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Molecular studies sometimes reveal evolutionary divergence within accepted species. Such findings can initiate taxonomic revision, as exemplified in the formerly recognized species  Ciona intestinalis . While an increasing number of studies have examined the ecology, reproductive barriers and genetics of  C .  intestinalis  and  C .  robusta , there are still much uncertainties regarding other species of this genus. Using experimental crosses and mitochondrial data, we investigated the evolutionary relationships among four native and introduced  Ciona spp ., found in sympatry in the Mediterranean Sea or English Channel. Outcome of 62 bi-parental reciprocal crosses between  C .  intestinalis ,  C .  robusta, C .  roulei  and  C .  edwardsi  showed that  C .  edwardsi  is reproductively isolated from the other taxa, which is in agreement with its distinct location in the phylogenetic tree. Conversely, hybrids are easily obtained in both direction when crossing  C .  intestinalis  and  C .  roulei , reinforcing the hypothesis of two genetically differentiated lineages but likely being from a same species. Altogether, this study sheds light on the evolutionary relationship in this complex genus. It also calls for further investigation notably based on genome-wide investigation to better describe the evolutionary history within the genus  Ciona , a challenging task in a changing world where biological introductions are shuffling species distribution.

Coupling molecular data and experimental crosses sheds light about species delineation: a case study with the genus Ciona