Sven van den Elsen

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

SSU ribosomal DNA-based monitoring of nematode assemblages reveals distinct seasonal fluctuations within evolutionary heterogeneous feeding guilds.

Soils are among the most complex, diverse and competitive habitats on Earth and soil biota are responsible for ecosystem services such as nutrient cycling, carbon sequestration and remediation of freshwater. The extreme biodiversity prohibits the making of a full inventory of soil life. Hence, an appropriate indicator group should be selected to determine the biological condition of soil systems. Due to their ubiquity and the diverse responses to abiotic and biotic changes, nematodes are suitable indicators for environmental monitoring. However, the time-consuming microscopic analysis of nematode communities has limited the scale at which this indicator group is used. In an attempt to circumvent this problem, a quantitative PCR-based tool for the detection of a consistent part of the soil nematofauna was developed based on a phylum-wide molecular framework consisting of 2,400 full-length SSU rDNA sequences. Taxon-specific primers were designed and tested for specificity. Furthermore, relationships were determined between the quantitative PCR output and numbers of target nematodes. As a first field test for this DNA sequence signature-based approach, seasonal fluctuations of nematode assemblages under open canopy (one field) and closed canopy (one forest) were monitored. Fifteen taxa from four feeding guilds (covering ∼ 65% of the free-living nematode biodiversity at higher taxonomical level) were detected at two trophic levels. These four feeding guilds are composed of taxa that developed independently by parallel evolution and we detected ecologically interpretable patterns for free-living nematodes belonging to the lower trophic level of soil food webs. Our results show temporal fluctuations, which can be even opposite within taxa belonging to the same guild. This research on nematode assemblages revealed ecological information about the soil food web that had been partly overlooked.

Rather than by direct acquisition via lateral gene transfer, GHF5 cellulases were passed on from early Pratylenchidae to root-knot and cyst nematodes

BackgroundPlant parasitic nematodes are unusual Metazoans as they are equipped with genes that allow for symbiont-independent degradation of plant cell walls. Among the cell wall-degrading enzymes, glycoside hydrolase family 5 (GHF5) cellulases are relatively well characterized, especially for high impact parasites such as root-knot and cyst nematodes. Interestingly, ancestors of extant nematodes most likely acquired these GHF5 cellulases from a prokaryote donor by one or multiple lateral gene transfer events. To obtain insight into the origin of GHF5 cellulases among evolutionary advanced members of the order Tylenchida, cellulase biodiversity data from less distal family members were collected and analyzed.ResultsSingle nematodes were used to obtain (partial) genomic sequences of cellulases from representatives of the genera Meloidogyne, Pratylenchus, Hirschmanniella and Globodera. Combined Bayesian analysis of ≈ 100 cellulase sequences revealed three types of catalytic domains (A, B, and C). Represented by 84 sequences, type B is numerically dominant, and the overall topology of the catalytic domain type shows remarkable resemblance with trees based on neutral (= pathogenicity-unrelated) small subunit ribosomal DNA sequences. Bayesian analysis further suggested a sister relationship between the lesion nematode Pratylenchus thornei and all type B cellulases from root-knot nematodes. Yet, the relationship between the three catalytic domain types remained unclear. Superposition of intron data onto the cellulase tree suggests that types B and C are related, and together distinct from type A that is characterized by two unique introns.ConclusionsAll Tylenchida members investigated here harbored one or multiple GHF5 cellulases. Three types of catalytic domains are distinguished, and the presence of at least two types is relatively common among plant parasitic Tylenchida. Analysis of coding sequences of cellulases suggests that root-knot and cyst nematodes did not acquire this gene directly by lateral genes transfer. More likely, these genes were passed on by ancestors of a family nowadays known as the Pratylenchidae.

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.