Experimental quantification of pollen with DNA metabarcoding using ITS1 and trnL

Abstract

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 highthroughput 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. Environmental DNA metabarcoding is a molecular method that consists of investigating environmental DNA samples made of complex mixtures of genomes from numerous organisms 1. Due to new sequencing technologies and bioinformatics tools, metabarcoding has been increasingly used to identify taxa in environmental samples 1 to monitor biodiversity 2-4 , to investigate ecosystem functioning 5 and interaction networks 6-8 , in both aquatic and terrestrial ecosystems. Nevertheless, its reliability in quantitative approaches, which depend on the match between counts of high-throughput sequence reads and the amount of sampled biological material 2 , is still the subject of debate 9,10. While taxon identification can reveal individual diet breadth 11 , species richness, and the composition of habitats 2 , communities 12 and ecological networks 4 , taxon quantification provides knowledge on species evenness in those habitats, communities and diets or on the level of individual or species specialization in networks, all of which is very useful in ecological studies. Research on pollination and knowledge of the quantities of pollen transported by pollinators allow for the estimation of plant-pollinator interaction strength and hence it gives a more realistic representations of networks than those made possible using traditional approaches such as observing visits to plants by pollinators 9,13. Metabarcoding has been used in pollen studies to identify pollen in honey 14-16 , insect loads 6-8,17 , insect nests 18 , airborne samples 19 , and to quantify pollen abundance across various sample types. Several studies found significant positive relationships between pollen abundance (estimated using light microscopy) or pollen DNA quantities, and the abundance or the frequencies of high-throughput sequencing reads in experimental samples 10,16,17,20,21 , airborne samples 22,23 , insect pollen loads 21,24-27 or in brood cells of solitary bees 28. Conversely, other studies found low or no significant pollen-sequence abundance relationships when using ITS2 markers applied to pollen provision in...

Using ITS2 metabarcoding and microscopy to analyse shifts in pollen diets of honey bees and bumble bees along a mass‐flowering crop gradient

“…Quantitative outcomes derived from ITS2 metabarcoding can be affected by contamination of the samples as well as DNA extraction and amplification biases. Pollen species, pollen counting methodology and the chosen marker may affect quantification as well (Baksay et al., 2020; Pornon et al., 2016). The number of pollen grains we used for metabarcoding is unknown and does exceed the number of pollen grains used for microscopy.…”

“…Due to the high amount of unexplained variation, especially for OSR pollen, further research is needed that takes potentially confounding factors into account, for instance pollen type identities, standardized amounts of pollen and defined compositions of mixed samples to confirm a general positive relationship between the quantitative outcomes of both methods (Baksay et al., 2020; Pornon et al., 2016; Richardson et al., 2015; Smart et al., 2017). Previous research has shown that the number of ITS2 reads does not reflect the actual number of pollen grains (Baksay et al., 2020; Pornon et al., 2016). Quantitative outcomes derived from ITS2 metabarcoding can be affected by contamination of the samples as well as DNA extraction and amplification biases.…”

“…We used metabarcoding of ITS2‐region PCR amplicons to quantify the richness of pollen collected by small and large honey bee and bumble bee colonies. In the present study, we used the advantages of metabarcoding techniques (e.g., high efficiency and resolution; Baksay et al., 2020; Bell et al., 2019) for qualitative high‐throughput identification of PCR‐amplified ITS2 sequences from plant genera present in pollen loads collected from honey bees and bumble bees to study pollen richness.…”

“…However, metabarcoding is restricted in its applicability in pollen quantification. Several factors may affect the quantification results obtained by next‐generation sequencing (NGS)‐based amplicon sequencing, such as DNA extraction, PCR (polymerase chain reaction) amplification or barcode copy number (Peel et al., 2019), which can lead to over‐ or underestimation of actual pollen counts (Baksay et al., 2020; Pornon et al., 2016; Richardson et al., 2015). The number of metabarcoding reads may be more likely to be positively related to microscopy‐based counts for commonly occurring taxa than for rare pollen taxa in the samples (Smart et al., 2017).…”

A pilot study of eDNA metabarcoding to estimate plant biodiversity by an alpine glacier core (Adamello glacier, North Italy)

“…Nowadays the integration of the DNA metabarcoding and the morphological approaches represent the best way to investigate the major part of the ecological sites 67 . Despite the accuracy of DNA metabarcoding analysis is strictly related to the reference database used and its quantification capability is still under study 68 , the advantages of the metabarcoding approach (overcoming the morphological approach limits, low cost and low time consumption) make it a fundamental method for ecological survey 6 , 53 .…”