The study area is the Site of National Interest (SNI) of Falconara Marittima (43°38’54’’N 13°21°35’’E), established under Law No. 179 of July 31, 2002, by Decree of the Minister of Environment and Territorial Protection of February 26th, 2003 (Corinaldesi et al., 2022; Figure 1 ). The SNI covers a marine-coastal area of about 1’200 ha and a territory of about 108 ha; it includes industrial settlements with high environmental impact, such as the API Raffineria S.p.A. and the disused area of the industrial plant “ex Montedison”, which produced phosphatic fertilizers from pyrite and phosphorite.

Although the SNI is included in the Area with High Risk of Environmental Crisis Falconara and Lower Esino Valley (AERCA) (ARPAM, https://www.arpa.marche.it/siti-di-interesse-nazionale), the anthropogenic impact is no longer detectable in the marine area and the marine sediment could be classified as “meso-eutrophic” (Pusceddu et al., 2009), a common condition in many areas of the Adriatic Sea (Bianchelli et al., 2016a; Bianchelli et al., 2018). However, in the terrestrial environment, soil and groundwater show contaminant levels above the thresholds (Legambiente, 2021). In the coastal area facing the SNI, there are 11 breakwater barriers, extending from the “ex Montedison” industrial plant to the mouth of the Esino River.

G. barbata (Fucales) lives in shallow and sheltered rocky environments and is considered a threatened species by the Barcelona Convention. This species is quite rare along the coasts of the Mediterranean Sea, 107 populations have been recorded in recent years along Italian coasts across the Central and Southern Tyrrhenian, Sardinian, Adriatic and Northern Ionian Seas (Tamburello et al., 2022). Local extinctions have been documented between 1979 and 2013–2016, whereas few populations naturally recovered by 2000–2008 (Tamburello et al., 2022).

The G. barbata specimens used for transplantation in the SNI were obtained from in-situ recruitment carried out at Passetto (Ancona), where during February 2021 3 artificial structures were positioned under reproductive adults of G. barbata (the donor population: Bianchelli et al., submitted). The structures were equipped with clay tiles characterized by rough surfaces to let the zygotes root and grow (Verdura et al., 2018).

In July 2021, the 3 structures with the G. barbata juveniles were moved from Passetto to the SNI of Falconara Marittima. The most distant breakwater barrier from the Esino River mouth was selected as receiving site since it corresponds to the lowest level of transport of sediment, freshwater, and nutrients.

A complementary action based on the installation of cages to exclude the herbivores due to the high grazing pressure in the area (Bianchelli et al., submitted) was also carried out. As a result, each experimental unit was composed of an artificial structure with G. barbata juveniles and the cage to exclude grazers. The cages were positioned at about 1-meter depth in the landward part of the barrier and were built with a galvanized iron mesh with 12 mm mesh and were 40 cm in length, 25 cm in width, and 20 cm in height and had a removable lid ( Figure 2 ). The experimental units were fixed to the artificial reef with the drill and stainless-steel screws.

On 21^st October 2021, following a strong storm, the cage of the third structure (structure 3) was uprooted and one of the five clay tails was lost. Since we observed that in the other two structures the algae’ heights reached the lid of the cages, the same day, the lid of the first cage (structure 1) was removed to let the algae grow and test the resistance of G. barbata grown against grazers.

The response variables chosen (G. barbata individuals’ height and canopy coverage, meio- and macrofaunal abundance and diversity) allowed us to monitor the growth of G. barbata over time and changes in the associated biodiversity. The height and the canopy coverage of the G. barbata were used to monitor its growth. The height was measured in 10 individuals for each experimental unit (abundance 5-15 cm^-2 individuals), whereas the canopy coverage was assessed as % over a standardized surface (corresponding to the area of the cage).

Meiofauna and macrofauna (investigated in terms of total abundance, richness of taxa, and taxonomic composition) were used as a proxy for the associated communities. Sampling for meio- and macrofauna analyses was carried out comparing assemblages under the structures with a substrate with no transplanted macroalgae (used as control), approximately every 2 months, from transplantation to SNI in July until January 2022. A modified manual corer with an inner diameter of 9 cm was used to take 3 replicated samples from the hard substrate nearby cages (1 replicate for each experimental unit) and 3 replicates far from them (as a reference). Samples were then collected in plastic bags (Danovaro and Fraschetti, 2002). The samples were then frozen at -20°C until the analyses at lab.

For the extraction of meio- and macrofauna, the samples were subjected to ultrasonic treatment (3 x 1 minutes) and filtered on 20 and 500 µm meshes, for separating meio- and macrofauna. For meiofauna, an extraction based on centrifugation in a density gradient, with Ludox HS40 gel solution was used (Heip et al., 1985; Danovaro, 2010). The samples were then placed in 50 ml falcon with 70% ethanol and Rose Bengal and kept refrigerated for 2-3 days to allow the staining of the sample. The abundance of organisms was determined by using the stereomicroscope with magnifications from 16 to 40X.

During the sampling survey in October 2021, individuals of P. marmoratus were photographed while eating G. barbata settled on the third structure, deprived of the cage after the sea storm. After 18 days, on the same structure, grazers completely removed G. barbata individuals. Since no herbivorous fish were observed along the breakwaters during the entire period, an experiment was conducted in the aquarium to investigate P. marmoratus grazing on the algae. To do this, in December 2021, 4 specimens of P. marmoratus, together with a small stone covered by numerous individuals of G. barbata less than 15 cm high, were taken from the donor site Passetto ( Figure 3 ).

The crabs and the rock with the algae were transferred inside a tank of 200 L, inside which was placed a rubber mat as a substrate to facilitate the movement of animals, which was not given any type of additional food. The photoperiod of the tank was set at seven hours of light and 17 h of dark, and the water temperature was 13°C.

The number of G. barbata individuals and their heights were measured across 20 days, every 4-5 days, for a total of 6 sampling times. Through an underwater camera, videos were also made taking photos at an interval of 30 seconds one from each other, to shoot crabs while eating G. barbata.

For algal growth, the experimental design considered 2 factors as source of variability: Time, fixed with 17 levels (corresponding to the sampling times), and Structure, fixed with 3 levels. The analysis considered 9-10 replicated measures each time in each structure. For the analysis of the associated biodiversity, the experimental design considered 2 factors as source of variability: Site (fixed, 2 levels: cages and control) and Time (fixed, 4 levels, corresponding to the sampling times), with 3 replicates. For algal growth and abundance in the experiment on grazing, the experimental design considered only Time as source of variance (fixed factor, with 6 levels).

The experimental designs were applied to permutational analyses of variance (PERMANOVA), in uni- (for algal growth, total meio- and macrofaunal abundance) and multivariate context (for taxonomic composition of the meio- and macrofaunal assemblages) and based on Euclidean distance and Bray-Curtis similarity matrices, respectively. Analyses on meio- and macrofaunal taxonomic composition were repeated considering the whole assemblages and considering only the rare taxa. When significant differences were observed, pair wise tests were performed to establish between which levels significant differences were present. To visualize differences in the taxonomic composition, bi-plot were also produced following CAP (Canonical Analysis of Principal coordinates) analysis.

All statistical analyses were done with the PRIMER7 software package.

During the recruitment phase (from March ‘21 to July ‘21) at Piscinetta del Passetto, the heights of G. barbata individuals significantly grew over time, with no difference among the structures ( Figure 4A ). After the transplant at SNI carried out in July ‘21, algae significantly grew until September ‘21, both in terms of height and canopy coverage (heights range 13.7-15.0 cm and canopy coverage 60.4-81.8%, Figures 4A, B ). In October ‘21 a storm destroyed one of the cages (Structure 3), resulting in the complete disappearance of the algae from the structure in one month ( Figure 4A , dark green dots). On that occasion, the cage lid was removed from Structure 1. In Structures 1 and 2, algal heights and canopy coverage did not significantly change over the following 3 months. At the beginning of January ‘23, another storm destroyed the remaining two cages, however, on that occasion the algae were not detached and their eight and canopy coverage did not change until the end of the experiment (end of January ‘22). At the end of the experiment, the algae heights, and their canopy coverage in the Structure 1 (without the lid) were higher than in the cage with the closed lid (Structure 2).

Meiofaunal abundance showed significant differences over time but not between cages and the control site ( Table 1A , Figure 5A ). The richness of meiofaunal taxa was higher in the control site in July ‘21 and underneath the cages in the last sampling time (January ‘22) ( Figure 5B ). The overall richness of taxa (i.e., cumulatively in control and cages sites) increased over time until November ‘21 and corresponded to the richness of taxa underneath the cages in the last sampling time ( Figure 5B ).

The assemblages were dominated by Nematoda (52-57% and 36-67% in control site and beneath the cages, respectively), Copepoda (11-37% and 9-30%), Polychaeta (2-5% and 3-10%), Bivalvia (2-32% and 2-44%) and Kinorhyncha (2% and 0-2%). The rare taxa (i.e., representing each <1% of the assemblages) observed were: Oligochaeta, Amphipoda, Ostracoda, Isopoda, Cumacea, Gasteropoda, Acarina, Tanaidacea and Hydrozoa in the control site; and Oligochaeta, Cladocera, Amphipoda, Ostracoda, Isopoda, Cumacea, Gasteropoda, Acarina, Tanaidacea, Hydrozoa and Picnogonida underneath the cages. Considering only the rare taxa, significant differences were observed between cages and control site ( Table 1A , Figure 5C ). In the last sampling time (January 2022) rare taxa were observed only underneath the cages with G. barbata.

Macrofaunal abundance did not show significant differences over time nor between cages and the control site ( Table 1B , Figure 6A ). The richness of macrofaunal taxa was higher in the control site only in July ‘21, where the values were higher underneath the cages in September and November 2021 ( Figure 6B ). The overall richness of taxa was higher than that observed in the control or cages ( Table 1B , Figure 6B ).

The assemblages were dominated by Polychaeta (21-49% and 27-64% in the control site and beneath the cages, respectively), Nematoda (7-41% and 4-53%), Bivalvia (3-21% and 0-13%) and Hydrozoa (0-50% and 0-55%). Significant differences were observed only among sampling times but not between cages and control site ( Table 1B , Figure 6C ).

The number of the algae individuals on the stone was 27 at the beginning of the experiment, decreased by ca. 50% after 12 days and the individuals were completely grazed after 30 days ( Figure 7A ). PERMANOVA analyses did not reveal any significant differences in the algal heights across time ( Table 1C , Figure 7B ). The recorded videos confirmed that P. marmoratus grazes on G. barbata detaching the individuals from the basal disc of the algae, irrespective of their height.

In this study, the SNI of Falconara Marittima, was selected for testing the possibility of rehabilitating the marine ecosystem through a transplant experiment of a population of G. barbata. Fucales forests can indeed be transplanted into degraded habitats where they were historically present, but also in areas where they had never been recorded in order to rehabilitate the habitat and increase its ecological value (Gianni et al., 2013).

Being interdicted to human activities for decades due to historical pollution and presenting nowadays characteristics indistinguishable from the surrounding non-interdicted areas, this SNI represents an optimal candidate as restoration site to implement combined passive and active restoration interventions (Corinaldesi et al., 2022; Fanelli et al., 2023).

Coastal infrastructures, such as breakwater barriers, ports, and offshore installations, are an ideal harsh substrate for the conservation of threatened marine species and can represent suitable substrates for “active restoration” interventions (Perkol-Finkel et al., 2012). In this regard, very few attempts were carried out in the Northern Adriatic Sea, where breakwater barriers represent a hard substrate different from the surrounding sandy habitats, using G. barbata on coastal defence structures, with encouraging results (Perkol-Finkel et al., 2012). However, in contrast to previous findings reporting a very low survival percentage (on average 20-30%, Perkol-Finkel et al., 2012), we obtained high survival rates, high levels of growth and canopy coverage. Differences between our and previous results may rely on the fact that we applied an approach integrating “passive” and “active” restoration, as an intervention continuum and not a mere action of transplant. In particular, the area in which we transplanted G. barbata was evaluated a priori as suitable for restoration as it no longer showed signs of pollution (Corinaldesi et al., 2022; Fanelli et al., 2023). We argue that this is a critical step to evaluate the success of “passive” restoration before implementing a transplant action on coastal infrastructures as breakwater barriers.

Moreover, we successfully managed the herbivory pressure using cages (see Paragraph 4.3), as also reported in previous studies (Perkol-Finkel et al., 2012; Orlando-Bonaca et al., 2021). The only factor threatening the success of the transplant was the autumnal storm that occurred in October, which destroyed one of the cages and allowed us to test the grazing pressure over G. barbata individuals of 15 cm height. This allows us to confirm that restoration must be considered a continuous process needing maintenance, in this case, the possible replacement of the lost cage. Also, the outcomes of previous studies highlighted that unpredictable climatic events are one of the major threats impairing the restoration process of G. barbata (Savonitto et al., 2021). The ongoing climate change, provoking temperature raising or stochastic and more and more frequent strong events (e.g., frequent storms, heatwaves), represents the biggest challenge to face when putting in force restoration interventions at the local level (Garrabou et al., 2022; Orlando-Bonaca et al., 2022). Climate change events can affect the survival and the reproduction of the target species but also impair the transplants themselves, destroying the infrastructures used to implement the reintroduction of the target populations (Savonitto et al., 2021; present study). Moreover, population vulnerability to warming would have to be considered before designing and investing in a long-term and successful restoration plan and climatic refugia should be considered as priority areas for habitat-forming species restoration (Verdura et al., 2021).

In this restoration experiment the total abundance, richness of taxa, and the taxonomic composition of meio- and macrofaunal assemblages were assessed underneath the structures with macroalgae and in the control (artificial reef’ substrate with no transplanted macroalgae), to assess potential biodiversity changes led by G. barbata. Meio- and macrofauna abundance did not show significant differences between the control and the restoration site but only changes over time. This is probably due to seasonal fluctuations and the effect of the sea storms occurring during the experiment (Danovaro and Fraschetti, 2002). However, in the last sampling period, the richness of meiofaunal taxa was higher underneath the structures with macroalgae than in the control site, whereas the macrofaunal richness of taxa was the same underneath the structures and in the control. Similarly, meio- and macrofauna taxonomic composition significantly changed over time and the only difference between structures with macroalgae and control sites was observed in the composition of meiofaunal rare taxa. Fucales forests typically attract and sustain high levels of benthic biodiversity, particularly related to meiofaunal and macrofaunal herbivores which feed on the macroalgae, but also other consumers, thus provoking an increase in the richness of taxa (Bianchelli et al., 2016b). Moreover, macroalgae form three-dimensional habitats, hosting a lot of associated species (as epiphytes), due to the high number of ecological niches available (Veiga et al., 2014). Indeed, our data indicate that the overall richness of taxa (i.e., cumulatively in control and underneath cages) was higher than those observed in each site, reflecting that G. barbata host taxa were otherwise absent.

Overall, our results let us hypothesize a positive response by meio- and macrofaunal assemblages, which could have a positive effect on the entire food web, due to their ecological role. However, a longer monitoring period is required to ascertain a consistent and durable response from the benthic community after the restoration intervention in terms of biodiversity enhancement.

Herbivores can drastically reduce algal and seagrass canopies, thus Sarpa salpa, Paracentrotus lividus and Arbacia lixula have been defined as important ecosystem modifiers (Tamburello et al., 2022 and citations therein). Since fluctuations in their populations cannot be easily controlled, grazers are considered one of the main constraints in macroalgal restoration actions (Orlando-Bonaca et al., 2021), and devices to prevent access by grazers (fish and sea urchins) and herbivore removal/culling should be considered (Gianni et al., 2018; Cebrian et al., 2021). Moreover, the knowledge of the critical threshold density of herbivores affecting recruitment success in macroalgae is still to be ascertained and will vary depending on environmental conditions (Cebrian et al., 2021). However, in this study, the main herbivores were represented by benthic meso- or macro-grazers (i.e., Pachigrapsus marmoratus), so the use of cages to protect the small juveniles of G. barbata was an effective complementary action to obtain macroalgal adults even in presence of high grazing pressure (Cebrian et al., 2021; Monserrat et al., 2023; Bianchelli et al., submitted).

In addition, we could estimate that when the algae reached 10-15 cm height (in January ‘22, Figure 4 ), they were grown enough to counteract the herbivory pressure. The use of cages to exclude grazers has indeed allowed the algae to grow and survive six months after the transplant in the SNI (except for structure 3, damaged by the sea storm after 3 months after the transplant). Especially in the early stages of the experiment, the presence of cages is essential to avoid grazing, and this has also been proven by other studies conducted on artificial reefs. In this regard, previous investigations (Perkol-Finkel et al., 2012) demonstrated that the use of cages with 1x1 mm mesh for the G. barbata restoration in artificial barriers allows the algae to survive and to have a growth comparable to natural populations, but with very low survival percentage. In the present study, we observed different factors possibly impairing the transplant success: i) the storm destroying one of the cages and ii) the lid of the cages, limiting the growth in height of individuals. Overall, our field results indicate that the G. barbata needs to reach at least a minimum height of ca. 10-15 cm and 80% in canopy coverage before removing the cages. Moreover, the laboratory experiment of grazing demonstrated that P. marmoratus (apparently the main grazer, Gianni et al., 2018) detach the algae from the basal disc. This means that, in our case, cages can be built without lids to avoid grazing and at the same time allow for algae growth. This could also impair the growth of epiphytes on the cages’ mesh; indeed, these species are in competition for resources with G. barbata and they could modify the irradiance and the water exchange conditions (Cebrian et al., 2021). Moreover, in future restoration interventions, it would be necessary to transplant a very large number of algae individuals to counteract the grazing pressure by P. marmoratus.

Despite the difficulties encountered mainly due to stochastic events (bad weather and sea storms) G. barbata survived and grew over time; this demonstrates that the cages play a fundamental role in the success of restoration interventions in artificial reefs, at least for the survival of juvenile algae and until they become resistant to grazing pressure.