Supplementary material 1 from: do Rosário Cruz AJ, Herrera-Mesías F, Hauprich A, Thissen D, Weigand AM (2025) beelibre.lu - Luxembourg's open library of wild bee species profiles, pollen data, DNA barcodes and bibliographic references. Research Ideas and Outcomes 11: e175726. https://doi.org/10.3897/rio.11.e175726

Beelibre protocol for pollen sampling and storage The Beelibre project follows a custom-made protocol for the sampling, preservation and storage of pollen samples from living specimens (Fig. 1). First, collect wild bee specimens using hand-netting, preferably with the assistance of experienced field workers or naturalists. At the beelibre project, this task is performed by trained specialists from the Zoology Department of the National Museum of Natural History of Luxembourg (MNHNL). After capture, place the bees in 2ml Eppendorf tubes and cold anesthetized them using ice packs, temporarily reducing their movement (“chill coma”). For small taxa, use 1.5 ml Eppendorf tubes, as this helps cool them more quickly. While the bees are incapacitated (usually, a few minutes after placing them on the ice packs), gently rub the pollen collecting structures of the anesthetized bees using the tip of cotton swabs, carefully extracting as much pollen as possible without harming the bee. Depending on their taxonomic group, these structures may be located either in their legs or in the underside of the abdomen. Cut the tip of the cotton swab used to collect the pollen and place it in a separated Eppendorf tube. Fill ¾ of each tube with 96% ethanol. All bees with pollen loads collected in this manner should be photographed in situ and assigned an individual code to maintain a backup record of their association with the pollen sample. Pollen samples must be labelled using the same identification number of their associated wild bee specimen, as well as their site and date of collection. Afterwards, release all wild bees that can be reliably identified at their finding spot. For optimal results, this step requires the assistance of an experienced parataxonomist. To minimize lethal sampling, only specimens that cannot be reliably identified in the field are to be taken back to the laboratory for further analysis. For long term storage, all pollen samples collected this way should be placed in a freezer, preferably at -20°C. Figure 1. Beelibre workflow for pollen sampling and storage. 1.- The bees are collected in the field and anaesthetized at low temperatures; 2.- Pollen loads are extracted directly from the scopae of foraging females using cotton swabs; 3.- Pollen samples are placed on Eppendorf tubes filled with 96% ethanol; 4.- Pollen samples are stored at -20 °C.