M.H.M. Holterman

A phylogenetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences

As a result of the scarcity of informative morphological and anatomical characters, nematode systematics have always been volatile. Differences in the appreciation of these characters have resulted in numerous classifications and this greatly confuses scientific communication. An advantage of the use of molecular data is that it allows for an enormous expansion of the number of characters. Here we present a phylogenetic tree based on 1215 small subunit ribosomal DNA sequences ( ca 1700 bp each) covering a wide range of nematode taxa. Of the 19 nematode orders mentioned by De Ley et al. (2006) 15 are represented here. Compared with Holterman et al. (2006) the number of taxa analysed has been tripled. This did not result in major changes in the clade subdivision of the phylum, although a decrease in the number of well supported nodes was observed. Especially at the family level and below we observed a considerable congruence between morphology and ribosomal DNA-based nematode systematics and, in case of discrepancies, morphological or anatomical support could be found for the alternative grouping in most instances. The extensiveness of convergent evolution is one of the most striking phenomena observed in the phylogenetic tree presented here – it is hard to find a morphological, ecological or biological characteristic that has not arisen at least twice during nematode evolution. Convergent evolution appears to be an important additional explanation for the seemingly persistent volatility of nematode systematics.

Small Subunit rDNA-Based Phylogeny of the Tylenchida Sheds Light on Relationships Among Some High-Impact Plant-Parasitic Nematodes and the Evolution of Plant Feeding

Cyst (Heteroderidae), root knot (Meloidogyne spp.), and lesion (Pratylenchus spp.) nematodes all belong to a single nematode order, Tylenchida. However, the relationships between and within these economically highly relevant groups, and their relatedness to other parasitic Tylenchida is unclear. We constructed a phylogeny of 116 Tylenchida taxa based on full length small subunit ribosomal DNA (small subunit [SSU] rDNA) sequences. Ancestral state reconstruction points at a gradual development of simple to more complex forms of plant parasitism. Good resolution was observed in distal clades that include cyst, root knot, and lesion nematodes, and monophyly of most families was confirmed. Our data suggest that root knot nematodes have evolved from an ancestral member of the genus Pratylenchus, but it remains unclear which species is closest to this branching point. Contrary to the notoriously polyphagous distal representatives, basal members of the genus Meloidogyne (and probably, their common ancestor) have narrow host ranges. Our analysis also shows that mitotic parthenogeny has arisen at least two times independently among root knot nematodes. In many cases resolution till species was observed, suggesting that SSU rDNA sequences have a potential for DNA barcode-based species identification with, due to the overall conserved nature of this gene, limited intra-species variation.

A ribosomal DNA‐based framework for the detection and quantification of stress‐sensitive nematode families in terrestrial habitats

Indigenous communities of soil‐resident nematodes have a high potential for soil health assessment as nematodes are diverse, abundant, trophically heterogeneous and easily extractable from soil. The conserved morphology of nematodes is the main operational reason for their under‐exploitation as soil health indicators, and a user‐friendly biosensor system should preferably be based on nonmorphological traits. More than 80% of the most environmental stress‐sensitive nematode families belong to the orders Mononchida and Dorylaimida. The phylogenetic resolution offered by full‐length small subunit ribosomal DNA (SSU rDNA) sequences within these two orders is highly different. Notwithstanding several discrepancies between morphology and SSU rDNA‐based systematics, Mononchida families (indicated here as M1–M5) are relatively well‐supported and, consequently, family‐specific DNA sequences signatures could be defined. Apart from Nygolaimidae and Longidoridae, the resolution among Dorylaimida families was poor. Therefore, a part of the more variable large subunit rDNA (≈ 1000 bp from the 5′‐end) was sequenced for 72 Dorylaimida species. Sequence analysis revealed a subclade division among Dorylaimida (here defined as D1–D9, PP1–PP3) that shows only distant similarity with ‘classical’ Dorylaimid systematics. Most subclades were trophically homogeneous, and — in most cases — specific morphological characteristics could be pinpointed that support the proposed division. To illustrate the practicability of the proposed molecular framework, we designed primers for the detection of individual subclades within the order Mononchida in a complex DNA background (viz. in terrestrial or freshwater nematode communities) and tested them in quantitative assays (real‐time polymerase chain reaction). Our results constitute proof‐of‐principle for the concept of DNA sequence signatures‐based monitoring of stress sensitive nematode families in environmental samples.

Small subunit ribosomal DNA-based phylogeny of basal Chromadoria (Nematoda) suggests that transitions from marine to terrestrial habitats (and vice versa) require relatively simple adaptations.

The phylum Nematoda is positioned the base of the superphylum Ecdysozoa and in terms of species number and abundance can be characterized as a highly successful group. Because of their relatively conserved morphology and many poor morphological descriptions, nematode taxonomy has been unstable for decades. However, the small and large subunit (SSU and LSU) ribosomal DNA (rDNA) sequences are remarkably powerful in resolving the internal relationships within this phylum. Studies on the basis of SSU rDNA sequences suggested a subdivision of the Nematoda into several numbered clades (Blaxter et al., 1998; Holterman et al., 2006). Over the last five years there has been a steep increase in the number of available sequences, and most recently Meldal et al. (2007) presented 46 new SSU rDNA sequences from marine taxa, a so far underrepresented group. For decades the hypothesis of Filipjev (1929) about the marine ancestry of nematodes has been widely accepted. Molecular analysis of Ecdysozoa on the basis of both small and large subunit ribosomal DNA sequence information seems to support Filipjev’s hypothesis (Mallatt and Giribet, 2006). Nematodes can be found in virtually all terrestrial, freshwater and marine habitats, and it is interesting to investigate on the basis of neutral molecular data where and how frequent fundamental habitat transitions occurred. We hypothesize that the taxonomic levels at which the transitions take place (class, order, family or genus level) are indicative for the number of physiological and/or morphological changes needed to

Structural and functional characterization of a novel, host penetration-related pectate lyase from the potato cyst nematode Globodera rostochiensis

SUMMARY The cell wall, a strong extraprotoplasmic layer surrounding plant cells that mainly consists of a variety of polysaccharides, constitutes a major barrier for potential parasites. Plant-parasitic nematodes are well equipped to overcome this barrier as they produce and secrete cell-wall-degrading enzymes. Expression profiling of various life stages of the potato cyst nematode Globodera rostochiensis revealed a novel pectate lyase gene (Gr-pel2, 759 bp). The Gr-PEL2 protein showed highest similarity to pectate lyases from the facultative plant-parasitic nematodes Bursaphelenchus mucronatus and B. xylophilus and the soil-inhabiting saprophytic Streptomyces and Frankia species (i.e. 40-42% identity and 58-60% similarity), whereas only a remote relatedness to the previously identified Gr-PEL1 was observed (i.e. 28% identity and 43% similarity). Transient expression of Gr-pel2 in leaves of Nicotiana benthamiana resulted in severe malformations of the infiltrated tissues, not relating to maceration and soft rot symptoms. Ca(2+) is known to be essential for pectate lyase activity, and the most likely calcium-binding site was identified in the Gr-PEL2 protein by combining homology modelling of the three-dimensional structure, site-directed mutagenesis and transient expression in leaves. A highly charged cleft in Gr-PEL2, which is likely to be involved in substrate binding and which is also significantly more hydrophobic in Gr-PEL1, was shown to be essential for protein activity. Our results underline the broad spectrum of pectate lyases and cell-wall-degrading enzymes necessary for successful parasitism by cyst nematodes.

Disparate gain and loss of parasitic abilities among nematode lineages

Plant parasitism has arisen time and again in multiple phyla, including bacteria, fungi, insects and nematodes. In most of these organismal groups, the overwhelming diversity hampers a robust reconstruction of the origins and diversification patterns of this trophic lifestyle. Being a moderately diversified phylum with ≈ 4,100 plant parasites (15% of total biodiversity) subdivided over four independent lineages, nematodes constitute a major organismal group for which the genesis of plant parasitism could be mapped. Since substantial crop losses worldwide have been attributed to less than 1% of these plant parasites, research efforts are severely biased towards this minority. With the first molecular characterisation of numerous basal and supposedly harmless plant parasites as well as their non-parasitic relatives, we were able to generate a comprehensive molecular framework that allows for the reconstruction of trophic diversification for a complete phylum. In each lineage plant parasites reside in a single taxonomic grouping (family or order), and by taking the coverage of the next lower taxonomic level as a measure for representation, 50, 67, 100 and 85% of the known diversity was included. We revealed distinct gain and loss patterns with regard to plant parasitism per se as well as host exploitation strategies between these lineages. Our map of parasitic nematode biodiversity also revealed an unanticipated time reversal in which the two most ancient lineages showed the lowest level of ecological diversification and vice versa.

Secreted venom allergen-like proteins of helminths: Conserved modulators of host responses in animals and plants

Despite causing considerable damage to host tissue at the onset of parasitism, invasive helminths establish remarkably persistent infections in both animals and plants. Secretions released by these obligate parasites during host invasion are thought to be crucial for their persistence in infection. Helminth secretions are complex mixtures of molecules, most of which have unknown molecular targets and functions in host cells or tissues. Although the habitats of animal- and plant-parasitic helminths are very distinct, their secretions share the presence of a structurally conserved group of proteins called venom allergen-like proteins (VALs). Helminths abundantly secrete VALs during several stages of parasitism while inflicting extensive damage to host tissue. The tight association between the secretion of VALs and the onset of parasitism has triggered a particular interest in this group of proteins, as improved knowledge on their biological functions may assist in designing novel protection strategies against parasites in humans, livestock, and important food crops.

Morphological and molecular data support the monophyletic nature of the genus Pratylenchoides Winslow, 1958 (Nematoda: Merliniidae) and reveal its intrageneric structuring

The genus Pratylenchoides has recently been transferred from the family Pratylenchidae to Merliniidae. To investigate further the relationship between these ‘Pratylenchus-like’ species (residing in the subfamily Pratylenchoidinae) and the subfamily Merliniinae, more than 500 soil samples were collected from various natural and agronomic habitats in the northern and north-western provinces of Iran. In this study, paratypes or populations of 22 species of Pratylenchoides, including the Iranian populations of P. alkani, P. crenicauda, P. erzurumensis, P. laticauda, P. nevadensis, P. ritteri and an undescribed species, were studied. Intra- and interspecies variation of the following characters were investigated: position of the pharyngeal gland nuclei, shape of female and male head, striation of female tail terminus, number of lateral lines at mid-body and in phasmid region for females, presence of intestinal fasciculi, and shape of sperm. Combining morphological and molecular data prompted us to propose two clusters of related Pratylenchoides species. One cluster includes P. crenicauda, P. variabilis and P. erzurumensis, whereas the second cluster consists of P. alkani, P. nevadensis and P. ritteri. Our data point to a sister positioning of P. magnicauda vis-a-vis all Pratylenchoides species included in this research. Analyses of SSU rDNA (for family and subfamily relationships) and partial LSU rDNA sequences (for intrageneric relationships) data revealed: i) the distal and nested positioning of all Pratylenchoidinae within the Merliniidae; ii) the single transition from ectoparasitism to migratory endoparasitism within the family Merliniidae corresponds with the current subfamily partitioning; and iii) support for the monophyletic nature of the genus Pratylenchoides.

A strategy in searching for stress tolerance-correlated characteristics in nematodes while accounting for phylogenetic interdependence

Biological indicators are highly relevant for assessing the condition of a soil as they are integrative; they reflect the overall impact of physical, chemical and biological changes. Indigenous soil organisms are preferable to other test organisms because the diversity and condition of indigenous soil organisms reflect both acute and chronic effects of soil disturbances. Nematodes are ubiquitous, speciose, easily extractable and present in extremely high numbers. Given the ever increasing amount of sequence data, DNA barcode-based community analysis will soon be possible and a next step would be to define objective criteria for the ecological grouping of soil nematodes. Here, we present a framework to ascertain which traits are correlated with a tolerance to stress. For this, a field study on the effects of pH and copper on nematode communities was re-analysed. Changes in abundances of individual genera were correlated with a number of potentially stress tolerance-related characteristics. The generalised least squares (GLS) method was used to account for the phylogenetic dependence of the data. Only the relationship between the ability to enter a survival stage and tolerance to copper at pH 6.1 was found to be significant, but the quantity of missing data probably had a negative impact on the analyses. This study did, however, clearly demonstrate the importance of accounting for the effects of phylogenetic dependence in the data. When the phylogeny was taken into account, we observed an average change in P value of 0.196 (and in some cases as much as 0.6) for the correlations of possible stress-related characteristics and Cu or pH tolerance. This research constitutes a proof-of-principle for a transparent method to relate stress tolerance to (ecological) characteristics. The usefulness of this powerful method should become even clearer when substantially higher numbers of individuals are analysed (as facilitated by using DNA barcodes) and when missing data are filled in