23 nucleotides and 17 gaps). Some di- and tri-nucleotides microsatellites, (TA)n and (TGG)n, were found in the population from Lecrín, Granada province (KT308854) contributing to sequence variation. Low homologies in the GenBank were found for ITS1 sequence, the closest species in relation to this marker were L. crassus (AF511414) and L. grandis (AF511419), with a similarity of 70% only. The partial 18S of L. indalus sp. nov. (KT308894-KT308895) closely matched (99% similarity) those for L. closelongatus (KJ802897), L. crassus (AY283158) and L. grandis (AY283165).
Above diagonal D2–D3 expansion segments of 28S rRNA and below diagonal internal transcribed spacer 1 (ITS1) region*.
Longidorus macrodorus Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, sp. nov. urn:lsid:zoobank.org:act:9A8C0479-3145-4781-B749-027654C7B8E2 Figs 4–6.
A) Pharyngeal region. B, C) Details of lip region. D) Vulval region. E-F) Female tails. G) Male tail. H) First-stage juvenile tail (J1).
A) Pharyngeal region. B–D) Female anterior regions. E) Detail of basal bulb. F) Vulval region. H-J) Female tails. K, L) Male tail with detail of spicules. M-P) First-, second-, third-, and fourth-stage juvenile (J1–J4) tails, respectively. Abbreviations: a = anus; af = amphidial fovea; dn = dorsal nucleus; spl = ventromedian supplements; svn = subventral nucleus. Scale bars A, K = 100 μm; B-J, L-P = 20 μm.
Holotype.
Adult female, collected from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) (38°22'33.9"N, 005°20'46.9"W), at La Grajuela, Córdoba province, Spain; collected by J. Martin Barbarroja, February 19, 2015; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number JAO6-01).
Paratypes.
Female, male and juvenile paratypes extracted from soil samples collected from the same locality as the holotype; mounted in pure glycerine and deposited in the following nematode collections: Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers JAO6-05-JAO6-20); one female and one male at Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (JAO6-02); one female and one male at Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RIT839); and one female and one male at USDA Nematode Collection, Beltsville, MD, USA (T-6630p); collected by J. Martin Barbarroja, February 19, 2015.
Diagnosis.
Longidorus macrodorus sp. nov. is a gonochoristic species characterized by a very long body (9.3–10.1 mm), assuming a straight to nearly straight body when heat relaxed; lip region conoid-narrowed continuous with body contour, 8.5–12.0 μm wide; guiding-ring located 45.5–55.0 μm from anterior end; very long odontostyle (183.0–210.0 μm); amphidial fovea pocket-shaped, symmetrically bilobed; vulva almost equatorial; female tail short, bluntly conoid, and bearing between three and four pairs of caudal pores; c’ ratio (0.5–1.0); males as frequently as females with long spicules (90.0–112.0 μm) and 17–25 ventromedian supplements; and specific D2–D3, ITS1 rRNA and partial 18S rRNA sequences (GenBank accession numbers KT308855-KT308856, KT308880-KT308881, and KT308896, respectively). According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], the new species has the following code: A7-B1-C45-D1-E2-F54-G12-H1-I2.
Etymology.
The species name refers to the primarily distinguishing character of the long odontostyle (from Greek macros = long, and dorus = stylet).
Description of taxa. Female.
Body very long and rather robust, sharply tapering towards anterior end, usually assuming a body straight or nearly so shape when heat relaxed. Cuticle very finely striated generally but mainly at the posterior extremity, 5.5 ± 0.7 (4.0–7.0) μm thick at mid-body but more thickened at tail tip where it is 13.5 ± 3.0 (8.5–17.0) μm thick, immediately anterior to anus. Lip region conoid-narrowed, anteriorly rounded, and continuous with body contour. Amphidial fovea pocket-shaped slightly symmetrically bilobed with lobes about equal length and extending about 2/3 part of distance between oral aperture and guiding-ring, openings obscure appearing as minute pores, not slit-like. Stylet guiding-ring single, located 5.3 ± 0.6 (4.1–6.0) times lip region diam. from anterior end. Lateral chord 26.2 ± (24.0–30.0) μm wide at mid-body or 20–27% of corresponding body diam. Odontostyle very long and robust straight or slightly arcuate, 3.9 ± 0.3 (3.4–4.0) times as long as distance between anterior end to guiding-ring, odontophore about 2/3 part of the odontostyle length, weakly developed with slightly enlarged at the base. Nerve ring encircling cylindrical part of pharynx at odontophore base, located 271.5 ± 10.3 (252.5–288.0) μm from anterior end. Anterior slender part of pharynx usually coiled in its posterior region. Basal bulb long and cylindrical, 182.2 ± 9.3 (166.0–197.0) μm long or ca one-fourth of neck length, and 36.5 ± 3.7 (28.0–45.0) μm in diam. Glandularium 156.9 ± 8.8 (144.5–172.0) μm long. Normal arrangement of pharyngeal glands [64, 65]: nuclei of the dorsal (DN) and subventral (SVN) glands situated at 26.2 ± 4.0 (21.0–33.0)% and 51.1 ± 3.1 (45.7–55.0)% of the distance from anterior end of pharyngeal bulb, respectively. Dorsal gland nucleus (DN) slightly larger than nuclei of two SVN (4.0–6.0 vs 3.5–5.0 μm in diam.). Cardia hemispherical, 18.7 ± 3.9 (14.5–25.0) μm long. Reproductive system with both genital branches equally developed, relatively short compared to body length, ranging between 622–1318 μm long, with reflexed ovaries very variable in length. Vulva in form of a transverse slit, located about mid-body, vagina perpendicular to body axis, extending to ca 2/3 corresponding body width, surrounded by well-developed muscles. Genital branches equally developed, 8.8 ± 1.6 (6.6–13.0), 8.8 ± 2.0 (6.5–14.0)% of body length, respectively. Uterus short, thick-walled, filled with sperm cells in most female specimens observed; well-developed sphincter between uterus and pars dilatata oviductus, usually containing numerous sperm cells too. Ovaries equally developed and very variable in length, 192–545 μm long, both of them with a single row of oocytes. Prerectum variable in length, 2170 ± 559.7 (1427–3045) μm long, and rectum 46.6 ± 7.9 (36.0–56.0) μm long, anus a small rounded slit. Tail short, bluntly conoid, rounded to almost hemispherical, bearing between three and four pairs of caudal pores.
Male.
Common, as frequently as female. Morphologically similar to female except for genital system, posterior region being more strongly coiled with slightly longer tail. Male genital tract diorchic with testes opposed, containing multiple rows of different stages of spermatogonia. Spicules massive, robust, and curved ventrally; lateral guiding pieces more or less straight or with curved proximal end. Tail convex-conoid, dorsally conoid, ventrally being almost straight with broad blunt terminus and thickened outer cuticular layer. One pair of adanal supplements and 17–25 mid-ventral supplements.
Juveniles.
Morphometrics obtained from juvenile specimens, and of the relative lengths of body, tail, and functional and replacement odontostyle, confirmed the presence of four juvenile stages (Table 6, Figs 4 and 6; [66, 67]). J1s were characterised by a bluntly rounded to cylindrical tail with a c’ ratio ≥ 1.2 (Table 6), an odontostyle very long, ca 120 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages.
Measurements, morphology and distribution.
Morphometric variability is described in Table 6, and morphological traits in Figs 4–7. Longidorus macrodorus sp. nov. was only found in the type locality in the rhizosphere of cultivated olive (Table 1, Fig 1).
A) Pharyngeal region. B, C) Details of lip region. D) Vulval region. E, F) Female tail. G) Male tail. H) First-stage juvenile tail (J1).
Relationships.
L. macrodorus sp. nov. can be differentiated from all known species of the genus by a combination of characters, but particularly by its stylet and odontostyle length (252–288, 183–210 μm, respectively), the longest in the genus. Nonetheless, according to this morphometric character, included on matrix code A (odontostyle length) [64, 65], L. macrodorus sp. nov. groups with L. ishigakiensis Hirata, 2002 [69] and L. tarjani Siddiqi, 1962 [70]. From L. ishigakiensis it differs mainly in having a longer body and odontostyle length (8.3–10.1 vs 5.3–6.9 mm, 183–210 vs 158–181 μm; respectively), lower a and c´ ratios (73.6–92.0 vs 106.0–130.0, 0.5–1.0 vs 1.0–1.2; respectively), higher c ratio (169.9–323.0 vs 133.0–169.0), amphidial pouch shape (symmetrically bilobed vs not bilobed, matrix code E2 vs E1), and presence vs absence of males. From L. tarjani the new species differs mainly by having a longer body and odontostyle length (8.3–10.1 vs 6.0–6.8 mm, 183–210 vs 178–182 μm; respectively), higher c ratio (169.9–323 vs 113–130), and lip region shape (rounded continuous vs set off from body contour, matrix code D1 vs D2). In addition, L. macrodorus sp. nov. is molecularly related to L. baeticus Gutiérrez-Gutiérrez, Cantalapiedra-Navarrete, Montes-Borrego, Palomares-Rius & Castillo, 2013 [19] from which it can be mainly differentiated by a slightly longer body length (8.3–10.1 vs 6.5–9.4 μm), a longer odontostyle length (183.0–210.0 vs 111.0–133.0 μm), and slightly higher c ratio (169.9–323.0 vs 180.0–286.2) [19].
Molecular divergence of the new species.
The sequence divergences between L. macrodorus sp. nov. and other congeneric species were significant, D2–D3 sequences (KT308855-KT308856) were 91% similar to L. baeticus (JX445106-JX445107), L. iuglandis (JX445105) and L. fasciatus (JX445108) (Table 5). No intraspecific variation for the D2–D3 segments was detected between the two studied samples. ITS1 sequences (KT308880-KT308881) region also agree with results obtained from D2–D3, these sequences were 75% similar to L. baeticus (JX445093), L. fasciatus (JX445097) and L. iuglandis (JX445099). Similarity values for the partial 18S of L. macrodorus sp. nov. sequence (KT308896) with those deposited in GenBank were high and matched closely with several sequences such, as L.vineacola (AY283169) and L. elongatus (EU503141).
Longidorus onubensis Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, sp. nov. urn:lsid:zoobank.org:act:A9BE98FF-58A2-4BA4-8DFF-309D31C7D64F Figs 4, 7 and 8.
A) Pharyngeal region. B) Female anterior region. C-F) Female lip regions. G) Detail of basal bulb. H-J) Female tails. K, L) Male tail with detail of spicules. M-P) First-, second-, third-, and fourth-stage juvenile (J1–J4) tails, respectively. Abbreviations: a = anus; af = amphidial fovea; spl = ventromedian supplements. Scale bars A-J, M-P = 20 μm; K, L = 10 μm.
Holotype.
Adult female, collected from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) (37°21'49.3"N, 006°39'56.8"W), Niebla, Huelva province, Spain; collected by J. Martin Barbarroja, January 21, 2012; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number ST5-13).
Paratypes.
Female, male and juvenile paratypes extracted from soil samples collected from the same locality as the holotype; mounted in pure glycerine and deposited in the following nematode collections: Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers ST5-02-ST5-12); one female and one male at Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RIT842); and one female and male at USDA Nematode Collection, Beltsville, MD, USA (T-6631p); collected by J. Martin Barbarroja, January 21, 2012.
Diagnosis.
Longidorus onubensis sp. nov. is a gonochoristic species characterized by a long and rather body (7.4–9.5mm), assuming an open C-shaped when heat relaxed; lip region broadly rounded to truncate, continuous or separated from body contour by slight depression, 14.0–16.5 μm wide; guiding-ring located 31–44μm from anterior end; long odontostyle (103–121 μm); amphidial fovea pocket-shaped with lobes of about equal length; vulva almost equatorial; female tail very short, broadly conoid to hemispherical, and bearing two or three pairs of caudal pores; c’ ratio (0.6–0.8); males frequent with long spicules (92–98 μm) and 14–16 ventromedian supplements; and specific D2–D3, ITS1 rRNA and partial 18S rRNA sequences (GenBank accession numbers KT308857-KT308858, KT308882-KT308883, and KT308897, respectively). According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], the new species has the following code (codes in parentheses are exceptions): A4-B2(3)-C34-D23-E2-F45-G2-H1-I2.
Etymology.
The species epithet refers to ‘Onuba’, the Roman name of the province of Huelva, where the type specimens were collected.
Description of taxa. Female.
Body long and rather robust, slightly tapering towards anterior end, usually assuming an open C-shaped when heat relaxed, almost straight anteriorly and more curved behind the vulva to single spirals. Cuticle appearing smooth, 4.5 ± 0.8 (3.5–6.0) μm thick, 11.1 ± 2.3 (8.5–13.5) μm thick at tail tip, and marked by very fine superficial transverse striate mainly in tail region. Lip region broadly rounded frontally and more so laterally, separated from body contour by slight depression. However, lip region truncate, slightly concave anteriorly and continuous with body contour shape, observed in some female specimens. Amphidial fovea pocket-shaped symmetrically bilobed, with lobes of about equal length, occupying more of 2/3 part of distance between oral aperture and guiding-ring. Labial papillae prominent. Stylet guiding-ring single, located 2.5 ± 0.3 (2.1–3.0) times lip region diam. from anterior end. Lateral chord ca 25 μm wide at mid-body or one-fourth of corresponding body diam. Odontostyle moderate long and robust, usually straight, 1.9 ± 0.1 (1.6–2.29) times as long as odontophore; odontophore weakly developed, posterior slightly enlarged with rather weak basal swellings. Nerve ring encircling cylindrical part of pharynx, 11.3 ± 0.6 (10.2–12.3) times body width at lip region far from anterior end. Anterior slender part of pharynx usually coiled in its posterior region. Basal bulb long and cylindrical, 149.7 ± 11.2 (135.0–173.0) μm long or ca one-third of neck length, 32.0 ± 3.7 (27.0–38.5) μm diam. Dorsal pharyngeal gland nucleus (DN) and ventro-sublateral pair of nuclei (SN) situated slightly posterior to normal arrangement of pharyngeal glands [64, 65], 34.8 ± 4.2 (30.3–39.5)%, and 56.7 ± 7.1 (52.0–69.0)% of distance from anterior end of pharyngeal bulb, respectively. Dorsal gland nucleus (DN) slightly larger than nuclei of two SVN (4.0–4.5 vs 3.5–4.0 μm in diam.). Glandularium 129.6 ± 11.8 (115.0–153.0) μm long. Cardia conoid-rounded, 12.3 ± 1.0 (11.5–13.5) μm long. Reproductive system with both genital branches equally developed, ranging between 456–989 μm long, with reflexed ovaries variable in length. Pars dilatata oviductus and uterus of about equal length, separated by a very strong and muscularised sphincter, on the external wall of which very cell body protrusions are present. Genital branches about equally developed, 7.4 ± 1.2 (6.1–9.4), 8.2 ± 1.5 (5.8–10.4)% of body length, respectively. Uterus wide and thick-walled, filled with little sperm cells in most female specimens observed. Ovaries equally developed 147–233 μm long, both of them with a single row of oocytes. Vulva in form of a transverse slit, approximately equatorial; vagina perpendicular to body axis, 42.0 ± 6.7 (30.0–50.5) μm long, or ca 42% of corresponding body width, surrounded by well-developed muscles. Prerectum very variable in length, 887.0 ± 331.1 (467.0–1155.0) μm long, and rectum 39.6 ± 4.4 (34.0–43.5) μm long, anus a small rounded slit. Tail very short, broadly conoid to hemispherical, with rounded terminus, bearing two or three pairs of caudal pores.
Male.
Common, but less frequent (40%) than female. Morphologically similar to female except for genital system, but with posterior region slightly curved ventrally and longer tail. Male genital tract diorchic with testes opposed, containing multiple rows of different stages of spermatogonia. Tail rounded, dorsally convex conoid, ventrally slightly concave with broad blunt terminus and thickened outer cuticular layer. Spicules arcuate, robust, ca 2 times longer than tail length, lateral guiding pieces more or less straight or with curved proximal end. One pair of adanal supplements and 14–16 midventral supplements.
Juveniles.
Morphologically similar to adults, but smaller. All four juvenile stages were found, being distinguishable by relative lengths of body and functional and replacement odontostyle (Table 7, Figs 4, 7 and 8; [66, 67]). J1s were characterised by a conoid-rounded tail, curved dorsally and slightly concave ventrally with a dorsal-ventral depression at hyaline region level, c’ ratio ≥ 1.5 (Table 7), an odontostyle length ca 58 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages.
Measurements, morphology and distribution.
Morphometric variability is described in Table 7 and morphological traits in Figs 4, 7 and 8. Longidorus onubensis sp. nov. was only found in the type locality from the rhizosphere of cultivated olive (Table 1, Fig 1).
Relationships.
According to the polytomous key by Chen et al. [64] and the supplement by Loof and Chen [65], and on the basis of sorting on matrix codes A (odontostyle length), B (lip region width), D (lip region shape), F (body length), and H (tail shape), L. onubensis sp. nov. is closed to L. goodeyi Hooper, 1961 [71], L. iuglandis Roca, Lamberti & Agostinelli, 1984 [72], L. oleae and L. vinearum. From L. goodeyi it differs mainly in having a longer body and odontostyle length (7.4–9.6 vs 5.6–7.7 mm, 103–121 vs 96–109 μm; respectively), higher c ratio (184.4–272.7 vs 99.0–188.0), and presence vs absence of males) [38, 71]. On the other hand, from L. iuglandis it differs mainly by a slightly longer body length (7.4–9.6 vs 5.4–8.3 mm) [19, 72]. From L. oleae it differs mainly by a smaller distance between guiding-ring from anterior end (28.5–38.5 vs 36.0–46.0 μm), a slightly narrower lip region width (14.0–16.5 vs 14.5–21.0 μm), and amphidial fovea shape (symmetrically vs asymmetrically bilobed) (S1 Table; [19]). Finally, L. onubensis sp. nov. differs mainly from L. vinearum in having a slightly smaller distance between guiding-ring from anterior end and spicules length (28.5–38.5 vs 32.5–47.0 μm, 92.0–98.0 vs 100.0–136.5 μm; respectively), and narrower lip region width (14.0–16.5 vs 18.0–28.0 μm) (S2 Table; [46, 73]).
Molecular divergence of the new species.
D2–D3 region of L. onubensis sp. nov. (KT308857-KT308858) was 95 and 94% similar to L. goodeyi (AY601581) and L. vinearum (KT308874-KT308877), respectively (Table 5). Intraspecific variation of D2–D3 segments detected amongst the studied individuals, consisted of one nucleotide and no indels (99% similarity). Similarly, intraspecific variation of the ITS1 for these sequences (KT308882-KT308883) was low, 99% similarity with 0 nucleotides differences and 3 gaps. ITS1 also showed some similarity (85%) with L. vinearum (KT308892-KT308893). Finally, the partial 18S of L. onubensis sp. nov. (KT308897) showed a high level of similarity (99%) with L. oleae (JX445119), L.vineacola (JX445123), and L. andalusicus (JX445118).
Longidorus silvestris Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, sp. nov. urn:lsid:zoobank.org:pub:C8230A9D-FD45-4AA4-9ABF-8445E8001CCC Figs 4, 9 and 10
A) Pharyngeal region. B, C) Details of lip region. D) Vulval region. E, F) Female tails. G) Male tail. H) First-stage juvenile tail (J1).
A) Female anterior region. B) Detail of basal bulb. C-F) Female lip regions. G) Detail of pharyngeal-intestinal junction. H) Vulval region. I-L) Female tails. M) First-stage juvenile lip region showing replacement odontostyle inside odontophore. N-Q) First-, second-, third-, and fourth-stage juvenile (J1–J4) tails, respectively. R, S) Male tail and detail of spicules. Abbreviations: a = anus; af = amphidial fovea; ca = cardias; gr = guiding-ring; n = nucleus; Rost = replacement odontostyle; sp = spicules. Scale bars = 20 μm.
Holotype.
Adult female, collected from the rhizosphere of wild olive (Olea europaea subsp. silvestris (Miller) Lehr) (36°06'34.4"N latitude, 5°42'39.5"W longitude), Tarifa, Cádiz province, Spain; collected by P. Castillo, May 1, 2012; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number AR27-19).
Paratypes.
Female, male and juvenile paratypes extracted from soil samples collected from the same locality as the holotype; mounted in pure glycerine and deposited in the following nematode collections: Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers AR27-01-AR27-15); one female at Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (AR27-16); one female at Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RIT838); and two females at USDA Nematode Collection, Beltsville, MD, USA (T-6632p); collected by P. Castillo, May 1, 2012.
Diagnosis.
Longidorus silvestris sp. nov. is a gonochoristic species characterized by a long and robust body (5.0–7.0 mm), assuming an open C-shaped when heat relaxed; lip region narrow, conoid-rounded, continuous with body contour, 9.5–11.5 μm wide; guiding-ring located 30.5–35.5 μm from anterior end; odontostyle 76.0–89.0 μm long; amphidial fovea pocket-shaped symmetrically bilobed; vulva equatorial; female tail short, hemispherical to blunty-conoid, bearing two or three pairs of caudal pores and c’ ratio (0.7–1.0); male extremely rare, only one male found, with spicules 69 μm long and 11 ventromedian supplements; and specific D2–D3, ITS1 rRNA and partial 18S rRNA sequences (GenBank accession numbers KT308859-KT308860, KT308884, and KT308898, respectively). According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], the new species has the following code (codes in parentheses are exceptions): A3(2)-B1-C3-D1-E2-F3-G2(1)-H1-I12.
Etymology.
The species name refers to the habitat (silvestris, silvestre = sylvan, living in the wild forest), where the type specimens were collected.
Description of taxa. Female.
Body robust, slightly tapering towards anterior end, usually assuming an open C-shaped when heat relaxed. Cuticle appears smooth, 3.2 ± 0.2 (3.0–3.5) μm thick, 13.6 ± 4.3 (8.0–19.0) μm thick at tail tip, and marked by very fine superficial transverse striae mainly in tail region. Lip region narrow, conoid-rounded, continuous with body contour. Amphidial fovea pocket-shaped symmetrically bilobed, extending about 3/4 part of anterior end-guiding ring distance. Labial papillae prominent. Guiding system with well-developed compensation sacs. Stylet guiding-ring single, located at 32.3 ± 1.6 (30.0–35.5) μm from anterior end. Odontostyle moderately long and narrow, 1.6 ± 0.2 (1.4–2.0) times as long as odontophore, straight or slightly arcuate; odontophore weakly developed, with rather weak basal swellings. Nerve ring encircling narrower part of pharynx. Pharynx consisting of an anterior slender narrow part 307–572 μm long, extending to a cylindrical, terminal pharyngeal bulb, well demarcated anteriorly, 103–155 μm long and occupying ca 22–40% of total pharyngeal length. Glandularium 110.6 ± 13.2 (92.0–136.55) μm long. Dorsal pharyngeal gland nucleus (DN) located at 35.3 ± 4.4 (28.4–42.2)%, nucleolus being slightly larger (2.0–4.5 vs 2.5–3.5 μm) than nucleoli of two ventrosublateral nuclei (SVN) situated at 57.8 ± 4.0 (53.2–64.4)% of distance from anterior end of pharyngeal bulb, respectively. Cardia well-developed, hemispherical, 17.3 ± 2.6 (15.0–21.0) μm long. Reproductive system with both genital branches equally developed, 7.9 ± 0.8 (6.1–9.3), 7.9 ± 0.9 (6.4–9.9)% of body length, respectively. Ovaries reflexed, variable in length, ca 72–110 μm long. Vulva in form of a transverse slit, located about mid-body, vagina perpendicular to body axis, 24.5 ± 2.6 (18.5–32.0) μm long, or 28–48% of corresponding max body width, surrounded by well-developed muscles. Uteri 456 ± 52.7 (372–578) μm long, without sperm cells in the female specimens examined and well-developed sphincter between uterus and oviduct. Prerectum short and variable in length, 414.3 ± 79.9 (266.0–489.0) μm long or ca 5–9% of body length. Rectum 31.4 ± 4.1 (26.5–37.0) μm long. Tail short, hemispherical to blunty-conoid shape, bearing two or three pairs of caudal pores.
Male.
Extremely rare, only one male specimen was found. Morphologically similar to female except for genital system and posterior region slightly curved ventrally Tail convex-conoid, ventrally slightly concave with broad blunt terminus and the thickened outer cuticular layer. Male genital tract diorchic with test opposed, containing multiple rows of different stages of spermatogonia. Spicules arcuate, robust, about 2 times longer than tail length, lateral guiding pieces more or less straight. One pair of adanal supplements preceded by a row of 10 ventromedian supplements.
Juveniles.
Morphologically similar to adults in most respects except for size and development reproductive system. All juvenile developmental stages were detected and distinguished by relative lengths of body and functional and replacement odontostyle (Table 8, Figs 4 and 10; [66, 67]), and the genital primordium. J1s characterised by a conoid-rounded tail, slightly curved dorsally and dorsal-ventral depression at hyaline region level, subdigitate (Fig 10), with a c´ ratio ca 2.5, odontostyle length ca 49 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages. However, morphology in second-, third- and fourth-stages (except for undeveloped genital structures) similar to that of female, including broadly conoid to hemispherical tail shape with rounded terminus, which becoming progressively shorter and stouter in each moult and shorter distance from anterior end to guiding-ring in each moult (Fig 10).
Measurements, morphology and distribution.
Morphometric variability is described in Table 8 and morphological traits in Figs 9 and 10. Longidorus silvestris sp. nov. was only found in type locality from the rhizosphere of wild olive (Table 1, Fig 1).
Relationships.
On the basis of body and odontostyle length, distance between guiding-ring from anterior body end, a, c and c´ ratios, amphidial fovea, or female tail shape, L. silvestris sp. nov. is very closely related to L. wicuolea sp. nov. from which it can be differentiated by a combination of these characters, but particularly in lip region shape (continuous vs separated from body contour by slight depression), and J1 tail shape (conoid-subdigitate vs conoid) (Figs 9 and 10). In addition, according to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], and on the basis of sorting on matrix codes A (odontostyle length), B (lip region width), C (distance of guiding-ring from anterior body end), F (body length), and H (tail shape), L. silvestris sp. can be related with L. belloi Andrés & Arias, 1988 [74], L. igoris Krnjaić, Lamberti, Krnjaić, Agostinelli & Radicci, 2000 [75], and L. moesicus Lamberti, Choleva & Agostinelli, 1983 [76]. From L. belloi it differs mainly in having a slightly shorter odontostyle length (76.0–89.0 vs 74.8–101.7 μm), slightly higher c´ ratio (0.7–1.0 vs 0.5–1.1), frequency of males (extremely rare vs common), amphidial fovea shape (symmetrically vs asymmetrically bilobed) and J1 tail shape (conoid-subdigitate vs conoid) [73, 74]. On the other hand, L. silvestris sp. nov. differs mainly from L. igoris by lower a ratio (73.0–101.4 vs 103.0–131.7) and J1 tail shape (conoid-subdigitate vs cylindrical) [75]. Finally, the new species differs mainly from L. moesicus in having lower a ratio (73.0–101.4 vs 96.0–147.0) and a shorter odontostyle length (76.0–89.0 vs 97.0–124.0 μm) [76, 77]
Molecular divergence of the new species.
Longidorus silvestris sp. nov. was closely related in D2–D3 (KT308859-KT308860) to L. wicuolea sp. nov. (KT308863-KT308866) with 98% similarity (Table 5). Intraspecific variation of D2–D3 detected between the two studied populations was low, 6 nucleotides and no indels. ITS1 also agree with the results obtained for D2–D3, this sequence (KT308884) was 90% similar to L. wicuolea sp. nov. (KT308887-KT308889). Finally, the partial 18S (KT308898) showed high homology with several sequences deposited in the GenBank, such as L. magnus (HM92921345, KT308902), L. vinearum (KT308903), L. lusitanicus (KT308901) and L. wicuolea sp. nov. (KT308900).
Longidorus vallensis Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, sp. nov. urn:lsid:zoobank.org:act:9C1B1CB3-F8CE-422B-BAE1-984B1BFE2173 Figs 4, 11 and 12.
A) Pharyngeal region. B, C) Details of lip region. D) Vulval region. E, F) Female tails. G) First-stage juvenile tail (J1).
A) Pharyngeal region. B-D) Female lip regions. E) Vulval region. F-J) Female tails. K) First-stage juvenile lip region showing replacement odontostyle inside odontophore. L-O) First-, second-, third-, and fourth-stage juvenile (J1–J4) tails, respectively. R) Male tail with detail of spicules. Abbreviations: a = anus; af = amphidial fovea; ca = cardias; gr = guiding-ring; n = nucleus; Rost = replacement odontostyle; sp = spicules. Scale bars = 20 μm.
Holotype.
Adult female, collected from the rhizosphere of wild olive (Olea europaea subsp. silvestris (Miller) Lehr) (36°37'57.3"N, 005°46'20.0"W), at San José del Valle, Cádiz province, Spain; collected by A. Archidona-Yuste, March 17, 2013; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number AR55-16).
Paratypes.
Female, male and juvenile paratypes extracted from soil samples collected from the same locality as the holotype; mounted in pure glycerine and deposited in the following nematode collections: Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers AR55-01-AR55-13); two females at Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (AR55-14); two females at Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RIT8340); and two females at USDA Nematode Collection, Beltsville, MD, USA (T-6633p); collected by A. Archidona-Yuste, March 17, 2013.
Diagnosis.
Longidorus vallensis sp. nov. is characterized by a long and thin body (6.2–8.7 mm), assuming an open C-shaped when heat relaxed; lip region anteriorly rounded separated from body contour by slight depression, 9.0–10.0 μm wide; guiding-ring located 25–30 μm from anterior end; odontostyle moderately long and narrow (71.5–85.0 μm); amphidial fovea pocket-shaped slightly symmetrically bilobed; vulva almost equatorial; female relatively tail short, convex-conoid to bluntly conoid, and bearing three pairs of caudal pores; c’ ratio (1.0–1.4); males not found; and specific D2–D3, ITS1 rRNA and partial 18S rRNA sequences (GenBank accession numbers KT308861-KT308862, KT308885-KT308886, and KT308899, respectively). According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], the new species has the following code (codes in parentheses are exceptions): A2(3)-B1-C2-D2-E2-F4(3)-G3-H2-I1.
Etymology.
The species epithet refers to San José del Valle, the name of the type locality, Cádiz province, where the type specimens were collected.
Description of taxa. Female.
Body long and thin, almost cylindrical, tapering in both extremities, especially in the anterior end. When heat relaxed, body usually assuming a spiral to an open C-shaped. Cuticle appearing smooth under low magnifications, 2.5 ± 0.8 (1.5–4.5) μm thick at mid body, but thicker (5.7 ± 1.4 (3.5–7.5) μm) and marked by very fine superficial transverse striate mainly in tail region, as shown by higher magnifications. Lip region anteriorly rounded, separated from body contour by slight depression. Amphidial fovea pocket-shaped slightly symmetrically bilobed. Labial papillae prominent. Stylet guiding-ring single, located 2.8 ± 0.1 (2.6–3.0) times lip region diam. from anterior end. Lateral chord 13.9 ± 1.9 (12.0–16.0) μm wide at mid-body or 20–30% of corresponding body diam. Odontostyle moderately long and narrow, straight or slightly arcuate, 1.8 ± 1.9 (1.5–2.0) times as long as odontophore, ca 3.0–3.5 μm wide towards its base; odontophore weakly developed, with rather weak basal swellings. Nerve ring encircling cylindrical part of pharynx, 2.2 ± 0.2 (1.9–2.7) times body width at neck base far from anterior end. Anterior slender part of pharynx usually coiled in its posterior region. Basal bulb relatively long and cylindrical, 118.5 ± 8.0 (106.5–135.0) μm long or ca one-third of neck length, 18.4 ± 2.0 (16.0–22.5) μm diam. Dorsal pharyngeal gland nucleus (DN) and ventro-sublateral pair of nuclei (SN) situated slightly posterior to normal arrangement of pharyngeal glands [64, 65], 34.1 ± 4.8 (27.8–40.7)%, 57.8 ± 5.0 (52.1–69.7)% of distance from anterior end of pharyngeal bulb, respectively. Dorsal gland nucleus (DN) slightly larger than nuclei of two SVN (2.5–3.5 vs 1.5–2.5 μm in diam.). Glandularium 102.3 ± 4.9 (95.0–113.0) μm long. Cardia conoid-rounded, 9.2 ± 2.6 (7.0–12.0) μm long. Reproductive system with both genital branches equally developed, very short compared with body length, ranging between 335–597 μm long, with reflexed ovaries variable in length. Vulva in form of a transverse slit, located about mid-body, vagina perpendicular to body axis, 23.0 ± 4.6 (16.0–30.0) μm long, or 30–50% of corresponding body width, surrounded by well-developed muscles. Genital branches equally developed, 5.6 ± 1.3 (4.3–8.2), 6.0 ± 1.3 (4.8–8.3)% of body length, respectively. Uteri short, without sperm cells in the female specimens examined. Anterior and posterior oviduct of similar size. Ovaries equally developed, 106–147 μm long, both of them with a single row of oocytes. Prerectum variable in length, 984.1 ± 133.2 (800–1194) μm long, and rectum 27.2 ± 2.6 (23.5–32.0) μm long, anus a small rounded slit. Tail relatively short, convex-conoid to bluntly conoid, with rounded terminus, bearing three pairs of caudal pores.
Juveniles.
Morphometrics obtained from juvenile specimens, and of the relative lengths of body, tail, and functional and replacement odontostyle, confirmed the presence of four juvenile stages (Table 9, Figs 4 and 12; [66, 67]). J1s were characterised by a conoid tail, dorso-ventrally curved with rounded terminus, and slightly depression at hyaline region level, c’ ratio ≥ 2.3 (Table 9); an odontostyle length ca 53 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages.
Measurements, morphology and distribution.
Morphometric variability is described in Table 9 and morphological traits in Figs 4, 11 and 12. In addition to the type locality, L. vallensis sp. nov. was found from one cultivated olive sample located in Córdoba province (Table 1, Fig 1).
Relationships.
According to the polytomous key by Chen et al. [64] and the supplement by Loof and Chen [65], and on the basis of sorting on matrix codes A (odontostyle length), B (lip region width), C (distance of guiding-ring from anterior body end), D (lip region shape), F (body length), and H (tail shape), L. vallensis sp. nov. groups with L. belloi, L. tabrizicus Niknam et al., 2010 [78] and L. wicuolea sp. nov. From L. belloi it differs mainly in having higher a and c´ ratio (125.1–149.8 vs 73.0–132.0, 1.0–1.4 vs 0.5–1.1; respectively), and the absence vs presence of males [73, 74]. On the other hand, L. vallensis sp. nov. differs from L. tabrizicus mainly by a longer body and odontostyle length (6.2–8.7 vs 4.1–6.1 mm, 71.5–85.0 vs 61.5–70.0 μm; respectively), higher a and c ratio (125.1–149.8 vs 81.5–135.0, 126.6–208.5 vs 91.0–155.0; respectively), and the absence vs presence of males [78]. Finally, from L. wicuolea sp. nov. differs mainly in having higher a ratio (125.1–149.8 vs 79.3–115.6) and slightly higher c´ ratio (1.0–1.4 vs 0.8–1.2) (Table 10, Figs 13 and 14). In addition, L. vallensis sp. nov. is molecularly related to L. rubi from which it can be mainly differentiated by a longer body length (6.2–8.7 vs 4.0–6.0 mm), higher c ratio (126.6–208.5 vs 70.0–126.9) and lower c´ ratio (1.0–1.4 vs 1.7–2.1) [19, 68].
A) Pharyngeal region. B, C) Details of lip region. D) Vulval region. E, F) Female tails. G) First-stage juvenile tail (J1).
A) Pharyngeal region. B-C) Female neck regions. D-F) Female lip regions. G) Detail of odontophore. H) Detail of pharyngeal bulb. I) Detail of cardias (pharyngeal-intestinal junction). J) Vulval region. K-N) Female tails. O-R) First-, second-, third-, and fourth-stage juvenile (J1–J4) tails, respectively. Abbreviations: a = anus; af = amphidial fovea; ca = cardias; gr = guiding-ring; dn = dorsal nucleus; svn = subventral nucleus; V = vulva. Scale bars = 20 μm.
Molecular divergence of the new species.
The sequence divergence between L. vallensis sp. nov. (KT308861-KT308862) and other congeneric species were significant. The closet species in relation to D2–D3 region were L. rubi (JX445116, 96% similarity) and L. indalus sp. nov. (KT308852-KT308854, 91% similarity) (Table 5). Low intraspecific variation was detected in the two studied populations, differing in 3 nucleotides and 0 gaps. ITS1 (KT308885-KT308886) also showed some similarity with L. rubi (JX445098, 81%). No more similarity values above 80% were found in GenBank. Intraspecific variations for ITS1 sequences were 22 nucleotides, and 4 indels. The partial 18S of L. vallensis sp. nov. (KT308899) matched closely, 99%, with several Longidorus species, such as L. rubi (JX445125), L. tabrizicus (FJ009678), L. closelongatus (KJ802897) and L. cretensis (KJ802898).
Longidorus wicuolea Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, sp. nov. urn:lsid:zoobank.org:act:53950FE4-AA33-4301-AFE7-D143C0FC24AE Figs 4, 13 and 14
Holotype.
Adult female, collected from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) (37°28'37.4"N, 005°42'26.7"W), at Carmona, Sevilla province, Spain; collected by A. Archidona-Yuste, May 13, 2015; mounted in pure glycerine and deposited in the nematode collection at Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection number JAO95-17).
Paratypes.
Female, male and juvenile paratypes extracted from soil samples collected from the same locality as the holotype; mounted in pure glycerine and deposited in the following nematode collections: Institute for Sustainable Agriculture (IAS) of Spanish National Research Council (CSIC), Córdoba, Spain (collection numbers JAO95-01-JAO95-16); one female at Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (JAO95-18); one female at Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RIT841); and two females at USDA Nematode Collection, Beltsville, MD, USA (T-6634p); collected by A. Archidona-Yuste, March 17, 2013.
Diagnosis.
Longidorus wicuolea sp. nov. is characterized by a long and robust body (6.1–8.7 mm), assuming an open C-shaped when heat relaxed; lip region anteriorly rounded, separated from body contour by a slight depression, 9.5–12.0 μm wide; guiding-ring located 27–33 μm from anterior end; odontostyle moderately long (77–94 μm); amphidial fovea pocket-shaped symmetrically bilobed; vulva almost equatorial; female relatively tail short, convex-conoid to bluntly conoid, and bearing two or three pairs of caudal pores; c’ ratio (0.8–1.2); males not detected; and specific D2–D3, ITS1 rRNA and partial 18S rRNA sequences (GenBank accession numbers KT308863-KT308866, KT308887-KT308889, and KT308900, respectively). According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], the new species has the following code (codes in parentheses are exceptions): A3(2)-B1(2)-C32-D2-E2-F4(3)-G2(1)-H12-I1.
Etymology.
The species epithet refers to the first letters of its host plants name, wild (wi) and cultivated (cu) olive (olea), where the type specimens were collected.
Description of taxa. Female.
Body long and robust, slightly tapering towards anterior end, usually assuming an open C-shaped when heat relaxed. Cuticle appears smooth, 3.8 ± 0.7 (2.5–5.0) μm thick, 6.5 ± 1.0 (5.5–8.0) μm thick at tail tip, and marked by very fine superficial transverse striae mainly in tail region. Lip region anteriorly rounded, separated from body contour by slight depression. Amphidial fovea pocket-shaped symmetrically bilobed, with lobes of about equal length, and extending about 3/4 part of anterior end-guiding ring distance. Labial papillae prominent. Guiding system with well-developed compensation sacs. Stylet guiding-ring single, located at 29.7 ± 1.6 (27.0–33.0) μm from anterior end. Odontostyle moderately long and narrow, 1.8 ± 0.2 (1.4–2.2) times as long as odontophore, straight or slightly arcuate; odontophore weakly developed, with rather weak basal swellings. Lateral chord ca 19% of corresponding body diam. Nerve ring encircling cylindrical part of pharynx, 1.7 ± 0.2 (1.4–2.0) times body width at neck base far from anterior end. Pharynx consisting of an anterior slender narrow part, extending to a terminal pharyngeal bulb, well demarcated anteriorly and cylindrical, 136.6 ± 9.8 (117.0–150.0) μm long, occupying ca 30% of total pharyngeal length, and 28.9 ± 3.0 (23.5–34.5) μm wide. Glandularium 11 ±0.6 ± 13.2 (92.0–136.55) μm long. Normal arrangement of pharyngeal glands [64, 65]: nuclei of the dorsal (DN) and subventral (SVN) pharyngeal gland located at 28.1 ± 2.8 (23.1–31.5), 56.8 ± 3.1 (50.9–61.6)% of distance from anterior end of pharyngeal bulb, respectively. Dorsal gland nucleus (DN) slightly larger than nuclei of two SVN (3.5–5.0 vs 3.0–4.5 μm in diam.). Cardia well developed, hemispherical to conoid, 14.1 ± 1.0 (12.5–15.5) μm long. Reproductive system with both genital branches equally developed, 7.4 ± 1.0 (5.7–9.8), 7.4 ± 0.8 (6.0–9.0)% of body length, respectively. Ovaries reflexed, very variable in length, ca 100–205 μm long. Vulva in form of a transverse slit, located about mid-body, vagina perpendicular to body axis, 30.9 ± 4.1 (20.0–36.0) μm long, or 30–50% of corresponding body width, surrounded by well-developed muscles. Uterus and oviduct of about equal length, without sperm cells in the female specimens examined. Ovaries equally developed ca 100–205 μm long, both of them with a single row of oocytes. Prerectum very variable in length, 11.1 ± 3.8 (6.4–16.3) times anal body diam., and rectum 1.6 ± 0.3 (1.3–2.0) times as long as anal body diam., anus a small rounded slit. Tail relatively short, convex-conoid to bluntly conoid, with rounded terminus, bearing two or three pairs of caudal pores.
Juveniles.
Morphologically similar to adults, but smaller. All four juvenile stages were found, being distinguishable by relative lengths of body and functional and replacement odontostyle (Table 10, Figs 4 and 14; [66, 67]). J1s were characterised by a conoid tail, dorso-ventrally curved with rounded terminus, and slightly depression at tip tail level, c’ ratio ≥ 1.7 (Table 10); an odontostyle length ca 52 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages.
Measurements, morphology and distribution.
Morphometric variability is described in Table 10 and morphological traits in Figs 4, 13 and 14. In addition to the type locality, L. wicuolea sp. nov. was extracted from one wild olive sample located in Huelva province, being distributed only in Western Andalusia (Table 1, Fig 1).
Relationships.
On the basis of body and odontostyle length, distance between guiding-ring from anterior body end, a, c and c´ ratios, amphidial fovea, or female tail shape, L. wicuolea sp. nov. is very closely related to L. silvestris sp. nov. from which it can be differentiated by a combination of these characters, but particularly in lip region shape (separated from body contour by slight depression vs anteriorly rounded continuous), and J1 tail shape (conoid vs conoid-subdigitate) (Figs 9, 10, 13 and 14). In addition, according to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], and on the basis of sorting on matrix codes A (odontostyle length), B (lip region width), C (distance of guiding-ring from anterior body end), D (shape of anterior region), F (body length), H (tail shape) and I (presence/absence of males), L. wicuolea sp. nov. can be related with L. henanus Xu & Cheng, 1992 [79] and L. vallensis sp. nov. From L. henanus it differs mainly in having a longer body and tail length (6.1–8.7 vs 3.8–7.0 mm, 37.5–56.0 vs 24.6–42.0 μm; respectively), a shorter odontostyle length (77.0–95.0 vs 90.5–104.0 μm) and a narrower lip region width (9.5–13.5 vs 13.2–18.0 μm) [79, 80, 81]. Finally, L. wicuolea sp. nov. differs basically from L. vallensis sp. nov. by lower a and c´ ratio (79.3–115.6 vs 125.1–149.8, 0.8–1.2 vs 1.0–1.4; respectively) (Table 9, Figs 11 and 12).
Molecular divergence of the new species.
D2–D3 sequences from L. wicuolea sp. nov. (KT308863-KT308866) differed with the closest related species, L. silvestris sp. nov. (KT308859-KT308860) by 13 nucleotides (98% similarity) and from L. magnus (JX445112) and L. vineacola (JX445110) by 60 nucleotides (92% similarity) [Table 5]. Intraspecific variation of D2–D3 segments detected between the two studied populations of L. wicuolea sp. nov. consisted of 6 nucleotides (99% similarity), and no indels. Similarly, the ITS1 (KT308887-KT308889) also showed a low intraspecific variability between the two studied populations with only 4 nucleotides (99% similarity). The closet ITS1 to that of L. wicuolea sp. nov. was L. silvestris sp. nov. (KT308884) consisting in 73 nucleotides and 37 gaps (90% similarity). The partial 18S of L. wicuolea sp. nov. (KT308900) closely matched with several species of Longidorus, some of them were L. magnus (HM92921345, KT308902), L. vinearum (KT308903), L. lusitanicus (KT308901) and L. silvestris sp. nov. (KT308898).
Morphology and morphometrics of known Longidorus species
Morphological and morphometrical data as well as molecular delineation (rDNA) of L. alvegus, L. intermedius, L. magnus, L. oleae and L. vineacola have been previously recorded within studies of dagger and needle nematodes infesting vineyards in southern Spain [19, 28]. The new records of these species from wild and cultivated olive in Granada and Sevilla provinces presented here extend the geographical distribution of these species (S1 and S2 Tables) in southern Spain [19]. Consequently, only D2–D3 sequences had been reported here for these samples. For other known species studied, representing the first molecular characterization and new records for olive or for Spain (viz. L. lusitanicus and L. vinearum), a brief description and a morphometric comparison with previous records and paratypes is provided below (S1–S3 Figs, S1 and S2 Tables).
Longidorus lusitanicus Macara 1985.
The gonochoristic population of Longidorus from wild olive at Sanlúcar de Barrameda (Cádiz province) agrees fairly well with studied paratypes and original description of L. lusitanicus. This population was characterised by a lip region expanded or distinctly offset by constriction, rounded laterally and almost flattened frontally; amphidial fovea pouch-shaped, distinctly asymmetrically bilobed; female tail conoid-rounded; and the same proportion of male specimens found (S1 Table, S2 Fig). Morphometrics were coincident with those provided in the original description, except for only minor differences in oral aperture-guiding ring distance, which may be due to few specimens originally studied, or geographical intraspecific variability [45]. This is the first report for Spain and confirms a wider distribution in the Iberian Peninsula, apart from original description in Portugal. According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], this species has the following code: A3 B34 C23 D4 E3 F234 G2 H1 I2.
D2–D3 segments of L. lusitanicus (KT308869) was 95% similar to L. vinearum (KT308874-KT308877), L. goodeyi (AY601581), L. magnus (JX445112) and L. onubensis sp. nov. (KT308857-KT308858). The ITS1 of L. lusitanicus (KT308891) showed some homology with L. onubensis sp. nov. (81% similarity) and scarce homology with other ITS1 sequences from Longidorus species available in GenBank. The partial 18S region of L. lusitanicus (KT308901), was very similar to several sequences of Longidorus spp., including L. vineacola (JX445153, AY283169), L. magnus (HM921345) and L. onubensis sp. nov. (KT308897).
Longidorus vinearum Bravo & Roca, 1995.
The four gonochoristic populations of L. vinearum from wild olive at Santa María de Trassierra (Córdoba province) agree fairly well with studied paratypes and original description of L. vinearum. The four studied populations were characterised by a robust and long body, lip region anteriorly rounded and separated from body contour by a very slight depression; amphidial fovea pouch-shaped, distinctly asymmetrically bilobed; female tail short, bluntly rounded to hemispherical with rounded terminus; and the common presence of male specimens (S2 Table, S3 Fig). Morphometrics of female, male and J1 specimens were coincident with those provided in the original description and rather similar to data reported subsequently for other populations of Portugal, except for minor differences in a ratio and length of spicules, which may be due to few specimens originally studied or geographical intraspecific variability [46, 73]. This is the first report for Spain and confirms a wider distribution in the Iberian Peninsula, apart from original description and other populations in Portugal. According to the polytomous key Chen et al. [64] and the supplement by Loof and Chen [65], this species has the following code: A45 B345 C34 D2 E3 F345 G12 H1 I2.
The closet species regarding D2–D3 segments of L. vinearum (KT308874-KT308877) were L. magnus (HM921361, JX445112, 96% similarity) and L. goodeyi (AY601581, 94%). ITS1 (KT308892-KT308893) region also showed some similarity with L. magnus (HM921340, 90% similarity), but no more similarity values above 80% were found in GenBank. The partial 18S of L. vinearum (KT308903) matched closely (99%) with several Longidorus spp., such as L. vineacola (JX445153, AY283169) and L. magnus (HM921345).
Phylogenetic relationships of the Longidorus spp.
The amplification of D2–D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial 18S rRNA yielded a single fragment of approximately 800 bp, 1000 bp, and 1500 bp, respectively, based on gel electrophoresis. Sequences from other species of Longidorus spp. obtained from National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) were used for further phylogenetic studies. Sequences for L. indalus sp. nov., L. lusitanicus, L. macrodorus sp. nov., L. onubensis sp. nov., L. silvestris sp. nov., L. vallensis sp. nov., L. vinearum, and L. wicuolea sp. nov., were obtained for these species in this study. On the other hand, sequences for L. alvegus (KT308867), L. intermedius (KT308868, KT308890), L. magnus (KT308870), L. oleae (KT308871) and L. vineacola (KT308872, KT308873) matched well with former sequences deposited in GenBank, extending the molecular diversity of these species to the newly studied areas.
Phylogenetic relationships among Longidorus species inferred from analyses of D2–D3 expansion segments of 28S, ITS1, and the partial 18S rDNA gene sequences using BI are given in Figs 15, 16 and 17, respectively. To facilitate discussion, clades that were well supported or are taxonomically well founded are labelled in roman numerals from I through VII (Fig 15). Poorly supported lineages are not explicitly labelled. The 50% majority rule consensus 28S rRNA gene BI tree of Longidorus and Paralongidorus spp. based in a multiple edited alignment including 133 sequences and 748 total characters consisted of six moderate to highly supported major clades in the genus (Fig 15). Clade I is well-supported (PP = 100%) comprising 16 species including nine reported in olives: L. vinearum (KT308874-KT308877), L. onubensis sp. nov. (KT308857-KT308858), L. silvestris sp. nov. (KT308857-KT308860), L. lusitanicus (KT308869), L. wicuolea sp. nov. (KT308863-KT308866) and L. macrodorus sp. nov. (KT308855-KT308856), L. magnus (JX445112, HM921361, KT308870), L. oleae (JX445103, KT308871), L. vineacola (JX445110-JX445111, KT308873-KT308874) and other Longidorus spp. from the Mediterranean Basin such as L. andalusicus (JX445101-JX445102), L. fasciatus (JX445108), L. iuglandis (JX445104- JX445105), L. crataegi (JX445114), L. baeticus (JX445106- JX445107), L. orientalis (GU001823, KJ802877), and L. goodeyi (AY601581) from UK. All these species shared a hemispherical, convex-conoid and short tail. Clade II is well-supported (PP = 100%) comprising ten species and including L. intermedius (AY601577, JX445117, KT308868). Longidorus vallensis sp. nov. (KT308861-KT308862), was phylogenetically related to L. rubi (JX445116) forming a well-supported clade (PP = 100%), and with L. alvegus (JX445115, HM921360, KT308867) which formed a sister-clade, however the BI values for this sister-clade is low. Finally, L. indalus sp. nov. (KT308852-KT308854) did not form supported clades with any of Longidorus species. Clade III is also well-supported (PP = 100%) and comprised all Paralongidorus species, except P. bikanerensis (JN032584), which clustered in the moderately supported (PP = 81%) clade IV with other species from different geographical origin. Clade V and VI are well-supported (PP = 100%) and comprised five species of Asiatic origin, and a basal well-supported (PP = 100%) clade VI with four species from different geographical origin (Fig 15).
Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are in bold letters. Scale bar = expected changes per site. A). Clades I & II. B). Clades III-VI.
Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are in bold letters. Scale bar = expected changes per site.
Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are in bold letters. Scale bar = expected changes per site.
Difficulties were experienced with alignment of the ITS1 sequences due to scarce homology, thus, related sequences were divided into two different groups in our study (Fig 16). The first group included 752 characters and 29 sequences comprising several Longidorus species also from the Mediterranean Basin and with hemispherical, convex-conoid and short tail, L. lusitanicus (KT308891), L. macrodorus sp. nov. (KT308880-KT308881), L. onubensis sp. nov. (KT308882-KT308883), L. silvestris sp. nov. (KT308857-KT308860), L. wicuolea sp. nov. (KT308884), L. vinearum (KT308892-KT308893), L. vallensis sp. nov. (KT308885-KT308886), and L. intermedius (KT308890) with a short body length (Fig 16). These results agree with those obtained for D2–D3 segments. This phylogenetic tree resolved two major well supported (PP = 100%) clades, L. vinearum, L. lusitanicus, L. onubensis sp. nov., L. silvestris sp. nov. and L. wicuolea sp. nov. were placed within the first major clade. Longidorus vinearum, L. lusitanicus and L. onubensis sp. nov. formed a high supported subclade (PP = 100%) with L. magnus (HM921340). Longidorus wicuolea sp. nov. was placed within another well supported subclade (PP = 100%) with L. silvestris sp. nov. and finally L. macrodorus sp. nov. was phylogenetically related to L. baeticus (JX445093). Second major clade was low-supported and was formed by five Longidorus species, L. vallensis sp. nov. was placed with L. rubi (JX445098) in a high-supported subclade (PP = 100%) and it was related to L. alvegus (HM921339) which formed another low-supported subclade. Finally, L. intermedius and L. elongatus (GU199044) formed a high-supported subclade (PP = 100%), occupying a basal position in the tree (Fig 16).
The second group of the ITS1 sequences included 1126 characters and 12 sequences comprising ten Longidorus species characterized by a medium to short body length, including L. indalus sp. nov. (KT308878-KT308879), L. profundorum (AJ549988), L. sturhani (FJ009680), L. crassus (AF511414), L. kuiperi (AM905257-AM905258), L. fragilis (AF511418) and L. breviannulatus (AF511413). Longidorus indalus sp. nov. clustered with L. profundorum in a high supported clade (PP = 100%) (Fig 16).
The 50% majority rule BI tree of a multiple alignment including 90 18S sequences and 1687 bp and as well as in the D2–D3 and ITS1 tree, L. lusitanicus (KT308901), L. macrodorus sp. nov. (KT308896), L. onubensis sp. nov. (KT308897), L. silvestris sp. nov. (KT308898), L. vinearum (KT308903) and L. wicuolea sp. nov. (KT308900) clustered within the same well-supported (PP = 100%) clade with Longidorus species from Mediterranean Basin and sharing a convex-conoid female tail shape such as L. andalusicus (JX445118), L. oleae (JX445119), L. vineacola (JX445123, AY283169), L. magnus (HM921345-KT308902), L. baeticus (JX445121), L. fasciatus (JX445122) and L. iuglandis (JX445120). Phylogenetic inferences based on 18S also suggest that L. vallensis sp. nov. and L. rubi are close-related species (PP = 100%). Finally, L. indalus sp. nov. (KT308894-KT308895) clustered in this case with L. dunensis (AY284819) with a low support (PP = 81%).
Discussion
The primary objective of this study was to unravel the biodiversity, distribution and molecular phylogeny of needle nematodes of the genus Longidorus associated with wild and cultivated olives in southern Spain. This was conducted in an extensive and systematic nematological survey that included 159 locations and 449 sampling sites. We found 40 Spanish populations of Longidorus spp. infesting olive soils. Our results demonstrate that the use of morphological studies together with rDNA molecular markers may decipher the specific biodiversity in this complex group of plant-parasitic nematodes. We described here six new Longidorus species, based on integrative taxonomy and the phylogenetic relationships of the genus Longidorus based on nuclear rDNA markers.
The comparative morphological and morphometrical study of the 40 Spanish populations of Longidorus spp. confirmed that diagnosis and identification of these species based solely on diagnostic morphometric features is quite complex since there is almost a continuous range of character measurements within populations as well as among species [8, 19]. The present results (including new and known species) enlarge the biodiversity of Longidorus in the Iberian Peninsula and agree with previous data obtained for the phylogeny and biogeography of the genus Longidorus in the Euro-Mediterranean region [19, 28, 82, 83], in which a dispersalist model was one of the primary explanations for the large groups of Longidorus species found in this region.
Considering the species richness of PPN associated with olive in different studies, the genus Longidorus is one of the most biodiverse with nine species (viz. L. africanus, L. belloi, L. closelongatus, L. cretensis, L. elongatus, L. macrosoma, L. oleae, L. pseudoelongatus, L. siddiqii, and L. vinearum) reported in several countries of the Mediterranean Basin such as Egypt, Greece, Jordan, Portugal and Spain [19, 35, 36, 73, 77, 84, 85, 86, 87, 88, 89]. Although all Longidorus spp. are obligate soil plant ecto-parasites of a wide range of wild and cultivated plants causing enlarged swellings of root tips, it is unlikely that these species could be detected in other wild and cultivated plants in the next future. The present results double the previous biodiversity of Longidorus species detected in olive worldwide, including six new species and two new records for wild and cultivated olives (L. alvegus and L. vineacola), as well as two additional new records for wild olives (L. intermedius and L. lusitanicus). The most recent major geological event having important effects for nematode biodiversity and distribution in Europe was the Quaternary glaciation which happened ca. 40,000 years ago. In Europe has been hypothesized that reduced species numbers in northern Europe is attributed to Quaternary glaciations, being the highly diverse nematofauna of the Mediterranean basin related to Miocene plate tectonics in that area [90]. Our study showed a great diversity in Southern Spain. However, because of no sampling North-South has been developed; more intensive studies are needed in northern areas in order to corroborate this hypothesis. The distribution of the 40 Longidorus populations collected in Andalusia showed that some of them revealed a certain geographic associations to western areas (viz. L. alvegus, L. intermedius, L. lusitanicus, L. onubensis sp. nov., L. vineacola, L. vinearum, L. wicuolea sp. nov.) and eastern regions (viz. L. indalus sp. nov.), while only L. magnus was detected in both areas (Fig 1). The present findings showed certain coincidences with the quantitative analysis of Longidorus spp. distribution carried out by Navas et al. [82], who recognized two main groups of species, the European-Atlantic and the Mediterranean. The widespread distribution of L. magnus may suggest a high ecological flexibility e.g. adaptability to a range of soil types, and reproduction sustained over a broad temperature range [91]. While other species seems to be better adapted to drier areas as it is the case for L. indalus sp. nov. in Eastern regions with markedly lower precipitation. Species showing a restricted distribution may be the result of isolation of populations in diverse biotopes which would result in reproductive isolation and hence the establishment of new species [91]. Also, although agricultural activities may result in the widespread dissemination of Longidorus species [91], the geographical distribution of Longidorus species in wild and cultivated olives in southern Spain suggest an established pattern related to ecological factors, on a geological timescale. These nematodes could have a lower dissemination by human activities than other plant-parasitic nematodes (i.e. cyst- or root-lesion nematodes) because of their sensitivity to fast desiccation, large body size, and the absence of survival-resistance forms. Unfortunately, little is known about the ecological requirements of Longidorus nematodes and elucidation of speciation and species biodiversity has currently to be approached on the groupings of morphometric characters [91]. Consequently, further research is needed in order to determine the influence of physico-chemical soil factors on the incidence and distribution of these nematodes in southern Spain and other wider areas.
Sequences of nuclear ribosomal RNA genes, particularly D2–D3 and ITS1, have proven to be a powerful tool for providing accurate species identification of Longidoridae [22, 25, 92]. However D2–D3 expansion region was more useful for establishing phylogenetic relationships among Longidorus species than ITS1 or 18S. The great diversity detected in the ITS1 suggests that a variety of poorly understood factors are involved in the fast evolution of this region in nematodes. Thus, ITS1 appears better suited for differentiation of species than for phylogenetic relationships within Longidorus. Our findings also confirm that partial 18S sequence does not have enough resolution to distinguish species, because different species showed a low nucleotide differences amongst them. Phylogenetic analyses based on D2–D3, ITS1, and partial 18S using BI resulted in a congruent position for the newly sequenced species of Longidorus spp. from Spain, which grouped in a separate clade, except for L. vallensis sp. nov. (KT308861-KT308862) and L. indalus sp. nov. (KT308852-KT308854) in the D2–D3, partial 18S, and ITS1 trees, which grouped separately (Figs 15, 16 and 17). Longidorus vallensis sp. nov. clustered in all ribosomal markers with L. rubi. However these species showed several morphological differences that made it difficult to establish a correspondence between morphological characters and the phylogenetic trees inferred from the molecular data. The majority of the species showed congruence in the phylogenetic relationships within these ribosomal markers using the DNA from the same individual. However L. indalus sp. nov. phylogenetic position was not congruent amongst the different ribosomal markers used here. This could be a result of different mutation rates within the different ribosomal markers, or difficulties in sequence alignment in ITS1 sequences. The phylogenetic relationships inferred in this study based on the D2–D3 and ITS1 sequences mostly agree with the lineages obtained by other authors [19, 25, 28, 93, 94]. Most of the newly and known described species in this research (viz. L. lusitanicus, L. macrodorus sp. nov., L. magnus, L. oleae, L. onubensis, L. silvestris sp. nov., L. vineacola, L. vinearum, L. wicuolea sp. nov.) grouped genetically in the same clade. These species shared a long body and odontostyle and can be considered as the most evolved species in the genus [14]. These traits could be related to the feeding habits of these nematodes, since longer stylets are better adapted to penetrate major woody plants roots persisting during the hot-dry summer conditions prevalent in Southern Spain and with long body sizes to move quickly deeper in the soil to avoid dry conditions in summer.
To confirm the correlation of the results obtained by conventional morphological approaches and new molecular methods is important for the proper understanding of the evolution of the genus Longidorus. The close relationship of the morpho-species groups detected in this and previous studies in Spain was also supported by molecular data (most of the species described were in the same clade), an observation that points to the Iberian Peninsula as a possible center of recent speciation [19], as it was suggested for other genera such as Xiphinema [5, 19, 22, 28], Trichodorus [95] or Rotylenchus species [11].
Conclusions
In summary, the present study establishes the importance of using integrative taxonomic identification highlighting the difficulty of a correct identification at species level within the genus Longidorus. This study also provides molecular markers for precise and unequivocal diagnosis of some species of Longidorus in order to differentiate virus vector or quarantine species. This and previous studies demonstrate that the genus Longidorus is clearly a complex group and much work remains to be done to elucidate species boundaries in this economically important group of PPN. Furthermore, similar intensive and extensive integrative studies on Longidorus species in several wider areas may help to elucidate if Longidorus species have originated in Southeast Africa and India, when these two areas were still united, and a later spread to Laurasia was followed by a main speciation of Longidorus in the Holarctic region, especially Europe, as hypothesised by Coomans [14]. This hypothesis is reinforced with the basal position of Asian species in D2–D3 region and partial 18S phylogenetic trees.
Supporting Information
S1 Fig. Light micrographs of Longidorus carpetanensis Arias et al., 1986 (A-F) and Longidorus unedoi Arias et al., 1986 (G-L) paratypes from the Nematode Collection of the Spanish National Museum of Natural Sciences-CSIC, Madrid, Spain. A-B, G-H) Female anterior regions.
C-D, I-J) Female tails. E-F, K-L) Male tails. Abbreviations: a = anus; gr = guiding-ring; spl = ventromedian supplements. Scale bars A-L = 20 μm.
https://doi.org/10.1371/journal.pone.0147689.s001
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S2 Fig. Light micrographs of Longidorus lusitanicus Macara, 1985 from wild olive at Sanlúcar de Barrameda (Cádiz province) (A-F), and paratypes from the Nematode Collection at the Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (G-L). A-C, G-I) Female lip regions.
J) Vulval region. D-E, K, L) Female tails. F) Male tail. Abbreviations: a = anus; af = amphidial fovea. (Scale bars = 20 μm).
https://doi.org/10.1371/journal.pone.0147689.s002
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S3 Fig. Light micrographs of Longidorus vinearum Bravo & Roca, 1995 from wild olive at Santa Mª de Trassierra (Córdoba province) (A-G), and paratypes from the nematode collection at the Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy (H-L). A-B, H-J) Female lip regions.
C) Vulval region. D, K, L) Female tails. E-G) Male tails and detail of spicules. Abbreviations: a = anus; af = amphidial fovea. (Scale bars = 20 μm).
https://doi.org/10.1371/journal.pone.0147689.s003
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S1 Table. Morphometrics of Longidorus lusitanicus Macara, 1985 and Longidorus oleae Gutiérrez-Gutiérrez et al., 2013 studied from southern Spain.
https://doi.org/10.1371/journal.pone.0147689.s004
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S2 Table. Morphometrics of Longidorus vinearum Bravo & Roca, 1995 populations studied from southern Spain.
https://doi.org/10.1371/journal.pone.0147689.s005
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Acknowledgments
This research is part of the PhD project of the first author. The authors thank Hava Rapoport for valuable suggestions and proofreading the manuscript, and J. Martín-Barbarroja and G. León Ropero (IAS-CSIC) for the excellent technical assistance.
Author Contributions
Conceived and designed the experiments: PC AAY JANC JEPR CCN. Performed the experiments: PC AAY JANC JEPR CCN. Analyzed the data: PC AAY JANC JEPR CCN. Contributed reagents/materials/analysis tools: AAY JEPR CCN. Wrote the paper: PC AAY JANC JEPR CCN.
References
- 1. Boucher G, Lambshead PJD. Ecological biodiversity of marine nematodes in samples from temperate, tropical, and deep sea regions. Conserv Biol. 1995; 9: 1594–1604.
- 2. Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, et al. A molecular evolutionary framework for the Phylum Nematoda. Nature. 1988; 392: 71–75.
- 3. Coomans A. Nematode systematics: past, present and future. Nematology. 2000; 2: 3–7.
- 4.
Siddiqi MR. Tylenchida parasites of plants and insects. 2nd ed. Wallingford, UK: CABI Publishing; 2000.
- 5.
Coomans A, Huys R, Heyns L, Luc M. Character analysis, phylogeny and biogeography of the genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae). Koninklijk Museum voor Midden-Afrika Tervuren, België. Ann Zool Wetenschappen. 2001; 287: 1–239.
- 6. Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K, et al. Cryptic species as a window on diversity and conservation. Trends Ecol Evol. 2006; 22: 148–155. pmid:17129636
- 7.
Subbotin SA, Moens M. Molecular taxonomy and phylogeny. In: Perry RN, Moens M, editors. Plant Nematology. Wallingford, UK: CABI; 2006; pp. 33–58.
- 8. Palomares-Rius JE, Cantalapiedra-Navarrete C, Castillo P. Cryptic species in plant-parasitic nematodes. Nematology. 2014; 16: 1105–1118.
- 9. Oliveira CMG, Ferraz LCCB, Neilson R. Xiphinema krugi, species complex or complex of cryptic species? J Nematol. 2006; 38: 418–428. pmid:19259458
- 10. Gutiérrez-Gutiérrez C, Palomares-Rius JE, Cantalapiedra-Navarrete C, Landa BB, Esmenjaud D, Castillo P. Molecular analysis and comparative morphology to resolve a complex of cryptic Xiphinema species. Zool Scr. 2010; 39: 483–498.
- 11. Cantalapiedra-Navarrete C, Navas-Cortés JA, Liébanas G, Vovlas N, Subbotin SA, Palomares-Rius JE, et al. Comparative molecular and morphological characterisations in the genus Rotylenchus: Rotylenchus paravitis n. sp., an example of cryptic speciation. Zool Anz. 2013; 252: 246–268.
- 12. Thorne G. Notes on free-living and plant parasitic nematodes. II. Higher classification groups of Dorylaimoidea. P Helm Soc Wash. 1935; 2: 96–98.
- 13. Micoletzky H. Die freilebenden Erd-Nematoden. Arch Naturg Berlin Abt. 1922; 87: 1–650.
- 14. Coomans A. Phylogeny of the Longidoridae. Russ J Nematol. 1996; 4: 51–60.
- 15.
Taylor CA, Brown DJF. Nematode Vectors of Plant Viruses. Wallingford, UK: CAB International; 1997.
- 16. MacFarlane SA, Neilson R, Brown DJF. Nematodes. Adv Bot Res. 2002; 36: 169–198.
- 17. MacFarlane SA. Molecular determinants of the transmission of plant viruses by nematodes. Mol Plant Pathol. 2003; 4: 211–215. pmid:20569381
- 18. Peneva VK, Lazarova SS, De Luca F, Brown DJ. Description of Longidorus cholevae sp. n. (Nematoda, Dorylaimida) from a riparian habitat in the Rila Mountains, Bulgaria. ZooKeys. 2013; 330: 1–26. pmid:24146553
- 19. Gutiérrez-Gutiérrez C, Cantalapiedra-Navarrete C, Montes-Borrego M, Palomares-Rius JE, Castillo P. Molecular phylogeny of the nematode genus Longidorus (Nematoda: Longidoridae) with description of three new species. Zool J Linn Soc. 2013; 167: 473–500.
- 20. Decraemer W, Robbins RT. The who, what and where of Longidoridae and Trichodoridae. J Nematol. 2007; 39: 295–297. pmid:19259501
- 21. Ye W, Szalanski AL, Robbins RT. Phylogenetic relationships and genetic variation in Longidorus and Xiphinema species (Nematoda: Longidoridae) using ITS1 sequences of nuclear ribosomal DNA. J Nematol. 2004; 36: 14–19. pmid:19262783
- 22. Gutiérrez-Gutiérrez C, Cantalapiedra-Navarrete C, Decraemer W, Vovlas N, Prior T, Palomares-Rius JE, et al. Phylogeny, diversity, and species delimitation in some species of the Xiphinema americanum-group complex (Nematoda: Longidoridae), as inferred from nuclear and mitochondrial DNA sequences and morphology. Eur J Plant Pathol. 2012; 134: 561–597.
- 23. De Luca F, Reyes A, Grunder J, Kunz P, Agostinelli A, et al. Characterization and sequence variation in the rDNA region of six nematode species of the genus Longidorus (Nematoda). J Nematol. 2004; 36: 147–152. pmid:19262800
- 24. Neilson R, Ye W, Oliveira CMG, Hübschen J, Robbins RT, Brown DJF, et al. Phylogenetic relationships of Longidoridae species (Nematoda: Dorylaimida) from North America inferred from 18S rDNA sequence data. Helminthologia. 2004; 41: 209–215.
- 25. He Y, Subbotin S, Rubtsova TV, Lamberti F, Brown DJF, Moens M. A molecular phylogenetic approach to Longidoridae (Nematoda: Dorylaimida). Nematology. 2005; 7: 111–124.
- 26. Palomares-Rius JE, Subbotin SA, Landa BB, Vovlas N, Castillo P. Description and molecular characterisation of Paralongidorus litoralis sp. n. and P. paramaximus Heyns, 1965 (Nematoda: Longidoridae) from Spain. Nematology. 2008; 10: 87–101.
- 27. De Luca F, Landa BB, Mifsud D, Troccoli A, Vovlas N, Castillo P. Molecular characterisation of Longidorus kuiperi Brinkman, Loof & Barbez, 1987 (Nematoda: Longidoridae) from the Mediterranean Basin. Nematology. 2009; 11: 155–160.
- 28. Gutiérrez-Gutiérrez C, Palomares-Rius J, Cantalapiedra-Navarrete C, Landa BB, Castillo P. Prevalence, polyphasic identification, and molecular phylogeny of dagger and needle nematodes infesting vineyards in southern Spain. Eur J Plant Pathol. 2011; 129: 427–453.
- 29. Pedram M, Niknam G, Robert RT, Ye W, Karegar A. Longidorus kheirii sp. n. Nematoda: Longidoridae) from Iran. Syst Parasitol. 2008; 71: 199–211. pmid:18815899
- 30. Belaj A, Muñoz-Díaz C, Baldoni L, Porceddu A, Barranco D, Satovic Z. Genetic diversity and population structure of wild olives from the North-western Mediterranean assessed by SSR markers. Ann Bot. 2007; 100: 449–458. pmid:17613587
- 31. Zohary D, Spiegel-Roy P. Beginning of fruit growing in the Old World. Science. 1975; 187: 319–327. pmid:17814259
- 32.
FAOSTAT. 2014; Available at: http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor.
- 33.
CAP-JA. Estadísticas agrarias (Agricultural statistics for Andalusia, Spain). Consejería de Agricultura y Pesca, Junta de Andalucía, Sevilla, Spain. 2014; Available at: http://www.juntadeandalucia.es/agriculturaypesca/portal/servicios/estadisticas/estadisticas/agrarias/superficies-y-producciones.html.
- 34.
MAGRAMA. Estadísticas agrarias (Agricultural Statistics for Spain). Ministerio de Agricultura, Alimentación y Medio Ambiente. 2014; Available at: http://www.magrama.gob.es/es/estadistica/temas/estadisticas-agrarias/agricultura/
- 35. Castillo P, Nico A, Navas-Cortés JA, Landa BB, Jiménez-Díaz RM, Vovlas N. Plant-parasitic nematodes attacking olive trees and their management. Plant Dis. 2010; 94: 148–162.
- 36. Ali N, Chapuis E, Tavoillot J, Mateille T. Plant-parasitic nematodes associated with olive tree (Olea europaea L.) with a focus on the Mediterranean Basin: A review. C R Biol. 2014; 337: 423–442. pmid:25103828
- 37. Palomares-Rius JE, Castillo P, Montes-Borrego M, Navas-Cortés JA, Landa BB. Soil properties and olive cultivar determine the structure and diversity of plant-parasitic nematode communities infesting olive orchards soils in Southern Spain. PLoS ONE. 2015; 10: e0116890. pmid:25625375
- 38. Lamberti F, Bleve-Zacheo T, Arias M. The Longidoridae of the Maltese Islands with the description of Longidorus magnus sp. n. and Xiphinema melitense sp. n. Nematol Mediterr. 1982; 10: 183–200.
- 39.
Peña Santiago R, Abolafia J, Liébanas G, Peralta M, Guerrero P. Dorylaimid species (Nematoda, Dorylaimida) recorded in the Iberian Peninsula and the Balearic islands: A compendium. Collection ‘Monographic Papers on Nematology’ n. 1. Jaén, Spain: Servicio de Publicaciones, Universidad de Jaén; 2003.
- 40. Palomares-Rius JE, Landa BB, Tanha Maafi Z, Hunt DJ, Castillo P. Comparative morphometrics and ribosomal DNA sequence analysis of Longidorus orientalis Loof, 1983 (Nematoda: Longidoridae) from Spain and Iran. Nematology. 2010; 12: 631–640.
- 41. Flegg JJM. Extraction of Xiphinema and Longidorus species from soil by a modification of Cobb´s decanting and sieving technique. Ann Appl Biol. 1967; 60: 429–437.
- 42.
Coolen WA (1979) Methods for extraction of Meloidogyne spp. and other nematodes from roots and soil. In: Lamberti F, Taylor CE, editors. Root-knot nematodes (Meloidogyne species). Systematics, biology and control. New York, USA: Academic Press; 1979. pp. 317–329.
- 43. Seinhorst JW. On the killing, fixation and transferring to glycerine of the nematodes. Nematologica. 1962; 8: 29–32.
- 44.
Jairajpuri MS, Ahmad W. Dorylaimida. Freeliving, predaceous and plant-parasitic nematodes. New Delhi, India: Oxford & IBH Publishing Co; 1992.
- 45. Macara A. Two new species of Longidorus (Nematoda: Longidoridae) associated with forest plants in Portugal. Nematologica. 1985; 31: 410–423.
- 46. Bravo MA, Roca F. Observations on Longidorus africanus Merny from Portugal with description of L. vinearum n. sp. (Nematoda: Longidoridae). Fund Appl Nematol. 1995; 18: 87–94.
- 47. Arias M, Andrés MF, Navas A. Longidorus carpetanensis sp. n. and L. unedoi sp. n. (Nematoda: Longidoridae) from Spain. Rev Nématol. 1986; 9: 101–106.
- 48. Castillo P, Vovlas N, Subbotin SA, Troccoli A. A new root-knot nematode, Meloidogyne baetica n. sp. (Nematoda: Heteroderidae), parasitizing wild olive in Southern Spain. Phytopathology. 2003; 93: 1093–1102. pmid:18944092
- 49.
Nunn GB. Nematode molecular evolution. Ph.D. Thesis, University of Nottingham. 1992.
- 50. Vrain TC, Wakarchuk DA, Levesque AC, Hamilton RI. Intraspecific rDNA Restriction Fragment Length Polymorphism in the Xiphinema americanum group. Fund Appl Nematol. 1992; 15: 563–573.
- 51. Cherry T, Szalanski AL, Todd TC, Powers TO. The internal transcribed spacer region of Belonolaimus (Nemata: Belonolaimidae). J Nematol. 1997; 29: 23–29. pmid:19274130
- 52. Holterman M, Van Der Wurff A, Van Den Elsen S, Van Megen H, Bongers T, Holovachov O, et al. Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Mol Phylogenet Evol. 2006; 23: 1792–1800.
- 53. Coomans A, De Ley IT, Jiménez LA, De Ley P. Morphological, molecular characterisation and phylogenetic position of Longidorus mindanaoensis n. sp. (Nematoda: Longidoridae) from a Philippine Avicennia mangrove habitat. Nematology. 2012; 14: 285–307.
- 54. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Resour. 2002; 30: 3059–3066.
- 55. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser. 1999; 41: 95–98.
- 56. Ronquist F, Huelsenbeck JP. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003; 19: 1572–1574. pmid:12912839
- 57. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9: 772.
- 58. Page RD (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci. 1996; 12: 357–358. pmid:8902363
- 59. Roca F, Pereira MJ, Lamberti F. Longidorus alvegus sp. n. (Nematoda, Dorylaimida) from Portugal. Nematol Mediterr. 1989; 17: 1–4.
- 60. Kozlowska J, Seinhorst JW. Longidorus elongatus and closely related species in the Netherlands and lower Saxony (Germany), with the description of two new species, L. cylindricaudatus and L. intermedius (Nematoda: Dorylaimida). Nematologica, 1979; 25: 42–53.
- 61. Sturhan D, Weischer B. Longidorus vineacola n. sp. (Nematoda: Dorylaimidae). Nematologica. 1954; 10: 335–341.
- 62.
Linnaeus C. Systema naturae regna tria naturae, secundum classes, ordines, genera, species cum characteribus differentiis, synonymis, locis. 10th ed. 1758.
- 63.
Pearse AS. Introduction to Parasitology. Baltimore: Springfield. 1942.
- 64. Chen QW, Hooper DJ, Loof PAA, Xu J. A revised polytomous key for the identification of species of the genus Longidorus Micoletzky, 1922 (Nematoda: Dorylaimoidea). Fund Appl Nematol. 1997; 20: 15–28.
- 65. Loof PAA, Chen Q. A revised polytomous key for the identification of the species of the genus Longidorus Micoletzky, 1922 (Nematoda: Dorylaimoidea). Supplement 1. Nematology. 1999; 1: 55–59.
- 66. Robbins RT, Brown DJF, Halbrendt JM, Vrain TC. Compendium of Longidorus juvenile stages with observation on L. pisi, L. taniwha and L. diadecturus (Nematoda: Longidoridae). Syst Parasitol. 1995; 32: 33–52.
- 67. Robbins RT, Brown DJ, Halbrendt JM, Vrain TC. Compendium of juvenile stages of Xiphinema species (Nematoda: Longidoridae). Russ J Nematol. 1996; 4: 163–171.
- 68. Romanenko ND. Redescription of Longidorus rubi Tomilin & Romanenko in Romanenko, 1993 (Nematoda: Longidoridae) associated with raspberry in the Poltava region, Ukraine. Russ J Nematol. 1998; 6: 185–187.
- 69. Hirata K. Description of a new species of Longidorus (Dorylaimida: Longidoridae) from Ishigaki Is., Japan. Jap J Nematol. 2002; 32: 31–36.
- 70. Siddiqi MR. Longidorus tarjani n. sp., found around oak roots in Florida. Nematologica. 1962; 8: 152–156.
- 71. Hooper DJ. A redescription of Longidorus elongates (De man, 1876) Thorne & Swanger 1936 (Nematoda: Dorylaimidae) and descriptions of five new species of Longidorus from Great Britain. Nematologica. 1961; 6:237–257.
- 72. Roca F, Lamberti F, Agostinelli A. Three new species of Longidorus (Nematoda, Dorylaimida) from Italy. Nematol Mediterr. 1984; 12: 187–200.
- 73. Bravo MA, Roca F. Two Longidorus species (Nematoda: Longidoridae) occurring in the rhizosphere of olive trees in North-eastern Portugal. Agron Lusitana 1998; 46: 101–121.
- 74. Arias M, Andrés MF. Longidorus belloi sp. n. (Nematoda: Longidoridae) from Spain. Rev Nématol. 1988; 11: 415–421.
- 75. Krnjaić D, Lamberti F, Krnjaić S, Agostinelli A, Radicci V. Three new longidorids (Nematoda: Dorylaimida) from Montenegro. Nematol Mediterr. 2000; 28: 235–248.
- 76. Lamberti F, Choleva B, Agostinelli A. Longidoridae from Bulgaria (Nematoda, Dorylaimida) with description of three new species of Longidorus and two new species of Xiphinema. Nematol Mediterr. 1983; 11: 49–72.
- 77. Tzortzakakis E, Archidona-Yuste A, Cantalapiedra-Navarrete C, Nasiou E, Lazanaki M, Kabourakis E, et al. Integrative diagnosis and molecular phylogeny of dagger and needle nematodes of olives and grapevines in the island of Crete, Greece, with description of Xiphinema cretense n. sp. (Nematoda, Longidoridae). Eur J Plant Pathol. 2014; 140: 563–590.
- 78. Niknam G, Pedram M, Nejad EG, Ye W, Robbins RT, Maafi Z. Morphological and molecular characterisation of Longidorus tabrizicus sp. n. and L. sturhani Rubtsova, Subbotin, Brown and Moens, 2001 (Nematoda: Longidoridae) from north-western Iran. Russ J Nematol. 2010; 18: 127–140.
- 79. Xu J, Cheng H. Longidorus litchii n. sp. and L. henanus n. sp. (Nemata: Longidoridae) from china. Fund Appl Nematol. 1992; 15: 517–523.
- 80. Zheng J, Peng D, Robbins RT, Brown DJF. Description of Longidorus hangzhouensis sp. n. (Nemata: Longidoridae) from Zhejiang province, new geographical records of L. henanus Xu & Cheng, 1992, and an identification key for Longidorus species occurring in China. Nematology. 2001; 3: 219–227.
- 81. Guo K, Shi H, Angelika M, Shi H, Zheng J. Molecular and morphological characterization of Longidorus henanus Xu & Cheng, 1992 (Nematoda: Dorylaimida) with all four juvenile developmental stages. Russ J Nematol. 2011; 19: 83–92.
- 82. Navas A, Andres MF, Arias M. Biogeography of Longidoridae in the Euromediterranea area. Nematol Mediterr. 1990; 18: 103–112.
- 83. Navas A, Baldwin JG, Barrios L, Nombela G. Phylogeny and biogeography of Longidorus (Nematoda: Longidoridae) in Euromediterranea. Nematol Mediterr. 1993; 21: 71–88.
- 84. Tarjan A. Plant-parasitic nematodes in the United Arab Republic. FAO Plant Prot Bull. 1964; 12: 49–56.
- 85. Lamberti F, Vouyoukalou E, Agostinelli A. Longidorids (Nematoda: Dorylaimoidea) occurring in the rhizosphere of olive trees in Western Crete, Greece. Nematol Mediterr. 1996; 24: 79–86.
- 86. Tzortzakakis E, Peneva V, Brown D, Avgelis A. A literature review on the occurrence of nematodes of the family Longidoridae in Greece. Nematol Mediterr. 2008; 36: 153–156.
- 87. Peña-Santiago R. Plant-parasitic nematodes associated with olive (Olea europea L.) in the province of Jaén, Spain. Rev Nématol. 1990; 13: 113–115.
- 88. Ibrahim I, Mokbel A, Handoo ZA. Current status of phytoparasitic nematodes and their host plants in Egypt. Nematropica. 2010; 40: 239–262.
- 89. Hashim Z. A preliminary report on the plant-parasitic nematodes in Jordan. Nematol Mediterr. 1979; 7: 177–186.
- 90. Tophan PB, Alphey TJW. Faunistic analysis of Longidorid nematodes in Europe. J Biogeogr. 1985; 12: 165–174.
- 91. Brown DJF, Halbrendt JM, Jones AT, Taylor CE, Lamberti F. An appraisal of some aspects of the ecology of nematode vectors of plant viruses. Nematol Mediterr. 1994; 22: 253–263.
- 92. Palomares-Rius JE, Cantalapiedra-Navarrete C, Gutiérrez-Gutiérrez C, Liébanas G, Castillo P. Morphological and molecular characterisation of Paralongidorus plesioepimikis n. sp. (Nematoda: Longidoridae) from southern Spain. Nematology 2013; 15, 363–378.
- 93. Amrei SB, Pedram M, Pourjam E, Elshishka M, Ghaemi R, Peneva V, et al. New data on Longidorus aetnaeus Roca, Lamberti, Agostinelli & Vinciguerra, 1986 (Nematoda: Longidoridae) from Iran and Ajaria (Georgia). Syst Parasitol. 2013; 85: 173–187. pmid:23673695
- 94. Subbotin SA, Rogozhin E, Chizhov V. Molecular characterisation and diagnostics of some Longidorus species (Nematoda: Longidoridae) from Russia and other countries using rRNA genes. Eur J Plant Pathol. 2014; 138: 377–390.
- 95. Decraemer W, Palomares-Rius JE, Cantalapiedra-Navarrete C, Landa BB, Duarte I, Almeida T, et al. Seven new species of Trichodorus (Diphtherophorina, Trichodoridae) from Spain, an apparent centre of speciation. Nematology. 2013; 15: 57–100.