ITS DNA barcoding reveals that Halophila stipulacea still remains the only non-indigenous seagrass of the Mediterranean Sea.
Non-indigenous species (NIS) are one of the major threats to the native marine ecosystems of the Mediterranean Sea. Halophila stipulacea was the only exotic seagrass of the Mediterranean until 2018, when small patches of a species morphologically identified as Halophila decipiens were reported in Salamina Island, Greece. Given the absence of reproductive structures during the identification and the taxonomic ambiguities known to lead to misidentifications on this genus, we reassessed the identity of this new exotic record using DNA barcoding (rbcL, matK and ITS) and the recently published taxonomic key. Despite their morphologic similarity to H. decipiens based on the new taxonomic key, the specimens showed no nucleotide differences with H. stipulacea specimens (Crete) for the three barcodes and clustered together on the ITS phylogenetic tree. Considering the high species resolution of the ITS region and the common morphological variability within the genus, the unequivocal genetic result suggests that the Halophila population found in Salamina Island most likely corresponds to a morphologically variant H. stipulacea. Our results highlight the importance of applying an integrated taxonomic approach (morphological and molecular) to taxonomically complex genera such as Halophila, in order to avoid overlooking or misreporting species range shifts, which is essential for monitoring NIS introductions.