Periodic Reporting for period 1 - AfterTheIce (After the Ice: using a hybrid zone as a window into the genes underlying species formation)
Reporting period: 2015-07-01 to 2017-06-30
This system is ideal for this novel integration between lab- and field-based approaches because: 1) these subspecies can be crossed in controlled laboratorial conditions, where they show phenotypes often found in model-organisms, such as hybrid male sterility; and 2) these subspecies have been hybridizing for 9,000 generations in nature, where they show that natural selection is targeting particular genes but not the whole genome. Yet, this system also shows a limitation that is common across many valuable non-model organisms: its genome is too large to be sequenced and assembled with modern technology. Hence, in AfterTheIce we established three main objectives: 1) building a nearly complete gene catalogue for a species lacking a reference genome, 2) determining which particular genes are targeted by selection in natural hybrid zones, and 3) determining if those same genes are implicated in hybrid male sterility in experimental hybrids.
By moving from lab to nature, this integrative framework not only provided unique insights on the field of speciation, but also provided a transferable framework that is applicable to emergent challenges of the modern society. For example, developing genomics tools for non-model organisms lacking reference genomes, which commonly occurs in species of economic interest, and establishing new genomic approaches to associate specific genes to important traits, such as sterility or adaptation to different habitats.
To build a complete gene catalogue for a species lacking a reference genome we have used RNA-sequencing methodologies to reconstruct all the protein coding genes in the grasshopper species. By using whole genome sequencing at low coverage, we could identify ~12,000 single copy genes with ~750,000 neutral mutations that are appropriate for population genomic analysis. Using this novel genomic toolkit we were able to infer the demographic history of this species. Our major findings are that: 1) the two subspecies have diverged some ~113,180 generations ago, predating the last glacial maximum, 2) after secondary contact, the hybrid zone has been permeable to gene flow, working as a bridge between the two, putatively “pure”, subspecies. A manuscript reporting these results is currently under preparation.
To determine the genes targeted by selection in natural hybrid zones we have sampled two independent transects across the hybrid zone. We developed a DNA capture approach that selects for all the ~12,000 single copy genes identified earlier. In the future, this methodology will allow us to identify which genes are currently being exchanged between subspecies, and most importantly which genes cannot be exchanged, i.e. are involved in genetic barriers between emerging species.
To determine if those same genes are implicated in hybrid male sterility we have started an experimental breeding program of these grasshoppers in controlled laboratory conditions. We successfully were able to grow larvae in the lab to adulthood, make crosses, and get eggs of F1 generation. In the future, this will allow us to explicitly test if genes resistant to gene flow in natural hybrid zones are implicated in hybrid male sterility and gonad dysfunction, using histology and gene expression studies.
Most of the results that are specific of the grasshopper system have been presented to the scientific community in conferences and workshops and to the general public in actions on the field site (Spain) and in the host institution (Denmark). The general methodology on how to develop genomic tools for non-model organisms lacking reference genomes have already been disseminated in two peer-reviewed papers (Pereira et al. 2016, da Fonseca et al 2016).
By applying this approach the grasshopper system, our results imply that hybrid male sterility can evolve rapidly (~100 thousand years) relatively to what is known from model organisms (several millions of years), suggesting that new species can emerge quickly. Moreover, our results demonstrate that natural hybrid zone, by acting as a selective filter to gene exchange, can serve as natural laboratories to identify the genes involved in sterility and other barriers between species.
More broadly, AfterTheIce offers a transferable approach that can be used to the large number of species that are characterized by large genomes, which largely remain unstudied. By applying this approach to plants of economical interest for example, we can start understanding how many genes are responsible to adaptation to different water regimes or can cause sterility, which has direct implications for agricultural breeding programs.