Spineless and overlooked: DNA metabarcoding of autonomous reef monitoring structures reveals intra- and interspecific genetic diversity in Mediterranean invertebrates.

The ability to gather genetic information using DNA metabarcoding of bulk samples obtained directly from the environment is crucial to determine biodiversity baselines and understand population dynamics in the marine realm. While DNA metabarcoding is effective in evaluating biodiversity at community level, genetic patterns within species are often concealed in metabarcoding studies and overlooked for marine invertebrates. In the present study, we implement recently developed bioinformatics tools to investigate intraspecific genetic variability for invertebrate taxa in the Mediterranean Sea. Using metabarcoding samples from Autonomous Reef Monitoring Structures (ARMS) deployed in three locations, we present haplotypes and diversity estimates for 145 unique species. While overall genetic diversity was low, we identified several species with high diversity records and potential cryptic lineages. Further, we emphasize the spatial scale of genetic variability, which was observed from locations to individual sampling units (ARMS). We carried out a population genetic analysis of several important yet understudied species, which highlights the current knowledge gap concerning intraspecific genetic patterns for the target taxa in the Mediterranean basin. Our approach considerably enhances biodiversity monitoring of charismatic and understudied Mediterranean species, which can be incorporated into ARMS surveys.

Evidence of genetic isolation between two Mediterranean morphotypes of Parazoanthus axinellae.

Coralligenous assemblages are among the most species-rich and vulnerable habitats of the Mediterranean Sea. Nevertheless, data on connectivity patterns on species inhabiting these habitats, crucial to define management and protection priorities, are largely lacking. Moreover, unreliable species-level taxonomy can confound ecological studies and mislead management strategies. In the northwestern Mediterranean two Parazoanthus axinellae morphotypes differing in size, color and preferred substrate are found in sympatry. In this study, we used COI and ITS sequence polymorphism to assess (1) the genetic divergence between the two morphotypes, (2) their connectivity patterns and (3) their phylogenetic position within the Parazoanthidae. Specimens of P. axinellae were sampled in 11 locations along the northwestern Mediterranean; in 6 locations, samples of the two morphotypes were collected in sympatry. Small genetic diversity and structure were found within morphotypes, while marked and consistent differentiation was detected between them. Moreover, the less widespread morphotype appeared to be closer to Pacific species as P. juanfernandezii and P. elongatus. Our findings confirmed the limited knowledge on Parazoanthus species complex, and how this gap can have important implication for the conservation strategies of this widespread and valuable genus in the Mediterranean Sea.

Mass mortality hits gorgonian forests at Montecristo Island.

Mediterranean gorgonian forests are species-rich habitats, and like many other marine habitats they are threatened by anthropogenic disturbances and mass mortality events. These mortality events have often been linked to anomalies in the temperature profiles of the Mediterranean region. On 5 September 2017, colonies of the gorgonians Eunicella singularis and Eunicella cavolini exhibited rapid tissue loss, down to a depth of 30 m along the steep cliffs of Montecristo Island, Tuscan Archipelago National Park, Tyrrhenian Sea, Italy. Interestingly, Montecristo has previously been identified as a reference site for the ecological quality assessment of the western Mediterranean benthic assemblages on rocky bottoms. The observed mortality event occurred during a period of increased sea temperature. By utilising a combination of high-resolution oceanographic analysis, forecast models and citizen science initiatives, we propose that an early warning system for the concomitance of heat waves and mortality events can be put in place. A temperature-based coral disease surveillance tool could then be established for the entire Mediterranean Sea. Such a tool would allow for the timely study of mass mortality phenomena and the implementation of prompt mitigation and/or restoration initiatives. Finally, this specific mortality event, in a Marine Protected Area, offers a unique opportunity to monitor and assess the resilience of gorgonian populations and associated benthic assemblages in the absence of other, more directly, anthropogenic disturbances such as pollution and land runoff.

Mediterranean Bioconstructions Along the Italian Coast.

Marine bioconstructions are biodiversity-rich, three-dimensional biogenic structures, regulating key ecological functions of benthic ecosystems worldwide. Tropical coral reefs are outstanding for their beauty, diversity and complexity, but analogous types of bioconstructions are also present in temperate seas. The main bioconstructions in the Mediterranean Sea are represented by coralligenous formations, vermetid reefs, deep-sea cold-water corals, Lithophyllum byssoides trottoirs, coral banks formed by the shallow-water corals Cladocora caespitosa or Astroides calycularis, and sabellariid or serpulid worm reefs. Bioconstructions change the morphological and chemicophysical features of primary substrates and create new habitats for a large variety of organisms, playing pivotal roles in ecosystem functioning. In spite of their importance, Mediterranean bioconstructions have not received the same attention that tropical coral reefs have, and the knowledge of their biology, ecology and distribution is still fragmentary. All existing data about the spatial distribution of Italian bioconstructions have been collected, together with information about their growth patterns, dynamics and connectivity. The degradation of these habitats as a consequence of anthropogenic pressures (pollution, organic enrichment, fishery, coastal development, direct physical disturbance), climate change and the spread of invasive species was also investigated. The study of bioconstructions requires a holistic approach leading to a better understanding of their ecology and the application of more insightful management and conservation measures at basin scale, within ecologically coherent units based on connectivity: the cells of ecosystem functioning.

Living upside down: patterns of red coral settlement in a cave.

BACKGROUND: Larval settlement and intra-specific interactions during the recruitment phase are crucial in determining the distribution and density of sessile marine populations. Marine caves are confined and stable habitats. As such, they provide a natural laboratory to study the settlement and recruitment processes in sessile invertebrates, including the valuable Mediterranean red coral Corallium rubrum. In the present study, the spatial and temporal variability of red coral settlers in an underwater cave was investigated by demographic and genetic approaches. METHODS: Sixteen PVC tiles were positioned on the walls and ceiling of the Colombara Cave, Ligurian Sea, and recovered after twenty months. A total of 372 individuals of red coral belonging to two different reproductive events were recorded. Basal diameter, height, and number of polyps were measured, and seven microsatellites loci were used to evaluate the genetic relationships among individuals and the genetic structure. RESULTS: Significant differences in the colonization rate were observed both between the two temporal cohorts and between ceiling and walls. No genetic structuring was observed between cohorts. Overall, high levels of relatedness among individuals were found. CONCLUSION: The results show that C. rubrumindividuals on tiles are highly related at very small spatial scales, suggesting that nearby recruits are likely to be sibs. Self-recruitment and the synchronous settlement of clouds of larvae could be possible explanations for the observed pattern.

Chasing genetic structure in coralligenous reef invertebrates: patterns, criticalities and conservation issues.

Conservation of coastal habitats is a global issue, yet biogenic reefs in temperate regions have received very little attention. They have a broad geographic distribution and are a key habitat in marine ecosystems impacted by human activities. In the Mediterranean Sea coralligenous reefs are biodiversity hot spots and are classified as sensitive habitats deserving conservation. Genetic diversity and structure influence demographic, ecological and evolutionary processes in populations and play a crucial role in conservation strategies. Nevertheless, a comprehensive view of population genetic structure of coralligenous species is lacking. Here, we reviewed the literature on the genetic structure of sessile and sedentary invertebrates of the Mediterranean coralligenous reefs. Linear regression models and meta-analytic approaches are used to assess the contributions of genetic markers, phylum, pelagic larval duration (PLD) and geographical distance to the population genetic structure. Our quantitative approach highlight that 1) most species show a significant genetic structure, 2) structuring differs between phyla, and 3) PLD does not appear to be a major driver of the structuring. We discuss the implication of these finding for the management and conservation, suggesting research areas that deserve attention, and providing recommendations for broad assessment and monitoring of genetic diversity in biogenic reefs species.

Inter- and Intraspecific Variability of Nitrogenated Compounds in Gorgonian Corals via Application of a Fast One-Step Analytical Protocol.

Gorgonian corals play a structural role in temperate and tropical biogenic reefs, forming animal forests and creating biodiversity hot spots. In the Mediterranean Sea, slow-growing and long-lived gorgonian species are threatened by human disturbances and global environmental changes and concern about their conservation is rising. Alkaloid metabolites have proven to be essential in protecting these species from environmental stressors. Traditional profiling methodologies to detect these metabolites require a large quantity of living tissue. Here, the chemodiversity of gorgonian alkaloids was investigated by applying a fast and effective protocol combining extraction and derivatization using small-scale tissue samples and GC/MS analysis. The method was effective in identifying and quantifying alkaloids and guanine-based compounds. Eight N-heterocyclic compounds were found in six Mediterranean gorgonians differing for types and quantity. The metabolomic profile was conservative in species of the Eunicella genus, with three species sharing the same pattern. Conversely, Paramuricea clavata displayed a noticeable spatial pattern of variation among colonies collected in different locations. The analytical approach presented here proved to be effective, allowing rare, endangered, and small-sized species to be screened rapidly for detection of new compounds in order to explore their biological and ecological functions.

Role of Recruitment Processes in Structuring Coralligenous Benthic Assemblages in the Northern Adriatic Continental Shelf.

Coralligenous biogenic reefs are among the most diverse marine habitats in the Mediterranean Sea. The northern Adriatic mesophotic coralligenous outcrops host very rich and diverse epibenthic assemblages. Several studies quantified the low temporal variability and high spatial heterogeneity of these habitats, while processes driving structuring and differentiation are still poorly understood. To shed light on these processes, temporal and spatial patterns of colonisation were investigated using travertine tiles deployed on three coralligenous outcrops, corresponding to the main typologies of benthic assemblages described in previous studies. Three years after deployment, assemblages colonising travertine tiles resembled the differentiation among sites revealed by the natural assemblages in terms of major ecological groups. Processes structuring and maintaining species diversity have been explored. Pioneer species with high reproduction rate, long distance larval dispersal and fast growth (e.g. the serpulid polychaete Spirobranchus triqueter and the bivalve Anomia ephippium), were the most abundant in the early stages of recruitment on the two outcrops further away from the coast and with lower sedimentation. Their success may vary according to larval availability and environmental conditions (e.g., sedimentation rates). At these sites early-stage lasted 10-12 months, during which even species from natural substrates began colonising tiles by settlement of planktonic propagules (e.g., encrusting calcareous Rhodophyta) and lateral encroachment (e.g., sponges and ascidians). On coastal outcrop, exposed to a higher sedimentation rates, tiles were colonised by fast-growing algal turfs. Resilience of northern Adriatic coralligenous assemblages, and maintenance of their diversity, appeared largely entrusted to asexual reproduction. Exploring the mechanisms that underlie the formation and maintenance of the species diversity is crucial to improve our understanding of ecological processes and to implement appropriate conservation strategies of the Adriatic coralligenous reefs.

Limited Genetic Connectivity between Gorgonian Morphotypes along a Depth Gradient.

Gorgonian species show a high morphological variability in relation to the environment in which they live. In coastal areas, parameters such as temperature, light, currents, and food availability vary significantly with depth, potentially affecting morphology of the colonies and the structure of the populations, as well as their connectivity patterns. In tropical seas, the existence of connectivity between shallow and deep populations supported the hypothesis that the deep coral reefs could potentially act as (reproductive) refugia fostering re-colonization of shallow areas after mortality events. Moreover, this hypothesis is not so clear accepted in temperate seas. Eunicella singularis is one of the most common gorgonian species in Northwestern Mediterranean Sea, playing an important role as ecosystem engineer by providing biomass and complexity to the coralligenous habitats. It has a wide bathymetric distribution ranging from about 10 m to 100 m. Two depth-related morphotypes have been identified, differing in colony morphology, sclerite size and shape, and occurrence of symbiotic algae, but not in mitochondrial DNA haplotypes. In the present study the genetic structure of E. singularis populations along a horizontal and bathymetric gradient was assessed using microsatellites and ITS1 sequences. Restricted gene flow was found at 30-40 m depth between the two Eunicella morphotypes. Conversely, no genetic structuring has been found among shallow water populations within a spatial scale of ten kilometers. The break in gene flow between shallow and deep populations contributes to explain the morphological variability observed at different depths. Moreover, the limited vertical connectivity hinted that the refugia hypothesis does not apply to E. singularis. Re-colonization of shallow water populations, occasionally affected by mass mortality events, should then be mainly fueled by larvae from other shallow water populations.

Benthic assemblages on artificial reefs in the northwestern Adriatic Sea: does structure type and age matter?

The use of artificial reefs is on the rise worldwide. While their fish aggregating effects are well known, the epibenthic assemblages have been poorly investigated. Two types of artificial reefs (pyramids of concrete slabs and bundles of concrete tubes) have been deployed out of the Po River Delta in 2006 and 2010. The epibenthic assemblages were investigated in 2009 and 2012. Benthic assemblages on both structure typologies were dominated by species tolerating high sedimentation rates. Dissimilarities were found among assemblages with different ages, and, in less extend, between reef typologies. Colonisation by Mytilus galloprovincialis and other major space occupiers did not follow a clear succession pattern and was not affected by reef typology. Species colonisation was likely driven by variability in environmental conditions and recruitment processes rather than by reef typology. This study suggests that environmental features of the deployment sites should be carefully considered in planning and designing artificial reefs, especially in eutrophic and turbid coastal waters, exposed to high river loads.

Ecological shifts in Mediterranean coralligenous assemblages related to gorgonian forest loss.

Mediterranean gorgonian forests are threatened by several human activities and are affected by climatic anomalies that have led to mass mortality events in recent decades. The ecological role of these habitats and the possible consequence of their loss are poorly understood. Effects of gorgonians on the recruitment of epibenthic organisms were investigated by manipulating presence of gorgonians on experimental panels at 24 m depth, for Eunicella cavolinii, and at 40 m depth, for Paramuricea clavata, at two sites: Tavolara Island (Tyrrhenian Sea) and Portofino Promontory (Ligurian Sea). After 4 months, the most abundant taxa on the panels were encrusting green algae, erect red algae and crustose coralline algae at 24 m depth and encrusting brown algae and erect red algae at 40 m depth. Assemblages on the panels were significantly affected by the presence of the gorgonians, although effects varied across sites and between gorgonian species. Species diversity and evenness were lower on panels with gorgonian branches. Growth of erect algae and recruitment of serpulid polychaetes were also affected by the presence of the gorgonians, primarily at Tavolara. Crustose coralline algae and erect sponges were more abundant on E. cavolinii panels at 24 m depth, while encrusting bryozoans were more abundant on P. clavata panels at 40 m depth. Effects of gorgonians on recruited assemblages could be due to microscale modification of hydrodynamics and sediment deposition rate, or by a shading effect reducing light intensity. Gorgonians may also intercept settling propagules, compete for food with the filter-feeders and/or for space by producing allelochemicals. Presence of gorgonians mainly limits the growth of erect algae and enhances the abundance of encrusting algae and sessile invertebrates. Therefore, the gorgonian disappearances may cause a shift from assemblages characterised by crustose coralline algae to filamentous algae assemblages, decreasing complexity and resilience of coralligenous bioconstructions.

Genetic structuring across marine biogeographic boundaries in rocky shore invertebrates.

Biogeography investigates spatial patterns of species distribution. Discontinuities in species distribution are identified as boundaries between biogeographic areas. Do these boundaries affect genetic connectivity? To address this question, a multifactorial hierarchical sampling design, across three of the major marine biogeographic boundaries in the central Mediterranean Sea (Ligurian-Tyrrhenian, Tyrrhenian-Ionian and Ionian-Adriatic) was carried out. Mitochondrial COI sequence polymorphism of seven species of Mediterranean benthic invertebrates was analysed. Two species showed significant genetic structure across the Tyrrhenian-Ionian boundary, as well as two other species across the Ionian Sea, a previously unknown phylogeographic barrier. The hypothesized barrier in the Ligurian-Tyrrhenian cannot be detected in the genetic structure of the investigated species. Connectivity patterns across species at distances up to 800 km apart confirmed that estimates of pelagic larval dispersal were poor predictors of the genetic structure. The detected genetic discontinuities seem more related to the effect of past historical events, though maintained by present day oceanographic processes. Multivariate statistical tools were used to test the consistency of the patterns across species, providing a conceptual framework for across-species barrier locations and strengths. Additional sequences retrieved from public databases supported our findings. Heterogeneity of phylogeographic patterns shown by the 7 investigated species is relevant to the understanding of the genetic diversity, and carry implications for conservation biology.

Quantifying spatial genetic structuring in mesophotic populations of the precious coral Corallium rubrum.

While shallow water red coral populations have been overharvested in the past, nowadays, commercial harvesting shifted its pressure on mesophotic organisms. An understanding of red coral population structure, particularly larval dispersal patterns and connectivity among harvested populations is paramount to the viability of the species. In order to determine patterns of genetic spatial structuring of deep water Corallium rubrum populations, for the first time, colonies found between 58-118 m depth within the Tyrrhenian Sea were collected and analyzed. Ten microsatellite loci and two regions of mitochondrial DNA (mtMSH and mtC) were used to quantify patterns of genetic diversity within populations and to define population structuring at spatial scales from tens of metres to hundreds of kilometres. Microsatellites showed heterozygote deficiencies in all populations. Significant levels of genetic differentiation were observed at all investigated spatial scales, suggesting that populations are likely to be isolated. This differentiation may by the results of biological interactions, occurring within a small spatial scale and/or abiotic factors acting at a larger scale. Mitochondrial markers revealed significant genetic structuring at spatial scales greater then 100 km showing the occurrence of a barrier to gene flow between northern and southern Tyrrhenian populations. These findings provide support for the establishment of marine protected areas in the deep sea and off-shore reefs, in order to effectively maintain genetic diversity of mesophotic red coral populations.

Comparing efficacy of different taxonomic resolutions and surrogates in detecting changes in soft bottom assemblages due to coastal defence structures.

Sandy shores on the West coast of the North Adriatic Sea are extensively protected by different types of defence structures to prevent coastal erosion. Coastal defence schemes modify the hydrodynamic regime, the sediment structure and composition thus affecting the benthic assemblages. This study examines the effectiveness in detecting changes in soft bottom assemblages caused by coastal defence structures by using different levels of taxonomic resolution, polychaetes and/or bivalves as surrogates and different data transformations. A synoptic analyses of three datasets of subtidal benthic macrofauna used in studies aimed at assessing the impact of breakwaters along the North Adriatic coast has been done. Analyses of similarities and correlations between distance matrices were done using matrices with different levels of taxonomic resolution, and with polychaetes or bivalves data alone. Lentidium mediterraneum was the most abundant species in all datasets. Its abundance was not consistently related to the presence of defence structures. Moreover, distribution patterns of L. mediterraneum were masking the structure of the whole macrofaunal assemblages. Removal of L. mediterraneum from the datasets allowed the detection of changes in benthic assemblages due to coastal defences. Analyses on different levels of taxonomic resolution showed that the level of family maintained sufficient information to detect the impacts of coastal defence structures on benthic assemblages. Moreover, the outcomes depended on the transformation used. Patterns of distribution of bivalves, used as surrogates, showed low correlations with the patterns of the total macrofaunal species assemblages. Patterns of polychaetes, if identified to the species or genus level showed higher correlations with the whole dataset. However, the identification of polychaetes to species and genus level is as costly as the identification of all macrobenthic taxa at family level. This study provided additional evidences that taxonomic sufficiency is a useful tool in environmental monitoring, also in investigations on the impacts of coastal defence structures on subtidal macrofauna. The use of coarser taxonomic level, being time-efficient, would allow improving sampling designs of monitoring programs by increasing replication in space and time and by allowing long term monitoring studies.

Allozyme genotypes and tolerance to copper stress in Hediste diversicolor (Polychaeta: Nereididae).

This study analysed the occurrence of genotypic shifts in laboratory populations of Hediste diversicolor (Polychaeta: Nereididae) exposed to copper stress. Specimens of H. diversicolor were collected at three sites, up to 10 km apart, in the estuarine area of the Pialassa lagoons (North Adriatic Sea, Italy) and were used in acute toxicity tests. Specimens were assigned to copper exposure (0.34 mg/l Cu2+) or control conditions. Each combination of Treatment and Site was replicated in two tanks containing 35 specimens of H. diversicolor. The genotypic structure of both dead and survived specimens was analysed by allozyme electrophoresis at six loci (ALD, FH, HBDH LDH, PGI, SDH). Under copper exposure, specimens with the genotypes ALD100/100 and PGI102/102 had significantly lower mortalities than other genotypes Results were consistent across the three sites, suggesting that, under laboratory conditions, effects of copper stress on H. diversicolor is related to individual genotypes at ALD and PGI loci.