An efficient iDNA method for surveying rainforest mammals with carrion flies

Metabarcoding of vertebrate DNA derived from invertebrates (iDNA) is a promising method for monitoring of mammal assemblages. However, optimization of field and laboratory methods for efficient and economical sampling and analysis is needed. We used metabarcoding of carrion flies to survey for mammals at Los Amigos Biological Station in the Peruvian Amazon. We used an extraction-free, direct PCR approach to metabarcode vertebrate DNA, whereby buffer from tubes that contained flies for 3-5 hours was used directly in PCR, negating the need for labor-intensive DNA extraction. We also examined two different pooling methods to investigate the efficacy of pooling multiple fly samples to economize sequencing of large numbers of samples. Metabarcoding detected 27 mammalian taxa from 9 orders and 18 families plus four anuran taxa from three families from 240 carrion flies collected over 3 days. Pooling of buffer from multiple flies prior to PCR resulted in failure to detect many species compared to sequencing of individual flies. However, pooling of PCR product after an initial PCR from individual flies, but prior to indexing, resulted in species detection nearly identical to sequencing of individual flies. These methodological findings will contribute to the feasibility of iDNA metabarcoding for vertebrate monitoring and biodiversity conservation.