Pin nematodes of the genus Paratylenchus are obligate ectoparasites of a wide variety of plants that are distributed worldwide. In this study, individual morphologically vouchered nematode specimens of fourteen Paratylenchus species, including P. aculentus, P. elachistus, P. goodeyi, P. holdemani, P. idalimus, P. microdorus, P. nanus, P. neoamblycephalus, P. straeleni and P. veruculatus, are unequivocally linked to the D2-D3 of 28S, ITS, 18S rRNA and COI gene sequences. Combined with scanning electron microscopy and a molecular analysis of an additional nine known and thirteen unknown species originating from diverse geographic regions, a total of 92 D2-D3 of 28S, 41 ITS, 57 18S rRNA and 111 COI new gene sequences are presented. Paratylenchus elachistus, P. holdemani and P. neoamblycephalus are recorded for the first time in Belgium and P. idalimus for the first time in Europe. Paratylenchus is an excellent example of an incredibly diverse yet morphologically minimalistic plant-parasitic genus, and this study provides an integrated analysis of all available data, including coalescence-based molecular species delimitation, resulting in an updated Paratylenchus phylogeny and the corrective reassignment of 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 COI gene sequences that were previously unidentified or incorrectly classified.

By performing cDNA AFLP on pre- and early parasitic juveniles, we identified genes encoding a novel type of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in the cyst nematode Heterodera schachtii. The proteins consist of three domains, a signal peptide for secretion, a mono-ubiquitin domain, and a short C-terminal positively charged domain. A gfp-fusion of this protein is targeted to the nucleolus in tobacco BY-2 cells. We hypothesize that the C-terminal peptide might have a regulatory function during syncytium formation in plant roots.

Summary Pratylenchus rwandae n. sp., a root-lesion nematode associated with maize (Zea mays) from Rwanda, is described. This new species is characterised by females of medium to large size (469-600 μm) having an offset lip region with three annuli, stylet of 13-14.6 μm long with prominent rounded or anteriorly concave knobs, short to long pharyngeal gland overlap of 10.9-34.7 μm long, variable number of lateral lines (4-14) in different regions of the body, lateral field consisting of smooth bands, oval to slightly rounded spermatheca, vulva located at 75-80% of the total body length, post-vulval uterine sac (PUS) 20.3-26.5 μm long, tail subcylindrical to conoid with variation in tail tip shape from rounded to truncate or indented with generally smooth tip, and male unknown. The results of the phylogenetic analyses based on sequences of the D2-D3 expansion regions of 28S, partial 18S and ITS of rDNA and COI of mitochondrial DNA indicate that P. rwandae n. sp. is a species within the Penetrans group and appears as a sister species to a group comprising P. convallariae, P. dunensis, P. fallax, P. oleae, P. penetrans, P. pinguicaudatus, and three other unidentified species. A comparison of important morphological characters of the closely related Pratylenchus spp. is provided.

BackgroundGlobin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles.ResultsIn the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection.ConclusionOur results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying selection subsequently dampened further evolution and facilitated fixation of the duplicated genes in the genome.

Hirschmanniella mucronata populations isolated from two Cambodian provinces were characterised using morphological, morphometric and molecular criteria. Examination of 1024 specimens from 60 different paddy fields revealed high intraspecific variation in morphology and morphometrics, especially in tail terminus shape and stylet length. Sequence results confirmed that morphologically divergent individuals represent a single species, suggesting that neglecting morphological variation has led to an overestimation of Hirschmanniella diversity in former studies. Phylogenetic analysis of the SSU, D2-D3, LSU and ITS1-5.8S-ITS2 regions revealed three concordant clades, H. mucronata having a sister relationship with H. kwazuna and H. loofi. Plotting the diagnostic features, including tail terminus shape, stylet length and lip region morphology on the phylogenetic framework, revealed that none of them supported the clades and represented convergent features. All three molecular markers were able to discriminate all Hirschmanniella species, but the D2-D3 region was the easiest, fastest and most successful region to be amplified. Species delimitation and the diagnostic features of Hirschmanniella were re-evaluated. Hirschmanniella abnormalis and H. exacta are considered to be junior synonyms of H. oryzae and H. mannai a species inquirenda. Finally, a list of valid species with indication of synonyms and a polytomous key are provided

A survey for slug-associated nematodes in five locations of East and West Flanders in Belgium revealed the presence of one new and six known slug-parasitic nematodes, Agfa flexilis (Dujardin, 1845), Alloionema appendiculatum (Schneider, 1859), Angiostoma dentiferum (Mengert, 1953), Angiostoma limacis (Dujardin, 1845), Angiostoma norvegicum (Ross et al., 2017) and Phasmarhabditis hermaphrodita (Schneider, 1859). Angiostoma norvegicum and P. hermaphrodita are recorded for the first time in Belgium. The six known species are documented by light microscopy (LM) microphotographs and informative DNA sequences. Angiostoma gandavensis n. sp. (Angiostomatidae), discovered from arionid slugs, is described based on light microscopy, scanning electron microscopy (SEM) and molecular data. Based on analyses of D2D3 expansion segment of 28S and 18S rDNA sequences, this new species is found to be related to A. limacis, A. norvegicum, A. margaretae (Ross et al., 2011) and A. milacis (Ivanova and Wilson, 2009). The new species can be distinguished from these others based on morphological characters such as the distinctive mucronate structures at the tail tip of both sexes, presence of lateral ala, reflexed female ovaries and the number and arrangement pattern of male genital papillae.

During a survey in the Botanical garden of Ghent University, a new species Rotylenchus rhomboides n. sp. and a population of Rotylenchus buxophilus were found. Rotylenchus rhomboides n. sp. is characterized by the presence of a rhomboid-like widening of the midridge of lateral field at the level of vulva, a feature previously unknown within the genus. The population of the new species, composed only by females, has a rounded labial region with 4 to 5 annuli, robust stylet (31-37 μ m long), short dorsal esophageal gland (9-19 μ m) overlap of the intestine, vulva located slightly posterior to mid-body, and hemispherical or rounded tail shape with large phasmids located 3 to 5 annuli anterior to the level of anus. The hierarchical cluster analysis based on morphological features indicated that the new species closely resembles R. corsicus, R. gracilidens, and R. rugatocuticulatus. The DNA analyses of the D2-D3 of 28S rDNA, ITS rDNA, and COI mtDNA sequences of Rotylenchus rhomboides n. sp. show a close relationship with R. buxophilus, R. goodeyi, R. laurentinus, R. pumilus, and R. incultus, all of which can also be differentiated from the new species by morphological features. The combination of morphological, morphometric, and molecular characteristics confirmed the new species and the first report of R. buxophilus on yam (Dioscorea tokoro) in Belgium.

Comprehensive morphological and molecular analyses revealed that published ITS sequences of the economically important plant-parasitic nematodePratylenchus goodeyiare actually sequences from distantly free-living bacterivorous ‘cephalobids’. We demonstrated that this incorrect labelling resulted in a cascade of erroneous interpretations, as shown by the reports of ‘P. goodeyi’ on banana in China and on cotton in India. This clearly illustrates the risk of mislabelled sequences in public databases. Other mislabelledPratylenchuscases are discussed to illustrate that this is not an isolated case. Herein,P. lentisn. syn. is considered a junior synonym ofP. pratensiswhileP. flakkensiswas for the first time linked to DNA sequences using topotype material. As taxonomic expertise is decreasing and sequence-based identification is growing rapidly, the highlighted problem may yet increase and a strong link between morphology and DNA sequences will be of crucial importance in order to prevent, or at least minimise, sequence-based misidentifications.