Ecological and evolutionary drivers of stingless bee honey variation at the global scale

Stingless bee honey (SBH) is a prime natural product consumed and used for diverse medicinal and traditional purposes by local communities across the (sub-)tropics. The drivers of its compositional variation within and among species remain poorly understood, although this could inform broader and less explored eco-evolutionary theories. In our study, we aimed to disentangle the roles of different drivers of SBH compositional variation at both continental and global scales. Using a comparative approach involving parallel analyses of honeys produced by Apis mellifera as reference points, we specifically aimed to characterize the relative importance of SBH variation in relation to stingless bee species and evolutionary history, environmental conditions and biogeography. We collected and analysed 100 honey samples from A. mellifera around the world and 150 samples from twenty-three genera of stingless bees equally distributed in the Afrotropics (n = 50), the Neotropics (n = 50) and the Indo-Malayan region/Oceania (n = 50). We performed honey profiling using H1-NMR spectroscopy to quantify 36 compounds grouped into five categories : sugars, organic acids, amino acids, fermentation markers and anti-microbial compounds. Our results showed a clear differentiation between the chemical composition and functional diversity of A. mellifera and stingless bee honeys, mainly due to the production of a range of bioproducts during sugar fermentation. The study of compositional variation of stingless bee honey showed that the role of ecological and evolutionary drivers and their joint effects varied within each tropical region, preventing the identification of a clear continental, phylogenetic or ecological pattern. Additionally, a significant part of the variation remained unexplained, presumably reflecting the various natural factors and human colony management that can affect honey properties. We provide the first global and comprehensive characterisation of stingless bee honey composition, a prerequisite for defining and accepting SBH in the different Codex Alimentarius. The chemical complexity of this product highlighted in this study requires either broad international standards or precise local quality labels. We also highlight the need for more interdisciplinary ethnographic studies on non-food uses of honeys, and to encourage trans-sectoral research adopting a holistic approach to investigate stingless bee honey characteristics.

Methods
Honey profiling by 1H‑NMR spectroscopy To characterize the compounds and properties of our honey samples, we used H1 Nuclear Magnetic Resonance spectroscopy (hereafter H1-NMR spectroscopy), a state-of-the-art analytical technique increasingly used alongside chemometrics statistical approaches for the qualitative and quantitative control of honeys, as well as to assess the botanical origin of honeys and to quantify their major constituting compounds (Schievano et al., 2012; Ohmenhaeuser et al., 2013). H1-NMR spectroscopy was carried out on all 240 samples described above at the laboratories of Quality Services International GmbH (QSI, Bremen, Germany) following the method described in Noiset et al, 2022. We quantified 36 compounds grouped in five categories : sugars (n=10), organic acids (n=3), amino acids (n=8), fermentation markers (i.e., all the compounds involved in sugar transformation trough alcoholic, acetic and lactic fermentation; n=10) and anti-microbial compounds (n=5) (Supplementary Table 3). Another set of physicochemical data of honey samples of Apis mellifera (n = 10) from Mexico analysed according to the protocol described previously at the QSI laboratories were pooled with our dataset of 90 Apis spp. samples; this provided a better balance between the number of honey bee and stingless bee samples analysed in this study. Statistical analyses All the statistical analyses presented here were performed in RStudio for R. We combined multivariate and model-based approach using R packages that are listed in the Scripts directory and produce plots that can be found in the Figure directory.