Experimental quantification of pollen with DNA metabarcoding using ITS1 and trnL

Although the use of metabarcoding to identify taxa in DNA mixtures is widely approved, its reliability in quantifying taxon abundance is still the subject of debate. In this study we investigated the relationships between the amount of pollen grains in mock solutions and the abundance of high-throughput sequence reads and how the relationship was affected by the pollen counting methodology, the number of PCR cycles, the type of markers and plant species whose pollen grains have different characteristics. We found a significant positive relationship between the number of DNA sequences and the number of pollen grains in the mock solutions. However, better relationships were obtained with light microscopy as a pollen grain counting method compared with flow cytometry, with the chloroplastic trnL marker compared with ribosomal ITS1 and with 30 when compared with 25 or 35 PCR cycles. We provide a list of recommendations to improve pollen quantification.