Genomic variation in montane bumblebees in Scandinavia: High levels of intraspecific diversity despite population vulnerability.

Populations of many bumblebee species are declining, with distributions shifting northwards to track suitable climates. Climate change is considered a major contributing factor. Arctic species are particularly vulnerable as they cannot shift further north, making assessment of their population viability important. Analysis of levels of whole-genome variation is a powerful way to analyse population declines and fragmentation. Here, we use genome sequencing to analyse genetic variation in seven species of bumblebee from the Scandinavian mountains, including two classified as vulnerable. We sequenced 333 samples from across the ranges of these species in Sweden. Estimates of effective population size (NE ) vary from ~55,000 for species with restricted high alpine distributions to 220,000 for more widespread species. Population fragmentation is generally very low or undetectable over large distances in the mountains, suggesting an absence of barriers to gene flow. The relatively high NE and low population structure indicate that none of the species are at immediate risk of negative genetic effects caused by high levels of genetic drift. However, reconstruction of historical fluctuations in NE indicates that the arctic specialist species Bombus hyperboreus has experienced population declines since the last ice age and we detected one highly inbred diploid male of this species close to the southern limit of its range, potentially indicating elevated genetic load. Although the levels of genetic variation in montane bumblebee populations are currently relatively high, their ranges are predicted to shrink drastically due to the effects of climate change and monitoring is essential to detect future population declines.

The Genomic Basis of Adaptation to High Elevations in Africanized Honey Bees.

A range of different genetic architectures underpin local adaptation in nature. Honey bees (Apis mellifera) in the Eastern African Mountains harbor high frequencies of two chromosomal inversions that likely govern adaptation to this high-elevation habitat. In the Americas, honey bees are hybrids of European and African ancestries and adaptation to latitudinal variation in climate correlates with the proportion of these ancestries across the genome. It is unknown which, if either, of these forms of genetic variation governs adaptation in honey bees living at high elevations in the Americas. Here, we performed whole-genome sequencing of 29 honey bees from both high- and low-elevation populations in Colombia. Analysis of genetic ancestry indicated that both populations were predominantly of African ancestry, but the East African inversions were not detected. However, individuals in the higher elevation population had significantly higher proportions of European ancestry, likely reflecting local adaptation. Several genomic regions exhibited particularly high differentiation between highland and lowland bees, containing candidate loci for local adaptation. Genes that were highly differentiated between highland and lowland populations were enriched for functions related to reproduction and sperm competition. Furthermore, variation in levels of European ancestry across the genome was correlated between populations of honey bees in the highland population and populations at higher latitudes in South America. The results are consistent with the hypothesis that adaptation to both latitude and elevation in these hybrid honey bees are mediated by variation in ancestry at many loci across the genome.

Population Genomics for Insect Conservation.

Insects constitute vital components of ecosystems. There is alarming evidence for global declines in insect species diversity, abundance, and biomass caused by anthropogenic drivers such as habitat degradation or loss, agricultural practices, climate change, and environmental pollution. This raises important concerns about human food security and ecosystem functionality and calls for more research to assess insect population trends and identify threatened species and the causes of declines to inform conservation strategies. Analysis of genetic diversity is a powerful tool to address these goals, but so far animal conservation genetics research has focused strongly on endangered vertebrates, devoting less attention to invertebrates, such as insects, that constitute most biodiversity. Insects' shorter generation times and larger population sizes likely necessitate different analytical methods and management strategies. The availability of high-quality reference genome assemblies enables population genomics to address several key issues. These include precise inference of past demographic fluctuations and recent declines, measurement of genetic load levels, delineation of evolutionarily significant units and cryptic species, and analysis of genetic adaptation to stressors. This enables identification of populations that are particularly vulnerable to future threats, considering their potential to adapt and evolve. We review the application of population genomics to insect conservation and the outlook for averting insect declines.

Genome Evolution of a Symbiont Population for Pathogen Defense in Honeybees.

The honeybee gut microbiome is thought to be important for bee health, but the role of the individual members is poorly understood. Here, we present closed genomes and associated mobilomes of 102 Apilactobacillus kunkeei isolates obtained from the honey crop (foregut) of honeybees sampled from beehives in Helsingborg in the south of Sweden and from the islands Gotland and Åland in the Baltic Sea. Each beehive contained a unique composition of isolates and repeated sampling of similar isolates from two beehives in Helsingborg suggests that the bacterial community is stably maintained across bee generations during the summer months. The sampled bacterial population contained an open pan-genome structure with a high genomic density of transposons. A subset of strains affiliated with phylogroup A inhibited growth of the bee pathogen Melissococcus plutonius, all of which contained a 19.5 kb plasmid for the synthesis of the antimicrobial compound kunkecin A, while a subset of phylogroups B and C strains contained a 32.9 kb plasmid for the synthesis of a putative polyketide antibiotic. This study suggests that the mobile gene pool of A. kunkeei plays a key role in pathogen defense in honeybees, providing new insights into the evolutionary dynamics of defensive symbiont populations.

Massive genome inversion drives coexistence of divergent morphs in common quails.

The presence of population-specific phenotypes often reflects local adaptation or barriers to gene flow. The co-occurrence of phenotypic polymorphisms that are restricted within the range of a highly mobile species is more difficult to explain. An example of such polymorphisms is in the common quail Coturnix coturnix, a small migratory bird that moves widely during the breeding season in search of new mating opportunities, following ephemeral habitats,1,2 and whose females may lay successive clutches at different locations while migrating.3 In spite of this vagility, previous studies reported a higher frequency of heavier males with darker throat coloration in the southwest of the distribution (I. Jiménez-Blasco et al., 2015, Int. Union Game Biol., conference). We used population genomics and cytogenetics to explore the basis of this polymorphism and discovered a large inversion in the genome of the common quail. This inversion extends 115 Mbp in length and encompasses more than 7,000 genes (about 12% of the genome), producing two very different forms. Birds with the inversion are larger, have darker throat coloration and rounder wings, are inferred to have poorer flight efficiency, and are geographically restricted despite the high mobility of the species. Stable isotope analyses confirmed that birds carrying the inversion have shorter migratory distances or do not migrate. However, we found no evidence of pre- or post-zygotic isolation, indicating the two forms commonly interbreed and that the polymorphism remains locally restricted because of the effect on behavior. This illustrates a genomic mechanism underlying maintenance of geographically structured polymorphisms despite interbreeding with a lineage with high mobility.

A genomic and morphometric analysis of alpine bumblebees: Ongoing reductions in tongue length but no clear genetic component.

Over the last six decades, populations of the bumblebees Bombus sylvicola and Bombus balteatus in Colorado have experienced decreases in tongue length, a trait important for plant-pollinator mutualisms. It has been hypothesized that this observation reflects selection resulting from shifts in floral composition under climate change. Here we used morphometrics and population genomics to determine whether morphological change is ongoing, investigate the genetic basis of morphological variation, and analyse population structure in these populations. We generated a genome assembly of B. balteatus. We then analysed whole-genome sequencing data and morphometric measurements of 580 samples of both species from seven high-altitude localities. Out of 281 samples originally identified as B. sylvicola, 67 formed a separate genetic cluster comprising a newly-discovered cryptic species ("incognitus"). However, an absence of genetic structure within species suggests that gene flow is common between mountains. We found a significant decrease in tongue length between bees collected between 2012-2014 and in 2017, indicating that morphological shifts are ongoing. We did not discover any genetic associations with tongue length, but a SNP related to production of a proteolytic digestive enzyme was implicated in body size variation. We identified evidence of covariance between kinship and both tongue length and body size, which is suggestive of a genetic component of these traits, although it is possible that shared environmental effects between colonies are responsible. Our results provide evidence for ongoing modification of a morphological trait important for pollination and indicate that this trait probably has a complex genetic and environmental basis.

Phylogenomics and evolutionary history of Oreobates (Anura: Craugastoridae) Neotropical frogs along elevational gradients.

Mountain ranges offer opportunities for understanding how species evolved and diversified across different environmental conditions. Neotropical frogs of the genus Oreobates (Anura: Craugastoridae) are adapted to highland and lowland habitats along the Andes, but many aspects of their evolution remain unknown. We studied their evolutionary history using ~18,000 exons enriched by targeted sequence-capture. Since capture success was very variable across samples, we evaluated to what degree differing data filtering produced robust inferences. The inferred evolutionary framework evidenced phylogenetic discordances among lowland species that can be explained by taxonomic misidentification or admixture of ancestral lineages. Highland species showed smaller effective populations than lowland frogs, probably due to greater habitat fragmentation in montane environments. Stronger genetic drift likely decreased the power of purifying selection and led to an increased proportion of nonsynonymous mutations in highland populations that could play an important role in their adaptation. Overall, our work sheds light on the evolutionary history and diversification of this group of Neotropical frogs along elevational gradients in the Andes as well as on their patterns of intraspecific diversity.

Genetic Barriers to Historical Gene Flow between Cryptic Species of Alpine Bumblebees Revealed by Comparative Population Genomics.

Evidence is accumulating that gene flow commonly occurs between recently diverged species, despite the existence of barriers to gene flow in their genomes. However, we still know little about what regions of the genome become barriers to gene flow and how such barriers form. Here, we compare genetic differentiation across the genomes of bumblebee species living in sympatry and allopatry to reveal the potential impact of gene flow during species divergence and uncover genetic barrier loci. We first compared the genomes of the alpine bumblebee Bombus sylvicola and a previously unidentified sister species living in sympatry in the Rocky Mountains, revealing prominent islands of elevated genetic divergence in the genome that colocalize with centromeres and regions of low recombination. This same pattern is observed between the genomes of another pair of closely related species living in allopatry (B. bifarius and B. vancouverensis). Strikingly however, the genomic islands exhibit significantly elevated absolute divergence (dXY) in the sympatric, but not the allopatric, comparison indicating that they contain loci that have acted as barriers to historical gene flow in sympatry. Our results suggest that intrinsic barriers to gene flow between species may often accumulate in regions of low recombination and near centromeres through processes such as genetic hitchhiking, and that divergence in these regions is accentuated in the presence of gene flow.

Population Genomics: How Do Cape Honey Bees Do Without Sex?

The Cape honey bee, Apis mellifera capensis, is able to produce female offspring asexually. This phenomenon has now been shown to have a simple genetic basis.

Female-biased gene flow between two species of Darwin's finches.

The mosaic nature of hybrid genomes is well recognized, but little is known of how they are shaped initially by patterns of breeding, selection, recombination and differential incompatibilities. On the small Galápagos island of Daphne Major, two species of Darwin's finches, Geospiza fortis and G. scandens, hybridize rarely and back-cross bidirectionally with little or no loss of fitness under conditions of plentiful food. We used whole-genome sequences to compare genomes from periods before and after successful interbreeding followed by back-crossing. We inferred extensive introgression from G. fortis to G. scandens on autosomes and mitochondria but not on the Z chromosome. The unique combination of long-term field observations and genomic data shows that the reduction of gene flow for Z-linked loci primarily reflects female-biased gene flow, arising from a hybrid-male disadvantage in competition for high-quality territories and mates, rather than from genetic incompatibilities at Z-linked loci.

Substantial Heritable Variation in Recombination Rate on Multiple Scales in Honeybees and Bumblebees.

Meiotic recombination shuffles genetic variation and promotes correct segregation of chromosomes. Rates of recombination vary on several scales, both within genomes and between individuals, and this variation is affected by both genetic and environmental factors. Social insects have extremely high rates of recombination, although the evolutionary causes of this are not known. Here, we estimate rates of crossovers and gene conversions in 22 colonies of the honeybee, Apis mellifera, and 9 colonies of the bumblebee, Bombus terrestris, using direct sequencing of 299 haploid drone offspring. We confirm that both species have extremely elevated crossover rates, with higher rates measured in the highly eusocial honeybee than the primitively social bumblebee. There are also significant differences in recombination rate between subspecies of honeybee. There is substantial variation in genome-wide recombination rate between individuals of both A. mellifera and B. terrestris and the distribution of these rates overlap between species. A large proportion of interindividual variation in recombination rate is heritable, which indicates the presence of variation in trans-acting factors that influence recombination genome-wide. We infer that levels of crossover interference are significantly lower in honeybees compared to bumblebees, which may be one mechanism that contributes to higher recombination rates in honeybees. We also find a significant increase in recombination rate with distance from the centromere, mirrored by methylation differences. We detect a strong transmission bias due to GC-biased gene conversion associated with noncrossover gene conversions. Our results shed light on the mechanistic causes of extreme rates of recombination in social insects and the genetic architecture of recombination rate variation.

Extreme Differences in Recombination Rate between the Genomes of a Solitary and a Social Bee.

Social insect genomes exhibit the highest rates of crossing over observed in plants and animals. The evolutionary causes of these extreme rates are unknown. Insight can be gained by comparing recombination rate variation across the genomes of related social and solitary insects. Here, we compare the genomic recombination landscape of the highly social honey bee, Apis mellifera, with the solitary alfalfa leafcutter bee, Megachile rotundata, by analyzing patterns of linkage disequilibrium in population-scale genome sequencing data. We infer that average recombination rates are extremely elevated in A. mellifera compared with M. rotundata. However, our results indicate that similar factors control the distribution of crossovers in the genomes of both species. Recombination rate is significantly reduced in coding regions in both species, with genes inferred to be germline methylated having particularly low rates. Genes with worker-biased patterns of expression in A. mellifera and their orthologs in M. rotundata have higher than average recombination rates in both species, suggesting that selection for higher diversity in genes involved in worker caste functions in social taxa is not the explanation for these elevated rates. Furthermore, we find no evidence that recombination has modulated the efficacy of selection among genes during bee evolution, which does not support the hypothesis that high recombination rates facilitated positive selection for new functions in social insects. Our results indicate that the evolution of sociality in insects likely entailed selection on modifiers that increased recombination rates genome wide, but that the genomic recombination landscape is determined by the same factors.

A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds.

BACKGROUND: The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map. RESULTS: Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor > 98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features. CONCLUSIONS: The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.

Social Parasitism in the Honeybee (Apis mellifera) Is Not Controlled by a Single SNP.

The Cape bee (Apis mellifera capensis) is a subspecies of the honeybee, in which workers commonly lay diploid unfertilized eggs via a process known as thelytoky. A recent study aimed to map the genetic basis of this trait in the progeny of a single capensis queen where workers laid either diploid (thelytokous) or haploid (arrhenotokous) eggs. A nonsynonymous single nucleotide polymorphism (SNP) in a gene of unknown function was reported to be strongly associated with thelytoky in this colony. Here, we analyze genome sequences from a global sample of A. mellifera and identify populations where the proposed thelytoky allele at this SNP is common but thelytoky is absent. We also analyze genome sequences of three capensis queens produced by thelytoky and find that, contrary to predictions, they do not carry the proposed thelytoky allele. The proposed SNP is therefore neither sufficient nor required to produce thelytoky in A. mellifera.

Chromosomal inversions associated with environmental adaptation in honeybees.

Chromosomal inversions can facilitate local adaptation in the presence of gene flow by suppressing recombination between well-adapted native haplotypes and poorly adapted migrant haplotypes. East African mountain populations of the honeybee Apis mellifera are highly divergent from neighbouring lowland populations at two extended regions in the genome, despite high similarity in the rest of the genome, suggesting that these genomic regions harbour inversions governing local adaptation. Here, we utilize a new highly contiguous assembly of the honeybee genome to characterize these regions. Using whole-genome sequencing data from 55 highland and lowland bees, we find that the highland haplotypes at both regions are present at high frequencies in three independent highland populations but extremely rare elsewhere. The boundaries of both divergent regions are characterized by regions of high homology with each other positioned in opposite orientations and contain highly repetitive, long inverted repeats with homology to transposable elements. These regions are likely to represent inversion breakpoints that participate in nonallelic homologous recombination. Using long-read data, we confirm that the lowland samples are contiguous across breakpoint regions. We do not find evidence for disruption of functional sequence by these breakpoints, which suggests that the inversions are likely maintained due to their allelic content conferring local adaptation in highland environments. Finally, we identify a third divergent genomic region, which contains highly divergent segregating haplotypes that also may contain inversion variants under selection. The results add to a growing body of evidence indicating the importance of chromosomal inversions in local adaptation.

The genomic basis of adaptation to high-altitude habitats in the eastern honey bee (Apis cerana).

The eastern honey bee (Apis cerana) is of central importance for agriculture in Asia. It has adapted to a wide variety of environmental conditions across its native range in southern and eastern Asia, which includes high-altitude regions. eastern honey bees inhabiting mountains differ morphologically from neighbouring lowland populations and may also exhibit differences in physiology and behaviour. We compared the genomes of 60 eastern honey bees collected from high and low altitudes in Yunnan and Gansu provinces, China, to infer their evolutionary history and to identify candidate genes that may underlie adaptation to high altitude. Using a combination of FST -based statistics, long-range haplotype tests and population branch statistics, we identified several regions of the genome that appear to have been under positive selection. These candidate regions were strongly enriched for coding sequences and had high haplotype homozygosity and increased divergence specifically in highland bee populations, suggesting they have been subjected to recent selection in high-altitude habitats. Candidate loci in these genomic regions included genes related to reproduction and feeding behaviour in honey bees. Functional investigation of these candidate loci is necessary to fully understand the mechanisms of adaptation to high-altitude habitats in the eastern honey bee.

Developing reduced SNP assays from whole-genome sequence data to estimate introgression in an organism with complex genetic patterns, the Iberian honeybee (Apis mellifera iberiensis).

The most important managed pollinator, the honeybee (Apis mellifera L.), has been subject to a growing number of threats. In western Europe, one such threat is large-scale introductions of commercial strains (C-lineage ancestry), which is leading to introgressive hybridization and even the local extinction of native honeybee populations (M-lineage ancestry). Here, we developed reduced assays of highly informative SNPs from 176 whole genomes to estimate C-lineage introgression in the most diverse and evolutionarily complex subspecies in Europe, the Iberian honeybee (Apis mellifera iberiensis). We started by evaluating the effects of sample size and sampling a geographically restricted area on the number of highly informative SNPs. We demonstrated that a bias in the number of fixed SNPs (FST = 1) is introduced when the sample size is small (N ≤ 10) and when sampling only captures a small fraction of a population's genetic diversity. These results underscore the importance of having a representative sample when developing reliable reduced SNP assays for organisms with complex genetic patterns. We used a training data set to design four independent SNP assays selected from pairwise FST between the Iberian and C-lineage honeybees. The designed assays, which were validated in holdout and simulated hybrid data sets, proved to be highly accurate and can be readily used for monitoring populations not only in the native range of A. m. iberiensis in Iberia but also in the introduced range in the Balearic islands, Macaronesia and South America, in a time- and cost-effective manner. While our approach used the Iberian honeybee as model system, it has a high value in a wide range of scenarios for the monitoring and conservation of potentially hybridized domestic and wildlife populations.

Whole genome SNP-associated signatures of local adaptation in honeybees of the Iberian Peninsula.

The availability of powerful high-throughput genomic tools, combined with genome scans, has helped identifying genes and genetic changes responsible for environmental adaptation in many organisms, including the honeybee. Here, we resequenced 87 whole genomes of the honeybee native to Iberia and used conceptually different selection methods (Samßada, LFMM, PCAdapt, iHs) together with in sillico protein modelling to search for selection footprints along environmental gradients. We found 670 outlier SNPs, most of which associated with precipitation, longitude and latitude. Over 88.7% SNPs laid outside exons and there was a significant enrichment in regions adjacent to exons and UTRs. Enrichment was also detected in exonic regions. Furthermore, in silico protein modelling suggests that several non-synonymous SNPs are likely direct targets of selection, as they lead to amino acid replacements in functionally important sites of proteins. We identified genomic signatures of local adaptation in 140 genes, many of which are putatively implicated in fitness-related functions such as reproduction, immunity, olfaction, lipid biosynthesis and circadian clock. Our genome scan suggests that local adaptation in the Iberian honeybee involves variations in regions that might alter patterns of gene expression and in protein-coding genes, which are promising candidates to underpin adaptive change in the honeybee.

Elevated Proportions of Deleterious Genetic Variation in Domestic Animals and Plants.

A fraction of genetic variants segregating in any population are deleterious, which negatively impacts individual fitness. The domestication of animals and plants is associated with population bottlenecks and artificial selection, which are predicted to increase the proportion of deleterious variants. However, the extent to which this is a general feature of domestic species is unclear. Here, we examine the effects of domestication on the prevalence of deleterious variation using pooled whole-genome resequencing data from five domestic animal species (dog, pig, rabbit, chicken, and silkworm) and two domestic plant species (rice and soybean) compared with their wild ancestors. We find significantly reduced genetic variation and increased proportion of nonsynonymous amino acid changes in all but one of the domestic species. These differences are observable across a range of allele frequencies, both common and rare. We find proportionally more single nucleotide polymorphisms in highly conserved elements in domestic species and a tendency for domestic species to harbor a higher proportion of changes classified as damaging. Our findings most likely reflect an increased incidence of deleterious variants in domestic species, which is most likely attributable to population bottlenecks that lead to a reduction in the efficacy of selection. An exception to this pattern is displayed by European domestic pigs, which do not show traces of a strong population bottleneck and probably continued to exchange genes with wild boar populations after domestication. The results presented here indicate that an elevated proportion of deleterious variants is a common, but not ubiquitous, feature of domestic species.