Identifying surviving Pinna nobilis after the Mass Mortality Event (MME) in the Mediterranean: Proposal of a low-risk methodology for collecting genetic samples

The critically endangered species, Pinna nobilis, has nearly disappeared from the Mediterranean Sea, primarily due to infection by the protozoan parasite Haplosporidium pinnae. However, some individuals survive in specific areas with favorable environmental conditions, such as water salinity and temperature. On the Spanish coast, the only two surviving populations are confined to the Ebro Delta and the Mar Menor coastal lagoon. Some resistant P. nobilis specimens have been found in the areas affected by mass mortality. It is sometimes difficult to distinguish between P. nobilis and P. rudis specimens by external morphological characteristics. Only genetic analysis allows unambiguous discrimination between the two species and even the detection of hybrid specimens. Biopsies, as a traditional sampling method for obtaining samples for genetic analysis, have proven to be an aggressive technique that causes high mortality rates among biopsied specimens, both in surviving specimens in natural environments and in stabled specimens kept in captivity ex situ. Therefore, it is important to look for sampling techniques that do not pose a risk to the health status of the specimens. This study focuses on the development of a novel methodology for obtaining samples for genetic studies. A survey of Pinna spp. individuals in open waters along the Mediterranean coast of spanish levantine area (western Mediterranean) was conducted. To identify the species of the individuals found and distinguish them from the closely related P. rudis, a new, less invasive methodology for genetic sampling was used. By collecting environmental DNA (eDNA) from the water inside the valves of 13 Pinna spp. individuals, a potential surviving specimen of Pinna nobilis was identified in the coast of Murcia (SE Spain).