Discussion
We found that a portfolio of four markers targeting three different
barcoding genes identified as many of our set of 103 marine and
freshwater taxa as possible to the species level and that no additional
taxa could be identified by applying an additional 15 markers to our DNA
reference pool. These same four markers identified the full set of 103
taxa to the family level. COI markers recovered the greatest number of
reference taxa and at the highest taxonomic resolution, but markers for
12S and 16S identified unique taxa missed by COI. These patterns offer
strong evidence of the benefits of a portfolio approach, but also
suggest that primer portfolios may need to be optimized for each target
taxonomic group or geographic region rather than considered broadly
transferable.
While we found an overall tendency towards lower read counts for taxa
added in lower input amounts, the patterns were highly inconsistent.
Accordingly, our read counts could not reliably be interpreted as even a
semi-quantitative proxy for relative abundance of a species in our
experimental tissue mixture, highlighting a key limitation for PCR-based
assays. Detection (presence/absence) of reference taxa from this complex
mixture became inconsistent with <1% of input tissue, which
points to a threshold of ~1% representation for robust
PCR-based detection in a heterogeneous sample. Interestingly, the
likelihoods of detecting species across the range of relative abundance
in the mixture was independent of whether fish tissues were analyzed
alone or after being diluted with either a plant-based or plant/animal
filler, probably thanks to the relative affinity of the markers tested
for fishes.