Dora Henriques

Genetic integrity of the Dark European honey bee (Apis mellifera mellifera) from protected populations: a genome-wide assessment using SNPs and mtDNA sequence data

Summary The recognition that the Dark European honey bee, Apis mellifera mellifera, is increasingly threatened in its native range has led to the establishment of conservation programmes and protected areas throughout western Europe. Previous molecular surveys showed that, despite management strategies to preserve the genetic integrity of A. m. mellifera, protected populations had a measurable component of their gene pool derived from commercial C-lineage honey bees. Here we used both sequence data from the tRNAleu-cox2 intergenic mtDNA region and a genome-wide scan, with over 1183 single nucleotide polymorphisms (SNPs), to assess genetic diversity and introgression levels in several protected populations of A. m. mellifera, which were then compared with samples collected from unprotected populations. MtDNA analysis of the protected populations revealed a single colony bearing a foreign haplotype, whereas SNPs showed varying levels of introgression ranging from virtually zero in Norway to about 14% in Denmark. Introgression overall was higher in unprotected (30%) than in protected populations (8%), and is reflected in larger SNP diversity levels of the former, although opposite diversity levels were observed for mtDNA. These results suggest that, despite controlled breeding, some protected populations still require adjustments to the management strategies to further purge foreign alleles, which can be identified by SNPs.

Signatures of selection in the Iberian honey bee (Apis mellifera iberiensis) revealed by a genome scan analysis of single nucleotide polymorphisms.

Understanding the genetic mechanisms of adaptive population divergence is one of the most fundamental endeavours in evolutionary biology and is becoming increasingly important as it will allow predictions about how organisms will respond to global environmental crisis. This is particularly important for the honey bee, a species of unquestionable ecological and economical importance that has been exposed to increasing human‐mediated selection pressures. Here, we conducted a single nucleotide polymorphism (SNP)‐based genome scan in honey bees collected across an environmental gradient in Iberia and used four FST‐based outlier tests to identify genomic regions exhibiting signatures of selection. Additionally, we analysed associations between genetic and environmental data for the identification of factors that might be correlated or act as selective pressures. With these approaches, 4.4% (17 of 383) of outlier loci were cross‐validated by four FST‐based methods, and 8.9% (34 of 383) were cross‐validated by at least three methods. Of the 34 outliers, 15 were found to be strongly associated with one or more environmental variables. Further support for selection, provided by functional genomic information, was particularly compelling for SNP outliers mapped to different genes putatively involved in the same function such as vision, xenobiotic detoxification and innate immune response. This study enabled a more rigorous consideration of selection as the underlying cause of diversity patterns in Iberian honey bees, representing an important first step towards the identification of polymorphisms implicated in local adaptation and possibly in response to recent human‐mediated environmental changes.

Reduced SNP Panels for Genetic Identification and Introgression Analysis in the Dark Honey Bee (Apis mellifera mellifera)

Beekeeping activities, especially queen trading, have shaped the distribution of honey bee (Apis mellifera) subspecies in Europe, and have resulted in extensive introductions of two eastern European C-lineage subspecies (A. m. ligustica and A. m. carnica) into the native range of the M-lineage A. m. mellifera subspecies in Western Europe. As a consequence, replacement and gene flow between native and commercial populations have occurred at varying levels across western European populations. Genetic identification and introgression analysis using molecular markers is an important tool for management and conservation of honey bee subspecies. Previous studies have monitored introgression by using microsatellite, PCR-RFLP markers and most recently, high density assays using single nucleotide polymorphism (SNP) markers. While the latter are almost prohibitively expensive, the information gained to date can be exploited to create a reduced panel containing the most ancestry-informative markers (AIMs) for those purposes with very little loss of information. The objective of this study was to design reduced panels of AIMs to verify the origin of A. m. mellifera individuals and to provide accurate estimates of the level of C-lineage introgression into their genome. The discriminant power of the SNPs using a variety of metrics and approaches including the Weir & Cockerham’s FST, an FST-based outlier test, Delta, informativeness (In), and PCA was evaluated. This study shows that reduced AIMs panels assign individuals to the correct origin and calculates the admixture level with a high degree of accuracy. These panels provide an essential tool in Europe for genetic stock identification and estimation of admixture levels which can assist management strategies and monitor honey bee conservation programs.

Revisiting the Iberian honey bee (Apis mellifera iberiensis) contact zone: maternal and genome-wide nuclear variations provide support for secondary contact from historical refugia.

Dissecting diversity patterns of organisms endemic to Iberia has been truly challenging for a variety of taxa, and the Iberian honey bee is no exception. Surveys of genetic variation in the Iberian honey bee are among the most extensive for any honey bee subspecies. From these, differential and complex patterns of diversity have emerged, which have yet to be fully resolved. Here, we used a genome‐wide data set of 309 neutrally tested single nucleotide polymorphisms (SNPs), scattered across the 16 honey bee chromosomes, which were genotyped in 711 haploid males. These SNPs were analysed along with an intergenic locus of the mtDNA, to reveal historical patterns of population structure across the entire range of the Iberian honey bee. Overall, patterns of population structure inferred from nuclear loci by multiple clustering approaches and geographic cline analysis were consistent with two major clusters forming a well‐defined cline that bisects Iberia along a northeastern–southwestern axis, a pattern that remarkably parallels that of the mtDNA. While a mechanism of primary intergradation or isolation by distance could explain the observed clinal variation, our results are more consistent with an alternative model of secondary contact between divergent populations previously isolated in glacial refugia, as proposed for a growing list of other Iberian taxa. Despite current intense honey bee management, human‐mediated processes have seemingly played a minor role in shaping Iberian honey bee genetic structure. This study highlights the complexity of the Iberian honey bee patterns and reinforces the importance of Iberia as a reservoir of Apis mellifera diversity.

Maternal diversity patterns of Ibero-Atlantic populations reveal further complexity of Iberian honeybees

Dissecting complex diversity patterns typically exhibited by organisms of hybrid zones is among the greatest challenges in evolutionary biology. This has been a long-standing goal of numerous Iberian honeybee studies. Herein, we further contribute to this goal by a fine-scale maternal survey of the Portuguese honeybee populations. In addition to confirm a predicted African ancestry of Portuguese populations and negligible levels of C-lineage introgression, our fine-scale survey revealed unparalleled levels of African maternal diversity and a remarkable north–south cline formed by African haplotypes of distinct ancestry. This survey further highlights the distinctiveness of Apis mellifera iberiensis by showing the existence of highly contrasting diversity patterns between the Atlantic and Mediterranean populations. Our findings further support an ancient natural invasion of the Iberian Peninsula possibly by Apis mellifera intermissa and also by Apis mellifera sahariensis. Protecting this African legacy is of unquestionable importance, especially with Iberia becoming warmer and dryer.

Mitochondrial DNA variation of Apis mellifera iberiensis: further insights from a large-scale study using sequence data of the tRNAleu-cox2 intergenic region

A large-scale survey of the Iberian honey bee (Apis mellifera iberiensis) diversity patterns, using sequence data of the tRNAleu-cox2 mitochondrial DNA (mtDNA) region, demonstrates that earlier studies based on the DraI test missed significant components of genetic variation. Based on results from this survey, existing haplotype names were revised and updated following a nomenclature system established earlier and extended herein for the intergenic region. A more complete picture of the complex diversity patterns of IHBs is revealed that includes 164 novel haplotypes, 113 belonging to lineage A and 51 to lineage M and within lineage A and 69 novel haplotypes that belong to sub-lineage AI, 13 to AII, and 31 to AIII. Within lineage M, two novel haplotypes show a striking architecture with features of lineages A and M, which based on sequence comparisons and relationships among haplotypes are seemingly ancestral. These data expand our knowledge of the complex architecture of the tRNAleu-cox2 intergenic region in Apis mellifera and re-emphasizes the importance of Iberia as a source of honey bee mtDNA diversity.

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.

Empirical comparison of microsatellite and SNP markers to estimate introgression in Apis mellifera mellifera

The genetic identity of the dark European honey bee, Apis mellifera mellifera is currently under pressure throughout most of its native range due to large scale commercial trade and replacement with honey bees of mainly Eastern European ancestry (C-lineage: Apis mellifera carnica and Apis mellifera ligustica). To counteract this process, numerous conservation efforts for the protection of native honey bees are sprouting across Europe. For the management of such protected areas and conservation breeding purposes, honey bee subspecies have been routinely identified through wing morphology and through DNA-hybrid tests using microsatellite markers. Currently, new methods are evolving including rapid innovations in single-nucleotide polymorphism (SNP) array technology and high-throughput sequencing. Here, we aim to quantify potential marker-specific biases of hybrid tests and give recommendations for applications in honey bee conservation management. Using an empirical dataset, we first assessed the accuracy of a recently developed reduced SNP panel to estimate C-lineage introgression in A. m. mellifera compared to whole-genome sequence (WGS) data. Using another independent data set, we estimated the differences in admixture proportions between the currently applied hybrid test based on microsatellites and the novel SNP test. We demonstrate that the SNP-based test which contains highly ancestry-informative markers is very efficient to estimate genome-wide ancestry. Furthermore, we report discrepancies between microsatellite and SNP-based admixture proportions. For conservation management, we, therefore, recommend the implementation of SNP-based hybrid tests to maintain high genetic variation within the breeding population, while minimizing influence of introduced honey bees.

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 populations 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 (Samβada, 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.