Species delimitation in the presence of strong incomplete lineage sorting and hybridization

Accurate species delimitation is essential to properly assess biodiversity, but also for management and conservation purposes. Yet, it is not always trivial to accurately define species boundaries in closely related species, as strong incomplete lineage sorting might still be present. Additional difficulties may be caused by hybridization, now evidenced as a frequent phenomenon. Here, we propose a three-step framework for species delimitation and divergence history inference: i) unsupervised species discovery based on multilocus genotypes; ii) species validation using the multi-species coalescent; iii) divergence scenario testing (including gene flow) using Approximate Bayesian Computation (ABC) methods. To test this framework, we used the brittle star cryptic species complex Ophioderma longicauda that encompasses six mitochondrial lineages, including broadcast spawners and internal brooders. 30 sequence markers (transcriptome-based, mitochondrial or non-coding) for 89 O. longicauda and outgroup individuals were used. First, multivariate analyses revealed six genetic clusters, which globally corresponded to the mitochondrial lineages, yet with many exceptions, suggesting ancient hybridization events and challenging traditional barcoding approaches. Second, multi-species coalescent-based analyses validated the six species and provided divergence time estimates, but the sole use of this method failed to accurately delimit species, highlighting the power of multilocus genotype clustering to delimit recently diverged species. Finally, Approximate Bayesian Computation showed that the most likely scenario involves hybridization between brooders and broadcasters. Our study shows that despite strong incomplete lineage sorting and complex speciation history, accurate species delimitation is possible using a three-step framework combining complementary methods.