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Herein, we investigated for the first time the microbiota from the hepatopancreas of the talitrid amphipod Orchestia montagui and the oniscidean isopod Tylos ponticus, two crustacean species associated with beached leaves of the seagrass Posidonia oceanica (Posidonia banquette). We carried out a metabarcoding analysis based on several hypervariable regions of the bacterial 16S rRNA gene. At the same time, three representative habitats of the supralittoral zone, namely leaf surface of Posidonia banquette, sandy shore and surface seawater were examined by means of bacterial metabarcoding analysis. Our data analyses indicated that the bacterial microbiota from the hepatopancreas and that from the marine habitats form distinct groups in terms of the taxonomic composition. Comparative analyses revealed that some bacteria were associated with the hepatopancreas of the syntopic O. montagui and T. ponticus but were not detected in the supralittoral habitats. This suggests that distinct components of the microbial community may be uniquely adapted within the hepatopancreas. The microbiota from the hepatopancreas of O. montagui was more complex than that of T. ponticus. However, the putative presence of polysaccharide-degrading bacteria in both species indicated symbiotic microbiomes that might be involved in the functionality of the hepatopancreas. A deeper understanding of halophilic hepatopancreatic symbionts in the supralittoral zone also has broad relevance to applied research, particularly to the biotechnological sector related to marine biomass conversion.

Species in the family Capulidae (Littorinimorpha: Capuloidea) display a wide range of shell morphologies. Several species are known to live in association with other benthic invertebrates—mostly bivalves and sabellid worms, but also other gastropods—and are believed to be kleptoparasitic filter feeders that take advantage of the water current produced by the host. This peculiar trophic ecology, implying a sedentary lifestyle, has resulted in highly convergent shell forms. This is particularly true for the genus Hyalorisia Dall, 1889, which occurs in deep water in the Caribbean and Indo-West Pacific provinces, with two nominal species recognized so far. Combining morphological, ecological and molecular data, we assessed the diversity of the genus, its phylogenetic position inside the family and its association with its bivalve host, the genus Propeamussium de Gregorio, 1884 (Pectinoidea), resulting in the description of nine new cryptic species. When sympatric, species of Hyalorisia are associated with different host species, but the same species of Propeamussium may be the host of several allopatric species of Hyalorisia.

Three new species of the neogastropod family Raphitomidae Bellardi, 1875, are described from the Croatian waters of the Adriatic Sea: Raphitoma petanii Prkic, Giannuzzi-Savelli & Pusateri n. sp., R. pusaterii Prkic & Giannuzzi-Savelli n. sp. and R. stanici Prkic, Giannuzzi-Savelli & Pusateri n. sp. The new species are diagnosed by teleoconch characters and by genetic data (COI sequences) in an integrative taxonomy framework. They belong to a group of species with microgranules on the external surface of all teleoconch whorls and all three have a multispiral protoconch indicating a planktotrophic larval phase. The first two species are so far known only from material found in Croatia. R. petanii Prkic, Giannuzzi-Savelli & Pusateri n. sp. and R. stanici Prkic, Giannuzzi-Savelli & Pusateri n. sp. are predominantly shallow-water species, whereas R. pusaterii Prkic & Giannuzzi-Savelli n. sp. has been so far collected only from deeper bottoms with presence of the red coral, Corallium rubrum (Linnaeus, 1758). Syntypes of Murex echinatusBrocchi, 1814 are figured and commented.

Blastocystis spp. is a common single-celled intestinal symbiont, comprising several genetic subtypes (ST) and transmissible by animal-to-animal, human-to-human, animal-to-human and, possibly, human-to-animal routes. This work was designed to explore the presence of Blastocystis in sympatric domestic and wild suids and their ability to carry zoonotic STs, in a condition of widespread opportunity to come in contact with the microorganism through their shared water and food resources, and other carriers. We sampled 42 and 37 stool samples from wild boars and domestic pigs, respectively. STs were first identified by PCR followed by Sanger sequencing. Sequences represented in double-band PCR products or in Sanger chromatograms displaying multiple peaks, were resolved by next generation sequencing (NGS). Twenty-six (61.9%) wild boar and 26 (70.2%) pig samples were PCR-positive, respectively. ST3, ST5 and ST15 were found in 3.8%, 38.4% and 80.8% of the positive wild boars and 11.5%, 88.5%, 11.5% of the positive pigs, respectively. ST1 was found only in pigs (3.8%). STs 5 and 15 were common in both groups of animals, but in reversed proportions, suggesting preferential colonization. We found significantly different ST distributions among wild boars and domestic pigs. This might indicate that lifestyle differences between the two populations influence their risk for contracting certain subtypes, or that ST5 and ST15 can colonize preferentially wild or domestic animals. Based on the STs described here, wild boars and domestic pigs can act as reservoirs with zoonotic potential. The ability of suids to carry zoonotic STs appears to be higher when using NGS than Sanger sequencing, and resolution of complex sequencing profiles is imperative before excluding the presence of STs of human concern.

Giannuzzi-Savelli, Riccardo, Pusateri, Francesco, Prkić, Jakov, Bartolini, Stefano, Russini, Valeria, Fassio, Giulia, Oliverio, Marco (2020): Revision of Mediterranean and NE Atlantic Raphitomidae (Gastropoda, Conoidea) 8: The genus Leufroyia Monterosato, 1884. Zoosystema 42 (22): 433-473, DOI: 10.5252/zoosystema2020v42a22

Poecilogony is the intraspecific variation in developmental mode, with larvae of different types produced by the same individual, population or species. It is very rare among marine invertebrates, and in gastropods has long been described only in a few opisthobranchs. The physiological and regulatory mechanisms underlying larval evolutionary transitions, such as loss of planktotrophy that occurred repeatedly in many caenogastropod lineages, are still largely unknown. We have studied the inter- v. intraspecific variation in larval development in the north-east Atlantic neogastropod genus Raphitoma Bellardi, 1847, starting with an iterative taxonomy approach: 17 morphology-based Preliminary Species Hypotheses were tested against a COI molecular-distance-based method (ABGD), and the retained species hypotheses were eventually inspected for reciprocal monophyly on a multilocus dataset. We subsequently performed an ancestral state reconstruction on an ultrametric tree of the 10 Final Species Hypotheses, time-calibrated by fossils, revealing that the interspecific changes were planktotrophy > lecithotrophy, and all have occurred in the Pleistocene, after 2.5 million years ago. This is suggestive of a major role played by Pleistocene Mediterranean oceanographic conditions – enhanced oligotrophy, unpredictable availability of water column resources – likely to favour loss of planktotrophy. Within this group of species, which has diversified after the Miocene, we identified one pair of sibling species differing in their larval development, Raphitoma cordieri (Payraudeau, 1826) and R. horrida (Monterosato, 1884). However, we also identified two Final Species Hypotheses, each comprising individuals with both larval developmental types. Our working hypothesis is that they correspond to one or two poecilogonous species. If confirmed by other nuclear markers, this would be the first documentation of poecilogony in the Neogastropoda, and the second in the whole Caenogastropoda. Although sibling species with different developmental strategies may offer good models to study some evolutionary aspects, poecilogonous taxa are optimally suited for identifying regulatory and developmental mechanisms underlying evolutionary transitions.

Three species of the neogastropod genus Columbella Lamarck, 1799 are recognised from the northeastern Atlantic and the Mediterranean. One is the common Mediterranean C. rustica (Linnaeus, 1758), with paucispiral protoconch, extending its range in the Atlantic South to Senegal and North to Portugal. Columbella adansoni Menke, 1853, with multispiral protoconch is restricted to the Macaronesian archipelagoes. A third species, also with multispiral protoconch, from West Africa is recognised through molecular methods, and the name C. xiphitella Duclos, 1840 is employed by correcting the original erroneous locality (“Californie”) to Gabon. Except for protoconch features, no major morphological characters are available to separate the three species; however diagnostic species-level differences in specific positions in the cytochrome c oxidase I (COI) sequences are present between all three species.

In marine environments, connectivity among populations of benthic invertebrates is provided primarily by dispersion of larvae, with the duration of pelagic larval phase (PLD) supposed to represent one of the major factor affecting connectivity. In marine gastropods, PLD is linked to specific larval development types, which may be entirely intracapsular (thus lacking a pelagic dispersal), or include a short pelagic lecithotrophic or a long planktotrophic phase. In the present study, we investigated two sibling species of the cosmopolitan neogastropod genus Columbella (commonly known as dove shells): Columbella adansoni Menke, 1853, from the Macaronesian Atlantic archipelagos, with planktotrophic development, and Columbella rustica Linnaeus, 1758, from the Mediterranean Sea, with intracapsular development. We expected to find differences between these two sister species, in terms of phylogeographic structure, levels of genetic diversification and spatial distribution of genetic diversity, if PLD was actually a relevant factor affecting connectivity. By analysing the sequence variation at the cytochrome c oxidase subunit I (COI) in 167 specimens of the two species, collected over a comparable geographic range, we found that Columbella adansoni, the species with planktotrophic development, and thus longer PLD, showed no phylogeographic structure, lower levels of genetic diversity, interpopulational variance lower than the intrapopulational one and no spatial structure in the distribution of the genetic diversity; Columbella rustica, the species with intracapsular development, thus with evidently lower dispersal abilities, showed a clear phylogeographic structure, higher levels of genetic diversity, high interpopulational and low intrapopulational variance, and a clear signature of global spatial structure in the distribution of the genetic diversity. Thus, in this study, two sibling species differing almost only in their larval ecology (and PLD), when compared for their genetic variation showed patterns supporting the hypothesis that PLD is a major factor affecting genetic connectivity. Therefore, it seems reasonable to expect that the ecological attributes of the marine communities - also in terms of the variation in larval ecology of the species involved - are taken into the due consideration in conservation actions, like the design of marine protected areas networks.

The systematics of several Eastern Atlantic conoidean species, traditionally ascribed to the genus RaphitomaBellardi, 1847, are revised on the basis of DNA sequence data from three gene regions (cytochrome c oxidase subunit I, 16S rRNA and 12S rRNA). We assign genus ranking to three major lineages (Raphitoma, CyrilliaKobelt, 1905 and LeufroyiaMonterosato, 1884) and suggest that two West African species belong in the subgenus Daphnella (Paradaphne) Laseron, 1954. A new classification, based on molecular systematics and critical study of morphology, is provided for all Eastern Atlantic and Mediterranean species that are currently ascribed to Raphitoma s.l. The genus ClathromangeliaMonterosato, 1884 is confirmed as belonging to Raphitomidae. Phylogenetic relationships and genetic distances suggest that Raphitoma maculosaHøisæter, 2016 and R. obesaHøisæter, 2016 may be divergent morphotypes of R. bicolor (Risso, 1826) and Cyrillia aequalis (Jeffreys, 1867), respectively.

The recent report of Nemoura lacustris Pictet, 1865 in Great Britain has raised doubts on its identity, given the isolation with respect to the Mediterranean and continental populations of this species. Using molecular analyses, we tested if populations from the United Kingdom and the Iberian Peninsula were conspecific and tested the hypotheses of a recent colonization event versus a more ancient origin for the British populations. Phylogenetic analysis of the mitochondrial marker COI allowed us to conclude that the United Kingdom specimens morphologically ascribed to N. lacustris were conspecific with populations from France and the Iberian Peninsula. Based on the genetic divergence of the two reciprocally monophyletic clades from the Iberian Peninsula and Great Britain/France, respectively, the present distribution of N. lacustris can be postulated as a relatively recent dispersal or introduction into Great Britain from France. Finally, we note the isolated position displayed by N. lacustris in the phylogenetic tree of Nemoura species based on COI sequences, as the sister to all included species of the genus. This isolated position corresponds with the specific morphology of N. lacustris genitalia and requires additional studies to ascertain clearer generic boundaries within the Nemouridae.

The thermophilic Aphaenogaster testaceopilosa-group is a taxon consisting of 14 species in Europe. Recently, this group was recently revisited using morphological characters suggesting different changes in the systematic of this group. For the first time, in this work we used a molecular approach using the DNA barcoding on the european members of the genus Aphaenogaster in order to: i) validate the current systematic of the A. testaceopilosa group in Italy, ii) test the efficiency of COI marker to delimit the species belonging to the genus Aphaenogaster. In the first phase, we morphologically identified 54 specimens of A. testaceopilosa group collected along the entire Italian range. In the second phase, fragments of COI and CAD markers were sequenced from phenotyped individuals. Phylogenetic reconstructions and genetic distances clearly showed six lineages (MOTUs) in agreement with morphological identification except for one specimen collected in a likely contact zone between Aphaenogaster picena and Aphaenogaster semipolita. Furthermore, this study confirms the powerful of DNA barcoding to support the morphological analysis to delimit the species belonging to Aphaenogaster testaceopilosa-group. Therefore, genetic data could be a valid tool to investigate other more problematics groups of this little-known genus.