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

Fylogenetische SSU rDNA-analyse van het fylum Nematoda

Nematoden vormen één van de meestgevarieerde en succesvolle diergroepen ter wereld. Ze zijn waarschijnlijk de meest talrijke dieren op aarde, komen in uiteenlopende milieus voor (zowel terrestrische als marien) en spelen een belangrijke rol in het ecosysteem. De verscheidenheid van voedingstypes en habitats maken deze groep ook erg interessant vanuit een evolutionair oogpunt. Ons onderzoek richt zich op het uitzoeken van de evolutionaire verwantschappen tussen nematoden aan de hand van het ribosomaal DNA - een neutraal gen dat niets te maken heeft met dier- of plantparasitisme - en de evolutie van kenmerken als voedingstypen, stresstrolerantie en de overgang van een marien leefmilieu naar het land. Nematodentaxonomie is een onderzoeksveld dat sinds het begin in beweging is geweest. De geconserveerde morfologie en de vaak moeilijk waarneembare kenmerken bemoeilijken de reconstructie van de evolutie van de nematoden. Dit heeft tot gevolg gehad dat de nematodensystemetiek steeds veranderde en er bijna net zoveel classificaties als taxonomen zijn. De laatste jaren is er veel veranderd door de opkomst van de moleculaire fylogenie. Het gebruik van DNA-sequenties - in het geval van vaak het small subunit ribosomal DNA (SSU rDNA)-gen - om de evolutie te traceren heeft geleid tot nieuwe inzichten en een hernieuwde interesses in nematodenevoluti

Comparison of two short DNA barcoding loci (COI and COII) and two longer ribosomal DNA genes (SSU & LSU rRNA) for specimen identification among quarantine root-knot nematodes (Meloidogyne spp.) and their close relatives

Root-knot nematodes (Meloidogyne spp.) are important pests of numerous crops worldwide. Some members of this genus have a quarantine status, and accurate species identification is required to prevent further spreading. DNA barcoding is a method for organism identification in non-complex DNA backgrounds based on informative motifs in short DNA stretches (˜600 bp). As part of the EU 7th Framework project QBOL, 15 Meloidogyne species were chosen to compare the resolutions offered by two typical DNA barcoding loci, COI and COII, with the distinguishing signals produced by two ribosomal DNA genes (small and large subunit rDNA; SSU¿˜¿1,700 and LSU¿˜¿3,400 bp). None of the four markers distinguished between the tropical species Meloidogyne incognita, M. javanica and M. arenaria. Taking P ID (Liberal) values =0.93 as a measure for species delimitation, the four mtDNA and rDNA markers performed well for the tropical Meloidogyne species complex, M. enterolobii, M. hapla, and M. maritima. Within cluster III A (Holterman et al. Phytopathology, 99, 227–235, 2009), SSU rDNA did not offer resolution at species level. Both mtDNA loci COI and COII did, whereas for LSU rDNA a longer fragment (=700 bp) is required. The high level of mitochondrial heteroplasmy recently reported for M. chitwoodi (Humphreys-Pereira and Elling Nematology, 15, 315–327, 2013) was not found in the populations under investigation, suggesting this could be a regional phenomenon. For identification of RKNs, we suggest the combined use of SSU rDNA with one of three other markers presented here

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 variatio

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 variatio