Serial founder effects slow range expansion in an invasive social insect.

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.

Post-invasion selection acts on standing genetic variation despite a severe founding bottleneck.

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.

Rapid evolution, rearrangements and whole mitogenome duplication in the Australian stingless bees Tetragonula (Hymenoptera: Apidae): A steppingstone towards understanding mitochondrial function and evolution.

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 duplication 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.

Abundant small RNAs in the reproductive tissues and eggs of the honey bee, Apis mellifera.

BACKGROUND: 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. RESULTS: 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 the semen of other animals. Intriguingly we also find abundant piRNAs that target the sex determination locus, suggesting that piRNAs may play a role in honey bee sex determination. CONCLUSIONS: We conclude that small RNAs may 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.

Reproductive plasticity and oogenesis in the queen honey bee (Apis mellifera).

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.

DNA methylation is not a driver of gene expression reprogramming in young honey bee workers.

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.

Adaptive, caste-specific changes to recombination rates in a thelytokous honeybee population.

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.

The role of epigenetics, particularly DNA methylation, in the evolution of caste in insect societies.

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?'

Parent-of-origin effects, allele-specific expression, genomic imprinting and paternal manipulation in social insects.

Haplo-diploidy and the relatedness asymmetries it generates mean that social insects are prime candidates for the evolution of genomic imprinting. In single-mating social insect species, some genes may be selected to evolve genomic mechanisms that enhance reproduction by workers when they are inherited from a female. This situation reverses in multiple mating species, where genes inherited from fathers can be under selection to enhance the reproductive success of daughters. Reciprocal crosses between subspecies of honeybees have shown strong parent-of-origin effects on worker reproductive phenotypes, and this could be evidence of such genomic imprinting affecting genes related to worker reproduction. It is also possible that social insect fathers directly affect gene expression in their daughters, for example, by placing small interfering RNA molecules in semen. Gene expression studies have repeatedly found evidence of parent-specific gene expression in social insects, but it is unclear at this time whether this arises from genomic imprinting, paternal manipulation, an artefact of cyto-nuclear interactions, or all of these. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

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?'

Global allele polymorphism indicates a high rate of allele genesis at a locus under balancing selection.

When selection favours rare alleles over common ones (balancing selection in the form of negative frequency-dependent selection), a locus may maintain a large number of alleles, each at similar frequency. To better understand how allelic richness is generated and maintained at such loci, we assessed 201 sequences of the complementary sex determiner (csd) of the Asian honeybee (Apis cerana), sampled from across its range. Honeybees are haplodiploid; hemizygotes at csd develop as males and heterozygotes as females, while homozygosity is lethal. Thus, csd is under strong negative frequency-dependent selection because rare alleles are less likely to end up in the lethal homozygous form. We find that in A. cerana, as in other Apis, just a few amino acid differences between csd alleles in the hypervariable region are sufficient to trigger female development. We then show that while allelic lineages are spread across geographical regions, allelic differentiation is high between populations, with most csd alleles (86.3%) detected in only one sample location. Furthermore, nucleotide diversity in the hypervariable region indicates an excess of recently arisen alleles, possibly associated with population expansion across Asia since the last glacial maximum. Only the newly invasive populations of the Austral-Pacific share most of their csd alleles. In all, the geographic patterns of csd diversity in A. cerana indicate that high mutation rates and balancing selection act together to produce high rates of allele genesis and turnover at the honeybee sex locus, which in turn leads to its exceptionally high local and global polymorphism.

Intergenerational transfer of DNA methylation marks in the honey bee.

The evolutionary significance of epigenetic inheritance is controversial. While epigenetic marks such as DNA methylation can affect gene function and change in response to environmental conditions, their role as carriers of heritable information is often considered anecdotal. Indeed, near-complete DNA methylation reprogramming, as occurs during mammalian embryogenesis, is a major hindrance for the transmission of nongenetic information between generations. Yet it remains unclear how general DNA methylation reprogramming is across the tree of life. Here we investigate the existence of epigenetic inheritance in the honey bee. We studied whether fathers can transfer epigenetic information to their daughters through DNA methylation. We performed instrumental inseminations of queens, each with four different males, retaining half of each male's semen for whole genome bisulfite sequencing. We then compared the methylation profile of each father's somatic tissue and semen with the methylation profile of his daughters. We found that DNA methylation patterns were highly conserved between tissues and generations. There was a much greater similarity of methylomes within patrilines (i.e., father-daughter subfamilies) than between patrilines in each colony. Indeed, the samples' methylomes consistently clustered by patriline within colony. Samples from the same patriline had twice as many shared methylated sites and four times fewer differentially methylated regions compared to samples from different patrilines. Our findings indicate that there is no DNA methylation reprogramming in bees and, consequently, that DNA methylation marks are stably transferred between generations. This points to a greater evolutionary potential of the epigenome in invertebrates than there is in mammals.

Queen pheromone modulates the expression of epigenetic modifier genes in the brain of honeybee workers.

Pheromones are used by many insects to mediate social interactions. In the highly eusocial honeybee (Apis mellifera), queen mandibular pheromone (QMP) is involved in the regulation of the reproductive and other behaviour of workers. The molecular mechanisms by which QMP acts are largely unknown. Here, we investigate how genes responsible for epigenetic modifications to DNA, RNA and histones respond to the presence of QMP in the environment. We show that several of these genes are upregulated in the honeybee brain when workers are exposed to artificial QMP. We propose that pheromonal communication systems, such as those used by social insects, evolved to respond to environmental signals by making use of existing epigenomic machineries.

Thermal adaptation in the honeybee (Apis mellifera) via changes to the structure of malate dehydrogenase.

In honeybees there are three alleles of cytosolic malate dehydrogenase gene: F, M and S. Allele frequencies are correlated with environmental temperature, suggesting that the alleles have temperature-dependent fitness benefits. We determined the enzyme activity of each allele across a range of temperatures in vitro The F and S alleles have higher activity and are less sensitive to high temperatures than the M allele, which loses activity after incubation at temperatures found in the thorax of foraging bees in hot climates. Next, we predicted the protein structure of each allele and used molecular dynamics simulations to investigate their molecular flexibility. The M allozyme is more flexible than the S and F allozymes at 50°C, suggesting a plausible explanation for its loss of activity at high temperatures, and has the greatest structural flexibility at 15°C, suggesting that it can retain some enzyme activity at cooler temperatures. MM bees recovered from 2 h of cold narcosis significantly better than all other genotypes. Combined, these results explain clinal variation in malate dehydrogenase allele frequencies in the honeybee at the molecular level.

A Single Gene Causes Thelytokous Parthenogenesis, the Defining Feature of the Cape Honeybee Apis mellifera capensis.

In honeybees, the ability of workers to produce daughters asexually, i.e., thelytokous parthenogenesis, is restricted to a single subspecies inhabiting the Cape region of South Africa, Apis mellifera capensis. Thelytoky has unleashed new selective pressures and the evolution of traits such as social parasitism, invasiveness, and social cancer. Thelytoky arises from an abnormal meiosis that results in the fusion of two maternal pronuclei, restoring diploidy in newly laid eggs. The genetic basis underlying thelytoky is disputed. To resolve this controversy, we generated a backcross between thelytokous A. m. capensis and non-thelytokous A. m. scutellata from the neighboring population and looked for evidence of genetic markers that co-segregated with thelytokous reproduction in 49 backcross females. We found that markers associated with the gene GB45239 on chromosome 11, including non-synonymous variants, showed consistent co-segregation with thelytoky, whereas no other region did so. Alleles associated with thelytoky were present in all A. m. capensis genomes examined but were absent from all other honeybees worldwide including A. m. scutellata. GB45239 is derived in A. m. capensis and has a putative role in chromosome segregation. It is expressed in ovaries and is downregulated in thelytokous bees, likely because of polymorphisms in the promoter region. Our study reveals how mutations affecting the sequence and/or expression of a single gene can change the reproductive mode of a population.

Paternally-biased gene expression follows kin-selected predictions in female honey bee embryos.

The Kinship Theory of Genomic Imprinting (KTGI) posits that, in species where females mate with multiple males, there is selection for a male to enhance the reproductive success of his offspring at the expense of other males and his mating partner. Reciprocal crosses between honey bee subspecies show parent-of-origin effects for reproductive traits, suggesting that males modify the expression of genes related to female function in their female offspring. This effect is likely to be greater in the Cape honey bee (Apis mellifera capensis), because a male's daughters have the unique ability to produce female offspring that can develop into reproductive workers or the next queen without mating. We generated reciprocal crosses between Capensis and another subspecies and used RNA-seq to identify transcripts that are over- or underexpressed in the embryos, depending on the parental origin of the gene. As predicted, 21 genes showed expression bias towards the Capensis father's allele in colonies with a Capensis father, with no such bias in the reciprocal cross. A further six genes showed a consistent bias towards expression of the father's allele across all eight colonies examined, regardless of the direction of the cross. Consistent with predictions of the KTGI, six of the 21 genes are associated with female reproduction. No gene consistently showed overexpression of the maternal allele.

Genetic Characterization of Exotic Commercial Honey Bee (Hymenoptera: Apidae) Populations in Thailand Reveals High Genetic Diversity and Low Population Substructure.

Domestication of animal species is often associated with a reduction in genetic diversity. The honey bee, Apis mellifera Linnaeus, 1758, has been managed by beekeepers for millennia for both honey and wax production and for crop pollination. Here we use both microsatellite markers and sequence data from the mitochondrial COI gene to evaluate genetic variation of managed A. mellifera in Thailand, where the species is introduced. Microsatellite analysis revealed high average genetic diversity with expected heterozygosities ranging from 0.620 ± 0.184 to 0.734 ± 0.071 per locus per province. Observed heterozygosities were generally lower than those expected under Hardy-Weinberg equilibrium, both locally and across the population as a whole. Mitochondrial sequencing revealed that the frequency of two evolutionary linages (C-Eastern European and O-Middle Eastern) are similar to those observed in a previous survey 10 yr ago. Our results suggest that Thai beekeepers are managing their A. mellifera in ways that retain overall genetic diversity, but reduce genetic diversity between apiaries.

Conserved numts mask a highly divergent mitochondrial-COI gene in a species complex of Australian stingless bees Tetragonula (Hymenoptera: Apidae).

Tetragonula carbonaria, Tetragonula davenporti, Tetragonula hockingsi and Tetragonula mellipes comprise a species complex of Australian stingless bee species known as the 'Carbonaria' group. The species are difficult to distinguish morphologically and the major species-defining characters relate to comb architecture and nest entrance ornamentation. The taxonomy of the group is further complicated by likely nuclear mitochondrial pseudogenes (numts) and inter-specific hybrids. Here we demonstrate the existence of COI numts and isolate and characterize the 'true' mt-COI gene in T. carbonaria and T. hockingsi. Numts were isolated from enriched-nuclear DNA extraction followed by PCR amplification and Sanger sequencing, and were recognized by the presence of deletions and/or premature stop codons in the translated sequences. The mt-COI sequences were obtained from NGS sequencing using purified mtDNA. In T. carbonaria, two numts (numt1 and numt2) were identified and a third (numt3) was identified in T. hockingsi. Numt2 and numt3 are similar (1.2% sequence divergence), indicating a recent common origin. The genetic distance between the mt-COI of the two Tetragonula species was higher than might be expected for closely related species, 16.5%, corroborating previous studies in which T. carbonaria and T. hockingsi were regarded as separate species. The three numts are more similar to the COI of other stingless bee species, including Australian Austroplebia australis and South American Melipona bicolor (81.7-83.9%) than to the mt-COI of their own species (70-71.4%). This is because the mt-COI of T. carbonaria and T. hockingsi differ greatly from other Meliponinae. Our findings explain some formerly puzzling aspects of Carbonaria biogeography, and misinterpreted amplifications.

Unique DNA Methylation Profiles Are Associated with cis-Variation in Honey Bees.

DNA methylation is an important epigenetic modification that mediates diverse processes such as cellular differentiation, phenotypic plasticity, and genomic imprinting. Mounting evidence suggests that local DNA sequence variation can be associated with particular DNA methylation states, indicating that the interplay between genetic and epigenetic factors may contribute synergistically to the phenotypic complexity of organisms. Social insects such as ants, bees, and wasps have extensive phenotypic plasticity manifested in their different castes, and this plasticity has been associated with variation in DNA methylation. Yet, the influence of genetic variation on DNA methylation state remains mostly unknown. Here we examine the importance of sequence-specific methylation at the genome-wide level, using whole-genome bisulfite sequencing of the semen of individual honey bee males. We find that individual males harbor unique DNA methylation patterns in their semen, and that genes that are more variable at the epigenetic level are also more likely to be variable at the genetic level. DNA sequence variation can affect DNA methylation by modifying CG sites directly, but can also be associated with local variation in cis that is not CG-site specific. We show that covariation in sequence polymorphism and DNA methylation state contributes to the individual-specificity of epigenetic marks in social insects, which likely promotes their retention across generations, and their capacity to influence evolutionary adaptation.