2.1 Study System and Experimental Set-up
The facultative biennial herb D. purpurea (Plantaginaceae)
produces long showy inflorescences (Fig. 1) of nectar-rich flowers that
open in sequence from the bottom up. The flowers are protandrous, with
anthers dehiscing following anthesis and the stigma becoming receptive
three to five days after anthesis. Animal pollination is required to
achieve full seed set (Nazir et al., 2008; Mackin et al .,
2021), and the primary pollinators in the UK are the long-tongued
bumblebees Bombus hortorum and B. pascuorum (Broadbent &
Bourke, 2012). In introduced populations in Central and South America,
hummingbirds are also important pollinators, in addition to bumblebees
(Mackin et al ., 2021). In those populations we also observe high
levels of nectar robbing by bumblebees (Riveros et al. 2006),Diglossa flower piercers, and hummingbirds. We have not observed
bumblebees making holes on the corollas; instead, bees and hummingbirds
are secondary robbers using the holes pierced by Diglossa birds.
In surveys in two localities in Colombia (Floresta N=50 and Choachí
N=60) we found that 36.2% and 47.8% (respectively) of all
post-anthesis flowers were robbed, with robbing making up to 14.1% to
19.4% of all visits to flowers in those two populations.
Nectar production and secretion begins the day before the first pair of
anthers dehisce, and peaks during anthesis and stigma maturation
(Percival and Morgan 1965). Nectar is produced in floral nectaries
located at the base of the ovary and escapes through modified stomata
that are permanently open (Gaffal et al ., 1998). Nectar sugar
concentration ranges from 16-27% and is predominately made up of
sucrose (78.5%) with some glucose and fructose sugars (Gaffal et
al ., 1998). In our study populations, a single flower secretes between
3.1 to 10.5µl of nectar over 24 hours without visitation (pers. obs).
We grew D. purpurea potted plants from seed collected from a wild
population near Portsmouth (UK) in 2017, that were grown until flowering
in summer 2019 and then transported to the University of Sussex campus
in Falmer. Between 16-18 plants were selected at random to be in one of
two treatment groups – “robbed” and “control” (non-robbed flowers).
To standardise the rate of natural pollination that all plants
experienced, plants were exposed for 3 hours each day to receive visits
by local bumblebees, and for the other period of 21 hours the
inflorescences were covered with a mesh bag to block visitation.
Plants in the robbed treatment group had all post-anthesis
(nectar-producing) flowers manually robbed by piercing a hole in the
proximal corolla tube with a microcapillary tube twice during the 3 hour
pollination period (once at the start, and then again after 90 minutes).
This rate of robbing is based on field observations in the non-native
range where D. purpurea flowers are robbed less than once per
hour, and on greenhouse trials on the experimental plants that showed
that nectar is replenished at a rate of 2.3µl per hour during three
hours following nectar depletion (N = 30 flowers over 10 plants).
Compared to other species tested, this is a fast rate of replenishment
(Castellanos et al ., 2002) as a single foxglove flower secretes
up to 10.5µl of nectar per day.
Bumblebees will often avoid plants that have recently been visited by
other floral visitors using olfactory and other cues (Stout et
al ., 2001), so we manually handled the inflorescences (both controls
and robbed) to mimic contact during simulated robbing. This entire
procedure was repeated daily for the period that plants flowered between
June 17 and July 3, 2019.