Human activities have an overwhelming impact on the natural environment, leading to a deep biodiversity crisis whose effects range from genes to ecosystems. We here analysed the effect of such anthropogenic impacts on bdelloid rotifers (Rotifera: Bdelloidea), for whom these effects are poorly understood. We targeted bdelloid rotifers living in lichen patches across urbanisation gradients in Flanders and Brussels (Belgium). Urbanisation was measured as the percentage of built-up area across different spatial scales, at circles from 50 m to 3,200 m of radius around the lichen. Urbanisation effects on biodiversity were assessed on abundance, species richness, and community-weighted mean body size of bdelloid rotifers, as well as on genetic diversity of one of the most common and widespread bdelloid species, Adineta vaga. Overall, no negative effect of urbanisation was found at any diversity level and at any spatial scale. Counterintuitively, built-up area quantified at the largest spatial scale had a positive effect on abundance. These results leave open the question of whether negative effects of urbanisation are present for bdelloid rotifers, or if such effects are only visible at even larger spatial scales.

Communities of bdelloid rotifers were sampled by collecting one lichen patch in each subplot. We selected lichens of the genus Xanthoria, for which bdelloid rotifer communities have been previously studied in Europe (Fontaneto et al. 2011). Lichens of the genus Xanthoria are among the most abundant in urban and rural areas, apparently unaffected by urbanisation levels (Nekrošienė 2012). Sampling was performed in June and July 2013. Suitable Xanthoria patches could be found in all but two subplots: total sample size is thus 79 lichens and not 81. 

The selection of the lichen was haphazard: the first suitable lichen patch encountered in each subplot was collected. Dry lichen thalli between 5 and 10 cm² were cut from the substrate with a knife and kept in zip lock bags. For each lichen sample, an area of 2.5 cm² was hydrated with distilled water in a plastic petri dish. 

All active bdelloid rotifers that recovered from dormancy within the four hours following hydration in the lab were sorted and identified to species level according to Donner (1965). Previous studies on bdelloid rotifers in these lichens (Fontaneto et al. 2011) revealed that animals start recovering between 10 and 40 minutes after hydration of the sample and that no more additional bdelloid rotifers usually recover after four hours. The very few dormant stages still found in the sample that did not recover after that time were considered dead, impossible to identify at any taxonomic rank, and excluded from the analyses.

All living bdelloids were isolated, counted, and identified to species level to obtain data on (1) abundance and (2) species richness. The other descriptor of community-level diversity was (3) community-level body size (Merckx et al. 2018b), calculated as community-weighted mean body size, which is the average of the species-specific body length for all sampled species in a community, weighted by species abundance in the community. Measurements of body length for the observed species, obtained from the observed animals, are reported in Merckx et al. (2018b).

The most common and abundant species in all samples, Adineta vaga, was selected to obtain metrics of genetic diversity. DNA was extracted from each of the individuals found in a separate extraction of bdelloids from the part of each lichen patch that was not used for the morphological identifications. We amplified the barcoding fragment of the mitochondrial marker cytochrome c oxidase subunit I, COI, using Folmer primers with optimised protocols for this species (Debortoli et al. 2016). COI sequences were trimmed to a total common length of 605bp and aligned using default settings in MAFFT v7 (Katoh et al. 2019), confirming correct amino acid translation. Species identity was confirmed through online BLAST searches (Ye et al. 2006). The number of A. vaga haplotypes for each population of the species in a lichen patch was used as a metric of genetic diversity.