Eusocial Hymenoptera are characterized by the presence of a remarkable reproductive division of labor between queens, who specialize in reproduction, and mostly sterile workers, who focus on tasks such as brood care, foraging, and nest defense (Wilson, 1971). In some species, however, workers can develop their ovaries and also lay unfertilized male eggs under certain conditions (Wilson, 1971). Nevertheless, the haplodiploid sex-determination system found in hymenopterans leads to workers being more related to their sisters (r = 0.75) than to their brothers (r = 0.25) or even to their own offspring (r = 0.50) in colonies headed by one single-mated queen. Due to this relatedness asymmetry, workers in such species would be expected to invest three times more in the production of new queens than in males, while the queen would be expected to invest equally in the production of both sexes, as she is equally related to sons and daughters (r = 0.50). Empirical data show that in several species this conflict over sex allocation is resolved in favor of the workers, since they care for the brood and can therefore manipulate the sex ratio to their own advantage (Hamilton, 1964; Trivers and Hare, 1976; Bourke and Franks, 1995; Wenseleers et al., 2021).

A striking exception to these predicted female-biased sex ratios is observed in the buff-tailed bumblebee Bombus terrestris (Hymenoptera: Apidae), where it is observed a highly male-biased population sex investment ratios (Bourke, 1997; Beekman and Van Stratum, 1998; Duchateau et al., 2004). The case of B. terrestris is broadly studied for investigating queen-worker conflict (Bloch and Hefetz, 1999; Bourke and Ratnieks, 2001; Alaux et al., 2004b; Amsalem et al., 2009) as this species constitute a perfect model study due to its colony features. First, it is a monogynous species with a singly mated queen (Schmid-Hempel and Schmid-Hempel, 2000) thus the conflict is expected to occur. Second, it is an annual species, thus a higher probability of an actual conflict between queen and workers, as both parties do not have interest in maintaining the colony in the later years (Bulmer, 1981). Third, the workers start to lay their own eggs in a specific moment of the colony lifecycle, called the “competition point”. This phase normally follows the “switch point” which occurs when the queen starts to lay haploid eggs for producing males (Duchateau and Velthuis, 1988; Lopez-Vaamonde et al., 2009), although some diploid eggs may also be laid after the switch point (Van der Blom, 1986; Van Doorn and Heringa, 1986; Duchateau and Velthuis, 1988). Over the years, several hypotheses have been put forward to try to explain this observed male-biased sex ratio. For example, the protandry hypothesis states that males produced early in the season have higher opportunities to mate than late-emerging males, and that colonies producing their males early would therefore gain greater fitness benefits (Bulmer, 1983; Bourke, 1997). However, the protandry hypothesis alone cannot explain the occurrence of a split sex ratio observed in this species, whereby some colonies specialize in the production of males and others in females (Meunier et al., 2008; Kümmerli and Keller, 2009). Indeed, B. terrestris presents two distinct social phenotypes correlated to differential reproductive strategies (Beekman and Van Stratum, 1998; Duchateau et al., 2004). In some colonies the queen “switches” to produce haploid male eggs early in the season (early switching colonies) when the worker/larvae ratio is small and specializes in male production, while other “late switching” colonies produce mostly queens toward the end of the season and start to produce males after queen larvae are detected (Duchateau and Velthuis, 1988; Alaux et al., 2004a,2005). As the two colony phenotypes were found with a frequency of 0.5 in nature (Duchateau and Velthuis, 1988), it was hypothesized that in half of the colonies, the mother queens could prevent the production of new queens by starting to lay haploid eggs earlier. In this scenario, in male-specialized colonies, workers would have no choice but to comply, given that they would have no diploid female eggs available to rear into queens and the replacement with workers’ eggs would not be profitable by the time the workers would be able to detect them (Bulmer, 1981; Pamilo, 1982; Bourke and Ratnieks, 2001). By contrast, in the remaining half of the colonies, the evolutionary interest of both the workers and the mother queen would coincide in benefiting from specializing in queen production (Bourke and Ratnieks, 2001). Although this mechanism seems plausible and a similar system was observed in some ant species (Helms, 1999), it still does not explain why workers in the late switching colonies begin to lay haploid male eggs at the onset of queen production (Bourke and Ratnieks, 2001; Alaux et al., 2004a,2005), resulting in a male-biased sex ratio at the population level (Goulson, 2010).

A further development of Bulmer’s model (Bulmer, 1983) proposes that the timing of the switch point, and consequently the split sex ratios, could be the result of an optimal sex allocation of resources since queens are 1.69–2.11 times more costly to produce than males (Beekman and Van Stratum, 1998; Duchateau et al., 2004). Therefore, colonies with more limited resources available could benefit from switching earlier and specializing in the production of the sex that is least costly to produce–males (Beekman and Van Stratum, 1998). However, in situations with overall low resource availability, such a mechanism would then cause the majority of colonies to produce mainly males, leading to a low reproductive success (Goulson, 2010), and so it is also hard to see how this theory would explain the male-biased sex ratios seen in bumblebees. In support of this, it was shown how environmental factors, including resources availability, have limited or no effect on reproductive decisions in B. terrestris (Schmid-Hempel and Schmid-Hempel, 1998; Baer and Schmid-Hempel, 2003).

Environmental stressors, such as climate change, do not only affect the availability of resources but can also lead to genetic variability constraints (Templeton et al., 1990; Gienapp et al., 2008; Hoffmann and Willi, 2008; Perez and Aron, 2020). A particularly important constraint in social insects is linked to the production of diploid males, which in extreme cases may result in the so-called “diploid male extinction vortex” (Zayed and Packer, 2005). In most hymenopteran species, including ants, bees, wasps, and sawflies, sex is determined by a complementary sex-determination system (CSD), in which heterozygous eggs at the CSD locus develop as females while hemizygous (haploid) or homozygous eggs develop as males (Whiting, 1943). Diploid male production comes at a large cost in social Hymenoptera, as they are produced at the expense of the production of new workers, thereby compromising colony growth (Duchateau et al., 1994; Van Wilgenburg et al., 2006), and on top of that, diploid males are also typically sterile or unable to produce viable offspring (Duchateau and Mariën, 1995; Beekman et al., 1999; Ayabe et al., 2004; Gerloff and Schmid-Hempel, 2005; Whitehorn et al., 2009). Nevertheless, some species have developed mechanisms to reduce the fitness costs of diploid male production to the colony. Such mechanisms are usually linked with inbreeding avoidance (Van Wilgenburg et al., 2006), but in some corbiculate bee species, different strategies have evolved to prevent colony collapse due to diploid male production. In the honeybee Apis mellifera, the queen mates with several males which spreads the risk of making a matched mating i.e., mating with a male carrying the same CSD allele (Page and Metcalf, 1982). In addition, workers are able to detect and cull diploid male larvae early during their development, therefore preventing a higher cost to the colony (Woyke, 1962, 1963). Stingless bees rear their brood in mass provisioned sealed cells and hence workers do not have access to the developing brood (Engels and Imperatriz-Fonseca, 1990). Nevertheless, it has been shown that matched mated queens are swiftly killed and replaced by one of their daughters once diploid males are detected (Vollet-Neto et al., 2017). Differently to honeybees, most of stingless bees and bumblebees are monandrous. Polyandrous species in fact have the advantage to dilute the effect of diploid males production due to a matched allele (Page, 1980; Ratnieks, 1990), while a monandrous mating system, such as in B. terrestris (Estoup et al., 1995; Schmid-Hempel and Schmid-Hempel, 2000), decreases the genetic variation and therefore increases the vulnerability to inbreeding (Chapman and Bourke, 2001). Due to the costs associated with inbreeding in bumblebees, the evolution favored mechanism of inbreeding avoidance, such as nestmate recognition and scent-marking during patrolling for a female (Foster, 1992). However, although B. terrestris males display scent-marking and patrolling behavior to find a partner (Brown and Baer, 2005; Goulson, 2010), it was shown that males do not avoid mating with related individuals (Gerloff and Schmid-Hempel, 2005; Bogo et al., 2018) to the detriment of certain colony traits such as the colony foundation success and the colony size, (Gerloff and Schmid-Hempel, 2005; Gosterit, 2016), although in some occasions inbred colonies can have a normal development (Duchateau et al., 1994; Bortolotti et al., 2020). Honeybees, stingless bees and bumblebees have been shown to be resilient to genetic bottlenecks with examples of successful populations being established from only a few colonies (Winston, 1996; Schmid-Hempel et al., 2007; Alves et al., 2011). These mechanisms reducing the costs of diploid males are likely to play an important role in the maintenance of populations with relatively lower diversity caused by genetic bottlenecks. Here, we use a combination of behavioral, chemical, and genetic data analysis to test if diploid male production causes a switch to early male production in the buff-tailed bumblebee B. terrestris. We hypothesize that the competition point, when workers begin to compete with the queen over male production, in early switching colonies could actually be a chance to produce haploid males and reduce the fitness cost of diploid male production.

The buff-tailed bumblebee B. terrestris is a remarkable model species to study colony-level reproductive strategies. A particularly interesting trait of this species is the existence of two distinct colony phenotypes with highly divergent reproductive patterns. Some of the colonies are characterized by the production of males early in the season and others by the late emergence of both virgin queens and males (Duchateau and Velthuis, 1988). Several hypotheses have been proposed in the past to explain the evolutionary mechanisms underlying these different colony phenotypes. Yet, to date, this phenomenon remains poorly understood. Here, we investigated if a diploid male production could be behind this bimodal pattern.

A striking outcome of our study is that all genotyped males belonging to the early switching type colonies from both the main stock and the experimental hybrid colonies obtained by mating males and queens from different populations of the commercial rearing facility were in fact diploid, thereby showing that early production of males was in fact caused by queens having made a matched mating with a male that by chance carried the same sex allele. The fact that males of early switching hybrid colonies were diploid as well indicates that this was not a consequence of strong inbreeding within the main stock of the commercial bumblebee rearing but a mating between individuals carrying by chance the same allele at the CSD locus. Although previous studies showed that colonies producing diploid males can have a normal development and reach a large colony size (Duchateau et al., 1994; Bortolotti et al., 2020), diploid males exact a large fitness cost, as in social Hymenoptera their production comes at a cost of the production of new workers or queens, and on top of that diploid males are usually sterile, bringing no fitness benefits to the colony (Cook and Crozier, 1995), or, when fertile, imposing a hefty fitness cost to a potential female mate which will then produce triploid sterile progeny (Ayabe et al., 2004; Darvill et al., 2012; Harpur et al., 2013). It is therefore in the colony’s interest to adopt strategies to reduce or avoid diploid male production. In the honeybee A. mellifera for example, workers can detect a quantitative shift in the pattern of tricosane, pentacosane, heptacosane, nonacosane, and the terpenoid squalene present on the cuticle of diploid males larvae (Santomauro et al., 2004), promptly triggering their cannibalization (Woyke, 1963). In two stingless bee species, Melipona quadrifasciata and Scaptotrigona depilis, the brood is reared in mass provisioned sealed cells and, consequently, diploid males cannot be recognized during their larval stage. Nevertheless, workers are able to detect the presence of adult diploid males in the colony, presumably by making use of chemical cues (Borges et al., 2012; Vollet-Neto et al., 2017), eliminating the matched mated queen and replacing her with one of her daughters (Ratnieks, 1990). According to the CSD system, when a matched mating occurs, 50% of the diploid eggs laid by the queen should result in homozygous males (Cook and Crozier, 1995; Heimpel and De Boer, 2008; Whidden and Owen, 2011). An interesting outcome of our study was that we only observed the expected 1:1 worker to diploid male ratio at the end of the experimental period, and that the first batch of brood still almost entirely consisted of workers, even though a priori one would expect workers and diploid males to be produced in a 1:1 ratio throughout the entire colony cycle (Whiting, 1943). This suggests that mother queens could be able to selectively remove diploid male brood in an early developmental stage during the early phases of the colony cycle. Mechanistically, this could be achieved by the detection of specific chemical cues emitted by either diploid male eggs or first instar larvae. Indeed, it has been shown that in B. terrestris, eggs have caste and colony-specific chemical signals which are largely composed of alkanes, alkenes and alkadienes (Ayasse et al., 1999). The precise mechanism preventing diploid male brood from developing into adults at the beginning of the colony cycle remains nevertheless an interesting topic for future studies.

Some morphological traits can be used as diagnostics to differentiate diploid from haploid males of B. terrestris such as body size (Duchateau and Mariën, 1995; Gerloff et al., 2003) and the wing venation pattern (Gerard et al., 2015; Bortolotti et al., 2021; Bogo et al., 2022). Here, we show that their cuticular hydrocarbon profiles can also be used to reliably distinguish adult diploid and haploid B. terrestris males with an accuracy of 98%. The CHC reveals that diploid (early switching colonies) and haploid (late switching colonies) males present no qualitative differences on their CHC profile, but shows significant quantitative differences, with some compounds being more expressed in diploid males while others being overrepresented in haploid individuals. Therefore, these specific differences in the chemical profile of diploid males could potentially be used by workers as cues to start a premature competition over the male parentage in the colony with the queen. At the end of our experiment, nearly 50% of the workers had developed ovaries in both colony types. Previous studies have shown that, despite the high levels of ovary activation in workers, only around 4.5% of the total males produced are worker sons (Alaux et al., 2004b; Lopez-Vaamonde et al., 2004a), which implies that the queen wins the competition over the male parentage (Bourke and Ratnieks, 2001). This could be an indication that workers have a limited potential to mitigate the fitness loss due to diploid male production. Nevertheless, worker matricide toward the end of the season is not uncommon in B. terrestris and allows workers to reproduce without interference by the mother queen (Van Honk et al., 1981; Van Doorn and Heringa, 1986; Bourke, 1994; Wenseleers et al., 2021), which could constitute an advantage for early switching colonies to produce workers’ haploid males instead of the queen’s diploid ones. Nevertheless, the difference in the frequency of matricide between early and late switching colonies is currently unknown (Goulson, 2010).

In conclusion, in this study we show that in our study population, early switching colonies of B. terrestris, might be resulting from queens mated with males that by chance carry the same sex allele. In our study the frequency of early switching colonies was relatively low, being observed in only 6% of the colonies, which is in accordance with a previous study (Lopez-Vaamonde et al., 2003). However, there is an overwhelming bias toward laboratory studies with respect to the proportions of the two colony types, which might not fully reflect the situation in natural conditions. While the frequency of early and late switching colonies was rarely assessed in the field, some studies show that the proportion of early switching colonies was much higher than what is observed in laboratory conditions (50% in Duchateau and Velthuis, 1988; 14% in Yeninar and Kaftanoglu, 1996). Unfortunately, the ploidy of the first male cohort emerging in early switching colonies was not determined in any of these occasions. In addition, the diploid brood was assumed to be females in experiments determining the ploidy of eggs (Lopez-Vaamonde et al., 2003, 2004b). Our results alone cannot preclude the existence of different populations with early switching colonies producing haploid males early in the season. Therefore, testing the generality of our findings in other B. terrestris populations is an interesting topic for future investigations.

The presence of diploid males is linked with the number of sex alleles in the population, which was estimated to be 24 for B. terrestris (Duchateau et al., 1994). However, this number could be an overestimate of the alleles actually available in wild that can vary from population to population. Furthermore, habitat fragmentation is another factor that increases population susceptibility to the loss of genetic diversity (Frankham et al., 2002; Keller and Waller, 2002). Many bumblebees population have suffered a sharp decline in recent decades, in which it is already possible to observe diploid males in wild populations at detectable frequencies (B. muscorum, Darvill et al., 2006; B. sylvarum, Ellis et al., 2006; B. florilegus, Takahashi et al., 2008; B. occidentalis, B. perplexus, Whidden and Owen, 2011). Therefore, the detection of diploid males in wild is an essential parameter in hymenopteran conservation genetics since high frequencies of homozygotes may signal genetic depletion resulting from matched mating, small habitat size, habitat fragmentation, loss of genetic diversity, or a combination of these factors (Packer and Owen, 2001).

Finally, our results suggest the existence of a mechanism by which a matched mated queen can prevent the emergence of diploid males during the first batch of brood but is no longer able to do so later on during the colony cycle. In addition, our data suggest that the early onset of the competition point (Bloch, 1999), in which workers engage in direct competition over the male parentage with the queen, might be a consequence of the premature appearance of males in the colony. Together, our findings demonstrate that, similarly to the other two groups of highly eusocial corbiculate bees (i.e., honeybees and stingless bees), the buff-tailed bumblebee B. terrestris might display a behavioral adaptation to prevent population decline due to diploid male production.

The datasets and R scripts generated during the current study are available in the Mendeley Data repository, doi: https://doi.org/10.17632/FDM8924TKP.3 (Di Pietro et al., 2021).

RCO and TW conceived the original idea. VDP, AVO, FW, TW, and RCO designed the study. AVO and FW selected and provided the colonies. VDP performed the experiments and performed the dissections. VDP and HMF performed the chemical analysis. VDP, RCO, HMF, and TW performed data analysis and interpretation. VDP, HMF, and RCO prepared the first draft. All authors contributed to revision, proofreading, and the approved version before submission.

AVO and FW were employed by company Biobest Group NV. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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