High-throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b-RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b-RAD protocols on non-model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b-RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade-off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective-base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b-RAD protocols on non-model organisms with different genome sizes, helping decision-making for a reliable and cost-effective genotyping.

Connectivity and local adaptation are two contrasting evolutionary forces highly influencing population structure. To evaluate the impact of early-life traits and environmental conditions on genetic structuring and adaptation, we studied two sympatric fish species in the Western Mediterranean Sea: Symphodus tinca and S. ocellatus. We followed an individual-based approach and measured early-life history traits from otolith readings, gathered information on environmental variables and obtained genome-wide markers from genotyping-by-sequencing (GBS). The two species presented contrasting population structure across the same geographic gradient, with high and significant population differentiation in S. ocellatus, mostly determined by oceanographic fronts, and low differentiation and no front effect in S. tinca. Despite their different levels of genetic differentiation, we identified in both species candidate regions for local adaptation by combining outlier analysis with environmental and phenotypic association analyses. Most candidate loci were associated to temperature and productivity in S. ocellatus and to temperature and turbulence in S. tinca suggesting that different drivers may determine genomic diversity and differentiation in each species. Globally, our study highlights that individual-based approach combining genomic, environmental and phenotypic information is key to identify signals of selection and the processes mediating them.

As species struggle to cope with rising ocean temperatures, temperate marine assemblages are facing major reorganization. Many benthic species have a brief but critical period dispersing through the plankton, when they are particularly susceptible to variations in temperature. Impacts of rising temperatures can thus ripple through the population with community‐wide consequences. However, responses are highly species‐specific, making it difficult to discern assemblage‐wide patterns in the life histories of different fish species. Here, we evaluate the responses to temperature in the early life histories of several fish species using otolith reconstructive techniques. We also assess the consequences of future warming scenarios to this assemblage. We sampled recent settlers of nine common species across a temperature gradient in the Mediterranean Sea and obtained environmental data for each individual. Using otolith microstructure, we measured early life traits including pelagic larval duration (PLD), growth rate, settlement size, hatching and settlement dates. We used a GLM framework to examine how environmental variables influenced early life‐history parameters. We show that increasing temperature results in considerable reduction in the dispersal potential of temperate fish. We find a nearly universal, assemblage‐wide decline in pelagic larval duration (PLD) of between 10% and 25%. This was because, with increasing temperature, larvae grew quicker to their settlement size. Settlement size itself was less affected by temperature and appears to be an ontogenetically fixed process. Given current estimates of ocean warming, there could be an assemblage‐wide reduction in larval dispersal of up to 50 km across the Mediterranean, reducing connectivity and potentially isolating populations as waters warm.