Step 1: Obtaining the pollen sample
Pollen can be obtained from a range of environmental samples. Most often
it is sampled from insects, either directly from the body (Chang et al.,
2018; Pornon et al., 2016; Suchan, Talavera, Saez, Ronikier, & Vila,
2019), or through provisions in nests (Gresty et al., 2018; McFrederick
& Rehan, 2016), honey (de Vere et al., 2017; Jones, Brennan, et al.,
2021), or honeycomb (Tanaka, Nozaki, Nakadai, Shiwa, & Shimizu-Kadota,
2020). Alternatively, pollen may be sampled from the air (Brennan et
al., 2019; Leontidou et al., 2018). How, where, and when a sample is
collected affects the number and diversity of target species sampled and
the plant richness and diversity captured. For example, when sampling
directly from a pollinator’s body, researchers should consider the
influence of body size and the pilosity of insects on the number and
diversity of pollen retained (Cullen et al., 2021). Likewise, capturing
foraging insects by hand-netting may be limited by surveyor bias. Some
sampling methods are specific to particular taxonomic groups (e.g.,
honey sampling or pollen trapping; Judd, Huntzinger, Ramirez, &
Strange, 2020). The temporal scale of a pollen sample should also be
considered. Pollen from an individual represents a single foraging trip,
whilst pollen trapping usually entails pooling samples so multiple trips
from multiple bees can be sampled over a short period. Pollen provisions
in solitary bee nests represent multiple trips by one individual. The
temporal scale of airborne samplers is dependent on study design. The
sampling period defines the knowledge which can be gained (e.g.,
sampling pollen from social bees with a long flight period during one
month of the year does not give a global picture of resource use). The
sampling period should also consider floral phenology, as pollen will
only be present when the plants are in flower.
The quantity of pollen required for the selected sequencing method, and
the quantity of pollen available per sample will determine whether
samples need to be pooled. Pooling of samples should be done with
caution, as it could decrease the accuracy of diet estimations relative
to the separate analysis of samples, especially for detecting species
that occur at relatively low abundance (Mata et al., 2019). The number
of pollen grains present in a sample will vary according to the sample
type. For pollen collected directly from the bodies of pollinators, this
will vary according to pollinator species. Estimates show that
Hymenoptera can carry relatively high numbers of pollen grains
(Apis mellifera and Bombus lucorum carry 2100-2500 pollen
grains; Bombus pascuorum , ~800 grains), Diptera
carry fewer (Syrphidae ~ 500 grains; Empididae
~80 grains), and Lepidoptera carry fewer again
(~20 grains), while the few Coleoptera observed carried
up to 200 grains (Pornon et al., 2016). The number of samples collected,
whether they are pooled, the number of sampling events across time and
space, and the replication level within each of these samples will
depend on the questions being addressed. Once the pollen samples are
obtained, they seem to be relatively resilient and easily storable over
extended periods under different conditions without introducing biases
(e.g., frozen, room temperature, silica gel dried; Quaresma et al.,
2021).
Control samples should be used to validate the methodology for each
study system. These include negative controls, ideally including
field-collected and laboratory blanks, to assess for contaminating DNA
from external sources, and positive controls or mock communities to
demonstrate that the sequencing method is effective, and if relevant,
quantitative, for the species in the study system. Contamination should
also be minimised through good laboratory practice. Extra precautions
can be taken for specific sample types, such as using separate
collection materials (i.e., nets, vials) to avoid cross-contamination
between specimens during collection (Pornon et al., 2016). For some
sample types, contamination will be unavoidable. For example, some types
of nest pollen from solitary bees may be contaminated by other plant DNA
during nest construction (Gresty et al., 2018; Alexander Keller et al.,
2015; McFrederick & Rehan, 2016). Finally, analysis of negative
controls will allow for detection and removal of contaminating sequences
during the bioinformatics step (e.g., Davis, Proctor, Holmes, Relman, &
Callahan, 2018).