Introduction
The importance of pollination for terrestrial ecosystem function and
services is well known. It is estimated that 78% of temperate flowering
plant species rely to some extent on the activity of animal pollinators
for reproduction (Ollerton et al. 2011). Many of these are critical for
maintaining ecosystem functions providing important services for humans
(e.g., increased water and air quality, prevention of soil erosion,
timber, fruit and nut production; Kearns et al. 1998, Ashman et al.
2004, Cardinale et al. 2012). In addition, insect pollinators are
critically important to global food crops, contributing to 65% of the
produced crop volume (Klein et al. 2007). All these ecosystem services
are often solely credited to bees (Woodcock 2012, Dicks et al. 2013) and
although they are obligate nectar and pollen foragers, this assumption
leaves out many other important flower visiting insects (Müller et al.
2006). In fact, there are more than 330,000 insect species such as
flies, beetles, wasps, moths and butterflies which may provide
pollination services that are often unaccounted for (Larson et al. 2001,
Wardhaugh 2015, Rader et al. 2016, Ollerton 2017). Such diverse flower
visitor assemblages have been shown to lead to evenness of pollination
services; resulting in higher fruit set and fruit weight with fewer
blemishes due to insufficient pollination (Nye and Anderson 1974,
Lopez-Medina et al. 2006, Hodgkiss et al. 2018).
This evenness of pollination service is the result of each species
having their own foraging preferences and tolerances, which leads to
more frequent and thorough visitation of flowers from different
pollinators, thus increasing the likelihood of conspecific pollen
transfer (Fontaine et al. 2005, Hoehn et al. 2008, Blüthgen and Klein
2011, Garibaldi et al. 2013, 2014, Rogers et al. 2014). For example,
some insect communities are abundant early in the growing season,
maintaining large populations for pollination services for only a week
or two, while others peak later in the season, whereas others have a
lower population density that is sustained throughout the season
(Bartomeus et al. 2013). Thus, there is always a reasonably large
pollinator community with potentially complementary and overlapping
population peaks (Garratt et al. 2018). Additionally, species have
different foraging strategies. For instance, honey bees tend to forage
along the tops of plants and travel in a linear fashion, while solitary
bees and flies tend to forage on lower branches in a random pattern
(Brittain et al. 2013, Brunet et al. 2019). The combination of these two
foraging behaviours ensures that the entire plant receives pollination
and therefore sets fruit evenly. Finally, different environmental
conditions can affect which insects are foraging. High winds and cloudy
days tend to keep honey bees inside their hives, while flies, bumblebees
and some solitary bees will continue to forage in the rain and the cold
(Morgan and Heinrich 1987, Brittain et al. 2013).
One of the leading justifications usually given for the exclusion of
non-bee species in pollination studies is that bees are often the most
efficient pollinators (Buchman and Nabhan 1996, Kennedy et al. 2013).
Bees have a nectar and pollen-dependent diet; as such, their behaviour
and foraging techniques often result in high pollen release and frequent
flower visiting (Sheffield 2014, Russo et al. 2017, Campbell et al.
2018). When considering non-bee pollinators, their average pollination
efficiency per flower visit may be comparatively low, but their ubiquity
can lead to high visitation frequency, resulting in equal, or greater,
pollen deposition than bees (Larson et al. 2001, Skevington and Dang
2002, Rader et al. 2009, Orford et al. 2015, Rader et al. 2016). This is
especially true when considering Diptera, which are particularly
speciose and abundant (Skevington and Dang 2002).
To ensure accurate estimation of relative contribution from all
crop-pollinating taxa, it is crucial to include non-bee pollinators in
crop-pollination surveys, pollination estimates, and
pollinator-management practices to avoid taxonomic bias. However, it can
be difficult to assign reliable species-level identification of non-bee
pollinators and the plant pollen they carry. Given the taxonomic breadth
of flower visitor communities, the number of taxonomic experts required
for accurate species-level identification is typically unobtainable.
Equally, the identification of plant pollen has traditionally been
achieved using light microscopy, comparing samples to an extensive
palynological collection. However, this method often restricts taxonomic
resolution to the genus or family level and requires extensive expertise
(Rahl 2008, Keller et al. 2015). DNA-based identification methods, such
as DNA barcoding (Hebert et al. 2003) and metabarcoding (Cristescu
2014), can generate sufficient taxonomic resolution to overcome those
barriers. The mitochondrial cytochrome c oxidase I (COI) gene is
the community-wide accepted standard DNA barcoding region for animals
(Hebert et al. 2003) while metabarcoding of the plastid barcode standard
gene region of rbcL (large subunit of RuBisCo) (Hollingsworth et
al. 2009) and the nuclear region ITS2 (internal transcribed spacer) has
been explored for its accuracy in the qualification and quantification
of pollen samples (e.g., Bell et al. 2019, Potter et al. 2019, de Melo
Moura et al. 2022).
For this study we aimed to identify a community of non-bee flower
visitors considered probable pollinators living on three fields of
strawberry (Fragaria spp.) crops in Southern Ontario, Canada. We
performed species-level identifications of the non-bee flower visiting
insects by using DNA barcoding. In addition, pollen loads collected from
the bodies of those flower visitors were quantified by particle
characterization and identified utilizing rbcL metabarcoding.
Subsequently, we assessed how non-bee flower visiting communities
changed across the season and in response to variation in local
environmental conditions, including temperature, humidity, solar
radiation, and wind speed.