The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes.

Rajidae (colloquially known as skates and rays) experienced multiple and parallel adaptive radiations allowing high species diversity and great differences of species composition between regional faunas. Nevertheless, they show considerable conservation of bio‐ecological, morphological and reproductive traits. The evolutionary history and dispersal of North‐east Atlantic and Mediterranean rajid fauna were investigated throughout the sequence analysis of the control region and 16S rDNA mitochondrial genes. Molecular estimates of divergence times indicated recent origin and rapid dispersal of the present species. Compared with the ancient origin of the family (Late Cretaceous), the present species diversity arose in a relatively narrow time‐window (12 Myr) from Middle Miocene to Early Pleistocene, likely by speciation processes related to dramatic geological and climatic events in the Mediterranean. Nucleotide substitution rates and phylogenetic relationships indicated Mediterranean endemic skates derived from sister species with wider distribution during Late Pliocene–Pleistocene. Skate phylogeny and systematics obtained using mitochondrial gene variation were largely consistent with those based on morpho‐anatomical data.

Running head: Allopatric cryptic speciation and population structuring in skates-2-ABSTRACT Aim Geographical (allopatric) speciation is a dynamic process whose footprints in the living world are a continuum of stages of increasing divergence. Geographical speciation can also contribute to the evolution of marine taxa. This study looked for two of these evolutionary stages (i.e. structured populations and sibling species) in the diversification patterns of two Atlantic skates (Raja, suborder Rajoidea) which exhibited high morphological and ecological conservativism. Location E Atlantic, Mediterranean, W Indian Methods Phylogeographical and population genetic analyses were performed by surveying DNA variation of 10 population samples assigned to the European Raja clavata and to the S African R. straeleni. Polymorphisms were detected by sequencing a mtDNA control region (CR) fragment and genotyping amplified fragment length polymorphism (AFLP) loci. Several statistical tests were used to explore genetic differentiation and population demography. Results CR haplotypes clustered in two clades consistent with taxon zoogeography. Mean sequence divergence between allopatric taxa amounted to 2% and Bayesian estimate of the time of the most recent common ancestor dated their separation between 0.155 and 1.3 Myr. Isolation-by-distance between European and S African demes was inferred from a significant correlation between coastal and genetic distances at AFLP loci. Null or low gene flow estimates suggested reproductive isolation between allopatric taxa. After separation, taxa have expanded moderately since 30-45 Kyr. Geographical subgrouping of CR haplotypes and significant genetic heterogeneity of samples at both markers featured the Atlantic and Mediterranean thornback skates, revealing pronounced levels of population structuring in this widely distributed taxon. Main conclusions In spite of the pronounced morpho-anatomical conservativism, R. clavata and R. straeleni are allopatric sibling species which diverged in the Pleistocene. A recent southward dispersal of European R. clavata-like elements along with the W African shelf with the budding of S African R. straeleni is ostensible. The tumultuous Quaternary palaeoclimatic history of equatorial and tropical Africa with the succession of glacial and interglacial palaeoenvironments could have promoted the repeated geographical isolation of local demes in relatively restricted refugial areas. Within the evolutionary trajectories experienced by Rajoidea, structured populations and allopatric sibling species could frequently represent intermediate frames of the microevolutionary animation proposed by Ernst Mayr to model allopatric speciation.

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