Species limits, interspecific hybridization and phylogeny in the cryptic land snail complex Pyramidula: The power of RADseq data.

Restriction site-associated DNA sequencing (RADseq) was used to jointly assess phylogenetic relationships, interspecific hybridization and species delimitation in the cryptic, non-model land snail complex Pyramidula. A robust phylogeny was inferred using a matrix of concatenated sequences of almost 1,500,000bp long, containing >97,000 polymorphic sites. Maximum likelihood analyses fully resolved the phylogenetic relationships among species and drastically improved phylogenetic trees obtained from mtDNA and nDNA gene trees (COI, 16S rRNA, 5.8S rRNA, ITS2 and 28S rRNA sequence data). The best species delimitation scenario was selected on the basis of 875 unlinked single nucleotide polymorphisms, showing that nine Pyramidula species should be distinguished in Europe. Applying D-statistics provided no or weak evidence of interspecific hybridization among Pyramidula, except for some evidence of gene flow between two species.