Antonio Archidona-Yuste

Unravelling the Biodiversity and Molecular Phylogeny of Needle Nematodes of the Genus Longidorus (Nematoda: Longidoridae) in Olive and a Description of Six New Species

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.

Description and molecular characterisation of Xiphinema herakliense n. sp. (Nematoda: Longidoridae) from wild and cultivated olives in Crete

Xiphinema herakliense n. sp. from wild olive and cultivated trees in Vathy Rema and other localities, Heraklion province of Crete, Greece, is described based on morphological and molecular characters. The new species is characterised by having differentiation in the tubular part of the uterus, body length 4.1 (3.3-5.5) mm, odontostyle 149 (135-163) μm long, odontophore 85 (71-98) μm long, female tail dorsally convex-conoid with subdigitate peg, and presence of functional males. The polytomous identification code of the new species is: A4, B2 + 3, C5a, D6(5), E5, F4(5), G3, H2, I3, J5, K5, L2. The new species belongs to morphospecies Xiphinema non-americanum Group 5 characterised by having two equally developed female genital branches with uterine differentiation, presence of pseudo-Z-organ with small granular bodies plus small spines (in low number) and crystalloid bodies along uterus, and a short, rounded tail ending in a peg. Xiphinema herakliense n. sp. appears closely related to X. barense, X. horvatovicae, X. lanceolatum, X. loteni, X. sphaerocephalum and X. thorneanum, from which it may be differentiated morphologically and/or molecularly. The results of the phylogenetic analyses based on the sequences of the D2-D3 expansion regions of the 28S rRNA gene and ITS1 rRNA genes are also supportive of its specific status.

Redescription and molecular characterisation of Xiphinema barense Lamberti et al., 1986 (Nematoda: Longidoridae) from wild olive trees in southern Italy

A population of Xiphinema barense from wild olive trees in Torre Pozzella, Brindisi province, southern Italy, is described using both morphological and molecular studies and compared with the description of the type specimens. The wild olive nematode population agrees very well with all morphometrics provided in the original description. However, detailed observations of the lumen of the tubular portion of the uterus in paratypes and specimens of the new population revealed a clear pseudo-Z-organ with small granules mixed with crystalloid bodies which were previously undetected. Photomicrographs of adult paratypes, which were lacking in the original description, and of specimens of the new population from wild olive trees are provided. The results of the phylogenetic analyses based on the sequences of the D2-D3 expansion regions of the 28S rRNA gene and ITS rRNA genes confirm the species differentiation and indicate the phylogenetic position of X. barense and its relationship with closely related species.

Molecular and morphological characterisation of Rotylenchus vitis n. sp. (Nematoda: Hoplolaimidae) infectinggrapevine in southern Spain.

Rotylenchus vitis n. sp., a new amphimictic species infesting soil and roots of commercial vineyards in southern Spain, is described. Rotylenchus vitis n. sp. is characterised by a truncate lip region with 7-9 annuli and continuous with the body contour, lateral fields areolated at pharyngeal region only, body without longitudinal striations, stylet length of 36-48 μm, vulva position at 47-57%, tail rounded to hemispherical with 13-21 annuli. Morphologically, this species is related to R. cazorlaensis, R. capitatus, R. elegans, R. fabalus, R. iranicus, R. labiodiscus, R. montanus and R. troncapitatus. The results of the phylogenetic analysis based on the sequences of the D2-D3 expansion regions of the 28S and ITS1-rRNA genes confirmed the species differentiation and the close molecular relationship between R. eximius and R. montanus, respectively.

The utility of mtDNA and rDNA for barcoding and phylogeny of plant-parasitic nematodes from Longidoridae (Nematoda, Enoplea)

The traditional identification of plant-parasitic nematode species by morphology and morphometric studies is very difficult because of high morphological variability that can lead to considerable overlap of many characteristics and their ambiguous interpretation. For this reason, it is essential to implement approaches to ensure accurate species identification. DNA barcoding aids in identification and advances species discovery. This study sought to unravel the use of the mitochondrial marker cytochrome c oxidase subunit 1 (coxI) as barcode for Longidoridae species identification, and as a phylogenetic marker. The results showed that mitochondrial and ribosomal markers could be used as barcoding markers, except for some species from the Xiphinema americanum group. The ITS1 region showed a promising role in barcoding for species identification because of the clear molecular variability among species. Some species presented important molecular variability in coxI. The analysis of the newly provided sequences and the sequences deposited in GenBank showed plausible misidentifications, and the use of voucher species and topotype specimens is a priority for this group of nematodes. The use of coxI and D2 and D3 expansion segments of the 28S rRNA gene did not clarify the phylogeny at the genus level.

Remarkable Diversity and Prevalence of Dagger Nematodes of the Genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) in Olives Revealed by Integrative Approaches

The genus Xiphinema includes a remarkable group of invertebrates of the phylum Nematoda comprising ectoparasitic animals of many wild and cultivated plants. Damage is caused by direct feeding on root cells and by vectoring nepoviruses that cause diseases on several crops. Precise identification of Xiphinema species is critical for launching appropriate control measures. We make available the first detailed information on the diversity and distribution of Xiphinema species infesting wild and cultivated olive in a wide-region in southern Spain that included 211 locations from which 453 sampling sites were analyzed. The present study identified thirty-two Xiphinema spp. in the rhizosphere of olive trees, ten species belonging to Xiphinema americanum-group, whereas twenty-two were attributed to Xiphinema non-americanum-group. These results increase our current knowledge on the biodiversity of Xiphinema species identified in olives and include the description of four new species (Xiphinema andalusiense sp. nov., Xiphinema celtiense sp. nov., Xiphinema iznajarense sp. nov., and Xiphinema mengibarense sp. nov.), and two new records for cultivate olives (X. cadavalense and X. conurum). We also found evidence of remarkable prevalence of Xiphinema spp. in olive trees, viz. 85.0% (385 out of 453 sampling sites), and they were widely distributed in both wild and cultivated olives, with 26 and 17 Xiphinema spp., respectively. Diversity indexes (Richness, Hill´s diversity, Hill´s reciprocal of D and Hill´s evenness) were significantly affected by olive type. We also developed a comparative morphological and morphometrical study together with molecular data from three nuclear ribosomal RNA genes (D2-D3 expansion segments of 28S, ITS1, and partial 18S). Molecular characterization and phylogenetic analyses allowed the delimitation and discrimination of four new species of the genus described herein and three known species. Phylogenetic analyses of Xiphinema spp. resulted in a general consensus of these species groups. This study is the most complete phylogenetic analysis for Xiphinema non-americanum-group species to date.

Molecular phylogenetic analysis and comparative morphology resolve two new species of olive-tree soil related dagger nematodes of the genus Xiphinema (Dorylaimida : Longidoridae) from Spain

Abstract. The genus Xiphinema constitutes a large group of ∼275 species of polyphagous, plant-ectoparasitic nematodes that are distributed almost worldwide. Some species affect agricultural crops by feeding directly on root cells as well as by transmitting nepoviruses. Despite their agricultural importance, species discrimination in Xiphinema is difficult, leading to potential misidentification. Integrative taxonomy, based on the combination of molecular analyses and morphology, constitutes a new insight into Xiphinema species identification. In this study we describe two new species of Xiphinema from the Iberian Peninsula (X. macrodora, sp. nov. and X. oleae, sp. nov.) associated with cultivated and wild olive trees. Both species have specific rRNA sequences. Morphologically, Xiphinema macrodora, sp. nov. is characterised by a very long body (7.2–8.7 mm), a very long odontostyle and odontophore (190–206 and 105–120 µm, respectively), and a well developed pseudo-Z-organ, comprising 8 to 12 sclerotised bodies. Xiphinema oleae, sp. nov. is characterised by an odontostyle and an odontophore 136–149 and 65–80 µm long, respectively, and a well developed Z-organ with refractive inclusions (3–5), variable in shape. Additionally, X. macrodora, sp. nov. has the longest body size, and the longest odontostyle and odontophore of any Xiphinema, whereas X. oleae, sp. nov. is the first species with a well developed Z-organ from the Iberian Peninsula.

Mitochondrial genome diversity in dagger and needle nematodes (Nematoda: Longidoridae)

Dagger and needle nematodes included in the family Longidoridae (viz. Longidorus, Paralongidorus, and Xiphinema) are highly polyphagous plant-parasitic nematodes in wild and cultivated plants and some of them are plant-virus vectors (nepovirus). The mitochondrial (mt) genomes of the dagger and needle nematodes, Xiphinema rivesi, Xiphinema pachtaicum, Longidorus vineacola and Paralongidorus litoralis were sequenced in this study. The four circular mt genomes have an estimated size of 12.6, 12.5, 13.5 and 12.7 kb, respectively. Up to date, the mt genome of X. pachtaicum is the smallest genome found in Nematoda. The four mt genomes contain 12 protein-coding genes (viz. cox1-3, nad1-6, nad4L, atp6 and cob) and two ribosomal RNA genes (rrnL and rrnS), but the atp8 gene was not detected. These mt genomes showed a gene arrangement very different within the Longidoridae species sequenced, with the exception of very closely related species (X. americanum and X. rivesi). The sizes of non-coding regions in the Longidoridae nematodes were very small and were present in a few places in the mt genome. Phylogenetic analysis of all coding genes showed a closer relationship between Longidorus and Paralongidorus and different phylogenetic possibilities for the three Xiphinema species.

Molecular characterization of pseudomonodelphic dagger nematodes of the genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) in Costa Rica, with notes on Xiphinema setariae Tarjan, 1964

Pseudomonodelphic dagger nematodes of the genus Xiphinema are characterized by having one of the genital branches reduced and lacking an ovary. They are usually reported from tropical regions. Nematode surveys conducted during rainy seasons in Costa Rica resulted in detection of several Xiphinema costaricense populations, but also other pseudomonodelphic and didelphic species of Xiphinema. We undertook detailed integrative morphometric and molecular studies using D2-D3 expansion segments of 28S rDNA, and ITS1-rDNA. From those studies, we also identified several populations of Xiphinema krugi and two populations of Xiphinema setariae with characteristics in agreement with those of the original and later descriptions of these species. The phylogenetic analyses of these species with other representatives of Xiphinema spp. indicated that pseudomonodelphic species are phylogenetically related (X. costaricense and X. krugi). On the basis of ITS1 sequences of X. costaricense and X. variegatum from Brazil, as well as similar morphology and morphometrics of both species, the latter is proposed here as a junior synonym of the former. Also, our morphometric data showed some intraspecific variability within X. setariae, which in combination with the molecular evidence, suggests that X. setariae and X. vulgare need to be considered as a single taxon.

Molecular diversity of bacterial endosymbionts associated with dagger nematodes of the genus Xiphinema (Nematoda: Longidoridae) reveals a high degree of phylogenetic congruence with their host.

Bacterial endosymbionts have been detected in some groups of plant‐parasitic nematodes, but few cases have been reported compared to other groups in the phylum Nematoda, such as animal‐parasitic or free‐living nematodes. This study was performed on a wide variety of plant‐parasitic nematode families and species from different host plants and nematode populations. A total of 124 nematode populations (previously identified morphologically and molecularly) were screened for the presence of potential bacterial endosymbionts using the partial 16S rRNA gene and fluorescence in situ hybridization (FISH) and confocal microscopy. Potential bacterial endosymbionts were only detected in nematode species belonging to the genus Xiphinema and specifically in the X. americanum group. Fifty‐seven partial 16S rRNA sequences were obtained from bacterial endosymbionts in this study. One group of sequences was closely related to the genus ‘Candidatus Xiphinematobacter’ (19 bacterial endosymbiont sequences were associated with seven nematode host species, including two that have already been described and three unknown bacterial endosymbionts). The second bacterial endosymbiont group (38 bacterial endosymbiont sequences associated with six nematode species) was related to the family Burkholderiaceae, which includes fungal and soil–plant bacterial endosymbionts. These endosymbionts were reported for the first time in the phylum Nematoda. Our findings suggest that there is a highly specific symbiotic relationship between nematode host and bacterial endosymbionts. Overall, these results were corroborated by a phylogeny of nematode host and bacterial endosymbionts that suggested that there was a high degree of phylogenetic congruence and long‐term evolutionary persistence between hosts and endosymbionts.