Phylogenetic relationships within major nematode clades based on multiple molecular markers

Nematodes are probably the most abundant Metazoans on our planet. They are present in densities of millions of individuals per square meter in soil and sediments. However, these inconspicuous animals are hardly known to the general public as most individuals are colorless and smaller than 1 mm. If people are aware of nematodes, it is because of the damage they inflict on humans and animals such as elephantiasis (<em>Wucheria bancrofti</em>) and ascariasis (in humans <em>Ascaris lubricoides</em>; in pigs <em>Ascaris suum</em>), or on plants such as potato (potato cyst nematode) and tomato (root-knot nematodes). To a far lesser extent it is known that majority of nematodes are key players in the soil food web, and as such they can be used as indicators for the biological condition of the environment they live in. This PhD thesis focuses on terrestrial nematodes belonging to four orders: Dorylaimida, Mononchida, Aphelenchida and Tylenchida, which represent animals of ecological and economical relevance. Members of families belonging to the first two are highly sensitive to the environmental disturbances, and are informative as biological indicators. The order Aphelenchida harbors numerous facultative plant-parasitic species. In the absence of a host plant, most of them are able to feed on fungi as an alternative food source. This is in contrast to the distal representatives of the order Tylenchida that are invariably obligate parasites of higher plants. Stress-sensitive nematode orders Dorylaimida and Mononchida have a high potential for soil health assessment. The SSU rDNA-based analysis of these two orders resulted in two highly distinct phylogenies. Relationships among the Mononchida, an order dominated by carnivorous nematodes, were to some extent in accordance with the classical nematode systematics. It is noted that the families Mylonchulidae, Mononchidae and Anatonchidae are not monophyletic. Nevertheless, it was possible to design family-specific primers for rDNA-based molecular detection. On the contrary, resolution of the SSU rDNA tree of the Dorylaimida was extremely poor, except for the plant-parasitic family Longidoridae and the mainly predaceous family Nygolaimidae. Analysis of a 1,000 bp fragment of the 5’ region of large subunit (LSU) rDNA resulted in an improved resolution. Twelve subclades were distinguished and this topology was only in slight agreement with the classical systematics of the suborder Dorylaimina. The poor resolution generated by SSU rDNA sequence analysis within this species-rich suborder is remarkable; it has not been observed in any other suborder in the phylum Nematoda. Possibly, Dorylaimina diversification is the result of rapid speciation events. A plant-parasitic lifestyle apparently accelerates the rate of change of rDNA genes. This was not only true for the obligate plant-parasitic Longidoridae, but also for the facultative plant-parasitic Aphelenchoididae<em>. </em>Most members of the genus <em>Aphelenchoides </em>are fungivores, but a few of them feed on higher plants as well. As they feed on aboveground parts of higher plants they are usually called ‘foliar nematodes’. Species such as <em>Aphelenchoides besseyi</em>, <em>A. fragariae </em>and <em>A. ritzemabosi </em>parasitize on ornamental plants in greenhouses and nurseries, and some field crops such as rice or strawberry. Moreover, <em>A. subtenuis </em>causes serious damage infecting flower bulbs<em>. </em>Identification of foliar nematode species, and the distinction between plant- parasitic species and other, mostly harmless, fungal feeding representatives of the genus <em>Aphelenchoides </em>is hampered by the scarcity of informative morphological characters and lack of well-established systematics. Based on nearly full-length SSU rDNA sequences, a phylogenetic tree was generated, where the four target species appeared as distinct, well-supported, monophyletic groups. The presence of species-specific DNA motifs made it possible to design PCR primers for the detection and quantification of the foliar nematode species in complex DNA backgrounds such as plant material and soil samples. The order Tylenchida is dominated by obligatory plant-parasitic nematode taxa and includes economically high-impact species such as the lesion (<em>Pratylenchus spp.</em>), root-knot (<em>Meloidgyne spp.</em>) and cyst (<em>Heterodera, Globodera</em>) nematodes. The sequence diversification of cellulases, a non-neutral, plant pathogenicity-related genes, was investigated. Unlike by far most other animals, nematodes do not depend on endosymbionts for the production of cell wall-degrading enzymes. Among a repertoire of these proteins, glycoside hydrolase family 5 (GHF5) cellulases are best studied. It is hypothesized that they were acquired by one or multiple horizontal gene transfer (HGT) events. Moreover, the nature of the donor - hypothesized to be a plant-parasitic soil bacterium - and the recipient, possibly a bacterivorous nematode is fully unclear. Using a range of primers, partial GHF5 cellulase sequences spanning the core catalytic domain were amplified and sequenced from basal <em>Meloidogyne, </em>and a range of <em>Pratylenchus </em>and <em>Hirschmanniella </em>species. Phylogenetic analysis of more than 100 partial GHF5 cellulase sequences resulted in a division of the enzymes’ catalytic domains into three types (A, B, C). Type B was numerically dominant, and notably the <em>P. thornei </em>cellulase was positioned sister to all type B root-knot nematode cellulases. Moreover, the overall topology of the catalytic domain B-type showed remarkable resemblance with trees based on rDNA sequences. This analysis suggests that most likely the cellulases were passed on by ancestors of a family nowadays known as the Pratylenchidae, and root-knot and cyst nematodes did not acquire these genes directly by lateral genes transfer. To further elucidate the relationship between a part of the family Pratylenchidae (namely the subfamilies Pratylenchinae and Hirschmanniellinae) and the Meloidogynidae, two neutral (= pathogenicity-unrelated) genes were taken into consideration: SSU rDNA and a part of the largest subunit of the RNA polymerase II gene (<em>rpb1</em>). Both morphological and molecular data seem to point at root-knot nematodes being a subclade branching from the migratory endoparasites <em>Pratylenchidae</em>. Extension of the SSU rDNA data set - more sequences from a broader range of species – did not result in a well- resolved relationship between the Pratylenchidae and the Meloidogynidae. A switch to another gene that was previously exploited to investigate relationships within the genus <em>Meloidogyne</em>, a fragment of the largest subunit of RNA polymerase II sequences, did not provide us with more robust information about the evolutionary transition between lesion and root-knot nematodes. The genus <em>Pratylenchus </em>comprises more than 100 species. It is referred to as a stenomorphic genus since only a few subtle characteristics are used for species identification. In the current study, only a subset of these species was taken to consideration; predominantly species that are relatively well-characterized as pathogens in agro-ecosystems. It was postulated that the <em>Pratylenchus </em>species closest to the basal root-knot nematodes should be sought among the less well-known and agronomically less relevant lesion nematode species.