Origins, diversity, and adaptive evolution of DWV in the honey bees of the Azores: the impact of the invasive mite Varroa destructor.

Deformed wing virus (DWV) is a honey bee virus, whose emergence from relative obscurity is driven by the recent host-switch, adaptation, and global dispersal of the ectoparasitic mite Varroa destructor (a highly efficient vector of DWV) to reproduction on honey bees (Apis mellifera). Our study examines how varroa affects the continuing evolution of DWV, using the Azores archipelago, where varroa is present on only three out of the eight Islands, as a natural experimental system for comparing different evolutionary conditions and trajectories. We combined qPCR of 494 honey bee colonies sampled across the archipelago with amplicon deep sequencing to reveal how the DWV genetic landscape is altered by varroa. Two of the varroa-free Islands were also free of DWV, while a further two Islands were intriguingly dominated by the rare DWV-C major variant. The other four Islands, including the three varroa-infested Islands, were dominated by the common DWV-A and DWV-B variants. The varroa-infested Islands had, as expected, an elevated DWV prevalence relative to the uninfested Islands, but not elevated DWV loads, due the relatively high prevalence and loads of DWV-C on the varroa-free Islands. This establishes the Azores as a stable refuge for DWV-C and provides the most convincing evidence to date that at least some major strains of DWV may be capable of not just surviving, but actually thriving in honey bees in the absence of varroa-mediated transmission. We did not detect any change in DWV genetic diversity associated with island varroa status but did find a positive association of DWV diversity with virus load, irrespective of island varroa status.

Varroa destructor shapes the unique viral landscapes of the honey bee populations of the Azores archipelago.

The worldwide dispersal of the ectoparasitic mite Varroa destructor from its Asian origins has fundamentally transformed the relationship of the honey bee (Apis mellifera) with several of its viruses, via changes in transmission and/or host immunosuppression. The extent to which honey bee-virus relationships change after Varroa invasion is poorly understood for most viruses, in part because there are few places in the world with several geographically close but completely isolated honey bee populations that either have, or have not, been exposed long-term to Varroa, allowing for separate ecological, epidemiological, and adaptive relationships to develop between honey bees and their viruses, in relation to the mite's presence or absence. The Azores is one such place, as it contains islands with and without the mite. Here, we combined qPCR with meta-amplicon deep sequencing to uncover the relationship between Varroa presence, and the prevalence, load, diversity, and phylogeographic structure of eight honey bee viruses screened across the archipelago. Four viruses were not detected on any island (ABPV-Acute bee paralysis virus, KBV-Kashmir bee virus, IAPV-Israeli acute bee paralysis virus, BeeMLV-Bee macula-like virus); one (SBV-Sacbrood virus) was detected only on mite-infested islands; one (CBPV-Chronic bee paralysis virus) occurred on some islands, and two (BQCV-Black queen cell virus, LSV-Lake Sinai virus,) were present on every single island. This multi-virus screening builds upon a parallel survey of Deformed wing virus (DWV) strains that uncovered a remarkably heterogeneous viral landscape featuring Varroa-infested islands dominated by DWV-A and -B, Varroa-free islands naïve to DWV, and a refuge of the rare DWV-C dominating the easternmost Varroa-free islands. While all four detected viruses investigated here were affected by Varroa for one or two parameters (usually prevalence and/or the Richness component of ASV diversity), the strongest effect was observed for the multi-strain LSV. Varroa unambiguously led to elevated prevalence, load, and diversity (Richness and Shannon Index) of LSV, with these results largely shaped by LSV-2, a major LSV strain. Unprecedented insights into the mite-virus relationship were further gained from implementing a phylogeographic approach. In addition to enabling the identification of a novel LSV strain that dominated the unique viral landscape of the easternmost islands, this approach, in combination with the recovered diversity patterns, strongly suggests that Varroa is driving the evolutionary change of LSV in the Azores. This study greatly advances the current understanding of the effect of Varroa on the epidemiology and adaptive evolution of these less-studied viruses, whose relationship with Varroa has thus far been poorly defined.

Niche modelling and landscape genetics of the yellow-legged hornet (Vespa velutina): An integrative approach for evaluating central-marginal population dynamics in Europe.

Genetic diversity is an important biological trait for a successful invasion. During the expansion across a new territory, an invasive species may face unprecedented ecological conditions that will determine its demography and genetic diversity. The first record of the yellow-legged hornet (Vespa velutina) in Europe dates back to 2004 in France, from where it has successfully spread through a large territory in the continent, including Italy, Spain and Portugal. Integrative approaches offer a powerful strategy to detect and understand patterns of genetic variation in central and marginal populations. Here, we have analysed the relationship between genetic diversity parameters inferred from 15 V. velutina nuclear DNA microsatellite loci, and geographical and environmental drivers, such as the distance to the introduction focus, environmental suitability and distance to native and invasive niche centroids. Our results revealed a central-marginal dynamic, where allelic richness decreased towards the edge of the expansion range. The low environmental suitability of the territories invaded by marginal populations could prevent a diverse population from establishing and reducing the genetic diversity in populations at the expansion edge. Moreover, Markov chain Monte Carlo analysis showed both geographical and environmental distances were influencing population genetic differentiation. This study highlights the importance of combining genetic analysis with geographical and environmental drivers to understand genetic trends of invasive species to new environment.

Development of a loop-mediated isothermal amplification assay for the rapid detection of Styphnolobium japonicum (L.) Schott as an adulterant of Ginkgo biloba (L.).

BACKGROUND: Species adulteration is a concern in herbal products, especially when plant substitutes of lower economic value replace valuable botanicals. Styphnolobium japonicum is well known as a potential adulterant of Ginkgo biloba, which is one of the most demanded medicinal plants due to its wide use in pharmaceuticals, food supplements, and traditional medicine. Despite bearing some resemblance to ginkgo's flavonol composition, S. japonicum lacks many of G. biloba's desired therapeutic properties. To prevent adulteration practices, it is crucial to implement rigorous quality control measures, including fast and simple diagnostic tools that can be used on-field. PURPOSE: This study aims to develop for the first time a species-specific loop-mediated isothermal amplification (LAMP) method for the fast identification of S. japonicum in ginkgo-containing products. METHODS: A set of four specific primers (SjF3, SjB3, SjFIP, and SjBIP) and loop primers (SjLF and SjLB) were designed for a LAMP based assay using the 5.8S partial sequence and the internal transcribed spacer 2 of nuclear ribosomal DNA of S. japonicum. RESULTS: The successful amplification of the LAMP assay was inspected through visual detection, with the highest intensity recorded at the optimal conditions set at 68 °C for 40 min. The primers showed high specificity and were able to accurately discriminate S. japonicum from G. biloba and 49 other species of medicinal plants. Furthermore, the proposed LAMP assay proved to be fast, selective, and highly sensitive, as demonstrated by the absolute and relative limits of detection, which were reached at 0.5 pg for S. japonicum DNA and 0.01 % S. japonicum in G. biloba, respectively. CONCLUSIONS: This novel approach allows easy identification and discrimination of S. japonicum as a potential adulterant of G. biloba, thus being a useful tool for quality control. Compared to chromatographic or PCR-based methods, the assay proved to be fast, sensitive and did not require expensive equipment, thus offering the possibly usage in field analysis.

Semi-automated sequence curation for reliable reference datasets in ITS2 vascular plant DNA (meta-)barcoding.

One of the most critical steps for accurate taxonomic identification in DNA (meta)-barcoding is to have an accurate DNA reference sequence dataset for the marker of choice. Therefore, developing such a dataset has been a long-term ambition, especially in the Viridiplantae kingdom. Typically, reference datasets are constructed with sequences downloaded from general public databases, which can carry taxonomic and other relevant errors. Herein, we constructed a curated (i) global dataset, (ii) European crop dataset, and (iii) 27 datasets for the EU countries for the ITS2 barcoding marker of vascular plants. To that end, we first developed a pipeline script that entails (i) an automated curation stage comprising five filters, (ii) manual taxonomic correction for misclassified taxa, and (iii) manual addition of newly sequenced species. The pipeline allows easy updating of the curated datasets. With this approach, 13% of the sequences, corresponding to 7% of species originally imported from GenBank, were discarded. Further, 259 sequences were manually added to the curated global dataset, which now comprises 307,977 sequences of 111,382 plant species.

Frequent Parasitism of Apis mellifera by Trypanosomatids in Geographically Isolated Areas with Restricted Beekeeping Movements.

Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae. Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques, through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L. passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae.

Honey bee (Apis mellifera) wing images: a tool for identification and conservation.

BACKGROUND: The honey bee (Apis mellifera) is an ecologically and economically important species that provides pollination services to natural and agricultural systems. The biodiversity of the honey bee in parts of its native range is endangered by migratory beekeeping and commercial breeding. In consequence, some honey bee populations that are well adapted to the local environment are threatened with extinction. A crucial step for the protection of honey bee biodiversity is reliable differentiation between native and nonnative bees. One of the methods that can be used for this is the geometric morphometrics of wings. This method is fast, is low cost, and does not require expensive equipment. Therefore, it can be easily used by both scientists and beekeepers. However, wing geometric morphometrics is challenging due to the lack of reference data that can be reliably used for comparisons between different geographic regions. FINDINGS: Here, we provide an unprecedented collection of 26,481 honey bee wing images representing 1,725 samples from 13 European countries. The wing images are accompanied by the coordinates of 19 landmarks and the geographic coordinates of the sampling locations. We present an R script that describes the workflow for analyzing the data and identifying an unknown sample. We compared the data with available reference samples for lineage and found general agreement with them. CONCLUSIONS: The extensive collection of wing images available on the Zenodo website can be used to identify the geographic origin of unknown samples and therefore assist in the monitoring and conservation of honey bee biodiversity in Europe.

A novel real-time PCR coupled with high resolution melting analysis as a simple and fast tool for the entomological authentication of honey by targeting Apis mellifera mitochondrial DNA.

Honey is one of the foods easily adulterated worldwide. Recently, the analysis of honeybee DNA has been proposed as a useful tool to authenticate the entomological origin of honey. However, the methods proposed so far require more than one polymerase chain reaction (PCR) and the use of agarose gels, making the authentication process laborious and lengthy. In this work, a novel real-time PCR coupled with high-resolution melting (HRM) analysis of a 150 bp fragment of the cytochrome c oxidase I (COI) gene is proposed as a fast and simple tool to assess honey's entomological origin by discriminating the mitochondrial DNA lineages of European honey bees (A, M and C lineages). In addition, the new tool allowed the differentiation of honeys produced by different mitotypes of C-lineage ancestry. The method showed high analytical performance and was able to successfully identify the entomological origin of honeys of known origin obtained from research apiaries/beekeepers. Therefore, it was applied to 44 commercial honeys from different countries. It confirmed the entomological authenticity of French PDO honeys that should be produced by the Corse ecotype A. m. mellifera. For the remaining honeys, the results were also in good agreement with the declared geographical origin. However, three honeys from Slovenia did not cluster with C2 mitotype A. m. carnica as expected, suggesting the mixture of honeys produced by honeybees of different mitotypes.

Complex population structure and haplotype patterns in the Western European honey bee from sequencing a large panel of haploid drones.

Honey bee subspecies originate from specific geographical areas in Africa, Europe and the Middle East, and beekeepers interested in specific phenotypes have imported genetic material to regions outside of the bees' original range for use either in pure lines or controlled crosses. Moreover, imported drones are present in the environment and mate naturally with queens from the local subspecies. The resulting admixture complicates population genetics analyses, and population stratification can be a major problem for association studies. To better understand Western European honey bee populations, we produced a whole genome sequence and single nucleotide polymorphism (SNP) genotype data set from 870 haploid drones and demonstrate its utility for the identification of nine genetic backgrounds and various degrees of admixture in a subset of 629 samples. Five backgrounds identified correspond to subspecies, two to isolated populations on islands and two to managed populations. We also highlight several large haplotype blocks, some of which coincide with the position of centromeres. The largest is 3.6 Mb long and represents 21% of chromosome 11, with two major haplotypes corresponding to the two dominant genetic backgrounds identified. This large naturally phased data set is available as a single vcf file that can now serve as a reference for subsequent populations genomics studies in the honey bee, such as (i) selecting individuals of verified homogeneous genetic backgrounds as references, (ii) imputing genotypes from a lower-density data set generated by an SNP-chip or by low-pass sequencing, or (iii) selecting SNPs compatible with the requirements of genotyping chips.

Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcoding.

Pollen metabarcoding is emerging as a powerful tool for ecological research and offers unprecedented scale in citizen science projects for environmental monitoring via honey bees. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. While in metabarcoding studies pollen has been preserved at - 20 °C (FRZ), this is not the best method for citizen scientists. Herein, we compared this method with ethanol (EtOH), silica gel (SG) and room temperature (RT) for preservation of pollen collected from hives in Austria and Denmark. After ~ 4 months of storage, DNAs were extracted with a food kit, and their quality and concentration measured. Most DNA extracts exhibited 260/280 absorbance ratios close to the optimal 1.8, with RT samples from Austria performing slightly worse than FRZ and SG samples (P < 0.027). Statistical differences were also detected for DNA concentration, with EtOH samples producing lower yields than RT and FRZ samples in both countries and SG in Austria (P < 0.042). Yet, qualitative and quantitative assessments of floral composition obtained using high-throughput sequencing with the ITS2 barcode gave non-significant effects of preservation methods on richness, relative abundance and Shannon diversity, in both countries. While freezing and ethanol are commonly employed for archiving tissue for molecular applications, desiccation is cheaper and easier to use regarding both storage and transportation. Since SG is less dependent on ambient humidity and less prone to contamination than RT, we recommend SG for preserving pollen for metabarcoding. SG is straightforward for laymen to use and hence robust for widespread application in citizen science studies.

A SNP assay for assessing diversity in immune genes in the honey bee (Apis mellifera L.).

With a growing number of parasites and pathogens experiencing large-scale range expansions, monitoring diversity in immune genes of host populations has never been so important because it can inform on the adaptive potential to resist the invaders. Population surveys of immune genes are becoming common in many organisms, yet they are missing in the honey bee (Apis mellifera L.), a key managed pollinator species that has been severely affected by biological invasions. To fill the gap, here we identified single nucleotide polymorphisms (SNPs) in a wide range of honey bee immune genes and developed a medium-density assay targeting a subset of these genes. Using a discovery panel of 123 whole-genomes, representing seven A. mellifera subspecies and three evolutionary lineages, 180 immune genes were scanned for SNPs in exons, introns (< 4 bp from exons), 3' and 5´UTR, and < 1 kb upstream of the transcription start site. After application of multiple filtering criteria and validation, the final medium-density assay combines 91 quality-proved functional SNPs marking 89 innate immune genes and these can be readily typed using the high-sample-throughput iPLEX MassARRAY system. This medium-density-SNP assay was applied to 156 samples from four countries and the admixture analysis clustered the samples according to their lineage and subspecies, suggesting that honey bee ancestry can be delineated from functional variation. In addition to allowing analysis of immunogenetic variation, this newly-developed SNP assay can be used for inferring genetic structure and admixture in the honey bee.

Authoritative subspecies diagnosis tool for European honey bees based on ancestry informative SNPs.

BACKGROUND: With numerous endemic subspecies representing four of its five evolutionary lineages, Europe holds a large fraction of Apis mellifera genetic diversity. This diversity and the natural distribution range have been altered by anthropogenic factors. The conservation of this natural heritage relies on the availability of accurate tools for subspecies diagnosis. Based on pool-sequence data from 2145 worker bees representing 22 populations sampled across Europe, we employed two highly discriminative approaches (PCA and FST) to select the most informative SNPs for ancestry inference. RESULTS: Using a supervised machine learning (ML) approach and a set of 3896 genotyped individuals, we could show that the 4094 selected single nucleotide polymorphisms (SNPs) provide an accurate prediction of ancestry inference in European honey bees. The best ML model was Linear Support Vector Classifier (Linear SVC) which correctly assigned most individuals to one of the 14 subspecies or different genetic origins with a mean accuracy of 96.2% ± 0.8 SD. A total of 3.8% of test individuals were misclassified, most probably due to limited differentiation between the subspecies caused by close geographical proximity, or human interference of genetic integrity of reference subspecies, or a combination thereof. CONCLUSIONS: The diagnostic tool presented here will contribute to a sustainable conservation and support breeding activities in order to preserve the genetic heritage of European honey bees.

Evaluation of Suppressed Mite Reproduction (SMR) Reveals Potential for Varroa Resistance in European Honey Bees (Apis mellifera L.).

In the fight against the Varroa destructor mite, selective breeding of honey bee (Apis mellifera L.) populations that are resistant to the parasitic mite stands as a sustainable solution. Selection initiatives indicate that using the suppressed mite reproduction (SMR) trait as a selection criterion is a suitable tool to breed such resistant bee populations. We conducted a large European experiment to evaluate the SMR trait in different populations of honey bees spread over 13 different countries, and representing different honey bee genotypes with their local mite parasites. The first goal was to standardize and validate the SMR evaluation method, and then to compare the SMR trait between the different populations. Simulation results indicate that it is necessary to examine at least 35 single-infested cells to reliably estimate the SMR score of any given colony. Several colonies from our dataset display high SMR scores indicating that this trait is present within the European honey bee populations. The trait is highly variable between colonies and some countries, but no major differences could be identified between countries for a given genotype, or between genotypes in different countries. This study shows the potential to increase selective breeding efforts of V. destructor resistant populations.

The honeybee (Apis mellifera) developmental state shapes the genetic composition of the deformed wing virus-A quasispecies during serial transmission.

The main biological threat to the western honeybee (Apis mellifera) is the parasitic mite Varroa destructor, largely because it vectors lethal epidemics of honeybee viruses that, in the absence of this mite, are relatively innocuous. The severe pathology is a direct consequence of excessive virus titres caused by this novel transmission route. However, little is known about how the virus adapts genetically during transmission and whether this influences the pathology. Here, we show that upon injection into honeybee pupae, the deformed wing virus type-A (DWV-A) quasispecies undergoes a rapid, extensive expansion of its sequence space, followed by strong negative selection towards a uniform, common shape by the time the pupae have completed their development, with no difference between symptomatic and asymptomatic adults in either DWV titre or genetic composition. This suggests that the physiological and molecular environment during pupal development has a strong, conservative influence on shaping the DWV-A quasispecies in emerging adults. There was furthermore no evidence of any progressive adaptation of the DWV-A quasispecies to serial intra-abdominal injection, simulating mite transmission, despite the generation of ample variation immediately following each transmission, suggesting that the virus either had already adapted to transmission by injection, or was unaffected by it.

Wing Geometric Morphometrics of Workers and Drones and Single Nucleotide Polymorphisms Provide Similar Genetic Structure in the Iberian Honey Bee (Apis mellifera iberiensis).

Wing geometric morphometrics has been applied to honey bees (Apis mellifera) in identification of evolutionary lineages or subspecies and, to a lesser extent, in assessing genetic structure within subspecies. Due to bias in the production of sterile females (workers) in a colony, most studies have used workers leaving the males (drones) as a neglected group. However, considering their importance as reproductive individuals, the use of drones should be incorporated in these analyses in order to better understand diversity patterns and underlying evolutionary processes. Here, we assessed the usefulness of drone wings, as well as the power of wing geometric morphometrics, in capturing the signature of complex evolutionary processes by examining wing shape data, integrated with geographical information, from 711 colonies sampled across the entire distributional range of Apis mellifera iberiensis in Iberia. We compared the genetic patterns reconstructed from spatially-explicit shape variation extracted from wings of both sexes with that previously reported using 383 genome-wide SNPs (single nucleotide polymorphisms). Our results indicate that the spatial structure retrieved from wings of drones and workers was similar (r = 0.93) and congruent with that inferred from SNPs (r = 0.90 for drones; r = 0.87 for workers), corroborating the clinal pattern that has been described for A. m. iberiensis using other genetic markers. In addition to showing that drone wings carry valuable genetic information, this study highlights the capability of wing geometric morphometrics in capturing complex genetic patterns, offering a reliable and low-cost alternative for preliminary estimation of population structure.

Hygroregulation, a key ability for eusocial insects: Native Western European honeybees as a case study.

Sociality has brought many advantages to various hymenoptera species, including their ability of regulating physical factors in their nest (e.g., temperature). Although less studied, humidity is known to be important for egg, larval and pupal development, and also for nectar concentration. Two subspecies of Apis mellifera of the M evolutionary lineage were used as models to test the ability of a superorganism (i.e. honeybee colony) to regulate the humidity in its nest (i.e. "hygroregulation hypothesis") in four conservation centers: two in France (A. m. mellifera) and two in Portugal (A. m. iberiensis). We investigated the ability of both subspecies to regulate the humidity in hives daily, but also during the seasons for one complete year. Our data and statistical analysis demonstrated the capacity of the bees to regulate humidity in their hive, regardless of the day, season or subspecies. Furthermore, the study showed that humidity in beehives is stable even during winter, when brood is absent, and when temperature is known to be less stable in the beehives. These results suggest that humidity is important for honeybees at every life stage, maybe because of the 'imprint' of the evolutionary history of this hymenopteran lineage.

Towards honey authentication: Differentiation of Apis mellifera subspecies in European honeys based on mitochondrial DNA markers.

Honey is the natural sweet substance produced by Apis mellifera honeybees in Europe. Depending on the country/region, the A. mellifera subspecies native to Europe belong to three different lineages: A (A. m. iberiensis), M (A. m. iberiensis and A. m. mellifera) and C (A. m. ligustica and A. m. carnica). In this work, two DNA-based approaches were developed with the aim of entomological authentication of European honeys. A cytb specific PCR assay was proposed to identify A-lineage honeybees, while a second method based on real-time PCR coupled to high resolution melting analysis targeting the COI gene was developed to differentiate C- and M-lineages honeybees. The proposed methodologies were validated successfully with honeys of known origin and applied to the entomological authentication of 20 commercial samples from different European countries. The results highlight the predominance of honeys from C-lineage honeybees in Europe, except in Iberian Peninsula countries (honey from A-lineage honeybees).

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.

High sample throughput genotyping for estimating C-lineage introgression in the dark honeybee: an accurate and cost-effective SNP-based tool.

The natural distribution of the honeybee (Apis mellifera L.) has been changed by humans in recent decades to such an extent that the formerly widest-spread European subspecies, Apis mellifera mellifera, is threatened by extinction through introgression from highly divergent commercial strains in large tracts of its range. Conservation efforts for A. m. mellifera are underway in multiple European countries requiring reliable and cost-efficient molecular tools to identify purebred colonies. Here, we developed four ancestry-informative SNP assays for high sample throughput genotyping using the iPLEX Mass Array system. Our customized assays were tested on DNA from individual and pooled, haploid and diploid honeybee samples extracted from different tissues using a diverse range of protocols. The assays had a high genotyping success rate and yielded accurate genotypes. Performance assessed against whole-genome data showed that individual assays behaved well, although the most accurate introgression estimates were obtained for the four assays combined (117 SNPs). The best compromise between accuracy and genotyping costs was achieved when combining two assays (62 SNPs). We provide a ready-to-use cost-effective tool for accurate molecular identification and estimation of introgression levels to more effectively monitor and manage A. m. mellifera conservatories.