ABSTRACT. Aim Explaining why some taxa are more diversified than their closest relatives remains one of the major challenges in evolutionary biology. However, much of Earth’s biodiversity is concentrated in few diverse non-vertebrate animal lineages, whose analysis of diversification dynamics is hampered by the lack of empirical data. The ERC HYPERDIVERSE project aims to understand the processes behind the evolutionary success of neogastropods, a hyperdiverse group of venomous marine molluscs.

Methods The first objective of the project is to clarify the systematics of the group, by applying integrative taxonomy to delimit species and speed up their description and by using an exon-capture approach to reconstruct a genus-level phylogeny. The second objective is to identify in the genomes and transcriptomes of selected species the toxins that they use to subdue their prey. By relying both on the reconstructed phylogeny and the estimated toxin diversity, we will then evaluate if the diversity of the neogastropods, or subgroups within neogastropods, is linked to the type and quantity of toxins they produce.

Results Hundreds of new species have been identified so far, and the phylogeny obtained as part of the project includes around 1,000 genera, sequenced for 1,100 exons, and most nodes are highly supported, revealing some unexpected relationships. The first chromosome-level genomes confirmed the presence of a whole genome duplication for the neogastropods, potentially linked to the apparition of new toxin-related genes, that might partly explain their evolutionary success.

Main conclusion The last two years of the project will be devoted to the analysis of the macroevolutionary patterns, relying both on the phylogeny to estimate diversification rate variation and on estimated toxin diversity to correlate it to the species diversity among lineages. But what is already clear from our results is the importance of a good knowledge of the species diversity and phylogenetic relationships, essential when tackling macroevolutionary questions.

Thematic Areas Macroevolution, Populations and Species

ABSTRACT. Aim Why some groups of organisms achieve a spectacular richness in both species and morphological diversity whereas others do not is a question which has been engaging evolutionary biologists for many decades. Indeed, evolutionary rates often vary among clades and lineages, with fast-evolving groups accumulating new species, phenotypic diversity or both faster than slow-evolving groups. Whether rates of speciation and phenotypic evolution are correlated is, however, an open question. Classical theory focussing on adaptive evolutionary divergence suggests that these rates should be associated and has received some empirical support. At the same time, other empirical studies have failed to find an association between rates of speciation and rates of phenotypic evolution.

Methods We move past previous efforts by using three traits – including principled projections of multivariate data. As focal clade we use wrasses, a hyperdiverse clade of marine fish comprising the families Labridae, Scaridae (parrotfish) and Odacidae. We build a new dated phylogeny of this group, and we study trait evolution and speciation using advanced geometric morphometric and statistical techniques.

Results We find: 1. substantial rate variation within this clade for both speciation and phenotypic evolution, 2. that the relationship between speciation rates and rates of phenotypic evolution depends on the trait considered and is significant only for a specific aspect of body shape which may be interpreted in terms of genetically-driven phenotypic integration, 3. that the strength of the relationship between rates of speciation and rates of phenotypic evolution, when present, varies between fast-evolving and slow-evolving lineages.

Main conclusion In addition to providing a deeper understanding of evolutionary patterns in wrasses, these results have broader implications for the way we address the general question of whether different rates are associated with each other.

ABSTRACT. Tanya Alessandro1, Rossella Annunziata1

1 Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, 80121, Naples, Italy

Email: tanya.alessandro@szn.it; rossella.annunziata@szn.it;

Abstract

Aim Environmental periodicities influence physiology and behavior of most living organisms on Earth, therefore sophisticated timing mechanisms have evolved to adapt to these periodicities. Molecular regulation of circadian rhythms has been described in animals as well as in plants, fungi and bacteria while information on non-chordate deuterostomes such as echinoderms is scarce and a detailed study of echinoderm larval transcriptomic and physiological activity during the light-dark cycle is missing.

Methods To explore the evolutionary conservation of the canonical circadian clock genes, we exploited the available genomic and transcriptomic databases of species belonging to the Ambulacraria clade. Phylogenetic analyses are ongoing and will shed light on their evolutionary conservation in deuterostomes. The transcriptomes of the sea urchin Paracentrotus lividus and the sea star Patiria miniata larvae entrained in 12L:12D will be examined by RNA-seq and Real Time-PCR. The spatial expression of the putative echinoderm circadian clock components during the development of sea urchin and sea star embryos up to the larval stage will be assessed through ISH and their involvement in the regulation of circadian rhythms will be tested through functional studies, using gene perturbation tools.

Results The majority of the considered circadian clock genes has been identified in the selected species. Interestingly, the well-conserved clock component Period was not found in none of the analysed echinoderm and hemichordates species, suggesting its loss in Ambulacraria during deuterostome evolution.

Main conclusion Our results highlight that the Ambulacraria core clock architecture might be organized differently from the animal circadian clocks so far investigated in metazoans. This study offers perspectives to elucidate timekeeping mechanisms in non-chordate deuterostomes, opening the way to introduce them as experimental system in the Chronobiology field.

Thematic Areas Macroevolution, Adaptation

ABSTRACT. Abstract Aim Longevity is a complex and poorly characterized trait: we do know that some species can live longer than others, but the molecular players responsible for the multiple independent occurrences of the extended longevity phenotype are largely unknown. In this context, bivalve molluscs can provide novel perspectives: the class Bivalvia shows indeed the highest lifespan disparity within Metazoa, ranging from 1 to 500+ years, and includes the longest-lived non-colonial animal species known so far, the clam Arctica islandica. Bivalves therefore represent important resources to provide insights into the evolution of extended longevity. Methods In this work, we leveraged transcriptomic resources spanning thirty bivalve species and we investigated genes with signature of convergent molecular evolution in bivalves showing extreme longevity. We analyzed, for the first time for such topic, networks of protein interactions among such genes, to have a more comprehensive view of the interplay of genes and pathways co-involved in the extended longevity phenotype. Results A large majority of genes showing convergent evolution in long-lived bivalves constitute a large, continuous network of connections. Such network is enriched for factors with experimental support for a role in longevity in other animal species. The network highlights that an integration of different genes and pathways is required for the extended longevity phenotype, and genes involved in cell proliferation control, translational machinery, and response to hypoxia seem to have a central role in lifespan extension. Main conclusions Our results suggest that the mechanisms underlying extended longevity are, at least partially, similar across metazoans: while some genes in the network have experimental support of a role in longevity in model species, and they are likely involved in longevity in bivalves, other genes in the network may represent new possible candidates with a role in extending lifespan, both in bivalves and other animals.

ABSTRACT. Aim The transition of aquatic species to terrestrial environments occurred repeatedly in all animal groups. Aquatic and terrestrial habitats are characterized by extremely different conditions and nearly every life history trait was affected during the conquest of dry land. Genomics has proven an effective tool for the study of terrestrialization thanks in particular to the discovery of gene groups involved in the water-to-land transition of several taxa. Although semi-terrestrial and terrestrial brachyuran crabs are a key group to understand the metabolic mechanisms involved in the transition from water to land, no genomic studies have so far considered patterns of land colonization in this taxon. True crabs (Decapoda; Brachyura) have been particularly successful in evolving semi-terrestrial and terrestrial life habits and count more than 20 phylogenetically unrelated families of Brachyura that conquered the land. We used genomic and transcriptomic tools to study the molecular traits involved in the terrestrialization process of brachyurans using the terrestrial crab Chiromantes haematocheir as a model species. Methods We sequenced and assembled a draft genome of Chiromantes haematocheir using both long- and short-reads sequencing approaches. We then performed transcriptome sequencing on C. haematocheir individuals kept in water or air-exposed. Expansion and contraction of differentially expressed genes were then investigated in malacostracan species using an annotated genome. Results and Conclusions The assembled genome of C. haematocheir had a size of 3.1Gb, an N50 of 200kb and a busco score of ~93%. Transcriptome analyses revealed differential expression in C. haematocheir kept in water versus those individuals exposed to air. Differentially expressed genes included those involved in the regulation of inducible factors, which play a role in sensing molecular oxygen. Analysis of expansion and contraction of these genes among malacostracan species is in progress and will be used to better understand patterns of genomic adaptation that led marine crabs to colonize terrestrial ecosystems.

ABSTRACT. Aim The caenogastropod superfamily Velutinoidea comprises >460 accepted extant species, currently classified into three families: Triviidae (293 species), Eratoidae (69 species) and Velutinidae (>100 species). This group of marine snails is almost exclusively associated to tunicates for feeding and reproduction, with a remarkable variation in shell morphology: the calcified and thick shell of triviids and eratoids is traditionally considered as plesiomorphic in the superfamily, while velutinids show a trend towards a fragile shell, often internal, frequently decalcified. We aim at studying with a phylogenetic approach on a taxonomically wide, multi-locus dataset the evolution of shell shape in the superfamily Velutinoidea.

Methods The multi-locus and single-gene datasets were assembled with sequences of mitochondrial and nuclear markers for 153 species belonging to eratoids, triviids, and velutinids. The partitions were analysed by Bayesian inference and Maximum Likelihood approach and compared to produce a robust phylogenetic hypothesis.

Results Our results suggested velutinids as a polyphyletic group (confirmed independently by nuclear and mitochondrial markers), with relevant implications for multiple shell reduction events that may have occurred at least three times in the history of Velutinoidea.

Main conclusion Contrary to what it is thought to be the most parsimonious route in shelled mollusc evolution (from an external strong shell to a vestigial internal one), Velutinoidea showed an unexpected great plasticity in shell evolution (with multiple shifts between these two states) possibly facilitated by an easiness in switching the genetic pathways regulating it. Several environmental conditions may have positively selected lineages with vestigial shells, like deep, cold and/or acid water.

ABSTRACT. AIM Bivalve molluscs hold a central role in many aspect of human economy and society, yet their sex determination system is largely overlooked. At present, the molecular mechanism by which sex is determined in bivalves is still unknown, and just few genes have been appointed as putatively involved. Nonetheless, their phylogenetic history and patterns of molecular evolution remain poorly understood.

METHODS Leveraging the growing amount of publicly available genome assemblies for bivalves, we performed an extensive survey of the Dmrt, Sox and Fox gene families, whose components have been frequently linked to the sex determination of bivalves. We also explored the pattern of amino acid evolution to provide additional support for their role in sex determination.

RESULTS Genes implicated in the male sex-determination cascade (i.e., Dmrt1L and SoxH) show high rate of amino acid evolution if compared to the entire set of bivalve orthologs. They also appear as novel acquisition of molluscs. Conversely, the gene associated to the female sex-determination cascade (i.e., FoxL2) does not exhibit any acceleration in the amino acid rate of evolution and is also shared with other Metazoa.

MAIN CONCLUSIONS In bivalves, putative male sex-determinant genes show a significantly higher rate of amino acid evolution if compared both to the putative female sex-determinant gene and to the majority of other ortholog sets. This is in line with what has been already shown for other well-studied organisms, where male-biased genes are evolving at a higher pace than female-biased and sex-unbiased genes. This work thus underlines the importance of broad comparative genomic studies to find more solid sex-determining gene candidates in species where the molecular basis of sex determination is unknown.

ABSTRACT. Aim: Differently from what observed in most Metazoa, Hox gene expression timing and localization during mollusc embryo development cannot be easily framed in a collinearity perspective. The aim of this study is to investigate the organization, diversity, and evolution of the Hox gene cluster in molluscs, focusing on the classes Bivalvia, Cephalopoda, and Gastropoda.

Methods: A broad characterization of the Hox gene cluster was performed by analysing genome assemblies of 35 species, representing 18 distinct families within the phylum Mollusca. Molecular evolution analyses were performed to estimate the selection pressures acting on Hox gene sequences. Additionally, the amino acid composition was explored within the Hox domain to identify specific residues characterizing each gene family.

Results: We found a coherent organization of Hox genes across the three classes, with a consistent genomic arrangement, following the order hox1-5, lox5, antp, lox4, lox2, post2, and post1, with the latter gene always located on the complementary strand. We found intriguing patterns in genic and intergenic distances within the Hox cluster, which varied depending on the gene family. The molecular evolution analyses provided evidence of negative/purifying selection acting on some sites within the Hox gene sequences and episodic diversifying selection in some tree branches.

Main conclusion: This study provided insights into the organization and diversity of Hox genes in the phylum Mollusca. The coherent genomic arrangement and conservation of Hox sequences, as well as the evidence of episodic diversifying selection across gene families, highlight the pivotal role of Hox genes in body plan development, but also the potential for contributing to the diversification of molluscan lineages. These findings open exciting perspectives to unravel the mechanisms that drive the expression and functional dynamics of Hox genes.

ABSTRACT. This work proposes to investigate the anatomy and molecular features of the gastrointestinal tract (i.e. gut) of the ascidian C. robusta, focusing on the adult stage. This protochordate shares several functional and morphogenetic traits with vertebrate models. Worthily, its non-duplicated and fully sequenced genome is available. Several similarities of Ciona and mouse gut have already been shown but the detailed molecular mechanisms of this regionalization together with its associated functionalities are still poorly understood.

We took a good look at the tissue architecture along the antero-posterior axis of Ciona gut. Then we performed an RNAseq analysis of the stomach and two different intestinal tracts to determine and to reconstitute the signalling pathways and molecular components expressed along the gastrointestinal tract. In particular, we used the well-known mouse Mus musculus as vertebrate model in order to compare and shed light on gut evolutionary and/or functional aspects.

The final goal of this research is to highlight some aspects of Ciona gut evolution and physiology. Our results can fit in a frame of evolutionary studies aimed to identify the molecular players acting to establish cell types distribution and their function in the different tissues. Such investigations can be of pivotal importance in defining major aspects of animal evolution.

ABSTRACT. In animals, three main RNA interference mechanisms have been described so far, which respectively maturate three types of small noncoding RNAs (sncRNAs): miRNAs, piRNAs and endo-siRNAs. The diversification of these mechanisms is deeply linked with the evolution of the Argonaute gene superfamily since each type of sncRNA is loaded by a specific Argonaute homolog protein. Moreover, other protein families play pivotal roles in the maturation of sncRNAs, like the DICER ribonuclease family, whose DICER1 and DICER2 paralogs maturate respectively miRNAs and endo-siRNAs. Among Metazoa, the distribution of these families has been only studied in major groups, and there are very few data for clades like Lophotrochozoa. Thus, we here inferred the evolutionary history of the animal Argonaute and DICER families including 43 lophotrochozoan species. Phylogenetic analyses along with newly sequenced sncRNA libraries depicted a loss of the endo-siRNA pathway along the Lophotrochozoa evolution, with the absence of DICER2 in Nematoda and Polyzoa, and with the absence of DICER2 and the Argonaute homolog in the rest of Trochozoa phyla. On the contrary, early diverging phyla, Platyhelminthes and Syndermata, showed a complete endo-siRNA pathway. On the other hand, miRNAs were revealed the most conserved and ubiquitous mechanism of the metazoan RNA interference machinery, confirming their pivotal role in animal cell regulation.

ABSTRACT. Aim The study of inflammatory mechanisms is essential to understanding the role of the immune system in the interaction with microbiota. The metamorphic stage 4 of juvenile development is the first filter-feeding stage in the life cycle of the tunicate ascidian Ciona robusta. This stage can be used to explore in vivo the activation of key components of the innate immune response in the whole animal after exposure to different pathogen-associated molecular patterns (PAMPs) at the early phase of immune system development and maturation.

Methods Transcriptional and targeted proteomic approaches were used to investigate the inflammatory response induced by exposure to stimuli of bacterial (lipopolysaccharide -LPS-, diacylated lipopeptide -Pam2CSK4) and fungal (zymosan).

Results Our findings show an interconnection between different pathogen recognition receptors pathways and indicate the upregulation of cytokine gene expression as markers of inflammation. A first protein-protein interaction map was constructed to predict potential molecular interactions, and to correlate changes observed at transcriptional and translational levels.

Main conclusion We gathered evidence in support of specific transcriptional and translational changes induced by PAMPs, and of possible interactions between molecules and pathways involved in the immune response, specifically TLRs and Dectin-1 pathways. In summary, this study depicts C. robusta as a unique chordate organism for studying factors and mechanisms that modulate immune activation and homeostasis, thus supporting its use as a viable experimental system in translational research and biotechnological approaches.

ABSTRACT. Aim To expand our knowledge of the immunogenomic landscape in chordates, we widened the spectrum of molecules that could be responsive to inflammatory stimuli in tunicate ascidians, the closest relatives of vertebrates. A homology search analysis of the tunicate ascidian Ciona robusta genome and proteome was performed by focusing on potentially immunocompetent molecules belonging to, or strictly correlated with, JAK-STAT signaling, Immunoglobulin and Dectin pathways.

Methods A structure-, function- and conservation-based bioinformatics approach was used to identify orthologs of human immune-response genes and proteins. Our study has been conducted using specific databases and bioinformatic tools (e.g., BLAST, InterPRO, Cytoscape, iTASSER-MTD, Jalview) to identify a list of molecules, whose expression has been validated in C. robusta juvenile stage. Action mechanism of such molecules has been predicted through analysis of protein functional domains and gene regulatory elements, protein-protein interactomes and phylogenetic trees to better understand their evolutionary history.

Results In a comparative approach to human immune-related pathways, the homology approach allowed to identify a high number of Gene Ontology terms, highly interconnected, corresponding to almost 50 uncharacterized genes containing CLECT, IRF and Ig domains. Several of these molecules are barely or absolutely uncharacterized in C. robusta. The immune-related activity of 11 immune-competent genes has been newly described and confirmed after inflammatory challenges. Finally, a protein-protein interactome network was built with the aim to identify the connections between molecular pathways involved in the immune response.

Main conclusion Trying to gain a wider view of the complex evolutionary puzzle at the intersection between invertebrates and vertebrates, our findings indicate that the number of immune-related genes in ascidians is higher than previously known. This study depicts C. robusta as a unique organism to gain insights into the evolution of the immunome.

ABSTRACT. The Venice lagoon is a delicate environment where Non-Indigenous Species (NIS) are abundant and human impact is high. This work represents a literature-based study of the ascidian biodiversity (Tunicata, Ascidiacea) in the lagoon, considering records from the eighteenth century. Although ascidians are the most abundant taxon at the climax of the ecological succession on hard substrata and some have a strong invasive potential, the knowledge on their biodiversity is fragmented. We used books provided by the Natural History Museums of Venice and Trieste, collection catalogues belonging to the Natural History Museums of Chioggia, Padua and Venice, and public online databases. We considered species lists, drawings, and descriptions. Species names were checked using the WORMS platform. We created a timeline from 1700 up to today, finding that, in the Venice lagoon, the first ascidian description dates to Stefano Chiereghin in 1784. Giuseppe Olivi's Zoologia Adriatica (1792) described Alcynium conicum and Alcyonium schlosseri, now known as Aplidium conicum and Botryllus schlosseri, respectively. However, the last comprehensive study was published by Brunetti in 1977, reporting five solitary and seven colonial species. Notably, Perophora multiclathrata and Styela plicata were later classified as NIS. Other NIS, including Botrylloides violaceus, Didemnum vexillum, and Styela clava, were later identified. Moreover, ascidian sampling contributed to solve controversial taxonomical issues, allowing the description of Ciona robusta and Ciona intestinalis (previously considered a single species) and of Botryllus schlosseri. Additionally, a new cryptic species, B. gaiae was described within B. schlosseri complex. Despite in the Lagoon of Venice ascidians are known since the eighteenth century, information on their biodiversity is fragmented. Therefore, in consideration of their ecological role, an urgent action is required for their monitoring, according to the PNRR aims.

ABSTRACT. Abstract Aim

Deep-sea ecosystems are among the biosphere's most extensive biomes, and polychaetes are the most abundant benthic invertebrates in deep-sea samples. Despite their important ecological role, and although the Mediterranean Sea is one of the most studied marine areas, the deep sea and its benthic macro- and meiofauna remain almost unexplored so far. To fill this gap, we are building a reference library of highly taxonomically accurate sequences for polychaetes from the deep Western Mediterranean Sea. We also aim to use this library in the study of environmental DNA obtained from the same study areas.

Methods

Two mitochondrial markers (16S rDNA and COI) were used to genotype 170 samples. Sequences were deposited in the Barcode Of Life Data System (BOLD); phylogenetic analyses were performed using maximum likelihood topologies with IQtree. Environmental DNA was extracted from 58 sediment samples of the Palinuro and Marsili Seamounts, Canyon Dohrn and the Salerno epibathyal plain using the DNeasy Powermax Soil Kit, to perform further analyses on the composition of the polychaete community through metabarcoding.

Results

During the construction of the BOLD library, in many cases the specimens did not match any available sequence, thus representing the first data deposited for their lineage. Phylogeographic analyses revealed that deep-sea polychaetes from the Western Mediterranean include both Lusitanian and Senegalese species, as well as potentially endemic species, confirming the composite biogeographical origin of the Mediterranean biota also in deep-sea environments.

Main conclusion The description of polychaete assemblages at the finest possible taxonomic level with integrative taxonomy and the subsequent use of eDNA will contribute to a more accurate assessment of their diversity and rate of endemicity. This is needed also to obtain reliable estimates of the effect of anthropogenic drivers of change on this poorly studied ecosystem.

ABSTRACT. Aim Host-parasite relationships provide very useful models to study adaptive processes. We investigated the interaction between parasitic marine gastropods, the Ovulidae, and their cnidarian preys. The Ovulidae is a family of caenogastropod that feed on octocorals (Anthozoa: Octocorallia: Alcyonacea) or, to a much lesser extent, on hexacorals (Anthozoa: Hexacorallia: Antipatharia, Scleractinia) and hydrocorals (Hydrozoa: Hydroidolina: Anthoathecata: Stylasteridae). A limited information is available on the phylogenetic relationships and the degree of specificity of the predator-prey relationships within this corallivorous lineage.

Methods To investigate ovulid/coral relationships in the context of their evolution, we generated the largest molecular dataset so far, comprising two mitochondrial (COI and 16S-rDNA) and one nuclear gene (28S-rDNA) from 524 specimens collected all over the world. To identify the coral host of the ovulid species, we integrated literature data with an empirical approach. For the former, each ovulid species present in the tree was related to the cnidarian identified in literature at the lowest possible taxonomic level. When the cnidarian host sample was available, morphological identification was conducted together with the amplification of the 16S molecular marker. Obtained sequences were matched against a reference database.

Results The obtained molecular phylogeny of the Ovulidae revealed the existence of groups that do not completely correspond to the currently subfamily arrangement. Moreover, some of the genera as traditionally conceived were not confirmed as monophyletic. Concerning trophic ecology, our results suggest that several ovulid lineages repeatedly shifted between octocorals and hexacorals, while only the pediculariines (Pedicularia and allied) are associated with hydrozoans (Stylasteridae).

Main conclusion The systematics of the ovulids should be redefined to better reflect the phylogenetic pattern emerging from the molecular analyses. Data on the coral prey of crucial species are still lacking (e.g. Pseudocypraea spp.), hampering a certain identification of the ancestral coral-host of Ovulidae.

Thematic areas Macroevolution, Adaptation

ABSTRACT. Aim Nitric oxide (NO) is a key signaling molecule in almost all organisms and is active in a variety of physiological and pathological processes. In non vertebrate chordates only one Nos gene is present, whereas in mammals three Nos gene copies have been identified, By extending studies to a non- vertebrate chordate, the ascidia Ciona robusta, we investigated the evolution among chordates of Nos/NO distribution and biological function. Results NOS/NO localization patterns during the different stages of Ciona larval development evidenced a highly dynamic localization profile that perfectly matches with the central role played by NO from the first phase of settlement induction to the next control of swimming behavior, adhesion to substrate, and progressive tissue resorption of metamorphosis. This specific and temporally controlled pattern of NO starts in the most anterior sensory structures of the early larva and progressively moves towards the caudal portion as larval development and metamorphosis proceeds. Conclusions Our comparative analysis of the function of Nos/NO among chordates from cephalochordates to tunicates, teleost fishes and amphibians provided a comprehensive set of data showing a high degree of conservation of No functions and adding new elements to our understanding of Nos evolution. Whereas in mammals NO acts mainly as neurotransmitter, vasodilator, and immune response mediator, in marine invertebrates the gas is involved in a variety of biological processes, including defence,, neurotransmission, swimming, feeding, symbiosis and development

ABSTRACT. Aim The swordfish, Xiphias gladius (L. 1758), belongs to order Perciformes and represents the only living species of genus Xiphias. The anatomical characteristic that distinguishes adult individuals is the upper jaw, which develops enormously to form a rostrum, of a considerable size. Despite the fully preserved external morphology, Di Natale et al. (1996) defined for the first time different internal structures describe as “with canals”, characterized by bony bundles running longitudinally and separated by tissue striations of lower density and “with chambers”, characterized by multiple chambers ranging from a few to dozens of units located in the central area of the sword. The aim of the present study is to verify the existence of an association between different internal rostrum morphologies with the occurrence of two mitochondrial haplogroups in Mediterranean specimens, as reported by several studies (Smith et al. 2015; Righi et al. 2020). Methods Genetic characterization was based on mitochondrial markers such as the CR (control region), NADH dehydrogenase subunit 2 (ND2), and cytochrome oxidase subunit 1 (COI); while the swordfish internal structures were inspected using x-ray or CT analysis. Results The haplotype networks obtained from the three mitochondrial markers support the presence of two distinct evolutionary lineages of X. gladius in the Mediterranean Sea, confirming the results described by various authors (see above). However, no relationship was found between the two clades and rostra morphologies when phylogenetic and genetic differentiation analyses were applied. Main conclusion Further alternative methodologies are currently employed to investigate the potential correlation between the genetic and morpho-anatomical diversity of the swordfish's rostra. From a genetic standpoint, we will be applying cutting-edge next-generation sequencing techniques, specifically 2bRAD. The availability of X-rays or CT scans will allow us to investigate the morpho-anatomical details of the rostra internal structure with specific programs for geometric morphometrics.

ABSTRACT. Introduction Sea spiders are marine chelicerates represented by ca. 1,400 extant species distributed worldwide: from littoral to abyssal waters and from tropical to polar regions. They nest within the Arthropoda tree of life as the sister to all the other chelicerates, which makes them a key lineage to understand arthropods’ early evolution. Aim While sea spiders are currently divided into eleven families (order Pantopoda), the fossil record testifies that their diversity was once much higher. With the aim to better understand their diversification through time, we assembled, for all we know, the most taxonomically complete molecular dataset of extant sea spiders with which, together with a reassessment of the fossil record, we inferred a Pantopoda evolutionary timeline. Methods The superalignment we inferred (22,384 bp), and with which we then estimated our phylogeny, consists of 12 mitochondrial protein-coding genes, the nuclear ribosomal gene 18S rRNA , and 99 UCEs of 173 species across all the extant families of Pantopoda. Subsequently, we revisited the key fossil calibrations that had been previously used to date the Pycnogonida tree of life. After reassessing such fossil evidence, we demonstrate that some of those calibrations were based on inaccurate taxonomic hypotheses. We used our updated hard minimum and soft maximum bounds of 5 fossil specimens to constrain the node ages of Arthropoda, Chelicerata, Colossendeidae, Phoxichilidioidea, and Ascorhynchidae in our phylogeny for timetree inference with MCMCtree. Main conclusions According to our estimated evolutionary timeline of sea spiders, the diversification of pantopods seems to have begun between the late Ordovician and the early Silurian. Nevertheless, future consistent sampling of extant sea spiders is required to better understand their diversity and interfamilial relationships, which will in turn help to shed light into the evolutionary history of the order Pantopoda. Thematic Areas Macroevolution, Biodiversity

ABSTRACT. At the north of Cap Corse, more than 100 meters under the sea, there are strange formations that have never been explained before: 1,417 perfectly circular rings, 20 meters in diameter. The Gombessa expedition team, led by biologist and photographer Laurent Ballesta, intends to resolve this mystery and discover its origin. In collaboration with 41 French and foreign scientists, Laurent Ballesta's team carried out a whole series of protocols aimed at understanding the origin and functioning of these rings: their age, their dynamics of change, but also to identify the associated biodiversity. Laurent will be presenting some of the results of this 4-years work.