2.2 Pollinator insects and plant characterization
Sampling activities were performed in October 2019, from 9:00 to 16:00
only with good climate conditions. Pollinator insects were sampled by
hand-collecting flower visitors through entomological nets along free
transects around and crossing over an area of continuous vegetation
about 50 m x 50 m at each sampling location. Pollinator insects were
captured after being observed foraging on flowers for a few seconds and
captures were performed during a fixed time of three hours to allow
unbiased comparison between sampling sites (similarly to Tommasi et al.,
2021 a). After collection, each insect was individually stored in a
clean tube and filled with 70% ethanol. A clean entomological net was
used in each sampling site to prevent cross contamination between
samples from different sites.
Insects identification was performed through morphological inspection
and via standard DNA barcoding (Supporting information - Table S2).
Specifically, insects were first sorted at the lowest possible taxonomic
level (family or genus) following morphological criteria and
identification keys (e.g. , Bratra, 1977; Gupta, 2003; Thompson,
1981). Morpho-species grouping was subsequently confirmed by analysing
representative individuals (1- 11 individuals representative for sex and
atoll of provenance) for each morpho-species through a standard DNA
barcoding approach using primers LCO1490 and HCO2198 (Folmer et al.,
1994) and the laboratory protocols described in Galimberti et al., 2021.
For doubtful identifications where two or more candidate DNA barcode
identification were equally likely, a detailed morphological scrutiny
was additionally performed by an expert (author PB). Flower species
richness was also estimated identifying all the flowering species
observed in the whole area involved in insect sampling and plants were
identified in the field or in the lab following Sujanapal & Sankaran
(2016). Sampling localities and details are available in BOLD under the
project code ZPLML
(https://www.boldsystems.org/index.php/MAS_Management_DataConsole?codes=ZPLML)
and are also reported in Supporting information - Table S1.