Primer biases in the molecular assessment of diet in multiple insectivorous mammals

Our understanding of trophic interactions of small insectivorous mammals
has been drastically improved with the advent of DNA metabarcoding. The
technique has continued to be optimised over the years, with primer choice
repeatedly being a vital factor for dietary inferences. However, the
majority of dietary studies examining the effect of primer choice often
rely on in silico analyses or comparing between species that occupy an
identical niche type. Here we apply DNA metabarcoding to empirically
compare the prey detection capabilities of two widely used primer sets
when assessing the diets of a flying (lesser horseshoe bat; Rhinolophus
hipposideros) and two ground dwelling insectivores (greater white-toothed
shrew; Crocidura russula and pygmy shrew; Sorex minutus). Although R.
hipposideros primarily rely on two prey orders (Lepidoptera and Diptera),
the unique taxa detected by each primer shows that a combination of
primers may be the best approach to fully describe bat trophic ecology.
However, random forest classifier analysis suggest that one highly
degenerate primer set detected the majority of both shrews’ diet despite
higher levels of host amplification. The wide range of prey consumed by
ground-dwelling insectivores can therefore be accurately documented from
using a single broad-range primer set, which can decrease cost and labour.
The results presented here show that dietary inferences will differ
depending on the primer or primer combination used for insectivores
occupying different niches (i.e. hunting in the air or ground) and
demonstrate the importance of performing empirical pilot studies for novel
study systems.