Identification of Multiple Loci Associated with Social Parasitism in Honeybees
[['In most colonies of the honeybee Apis mellifera , the queen is the only reproductively active female and it produces pheromones that inhibit ovary activation in the workers [1 , 2] , keeping them sterile .', 'Under some circumstances , particularly when the colony lacks a queen , workers may activate their ovaries and lay unfertilized haploid eggs via a process called arrhenotokous parthenogenesis .', 'Due to the haplodiploid sex determination of honeybees ( and other hymenoptera ) these haploid eggs develop into male drones .', 'The Cape bee ( Apis mellifera capensis Escholtz ) is a subspecies of the honeybee Apis mellifera that inhabits the far south of South Africa [3–5] .', 'The Cape bee differs from other honeybee subspecies in that unmated females , particularly workers , frequently produce female progeny that develop from unfertilized eggs through thelytokous parthenogenesis .', 'Thelytoky is a form of meiosis whereby two daughter nuclei that form in the oocyte fuse to produce a viable diploid egg [6] .', 'This abnormal mechanism of restoring diploidy in unfertilized eggs occurs only occasionally in normally arrhenotokous populations ( <1% of eggs ) but is the norm in reproducing Cape bee workers ( >99% of eggs ) [7] .', 'The Cape bee exhibits a number of other traits that distinguish it from other subspecies and lead it to behave as a social parasite , whereby workers invade the nests of foreign colonies , reproduce and utilize their resources .', 'Cape bee workers have significantly larger ovaries that are more readily activated and contain more egg-producing ovarioles than worker bees of other subspecies ( 10–20 vs . 1–5 ) [3 , 8] .', 'Parasitic egg-laying Cape bee workers produce queen pheromones , allowing them to assert reproductive dominance over other workers [9] and have a significantly increased lifespan of 3–5 months compared to 6 weeks in non-parasitic workers [10] .', 'These traits likely increase the evolutionary advantage of parthenogenesis , which allows this mode of selfish reproduction to become established .', 'As with other honeybee subspecies , Cape bee workers rarely reproduce if a queen is present ( ~0 . 1% of drones are produced from worker-laid eggs in a normal honeybee colony [11] ) .', 'However , during reproductive swarming in Cape bees as many as 10% of young workers are produced by egg-laying workers [12] .', 'In between 40% to 60% of young queens produced during swarming are the offspring of workers [13 , 14] .', 'The genetic basis of thelytoky and its associated behavioral and physiological traits in the Cape bee is still largely unclear .', 'Asexual reproduction by thelytokous parthenogenesis is relatively common in other species of Hymenoptera [15] .', 'In the parasitoid wasp Lysiphlebus fabarum , where different forms exhibit arrhenotokous or thelytokous parthenogenesis , a single locus determines this difference [16] .', 'Thelytokously reproducing individuals of this species are all homozygous for an allele at a particular microsatellite locus , which is very rare in arrhenotokously reproducing individuals .', 'In the Cape bee , the genetic basis of thelytoky has been investigated using backcrosses of hybrids formed by crossing A . m .', 'capensis with A . m .', 'carnica [17] .', 'The pattern of segregation in such crosses is generally consistent with inheritance of thelytoky being determined by a single recessive locus , which has been termed thelytoky ( th ) , although more complex modes of inheritance incorporating more loci are also compatible with this inheritance pattern .', 'This putative locus has been mapped to an interval on chromosome 13 [18] and a 9 bp deletion in this region has been proposed as the causative variant [19] , however , a subsequent study failed to replicate this association [20] and detected the presence of the deletion in populations of other subspecies where thelytoky is absent ( see Materials and Methods for more details ) .', 'Cape bees are genetically extremely similar to other African subspecies of honeybees , including A . m .', 'adansonii and A . m .', 'scutellata [21] suggesting a recent common ancestor and/or high levels of gene flow .', 'Social parasitism in Cape bees should therefore represent a recently derived adaptation that has likely experienced positive selection .', 'The distribution of Cape bees shows strong concordance to the biodiverse Fynbos ecoregion of South Africa , which suggests that the traits specific to the Cape bee are evolutionarily advantageous in this region [5] .', 'Cape bee workers reproduce by thelytokous parthenogensis in the absence of a queen , during reproductive swarming or when invading the colony of a different subspecies .', 'The propensity to do this is a trait that is fixed or close to fixation in the Cape bee population [3 , 22] .', 'However , in their native range most reproduction in Cape bees is sexual , so patterns of genetic diversity therefore resemble those in other sexual outcrossing populations [21] .', 'We therefore expect genetic variants responsible for traits associated with social parasitism to be located in regions of high differentiation with populations of other subspecies and associated with signals of selection in Cape bees .', 'What is the genetic basis of social parasitism in Cape bees ?', 'Social parasitism is facilitated by a range of different traits related to ovary development , behaviour and abnormal meiosis .', 'Does a single thelytoky locus act as a “master switch” to enable this suite of phenotypes as previously suggested [17–19] , or are each of these traits determined by an independent set of loci ?', 'To what extent are Cape bees genetically distinct from other African honeybee populations ?', 'Was the emergence of social parasitism associated with increased positive selection in the Cape bee population ?', 'Here we use population-scale genome sequencing to address these questions .', 'We developed a method to detect selection in Cape bees compared to other African populations that combines detecting SNPs with high differentiation between populations with long haplotype tests of selection [23 , 24] .', 'Here we apply this approach to a dataset of 10 whole genome sequences of Cape bees , sampled from locations were workers are known to produce nearly exclusively female offspring [25] , compared to 20 genome sequences of other sub-Saharan African honeybees that do not reproduce thelytokously .', 'We detect a number of genomic regions with strong signals of selection in the Cape bee , which are likely to underlie the unique suite of traits that facilitate social parasitism in this subspecies .'], ['We aimed to identify genetic variants associated with strong differentiation between the Cape bee and other African bees due to selection in the Cape bee in favor of traits involved in social parasitism .', 'We analyzed whole genome sequences from 10 Cape bees ( A . m . capensis ) sampled from two localities in southern South Africa: Port Elizabeth to the east ( n = 5 ) and Cape Town to the west ( n = 5 ) .', 'We compared these with sequences from two other African subspecies: A . m .', 'scutellata collected in Pretoria ( n = 10 ) and A . m .', 'adansonii collected in Nigeria ( n = 10; Fig 1 ) .', 'Genetic differentiation between these three African populations ranges between FST = 0 . 051–0 . 056 ( Fig', '1 ) and A . m .', 'scutellata appears to be nearly equidistant genetically between the Cape bee in the south and A . m .', 'adansonii in the north .', 'This level of divergence is consistent with a recent common ancestor and/or pervasive gene flow among sub-Saharan populations .', 'We considered the geographically widespread A . m .', 'scutellata and A . m .', 'adansonii populations as a single African background population and attempted to identify alleles under selection in the Cape bees compared to this background population .', 'FST between the Cape bee population and the background population is 0 . 044 and the allele frequency spectrum is dominated by variants segregating at similar frequencies: 93 . 3% of SNPs ( n = 5 . 89 x 106 ) have FST values below 0 . 1 , whereas only 0 . 33% of SNPs ( n = 20 , 460 ) have FST values above 0 . 3 ( S1A Fig ) .', 'There are 45 SNPs fixed for different variants between the Cape bees ( n = 10 ) and other African bees ( n = 20 ) , which are associated with one gene accession on chromosome 1 corresponding to Ethr ( ecdysis-triggering hormone receptor ) and three predicted genes on chromosome 11 ( hypothetical proteins GB44917 , GB45238 and GB45239; S1 Table ) .', 'When the two Cape bee subpopulations are compared against other African populations separately , we observed 423 such fixed SNPs in the ( western ) Cape Town population ( n = 5 ) , which are associated with 38 gene accessions ( S1B Fig; S1 Table ) .', 'However , in the ( eastern ) Port Elizabeth population ( n = 5 ) , we detect only 60 fixed SNPs ( associated with five gene accessions ) .', 'FST between the African background population and the Cape Town population ( 0 . 070 ) is slightly higher than the African background population and the Port Elizabeth population ( 0 . 063 ) .', 'The Cape Town population also has a general excess of high FST variants .', 'Between the two comparisons against the African background population , the analysis with the Cape Town subpopulation include 70% or more of the variants segregating at FST>0 . 5 ( S1B and S1C Fig ) .', 'Colonies with the entire suite of Cape-bee-specific phenotypes are more common in the Cape Town region , whereas they occur less frequently in Port Elizabeth [3 , 22] and hence the higher number of fixed SNPs in the Cape Town region may reflect additional variants associated with these phenotypes .', 'Natural selection is expected to drive adaptive genetic variants towards fixation together with linked neutral variation , generating a pattern of increased population differentiation and higher haplotype homozygosity around selected variants [26] .', 'We sought to identify genetic variants with high genetic differentiation due to recent selection specifically in the Cape bee population , reasoning that such variants likely underlie traits associated with social parasitism .', 'To do this , we integrated SNP-level FST estimates with haplotype structure analyses .', 'We implemented the population branch statistic ( PBS ) [27] to detect genomic regions that diverged rapidly on the Cape bee lineage compared to other African populations .', 'We also scanned genome variation for genetic variants linked to long haplotypes in the Cape bee population using the cross-population extended haplotype homozygosity statistic ( XP-EHH ) [23] ( implemented in the program selscan [28] ) .', 'For both PBS and XP-EHH estimates , values greater than zero are associated with selection signals in the Cape bees , whereas negative values indicate selection in the African background population .', 'Across all SNPs in the dataset we find that the distribution of these values are centered around zero ( mean XP-EHH = 0 . 007; mean PBS = 0 . 039 ) , but that the most extreme XP-EHH and PBS scores are biased towards stronger signals in the Cape bees rather than the background population: for the PBS , the upper and lower 99 . 9% percentiles of the empirical distributions are 1 . 27 and -0 . 34 , respectively , and for the XP-EHH theses values are 4 . 12 and -3 . 46 ( S2A and S2B Fig ) .', 'Low FST SNPs ( FST<0 . 3; >99% of SNPs ) appear to be largely unbiased between the two groups with respect to the PBS and XP-EHH statistics ( scores are close to zero ) , whereas high FST SNPs tend to have high values of these statistics in Cape bees: the average PBS and EHH scores of SNPs with FST>0 . 85 are within the top 0 . 5% of these statistics ( p<0 . 01 , bootstrap; S2C–S2E Fig ) .', 'Moreover , variants segregating at the highest frequency differences are not randomly distributed but tend to be clustered in regions with the strongest linked signatures of selection: regional FST drops to the top 1% level after about 50kbp for SNPs at FST>0 . 9 , compared to about 20kbp for SNPs at FST = 0 . 8–0 . 9 , and these patterns decay similarly for PBS and XP-EHH ( S3A–S3C Fig ) .', 'These analyses suggest that more variants are associated with selection signals in the Cape bee population compared to the background population .', 'Many of these variants may underlie the unique set of derived characteristics associated with thelytokous parthenogenesis and social parasitism in the Cape bee .', 'In order to test the robustness of these observations , we performed two additional reciprocal comparisons: one comparing A . m .', 'scutellata to the remaining two African populations , and the other comparing A . m .', 'adansonii to the remaining populations .', 'For each of the comparisons , we estimated FST , PBS , and XP-EHH statistics in the same way as in the original genome scan ( Fig 2A ) .', 'The distribution of FST in the additional comparisons has fewer peaks than in the Cape Bee , indicating fewer signals of selection specifically in these populations ( SNPs with FST>0 . 8 occur on 13 chromosomes comparing Cape bees against the other African populations , but only on one and six chromosomes respectively in the corresponding A . m . scutellata and A . m . adansonii scans; S4A–S4C Fig ) .', 'In some cases we detected partially overlapping signals ( for instance around position 14 , 000 , 000 on chromosome', '1 ) such that some accessions had outlier SNPs associated with more than one subspecies .', 'However , among the gene candidates identified ( see section Characterization of candidate genes below ) , 95% and 99% of the accessions had higher overall PBS signals in the Cape bees than in A . m .', 'scutellata or A . m .', 'adansonii , respectively , consistent with an excess of uniquely derived variants and stronger selection in the Cape bees in most cases .', 'In the comparison with A . m .', 'scutellata , there are extremely few SNPs with high FST ( FST>0 . 80; 7 SNPs , compared to 730 SNPs in the Cape bee comparison ) .', 'These SNPs have mildly elevated PBS values , but do not appear to be associated with signals of selection in A . m .', 'scutellata according to the XP-EHH test ( Fig 2B–2D ) .', 'In the comparison with A . m .', 'adansonii , a large number of SNPs have high FST , but they are restricted to a few genomic regions .', 'It is likely that many of these SNPs lie within regions of structural variation such as inversions that differ in frequency in the A . m .', 'adansonii population , as high-FST SNPs are restricted to a few large blocks in this comparison .', 'For instance , 84% of 1 , 241 SNPs with FST>0 . 8 are located to two 0 . 5Mb blocks on chromosome 7 and a 1Mb block on chromosome 11 ( S4C Fig ) .', 'However , PBS is only marginally biased towards increased divergence in A . m .', 'adansonii at high-FST SNPs in this comparison .', 'Furthermore , XP-EHH values are below zero , indicating that differentiation at these variants is more strongly associated with selection in the background population ( A . m . capensis and A . m . scutellata ) ( Fig 2B–2D ) .', 'Taken together , these observations confirm that despite similar levels of genetic differentiation overall , the number of genetic variants that are highly differentiated due to selection in the Cape bee population is much higher than in other African populations .', 'These genetic variants are therefore likely to underlie the suite of traits connected to social parasitism .', 'Since the western Cape Town and eastern Port Elizabeth Cape bee subpopulations showed different levels of fixation against the background population , we also queried these two populations separately for linked signals of haplotype divergence and diversity using three sets of outlier SNPs: those identified as 0 . 1% outliers for FST against the background population and with minimal evidence for extended haplotype homozygosity in the Cape bees ( XP-EHH>0 ) across the complete dataset and those identified for the same criteria , but for each subpopulation individually .', 'The associated haplotype-level signals are consistently stronger in the western Cape Town subpopulation compared with the Port Elizabeth subpopulation for all three sets ( S5A–S5C Fig ) , congruent with greater fixation of Cape-bee-specific traits in the western region [3] .', 'In order to identify variants specifically under selection in Cape bees , we estimated a composite selection score ( CSS ) [24] based on FST and XP-EHH scores ( see Materials and Methods ) .', 'We did not include PBS in this score as the magnitude of PBS is expected to correlate with FST under neutrality .', 'However , a strong association between FST and XP-EHH is not expected according to the results of coalescent simulations [29] .', 'In our dataset , we find a positive and significant but weak correlation between the absolute values of the XP-EHH statistic and FST ( R2 = 0 . 07; p<1e-5 ) , consistent with the simulated data .', 'The genome-wide distribution of FST , XP-EHH and PBS are shown in Fig 3A–3C .', 'The distribution of CSS scores of all six million SNPs is shown in Fig 3D .', 'We found that SNPs with the top 2000 CSS scores were significantly enriched within coding sequence by a factor of 2 . 3x ( p<1e-5; Fischer’s exact test ) compared to the expected .', 'Likewise , SNPs are enriched within UTR regions and introns by 1 . 9x and 1 . 3x respectively ( p<1e-5 in both cases ) .', 'Taken together , this enrichment indicates that extreme signals are centered on gene bodies and that coding variants in particular are more commonly targets of selection than non-coding intergenic ones , which are underrepresented in the top SNPs ( 0 . 57x the expected amount; p<1e-5 ) .', 'Due to the association of outlier SNPs with gene bodies and the difficulty in characterizing the functional relevance of intergenic SNPs in gene deserts , we focused downstream analyses on outlier variants located close to genes .', 'We selected a conservative set of top-ranking SNPs to produce a set of putative candidate genes .', 'Among the top 1000 SNPs in the full dataset , 873 SNPs ( 87 . 3% ) occur within 8kbp of a gene , compared to 72% overall , a gene-centric overrepresentation ( p<1e-5 ) consistent with the enrichment reported above .', 'We selected these 873 SNPs plus the next 137 for analyses based on the top 1000 SNPs located within 8 kb of a gene ( CSS = 3 . 04–5 . 52; 0 . 022% of all SNPs <8 kb from a gene; S2 Table ) .', 'These make up 87 . 7% of the top 1140 SNPs .', 'The average FST of these SNPs is 0 . 67 , indicating that although they were selected on the basis of both FST and XP-EHH , they typically have high allele frequency differences between the Cape bees and the African background population .', 'Indeed , among these SNPs , the average frequency of the major allele in the Cape bees is about 90% , compared to only 20% in the African background and 18% in the European sample used as outgroup for the PBS ( S2 Table ) .', 'The frequency of the major Cape bee allele was below 0 . 2 in 63% and 80% of SNPs in the African background and European populations , respectively .', 'This demonstrates that putative candidate variants for social parasitism are typically rare outside of the Cape bee population .', 'We next queried the 1000 SNPs against the current honeybee gene annotation models and found them to be located to peaks that associate with 97 gene accessions ( S3 Table ) .', 'These gene accessions have average CSS levels across the full gene bodies within the top 1% percentile of genes ( CSS97 = 1 . 654 vs CSS1% = 1 . 546; S6 Fig ) .', 'We queried the Drosophila orthologues ( n = 61 ) of this gene set for enriched gene ontology terms using the GOrilla platform [30] and detected 16 significantly enriched biological processes and molecular functions after controlling for multiple testing [31] ( p<0 . 05; S7A and S7B Fig ) .', 'We used the REVIGO tool [32] to reduce redundancy in the list of GO terms , removing 4 terms ( S4 Table ) .', 'Among the remaining GO-terms , we detect significant enrichment of genes associated with ecdysteroid metabolic processes ( GO:0042445; n = 5; p = 6 . 15e-3; S4 Table ) .', 'The genes we detected within this category include orthologs of three glucose-methanol-choline ( GMC ) oxidoreductases ( GMCOX4/6/9 ) located in a conserved cluster on chromosome 1 [33] , and also Npc1a and foxo ( S4 Table; S7A Fig ) .', 'Ecdysteroid signaling is a hormonal process that is correlated with development and ovary maturation in honeybees [34–36] .', 'The corresponding top 1000 SNPs from the two other reciprocal comparisons ( scutellata vs the rest and adansonii vs the rest ) did not reveal any significantly enriched biological process GO terms .', 'It is possible that genes associated with particular biological functions may be under selection but do not have clearly identifiable outlier variants under our criteria .', 'In order to address this question , we analyzed a ranked list of all accessions with Drosophila orthologues ( n = 6 , 253 ) .', 'We used the gene-wide average CSS scores to rank all genes and provided the list to GOrilla to perform analyses of enriched GO terms among top accessions while controlling for multiple testing .', 'We did not detect any significantly enriched GO term with FDR-corrected p<0 . 05 using this method .', 'To further address whether selection may be targeting genes specifically associated with meiosis and abnormal chromosome segregation ( i . e . thelytoky candidates ) , we collected all genes associated with GO terms related to meiosis or chromosome segregation .', 'Genes of GO terms matching key words “meiotic” or “meiosis” ( 56 GO-terms; 126 genes ) and “segregation” ( 10 GO-terms , 74 genes ) were compiled into a meiosis-list .', 'Each gene was counted only once despite the number of terms it was associated with , resulting in a list of 161 genes .', 'In the same way , we compiled a list of 55 ecdysteroid-related genes ( i . e . ovary development candidates ) , associated with GO terms matching “ecdysteroid” ( 10 GO-terms; 20 genes ) or “ecdysone” ( 12 GO-terms , 41 genes; ecdysone is an ecdysteroid hormone ) .', 'There is only a single shared gene between the two lists , so effects of multiple testing should be expected to be negligible .', 'The average CSS score among all honeybee accessions is 0 . 383 .', 'The mean CSS of the “meiosis-genes” is 0 . 377 .', 'After randomly sampling 161 genes across the genome 2000 times we find that this minor decrease of about 1 . 5% is not significant at the 0 . 05 level .', 'For the “ecdysteroid-genes” the mean CSS is 0 . 410 , a small increase of about 7% but not significant at the 0 . 05 level using the same resampling procedure .', 'These alternative GO analyses fail to detect broad patterns of selection on particular biological functions .', 'For instance , while we find that some of the clearest signals of selection based on outlier variants are associated with a small subset of genes involved in ecdysteroid metabolism , we do not find evidence for widespread selection signals across all genes associated with ecdysteroid metabolism .', 'Taken together , the results of the GO analyses are consistent with a scenario in which selection in the Cape bees has targeted a restricted set of loci ( i . e . <1% of genes ) with diverse functions , rather than broadly affecting genes with similar functions across the whole genome .', 'We further filtered the set of 97 candidate genes down to the subset of 25 accessions that had at least one top CSS SNP with an FST>0 . 8 ( nSNPs = 393; Table 1 ) .', 'At these SNPs , the average frequency of the major allele in the Cape bees was 92% , but only 1 . 4% or 1 . 9% in the African background or European samples , respectively ( S2 Table ) .', 'The frequency of the major Cape bee allele was found to be below 0 . 2 at >97% of these SNPs in either of the two populations .', 'These 25 genes therefore contain the most highly differentiated variants with signals of selection between the Cape bees and the other African bees and those variants are also typically very rare also outside of Africa .', 'Out of the original top 1000 SNPs , 762 SNPs are associated with these 25 genes and the average CSS score across these genes are within the top 0 . 5% of all genes in the genome ( CSS25 = 2 . 175 vs CSS0 . 5% = 1 . 912; S6 Fig ) , indicating that these genes have comprehensive signals of selection .', 'Of these 25 accessions , 16 genes belong to the set of 38 accessions with fixed variants between the western Cape Town subpopulation and African background population and 4 genes belong to the set of 5 with fixed variants between the eastern Port Elizabeth and the background population .', 'These are listed in Table 1 and highlighted in blue in Fig 3A–3D ( see S8 Fig for candidate gene plots ) .', 'The top 762 SNPs associated with the 25 genes include 21 non-synonymous variants , affecting the coding sequence of 11 genes .', 'Some of the 25 genes are located close together such that selection signals appear to extend across more than one accession .', 'From manually inspecting these data we assigned these 25 genes into 12 distinct putative selective sweeps with strong evidence of selection in the Cape bee , including highly differentiated SNPs , which we labeled as A–L according to their location in the genome ( Table 2 ) .', 'Corresponding plots of XP-EHH , FST , PBS , and the combined CSS around these sweeps are shown in S8 Fig . The three putative selective sweeps with highest CSS values are on chromosomes 11 ( locus H ) , 1 ( locus B ) , and 6 ( locus D ) and overlap with 14 of the accessions , with outlier signals spanning at least three genes each .', 'The three SNPs with the highest CSS scores in the dataset are located in locus H on chromosome 11 ( ~40kbp wide ) , which contains 36 of the 45 fixed SNPs in the Cape bee sample relative to the African background population .', 'The CSS scores are centered on the gene accession GB45239 , which contains the non-synonymous SNP with the highest selection score in the dataset ( CSS = 4 . 07 ) .', 'This gene is not characterized in the honeybees but has peptide sequence similarity to a structural maintenance of chromosomes ( SMC ) domain ( COG1196; p = 6 . 82e-04; TIGR02168; p = 2 . 43e-03 ) in the NCBI Conserved Domain Database [37] .', 'The role in chromosome integrity may indicate that this gene is involved in thelytokous meiosis in the Cape bee .', 'Locus B on chromosome 1 is ~110kbp wide and encompasses 7 genes .', 'However , the 9 fixed SNPs are centered at an intron of the Ethr gene , which codes for the receptor for the neuropeptide ecdysis-triggering hormone ( ETH ) .', 'ETH is essential for initiating ecdysis but also regulates juvenile hormone ( JH ) synthesis in insects [38 , 39] .', 'Notably , the region also spans genes coding for a StAR-like protein , an intracellular cholesterol transporter facilitating ovarian steroid hormone synthesis in vertebrates and insects [40–44] , and ebony , which makes N-β-alanyl-dopamine from dopamine , a process associated with yellow cuticular pigmentation instead of the brown/black tan resulting from dopamine melanization [45 , 46] .', 'This region therefore contains candidates for larval development , ovary development and pigmentation , all of which differ in the Cape bee compared to other subspecies of A . mellifera .', 'Locus D on chromosome 6 ( ~40kbp wide; 5 genes ) is centered on β-glucosidase , a digestive enzyme associated with carbohydrate metabolism , including cellulose digestion .', 'In the honeybee , the gene codes for an enzyme that hydrolyses small mono-glycosides , with a particular preference for phenyl-glycosides such as β-pNPG [47–49] .', 'While the EHH signal decays rapidly around the β-glucosidase gene , neighboring SNPs around the accession also have unusually high FST values .', 'To either side of the accession are the UDP glycosyltransferase ( UGT ) genes encoding the two proteins UDP-glucuronosyltransferase 1-3-like and UDP-glucuronosyltransferase 2B13-like .', 'UGT enzymes catalyze reactions that metabolize oligosaccharides and conjugate sugars to a broad range of lipophilic compounds ( see GO analysis results above ) .', 'β-glucosidase has been shown to have a role in chemical signaling in social insects [50–52] .', 'In the noctuid moth Mamestra configurata , β-glucosidase produces a glucoside precursor of the phenylethanol sex pheromone [50] .', '2-phenylethanol has been suggested to advertise ovary status between young unmated honeybee queens and workers during the queen elimination phase [53] , but the role of β-glucosidase for producing the pheromone has not yet been studied in the honeybee .', 'We find additional signatures of selection on loci outside of these three largest sweeps .', 'High CSS scores across accession GB40769 “dehydrogenase/reductase SDR family member 11-like” ( LOC412458 ) on chromosome 1 ( locus A ) .', 'SDRs make up large families of NADPH-dependent oxidoreductases with central metabolic functions [54] .', 'The Drosophila orthologue is CG9360 , a NADP+-dependent farnesol dehydrogenase ( FOHSDR; KEGG: K15890 ) [55] .', 'The orthologue detected in the closely related honeybee Apis florea using BLASTp [56 , 57] is also annotated as “farnesol dehydrogenase-like” ( E-value = 1e-140; 88% identity; XP_003690738 . 1 ) .', 'This gene may therefore code for a farnesol dehydrogenase enzyme in bees .', 'FOHSDR acts in the JH biosynthesis pathway and oxidizes the sesquiterpene alcohol farnesol into farnesal , a precursor of JH [39 , 58] .', 'JH has an important function regulating ovary development and transition to adulthood [36] .', 'Locus C , on chromosome 2 encompasses GB50742 ( LOC102655146 ) , annotated as “Bardet-Biedl syndrome 1 protein-like” ( BBS1 ) , a member of the BBSome protein complex , which is an essential protein transporter for cilium assembly and localizes near the primary cilium and centrosome in the cell [59 , 60] , which suggests it may be a candidate for thelytoky .', 'We detect a CSS peak in the intron of GB54634 ( LOC725260; Drosophila orthologue: CG9896 ) on chromosome 7 ( locus E ) .', 'This gene encodes a protein that is currently uncharacterized across the animal kingdom: searches with NCBI BLASTp and in the Conserved Domain Database did not identify any functionally annotated orthologues or known domains .', 'Locus F encompasses accession GB43519 ( LOC411614 ) on chromosome 11 , corresponding to the Neuropeptide Y-like ( NPY; the insect orthologue is NPF ) receptor snpfR .', 'NPF is a neurohormone that regulates feeding motivation and behaviors in animals and is up-regulated in honeybee foragers compared to nurse bees [61] .', 'We hypothesize that this gene is involved in lack of foraging in the Cape bee that occurs during social parasitism .', 'On the same chromosome ( locus G ) , we also find outlier SNPs across accession GB44980 ( LOC409260 ) , which codes for an “epidermal retinol dehydrogenase 2-like” protein .', 'Retinol dehydrogenases oxidate retinol into retinal and are important for vitamin A metabolism and the production of photopigments and retinoic acid , essential for vision , neural development , organogenesis and reproduction across animal taxa [62–64] .', 'Locus J is centered on GB49919 ( LOC724687 ) on chromosome 13 , which codes for a protein with a Guanylate-kinase-associated protein domain ( GKAP ) and shows high similarity towards insect sequences annotated as disks large-associated protein 5-like ( DLGAP5 ) .', 'The Drosophila orthologue is MARS ( FBgn0033845 ) , which is the fly version of the vertebrate DLGAP5-gene ( syn . HURP ) .', 'In both the fly and vertebrates , this protein promotes microtubular stability in mitotic and meiotic spindles prior to cell division [65–68] , making it a good candidate for involvement in thelytoky in the Cape bee .', 'Close to the end of chromosome 16 , we detect a clear selection signature across accession GB45973 ( locus L ) .', 'This gene codes for the Dopa decarboxylase ( Ddc ) enzyme , which converts L-dopa to dopamine , a neurotransmitter with diverse functions in insects , including roles in aversive learning and memory formation , innate immunity , and ovary development [69–71] .', 'We next analyzed patterns of genetic variation in the 12 putative selective sweep regions ( Table 2 ) .', 'Cape bee haplotypes with strongly selected alleles can be expected to have reduced levels of genetic diversity compared to unselected Cape and African haplotypes .', 'I
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['In colonies of the honeybee Apis mellifera , the queen is usually the only reproductive female , which produces new females ( queens and workers ) by laying fertilized eggs .', 'However , in one subspecies of A . mellifera , known as the Cape bee ( A . m . capensis ) , worker bees reproduce asexually by thelytoky , an abnormal form of meiosis where two daughter nucleii fuse to form single diploid eggs , which develop into females without being fertilized .', 'The Cape bee also exhibits a suite of phenotypes that facilitate social parasitism whereby workers lay such eggs in foreign colonies so their offspring can exploit their resources .', 'The genetic basis of this switch to social parasitism in the Cape bee is unknown .', 'To address this , we compared genome variation in a sample of Cape bees with other African populations .', 'We find genetic divergence between these populations to be very low on average but identify several regions of the genome with extreme differentiation .', 'The regions are strongly enriched for signals of selection in Cape bees , indicating that increased levels of positive selection have produced the unique set of derived phenotypic traits in this subspecies .', 'Genetic variation within these regions allows unambiguous genetic identification of Cape bees and likely underlies the genetic basis of social parasitism .', 'The candidate loci include genes involved in ecdysteroid signaling and juvenile hormone and dopamine biosynthesis , which may regulate worker ovary activation and others whose products localize at the centrosome and are implicated in chromosomal segregation during meiosis .', 'Functional analysis of these loci will yield insights into the processes of reproduction and chemical signaling in both parasitic and non-parasitic populations and advance understanding of the process of normal and atypical meiosis .']
['Honeybee colonies are mainly composed of female worker bees that are unable to lay eggs and reproduce; a task that is performed by a single queen bee .', 'However , in a subspecies of the Western honeybee ( Apis mellifera ) known as the Cape bee ( A . m . capensis ) , worker bees can lay eggs produced by an abnormal form of meiotic cell division known as thelytoky .', 'These eggs are produced asexually and develop into female worker bees , which are then able to reproduce in the same way and hence to propagate this trait .', 'Cape bee workers also display a variety of characteristics that enable them to invade foreign colonies , reproduce and feed off their resources , a behavior known as social parasitism .', 'The genetic basis of these traits is unknown .', 'Here we compare whole genome sequences of Cape bees with other honeybee subspecies and identify the genes that likely control this reproductive strategy .', 'Although Cape bees are extremely genetically similar to other African bees , we identify genetic variants that make it possible to unambiguously distinguish them .', 'In contrast to previous studies , our results suggest that multiple genes are involved in social parasitism , including those involved in hormonal signaling that may cause worker bees to activate their ovaries and those involved in chromosomal segregation , which may cause the switch between normal meiosis and thelytoky .', 'Our results give insights into how genetic variants that facilitate asexual reproduction are able to invade a sexually reproducing population .']
['invertebrates', 'ecology and environmental sciences', 'honey bees', 'population genetics', 'invertebrate genomics', 'animals', 'population biology', 'bees', 'genomic signal processing', 'hymenoptera', 'parasitism', 'genetic loci', 'animal genomics', 'insects', 'arthropoda', 'community ecology', 'signal transduction', 'haplotypes', 'cell biology', 'ecology', 'trophic interactions', 'genetics', 'biology and life sciences', 'species interactions', 'genomics', 'evolutionary biology', 'cell signaling', 'organisms']