Stingless bees in the genus Tetragonula are social insects with a fully sterile worker caste, and are therefore well-placed to provide insights into the genomic changes associated with 'superorganismal' life histories. Here we assemble the genome of Tetragonula carbonaria and characterise the population structure and divergence of both T. carbonaria and its cryptic congener T. hockingsi in eastern Australia, revealing three distinct populations for T. carbonaria and two partially differentiated subpopulations for T. hockingsi. We then combine our genomic results with RNA-seq data from different T. carbonaria castes (queens, males, workers) to test two hypotheses about genomic adaptations in social insects: the 'Relaxed Constraint' hypothesis, which predicts indirect, and therefore relaxed, selection on worker-biased genes; and the 'Adapted Worker' hypothesis, which predicts intensified positive selection on worker genes due to their evolutionarily novel functions. Although we do not find a direct signal of either weaker purifying selection or elevated positive selection in worker-biased genes based on deviations from neutral expectations of nucleotide change between the two species, other evidence does support a model of relaxed selection on worker-biased genes: such genes show higher nucleotide diversity and greater interspecies divergence than queen-biased genes. We also find that differentially caste-biased genes exhibit distinct patterns of length, GC content and evolutionary origin. These findings, which converge with patterns found in other social insects, support the hypothesis that social evolution produces distinct signatures in the genome. Overall, Tetragonula bees emerge as a valuable model for studying the genomic basis of social complexity in insects.

Honey bees ( Apis spp.) are important pollinators in many natural and agro-ecosystems across the world. Effective means of surveying wild populations are therefore key to their conservation and management. One available survey method infers honey bee colony density from the genotype of drones (males) sampled from sites known as Drone Congregation Areas (DCAs). While this approach has been commonly used for the Western honey bee ( A. mellifera ), its feasibility for other Apis species is unknown. Here, we investigate drone congregation behaviour in the Asian honey bee Apis cerana in north-east Australia and its suitability for inferring colony density. As this A. cerana population is invasive, surveys in this case can aid in monitoring the population’s growth and spread. Over 5 years, we identified 30 DCAs, many of which were stable across time. DCAs were sheltered areas beside tree-lines or openings in the forest canopy. A. cerana drones joined DCAs during 1–2-h afternoon intervals and could be sampled at heights of 2–24 m via adhesive lines attached to helium balloons carrying lures coated in queen pheromone. Drones were more likely to be present at a DCA as temperature increased, though abiotic factors did not predict overall drone abundance. Drones could be sampled in low numbers even where colony density was extremely low. Based on the genotyping and inferred sibship of drones sampled at DCAs between 2016 and 2021, we estimate population density in Australia’s A. cerana to be in the range 1.1–8.1 colonies/km 2 . This extrapolates to a total population size in the range 11,000–83,000 colonies, with more refined estimates requiring better knowledge of drone flight distance and the effect of habitat on colony density. We conclude that population surveys based on drones from DCAs are possible for A. cerana and propose that this approach be part of a toolkit of methods used to monitor Asian honey bee populations in both their native and invasive ranges.

Invasive populations often experience founder effects: a loss of genetic diversity relative to the source population, due to a small number of founders. Even where these founder effects do not impact colonization success, theory predicts they might affect the rate at which invasive populations expand. This is because secondary founder effects are generated at advancing population edges, further reducing local genetic diversity and elevating genetic load. We show that in an expanding invasive population of the Asian honey bee (Apis cerana), genetic diversity is indeed lowest at range edges, including at the complementary sex determiner, csd, a locus that is homozygous-lethal. Consistent with lower local csd diversity, range edge colonies had lower brood viability than colonies in the range centre. Further, simulations of a newly-founded and expanding honey bee population corroborate the spatial patterns in mean colony fitness observed in our empirical data and show that such genetic load at range edges will slow the rate of population expansion. Invasive populations often have low genetic diversity because they originated from a small number of founding individuals. This study shows that in an invasive honey bee, one consequence of low genetic diversity is a reduced rate of population expansion due to serial founder effects at range edges.

Invasive populations often have lower genetic diversity relative to the native-range populations from which they derive.1,2 Despite this, many biological invaders succeed in their new environments, in part due to rapid adaptation.3,4,5,6 Therefore, the role of genetic bottlenecks in constraining the adaptation of invaders is debated.7,8,9,10 Here, we use whole-genome resequencing of samples from a 10-year time-series dataset, representing the natural invasion of the Asian honey bee (Apis cerana) in Australia, to investigate natural selection occurring in the aftermath of a founding event. We find that Australia's A. cerana population was founded by as few as one colony, whose arrival was followed by a period of rapid population expansion associated with an increase of rare variants.11 The bottleneck resulted in a steep loss of overall genetic diversity, yet we nevertheless detected loci with signatures of positive selection during the first years post-invasion. When we investigated the origin of alleles under selection, we found that selection acted primarily on the variation introduced by founders and not on the variants that arose post-invasion by mutation. In all, our data highlight that selection on standing genetic variation can occur in the early years post-invasion, even where founding bottlenecks are severe.

The extreme conservation of mitochondrial genomes in metazoans poses a significant challenge to understanding mitogenome evolution. However, the presence of variation in gene order or genome structure, found in a small number of taxa, can provide unique insights into this evolution. Previous work on two stingless bees in the genus Tetragonula (T. carbonaria and T. hockingsi) revealed highly divergent CO1 regions between them and when compared to the bees from the same tribe (Meliponini), indicating rapid evolution. Using mtDNA isolation and Illumina sequencing, we elucidated the mitogenomes of both species. In both species, there has been a dupli-cation of the whole mitogenome to give a total genome size of 30,666 bp in T. carbonaria; and 30,662 bp in T. hockingsi. These duplicated genomes present a circular structure with two identical and mirrored copies of all 13 protein coding genes and 22 tRNAs, with the exception of a few tRNAs that are present as single copies. In addition, the mitogenomes are characterized by rearrangements of two block of genes. We believe that rapid evolution is present in the whole Indo-Malay/Australasian group of Meliponini but is extraordinarily elevated in T. carbonaria and T. hockingsi, probably due to founder effect, low effective population size and the mitogenome duplication. All these features -rapid evolution, rearrangements, and duplication -deviate significantly from the vast majority of the mitogenomes described so far, making the mitogenomes of Tetragonula unique opportunities to address fundamental questions of mitogenome function and evolution.

In the western honey bee (Apis mellifera) populations, cytosolic malate dehydrogenase (cMDH) allele frequencies are correlated with the environmental temperatures.The Slow and Fast alleles predominate in hot climates; the Medium allele predominates in cold climates.We wondered whether natural selection has had anything to do with the Medium allele in the imported exotic A. mellifera population in Thailand.We genotyped workers (n = 1032) from 86 commercial colonies from three regions.Three alleles: Fast, Medium, and Slow, were detected.Over 96% of the Thai A. mellifera are either homozygous for the Slow or heterozygous for the Fast allele; and the Medium allele has the lowest frequency (c.a.10%) in all sampled population.This might be indicative of selection against the Medium allele.However, as the cMDH allele frequencies in the Thai A. mellifera are similar to those seen in the source populations in the United States and Northern Italy, it might also be that the observed frequencies are reflective of frequent imports from the United States, and that natural selection is unable to overcome the effects of migration.

Pollinator conservation is aided by knowledge of dispersal behavior, which shapes gene flow and population structure. In many bees, dispersal is thought to be male-biased, and males’ movements may be critical to maintaining gene flow in disturbed and fragmented habitats. Yet male bee movements are challenging to track directly and male dispersal ability remains poorly understood in most species. Here, we combine field manipulations and models to assess male dispersal ability in a stingless bee (Tetragonula carbonaria). We placed colonies with virgin queens at varying distances apart (1–48 km), genotyped the males that gathered at mating aggregations outside each colony, and used pairwise sibship assignment to determine the distribution of likely brothers across aggregations. We then compared simulations of male dispersal to our observed distributions and found best-fit models when males dispersed an average of 2–3 km (>2-fold female flight ranges), and maximum of 20 km (30-fold female flight ranges). Our data supports the view that male bee dispersal can facilitate gene flow over long-distances, and thus play a key role in bee populations’ resilience to habitat loss and fragmentation. In addition, we show that the number of families contributing to male aggregations can be used to estimate local stingless bee colony densities, allowing population monitoring of these important tropical pollinators.

SUMMARYInsect sociality is a major evolutionary transition based on the suppression of worker reproduction in favor of the reproductive monopoly of the queen. In the honey bee (Apis mellifera) model organism, the development of the two female caste phenotypes, queen and worker, is triggered by differences in their larval diets. However, the mechanistic details underlying their respective developmental trajectories, as well as the maintenance of sterility in the adult workers, are still not fully understood. Here we show that the long non-coding RNA lncov1 interacts with the Tudor staphylococcus nuclease (Tudor-SN) protein to form a regulatory module that promotes apoptosis in the ovaries of worker larvae. In adult workers, the lncov1/Tudor-SN module responds positively to environmental cues that suppress reproductive capacity. As lncov1 is considerably conserved in the Apidae, we propose that, by promoting worker sterility, the lncov1/Tudor-SN module has likely played critical roles in the social evolution of bees.

Australia has an abundance of feral honey bee colonies. Understanding their densities is important to assess their current economic and ecological impact and the need for mitigation should the mite Varroa destructor become established. Inferring colony density based on the genotypes of honey bee drones (males) caught in a Williams trap has been identified as a promising approach. This method assumes that (a) drones are attracted to the trap from an area bounded by the drone flight range, (b) sufficient colonies present within that radius are represented in a sample and (c) colonies that do not produce drones are small and of little ecological consequence. Here, we investigate whether known feral colonies were represented in drone samples and whether drone contribution per colony correlated with the relative colony size or the distance between the colony and the trap. We found that one-third of known colonies were not represented in the drone sample, and this proportion did not correlate with colony size or distance. For colonies that contributed at least one drone, there was a correlation between the number of drones caught per colony and the distance of the colony from the DCA, and at distances beyond 0.9 km, there was substantial non-detection. Further work is needed to determine an appropriate correction factor that converts the estimated number of colonies represented in a drone sample to colony density.

Invasive populations often experience founder effects: a loss of genetic diversity relative to the source population, due to a small number of founders. Even where these founder effects do not impact colonization success, theory predicts they might affect the rate at which invasive populations expand. This is because additional, serial founder effects occurring at advancing population edges can further reduce local genetic diversity, leading to high genetic load at range edges. Such a range edge effect should be particularly strong in populations of hymenopteran insects due to their sex determination system, whereby homozygosity at a single critical sex locus produces ’diploid males’ with low to zero fitness. We tested this prediction in an invasive population of the Asian honey bee (Apis cerana). The Apis sex locus (csd) is well-characterized and homozygosity at csd is lethal. We show that genetic diversity is lowest at range edges, both at neutral loci and particularly at the fitness-critical sex locus. Consistent with lower local csd diversity, range edge colonies also had higher proportions of inviable brood than colonies in the range centre. We then use simulations of a newly-founded and expanding honey bee population to corroborate the spatial patterns in mean colony fitness we find in our empirical data, and to show that genetic load at the sex locus in such cases will slow the rate of population expansion. In all, our study highlights how the fitness effects of reduced genetic diversity, caused by colonizing bottlenecks, can be amplified at expanding range edges by serial founder effects and curb a population’s rate of spread.

In the honey bee (Apis mellifera), queen and worker castes originate from identical genetic templates but develop into different phenotypes. Queens lay up to 2000 eggs daily whereas workers are sterile in the queen's presence. Periodically queens stop laying: during swarming, when resources are scarce in winter, and when they are confined to a cage by beekeepers. We used confocal microscopy and gene expression assays to investigate the control of oogenesis in the ovaries of honey bee queens that were caged inside and outside the colony. We find evidence that queens use a different combination of 'checkpoints' to regulate oogenesis compared to honey bee workers and other insect species. However, both queen and worker castes likely use the same programmed cell death pathways to terminate oocyte development at their caste-specific checkpoints. Our results also suggest that a key factor driving the termination of oogenesis in queens is nutritional stress. Thus, queens may regulate oogenesis via the same regulatory pathways that were utilised by ancestral solitary species but likely have adjusted physiological checkpoints to suit their highly-derived life history.

Eusocial insects can be defined as those that live in colonies and have distinct queens and workers. For most species, queens and workers arise from a common genome, and so caste-specific developmental trajectories must arise from epigenetic processes. In this review, we examine the epigenetic mechanisms that may be involved in the regulation of caste dimorphism. Early work on honeybees suggested that DNA methylation plays a causal role in the divergent development of queen and worker castes. This view has now been challenged by studies that did not find consistent associations between methylation and caste in honeybees and other species. Evidence for the involvement of methylation in modulating behaviour of adult workers is also inconsistent. Thus, the functional significance of DNA methylation in social insects remains equivocal. This article is part of the theme issue ‘How does epigenetics influence the course of evolution?’

Epigenetics is the study of changes in gene activity that can be transmitted through cell divisions but cannot be explained by changes in the DNA sequence. Epigenetic mechanisms are central to gene regulation, phenotypic plasticity, development and the preservation of genome integrity. Epigenetic mechanisms are often held to make a minor contribution to evolutionary change because epigenetic states are typically erased and reset at every generation, and are therefore, not heritable. Nonetheless, there is growing appreciation that epigenetic variation makes direct and indirect contributions to evolutionary processes. First, some epigenetic states are transmitted intergenerationally and affect the phenotype of offspring. Moreover, bona fide heritable ‘epialleles' exist and are quite common in plants. Such epialleles could, therefore, be subject to natural selection in the same way as conventional DNA sequence-based alleles. Second, epigenetic variation enhances phenotypic plasticity and phenotypic variance and thus can modulate the effect of natural selection on sequence-based genetic variation. Third, given that phenotypic plasticity is central to the adaptability of organisms, epigenetic mechanisms that generate plasticity and acclimation are important to consider in evolutionary theory. Fourth, some genes are under selection to be ‘imprinted' identifying the sex of the parent from which they were derived, leading to parent-of-origin-dependent gene expression and effects. These effects can generate hybrid disfunction and contribute to speciation. Finally, epigenetic processes, particularly DNA methylation, contribute directly to DNA sequence evolution, because they act as mutagens on the one hand and modulate genome stability on the other by keeping transposable elements in check. This article is part of the theme issue ‘How does epigenetics influence the course of evolution?'

Polyandrous social insects such as the honey bee are prime candidates for parental manipulation of gene expression in offspring. Although there is good evidence for parent-of-origin effects in honey bees the epigenetic mechanisms that underlie these effects remain a mystery. Small RNA molecules such as miRNAs, piRNAs and siRNAs play important roles in transgenerational epigenetic inheritance and in the regulation of gene expression during development. Here we present the first characterisation of small RNAs present in honey bee reproductive tissues: ovaries, spermatheca, semen, fertilised and unfertilised eggs, and testes. We show that semen contains fewer piRNAs relative to eggs and ovaries, and that piRNAs and miRNAs which map antisense to genes involved in DNA regulation and developmental processes are differentially expressed between tissues. tRNA fragments are highly abundant in semen and have a similar profile to those seen in semen in other animals. Intriguingly we find abundant piRNAs that target the sex determination locus, suggesting that piRNAs may play a role in honey bee sex determination. We conclude that small RNAs play a fundamental role in honey bee gametogenesis and reproduction and provide a plausible mechanism for parent-of origin-effects on gene expression and reproductive physiology.### Competing Interest StatementThe authors have declared no competing interest.

Male honey bees trapped at Drone Congregation Areas (DCAs) can be used to infer the number of colonies from which drones were derived, and thereby colony density in the environment. Crucial to the accuracy of this method is precise grouping of males into brother groups based on genetic markers, and a sample size that is sufficient so that all colonies in the area are included in the sample. The optimal sample size is a trade-off between cost and accuracy and cannot be known prior to sampling. Therefore, follow-up surveys may be necessary if the data indicate that the first sample was too small. However, the effect of multiple sampling on allele frequency estimates and the accuracy of the method is poorly understood. Here we trapped drones from two independent DCAs every month over 2 and 2.5 years. We analysed our data using the sibship grouping programme COLONY in three ways: (i) using data from the entire year, and counting the number of colonies identified in each month; (ii) using monthly data with allele frequencies from the entire season; and (iii) using data from each month separately. Although there were significant changes in allele frequencies over the year, these changes had no material effect on classifications of drones into families. Therefore, multiple samples can provide more robust estimates of family groupings due to the larger sample size and can be used with confidence where required.

Soley (1) argues that our study (2) does not prove that “transgenerational [epigenetic] inheritance serv[es] an adaptive role in evolution” in honey bees. This view misrepresents our intent. Our main findings are that in honey bees 1) DNA methylation profiles are much more similar between fathers and daughters than between unrelated males and females of different generations and 2) DNA methylation profiles are conserved across somatic and germline tissues (2). We are agnostic as to whether DNA methylation … [↵][1]1To whom correspondence may be addressed. Email: boris.yagound{at}sydney.edu.au. [1]: #xref-corresp-1-1

In the honey bee (Apis mellifera), queen and worker castes originate from identical genetic templates but develop into different phenotypes. Queens lay up to 2,000 eggs daily whereas workers are sterile in the queen’s presence. Periodically queens stop laying; during swarming, when resources are scarce in winter and when they are confined to a cage by beekeepers. We used confocal microscopy and gene expression assays to investigate the control of oogenesis in honey bee queen ovaries. We show that queens use different combination of ‘checkpoints’ to regulate oogenesis compared to honey bee workers and other insect species. However, both queen and worker castes use the same programmed cell death pathways to terminate oocyte development at their caste-specific checkpoints. Our results also suggest that the termination of oogenesis in queens is driven by nutritional stress. Thus, queens may regulate oogenesis via the same regulatory pathways that were utilised by ancestral solitary species but have adjusted physiological checkpoints to suit their highly-derived life history. Summary statement Honey bee queens regulate oogenesis using a different combination of ‘checkpoints’ to workers, but both castes use the same molecular pathways.

The presence of DNA methylation marks within genic intervals, also called gene body methylation, is an evolutionarily-conserved epigenetic hallmark of animal and plant methylomes. In social insects, gene body methylation is thought to contribute to behavioural plasticity, for example between foragers and nurse workers, by modulating gene expression. However, recent studies have suggested that the majority of DNA methylation is sequence-specific, and therefore cannot act as a flexible mediator between environmental cues and gene expression. To address this paradox, we examined whole-genome methylation patterns in the brains and ovaries of young honey bee workers that had been subjected to divergent social contexts: the presence or absence of the queen. Although these social contexts are known to bring about extreme changes in behavioral and reproductive traits through differential gene expression, we found no significant differences between the methylomes of workers from queenright and queenless colonies. In contrast, thousands of regions were differentially methylated between colonies, and these differences were not associated with differential gene expression in the subset of genes examined. Methylation patterns were highly similar between brain and ovary tissues and only differed in nine regions. These results strongly indicate that DNA methylation is not a driver of differential gene expression between tissues or behavioral morphs. Finally, despite the lack of difference in methylation patterns, queen presence affected the expression of all four DNA methyltransferase genes, suggesting that these enzymes have roles beyond DNA methylation. Therefore, the functional role of DNA methylation in social insect genomes remains an open question.

The ability to clone oneself has clear benefits-no need for mate hunting or dilution of one's genome in offspring. It is therefore unsurprising that some populations of haplo-diploid social insects have evolved thelytokous parthenogenesis-the virgin birth of a female. But thelytokous parthenogenesis has a downside: the loss of heterozygosity (LoH) as a consequence of genetic recombination. LoH in haplo-diploid insects can be highly deleterious because female sex determination often relies on heterozygosity at sex-determining loci. The two female castes of the Cape honeybee, Apis mellifera capensis, differ in their mode of reproduction. While workers always reproduce thelytokously, queens always mate and reproduce sexually. For workers, it is important to reduce the frequency of recombination so as to not produce offspring that are homozygous. Here, we ask whether recombination rates differ between Cape workers and Cape queens that we experimentally manipulated to reproduce thelytokously. We tested our hypothesis that Cape workers have evolved mechanisms that restrain genetic recombination, whereas queens have no need for such mechanisms because they reproduce sexually. Using a combination of microsatellite genotyping and whole-genome sequencing we find that a reduction in recombination is confined to workers only.