Keep It Simple: Improving the Ex Situ Culture of Cystoseira s.l. to Restore Macroalgal Forests.

Brown algae from genus Cystoseira s.l. form dense underwater forests that represent the most productive areas in the Mediterranean Sea. Due to the combined effects of global and local stressors such as climate change, urbanization, and herbivore outbreaks, there has been a severe decline in brown algal forests in the Mediterranean Sea. Natural recovery of depleted sites is unlikely due to the low dispersal capacity of these species, and efficient techniques to restore such habitats are needed. In this context, the aims of our study were (1) to improve and simplify the current ex situ laboratory protocol for the cultivation of Gongolaria barbata by testing the feasibility of some cost-effective and time-efficient techniques on two donor sites of G. barbata and (2) to evaluate the survival and growth of young thalli during the laboratory phase and during the most critical five months after out-planting. Specifically, the following ex situ cultivation methods were tested: (A) cultivation on clay tiles in mesocosms with culture water prepared by three different procedures (a) filtered seawater with a 0.22 µm filter membrane, (b) filtered seawater with a 0.7 µm filter membrane (GF), and (c) UV-sterilized water, and (B) cultivation on clay tiles in open laboratory systems. After two weeks, all thalli were fixed to plastic lantern net baskets suspended at a depth of 2 m in the coastal sea (hybrid method), and the algal success was monitored in relation to the different donor sites and cultivation protocol. The satisfactory results of this study indicate that UV-sterilized water is suitable for the cultivation of G. barbata in mesocosm, which significantly reduces the cost of the laboratory phase. This opens the possibility of numerous and frequent algal cultures during the reproductive period of the species. Additionally, if the young thalli remain in the lantern net baskets for an extended period of several months, they can grow significantly in the marine environment without being exposed to pressure from herbivorous fish.

Halimeda tuna (Bryopsidales, Ulvophyceae) calcification on the depth transect in the northern Adriatic Sea; carbonate production on the microscale of individual segments.

Halimeda tuna (J. Ellis & Solander) J.V. Lamouroux is the only Halimeda species found in the Mediterranean Sea, and it is an important habitat former. In the northern Adriatic, H. tuna is among the ten most abundant seaweeds in the upper-infralittoral belt in spring and autumn. The modular thalli consist of serially arranged calcified segments. Calcification is closely related to photosynthesis, which causes alkalinization of the inter-utricular space and triggers aragonite formation. Understanding of the complex patterns of segment shape plasticity in relation to CaCO3content at different depth levels is still incomplete. Geometric morphometrics was used to investigate H. tuna segment shape variation on the depth transect at Cape Madona Nature Monument in the northern Adriatic Sea. The position on the thallus and the CaCO3 content of each studied segment were recorded, allowing slight changes in mineral content to be detected at the microscale of the segments. Our results showed that shape, size, or asymmetry of H. tuna segments were not significantly affected by depth. On the other hand, plants that grew deeper were generally more calcified. The apical and subapical segments contributed to the increase in CaCO3 content at the deeper sites, whereas the basal segments did not. This indicates that reniform or oval segments positioned apically or subapically play a key role in calcification of H. tuna in Mediterranean ecosystems.

Diversity of Molluscan Assemblage in Relation to Biotic and Abiotic Variables in Brown Algal Forests.

Canopy-forming macroalgae, mainly those belonging to the order Fucales, form the so-called brown algal forests, which are among the most productive assemblages in shallow coastal zones. Their vertical, branching canopies increase nearshore primary production, provide nursery areas for juvenile fish, and sustain understory assemblages of smaller algae and both sessile and vagile fauna. The majority of benthic invertebrates inhabiting these forests have larval stages that spend some time floating freely or swimming in the plankton. Therefore, canopy-forming macroalgae play an important role as species collectors related to larval supply and hydrodynamic processes. During the past several decades, brown algal forests have significantly reduced their extension and coverage in the Mediterranean basin, due to multiple interacting natural and anthropogenic pressures, with negative consequences also for the related fauna. The aim of this research was to examine how differences in macrophyte abundance and structure, as well as environmental variables, affect the associated molluscan communities in the shallow northern Adriatic Sea. Sampling sites with well-developed vegetation cover dominated by different canopy-forming species were selected in the shallow infralittoral belt of the northern Adriatic Sea in the spring-summer period of the years 2019 and 2020. Our results confirm the importance of algal forests for molluscan assemblage, with a total of 68 taxa of molluscs found associated with macrophytes. Gastropods showed the highest richness and abundance, followed by bivalves. Mollusc richness and diversity (in terms of biotic indices) were not related with the degree of development of canopy-forming species (in terms of total cover and total volume), nor with the ecological status of benthic macroalgae at different depths. On the contrary, the variability in molluscan taxa abundances was explained by some environmental variables, such as temperature, pH, light, and nitrates concentration.

Evaluating Seagrass Meadow Dynamics by Integrating Field-Based and Remote Sensing Techniques.

Marine phanerogams are considered biological sentinels or indicators since any modification in seagrass meadow distribution and coverage signals negative changes in the marine environment. In recent decades, seagrass meadows have undergone global losses at accelerating rates, and almost one-third of their coverage has disappeared globally. This study focused on the dynamics of seagrass meadows in the northern Adriatic Sea, which is one of the most anthropogenically affected areas in the Mediterranean Sea. Seagrass distribution data and remote sensing products were utilized to identify the stable and dynamic parts of the seagrass ecosystem. Different seagrass species could not be distinguished with the Sentinel-2 (BOA) satellite image. However, results revealed a generally stable seagrass meadow (283.5 Ha) but, on the other hand, a stochastic behavior in seagrass meadow retraction (90.8 Ha) linked to local environmental processes associated with anthropogenic activities or climate change. If systemized, this proposed approach to monitoring seagrass meadow dynamics could be developed as a spatial decision support system for the entire Mediterranean basin. Such a tool could serve as a key element for decision makers in marine protected areas and would potentially support more effective conservation and management actions in these highly productive and important environments.

Spatial and temporal distribution of trace elements in Padina pavonica from the northern Adriatic Sea.

Chemical pollution is a major environmental concern especially in coastal areas, having adverse impacts on marine organisms and ecosystem services. Macroalgae can accumulate trace elements, but available studies are restricted to a limited number of elements and species. The goal of this research was to assess, seasonally, the concentrations of 22 elements in the brown alga Padina pavonica from monitoring sampling sites in Slovenian waters. The concentration of most elements in thalli differed significantly between spring and autumn, with generally higher levels in autumn samples. However, it was not possible to correlate these concentrations with the ecological status of macroalgae. The maximum values set by European regulations for the potentially hazardous As, Cd and Hg in food and feed were never exceeded, while Pb concentrations were slightly higher. The results show that P. pavonica can act as an effective bioindicator of chemical pollution.

First Restoration Experiment for Gongolaria barbata in Slovenian Coastal Waters. What Can Go Wrong?

The global decline of brown algal forests along rocky coasts is causing an exceptional biodiversity loss. Regardless of conservation efforts, different techniques have been developed for large-scale restoration strategies in the Mediterranean Sea. In this study we tested ex situ pilot restoration of Gongolaria barbata (=Treptacantha barbata) for the first time in Slovenian coastal waters. Healthy apical fronds of the species were collected and the development of recruits on clay tiles was followed under laboratory conditions for 20 days. Despite the experimental difficulties experienced, especially due to the lack of antibiotics to prevent the growth of the biofilm, G. barbata recruits were outplanted in the sea on two concrete plates with 48 tiles each, protected by purpose-built cages to avoid grazing by herbivorous fish. The high survival rate of juveniles after four months in the field (89% of the tiles on the plate that was constantly protected) suggests that outplanting G. barbata is an operable approach for restoration efforts in the northern Adriatic Sea. Our first experiment in Slovenian coastal waters provides new information for the optimization of the best practices during the laboratory cultivation and addresses the early steps of restoration and introduction of young thalli in the natural environment.

Non-indigenous macrophytes in Central Mediterranean ports, marinas and transitional waters: Origin, vectors and pathways of dispersal.

Non-indigenous species are confirmed to be among the biggest threats for marine biodiversity. Among them, non-indigenous macrophytes (NIM) are well known to have local negative effects, especially in coastal ecosystems. Since transitional waters (TWs), ports and also marinas are recognized as very vulnerable coastal ecosystems, greatly subjected to biological invasions, the present study analysed the available scientific literature on NIM in such areas in the Central Mediterranean Sea (CMED), in the period 1970-2019. The analysis underlined that 27 NIM were recorded in CMED TWs, marinas and ports. Around 37% of them (10 species) are marked as invasive, while about 11% (3 species) are considered as potentially invasive. Maricultural activities resulted the key vector of introduction, while the primary pathway of NIM dispersion is related to maritime activities. The paper also discusses why in CMED TWs, marinas and ports, currently, less NIM were detected than in similar Adriatic areas.

Seagrass Cymodocea nodosa and seaweed Ulva lactuca as tools for trace element biomonitoring. A comparative study.

This study compared the capacity of the seagrass Cymodocea nodosa and the green alga Ulva lactuca to sequester the trace elements Cd, Cr, Cu, Ni, Pb and Zn, in seawater and benthic sediments, under the same environmental conditions. Samples of C. nodosa leaves and U. lactuca thalli were collected in four sites off the coast of Sicily (Italy), with varying degrees of anthropogenic impact. C. nodosa accumulated generally higher levels of trace elements, suggesting a better performance in marine phytoremediation. In turn, C. nodosa and U. lactuca showed a similar performance regarding the bioindication of trace elements. Since salinity, temperature and pH had relatively constant values, their role in the accumulation processes in both species should be considered as secondary. Although C. nodosa and U. lactuca fulfill several criteria as bioindicators, including widespread distribution, abundance and tolerance to toxicity, their regular use in biomonitoring programs is still limited.

Marine plastics: What risks and policies exist for seagrass ecosystems in the Plasticene?

Plastic debris is nowadays a well-recognized global threat to marine ecosystems, due to its increasing abundance and pervasiveness. Although the effects of marine plastics on animal wildlife is generally documented, the available studies of plastics affecting seagrasses and their associated biota are relatively scarce. This makes the degree of risk uncertain as regards seagrass ecosystems affected by plastic debris, but also it results in several critical knowledge gaps such as to what extent plastic debris can affect food webs supported by seagrasses. In the age of plastics, the Plasticene, the international political agenda shows great interest in this matter. However, to date, no conservation policies have specifically targeted the protection of seagrasses from plastics debris. Future actions should therefore move in two directions: prompting a radical shift in plastics consumption, and shedding further light on plastics-biota interactions in the marine environment.

Comparative assessment of trace element accumulation and biomonitoring in seaweed Ulva lactuca and seagrass Posidonia oceanica.

The present research compared the capability of the green seaweed Ulva lactuca and the seagrass Posidonia oceanica to accumulate and biomonitor the levels of six trace elements: Cd, Cr, Cu, Ni, Pb, Zn. The concentrations of these elements were analyzed in seawater, bottom sediments, U. lactuca thalli and P. oceanica leaves, in four sites of Sicily (Italy) with different levels of pollution. The results showed that P. oceanica is able to accumulate greater concentrations of trace elements compared to U. lactuca. Both species, instead, acted equally as good bioindicators of marine pollution, showing significant correlations with the levels of trace elements in the surrounding environment. The use of P. oceanica and U. lactuca as bioindicators is still limited in biomonitoring programs, and should be further encouraged given the ever-increasing marine pollution.

Non-indigenous macrophytes in Adriatic ports and transitional waters: Trends, taxonomy, introduction vectors, pathways and management.

The Adriatic Sea is considered as a hotspot of marine biodiversity but, due to intense human activities (e.g. maritime transport, mariculture), it is also a recipient area of non-indigenous species (NIS). This study analyzed the scientific literature on non-indigenous macrophytes (NIM) recorded in ports, marinas and transitional waters (TWs) of the Adriatic Sea, in the period 1987-2018. The results show that 51 out of 118 Mediterranean NIM are found in Adriatic ports and TWs. The Venice Lagoon is the main Adriatic recipient area of NIM. Mariculture is the main vector of introduction, whereas the principal pathways of NIM dispersal are shipping and shellfish transfer. The largest group of NIM is of temperate northern Pacific origin. This study corroborated the fact that ports and TWs act as dispersal hubs and invasion hotspots. The importance of early detection of NIM at such sites should be considered in any environmental management strategy.

Port Baseline Biological Surveys and seaweed bioinvasions in port areas: What's the matter in the Adriatic Sea?

One of the objectives of the BALMAS project was to conduct Port Baseline Biological Surveys of native and non-indigenous benthic flora in 12 Adriatic ports. Samples of macroalgae growing on vertical artificial substrates were collected in spring and autumn 2014 and/or 2015. A total number of 248 taxa, 152 Rhodophyta, 62 Chlorophyta, and 34 Ochrophyta, were identified. Of these, 13 were non-indigenous seaweeds, mainly filamentous macroalgae, that were probably introduced through hull fouling. Some of these taxa had already been described in the study areas, others were recorded for the first time, a few were no longer detected at sites where they had previously been recorded (e.g. Sargassum muticum). Some other NISS reported for the Adriatic Sea, were not collected at any sampling site (i.e. Caulerpa cylindracea, Codium fragile). Possible reasons for the absence of these species are discussed.

Strategy of port baseline surveys (PBS) in the Adriatic Sea.

Port baseline surveys (PBS) provide species inventories in and around ports, with a focus on non-indigenous species that may have been introduced by vessels, primarily via ballast water. PBS are an essential tool to support effective management strategies for non-indigenous as well as native harmful aquatic organisms and pathogens (HAOP). This paper describes the methodology of PBS that were conducted in 12 Adriatic ports. The PBS employed existing protocols that were adapted to meet the characteristics of the Adriatic sites. Their results are reported in several papers included in this special issue, each of which is devoted to a specific community. An overview of existing surveys protocols - which provide valuable support to decision-making and to design effective monitoring of non-indigenous species - is also supplied.

Perspectives on using marine species as bioindicators of plastic pollution.

The ever-increasing level of marine pollution due to plastic debris is a globally recognized threat that needs effective actions of control and mitigation. Using marine organisms as bioindicators of plastic pollution can provide crucial information that would better integrate the spatial and temporal presence of plastic debris in the sea. Given their long and frequent migrations, numerous marine species that ingest plastics can provide information on the presence of plastic debris but only on large spatial and temporal scales, thus making it difficult to identify quantitative correlations of ingested plastics within well-defined spatio-temporal patterns. Given the complex dynamics of plastics in the sea, the biomonitoring of marine plastic debris should rely on the combination of several bioindicator species with different characteristics that complement each other. Other critical aspects include the standardization of sampling protocols, analytical detection methods and metrics to evaluate the effects of ingested plastics in marine species.

Chemical elements in Mediterranean macroalgae. A review.

This review analyzes the state of knowledge on the capacity of Mediterranean macroalgae to accumulate, tolerate and biomonitor macro- and micro-elements present in seawater and sediments. The results show that the investigated macroalgal species amount to c. 5.0% of all Mediterranean native Rhodophyta, Chlorophyta and Ochrophyta. The most commonly analyzed elements in algae were Pb, Cd, Zn and Cu, whereas very few studies were available for macroelements such as Ca and K. Uptake mechanisms and the factors influencing accumulation are overall well known, but the available studies are restricted to a limited number of chemical elements and algal species. This review also shows that macroalgae can accumulate and tolerate high concentrations of chemical elements, and can act as effective bioindicators of marine pollution. Phytotoxic limits for algal species are not clearly identified by the available studies and require further investigation. Future research should focus on the development of protocols for long-term biomonitoring programmes through the use of macroalgae. Future studies also need to investigate the biomagnification of toxic trace elements in macroalgae-based food webs.

Trace elements in Mediterranean seagrasses and macroalgae. A review.

This review investigates the current state of knowledge on the levels of the main essential and non-essential trace elements in Mediterranean vascular plants and macroalgae. The research focuses also on the so far known effects of high element concentrations on these marine organisms. The possible use of plants and algae as bioindicators of marine pollution is discussed as well. The presence of trace elements is overall well known in all five Mediterranean vascular plants, whereas current studies investigated element concentrations in only c. 5.0% of all native Mediterranean macroalgae. Although seagrasses and macroalgae can generally accumulate and tolerate high concentrations of trace elements, phytotoxic levels are still not clearly identified for both groups of organisms. Moreover, although the high accumulation of trace elements in seagrasses and macroalgae is considered as a significant risk for the associated food webs, the real magnitude of this risk has not been adequately investigated yet. The current research provides enough scientific evidence that seagrasses and macroalgae may act as effective bioindicators, especially the former for trace elements in sediments, and the latter in seawater. The combined use of seagrasses and macroalgae as bioindicators still lacks validated protocols, whose application should be strongly encouraged to biomonitor exhaustively the presence of trace elements in the abiotic and biotic components of coastal ecosystems.

Trace elements in Mediterranean seagrasses: Accumulation, tolerance and biomonitoring. A review.

This study investigated the state of the art on trace elements in Mediterranean seagrasses, and their close environment (seawater and sediment). The analyzed species were Posidonia oceanica, Cymodocea nodosa, Halophila stipulacea, Zostera marina and Zostera noltei. All these species showed high tolerance to pollution and high capacity of accumulation of trace elements. Seagrasses also showed similar patterns of accumulation: the highest concentrations of As, Hg and Pb were found in the roots, whereas those of Cd, Cr, Cu, Mn, Ni and Zn were found in the leaves. Phytotoxic levels in seagrasses are unknown for most trace elements. The accumulation of trace elements in seagrasses is widely recognized as a risk to the whole food web, but the real magnitude of this risk is still uncertain. Seagrasses are known to act as trace element bioindicators, but this potential is still poorly valued for the creation of biomonitoring networks.

A new index (MediSkew) for the assessment of the Cymodocea nodosa (Ucria) Ascherson meadow's status.

In the Mediterranean region Cymodocea nodosa is widely distributed throughout shallow sites. Therefore, a correct assessment of the status of its meadows is of great importance for the implementation of the Water Framework Directive (WFD), the Marine Strategy Framework Directive (MSFD), and the Habitat Directive (HD), especially for areas where Posidonia oceanica meadows (the most frequently used indicator in the Mediterranean Sea) are rare or not present. The previously proposed index for the evaluation of the status of C. nodosa meadows (CymoSkew) is based on ln-transformed relative frequencies of photosynthetic leaf lengths, which we believe is statistically questionable. Therefore, we further improved the methodology and developed a new index named MediSkew, where ln-transformation is applied to raw data of leaf lengths. More specifically, the index is a combination of two metrics, both based on C. nodosa leaf length: deviation from the reference median length (Medi-) and skewness of the length frequency distribution (-Skew), though greater importance was assigned to the first. To develop the classification criteria for the assessment of the status, also a Pressure Index for Seagrass Meadows (PISM), for the evaluation of pressure-impact relationships, was developed. The MediSkew is meant to be a rapid screening method for wide areas, therefore the index should be tested for the assessment of the status of C. nodosa meadows throughout Mediterranean coastal waters.

Benthic macrophytes as a tool for delineating, monitoring and assessing ecological status: the case of Slovenian coastal waters.

Benthic macrophytes from 51 sites in the upper infralittoral zone along the Slovenian rocky coast were sampled (randomly stratified) in order to (1) confirm water body (WB) boundaries, (2) select sampling points for a surveillance monitoring programme, and (3) assess preliminary Ecological Status Classes (ESC) within the European Water Framework Directive (WFD). Coverage data of macrophytes were analyzed by using cluster analysis, Simper analysis and the Ecological Evaluation Index (EEI). The vegetation was rather homogenously classified into two Cystoseiretum crinitae subassociations: Halopithetosum incurvae, and Cystoseiretosum compressae, and into Cystoseiretum barbatae association. Seven monitoring sites at a kilometer scale were selected in two significant sized WBs following the mixing zone principle and the EEI successional model. The preliminary assessment of the ESC with the EEI was in agreement with existing human pressures in the area. In view of the present results, benthic macrophytes and EEI could be valuable tools for the implementation of the WFD within the Mediterranean eco-region.