quaddRAD: a new high‐multiplexing and PCR duplicate removal ddRAD protocol produces novel evolutionary insights in a nonradiating cichlid lineage

<jats:title>Abstract</jats:title><jats:p>The identification of thousands of variants across the genomes and their accurate genotyping are crucial for estimating the genetic parameters needed to address a host of molecular ecological and evolutionary questions. With rapid advances of massively parallel high‐throughput sequencing technologies, several methods have recently been developed to access genomewide data on population variation. One of the most successful and widely used techniques relies on the combination of restriction enzymes and sequencing‐by‐synthesis: restriction‐site‐associated <jats:styled-content style="fixed-case">DNA</jats:styled-content> sequencing (RADSeq). We developed a new, more time‐ and cost‐efficient double‐digest <jats:styled-content style="fixed-case">RAD</jats:styled-content> paired‐end protocol (quadd<jats:styled-content style="fixed-case">RAD</jats:styled-content>) that simplifies and speeds up the identification of <jats:styled-content style="fixed-case">PCR</jats:styled-content> duplicates and permits large‐scale multiplexing. Assessing its performance on a technical data set, we also applied the quadd<jats:styled-content style="fixed-case">RAD</jats:styled-content> method on population samples of a Neotropical cichlid fish lineage (<jats:italic>Archocentrus centrarchus</jats:italic>) to assess its genetic structure and demographic history. While we identified allopatric interlake genetic divergence, most likely driven by drift, no signature of sympatric divergence was detected. This differs from what has been observed in the clade of Midas cichlids (<jats:italic>Amphilophus citrinellus</jats:italic> spp.), another cichlid lineage that inhabits the same lakes and shares a similar demographic history, but has evolved into small‐scale adaptive radiations via sympatric speciation. We demonstrate that quadd<jats:styled-content style="fixed-case">RAD</jats:styled-content> is a robust and efficient method for genotyping a massive number and widely overlapping set of loci with high accuracy. Furthermore, the results on <jats:italic>A. centrarchus</jats:italic> open new research avenues providing an ideal system to investigate genome‐level mechanisms that could alter the speciation potential of different but closely related cichlid lineages.</jats:p>