Toon Janssen

Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution.

The polyphagous parthenogenetic root-knot nematodes of the genus Meloidogyne are considered to be the most significant nematode pest in sub-tropical and tropical agriculture. Despite the crucial need for correct diagnosis, identification of these pathogens remains problematic. The traditionally used diagnostic strategies, including morphometrics, host-range tests, biochemical and molecular techniques, now appear to be unreliable due to the recently-suggested hybrid origin of root-knot nematodes. In order to determine a suitable barcode region for these pathogens nine quickly-evolving mitochondrial coding genes were screened. Resulting haplotype networks revealed closely related lineages indicating a recent speciation, an anthropogenic-aided distribution through agricultural practices, and evidence for reticulate evolution within M. arenaria. Nonetheless, nucleotide polymorphisms harbor enough variation to distinguish these closely-related lineages. Furthermore, completeness of lineage sorting was verified by screening 80 populations from widespread geographical origins and variable hosts. Importantly, our results indicate that mitochondrial haplotypes are strongly linked and consistent with traditional esterase isozyme patterns, suggesting that different parthenogenetic lineages can be reliably identified using mitochondrial haplotypes. The study indicates that the barcode region Nad5 can reliably identify the major lineages of tropical root-knot nematodes.

Molecular phylogeny of Kalyptorhynchia (Rhabdocoela, Platyhelminthes) inferred from ribosomal sequence data

With 556 species described to date, Kalyptorhynchia includes about one‐third of all species of rhabdocoel flatworms. In this study, we present the first molecular phylogenetic analysis of this taxon. The final analysis comprises 110 species. These represent 11 of the 17 known families. The largest family (241 known species), Polycystididae, is represented by nine of 10 subfamilies and 33 of the 58 genera. Sequence data from 18S rDNA and 28S rDNA were analysed using maximum likelihood and Bayesian approaches. Of the two taxa traditionally recognised within Kalyptorhynchia, Eukalyptorhynchia and Schizorhynchia, only Schizorhynchia is monophyletic. All eukalyptorhynch families, except Cicerinidae, are monophyletic. On the other hand, two of the three schizorhynch families included are not monophyletic. Within Polycystididae, the traditional taxonomy was not reflected in our phylogenetic analyses and most subfamilies are polyphyletic. These results suggest that current classification, mostly based on characters of the genital system, suffers from homoplasy. Where possible, a revised classification, taking into account these new findings, is given.

Integrative taxonomy of root-knot nematodes reveals multiple independent origins of mitotic parthenogenesis

During sampling of several Coffea arabica plantations in Tanzania severe root galling, caused by a root-knot nematode was observed. From pure cultures, morphology and morphometrics of juveniles and females matched perfectly with Meloidogyne africana, whereas morphology of the males matched identically with those of Meloidogyne decalineata. Based on their Cox1 sequence, however, the recovered juveniles, females and males were confirmed to belong to the same species, creating a taxonomic conundrum. Adding further to this puzzle, re-examination of M. oteifae type material showed insufficient morphological evidence to maintain its status as a separate species. Consequently, M. decalineata and M. oteifae are synonymized with M. africana, which is herewith redescribed based on results of light and scanning electron microscopy, ribosomal and mitochondrial DNA sequences, isozyme electrophoresis, along with bionomic and cytogenetic features. Multi-gene phylogenetic analysis placed M. africana outside of the three major clades, together with M. coffeicola, M. ichinohei and M. camelliae. This phylogenetic position was confirmed by several morphological features, including cellular structure of the spermatheca, egg mass position, perineal pattern and head shape. Moreover, M. africana was found to be a polyphagous species, demonstrating that “early-branching” Meloidogyne spp. are not as oligophagous as had previously been assumed. Cytogenetic information indicates M. africana (2n = 21) and M. ardenensis (2n = 51–54) to be a triploid mitotic parthenogenetic species, revealing at least four independent origins of mitotic parthenogenesis within the genus Meloidogyne. Furthermore, M. mali (n = 12) was found to reproduce by amphimixis, indicating that amphimictic species with a limited number of chromosomes are widespread in the genus, potentially reflecting the ancestral state of the genus. The wide variation in chromosome numbers and associated changes in reproduction modes indicate that cytogenetic evolution played a crucial role in the speciation of root-knot nematodes and plant-parasitic nematodes in general.

Molecular characterization and species delimiting of plant-parasitic nematodes of the genus Pratylenchus from the penetrans group (Nematoda: Pratylenchidae)

Root-lesion nematodes of the genus Pratylenchus are an important pest parasitizing a wide range of vascular plants including several economically important crops. However, morphological diagnosis of the more than 100 species is problematic due to the low number of diagnostic features, high morphological plasticity and incomplete taxonomic descriptions. In order to employ barcoding based diagnostics, a link between morphology and species specific sequences has to be established. In this study, we reconstructed a multi-gene phylogeny of the Penetrans group using nuclear ribosomal and mitochondrial gene sequences. A combination of this phylogenetic framework with molecular species delineation analysis, population genetics, morphometric information and sequences from type location material allowed us to establish the species boundaries within the Penetrans group and as such clarify long-standing controversies about the taxonomic status of P. penetrans, P. fallax and P. convallariae. Our study also reveals a remarkable amount of cryptic biodiversity within the genus Pratylenchus confirming that identification on morphology alone can be inconclusive in this taxonomically confusing genus.

The pitfalls of molecular species identification : a case study within the genus Pratylenchus (Nematoda: Pratylenchidae)

Comprehensive morphological and molecular analyses revealed that published ITS sequences of the economically important plant-parasitic nematode Pratylenchus goodeyi are 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 mislabelled Pratylenchus cases are discussed to illustrate that this is not an isolated case. Herein, P. lentis n. syn. is considered a junior synonym of P. pratensis while P. flakkensis was 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.

mtCOI successfully diagnoses the four main plant-parasitic Aphelenchoides species (Nematoda: Aphelenchoididae) and supports a multiple origin of plant-parasitism in this paraphyletic genus

Composed mostly of fungivorous species, the genus Aphelenchoides also comprises 14 plant-parasitic species. The most common and devastating, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis have been reported on more than 900 plant species. The combination of low inter-specific and high intra-specific morphological variability makes morphology-based identification extremely difficult within this genus, and has led to molecular tools being employed to ensure accurate diagnoses. rDNA markers are widely used for the identification of nematodes while the Cytochrome Oxidase I gene (COI) remains relatively unexplored despite its role as the standard barcode for almost all animal groups. To explore its suitability as a diagnostic tool, we studied a fragment of the mtCOI region of the four main plant-parasitic Aphelenchoides within a phylogenetic framework. We generated 69 mtCOI and 123 rDNA sequences of diverse Aphelenchoides taxa; 67 belong to the main plant-parasitic species including the first mtCOI sequence of A. fragariae and the first mtCOI and 28S sequences of A. subtenuis. mtCOI had a similar success rate for PCR amplification. Phylogenetic trees based on the three studied markers are largely in agreement with one another, validating their use for Aphelenchoides diagnosis; additionally, we were able to locate several misidentified sequences of plant-parasitic Aphelenchoides in existing databases. The concatenated analysis from the three markers resulted in a more robust insight into the phylogeny and evolution of Aphelenchoides, revealing that plant-parasitism has evolved independently at least three times within this genus, presumably from fungal-feeding ancestors.

Two new species of Carcharodorhynchus Meixner, 1938 (Platyhelminthes: Rhabdocoela: Schizorhynchidae) from Brazil and Lanzarote

Two new turbellarian species of the kalyptorhynch taxon Carcharodorhynchus (Rhabdocoela: Schizorhynchidae) are described. C. brasiliensis n. sp. from Brazil differs from its congeners by the fact that only the ventral lip of the slightly asymmetrical proboscis bears denticles, arranged into two rows. It is also characterized by a unique combination of features: 10–12 testes arranged in two rows rostrally from the pharynx, elongated seminal vesicles, and the copulatory organ proper being a short cirrus, the distal tip of which is surrounded by a short sclerotized hood. C. worsaae n. sp. stands unique within the genus in that the male system has a long, double-walled stylet, which drains sperm and prostate secretion. Moreover, C. worsaae n. sp. has a markedly asymmetrical proboscis, with the dentition of the largest lip consisting of up to six rows of small denticles which are smallest proximally near the juncture with the smaller lip. The shorter lip has only large teeth. C. brasiliensis n. sp. is the first species of Carcharodorhynchus to be described from Brazil and the second for South America. C. worsaae is the first species of the taxon to be described from the West African coast.

An early record of Meloidogyne fallax from Ireland

Abstract Root-knot nematodes, Meloidogyne spp., cause huge economic losses worldwide. Currently, three Meloidogyne spp. are present on the quarantine A2 list of EPPO, Meloidogyne chitwoodi, Meloidogyne fallax and Meloidogyne enterolobii. As a quarantine organism, Meloidogyne fallax has been detected in England and Northern Ireland on sport turf in 2011, and in England on leek in 2013. However, its presence in Ireland has probably been overlooked since 1965, when Mr. John F. Moore and Dr. Mary T. Franklin had detected a new Meloidogyne species for that time. While the relevant data was recorded and a preliminary manuscript describing the species was prepared but never submitted for publication, and together with the original slides, pictures and drawings, it was restudied recently. We compared the population of Irish Meloidogyne sp. to other similar Meloidogyne spp. Careful observation and comparison shows that it belongs to Meloidogyne fallax. The characters found to be common for Irish Meloidogyne sp. and Meloidogyne fallax are female stylet length (14.6 μm) with oval to rounded basal knobs, oval shaped perineal pattern with moderately high dorsal arch, slender stylet in males (18.5 μm) with set off and rounded basal knobs, slightly set off male head with one post-labial annule and incomplete transverse incisures, and second-stage juveniles with large and rounded stylet basal knobs, and a gradually tapering tail (46.9 μm) with a broadly rounded tip and a clearly delimitated smooth hyaline part sometimes marked by constrictions (12.9 μm). The host test and gall formation also correspond to Meloidogyne fallax. The identification could not be additionally supported by molecular analysis, as we were unable to extract DNA from the old permanent slides. Nevertheless, our study reveals that the Meloidogyne species detected in Ireland in 1965 belongs to Meloidogyne fallax.