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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.

We report the discovery of the Indo-Pacific sacoglossan Elysia nealae Ostergaard, 1955 from northeastern Sardinia (Central Tyrrhenian Sea). This is the first record of this species in the western Mediterranean Sea and only the second for the whole of the Mediterranean Sea following a report from Cape Epanomi, Greece. We discuss the identification of this species as well as the expansion of its geographical range. Data on the ecology and behavior of E. nealae leads us to hypothesize that the increase in the Mediterranean’s water temperature due to climate change has favored this non-native species and contributed to its expanded distribution.

The mollusc fauna of the Mediterranean Sea is still considered as the best-known marine mollusc fauna in the world. The previous modern checklists of marine Mollusca were produced by joint teams of amateurs and professionals. During the last years the Italian Society of Malacology (Società Italiana di Malacologia – S.I.M.) maintained an updated version of the Mediterranean checklist, that served as the backbone for the development of the new Italian checklist. According to the current version (updated on April 1st, 2021), 1,777 recognised species of marine molluscs are present in the Italian Economic Exclusive Zone, including also the Tyrrhenian coasts of Corsica and the continental shelf of the Maltese archipelago. The new checklist shows an increase of 17% of the species reported in the 1995 Checklist. This is largely (yet not solely) due to the new wave of studies based on Integrative Taxonomy approaches. A total of 135 species (7.6%) are strictly endemic to the Italian waters; 44 species (2.5%) are alien and correspond to the 28% of the Mediterranean alien marine molluscs. All eight extant molluscan classes are represented. The families represented in the Italian fauna are 307, an increase of 14.6% from the first checklist, partly due to new records and partly to new phylogenetic systematics. Compared with the whole Mediterranean malacofauna, the Italian component represents 71% in species and 61% in families, which makes it a very remarkable part of the Mediterranean fauna.

The genus Nemesis Furfaro & Mariottini, 2021, was recently introduced for an independent lineage of aeolid nudibranchs, and Dondice banyulensis Portmann & Sandmeier, 1960, established as its type species. Anyway, the presence of a senior homonym, Nemesis Risso, 1826, was evidently missed. In fact, in 1826, Risso established this genus for a group of Copepoda (Arthropoda, Crustacea) and according to the Principle of Priority (ICZN) only the senior homonym may be used as a valid name. Therefore, a new replacement name is here proposed. Furthermore, the genus name Nanuca Er. Marcus, 1957, has priority over Dondice Er. Marcus, 1958 and consequently, the species in this clade should be classified under Nanuca, mostly as new combinations.

Integrative taxonomy is an evolving field of multidisciplinary studies often utilised to elucidate phylogenetic reconstructions that were poorly understood in the past. The systematics of many taxa have been resolved by combining data from different research approaches, i.e., molecular, ecological, behavioural, morphological and chemical. Regarding molecular analysis, there is currently a search for new genetic markers that could be diagnostic at different taxonomic levels and that can be added to the canonical ones. In marine Heterobranchia, the most widely used mitochondrial markers, COI and 16S, are usually analysed by comparing the primary sequence. The 16S rRNA molecule can be folded into a 2D secondary structure that has been poorly exploited in the past study of heterobranchs, despite 2D molecular analyses being sources of possible diagnostic characters. Comparison of the results from the phylogenetic analyses of a concatenated (the nuclear H3 and the mitochondrial COI and 16S markers) dataset (including 30 species belonging to eight accepted genera) and from the 2D folding structure analyses of the 16S rRNA from the type species of the genera investigated demonstrated the diagnostic power of this RNA molecule to reveal the systematics of four genera belonging to the family Myrrhinidae (Gastropoda, Heterobranchia). The “molecular morphological” approach to the 16S rRNA revealed to be a powerful tool to delimit at both species and genus taxonomic levels and to be a useful way of recovering information that is usually lost in phylogenetic analyses. While the validity of the genera Godiva, Hermissenda and Phyllodesmium are confirmed, a new genus is necessary and introduced for Dondice banyulensis, Nemesis gen. nov. and the monospecific genus Nanuca is here synonymised with Dondice, with Nanuca sebastiani transferred into Dondice as Dondice sebastiani comb. nov.

Species delimitation is particularly challenging in Mediterranean nudibranchs because morphological distinction between closely related species is subtle and several cryptic species have been discovered. In this study, we apply an integrative approach to investigate taxonomic distinction of Flabellina cavolini and F.gaditana and assess their genetic and morphologic distinction. We combined a comprehensive phylogeographic survey from across their distribution range and compared genetic variation at mitochondrial and nuclear markers with the variation in colour pattern of cerata that is used to discriminate these two species. Phylogenetic, phylogeographic and species delimitation analyses consistently identify two evolutionary and taxonomic units that do not match with the morphospecies, demonstrating that Flabellina cavolini and F.gaditana are indeed two cryptic, closely related, species and that colour pattern of cerata is not a valid diagnostic character. This study emphasizes how the comparison of range-wide patterns of phylogenetic and morphological variation, with the inclusion of samples from the type localities, is essential for species delimitation of Mediterranean nudibranchs. Moreover, this study demonstrates the occurrence of Flabellina gaditana in the Mediterranean Sea, allowing to extend the known distribution range of the species, that was previously restricted to the eastern Atlantic Ocean, as well as increasing our inventory of Mediterranean nudibranch diversity.

The diversity of Mediterranean nudibranchs has yet to be thoroughly studied: new species are constantly described, and molecular approaches have revealed some cryptic species. A new facelinid species has been discovered based on specimens collected from the Tyrrhenian Sea (Mediterranean Sea). Integrative results of molecular analyses and of anatomical investigations support the description of Dondice trainitoi sp. nov. The characteristic chromatic body pattern and the black epithelium covering the masticatory jaws allow an unambiguous identification of the new taxon. Preliminary phylogenetic analyses based on multi-locus molecular markers (nuclear H3 gene and mitochondrial markers 16S rDNA and COI) surprisingly revealed paraphyly of the genus Dondice and the need of further studies including more taxa assigned to the currently accepted family Facelinidae. Furthermore, following an integrative taxonomy approach, considerations on the ecological behaviour characterizing most of the species involved in this study provide useful insights for understanding the evolutionary history of this facelinid group.

The Atlantic-Mediterranean Bursa scrobilator (Linnaeus, 1758) complex is represented by three morphologically distinct forms, two that are extant corresponding to the nominal taxa Murex scrobilator Linnaeus, 1758 and Ranella coriacea Reeve, 1844, and one fossil corresponding to Murex nodosus Borson, 1825. The complex is here reviewed integrating morphological, molecular (mitochondrial COI marker) and paleontological data. The ‘scrobilator’ and ‘coriacea’ forms are proposed as two subspecies: Bursa scrobilator scrobilator (Linnaeus, 1758) with an Atlantic-Mediterranean distribution, and Bursa scrobilator coriacea (Reeve, 1844) with a West African range. The evolutionary relationships of the two recent subspecies with their most probable ancestor, the Mediterranean Miocene fossil B. nodosa, are also discussed.

The first record from Sicily of the introduced facelinid nudibranch Godiva quadricolor allowed the detection of trophic relationships with the polycerid Polycera hedgpethi, another non-native nudibranch, and with two bryozoan species, namely the naturalized Cheilostomatida Bugula neritina and the cryptogenic Ctenostomatida Amathia verticillata. The settlement of both nudibranchs was presumably promoted by a trophic shift of P. hedgpethi from the natural prey B. neritina towards the largely available and not exploited A. verticillata. This short food web, without evident links with native fauna and having G. quadricolor as the top predator, is described. A DNA barcoding approach was used to confirm the identity of this facelinid species and to explore the possible genetic divergence occurring among the samples analysed.

Recent molecular studies revealed high level of endemism and numerous cryptic species within opisthobranchs, with Mediterranean taxa clearly understudied. Here we used genetic data from both mitochondrial and nuclear gene fragments as well as morphological data from taxonomically relevant characters to investigate the phylogenetic relationships and systematics of Mediterranean taxa of the Flabellinidae and Piseinotecidae families. Phylogenetic analyses based on Bayesian and Maximum-Likelihood methods indicate that Flabellinidae and Pisenotecidae taxa and species within the genera Flabellina, Calmella and Piseinotecus do not form monophyletic clades. These results are supported by our morphological analyses which allowed the re-evaluation of the triseriate radula condition in Pisenotecidae and Calmella taxa and their inclusion in the genus Flabellina as Flabellina gaditana comb. nov. (synonym of F. confusa), Flabellina gabinierei comb. nov. and Flabellina cavolini comb. nov. Species delimitation and barcoding gap analyses allowed uncovering cryptic species within Flabellina gracilis (Alder and Hancock, 1844), F. trophina (Bergh, 1890), F. verrucosa (M. Sars, 1829) and F. ischitana Hirano and Thompson, 1990, the latter with an Atlantic form which is under description. This study corroborates the relevance of combining molecular and morphological data from multiple populations and species in the assessment of nudibranch diversity and classification.

The southernmost record of the Mediterranean nudibranch Felimida elegantula (Philippi, 1844) is reported here. A single specimen, with a colour pattern that hampered an easy morphological identification, was collected at the Kerkennah Islands (Tunisia). DNA-barcoding analysis (with the cytochrome c oxidase subunit I genetic marker) unambiguously confirmed its identification, and radular and anatomical features conformed with those previously described. With this record, the chromatic polymorphism of F. elegantula is demonstrated to be extremely large and the known range of the species is extended to the African coast of Tunisia.

Specimens of the nudibranch genus Calma were observed under boulders at two Croatian localities while feeding on gobiid eggs. Some ambiguous morphological features compared with the original descriptions of the known species of the genus, C. glaucoides and C. gobioophaga hampered easy identification. Genetic data (COI and 16S sequences) confirmed the distinction between the two species of the genus Calma, and allowed unambiguous identification of the Croatian specimens as Calma gobioophaga. This is the first record of this species for the Mediterranean and extends its distribution range remarkably. Finally, the eggs consumed by the Croatian C. gobioophaga specimens have been taxonomically identified by using the 12S rDNA marker as Gobius cobitis.

The nudibranch Diaphorodoris luteocincta (M. Sars, 1870) shows two colour morphotypes defined as D. luteocincta var. alba and D. luteocincta var. reticulata, which are easy to identify and which share an overlapping distribution in the Med-iterranean Sea and the North-Eastern Atlantic Ocean. Their systematics has long been discussed by several authors until recently when a molecular study proposed the two varieties as intraspecific colour variability occurring within D. lu-Teocincta species. In order to solve their ranking status, we have carried out a morphological study on anatomical charac-Ters and molecular analyses on the mitochondrial markers (COI and 16S rDNA) and the nuclear H3 gene. Results proved the usefulness of the integrative taxonomy approach in assessing species delimitation; in fact Diaphorodoris alba stat. nov. and D. luteocincta were revealed to be two different species. D. luteocincta var. reticulata is confirmed as synonym of D. luteocincta s.str. A hypothesis on phylogenetic relationship among most of the currently recognised species of the genus Diaphorodoris Iredale & O'Donoghue, 1923 is also here presented.

The state of knowledge of the alien marine Mollusca in Italy is reviewed and updated. Littorina saxatilis (Olivi, 1792), Polycera hedgpethi Er. Marcus, 1964 and Haminoea japonica Pilsbry, 1895 are here considered as established on the basis of published and unpublished data, and recent records of the latter considerably expand its known Mediterranean range to the Tyrrhenian Sea. COI sequences obtained indicate that a comprehensive survey of additional European localities is needed to elucidate the dispersal pathways of H. japonica. Recent records and interpretation of several molluscan taxa as alien are discussed both in light of new Mediterranean (published and unpublished) records and of four categories previously excluded from alien species lists. Within this framework, ten taxa are no longer considered as alien species, or their records from Italy are refuted. Furthermore, Trochocochlea castriotae Bellini, 1903 is considered a new synonym for Gibbula albida (Gmelin, 1791). Data provided here leave unchanged as 35 the number of alien molluscan taxa recorded from Italy as well as the percentage of the most plausible vectors of introduction, but raise to 22 the number of established species along the Italian shores during the 2005–2010 period, and backdate to 1792 the first introduction of an alien molluscan species (L. saxatilis) to the Italian shores.