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The change in allele frequencies within a population over time represents a fundamental process of evolution. By monitoring allele frequencies, we can analyze the effects of natural selection and genetic drift on populations. To efficiently track time-resolved genetic change, large experimental or wild populations can be sequenced as pools of individuals sampled over time using high-throughput genome sequencing (called the Evolve & Resequence approach, E&R). Here, we present a set of experiments using hundreds of natural genotypes of the model plant Arabidopsis thaliana to showcase the power of this approach to study rapid evolution at large scale. First, we validate that sequencing DNA directly extracted from pools of flowers from multiple plants -- organs that are relatively consistent in size and easy to sample -- produces comparable results to other, more expensive state-of-the-art approaches such as sampling and sequencing of individual leaves. Sequencing pools of flowers from 25-50 individuals at ∼40X coverage recovers genome-wide frequencies in diverse populations with accuracy r > 0.95. Secondly, to enable analyses of evolutionary adaptation using E&R approaches of plants in highly replicated environments, we provide open source tools that streamline sequencing data curation and calculate various population genetic statistics two orders of magnitude faster than current software. To directly demonstrate the usefulness of our method, we conducted a two-year outdoor evolution experiment with A. thaliana to show signals of rapid evolution in multiple genomic regions. We demonstrate how these laboratory and computational Pool-seq-based methods can be scaled to study hundreds of populations across many climates.
In recent years, the popularity of rock-climbing has grown tremendously, setting an increasing pressure on cliff habitats. Climbing may be particularly harmful in the Mediterranean biome due to its appropriate environmental conditions for climbing. A few studies have identified the effect of climbing on plant diversity at a small-scale (namely locally or even just in specific climbing areas). However, no studies exist assessing the potential risk of rock-climbing on a broad-scale (e.g., regional or national). The study aims to identify the priority locations and priority cliff plant species in Spain to focus future study efforts. Spain was selected because it is a plant biodiversity hotspot, with a great diversity of endemic and endangered species, and one of the most popular destinations for climbers. We used a geographic information system-based approach to model the spatial concurrence among Spanish climbing areas (and climbing intensity), natural protected areas (NPAs), and distribution of threatened cliff plants (and their IUCN threat category). We found that 53.5% of climbing areas in Spain are located within a NPA, most of them falling into NPAs of medium protection level. We mapped 151 threatened cliff plants, identifying four medium priority Mediterranean locations and eight priority species in which future research efforts should be focused. High-priority study locations are absent in Spain according to our spatial modeling. For the first time on a national scale, this study identifies areas in which climbing represents a potential threat for cliff habitats and threatened plants. These findings contribute to designing field studies on the effects of rock-climbing on Mediterranean cliffs, laying the groundwork for a sustainable, yet challenging, balance between the protection of these unique habitats and rock-climbing.