Identification of honey bee populations from the Azores: insights from wing geometric morphometrics

The geometric morphometrics of the wings has been an important method for the identification and evaluation of honey bee diversity patterns around the world. Honey bee populations of the Macaronesian archipelagos of Canaries and Madeira have been intensively surveyed for diversity using a variety of genetic markers. In contrast, honey bee populations inhabiting the Azorean archipelago have been largely undersampled. To fill this gap, we sampled 473 colonies from across the Azores and assessed diversity patterns using a geometric morphometrics approach. A total of 5 forewings were collected per colony, mounted in a slide and photographed with a stereomicroscope. Additionally, the forewings representing 711 colonies of A. m. iberiensis, 11 A. m. ligustica, 15 A. m. carnica and 12 A. m. caucasia were used as reference samples. To extract shape information, 19 anatomical landmarks were plotted across the veins’ intersections in the wing structures of all individuals. The analyses of wing shape were performed in MorphoJ using the Procrustes superimposition method. Shape differences were investigated through multivariate statistical analysis and Mahalanobis and Procrustes distances were used to construct a dendrogram of the morphological proximity. Results revealed the power of landmark-based methods to discriminate different honey bee populations from the Azores, and also to distinguish them from the subspecies of the reference collection. The wing geometric morphometrics patterns showed that while, overall, populations from the Azores exhibited a closer relationship with A. m. iberiensis, some populations, especially those from the islands of Graciosa, but also Terceira and Pico tended to cluster closer to A. m. ligustica, A. m. carnica. Several non-mutually exclusive factors can contribute to the observed wing patterns such as the recent human-mediated introductions of subspecies from Eastern Europe, and the founder effect resulting from honey bee introductions in historical times. Moreover, the particular insular environment and the barrier to gene flow due to geographical isolation possibly shaped the diversity patterns currently observed in the Azores.info:eu-repo/semantics/publishedVersio

Estrutura populacional da abelha ibérica (Apis mellifera iberiensis) inferida por análise de morfometria geométrica em obreiras

A abelha ibérica (Apis mellifera iberiensis) é uma das subespécies de abelha melífera mais estudadas no mundo no que concerne aos padrões de diversidade genética. Esses estudos têm revelado resultados incongruentes e dependentes do tipo de marcador genético utilizado. No sentido de contribuirmos para uma melhor compreensão e representação dos padrões espaciais de diversidade genética da abelha ibérica e forças evolutivas subjacentes, recentemente amostramos na Península Ibérica 711 colónias ao longo de três transectos: um na costa atlântica, um na costa mediterrânica e outro no centro. Contrariamente aos microssatélites, que são incapazes de detetar estrutura genética, os marcadores mitocondriais (região intergénica tRNAleu-cox2) e nucleares (polimorfismos de nucleótido simples, SNPs) revelaram a existência de um cline com orientação nordeste-sudoeste, padrão consistente com um processo de contacto secundário pós-glacial (Chávez-Galarza et al. 2015). No seguimento destes resultados, queríamos testar o poder da morfometria geométrica na captura do padrão clinal previamente encontrado. Para tal, examinamos a asa anterior direita de cinco obreiras de cada uma das 711 colónias. Em cada asa foram marcados 19 pontos nas interseções das veias e, após alinhamento Procrustes, foi calculada a distância entre todos os pontos. A matriz de distâncias obtida foi usada para inferir a estrutura populacional aplicando uma análise multivariada espacial baseada na análise de componentes principais e na autocorrelaçao de Moran. Surpreendentemente, os nossos resultados mostram a presença de dois grupos com uma distribuição espacial totalmente congruente com o mtDNA e os SNPs, indicando que a morfometria geométrica é capaz de capturar padrões genéticos moldados por processos evolutivos complexos. A adição do componente geográfico nas análises multivariadas contribuiu consideravelmente para os resultados e deverá ser incorporada em futuras análises morfométricas populacionais.info:eu-repo/semantics/publishedVersio

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 fromspatially-explicit shape variation extracted fromwings 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, o ering a reliable and low-cost alternative for preliminary estimation of population structure.This research was funded by 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). FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) provided funds for TMF (2011/07857-9) and JSGT (2011/02434-2).info:eu-repo/semantics/publishedVersio

Historical and contemporaneous human-mediated processes left a strong genetic signature on honey bee populations from the Macaronesian archipelago of the Azores

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/publishedVersio

Does geometric morphometrics provide congruent results with SNP data? The case of Iberian honey bee (Apis mellifera iberiensis)

While molecular markers are usually preferred to infer population structure, geometric morphometrics is a cheap method that has been widely applied to the wings of female honey bees to identify subspecies or lineages and can be used alternatively or complementarily to molecular markers. However, the power of geometric morphometrics to capture the signature of complex evolutionary processes has not been tested in honey bees. In this study, we applied geometric morphometrics, combined with geographical information, to the right forewings of female individuals from 711 colonies distributed along the Iberian Peninsula, which contains a complex population structure. The results were further compared with those obtained using 383 SNPs. Our data showed that geometric morphometrics provided a similar spatial structure of SNPs data (r=0.90). Our findings reinforce the power of spatially explicit wing geometric morphometrics data to capture the signature of complex evolutionary processes. Thus, this method could be used as a low-cost alternative for preliminary estimation of population structure.info:eu-repo/semantics/publishedVersio

Hygienic behavior of africanized honey bees Apis mellifera directed towards brood in old and new combs during diurnal and nocturnal periods

Hygienic behavior in honey bees, Apis mellifera, is measured by determining the rate at which the bees uncap and remove dead sealed brood. We analyzed individual behavior of house-cleaning Africanized honey bees in order to focus on some poorly understood aspects of hygienic behavior. Two observation hives, each with approximately 3,000 individually marked bees, were used in this study. The efficiency of hygienic behavior was evaluated in hygienic and non-hygienic strains of bees using two types of combs (new and old), as well as at different periods of the day (night and day). We also recorded the age of workers that performed this task of removing dead brood. In both strains, the workers that performed tasks related to hygienic behavior were within the same age cohort; we found no influence of age on the amount of time dedicated to the task, independent of the type of comb or period of the day. The total time from perforation of the cell capping until the dead brood had been completely removed, and was significantly shorter during daytime than at night. Hygienic behavior directed towards dead brood in new combs was also significantly more efficient (faster) than for brood in old combs. The type of comb had significantly more effect than did the time of day. We conclude that the type of comb and time of day should be taken into consideration when evaluating hygienic behavior in honey bees.CAPESFAPESP 2011/07857-9NAP Biocomp/US

Geometric morphometrics of wings of drones and workers support a process of secondary contact in the Iberian honey bee (Apis mellifera iberiensis)

A recent survey of the Iberian honey bee genetic patterns using concurrently mtDNA and nuclear (SNPs) markers revealed the presence of a concordant southwestern-northeastern cline in Iberia, supporting a post-glacial secondary contact process. Here, we followed up those findings with geometric morphometrics of wings. The aim was two-fold: (1) evaluating the effectiveness of this approach in capturing the clinal pattern and (2) comparing the effectiveness of drones and workers in detecting the clinal pattern. To that end, we used a fine-scale sample of 711 colonies taken across three Iberian north-south transects. For each colony, we recorded the geographical coordinates and collected the right forewing of five workers and five drones. We plotted 19 landmarks in the forewing venation of over 7100 individuals, and after the Procrustes alignment, the distances between landmarks were calculated. The distance matrix was used to infer population structure by applying a spatial multivariate analysis based on principal component analysis and Moran’s autocorrelation. A correlation analysis was performed between the first spatial component of drones and workers with a SNP dataset. Interestingly, the spatial patterns inferred from the wings of both workers and drones, which were greatly concordant, displayed the presence of two clusters with a spatial distribution overlapping with mtDNA and SNP data. Our findings suggest that geometric morphometrics is able to detect the signature of complex evolutionary processes. CN 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/publishedVersio

Evaluation of inexpensive pollen substitute diets through quantification of haemolymph proteins

Throughout much of South and Central America, Africanized honey bees tend to abscond during dearth periods. Consequently, there has been much interest in finding ways to secure colonies by artificial feeding. Based on locally-available ingredients, we tested five different protein diets against bee bread and sucrose syrup by measuring the amount of protein in the haemolymph of caged, newly emerged Africanized honey bees fed exclusively on one of these diets for seven days. The diets contained one or several of the following ingredients: sucrose, soy meal, rice meal, sugar-cane-alcohol distillery yeast, wheat meal, soy milk powder, and ground lentils. Sucrose, in the form of sugar syrup, was used as a protein-free control. One of the diets, which included soy milk powder as a major protein source, instead of soy meal, resulted in low haemolymph protein levels, similar to that of the sucrose diet. All of the other protein diets raised the haemolymph protein levels significantly above that of the newly emerged bees (approximately 20 – 28 versus an initial 14 mg/ml haemolymph). The haemolymph protein levels of bees fed with these pollen substitute diets were similar to those of bees fed on bee bread. The initial protein levels in the newly emerged bees were considerably higher than in previous studies done in Brazil, apparently because our study was conducted during the spring, when natural food sources are relatively abundant. Nevertheless, it was still possible to objectively compare the diets under these conditions.FAPESP 2007/07701-3CNPqResearch Center on Biodiversity and Computing (Biocomp-USP

A wing geometric morphometrics approach applied to the wings of Apis mellifera L. from the Azores

Populações de abelha melífera (Apis mellifera) foram avaliadas pela primeira vez no arquipélago dos Açores usando a morfometria geométrica aplicada à forma das asas. As asas de abelhas de 473 colónias (5 por colónia) foram montadas numa lâmina e fotografadas. Para além destas, asas de abelhas de 711 colónias de A. m. iberiensis, 11 de A. m. ligustica e 15 de A. m. carnica foram usadas como subespécies de referência. Para extrair informação sobre a forma, 19 marcos anatómicos foram colocados nas intersecções das veias ao longo da estrutura das asas de todos os indivíduos. As análises da forma da asa foram realizadas no MorphoJ usando o método de sobreposição de Procrustes. As diferenças na forma da asa entre indivíduos foram analisadas recorrendo a métodos de estatística multivariada. Os resultados obtidos revelaram o poder dos métodos da morfometria geométrica baseados em marcos anatómicos para discriminar as diferentes populações de abelha melífera dos Açores, e também permitiram distingui- -las das subespécies de referência. Os padrões morfométricos das asas mostraram que as populações dos Açores exibem uma relação próxima com A. m. iberiensis, enquanto algumas populações, especialmente as da Graciosa, tendem a agrupar com A. m. ligustica e A. m. carnica. O efeito fundador resultante de introduções em tempos históricos juntamente com o ambiente insular, a barreira ao fluxo génico associada ao isolamento geográfico, e o fluxo génico recente associado à atividade apícola possivelmente moldaram os padrões de diversidade observados nos Açores.Honey bee (Apis mellifera) colonies sampled across 8 islands of the Azores were assessed for the first time using a geometric morphometrics approach of the wing shape. The wings of 473 colonies (5 individuals per colony) from the Azores were mounted in a slide and photographed. Additionally, the wings of 711 colonies of A. m. iberiensis, 11 colonies of A. m. ligustica, and 15 colonies of A. m. carnica were used as reference samples. To extract shape information, 19 anatomical landmarks were plotted in the vein intersections across the wing structures of all individuals. The analyses of wing shape were performed in MorphoJ using the Procrustes superimposition method. Shape differences were investigated through multivariate statistical analysis. The results obtained with geometric morphometrics revealed the power of landmark-based methods to discriminate different honey bee populations from the Azores, and also to distinguish them from the subspecies of the reference collection. The wing morphometric patterns showed that while, overall, populations from the Azores exhibited a closer relationship with A. m. iberiensis, some populations, especially those from Graciosa, tend to be clustered closer to A. m. ligustica and A. m. carnica. The founder effect resulting from introductions of A. mellifera in historical times together with the particular insular environment, the barrier to gene flow due to geographical isolation and the contemporary human-assisted gene flow associated with the beekeeping activity have possibly shaped the diversity patterns observed today in the Azores.info:eu-repo/semantics/publishedVersio