ABSTRACT. When faced with stressful conditions, such as lack of food or physical damage, the medusae of Turritopsis dohrnii avoid death by reverting to the polyp stage, passing through a short-lived cyst stage. In the cyst stage, cellular transdifferentiation occurs. Because of its unique potential for rejuvenation, and because this life cycle reversal can be induced under controlled laboratory conditions and in 24-48 hours, T. dohrnii represents an unparalleled model system to investigate the molecular mechanisms that control stage-specific cell differentiation. We present the assembled transcriptome of the T. dohrnii life cycle stages. We perform time-series differential gene expression analyses to identify genes enriched in the cyst during the reverse developmental sequence (i.e., from wild polyp to medusa, cyst, and reversed polyp). Using pairwise differential gene expression analyses, we compare the benthic polyp and planktonic medusa's expression profiles and those of the polyps generated by different developmental pathways (colonial polyp generated by asexual budding, and polyp generated from the cyst through reverse development). We conduct a preliminary screening of our RNA-seq libraries and transcriptome assembly to test whether homologs of the Yamanaka and Thompson transcription factors (Oct4, Sox2, Klf4, c-Myc, Nanog) that govern the induction of pluripotency in humans are present in T. dohrnii. Finally, we investigate whether Sirtuin proteins, telomerase and regulators of telomere elongations, heat shock proteins, Yamanaka transcription factors, and proteins in the AMPK signaling pathway, all canonically involved in processes of tissue regeneration, cell plasticity, and aging in vertebrates and Metazoa, play a role in T. dohrnii's reverse development.

ABSTRACT. Aim Conservation actions aim to preserve and recover animal and plant species using in-situ or ex-situ strategies. Conservation genetics can provide important insights into the dynamics of endangered populations facilitating their management. This project shows how the use of traditional markers and new generation sequencing can benefit conservation management, focusing on the case studies of the loggerhead (Caretta caretta) and the green turtle (Chelonia mydas).

Methods We used microsatellites and mtDNA to assess the outcome of a reintroduction program of green turtles in the Cayman Islands and the status of the reintroduced wild population. We performed parentage analysis between wild and captive population, tested for loss of genetic variability and assessed population structure. Then we explored the potential of 2b-RAD genomic sequencing, studying the population structure and local adaptation of 10 Eastern Mediterranean loggerhead turtle rookeries.

Results In the Cayman Island case study, we found that 90% of adult wild females and 80% of wild F1 hatchlings were related to the captive population, proving the program successful. This relatedness did not affect negatively the fitness of the wild population. Moreover, we found that after only one generation, genetic differentiation between the populations was significant. In the Mediterranean case study, we found a high level of population structure and the PCoA showed three main groups of loggerhead turtle rookeries. Atmospheric temperature and geographic location of the rookery showed a significant impact on population structure, as outlier loci were found associated with these predictors. Finally, different effective population sizes denote that the studied populations belong to different classes of extinction risk.

Main Conclusion Our results show the key role of genetics and genomics studies in conservation management. These analyses, in fact, can uncover details crucial for decision making in both in-situ and ex-situ conservation.

ABSTRACT. Coral reefs are known to host the largest diversity of symbiotic associations in the marine environment. Scleractinian corals provide habitats for a broad variety of cryptofauna, which in turn may contribute to the overall functioning of coral symbiomes. Among these invertebrates, hydrozoans belonging to the genus Zanclea represent an increasingly known and ecologically important group of coral symbionts. In this study, about 300 Zanclea colonies associated with scleractinian corals from the Indo-Pacific and Caribbean regions were analysed using an integrative approach. Specifically, molecular phylogenetics, species delimitation techniques, morphological analyses and comparative phylogenetic methods were used to shed light on the evolution and biogeography of the group. Overall, we found a high genetic diversity of hydrozoans despite their conserved morphology. However, preliminary results suggest that the obtained genetic clades may be successfully distinguished using statistical treatment of morphometric data and green fluorescence patterns, other than DNA data. The majority of clades were associated with one or two coral genera belonging to the Complex clade, whereas the remaining ones were generalists associated with both Complex and Robust corals. Despite the observed specificity patterns, no congruence between Zanclea and coral phylogenies was observed, suggesting a lack of coevolutionary events. Most clades had a wide distribution across the Indo-Pacific, including a generalist one extending also in the Caribbean, while two host-specific clades were possibly endemics to the Red Sea, confirming the importance of this peripheral region as an endemicity hotspot. Ancestral state reconstructions suggested that the most recent common ancestor of all extant coral-associated Zanclea was a specialist species with a perisarc, occurring in what is now known as the Indo-Pacific. Ultimately, a mixture of geography- and host-related diversification processes is likely responsible for the observed enigmatic phylogenetic structure of coral-associated Zanclea.

ABSTRACT. Estimating patterns of gene flow during the early stages of speciation is central to understanding whether reproductive isolation arises via gradual erosion of gene flow or through stages of strict isolation and secondary contact. Such scenarios can be tested by comparing the joint allele frequency spectrum (jAFS) of a set of populations to jAFS simulated under scenarios of isolation with migration (IM) and secondary contact (SC). However, the potential effect of unaccounted demographic events (such as expansions and bottlenecks) on model choice and parameter estimation remains unexplored. Using simulations, we demonstrate that under realistic scenarios of population divergence with constant gene flow, failure to account for population size changes in a daughter population or the ancestral population leads to overestimated divergence time and to a bias towards the choice of SC models. On the other hand, when the simulations included long periods of strict isolation the correct gene flow scenario was usually retrieved. We illustrate these issues reconstructing the demographic history of North Sea and Baltic Sea turbots (Schopthalmus maximus) by testing 16 IM and 16 SC scenarios, modelling changes in Ne as well as the effects of linked selection and heterogeneous migration rates across the genome. As in the simulated data, failure to account for changes in Ne resulted in selecting SC models with a long period of strict isolation and divergence times preceding the formation of the Baltic Sea. In contrast, models accounting for Ne changes suggest the Baltic Sea turbot population originated from a very recent (<6 kya) invasion and diverged with constant gene flow from the North Sea. The results have implications for the study of speciation, high-lighting the potential effects of ancestral size changes and bottlenecks on choices between competing scenarios. In general, extreme caution should be exercised when interpreting results of demographic model comparisons.

ABSTRACT. In the last century, species across the globe have experienced the effects of environmental and anthropogenic changes, and genetic analyses of historical collections represent a unique resource for tracking their impact on the genetic make-up of species. In this study we used a retrospective genomic approach to study the population of tiger sharks (Galeocerdo cuvier) in Eastern Australia. Due to the high mobility of the tiger shark, which is a globally distributed apex predator, we expected to find a single panmictic population at such local scale and as previously reported, but we were alert to the possibility of significant changes to the population over the last century as a consequence of documented population reductions. The dataset used consisted of 116 unique samples originated from northeastern and eastern Australia, extending from the Gulf of Carpentaria to the Tasman Sea, and that were caught over a time-span of 80 years (1939-2015). Both the historical (jaws and vertebrae) and contemporary (fin clips) samples were subject to DNA capture using 20.000 gene associated “baits” from which ~2000 validated SNPs were successfully genotyped. Overall, we documented a significant change in genetic composition of tiger sharks over time and space. Our data is most compatible with a hypothesis of two distinct population groups, with varying contributions to spatiotemporal samples, and a population almost completely absent from contemporary and most northern samples. Findings that are possibly associated with differences in direct or indirect exploitation rates. This study shows that archived samples used in combination with modern genomic tools, can reveal temporal changes in biodiversity, which otherwise would have remained unnoticed. Such results highlights the importance of using genomic data to comprehend past evolutionary responses, as a key tool to inform decisions for the protection and management of elasmobranchs’ populations.

ABSTRACT. Aim Social interactions are fundamental in nature, but little is known about the neural mechanisms establishing the social relationships between two species of reef fish. Marine fishes offer a great opportunity to understand these interactions as some species depend on others through symbiotic relationships. In particular, the cleaner fish Labroides dimidiatus allows us to study mutualistic cleaning interactions displaying sophisticated social behavior while interacting with other species known as ‘clients’, that can benefit from ectoparasite reduction and stress relief.

Methods To understand this essential inter-specific behavior, this study identified using transcriptomics, relevant molecular pathways related to social behavior in the fore-, mid- and hindbrain regions of the cleaner fish and one of its potential clients (Acanthurus leucosternon).

Results Comparing the brains of interacting fish vs fish alone in aquariums, we found that the social interaction of L. dimidiatus with its client fish involved several social neural systems such as the dopaminergic, glutamatergic and GABAergic pathways as well as social decision-making genes. In addition, we found immediate early gene responses related to social plasticity and transcription factor activity such as c-jun, c-fos and CREB indicating neural activation during interaction. The client fish on the other hand had less differential gene expression across the brain and genes were functionally involved in hormonal activity. For both species most of the neural activation during interaction was produced and regulated in the hindbrain and forebrain region.

Main conclusions These results suggest an important implication of hormone systems and the activation of immediate early genes during the social interaction of L. dimidiatus and A. leucosternon. Analysing the role of these molecular mechanisms in the brain provides key information on crucial inter-specific behaviours and can shed light on the key mechanisms of vertebrate mutualism among marine species.

ABSTRACT. Aim One fundamental aspect of environmental adaptation is the synchronic adjustment of physiological activities with environmental cues driven by the circadian-clock system. Recent studies have shown that epigenetic and epitranscriptomic mechanisms (e.g. RNA methylation) regulate the circadian clock in several organisms, but only few studies have focused on plants. Methylation of RNA bases (m6A) is the most prevalent modification, carried out by proteins responsible of adding (writers) and deleting (erasers) methylation marks. Here, we aimed at characterizing m6A-related genes in seagrasses and get insights into their role in regulating circadian rhythms.

Methods We provided a first inventory of m6A-related genes in two seagrass species (Cymodocea nodosa and Zostera marina) exploiting available genome and transcriptome data and comparing the results with terrestrial model plants. We further investigated daily changes in global m6A RNA methylation and transcript levels of writers and erasers genes over a 24h cycle, across species and latitudes, via RT-qPCR.

Results Z. marina and C. nodosa possess the entire repertoire of m6A genes, with an expansion of a writer (FIP37) gene family. C. nodosa was redundant in transcripts encoding for erasers. Our analyses also revealed a striking rhythmicity of writer and eraser genes and concomitant m6A levels in both species. M6A methylation always peaked toward the dark period, suggesting a role in driving transcriptome rearrangements during the night. In general, the observed patterns were asynchronous between species at the same location, while being were very similar in populations growing at contrasting latitudes, suggesting a regulation by endogenous species-specific cues more than by external stimulus.

Main conclusion RNA methylation could widely participate in the daily regulation of gene expression and circadian-clock functions in seagrasses, potentially affecting the photo-biological behaviour of the species and their ability to spread across different latitudes and photoperiodic regimes.

ABSTRACT. The ocean is Earth’s last frontier. It comprises most of its habitable volume, is home to a unique and extraordinary diversity of plants, animals, and microbes; regulates its climate; and provides food and livelihood for billions of people. Our future is inextricably linked to the ocean, and to maintaining the flow of critical and irreplaceable services healthy oceans provide. While much investigation and discussion is focused on impacts, there is a critical need and opportunity to develop solutions based in oceans. I will present and discuss ocean-based solutions to two grand challenges, climate and food and nutritional security, highlighting knowns and unkowns in their potential for deployment at scale, and opportunities for expanding global capacity for ocean solutions.

ABSTRACT. Adaptation and speciation are key processes that determine biological diversity and its distribution in space and time. Speciation requires the evolution of barriers to gene flow between diverging populations. The mechanisms underlying these processes are often unclear, particularly when they occur in the face of gene flow. An ideal model to understand the factors enabling adaptive divergence with gene flow is the marine snail Littorina saxatilis. It shows repeated adaptive divergence between crab-adapted and wave-adapted ecotypes. They are ecologically, morphologically and behaviourally distinct but hybridize in the contact zones. Here, we take advantage of these hybrid zones to uncover the genomic architecture of parallel adaptive divergence with gene flow in Sweden and Spain, the extremes of L. saxatilis distribution in Europe. Our preliminary analyses of crab-hybrid-wave transects using low coverage whole genome resequencing showed clinal variation in Sweden but two discrete groups in Spain. This result suggested that crab and wave are at a more advanced stage of speciation at the southern end. By studying both locations, we are able to understand how reproductive isolation emerge and population diverge. Notably, genomic divergence between crab and wave do not fully correspond to habitat transition in both countries. This pattern indicates that ecologically diversifying selection might not be the single evolutionary force at play in L. saxatilis’ adaptive polymorphism. Further analyses including experimental fieldwork and ecological niche modelling will clarify the relative role of environmental and genetic mechanisms in a natural system such as L. saxatilis, which is crucial to obtain a more complete and comprehensive understanding of the mechanisms underlying (adaptive) divergence. Our findings will shed light into the factors and interactions that promote speciation-with-gene-flow in nature.

ABSTRACT. Aim Cassiopea andromeda is a mixotrophic Mediterranean alien species found in shallow water environments as lagoons and harbors. In the present study we investigated the short time reaction in the trophic strategy of Cassiopea jellyfish from a low irradiance to two different high irradiance conditions, simulating potential changes from eutrophic and turbid habitats to meso/oligotrophic waters. Understanding the physiological response mechanisms may allow to explain the adaptive potential of this non indigenous species in new habitats.

Methods Jellyfish were at first acclimated to very low light condition and then moved for one week to two different higher light intensities. Photosynthesis measurements of Cassiopea sp. exposed to the two different PAR were carried out together with data obtained from PAM measurements. These data were complemented with measurements of symbiont density, chlorophyll a, and protein content to give a detailed frame of Cassiopea sp. autotrophic strategy.

Results The short time response to different light treatment showed similar photosynthetic parameters in Cassiopea sp. The high photosynthetic rates were confirmed also by the RLCs. No significant differences between the two conditions were observed concerning symbiont abundance. However, jellyfish exposed to lower light intensity showed a higher content in chlorophyll a and c, and thus a higher Ci.

Main conclusion No matter the acclimation at low irradiances, Cassiopea sp. showed high photosynthetic performances, demonstrating the ability of the species to adapt to fast changes in abiotic condition, such as light exposure. This ability, in addition to Cassiopea dual nutrition mode, makes this species a candidate survivor and potential winner in a changing oceans scenario against native species struggling these sudden changes.

ABSTRACT. Ocean acidification is one of the many consequences of climate change. Various studies suggest that marine organisms’ behaviour will be impaired under high CO2. Here, we show that the cognitive performance of the cleaner wrasse, Labroides dimidiatus, has not suffered from the increase of CO2 from pre-industrial levels to today, and that the standing variation in CO2 tolerance offers potential for adaptation to at least 750 µatm. We acclimated cleaners over 30 days to five levels of pCO2, from pre-industrial to high future CO2 scenarios, before testing them in an ecologically relevant task—the ability to learn to prioritise an ephemeral food source over a permanent one. Fish learning abilities remained stable from pre-industrial to present-day pCO2. While performance was reduced under mid (750 µatm) and high CO2 (980 µatm) scenarios, under the former 36% of cleaners still solved the task. The presence of tolerant individuals reveals the potential for adaptation, as long as selection pressure on cognitive performance is strong. However, the apparent absence of high CO2 tolerant fish, and potentially synergistic effects between various climate change stressors, renders the probability of further adaptation unlikely.

ABSTRACT. Aim: Phototransduction is the signaling mechanism that underlies vision. The cascade has been described for two types of visual opsins: ciliary opsins common to vertebrates and rhabdomeric opsins common to arthropods. A third type of visual opsin is cnidop or cnidarian opsin whose cascade is not well known but are closely related to ciliary opsins. Hydra vulgaris, which lack eyes but exhibit light-dependent behavior, can be used to investigate candidate genes involved in cnidarian phototransduction.

Methods: We used reciprocal BLAST and gene ontology term annotations to identify candidate phototransduction genes in a reference genome and transcriptome. For opsin genes, we generated a phylogenetic tree that included other cnidarian species. We also used RNA-seq data from adult Hydra hypostome, tentacles, body column, foot, and budding zone in addition to time points in hypostome regeneration and during budding to quantify gene expression patterns.

Results: We identified a total of 45 opsin genes in Hydra. We determined that opsin genes are evolving by lineage-specific duplications and potentially by tandem duplications. We identified two opsin genes outside of the previously determined Hydra opsin groups; these genes possibly function in nematocytes and mucous gland cells, respectively. We found two opsin genes that have similar expression patterns to genes annotated with phototransduction functions.

Main Conclusion: We propose that the H. vulgaris phototransduction cascade could have similar components to both ciliary and rhabdomeric cascades.

Thematic Areas: Adaptation

ABSTRACT. Biomineralization is the process by which organisms form minerals. Coral (Cnidaria: Anthozoa) biomineralization has been extensively studied in members of the order Scleractinia, primarily due to their role as framework-builders of coral reefs. However, from a biomineralogical perspective, scleractinians are quite uniform, as they all form aragonite exoskeletons. Contrarily, subclass Octocorallia (soft corals) produce diverse skeletons, including structures composed of different calcium carbonate (CaCO3) polymorphs, i.e., calcite and aragonite. However, how corals can biologically control different polymorphs deposition remains elusive. This has important evolutionary implications, as changes in seawater chemistry through Earth’s history, especially the Mg/Ca ratio, appear to favour the deposition of different polymorphs. Here, we combined transcriptomics and proteomics to characterize the skeletal proteome of two calcite and one aragonite-forming octocoral and one scleractinian species. We found low proteome overlap between octocorals depositing different polymorphs, despite instances of shared proteins (e.g., galaxins and carbonic anhydrases). We additionally recreated proposed ‘Calcite Sea’ conditions (i.e., Mg/Ca of 2.5 and 1.5) in marine aquaria to culture the aragonitic octocoral Heliopora coerulea and scleractinian M. digitata. This revealed a species-specific response with H. coerulea maintaining the ability to produce aragonite, while up to 10% calcite was observed in M. digitata skeletons. RNA sequencing showed expression changes for calcium channels, pointing to possible responses of H. coerulea to lower Mg/Ca ratios. In conjunction, these data provide insights on how the diversity of coral skeletons evolved and may inform predictions about the response of these ecologically important organisms to future changes in ocean chemistry.

ABSTRACT. Aim. Pelagic tunicates (Appendicularia, Thaliacea) play a key role in the ocean food web and carbon export towards lower compartments. Their global distribution is poorly known and we still possess an incomplete knowledge of how abiotic and biotic factors influence their diversity and abundance. Herein, we used metabarcoding data to build the first atlas of pelagic tunicates in oceans., We dedicated a specific focus on the response to local conditions and on the interaction with local planktonic communities.

Methods. We took advantage of the TARA Ocean V9 database to build a tunicates-specific V9 dataset. To do so, a new reference database was developed. OTUs delimitation was performed as a phylogenetic-based approach, which minimizes the uncertainties in the taxonomic assignation. To investigate the role of biotic and abiotic variables in triggering pelagic tunicates distribution, we used a machine learning approach (Boosted Regression Tree - BRT).

Results. Herein, the global distribution and abundance of 48 pelagic tunicate genera in the open ocean are presented. Pelagic tunicate abundance is associated with levels of Photosynthetically Active Radiation (PAR) and with seawater temperature. Moreover, biotic interactions with potential organic food and competitors contribute in determining the abundance of pelagic tunicates in the ocean. Tentatively, we have modelled Appendicularia and Thaliacia distribution in the framework of climate change. Preliminary results indicate that global warming may trigger an increase in pelagic tunicate abundance in the ocean.

Main conclusions. Tunicates are among the major component of macro-zooplankton, thus playing a fundamental role in the oceanic foodweb. We firstly created a global oceanic distribution map of pelagic tunicates, and determined the relative importance of various environmental drivers. Finally, we provide hints into how climate changing-related ocean warming may shape the future distribution of this important group of zooplankton.

ABSTRACT. Aim Ocean acidification can have wide-ranging impacts on marine ecosystems. While short-term studies have reported negative effects of future predicted CO2 levels on a variety of marine organisms, some populations might have the potential to acclimate and/or adapt to elevated CO2 levels across generations. Understanding the capacity of marine organisms to adapt to future CO2 levels will enable us to better predict the biological impacts of ocean acidification. This study therefore focuses on the effects of 1) variation in parental tolerance and 2) parental exposure to elevated CO2, on the molecular responses in the gills of the offspring to elevated CO2 in a coral reef fish.

Methods Adult Acanthochromis polyacanthus were collected from the Great Barrier Reef and classified as either having sensitive or tolerant behavioural phenotype based on their response to chemical alarm cues after elevated CO2 exposure. Breeding pairs of each phenotype were kept in control or elevated CO2 conditions with the resulting offspring reared either in control and elevated CO2 conditions. RNA was extracted from the gills of the offspring, sequenced, and analyzed.

Results Cross-generational exposure to elevated CO2 had a significant impact on gene expression patterns of the offspring. Offspring from parents exposed to elevated CO2 exhibited approximately half the differentially expressed genes compared with offspring in the within-generation elevated CO2 treatment. The parental phenotype also influenced gene expression, with 732 and 183 genes differentially expressed in the control and within-generation CO2 treatment respectively, when comparing directly between offspring of the two parental phenotypes.

Main conclusion Fish whose parents were exposed to elevated CO2 had an enrichment of genes involved in ion regulation and homeostasis related functions and decreased expression of stress response genes compared to the within-generation treatment.

Thematic areas Adaptation, Population and Species

ABSTRACT. ABSTRACT Aims High levels of pollutants in anthropogenically-modified marine environments may exert high pressure on maximum physiological responses of sessile organisms acting as powerful agents of selection. Thus, an organism’s acclimation or adaptive potential could play an important evolutionary role by enabling or conditioning species tolerance to stressful environmental conditions. Temperature modulates marine ectotherm physiology, influencing survival, abundance and species distribution. While native species could be susceptible to ocean warming, thermal tolerance might favour the spread of non-native species. Determining the success of invasive species in response to climate change is confounded by the cumulative, synergistic or antagonistic effects of environmental drivers, which vary at a geographical and temporal scale.

Methods We investigated the developmental performance of early life stages of the ascidian Ciona intestinalis (derived from populations of anthropogenically-impacted and control sites) to an extreme weather event (i.e. marine heat wave). Fertilisation rate, embryo and larval development, settlement, metamorphosis success and juvenile heart beat rate were assessed as experimental endpoints.

Results With the exception of fertilization and heart beat rates, temperature influenced all analysed endpoints. C. intestinalis derived from control sites were the most negatively affected by increased temperature conditions. Opposingly, C. intestinalis from anthropogenically impacted sites showed an overall positive response to thermal stress, with a higher proportion of larvae development, settlement and metamorphosis success being observed under increased temperature conditions. No differences were observed for heart beat rates between sampled populations and experimental temperature conditions. Moreover, interaction between temperature and populations was statistically significant for embryo and larvae development, and metamorphosis.

Main conclusion We hypothesize that selection resulting from anthropogenic forcing could shape stress resilience of species in their native range and subsequently confer them advantageous traits underlying their invasive potential.

Thematic Areas Adaptation, Populations and Species

ABSTRACT. Aim Marine Heat Waves (MHWs) have been increasing in intensity and frequency since the past century with impacts to marine ecosystems. Their effects on the seagrass Posidonia oceanica need to be deeply explored in order to predict its performance in the future scenario. Here the phenological change of the seagrass at MHWs of different intensity was estimated in a field experiment, testing if the origin of the plant may influence the resilience to the heat stress.

Methods In order to simulate MHWs in the field, the Fiume Santo thermal power plant (North West Sardinia), which releases sea water up to 8°C warmer than the natural, was chosen. Because the released water remains stratified on the surface, specific structures to expose P. oceanica plants at 1 m deep were used. P. oceanica shoots taken from two donor sites (hot vs cold, North and West Sardinian coast, respectively) were transplanted in a common garden set up by three areas in front of the power plant, so that at each of them a different MHW intensity was simulated (high, medium and control temperature). After 8, 19 and 40 days of exposure some phenological variables were measured. The experiment was conducted in summer 2020.

Results Overall, important effects of the three heat treatments on the transplanted plants were highlighted as there was a decrease in most of the variables considered. However, a different performance between plants collected in the two donor sites was found, with the former showing a higher resistance to the treatments.

Main conclusion The impact on P. oceanica performance of the simulated MHWs in the field suggests that the future summer temperature due to climate change projection will threat the plants, but that the impact will be context-dependent in relation to the pre-adaptation of the plants to the local environmental conditions.