Introduction
The concept of extended phenotype lies in the idea that a gene exerts effects beyond the organism itself, by creating physical structures, modifying the environment or manipulating other organisms to increase its reproductive success. Ever since Dawkins introduced the theoretical framework of “extended phenotype” (Dawkins, 1982), contemporary evolutionary biology has demonstrated its influence by providing numerous examples of complex relationships and adaptations in all taxonomic groups (Camargo & Pedro, 2003; Whitham et al., 2003; Cui et al., 2020; Fernandez et al., 2022; DeLong et al., 2022). As the introduction of this conceptual framework stimulated both heated debates and a great deal of research activity (Dawkins, 2004; Hunter, 2018), its incorporation into quantitative and empirical approaches in an attempt to bridge the gap between genotypes and environments towards an integrated theory of evolution remains a challenge. The environment, usually seen as an independent component of phenotypic variation in quantitative genetics, is instead viewed as a variable affected by the genotype (Bailey, 2012; Edelaar et al., 2023). The investigation into extended phenotypes, including behavioral manipulation or housing structure and associated microorganisms, has many potential applications in the fields of ecology and agriculture (Woods et al., 2021; Favela et al., 2023). This is particularly relevant for keystones species (“whose impact on its community or ecosystem is large, and disproportionately large relative to its abundance” (Power et al., 1996)) such as social insects (Elizalde et al., 2020). In the case of social bees, their ecological and economical importance triggered research on their related extended phenotype, mainly focusing on nesting (Smith et al., 2015), foraging and communication (Hughes, 2008; Schaedelin & Taborsky, 2009) yet overlooking their most renowned product, honey. Honey production combines the features of the extended phenotype with a well-defined genetic basis, from foraging (Page et al., 2000) to nectar collection and processing by bee enzymes (Kunieda et al., 2006; Vannette et al., 2015) and symbiotic microorganisms (de Paula et al., 2021). Further, this contributes to the colony fitness not only as stored source of carbohydrates, but also for improving immunity and regulating temperature, development and adult longevity (Berenbaum & Calla, 2021).
Although the production of honey has been the hallmark of Apis mellifera in Europe, the well-known Western honey bee spread across the entire world, another tribe of social bees called stingless bees (Apidae, Subfamily Meliponinae) are found exclusively in the (sub-)tropical regions and produce honey used for its nutritional and medicinal properties (Chuttong et al., 2018; Pimentel et al., 2021; Noiset et al., 2022; Héger et al., 2023; Vit et al., 2024). Approximately 600 species of stingless bees belonging to 45 genera have been described to this date (Engel et al., 2023), making Meliponinae the largest group of corbiculate bees. Despite this great diversity, stingless bees and their products have been relatively less studied than honey bees and have mainly been investigated in a few Neotropical countries (Brazil & Mexico in the lead), neglecting other Neotropical countries, the Afrotropics and the Indomalayan, Papuasian, and Australian (IPA) region (Nordin et al., 2018). Stingless bee honey (SBH) has higher water content than honey from A. mellifera , which allows the persistence of hydrophilous symbiotic microorganisms involved in sugar fermentation (Menezes et al., 2013). This is a key process for the preservation of honey that results in a less sweet and more acidic product (Biluca et al., 2016) but also in the production of a range of unique by-products associated with health benefits (Fletcher et al., 2020). Additionally, honey microbiota and phytochemicals transferred from honey pots made of cerumen (a mix of wax and propolis) contributes to its medicinal properties by introducing antimicrobial compounds to reduce the proliferation of competing microorganism (Ngalimat et al., 2020; Chuttong et al., 2023). Therefore, SBH may be the extended phenotype of the hive but also the one of the microorganisms hosted by honey and bees. These microorganisms benefits as well from this aqueous solutions that has been proposed to be the natural habitat of some osmophilic yeasts (Matos et al., 2020).
The understanding of the compositional variability of SBH is still limited due to the numerous sources of variation interacting with each other. Biochemical reactions in stingless bee honey vary according to the bee species producing enzymes, processing dynamics and the microbiota (Souza et al., 2021). Honey microbiota varies according to the foraging patterns, nectar microbiota, environment, hive and the bee species trough vertical and horizontal transmission, the relative importance of each has yet to be defined (Cerqueira et al., 2024). Stingless bee also visits a wide variety of (sub-)tropical plants (Bueno et al., 2023) whose nectar and microbiota composition vary, adding significant levels of phytochemicals and amino acids to honey (Shamsudin et al., 2019; Biluca et al., 2019). Nectar composition itself is influenced by ecological (water availability, temperature, soil factors, interactions with herbivores and nectar robbers) and evolutionary drivers such as plant phylogenetic constraints and pollination syndrome (Nicolson & Thornburg, 2007; Parachnowitsch et al., 2019). Foraging activity of stingless bees also include resin collection to build nests and honey pots which also influence honey composition and properties (Kegode et al., 2023; Villagomez et al., 2024; Nakamura et al., 2024). Beyond these complex factors, the lack of scientific research on the physico-chemical characteristics and bio-functional properties of SBH from the Afrotropics and the Indomalayan, Papuasian, and Australian regions hinder the exploration of the evolutionary ecology hypothesis regarding SBH.
To fill these gaps, our study aimed to work at the global level to i) characterize the differences between the composition of honeys from stingless bees and honey bees using advanced chemometric methods, ii) investigate patterns of compositional variation among tropical regions and finally iii) disentangle the roles of evolutionary history and environmental conditions on honey properties. Ultimately, we aim to provide the basis of a theoretical framework to understand the drivers of compositional variability of SBH.