The introduction rate of alien species in the Mediterranean Sea is rapidly growing, and their taxonomical identification is increasingly challenging. This uncertain identification often leads to an incorrect estimation of the number of alien species, their route of introduction, and their potential negative effects. This is particularly true for some bivalves, which are characterized by high variation in their shells, resulting in uncertain morphological identification. This is the case for two alien bivalves, i.e., an Isognomonidae and a Malleidae species, both characterized by confused historical colonization records in the Mediterranean Sea, misidentifications, and controversial and changing nomenclatures that have insofar negatively affected our knowledge on their geographical distributions. In this respect, molecular approaches provide a strategy that is especially useful when traditional taxonomy fails, and DNA barcoding is a powerful and well-known tool to obtain reliable identifications through efficient molecular markers. In this work, we used the 16S rRNA marker to assess the preliminary identification of Isognomon sp. and Malleus sp. specimens from different localities in the Southern Mediterranean Sea. Bayesian inference (BI) and maximum likelihood (ML) methods were applied to test the monophyly of the phylogenetic linages and to clarify their taxonomic positions, allowing a complete overview of the colonization and spreading of these two alien bivalves in the Mediterranean Sea. In particular, the Isognomon sp. specimens were identified as the Atlantic I. bicolor, highlighting that previously suggested invasive migration patterns, (i.e., the Lessepsian migration), must be reconsidered with stronger critical attention in light of currently occurring global changes.
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