Hybrid zones (HZ) have been described as natural laboratories for evolutionary studies. In that sense, Iberia may be considered as
one of the top laboratories in Europe and we argue that the Iberian honey bee (IHB) is its top HZ study model.This proposal brings to
bear carefully designed, thorough sampling, assays of mtDNA variation, unique assays of nuclear haploid phase information, and
whole genome screening of nuclear genetic variation made possible by SNP analysis validated in the HB genome project.The
proposed is a carefully constructed succession of high throughput molecular approaches that compare mitochondrial and nuclear
genomic information in a spatial and temporal context.The constructed data set is uniquely possible for this model organism. The
succession of analyses (see Fig.1) is designed to uniquely tease from the data, the several evolutionary forces, some due to manÂ´s
actions and others due to selection, dispersal and chance that have forged the spatial and temporal variation we see today in IHBs.
HZs are shaped by evolutionary forces acting on the whole genome and on specific regions of chromosomes. Dissecting genome-wide
(neutral) from genome-specific effects (adaptive) is a goal of central importance as it leads to more robust inferences of demographic
history and to identification of adaptive divergence. The combination of population genomics (PG) and landscape genetics (LG)
provide a powerful framework in which to address this issue. Examining genome scans using a LG approach will assist in: 1)
unravelling the relative importance of processes underlying spatial structure by comparing distributions of neutral and selected
markers, 2) detecting potential adaptive variation, by sampling across gradients, 3) identifying potential causes and consequences of
adaptive variation by finding environmental correlates of spatial genetic variation. The power of this approach is further increased for
model organisms as genomic resources enable a more thorough exploration of the molecular basis of adaption. Thus, it is expected
that novel insights into the evolutionary forces shaping HZs will be gained by using this integrated framework.
The IHB provides a challenging and at the same time a powerful model in which to examine the evolutionary forces underlying HZs.
Previous studies suggest that Iberia was a major refuge during ice ages and a zone of secondary contact between 2 divergent HB
lineages. These historical events, coupled with contemporaneous human-mediated processes, have produced a complex pattern of
variation which has yet to be reliably dissected. With the publication of the HB genome and development of high-density SNP
markers, powerful tools are now available to dissect the relative importance of neutral and adaptive forces in shaping the IHB HZ.
The power provided by these tools is further increased in HBs because the hurdles of genetic analysis requiring phased data are
circumvented by using haploid males.
This study seeks to promote an integrative approach to the evolutionary history of the IHB HZ by examining genome scans in a LG
context and by developing multilocus nuclear-haplotype based genealogies. The powerful combination of the cutting edge tools and
approaches employed promises to revolutionize our current view of the IHB hybrid zone. This study comprises 5 major parts: 1) a
fine-resolution IHB collection will be made by sampling across 3 latitudinal gradients in Iberia, 2) mtDNA surveys of the Portuguese
HBs will be performed to complete assessment of IHBs variation of previous studies, 3) genome scans will be performed on the
complete IHB sample employing 1536 SNPs, 4) the SNP data will be analyzed using a battery of PG and LG tools to identify, spatially
represent and interpret patterns of neutral and adaptive variation, and explore the molecular basis of adaptive divergence, 5)
sequence-based nuclear haplotype genealogies will be developed for 5 loci with the strongest selection signature, providing temporal
resolution to infer relative ages and historical relationships of alleles as well as a powerful framework for testing sophisticated
At the end of the study, we expect to: 1) provide a robust inference of the IHB demographic history, 2) gain a fundamental
understanding about the relative role of neutral forces and selection in shaping IHBs diversity, and 3) develop a more complete
understanding of the molecular basis of local adaption of IHBs. The employed approach will be a novelty in HB studies, while analysis
of the Portuguese HBs will provide the first genetic data for mainland Portugal. Thus, we are certain that unparalleled insights into
the dynamics of evolutionary phenomena of the IHB HZ, and perhaps of other HZs, will be gained.
The team comprises a wide range of expertises including population genetics, phylogeography, molecular biology, and landscape
ecology, which encompasses the full scope of the project. Some team members are well grounded in HB genetics and in the study of
HB hybrid zones of the USA and Iberia.