Quantitative Amplicon Sequencing for Meta-DNA Analysis Reveals Patterns
in Pollen Use by Bees
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.