DNA metabarcoding using nrITS2 provides highly qualitative and quantitative results for airborne pollen monitoring

Airborne pollen monitoring is of global socio-economic importance as it provides information on presence and prevalence of allergenic pollen in ambient air. Traditionally, this task has been performed by microscopic investigation, but novel techniques are being developed to automate this process. Among these, DNA metabarcoding has the highest potential of increasing the taxonomic resolution, but uncertainty exists about whether the results can be used to quantify pollen abundance. In this study, it is shown that DNA metabarcoding using trnL and nrITS2 provides highly improved taxonomic resolution for pollen from aerobiological samples from the Netherlands. A total of 168 species from 143 genera and 56 plant families were detected, while using a microscope only 23 genera and 22 plant families were identified. NrITS2 produced almost double the number of OTUs and a much higher percentage of identifications to species level (80.1%) than trnL (27.6%). Furthermore, regressing relative read abundances against the relative abundances of microscopically obtained pollen concentrations showed a better correlation for nrITS2 (R(2) = 0.821) than for trnL (R(2) = 0.620). Using three target taxa commonly encountered in early spring and fall in the Netherlands (Alnus sp., Cupressaceae/Taxaceae and Urticaceae) the nrITS2 results showed that all three taxa were dominated by one or two species (Alnus glutinosa/incana, Taxus baccata and Urtica dioica). Highly allergenic as well as artificial hybrid species were found using nrITS2 that could not be identified using trnL or microscopic investigation (Alnus × spaethii, Cupressus arizonica, Parietaria spp.). Furthermore, perMANOVA analysis indicated spatiotemporal patterns in airborne pollen trends that could be more clearly distinguished for all taxa using nrITS2 rather than trnL. All results indicate that nrITS2 should be the preferred marker of choice for molecular airborne pollen monitoring.