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.

During nematode surveys in southern Spain and Italy 14 populations of Xiphinema species tentatively identified as Xiphinema americanum-group were detected. Morphological and morphometrical studies identified three new species and six known Xiphinema americanumgroup species, viz.: Xiphinema parabrevicolle n. sp., Xiphinema parapachydermum n. sp., Xiphinema paratenuicutis n. sp., Xiphinema duriense, Xiphinema incertum, Xiphinema opisthohysterum, Xiphinema pachtaicum, Xiphinema rivesi, and Xiphinema santos. The Xiphinema americanum-group is the most difficult Xiphinema species group for diagnosis since the morphology is very conservative and morphometric characters often overlap. This group includes vectors of several important plant pathogenic viruses that cause significant damage to a wide range of agricultural crops. Molecular characterisation of these species using D2-D3 expansion regions of 28S rRNA, 18S rRNA, ITS1-rRNA and the protein-coding mitochondrial gene, cytochrome oxidase c subunit 1 was carried out and maximum likelihood and Bayesian inference analysis were used to reconstruct phylogenetic relationships among these species and with other Xiphinema americanum-group species.

The Xiphinema americanum-group constitutes a complex of about 55 species of polyphagous plant-ectoparasitic nematodes with a worldwide distribution. This group of plant-parasitic nematodes is one of the most difficult dagger nematode species complexes for diagnosis because the morphology is very conservative and morphometric characters often overlap. We conducted nematode surveys in cultivated and wild olives in southern Spain from 2012 to 2014, from which we identified 16 nematode populations of the X. americanum-group, five of which were tentatively identified as belonging to three new species and are described herein as Xiphinema plesiopachtaicum sp. nov., Xiphinema vallense sp. nov., and Xiphinema astaregiense sp. nov., and 11 populations belonging to nine known species: Xiphinema brevisicum, Xiphinema duriense, Xiphinema incertum, Xiphinema luci, Xiphinema madeirense, Xiphinema opisthohysterum, Xiphinema pachtaicum, Xiphinema parapachydermum, and Xiphinema rivesi. A phenetic study based on multivariate factor analyses was developed to compare some of these related species by using morphometric features. In the factor analysis the first four factors accounted for 73.1% of the total variance of the selected characters, identifying body length, body length/maximum body width (a), body length/ pharyngeal length (b), body length/tail length (c), and tail length/body width at anus (c′) ratios, distance from anterior end to vulva as percentage of body length (V), stylet length, oral aperture-guiding ring distance, and lip region width as key morphometric characters to differentiate a restricted set of species within the X. pachtaicumsubgroup that includes X. plesiopachtaicum sp. nov. and X. vallense sp. nov. Multivariate analysis of variance using these specific characters allowed to differentiate species in the X. pachtaicum complex or groups of them using morphometric characters (body length, a, b, c, c′, V, stylet length, lip region width, oral apertureguiding ring distance, female tail length, and hyaline region length). Phylogenetic analyses based on nuclear ribosomal DNA genes [D2-D3 expansion segments of large ribosomal subunit 28S, and internal transcribed spacer 1 (ITS1)] and the protein-coding mitochondrial gene, cytochrome c oxidase subunit 1 (coxI) were congruent, showing two main clades separating most of the species of X. americanum-subgroup 'sensu stricto' from the X. pachtaicumsubgroup. Agreement between phylogenetic trees and some morphological characters (viz. total stylet length, vulva position, and a ratio) were tested by reconstruction of their histories on rRNA-based trees using parsimony and Bayesian approaches. Thus, integrative taxonomy, based on a combination of multivariate morphological and molecular analyses constitutes a new insight into the identification of X. americanum-group species.

P. (2010). Molecular analysis and comparative morphology to resolve a complex of cryptic Xiphinema species. -Zoologica Scripta, 39, 483-498.During nematode surveys in cultivated and natural environments in southern Spain nine populations of parthenogenic Xiphinema species tentatively identified as Xiphinema cf. pyrenaicum and one population morphologically close to Xiphinema turcicum were detected. Surveys in southern France also identified one population resembling X. pyrenaicum. We developed a comparative study among these related Xiphinema species, including topotypes of two species of this group previously synonymized, viz. Xiphinema hispanum and Xiphinema sphaerocephalum, 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). Morphological and morphometrical results identified eight of the Spanish populations as Xiphinema nuragicum (previously synonymized with X. pyrenaicum) whereas the ninth population was identified as Xiphinema adenohystherum (also synonymized with X. pyrenaicum). The species X. adenohystherum, X. nuragicum, X. pyrenaicum, and X. sphaerocephalum were shown to be morphologically almost indistinguishable but clearly separated by phylogenetic analyses, thus constituting a complex of cryptic species. Consequently, X. adenohystherum, X. nuragicum, and X. sphaerocephalum were re-established as valid species. Similarly, X. hispanum (morphologically similar to X. aceri) was also shown as a valid species. Xiphinema turcicum, morphologically related to X. pyrenaicum complex by its rounded tail, uterus with a pseudo-Z-differentiation and small spines, was phylogenetically distant to these species based on D2-D3 expansion segments of 28S and ITS1, which suggests a morphological convergence in their evolution.

The phylum Nematoda includes the genus Longidorus, a remarkable group of invertebrates that are polyphagous root‐ectoparasites of many plants including various agricultural crops and trees. Damage is caused by direct feeding on root cells as well as by transmitting nepoviruses. Species discrimination in Longidorus is complicated by phenotypic plasticity (intraspecific variability and minor interspecific differences) leading to potential misidentification. We conducted nematode surveys in cultivated and natural environments in southern Spain that detected 11 species of Longidorus. We developed a comparative study amongst these related species by considering morphological and morphometric features together with molecular data from nuclear ribosomal RNA genes [D2‐D3 expansion segments of large ribosomal subunit (28S), internal transcribed spacer 1 (ITS1), and partial small ribosomal subunit (18S)]. The results of our molecular and phylogenetic analyses confirmed the morphological hypotheses and allowed the delimitation and discrimination of three new species of the genus, described herein as Longidorus baeticus sp. nov., Longidorus oleae sp. nov., and Longidorus andalusicus sp. nov., and eight known species (Longidorus alvegus, Longidorus crataegi, Longidorus fasciatus, Longidorus intermedius, Longidorus iuglandis, Longidorus magnus, Longidorus rubi, and Longidorus vineacola). Phylogenetic analyses of Longidorus spp. based on the three molecular markers resulted in a general consensus of these species grouping, as lineages were maintained for the majority of species (i.e. species with a conoid‐rounded lip region, amphidial fovea asymmetrically bilobed, female tail bluntly rounded), but not in some others (i.e. positions of L. crataegi, L. intermedius, and L. rubi were quite variable). To date, this is the most complete phylogenetic analysis for Longidorus and Paralongidorus species, with the highest number of species included. No correspondence between phylogenetic trees and morphological characters was found for ribosomal markers, with the exception of amphidial shape. Thus, polyphasic identification, based on integration of molecular analysis with morphology, is a tool beyond doubt in Longidorus identification. © 2013 The Linnean Society of London

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

In previous studies, fifteen species of Paratylenchus, commonly known as pin nematodes, have been reported in Spain. These plant-parasitic nematodes are ectoparasites with a wide host range and global distribution. In this research, 27 populations from twelve Paratylenchus species from 18 municipalities in Spain were studied using morphological, morphometrical and molecular data. This integrative taxonomic approach allowed the identification of twelve species, four of them were considered new undescribed species and eight were already known described. The new species described here are P. caravaquenus sp. nov., P. indalus sp. nov., P. pedrami sp. nov. and P. zurgenerus sp. nov. As for the already known described species, five were considered as first reports for the country, specifically P. enigmaticus, P. hamatus, P. holdemani, P. israelensis, and P. veruculatus, while P. baldaccii, P. goodeyi and P. tenuicaudatus had already been recorded in Spain. This study provides detail morphological and molecular data, including the D2-D3 expansion segments of 28S rRNA, ITS rRNA, and partial mitochondrial COI regions for the identification of different Paratylenchus species found in Spain. These results confirm the extraordinary cryptic diversity in Spain and with examples of morphostatic speciation within the genus Paratylenchus.

The occurrence and geographic distribution of longidorid nematode species inhabiting the rhizosphere of grapevine plants in southern Spain were investigated. Nematode surveys were conducted on 77 vineyards during the spring seasons of 2006, 2007 and 2008 in the main Andalusian grapevine-growing areas, including the provinces of Cádiz, Córdoba, and Huelva. Morphological and morphometrical studies identified two Longidorus and nine Xiphinema species, viz.: Longidorus alvegus, L. magnus, Xiphinema adenohystherum, X. hispidum, X. index, X. italiae, X. lupini, X. nuragicum, X. pachtaicum, X. rivesi, and X. turcicum. Overall, frequencies of infestation were, in decreasing order: X. pachtaicum 90.8%, X. index 30.3%, X. italiae 13.2%, L. magnus 11.8%, X. hispidum 7.9%, X. lupini 3.9%, L. alvegus and X. rivesi 2.6%, and X. adenohystherum, X. nuragicum and X. turcicum 1.3%. Xiphinema hispidum, X. lupini, L. alvegus and L. magnus were compared with nematode type specimens and are reported for the first time in Spain. Furthermore, the male of L. alvegus is described for the first time in the literature. Molecular characterisation of these species using D2-D3 expansion regions of 28S rRNA, 18S rRNA and ITS1-rRNA was carried out and maximum likelihood and Bayesian inference analysis were used to reconstruct phylogenetic relationships among these species and with other longidorids. The monophily of the genera Xiphinema and Longidorus was accepted and the genera Paralongidorus and Xiphidorus were rejected by the ShimodairaHasegawa test based on tree topologies.