**4. Discussion**

The results of this study highlight the potential of small primers, especially diffused transplants of single rhizomes, for a rapid colonization of areas that have the suitable ecological conditions. The transplants of aquatic angiosperms by single rhizomes do not require large efforts and expertise and favors the diffusion of plants throughout the area. Therefore, the involvement of motivated groups of people such as fishermen, hunters, and sport associations who frequently spend part of their time in the lagoon was a grea<sup>t</sup> achievement for lagoon recovery. In the past, several transplants of sods measuring some square meters [20–22] were carried out at the edges of canals or in relatively deep areas. Such operations required grea<sup>t</sup> efforts, and also the use of large boats and mechanical means that cannot operate in shallow waters. The Project Life12 NAT/IT/000331—SERESTO (www.lifeseresto.eu) tested the possibility of recolonizing a large and shallow lagoon area (approximately 36.6 km2), where the aquatic angiosperms had almost completely disappeared, through small, widespread transplants. The proposed strategy achieved a large-scale restoration through the transplants of a small number of plants (approximately 40 m<sup>2</sup> during the entire project), with advantages in terms of costs and impact on the donor sites.

Intense monitoring of plant growth and environmental parameters over the whole area during one year allowed to understand the reasons behind achievements and/or failures. Results showed that aquatic angiosperms took roots only in areas where nutrient concentrations were not too high and where the tionitrophilic macroalgae, especially Ulvaceae, Cladophoraceae, and Gracilariaceae prevailed, both sods and rhizomes chocked and died. Angiosperm transplants failed near river outflows because waters were turbid and rich in nutrients. The choice to transplant angiosperms as well in these areas and to monitor the environmental parameters allowed to identify a series of critical values that must be taken into account should these techniques be successfully applied to other areas. First of all, as reported in other studies [7], the presence and growth of aquatic angiosperms depended on the species considered, degree of trophic conditions, and clearness of the waters. The lagoon areas we studied are colonized by five species: three seagrasses (*Cymodocea nodosa* Ucria (Ascherson), *Zostera marina* Linnaeus, and *Zostera noltei* Hornemann) and two aquatic angiosperms (*Ruppia cirrhosa* Petagna (Grande) and *Ruppia maritima* Linnaeus) [23]. All of them have different ecological features. *Cymodocea nodosa* is a subtropical species that colonizes areas characterized by high salinity, coarse grain sediments, and low nutrient concentrations. It prefers the areas which are strongly influenced by marine waters near the lagoon mouths or high-flow canals, but it can also be present in choked areas [7,23] provided there are the conditions quoted above. However, in the shallow choked areas, where water renewal is low, it reaches much smaller sizes than in areas vivified by the sea. *Zostera marina* prefers areas characterized by fine sediments and high water exchange, because it is a species very sensitive to high temperatures. It reaches the highest growth in April–May, afterwards it decreases and disappears almost completely when the water temperature remains above 26–28 ◦C for long periods. *Zostera noltei* and *Ruppia cirrhosa* prefer shallow choked areas. The former grows mainly along saltmarsh edges and the latter in any shallow bottom, provided the waters are clear. Both species colonize areas with fine sediments where temperature and salinity are very variable, but *Ruppia cirrhosa* prefers the most choked areas and sediments with a higher percentage of fines. Finally, *Ruppia maritima* is the species which most easily adapts to extreme variations in salinity and temperature. It usually grows in the pools of salt marshes, which are in-flowed by new water only during particularly high tides [23].

Our transplants involved the first four species, particularly *Z. marina* and *Z. noltei,* whereas *R. cirrhosa* and *C. nodosa* were only transplanted in some stations with suitable ecological conditions. All the species took root, but *Z. noltei* was the most successful. It rapidly colonized all the saltmarsh edges of the whole transplanting area, where new plants were also produced through natural seed dispersion. *Zostera marina* took root in areas with a negligible tionitrophilic algal biomass, where good water renewal kept temperatures moderate, preventing the formation of sulfides that are very toxic and lead to the species local extinction [24]. Transplants of *Z. marina* carried out in the autumn were a grea<sup>t</sup> achievement, because they formed numerous patches, even though in the presence of the high summer temperatures (>26–28 ◦C), which were also recorded by [25] in the Denmark coasts, they started to regress and in some areas, disappeared altogether. Temperature was the most critical factor for the survival of this species. Some authors [26] who had carried out in-culture experiments fond that the best temperature for the growth of *Z. marina* was <25 ◦C. At 27 ◦C, the growth decreased significantly and at higher temperatures it died. This explains why it is widely distributed in the cold waters of the northern part of the Atlantic Ocean and the Pacific Ocean, up to the Arctic Circle. In the Mediterranean Sea, it is a relict species of the ancient Tetide Ocean that colonizes mainly areas characterized by moderate temperatures and salinities [10–25,27] such as the northern Adriatic Sea, the Aegean Sea, the Black Sea, and some areas of the Spanish and French coasts [28]. *Ruppia cirrhosa* was transplanted into 12 stations. In the three decades previous to transplant activities, there was no sign of the presence of this species in the lagoon which is open to tidal expansion, whereas it was abundant in the closed fishing ponds [28,29]. The year after transplanting its spread was explosive, as it rapidly colonized all the choked shallow areas, while no rooting was observed in areas vivified by large canals.

*Cymodocea nodosa* was transplanted only in a station and as expected, its spread was very limited (stations 7 and 17) because of the ecological conditions of the transplant areas.

The establishment and growth of these aquatic angiosperms are strongly a ffected by the excess of nutrients in the water column, surface sediment, and SPM, especially by the phosphorus compounds (Table 1) as reported by [30]. High sediment moisture and porosity also counteract plant rooting because sediments are less compact, anoxic, and rich of organic nutrients. In addition, sediments characterized by high moisture usually contain a high amount of ammonium [31] and sulfides [32,33] that act as phytotoxins and hamper plant rooting.

Despite the short period considered, the responses of some indices used to assess the ecological status were positive. The macrophyte quality index (MaQI) and habitat fish biotic index (HFBI) only one year after transplanting showed the same positive correlations with the presence of aquatic plants as by Spearman's non-parametric coe fficients and PCA. On the contrary, M-AMBI did not respond positively to the presence of aquatic plants. In fact, the benthic macrofauna used for the index application lives inside the sediment, which requires longer time to change its physico-chemical characteristics than the water column. Therefore, we expect that benthic macrofauna will respond to the changes with a delay of a few years.

According to [34–36], pristine TWS should be colonized by aquatic angiosperms. These plants favor the maintenance of good ecological conditions. They control the change of water and sediments pH, favoring the development of calcareous algae, trap large amounts of CO2 [37,38], and prevent sediment erosion and dispersion, favoring sedimentation processes. In addition, angiosperm meadows are natural nurseries where the fish macrofauna finds food and shelter [39–41]. Finally, the small aquatic angiosperms like *Z. noltei* and *Ruppia* spp. that colonize the emerging seabed at low tide are also the best environment for birds such as ducks, flamingos, and herons that feed on both plants and the organisms typical of those areas. Therefore, the recovery of TWS through recolonization of environments with aquatic angiosperms is of fundamental importance. But, the success of transplants largely lies also on the cooperation of the population interested in safeguarding the environments they attend for work or leisure.

**Author Contributions:** Conceptualization, A.S., P.F., A.B. (Andrea Bonometto) and R.B.B.; Formal analysis, A.B. (Alessandro Buosi), Y.T., A.-S.J., A.A.S., L.S., E.P., F.R., D.B., C.G., F.O. and F.C.; Funding acquisition, A.S., A.B. (Andrea Bonometto) and R.B.B.; Investigation, A.S. and P.F.; Methodology, A.S. and A.B. (Andrea Bonometto); Project administration, A.S. and C.F.; Resources, A.S. and A.B. (Andrea Bonometto); Supervision, A.S.; Visualization, A.S. and A.B. (Andrea Bonometto); Writing—original draft, A.S.; Writing—review and editing, A.S., A.B. (Alessandro Buosi), Y.T., A.-S.J., C.F., A.A.S., P.F., L.S., A.B. (Andrea Bonometto), E.P., F.R., D.B., C.G., F.O., F.C. and R.B.B.

**Funding:** This research was funded by the European Union's LIFE+ financial instrument (grant LIFE12 NAT/IT/000331—LIFE SERESTO, which contributes to the environmental recovery of a Natura 2000 site, SIC IT3250031—Northern Venice Lagoon).

**Acknowledgments:** The authors thank Orietta Zucchetta for reviewing the English language and are grateful to the anonymous reviewers that have revised the paper for useful suggestions to improve the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
