RAD sequencing enables unprecedented phylogenetic resolution and objective species delimitation in recalcitrant divergent taxa

• Unambiguously resolved phylogenetic relationships among recalcitrant taxa with divergences >80 million years.
• Objectively inferred robust species boundaries in a deep-sea octocoral genus by testing alternative delimitation hypotheses.
• Present substantial evidence rejecting the current morphological species delimitation model for the genus.
• Inferred the presence of cryptic deep-sea species associated with environmental variables.
• Argue that the suitability limits of RAD-seq for phylogenetic inferences are contingent on taxon-specific factors.

Species delimitations is problematic in many cases due to the difficulty of evaluating predictions from species hypotheses. In many cases delimitations rely on subjective interpretations of morphological and/or DNA data. Species with inadequate genetic resources needed to answer questions regarding evolutionary relatedness and genetic uniqueness are particularly problematic. In this study, we demonstrate the utility of restriction site associated DNA sequencing (RAD-seq) to objectively resolve unambiguous phylogenetic relationships in a recalcitrant group of deep-sea corals with divergences >80 million years. We infer robust species boundaries in the genus Paragorgia by testing alternative delimitation hypotheses using a Bayes Factors delimitation method. We present substantial evidence rejecting the current morphological species delimitation model for the genus and infer the presence of cryptic species associated with environmental variables. We argue that the suitability limits of RAD-seq for phylogenetic inferences cannot be assessed in terms of absolute time, but are contingent on taxon-specific factors. We show that classical taxonomy can greatly benefit from integrative approaches that provide objective tests to species delimitation hypotheses. Our results lead the way for addressing further questions in marine biogeography, community ecology, population dynamics, conservation, and evolution.

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