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The fan mussel, Pinna nobilis, represents the largest bivalve endemic to the Mediterranean Sea. Since 2016, dramatic mass mortality of this species has been observed in several areas. The first surveys suggested that Haplosporidium pinnae (currently considered species-specific) was the main etiological agent, but recent studies have indicated that a multifactorial disease may be responsible for this phenomenon. In this study, we performed molecular diagnostic analyses on P. nobilis, P. rudis, and bivalve heterologous host species from the island of Sardinia to shed further light on the pathogens involved in the mass mortality. The results support the occurrence of a multifactorial disease and that Mycobacterium spp. and H. pinnae are not necessarily associated with the illness. Indeed, our analyses revealed that H. pinnae is not species-specific for P. nobilis, as it was present in other bivalves at least three years before the mass mortality began, and species of Mycobacterium were also found in healthy individuals of P. nobilis and P. rudis. We also detected the species Rhodococcus erythropolis, representing the first report in fan mussels of a bacterium other than Mycobacterium spp. and Vibrio spp. These results depict a complicated scenario, further demonstrating how the P. nobilis mass mortality event is far from being fully understood.

Comparison of the electrostatic potential surface of the Centaurus and BA.2 Spike receptor binding domains (RBDs). Red and blue colors indicate negative and positive potential, respectively. The color scale ranges from −5.0 to +5.0 kT/e. The RBD is oriented with the ACE2 interface in the front. Position of the R493 in BA.2 is marked by the white arrow. Calculations have been carried out with the software PROPKA3 12 while in-silico mutagenesis by means of PyMOL Molecular Graphics System v.2 (available at https://pymol.org/2/). The conformation of the side chains of the mutagenized structures was optimized by the application of the software FoldX 5.0. 13

Two samples of Aphanius fasciatus collected in the Pilo pond (northern Sardinia, Italy) and in an adjacent pool of small surface area were analysed morphologically (235 individuals) and genetically (a subsample of 58 individuals). The aims of the present study were (i) to test the hypothesis that different predation pressures may be associated with morphological and/or genetic differences between samples from each habitat and (ii) to assess the level of divergence between the two populations. Morphological analysis was based on the relative size of fins because it has been shown to be associated with predation pressure. The relative caudal fin area (caudal fin area/total body surface) was smaller in specimens from the pool, in both males and females, whereas no differences were found for the dorsal and anal fin areas. Caudal fins with higher aspect ratio (fin depth/fin length) were found in fish from the pool but not in the pond, due to a higher fin depth. We hypothesised that specimens from the pool would show smaller caudal fin area, since they are subject to lower predation pressure. Random amplification of polymorphic DNA (RAPD) analysis revealed a relatively high degree of both within-and between-sample genetic heterogeneity. The pond and pool samples exhibited heterozygosities, which did not differ significantly by t-test. Between-sample genetic divergence was highlighted by the coancestry coefficient (h = 0.301 ± 0.059, P < 0.001) and analysis of molecular variance (AMOVA) (variance between sites = 41%, P < 0.001). Genetic divergence between sites with a relatively high genetic diversity within both samples suggested that the population in the pool did not originate from a single colonisation event with a small number of founders. The genetic divergence between the two populations is consistent with their differences in fin size.

Patella ferruginea Gmelin, 1791 is an endangered marine gastropod endemic to the Western Mediterranean. Its range is restricted to the Sardinian-Corsican region (SCR), North Africa, a few scattered sites in Southern Spain, and Sicily. Inter-simple sequence repeat (ISSR) markers and three different mitochondrial DNA (mtDNA) regions, Cytochrome c Oxidase subunit I, 12S (small-subunit ribosomal RNA gene) and 16S (large-subunit ribosomal RNA gene), were used to investigate the presence of genetic population structuring. The mtDNA sequences showed very low levels of genetic differentiation. Conversely, ISSRs showed the presence of two main genetic groups, corresponding to Spain, North Africa and Sicily and the SCR. The SCR was further split into two subgroups. The ISSR results suggest that, on a regional scale, the genetic structure of P. ferruginea is mainly determined by the restriction of gene flow by dispersal barriers. On a more local scale human harvesting may play a crucial role in population structuring by increasing the effect of genetic drift.

Pseudomonocelis ophiocephala (Schmidt, 1861), a mesopsammic proseriate, is common in a variety of shallow water habitats in the Mediterranean. The genetic relationships between morphologically indistinguishable populations across the Mediterranean were surveyed by means of combined allozyme and Random Amplified Polymorphic DNA (RAPD) analysis. Genetic distances, UPGMA cluster analysis and F -statistics based on 27 allozyme genetic loci and 68 RAPD primer fragments were consistent in showing that the taxon Pseudomonocelis ophiocephala is a complex of sibling species, consisting of four taxonomic units. The four species differ in distribution and habitat: sibling A is widespread in lower intertidal habitats of the Mediterranean, in well-sorted, medium-to coarse-grained sand; siblings B, C and D show a restricted distribution (Corsican-Sardinian region and Elba Island, west and east coast of Greece, respectively) in low-energy marine habitats. Given that the species of the complex are morphologically indistinguishable, the type material is absent, and that there are two siblings in the type locality (Corfu Island, Greece), a neotype is designated for P. ophiocephala . The three further siblings are named; species descriptions are based on non-morphological characters (karyotype, allozymic and RAPD patterns). Distributions and reconstruction of phylogenetic relationships based on allozyme data suggest that both allopatric and ecological speciation have played a role in the evolutionary history of the species complex.

Only a few species belonging to the Proseriata (Platyhelminthes) show a parenchymatic pigmentation, which may aid identification. Among these, Pseudomonocelis agilis has a yellowish body and is provided with a reddish-brown girdle in front of the statocyst. The species is known for limited areas of northern Europe and the Mediterranean. The present study was conducted to assess both the taxonomic status of populations attributed to the species across the unusually wide range for an interstitial flatworm, which lacks an obvious means of dispersal, and the levels of genetic variability within and among populations, by employing an integrative approach that included the analyses, on six populations, of three molecular markers (small subunit ribosomal 18S-like gene, inter-simple sequence repeat, allozymes), karyotypes, and 11 morphological characters. Furthermore, crossbreeding experiments were carried out on the Mediterranean populations. The results obtained revealed the existence of four highly divergent genotypic clusters, accompanied by karyological differences, with complete intersterility among the clusters tested. The combination of approaches adopted strongly supports the conclusion that the wide-ranging European pigmented species P. agilis is actually composed of four species: P. agilis in the Baltic area; Pseudomonocelis cetinae in the Adriatic; and Pseudomonocelis sp. nov. A and Pseudomonocelis sp. nov. B in the western and eastern Mediterranean, respectively. The latter two species are morphologically indistinguishable for the parameters essayed. Reconstruction of the phylogenetic relationships of these taxa, including congeneric and consubfamilial outgroups, showed that pigmentation is a plesiomorphic condition for the genus Pseudomonocelis and that Pseudomonocelis sp. nov. A shares a previously undetected, sister-group relationship with species of the unpigmented P. ophiocephala complex. The present study thus depicts complex speciation processes in a mesopsammic species, which involves allopatric divergence operating on different scales and ecological shifts, and highlights that the contribution of microturbellarians to marine biodiversity may be seriously underestimated.

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