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In this study, honey bees fromthe Macaronesian archipelago of the Azoreswere extensively surveyed to unveil diversity patterns. A total of 638 colonies were analyzed over two time periods using mtDNA and wing geometric morphometrics. The genetic composition revealed to be heterogeneous and related to historical and contemporary human-mediated introductions. The close relationship of Azorean populations with those from northern Portugal supports historical introductions by Portuguese settlers. The African sublineage AIII prevailed on five islands, contrasting with three islands where C haplotypes were dominant. On Pico and Graciosa, C haplotypes are due to recent imports of commercial queens. On Faial, the sudden replacement of AIII by C haplotypes coincided with arrival of Varroa destructor . This study deepens the current understanding of Macaronesian honey bees, suggesting that they are variants of the Iberian honey bee with differential levels of Cderived introgression. CASM was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Financial support was provided through the program COMPETE 2020 – POCI (Programa Operacional para a Competividade e Internacionalização) and by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of the project BeeHappy (POCI-01-0145-FEDER-029871). info:eu-repo/semantics/publishedVersion

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.

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.

The tRNA -COX2 intergenic region of the mitochondrial DNA has been used for assessing diversity in honey bee (Apis mellifera L.) populations worldwide. However, differential mutation rates in different partitions of the mitogenome may produce incongruent results. In this study, we sequenced 123 mitogenomes of 7 subspecies from lineages A, M, and C. This allowed generating a comprehensive dataset to investigate the phylogenetic and phylogeographic congruence among the mitogenome, individual genes, and the tRNA -COX2 region. We showed that the diversity patterns inferred from the tRNA -COX2 marker are not fully paralleled by those obtained with the mitogenome and the individual genes; while the three lineages are supported by these, the African sub-lineages and the haplotypes are not. Thus, conclusions drawn from the tRNA -COX2 region need to be taken with caution and this marker may not be appropriate to infer phylogenetic relationships between honey bee colonies. leu leu leu leu, We are deeply indebted to John C. Patton, Phillip San Miguel, Paul Parker, and Rick Westerman, Univer sity of Purdue, for resequencing the whole genomes, and to Pilar de la Rúa and to Carlo Amodeo for providing A. m. sahariensis and A. m. siciliana samples, respectively. Analyses were performed using computational resources at the Polytechnic Institute of Bragança. DH was supported by a PhD scholarship (SFRH/BD/84195/2012) from the Portuguese Science Foundation (FCT). This work was supported by FCT through the programs COMPETE/QREN/EU (PTDC/BIA-BEC/099640/2008) and the 2013-2014 BiodivERsA/ FACCE-JPI joint call for research proposals, with the national funders FCT (Portugal), CNRS (France), and MEC (Spain).

In Europe, several translocation experiments suggested that native populations of Apis mellifera are adapted to local climate and flora. However, so far, no study has been conducted on the Iberian honeybee, Apis mellifera iberiensis. The goal of this study was to assess the existence of genotype-environment interaction (GEI), and consequently local adaptation, in the Iberian honeybee. In 2015 two apiaries were set up, each one with 36 colonies (18 of the origin Bragança and 18 of the origin Vila do Bispo), in two latitudinal extremes of Portugal: Bragança (north) and Vila do Bispo (south). Several traits of the 36 colonies were measured for almost 2 years, including: number of brood and pollen cells, honey yield, survival, and Varroa destructor infestation. The analyses were performed using t-Student and Mann-Whitney tests to compare those traits between the two origins in the same apiary and the same origin between the two apiaries. The survival analysis was performed using the Cox proportional hazard model in R. Colonies of the southern origin Vila do Bispo showed a tendency to collect more pollen and consequently they produced a higher number of brood cells, had a higher varroa infestation level and a lower survival rate than colonies of the origin Bragança in both locations. Honey yield was the only trait that showed existence of GEI, and therefore local adaptation, since the local honeybees had a higher honey production in their apiary of origin. Additionally, the differences between the two origins were sharper in more favourable environments where the honeybees can better express their genetic potential. Our findings highlight the importance of protecting local honeybee diversity in a period of increasing selection pressures such as climate change, agricultural land overuse and novel pathogens and parasites. Thisresearchwas funded through the 2013-2014~'BiodivERsA/FACCE-JPI Joint call for research proposals, with the national funders FCT(Portugal), CNRS (France), and MEC(Spain). info:eu-repo/semantics/publishedVersion

12 Páginas 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. Abstract. Introduction. Material and methods. Results. Discussion. References.

Honey bee colony data collected continuously together with climate data are of great importance because they provide the opportunity to understand colony phenology. Continuous monitoring of honey bee colonies initiated long time ago with Gates (1914) and Hambleton (1925), when they assessed weather effects on hive weight using mechanical scales. Currently, the study of colony dynamics has been intensified with development of new technologies such as electronic scales, hygro-buttons, thermo-buttons, and computer-assisted digital image analysis of brood combs. Studies of colony dynamics are of great interest in Portugal because of large climatic (and flora) differences between the two latitudinal extremes and because of distinct genetic backgrounds of the native subspecies, Apis mellifera iberiensis (Pinto et al. 2013). In this study we will compare the temporal dynamics of colony weight and nest temperature and humidity of 12 colonies, which have been continuously monitored since July of 2015 with electronic scales and thermo-hygro-buttons, set up in apiaries located in two latitudinal extremes of Portugal. These colony variables will be correlated with climatic data (temperature, humidity, wind speed, and rain) obtained from automatic weather stations installed in the two apiaries. This research is funded through the 2013-2014 BiodivERsA/FACCE-JPI Joint call for research proposals, with the national funders FCT (Portugal), CNRS (France), and MEC (Spain). info:eu-repo/semantics/publishedVersion

The Iberian honey bee has been one of the most surveyed subspecies for genetic diversity. Yet, previous studies have missed an important component of Iberian honey bee variation harbored by populations inhabiting the Atlantic side of the Iberian Peninsula. Herein, we provide a fuller picture of the Iberian honey bee maternal diversity by revealing 16 novel haplotypes detected in honey bees from Portugal. Of the 16 haplotypes, all of African ancestry, 15 belong to the Atlantic sub-lineage AIII while only one fits the most common sub-lineage AI. This level of new variation is remarkable as it represents a 59% increase in the widerange African lineage and a 188% in sub-lineage AIII. Our findings further highlight the complexity of the Iberian honey bee diversity patterns and reinforce the importance of this southernmost European territory as a reservoir of Apis mellifera genetic diversity, a resource increasingly important in a rapidly changing and demanding world. Fundação para a Ciência e Tecnologia

Genome-wide scan approaches to detect signatures of selection have become popular tools to study local adaptation at the molecular level and single nucleotide polymorphisms (SNPs) are seemingly the most appropriate marker for that endeavor. SNPs (i) provide a genome-wide coverage, (ii) generate high quality data, (iii) are suitable for automated high throughput technologies allowing genotyping of hundreds to thousands of loci in many individuals, and (iv) are commonly observed in functional genes. In this work, a total of 711 individuals of A. m. iberiensis were collected in 23 sites across three north-south transepts in the Iberian Peninsula. This collection was genotyped for 383 SNPs using the GoldenGate assay of Illumina. Then, the SNP dataset was examined in search for the footprints of selection with the following five different methods: (i) coalescence - LOSITAN and ARLEQUIN, (ii) Bayesian - BAYESFST and BAYESCAN, and (iii) Spatial Analysis - matSAM. These methods identified 10 loci exhibiting strong selection signal. The 10 loci were then located in the honey bee genome and their putative functions were determined using the honey bee genomic resources. The 10 loci were mapped to putative genes with diverse biological functions, which included vision, detoxification metabolism, and immunity. Fundação para a Ciência e Tecnologia

The Iberian honey bee (Apis mellifera iberiensis) has been intensely surveyed for genetic variation with different markers ranging from morphology, allozymes, mitochondrial DNA (mt DNA), to microsatellites. Some of these markers have revealed non-concordant complex patterns of genetic variation, which led scientists to evoke competing hypotheses for the origin of Iberian honey bees. While complex patterns and underlying historical processes are typical of hybrid zones, the use of more powerful molecular and analytical tools and the fine-scale sampling promised to help dissecting the complexities of the Iberian hybrid zone. In this study, we conducted a genome-wide sampling by genotyping over 384 SNPs (single nucleotide polymorphisms) and sequenced an intergenic fragment of the mt DNA in 711 georeferenced honey bee samples collected across three North-South transects in the Iberian Peninsula. Both mt DNA and SNP datasets were analyzed using spatial tools to represent the structure generated by both types of molecular markers. We found concordant spatial patterns between markers which led to rejection of the standing hypothesis of recent human introductions and selection as the processes shaping Iberian honey bees patterns. This study shows that the fine-scale genomic and spatial analyses can reveal patterns which would otherwise had been undetected. Fundação para a Ciência e Tecnologia

Over the last 20 years Iberian honey bees (IHB) have been scrutinized with a variety of genetic markers including morphology, allozymes, mtDNA, and microsatellites. The complex patterns and processes revealed have yet to be fully understood. While mtDNA is useful to reveal the maternal component, biparental markers capture genome-wide effects (admixture, expansions, and contractions) and locus-specific effects (selection). Accordingly, we expect to dissect out, through genome-wide scans, neutral and selection effects. In this study, the Illumina GoldenGate Assay was used to genotype SNPs across the complete genome of IHBs. The SNP data set was analyzed for evidence of selection using a frequentist, FDIST, and a Bayesian method, BayeScan. Additionally, a spatial analysis method (MatSAM) was used to test for associations between SNPs and climatic variables. With these approaches we detected 8 loci with a strong signal of directional selection. The genome positions and putative gene functions, given in the HB genomic resources, suggest that these putatively selected loci are strong candidates for selection. The spatial representation of these same loci reveals a clinal pattern of variation, further supporting selection. Our approach allows for a rigorous consideration of selection as the underlying cause of observed patterns of diversity and contrast with previous studies where evocation of selection was merely speculative. Fundação para a Ciência e Tecnologia

Identification of population structure, a primary goal in population genetics, is easily performed because there is a number of methods available, implemented by user-friendly software packages. However, the user must be cautious when inferring population structure because spurious results may be obtained when there is strong linkage disequilibrium. With recent development of high-density SNPs we have now more power to interrogate the honey bee genome. However, the greater the number of loci genotyped the greater the chance of scoring loci that are linked. In addition, events such as population bottleneck, small effective population size, genetic drift, and admixture may also generate strong linkage disequilibrium. According to Kaeuffer et al. (2007), correlation rLD is the best way to deal with linkage disequilibrium. These authors recommend removing loci with rLD higher than 0.5 when inferring structure. In this study we used the GoldenGate Assay of Illumina to genotype over 1221 loci in individuals sampled from populations of A.m. iberiensis and A.m. mellifera. In this dataset we used the genetic distance between SNPs and rLD to test the effect of linkage in the number of clusters and the introgression level inferred by the clustering method implemented in the software STRUCTURE. Fundação para a Ciência e Tecnologia

The Honey bee, Apis mellifera L., occurs naturally in Africa, Middle East and Europe. The adaptation to a diversity of ecological conditions has led to evolution of over 24 subspecies. The honey bee subspecies that occurs in the Iberian Peninsula is Apis mellifera iberiensis, which is originated by natural hybridization between lineage A (African) and lineage M (western European). The objective of this study was to unravel the population structure of A. m. iberiensis by carrying out a genome wide analysis using SNPs. Over 711 individuals sampled across three transects (one along the Atlantic Coast, one along the Mediterranean coast, and one central) in the Iberian peninsula were genotyped for 1536 SNPs using the golden gate assay of Illumina. The genetic structure was analyzed by a Bayesian clustering method. It was observed a north – south cline in the three transects and the largest difference was exhibited between the Atlantic populations and the other two transects.