Metabarcoding of vertebrate DNA derived from carrion flies has been proposed as a promising tool for biodiversity monitoring. To evaluate its efficacy, we conducted metabarcoding surveys of carrion flies on Barro Colorado Island (BCI), Panama, which has a well-known mammal community, and compared our results against diurnal transect counts and camera-trapping. We collected 1084 flies in 29 sampling days, conducted metabarcoding with mammal-specific (16S) and vertebrate-specific (12S) primers, and sequenced amplicons on Illumina MiSeq. For taxonomic assignment, we compared BLAST with the new program PROTAX, and we found that PROTAX improved species identifications. We detected 20 mammal, four bird, and one lizard species from carrion fly metabarcoding, all but one of which are known from BCI. Fly metabarcoding detected more mammal species than concurrent transect counts (29 sampling days, 13 species) and concurrent camera-trapping (84 sampling days, 17 species), and detected 67% of the number of mammal species documented by eight years of transect counts and camera-trapping combined, although fly metabarcoding missed several abundant species. This study demonstrates that carrion fly metabarcoding is a powerful tool for mammal biodiversity surveys, and has the potential to detect a broader range of species than more commonly used methods.

protaxscriptsDirectory containing perl scripts used to run PROTAXmammal16S_OTUFasta file containing all mammal 16S OTU sequencesmammal12S_OTUFasta file containing all mammal 12S OTU sequencesmammal16S_refseqsFasta file containing mammal 16S reference sequences used in training PROTAXmammal12S_refseqsFasta file containing mammal 12S reference sequences used in training PROTAXMammalia_taxonomyFull Linnean taxonomy of mammal speciesprotax_trainBASH commands used to train unweighted PROTAX modelprotax_classifyBASH commands used to classify OTU sequences using unweighted PROTAX modelprotax_weighted_trainBASH commands used to train weighted PROTAX modelprotax_weighted_classifyBASH commands used to classify OTU sequences using weighted PROTAX modelBCI_sp_listList of species known to be present on BCIpanama_sp_listList of species known from Panama