DNA Taxonomy Confirms the Identity of the Widely-Disjunct Mediterranean and Atlantic Populations of the Tufted Ghost Crab Ocypode cursor (Crustacea: Decapoda: Ocypodidae).

The distribution area of the tufted ghost crab Ocypode cursor includes two widely separate sub-areas, i.e. the tropical and subtropical Atlantic coasts of Africa and Macaronesia, and the central-eastern Mediterranean basin. The current disjunct distribution of the species is possibly the remnant of a previous wider and continuous distribution area that was fragmented during the Pleistocene, with the disappearance of the species from the temperate Atlantic Ocean and the western Mediterranean basin, and its survival in the warmer areas of the eastern Atlantic and the Mediterranean Sea. Such disjunction is thus compatible with an ancient isolation between the Mediterranean and Atlantic populations of the species, which could in fact constitute two well-characterized independent evolutionary lineages, or even two cryptic species. Unexpectedly, the sequencing of a fragment of the mtDNA COI gene from Mediterranean and Atlantic Ocypode cursor allopatric populations showed the cohesion of the species throughout its distribution range, and the nesting of Mediterranean populations within the single Atlantic population studied. This pattern is hereby tentatively ascribed to an incomplete lineage sorting due to the large population sizes of both the Atlantic and Mediterranean subpopulations of the species. The current westward expansion of the species in the Mediterranean Sea originating from the Levantine basin, due to ongoing regional sea warming, follows a typical phalanx dispersal mode.

The DNA barcoding of Mediterranean combtooth blennies suggests the paraphyly of some taxa (Perciformes, Blenniidae).

A dataset including novel and publicly available mtDNA COI sequences of 14 Mediterranean combtooth blenny species belonging to nine genera was assembled in order to provide a reference dataset for DNA barcoding studies. Some inconsistencies in the current taxonomy of some genera were observed. In particular, the monophyly of the genera Parablennius and Salaria is not supported by the present dataset and the absence of reciprocal monophyly between the morphospecies Salaria basilisca and S. pavo questions their status and stresses the need of a revision of the genus Salaria.

On the Occurrence of the Invasive Freshwater Limpet Ferrissia californica (Mollusca: Gastropoda: Planorbidae) in Japan.

Two members of the genus Ferrissia Walker, 1903, namely Ferrissia nipponica (Kuroda, 1949) and F. japonica Habe and Burch, 1965, have been reported to occur in Japan, but due to a lack of molecular data, doubts have been expressed as to their validity. Furthermore, the possible presence of allochthonous Ferrissia taxa has been stated under a variety of names, so that even now there is no consensus on their presence and identity. Recently, freshwater limpets belonging to the planorbid genus Ferrissia were collected in an irrigation trough on the Izu island of Hachijojima, Tokyo, Japan. Molecular identification of the collected specimens, based on a fragment of the large ribosomal subunit 16S (mtDNA), unequivocally proved that they belong to the allochthonous, invasive species Ferrissia californica (Rowell, 1863), the protagonist of a relentless cryptic invasion worldwide to which can Japan can now be added. The shells of the collected specimens show some scars, which bear testimony to the fragile nature of the shell and, possibly, to the paucity of calcium in the inland waters of Hachijojima.

Distribution and Genetic Lineages of the Craspedacusta sowerbii Species Complex (Cnidaria, Olindiidae) in Italy.

Olindiid freshwater jellyfishes of the genus Craspedacusta Lankester, 1880 are native to eastern Asia; however, some species within the genus have been introduced worldwide and are nowadays present in all continents except Antarctica. To date, there is no consensus regarding the taxonomy within the genus Craspedacusta due to the morphological plasticity of the medusa stages. The species Craspedacusta sowerbii Lankester, 1880 was first recorded in Italy in 1946, and until 2017, sightings of the jellyfish Craspedacusta were reported for 40 water bodies. Here, we shed new light on the presence of the freshwater jellyfishes belonging to the genus Craspedacusta across the Italian peninsula, Sardinia, and Sicily. First, we report 21 new observations of this non-native taxon, of which eighteen refer to medusae sightings, two to environmental DNA sequencing, and one to the finding of polyps. Then, we investigate the molecular diversity of collected Craspedacusta specimens, using a Bayesian analysis of sequences of the mitochondrial gene encoding for Cytochrome c Oxidase Subunit I (mtDNA COI). Our molecular analysis shows the presence of two distinctive genetic lineages: (i) a group that comprises sequences obtained from populations ranging from central to northern Italy; (ii) a group that comprises three populations from northern Italy-i.e., those from the Lake Levico, the Lake Santo of Monte Terlago, and the Lake Endine-and the single known Sicilian population. We also report for the first time a mtDNA COI sequence obtained from a Craspedacusta medusa collected in Spain.

Disentangling cryptic species in Parastenocarididae (Copepoda: Harpacticoida) with an integrative approach: the case of Stammericaris similior sp. nov. and Stammericaris destillans Bruno & Cottarelli 2017.

Stammericaris similior sp. nov. is described combining light microscopy, scanning electron microscopy, and genetic barcoding. The new species was collected from rimstone pools in Scrivilleri Cave, a cave in Sicily with so far unexplored microcrustacean fauna. The new species is particularly interesting because it is morphologically very similar to Stammericaris destillans, an epikarstic parastenocaridid endemic to a different Sicilian cave; however, the phylogenetic analysis based on the mitochondrial COI gene of sixteen parastenocaridids shows that these two Stammericaris are two distinct species, with an uncorrected p-distance of 22.9, and the sequences of Stammericaris similior sp. nov. cluster together in a well-supported monophyletic clade, with two different haplotypes. To our knowledge, the presence of different species of almost identical morphology had not been recorded before for the genus Stammericaris. The integrated molecular and morphological analysis, the latter conducted with the support of SEM, allows disentangling the affinities of the new species and identifying a few distinctive characters: the males of the new species are characterized by the caudal rami shorter than the anal somite; the morphology of the P3, which is thin and slightly arched, with three proximal spinules on exp-1; the peculiar structure of the P4 enp; the P4 basis ornamented with two spinules of different length, the one closest to the endopod being the shortest one, and a half-moon shaped lamella. The new species differs from S. destillans for its larger size, the presence of: three spinules, instead of two, on the P3 exp-1; the half-moon shaped lamella on the P4 basis; a row of spinules along the inner margin of P4 exp-1. We also provide data on the ecology and distribution of the new species, a list of the other copepod species collected, and a dichotomic key for the males of all species presently assigned to the genus.

Morphological and molecular diversity patterns of the genus Tropodiaptomus Kiefer, 1932 (Copepoda, Calanoida, Diaptomidae) in Thailand.

Tropodiaptomus is one of the most specious genera in the family Diaptomidae, but it is often rare in terms of distribution and abundance. Moreover, Tropodiaptomus species show a noteworthy variability in some of the morphological characters considered of prime importance in diaptomid taxonomy, and the presence of cryptic or pseudocryptic species is likely. Thus, through a geographically-wide sampling in Thailand, we aimed to investigate the local diversity of the genus and to compare the morphological and molecular diversity pattern based on mitochondrial and nuclear genes sequences. DNA taxonomy was also implemented in order to check whether the Tropodiaptomus lineages were independent species according to the "evolutionary genetic species concept". Six Tropodiaptomus morphospecies were found, three of which are putative species new to Science pending a formal description. The finding of such a high incidence of undescribed species stresses the existence of a significant "Linnean shortfall" affecting Thai diaptomids. The molecular results showed that most of the studied species could be identified consistently with their morphology-based taxonomy. However, Tropodiaptomus vicinus and T. cf. lanaonus showed a high level of genetic diversity, suggesting that traditional morphological techniques might be inadequate for correctly assessing their taxonomical status.

Unveiling the egg microbiota of the loggerhead sea turtle Caretta caretta in nesting beaches of the Mediterranean Sea.

Microbes have central roles in the development and health of animals, being the introduction of specific microbial species a potential conservation strategy to protect animals from emerging diseases. Thus, insight into the microbiota of the species and their habitats is essential. In this manuscript, we report for the first time the bacterial composition of all the components (eggshells of hatched and unhatched eggs, internal content of unhatched eggs, intestinal content of hatchling and pipping sea turtles, and sand) of three nesting beaches of Caretta caretta along the Italian coasts of the Mediterranean Sea. The analysis of 26 amplicon samples was carried out using next-generation sequencing analysis, targeting V3-V4 regions of the bacterial 16S rRNA gene. Samples featured mainly Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes, whose percentages depended on the sample type. Our results showed that, although from different sampling sites, the internal content of the unhatched eggs, intestinal content of hatchling and pipping sea turtles share the microbiota, which was yet different from that of eggshells and sand of the same nesting beach. This study suggests the maternal and environmental influence alongside a protective role of eggshells in shaping the egg microbiota of Caretta caretta sea turtles.

The European Pine Marten Martes martes (Linnaeus, 1758) Is Autochthonous in Sicily and Constitutes a Well-Characterised Major Phylogroup within the Species (Carnivora, Mustelidae).

No molecular data are currently available for the Sicilian populations of the European pine marten Martes martes, thus preventing any sound inference about its native or non-native status on the island, as well as the local phylogeography of the species. In order to investigate these issues, we sequenced two mtDNA markers in road-killed specimens collected in Sicily. Both markers consistently demonstrated the existence of a well-characterised Sicilian clade of the species, which is endemic to the island and constitutes the sister group of a clade including the Mediterranean and Central-North European major phylogroups of the European pine marten. Such evidence supports the autochthony of Martes martes in Sicily and points to a natural Pleistocene colonisation of the island followed by isolation. The occurrence of a, to date undetected, major phylogroup of the species in Sicily calls for the dedicated monitoring of the Sicilian populations of the species in order to preserve this evolutionarily significant unit.

Conservation state of two paintings in the Santa Margherita cliff cave: role of the environment and of the microbial community.

The conservation of ancient paintings sited in humid environments is an actual challenge for restorers, because it needs the knowledge of the materials the paintings are made up and of their interaction with a peculiar surrounding environment; thus, tailored procedures and strategies aimed at restoring and preserving paintings are necessary. Santa Margherita's cave in Castellammare del Golfo (Trapani, Italy) is a natural cave, containing the remains of paintings, in a poor state of conservation, belonging to an ancient church dated back to the Middle Age. The present manuscript reports the monitoring of environmental conditions (i.e., temperature and humidity) in a full year, as well as a study on the materials constituting the stone support and the paintings together with a survey of the microbial community. The findings allow us to define the causes that mainly involve the degradation of the paintings. In detail, the degradation of the east and the west walls occurred differently because of the exposure to the sea aerosol, which influenced the salt composition, also contributing to diversifying the bacterial community. Some specific actions to plan the conservation and restoration of paintings and to preserve the site are suggested.

A Comparative Analysis of Aquatic and Polyethylene-Associated Antibiotic-Resistant Microbiota in the Mediterranean Sea.

In this study, we evaluated the microbiome and the resistome profile of water and fragments of polyethylene (PE) waste collected at the same time from a stream and the seawater in a coastal area of Northwestern Sicily. Although a core microbiome was determined by sequencing of the V3-V4 region of the bacterial 16S rDNA gene, quantitative differences were found among the microbial communities on PE waste and the corresponding water samples. Our findings indicated that PE waste contains a more abundant and increased core microbiome diversity than the corresponding water samples. Moreover, PCR analysis of specific antibiotic resistance genes (ARGs) showed that PE waste harbors more ARGs than the water samples. Thus, PE waste could act as a carrier of antibiotic-resistant microbiota, representing an increased danger for the marine environment and living organisms, as well.

Comparison of the Intestinal Microbiome of Italian Patients with Multiple Sclerosis and Their Household Relatives.

Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system, caused by a combination of genetic and environmental factors. In recent years, a role in MS pathogenesis was assigned to the gut microbiota. However, different signatures of gut dysbiosis have been shown to depend on environmental factors, like diet and lifestyle. In this study, we compared the gut microbiome in MS patients and their household healthy relatives sharing lifestyle and environmental factors. Faecal metagenomic DNA was extracted and the V3-V4 regions of the conserved bacterial 16S ribosomal RNA gene were amplified and sequenced. While overall bacterial communities were similar, specific families differed between healthy and MS subjects. We observed an increase in Ruminococcaceae, Christensenellaceae, Desulfovibrionaceae, Clostridiales, and Family XIII in MS patients, while Bacteroidaceae, Tannerellaceae, Veillonellaceae, and Burkholderiaceae were more abundant in healthy controls. In addition, principle coordinate analysis showed that the gut microbiome of all MS patients formed a cluster being less diverse than the household relatives and that gut microbiota of MS patients with EDSS 4.5-7 formed a distinct cluster in respect to their controls. Overall, our study is consistent with the hypothesis that MS patients have gut microbial dysbiosis and evidenced the importance of environmental factors in shaping the gut microbiome.

Can phthalates move into the eggs of the loggerhead sea turtle Caretta caretta? The case of the nests on the Linosa Island in the Mediterranean Sea.

During the monitoring of Caretta caretta nests on the island of Linosa, 30 unhatched eggs from four nests were collected to study the presence of phthalates in their three components (shell, yolk, and albumen). Four phthalates, namely diethyl (DEP), dibutyl (DBP), di-(2-ethylhexyl) (DEHP), and dioctyl (DOTP) phthalic acid esters (PAE), which are widely used as additives in plastics, were detected in all egg components. The most frequently found phthalate was DBP, followed by DEHP in eggshell and yolk. Dimethyl- (DMP) and butylbenzyl-phthalate (BBP) were below the limits of detection for all samples. The high total phthalate recorded in the yolk suggests that contamination could arise by vitellogenesis. PERMANOVA analysis (p = 0.01) confirmed significant differences in the PAEs contamination profiles in the eggs from the four nests. This study confirms the negative impact of plastic related compounds posing questions about the potential adverse effects on organisms and their conservation status.

Molecular Cytogenetic Characterization of the Sicilian Endemic Pond Turtle Emys trinacris and the Yellow-Bellied Slider Trachemys scripta scripta (Testudines, Emydidae).

Turtles, a speciose group consisting of more than 300 species, demonstrate karyotypes with diploid chromosome numbers ranging from 2n = 26 to 2n = 68. However, cytogenetic analyses have been conducted only to 1/3rd of the turtle species, often limited to conventional staining methods. In order to expand our knowledge of the karyotype evolution in turtles, we examined the topology of the (TTAGGG)n telomeric repeats and the rDNA loci by fluorescence in situ hybridization (FISH) on the karyotypes of two emydids: the Sicilian pond turtle, Emys trinacris, and the yellow-bellied slider, Trachemys scripta scripta (family Emydidae). Furthermore, AT-rich and GC-rich chromosome regions were detected by DAPI and CMA3 stains, respectively. The cytogenetic analysis revealed that telomeric sequences are restricted to the terminal ends of all chromosomes and the rDNA loci are localized in one pair of microchromosomes in both species. The karyotype of the Sicilian endemic E. trinacris with diploid number 2n = 50, consisting of 13 pairs of macrochromosomes and 12 pairs of microchromosomes, is presented here for first time. Our comparative examination revealed similar cytogenetic features in Emys trinacris and the closely related E. orbicularis, as well as to other previously studied emydid species, demonstrating a low rate of karyotype evolution, as chromosomal rearrangements are rather infrequent in this group of turtles.

New insights into the gut microbiome in loggerhead sea turtles Caretta caretta stranded on the Mediterranean coast.

Caretta caretta is the most common sea turtle species in the Mediterranean Sea. The species is threatened by anthropomorphic activity that causes thousands of deaths and hundreds of strandings along the Mediterranean coast. Stranded turtles are often cared for in rehabilitation centres until they recover or die. The objective of this study was to characterize the gut microbiome of nine sea turtles stranded along the Sicilian coast of the Mediterranean Sea using high-throughput sequencing analysis targeting V3-V4 regions of the bacterial 16S rRNA gene. Stool samples were collected from eight specimens hosted in the recovery centre after a few days of hospitalization (under 7) and from one hosted for many weeks (78 days). To better explore the role of bacterial communities in loggerhead sea turtles, we compared our data with published fecal microbiomes from specimens stranded along the Tuscan and Ligurian coast. Our results highlight that, despite the different origin, size and health conditions of the animals, Firmicutes, Bacteroidetes and Proteobacteria constitute the main components of the microbiota. This study widens our knowledge on the gut microbiome of sea turtles and could be helpful for the set up of rehabilitation therapies of stranded animals after recovery in specialized centres.