INTRODUCTION
Discovering how the interactions of plants and pollinators play out is
critical to understanding how they mutually rely on each other, and
molecular methods are an increasingly common toolkit in this endeavor.
In particular, identifying the source of DNA in mixed-DNA samples has
become increasingly popular with the development of a range of
metabarcoding methods. These approaches rely on single-locus PCR
amplicons, leveraging the power of high-throughput DNA sequencing to
compare reads to a database of putative DNA sources. The applications of
metabarcoding are manifold, including analyses of microbiomes and diets
(Sousa et al. 2019), as well as environmental DNA analysis to quantify
community composition (Deiner et al. 2017, Sousa et al. 2019). Such
molecular approaches have also recently been applied to plant
identification from the pollen found on bees to determine which species
of flowers they have visited (Wilson et al. 2010; Galimberti et al.
2014; Sickel et al. 2015; Bell et al. 2016, Bell et al. 2017).
Historically, pollen has been identified using microscopic morphological
differences to distinguish different plant species (Martin and Harvey
2017). However, for some plant taxa, morphological similarity of pollen
between distinct species makes this method impossible. In these
situations, pollen grains taken from wild bees can be molecularly
interrogated to identify the species of plant they are from (Mitchell et
al. 2009, Galliot et al. 2017, Lucas et al. 2018).
Molecular methods for identifying pollen origin have been generally
restricted to making inferences about the presence or absence of a
source plant species from pollen (via metabarcoding techniques; Bell et
al. 2017). This is in part because previous methods have been unable to
reliably quantify relative abundance of mixed pollen samples for several
reasons, discussed in detail in Bell et al. (2019). One reason is that
when using plastid DNA in metabarcoding (e.g. Galimberti et al. 2014),
it is unclear how the abundance of chloroplast DNA (cpDNA) is related to
overall pollen abundance; if the ratio of cpDNA and pollen grains is not
one-to-one, this could bias estimates of relative abundance (Richardson
et al. 2015, Bell 2019). Another bias of pollen metabarcoding approaches
has to do with how polymerase chain reaction (PCR) amplifies target
markers (Bell et al. 2017). In PCR, the final concentration of amplicon
DNA after a full PCR protocol is not necessarily directly correlated to
input DNA concentration. This is because most PCR protocols will take
the amplification process into the “plateau phase”, usually after
approximately 30 thermal cycles. At the plateau stage, amplicon
concentration may be a function of exhausted reaction reagents rather
than original concentration of input DNA - the desired information.
Because of this, studies using the “plateau abundance” of amplicons
could be subject to PCR bias, especially in cases of low abundance
(rarity) in the sample.
Though the potential weakness of using PCR to quantify amplicons has
been raised by previous authors (e.g. Richardson et al. 2015; Bell et al
2017; Bell et al. 2019), it is still of interest to use molecular
methods to estimate the relative abundance of different pollen sources
found in bees’ scopae or corbiculae (that is, in their pollen-carrying
morphological structures). This is in part because understanding
resource use among mutualists such as plants and pollinators is
fundamental to determining the extent to which they may rely on each
other for population persistence (Roulsten and Gooddell 2011), support
ecosystem functioning (Lucas et al. 2018), and mediate interspecific
competition and coexistence (Johnson 2019). In fact, as Bell et al.
(2019) noted, the ability to molecularly determine species abundances in
mixed-pollen samples may be ‘groundbreaking’ for understanding
plant-pollinator communities, because successful pollen transport
between plants determines plant seed production.
Plant-pollinator interactions can provide a predictive framework for how
pollinators introduce reproductive interference and/or fitness benefits
to co-flowering plants (James 2020). The effects of pollinator
visitation on plant seed set are determined by the extent to which
pollinators (1) carry a mix of pollen on their bodies while foraging
(constancy) and (2) carry rare versus common species in their pollen
balls (preference). During a single pollen collection bout, bees can
visit multiple flower species, a behavior known as inconstancy, or visit
the same flower species, known as constancy (Kunin and Iwasa 1996).
Pollinator inconstancy exposes plants to reproductive interference via
heterospecific pollen transfer, which has been shown to drive lower seed
production and fitness in plants (Mitchell et al. 2009; Carvalheiro et
al. 2014; Arceo-Gómez et al. 2019). Pollinator preference is a measure
of flower choice by pollinators. If a strongly competitive plant is
preferred by pollinators, pollinator preference might exacerbate
competitive exclusion, but if a weakly competitive plant is preferred by
pollinators, preference could mitigate competitive exclusion. Though
pollinator-mediated plant interactions are most often understood through
the lens of pollinator behavior, ample evidence suggests that linking
pollinator visitation to plant seed set is problematic: not all
plant-pollinator contacts result in pollen transfer (Mayfield 2001;
Popic et al. 2013; Ballantyne et al. 2015, 2017; Barrios et al. 2016).
Because of this, the relative abundances of pollen on bees could provide
valuable information about how pollinators mediate plant interactions.
Here, we develop and use a novel high throughput amplicon sequencing
method to quantify the relative abundance of different pollen sources on
bee pollinators visiting a group of sympatric winter annual plants in
the genus Clarkia (Onagraceae). This group of plants – C.
cylindrica ssp. clavicarpa (Jeps.) Lewis & Lewis, C.
speciosa ssp. polyantha Lewis & Lewis, C. unguiculataLindl., C. xantiana ssp. xantiana A. Gray — are
sympatric in the woodland-chaparral areas of the southern foothills of
the Sierra Nevada mountain range (from here, we do not use their
subspecies epithets). The four species of Clarkia rely on a small
group of bee pollinators specialized on the genus Clarkia rather
than any one species (MacSwain et al. 1973; Moeller 2005). Though theseClarkia have distinct adult phenotypes, their pollen grains are
morphologically indistinguishable. The Clarkia also co-occur with
each other more often than they occur alone in plant communities in
their range of sympatry, and assemblages can contain one to four species
of Clarkia (Eisen and Geber 2018). Finally, Clarkia bloom
much later in the growing season than the vast majority of co-occurring
flowering annual plants, and as such are often the only flowering plants
where they occur.
A previous study of the bee visitors to one of the four species of
interest to this study, C. xantiana , showed that though 49
species of bee visit C. xantiana, there were only 12 likely
“core pollinators” of the species, nine of which carry almost
exclusively Clarkia pollen (Moeller 2005). Studies including the
other three species of Clarkia in their range of sympatry withC. xantiana have found that there are three consistently common
pollinator taxa in multi-species Clarkia assemblages (Singh 2013,
James 2020). The most common pollinator, Hesperapis regularis(Melittidae) has been shown to preferentially visit C. xantiana .
Preferences of bees in the Lasioglossum genus (Halictidae), the
second most common pollinator taxon, is unclear: they have been shown to
visit all Clarkia species at relatively the same rates (Singh
2014) or preferentially visit C. xantiana and C.
cylindrica (James, 2020). The unresolved nature of Lasioglossumpreferences are at least partially explained by the fact that it is
difficult to identify different Lasioglossum species when
observing them on the wing, as in (James, 2020). Despite preferences,Hesperapis regularis and Lasioglossum species visitC. cylindrica, C. unguiculata, and C. xantiana regularly,
and are inconstant when foraging in diverse arrays and thus likely to
transfer incompatible pollen between plants (James 2020). The final
most-common bee pollinator in the system, Diadasia angusticeps(Apidae), is behaviorally more specialized on one Clarkiaspecies, C. speciosa , and rarely visits the other species ofClarkia (Singh 2013, James 2020).
Critically, experimental evidence in this system has linked the
behavioral inconstancy and preference of pollinators with Clarkiaseed production (James 2020). Clarkia speciosa exhibits low
pollen limitation to reproduction, which may be explained by the
constancy and preference behavior of Diadasia angusticeps. The
other three species exhibit higher pollen limitation to reproduction,
which may be due to the inconstancy of Hesperapis regularis andLasioglossum sp . However, because pollinator visitation does not
equate with pollen transfer, it remains unknown if pollinator preference
and constancy in the Clarkia system in fact determine plant
interactions.
The wealth of natural history knowledge and the morphological similarity
of Clarkia pollens make the Clarkia system ideal for
developing a method that can both identify and quantify different
species in pollen samples. In this paper, we develop a method that we
call “quantitative amplicon sequencing” or “qAMPseq” to quantify the
relative abundance of Clarkia pollen in pollen balls from wild
bees. Quantitative amplicon sequencing uses the amplification curve of
PCR as a backbone for quantification: it targets single nucleotide
polymorphisms private to each species, and then uses PCR to amplify
these regions with the goal of post-amplification sequencing, as in
metabarcoding. Critically, PCR amplification is stopped before
saturation at four different times so one can estimate when each
species’ amplification curve crosses a critical threshold (as in
quantitative PCR or qPCR; Figure 2). The estimate of when the curve
crosses the critical threshold point is then used to estimate the
relative abundance of each species in each sample. If
previously-observed trends in bee behavior match what they carry in
their pollen balls (James 2020), then we predict the inconstant bees,Hesperapis regularis and Lasioglossum sp. , will carry
multiple species of pollen at once. In addition, because these
particular bee taxa have an established behavioral preference forC. xantiana , we expect that they will carry more C.
xantiana than other Clarkia species. We also predict that the
pollen on Diadasia angusticeps will contain only C.
speciosa pollen. Finally, because Hesperapis andLasioglossum sp. are the most common taxa in this region
of Clarkia sympatry, we predict an overall pollinator preference
for C. xantiana pollen.