The presence of a guard vicariously drives split sex ratios in a facultatively social bee

Split sex ratios provide broad insights into how reproductive strategies evolve and historically have special relevance to the evolution of eusociality. Yet almost no attention has been directed to situations where split sex ratios may potentially decrease the payoffs for worker-like behaviour, increasing selective thresholds for eusociality. We examined sex ratios in a facultatively social colletid bee, Amphylaeus morosus. Sex ratios in this bee vary strongly with the presence of a nest guard and in a pattern that does not conform to assumptions of previous models in which split sex ratios facilitate altruism. While the production of daughters was constant across social and solitary nests, mothers produced more brood when a non-reproductive guard was present, but these extra brood were all male. This leads to split sex ratios, vicariously driven by guards that are unable to manipulate sex ratios in their favour. Importantly, if guarding becomes more common in a population this would lead to an excess of males and lower the genetic value of these extra males to guards, effectively putting a brake on selection for worker-like behaviour.Funding provided by: Holsworth Wildlife Research EndowmentCrossref Funder Registry ID: http://dx.doi.org/10.13039/100008190Award Number:Nests of Amphyaleus morosus (n = 298) were collected from the Dandenong Ranges, Victoria, Australia throughout the reproductive season. Nests were sampled across five consecutive years (2017-2021; equating to four reproductive seasons) and seven separate collections. To assess patterns of investment sex ratios in colonies of A. morosus, brood sex, wet weight, and brood cell position were recorded. Pupae were weighed on a Thermoline precision balance to ± 0.1 mg. The numerical sex ratio (NSR) was calculated as the number of male brood divided by the total number of brood that reached pupation to a point that they could be reliably sexed (NSR = Σmale brood/ Σmale brood + female brood). Investment sex ratio (ISR) was calculated as a product of the numerical sex ratio and the pupal weight ratio was calculated from mean brood sex pupal weight and was used to test whether the observed numerical sex ratio deviated from the investment a mother allocates to each sex. Brood that had died before nests were opened were not included in pupal weight measurements but were used for numerical sex ratio calculations. To examine sex allocation patterns across cell positions in the nest, cell position was coded so that 'cell 1' corresponded to the first cell provisioned (furthest from the nest entrance), following Hearn et al. (2022). For some analyses, brood that reached adulthood were pooled across all nests and the pupal sex was treated as a binomial response variable (female = 0, male = 1). For all analyses, we define solitary nests as nests containing one or no adult female at the time of nests collection and social nests as colonies containing two adult females or genome-inferred social nests (see Hearn et al. (2022)), but recognise that social nesting is likely to be more common than our nest census data suggests (SI Appendix). All statistical analyses were performed in SPSS version 27.0 (IBM, Armonk, NY) and R version 4.0.4 (R Core Team 2018). Tests of normality and homoscedasticity were assessed using a Shapiro–Wilk test and Levene's test. A Chi-square goodness of fit test was used to determine if observed numerical sex ratio significantly differed from an expected null hypothesis of equal investment. Where necessary we used arcsine transformed values of sex ratio and the corresponding confidence intervals