In 2014, Berghia marinae Carmona, Pola, Gosliner & Cervera, 2014 from Senegal was described along with the revision of the genus Berghia Trinchese, 1877. In this study, we establish a second record for the senegalese species B. marinae in the Mediterranean Sea, 4,000 Km away from its type location. The morphological mismatch from the original description hampered its identification, and thus, a molecular approach was needed. Multilocus phylogenetic trees were inferred from Maximum-likelihood and Bayesian analyses based on partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes, and the nuclear gene histone-3. Species delimitation analyses were performed to support the phylogenetic results and a new morphological description is provided complementing earlier information on this barely known species.

Population genomic studies have increased in the last decade, showing great potential to understand the evolutionary patterns in a great variety of organisms, mostly relying on RAD sequencing techniques to obtain reduced representations of the genomes. Among them, 2b-RAD can provide further secondary reduction to adjust experimental costs by using base-selective adaptors, although its impact on genotyping is unknown. Here we provide empirical comparisons on genotyping and genetic differentiation when using fully degenerate or base-selective adaptors and assess the impact of missing data. We built libraries with the two types of adaptors for the same individuals and generated independent and combined datasets with different missingness filters (presence in 100%, 75% and 50% of the libraries). Our approach, by exploring locus-by-locus, found 92% of identical genotypes between the two libraries of the same individual when using loci shared among 100% of the libraries, which decreased to 35% when working with loci present in at least 50% of them. We show that missing data is a major source of individual genetic differentiation. The loci discordant by genotyping were in low frequency (7.67%) in all filtered files. Only 0.96% of them were directly attributable to base-selective adaptors reducing heterozygosity. An underestimation of heterozygosity in 6.44% of genotypes was found in libraries generated with fully degenerated adaptors, of which ca. 70% had <10 reads per locus indicating that sufficient read depth should be ensured for a correct genotyping. Base-selective adaptors reduce the number of loci, increase their coverage, and provide a reliable genotyping with no heterozygosity underestimation at a lower sequencing cost. We demonstrate that missing data is a major concern for a correct identification of genetic differentiation, and should not be neglected in genomic studies. Thus, we conclude that 2b-RAD libraries using base-selective adaptors are a robust tool to use in population genomics regardless the target species genome size, promoting their use in future studies.

The genus Dendrodoris is ascribed to the family Dendrodorididae, whose members are characterized lacking spicules in the dorsum as well as a radula, one of the most important taxonomic traits to distinguish and define nudibranch species. The absence of this taxonomic character makes difficult to delineate species based on morphologic traits, and consequently, several cryptic species may remain unreported resulting in a potential underestimation of the real diversity of the genus. Species delimitation analyses based on molecular data constitute an important tool to unveil cryptic species, especially when morphology may be misleading. However, for the genus Dendrodoris, only two molecular studies have been performed to date, both based on Indo-pacific species and the molecular marker COI. In the present study, we performed a multilocus approach using the molecular markers COI, 16S and H3 based on 47 Dendrodoris specimens from the North Atlantic Ocean and Mediterranean Sea. As a result, we detected monophyly for five of the previously accepted species plus the presence of an unidentified one. Using morphological and anatomical data from 14 additional individuals and a review of the literature, we verified this unidentified species is the previously synonymized species Dendrodoris temarana Pruvot-Fol, 1953, and we redescribe it morphologically (chromatic pattern, gills, rhinophores and anal papilla), anatomically (neural, circulatory, digestive and reproductive systems), and biologically herein. D. temarana is differentiated by its chromatic pattern, previously thought to be intraspecific variation of D. grandiflora (Rapp, 1827), D. limbata (Cuvier, 1804) and D. herytra Valdés & Ortea in Valdés, Ortea, Avila & Ballesteros, 1996, and therefore it is considered a pseudocryptic species. This study increases the number of valid species in the North Atlantic and Mediterranean to nine and confirms the importance of integrative taxonomic approaches to resolve the identification of cryptic species in complex groups.

Despite their recognized primary importance, marine coastal ecosystems around the globe are currently under threat, being subject to continuous local and global anthropogenic stressors. In this frame, understanding the response of coastal habitat-forming species to multiple stressors and their resilience is fundamental for the sustainable management of coastal ecosystems. In the present study, to provide some glimpses in this direction, we explored the response of the Anemonia viridis-associated microbiota to the combined anthropogenic stressors, which typically affect touristic hotspots at Mediterranean coastal sites. To this aim, two case studies have been carried out, the first in the Riccione coastal site (Italy, Center Mediterranean) and the second at Cap de Creus (Spain, North-western Mediterranean), where the A. viridis microbiota was assessed under the conditions of both high and low anthropogenic pressure. According to our findings, the A. viridis microbiota showed a relevant degree of plasticity in response to combined anthropogenic and environmental stressors, with changes that also mirrored variations in the surrounding seawater, thus indicating a close connection with the environment, from which potential symbiotic partners are selected. However, this potentially adaptive process also has a limitation, as observed in the highly anthropogenic impact site of Cap de Creus, where A. viridis-associated microbiota appeared completely unstructured, as demonstrated by an increased dispersion according to the Anna Karenina principle. This raises the question about the resilience of the A. viridis-associated microbiota under combined climate and anthropogenic threats, as well as of the anthropogenic factors driving the observed dysbiosis changes.

Population genomic studies are increasing in the last decade, showing great potential to understand the evolutionary patterns in a great variety of organisms, mostly relying on RAD sequencing techniques to obtain reduced representations of the genomes. Among them, 2b-RAD can provide further secondary reduction to adjust study costs by using base-selective adaptors, although its impact on genotyping is unknown. Here we provide empirical comparisons on genotyping and genetic differentiation when using fully degenerate and base-selective adaptors and assess the impact of missing data. We built libraries with the two types of adaptors for the same individuals and generated independent and combined datasets with different missingness filters according to their presence (100%, 75% and 50%). Exploring locus-by-locus, we found 92% of identical genotypes between both libraries of the same individual when using loci present in 100% of the samples, which decreased to 35% when working with loci present in at least 50% of them. We show that missing data is a major source of individual genetic differentiation. The loci discordant by genotyping were in low frequency (7.67%) in all filtered files. Only 0.96% were directly attributable to base-selective adaptors, and 6.44% underestimated heterozygosity in NN libraries, of which ca. 70% had <10 reads per locus indicating that sufficient read depth should be ensured for a correct genotyping. Our work confirms that 2b-RAD libraries using base-selective adaptors are a robust tool to use in population genomics of species with large genome sizes.