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Quantitative Amplicon Sequencing for Meta-DNA Analysis Reveals Patterns in Pollen Use by Bees
  • Aubrie James,
  • Monica Geber,
  • David Toews
Aubrie James
The University of Queensland
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Monica Geber
Cornell University
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David Toews
Pennsylvania State University
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Abstract

An underdeveloped but potentially valuable molecular method in ecology is the ability to quantify the frequency with which foraging pollinators carry different plant pollens. Thus far, DNA metabarcoding has only reliably identified the presence/absence of a plant species in a pollen sample, but not its relative abundance in a mixed sample. Here we use a system of four congeneric, co-flowering plants in the genus Clarkia and their bee pollinators to (1) develop a molecular method to quantify different Clarkia pollens found on foraging bees; and (2) determine if bee pollinators carry Clarkia pollens in predictable ways, based on knowledge of their foraging behaviors. We develop a molecular method we call quantitative amplicon sequencing (qAMPseq) which varies cycling number (20, 25, 30, and 35 cycles) in polymerase chain reaction (PCR), individually indexing the same samples in different cycle treatments, and sequencing the resulting amplicons. These values are used to approximate an amplification curve for each Clarkia species in each sample, similar to the approach of quantitative PCR, which can then be used to estimate the relative abundance of the different Clarkia species in the sample. Using this method, we determine that bee visitation behaviors are generally predictive of the pollens that bees carry while foraging. We also show that some bees carry multiple species of Clarkia at the same time, indicating that Clarkia likely compete via interspecific pollen transfer. In addition to adding a ‘missing link’ between bee visitation behavior and actual pollen transfer, we suggest qAMPseq as another molecular method to add to the developing molecular ecology and pollination biology toolbox.

Peer review status:UNDER REVIEW

16 Apr 2020Submitted to Molecular Ecology
17 Apr 2020Assigned to Editor
17 Apr 2020Submission Checks Completed
12 May 2020Review(s) Completed, Editorial Evaluation Pending