High Genetic Diversity of Hirudo verbana Carena, 1820 (Annelida: Hirudinea: Hirudinidae) in Romania Confirms That the Balkans Are Refugia Within Refugium

This study presents the first data on the distribution of the Mediterranean medicinal leech Hirudo verbana in Romania, as well as the first molecular characterization of the Romanian populations, using mitochondrial DNA markers. In total, 133 specimens from 19 populations in Romania were surveyed for mitochondrial COI and 12S markers. Available GenBank sequences from other localities throughout Europe were included to place the Romanian populations in a broader phylogeographical context. The Romanian H. verbana samples belong to 23 COI haplotypes, 4 of which are previously known from Serbia and Ukraine, while 19 haplotypes are herein reported for the first time. For 12S, the number of haplotypes identified was 12, from which only 5 are unique to Romania. The Romanian H. verbana cluster within the previously reported eastern phylogroup, known to occur from Serbia to North Caucasus, Turkey, and Uzbekistan. The mismatch distribution analysis and the results of neutrality tests suggest that the Romanian H. verbana currently undergoes a population dispersal. This study also revealed that the lower Danube basin is a hotspot of genetic diversity of H. verbana, where different lineages from the Balkan and Anatolian glacial refugia mixed in the postglacial period. Species Distribution Models (SDMs) indicated that the main ecological variables influencing the current distribution of H. verbana are wetland coverage and elevation. Following the IUCN Red List categories and criteria, the present conservation status of the Mediterranean medicinal leech H. verbana in Romania was assessed as Vulnerable (VU). Our study contributes towards a more refined understanding of the genetic diversity of H. verbana and of the factors that led to its current geographical distribution in Romania and Europe.