**3. Results**

Figure 3a shows per station, the average diameter of the patches formed by sods and rhizomes 12 months after transplants. The growth was very variable due to the different environmental conditions of the stations. The maximum average diameter of the patches was 143 cm at station 14. Patches over 100 cm in diameter were also found in six other stations (9, 11, 12, 15, 18, 33). In three stations, the sods disappeared completely despite frequent new transplants. In the other stations, the diameter increase was intermediate.

**Figure 3.** (**a**) Average diameters of the patches. Vertical bars represent the diameter of the transplanted sods; (**b**) Total surface of patches, formed by the growth of sods and rhizomes at the 35 sampling sites after 12 months. Only patches with a diameter >20 cm were considered.

The total surface of the patches is shown in Figure 3b. In most stations, there was no correspondence between the diameter and the surface of the patches. The patch surface, in fact, after one year did not distinguish between the sods and rhizomes that grew mixed. In some cases, the total patch surface was the result of a large quantity of patches, in other cases its size depended only on the number of those that had survived. Therefore, results were very different. The maximum growth occurred at station 18 with a colonization of 52% (52 m<sup>2</sup> out of 100 m2) and a patch diameter that was among the biggest (133 cm). In contrast, at station 14, just less than 1 km away, the coverage was only 16.4%, even though the diameter of the patches was bigger (143 cm). No angiosperm rooting was observed at stations 1, 5, 6, and 16. Stations 1, 5, and 6 were affected by river outflows, whereas station 16 showed a higher trophic level than the other stations, which was probably due to the proximity of the Cavallino urban center.

The main metrics of sods and rhizomes one year after transplants are shown in Figure 4. The number of survived sods was, on average, 72 ± 38% with high differences between the stations. All the transplanted sods took root in 20 stations out of 35. No angiosperm rooting was found in four stations (1, 5, 6, and 16), whereas in the other stations, their rooting varied widely. On average, rhizome rooting was lower (37 ± 25%) than that of sods, but the number of single rhizome transplants per station was much higher (637) than the number of sods, and so was their potential for developing new rooting.

**Figure 4.** Percentage, patch diameter, and daily growth of survived sods and rhizomes.

The average diameter of the patches formed by the surviving sods was 68 ± 44 cm, whereas rhizomes exhibited, on average, a diameter of 40 ± 23 cm, a value very similar to that of sods if we take into account that the diameter of transplanted sods was 30 cm. Moreover, some rhizomes took root also at stations 6 and 16, whereas at station 23 some survived rhizomes formed patches with a diameter shorter than 20 cm. The stations 14 and 18 produced the largest patches.

By comparing growth, rhizomes showed values higher than sods: 0.19 ± 0.13 cm day-<sup>1</sup> vs.0.14 ± 0.12 cm day−1. It is interesting to observe that the rhizomes transplanted at station 14 showed the highest growth with 0.68 cm day−<sup>1</sup> and formed patches of approximately 1 m in diameter.

The analysis of nutrient concentrations in the samples of surface sediment collected in all 35 stations during the spring transplants shows that the area is divided into two parts: the one situated northwest of the San Felice canal is characterized by high nutrient concentrations due to river outflows; the other which is on the southeastern side of the canal is characterized by a higher seawater renewal and no river outflow (Figure 5).

**Figure 5.** Concentration of organic phosphorus, total nitrogen, and organic carbon in surface sediments of the transplant area. Black dots represent transplant stations.

Stations 5 and 6 were the most affected by high nutrient concentrations that favored the growth of tionitrophilic macroalgae and phytoplankton blooms which prevent the rooting of aquatic angiosperms. In the northwestern area of the San Felice canal, the concentration of organic phosphorus (OP), total nitrogen (TN), and organic carbon (OC) were one order of magnitude higher than in the southeastern area.

Similarly, the analysis of the mean concentrations of nutrients in the water column of the eight stations selected for monthly monitoring shows that reactive phosphorus (RP), dissolved inorganic nitrogen (DIN), and silicates (Si) were significantly higher at stations 5 and 1 than in the others (Figure 6).

**Figure 6.** Concentration of reactive phosphorus, dissolved inorganic nitrogen, and silicates in the eight stations monitored monthly during one year.

On average, RP showed a concentration of 0.17 ± 0.6 μM with the highest value at station 5 (0.32 μM) near the mouth of the Siloncello river. The same results were found for DIN that averagely was 14.3 ± 8.8 μM, but at station 5, it was approximately twice as high (27.8 μM). Silicates were slightly higher at station 1 (17.0 μM) and the mean value was 10.9 ± 3.8 μM.

Salinity confirms the influence of the Siloncello freshwaters. Stations 5 and 1 exhibited values of approximately 21.0 and 22.4 psu, respectively, against 26.8 to 29.2 recorded in the others (Figure 7). Total chlorophyll-*a* (Chl*-a*) also showed the highest value (approximately 1.3 μg <sup>L</sup>−1), although, on average, it was very low (0.94 ± 0.29 μg <sup>L</sup>−1). The particulate collected by sedimentation traps (SPM) highlighted the environmental differences between the stations more clearly than Chl*-a*, because the sedimentation rates were collected and recorded constantly during the whole sampling year. Also, this parameter showed the highest value at station 5 (635 g dw m<sup>−</sup><sup>2</sup> <sup>d</sup>−1) accounting for approximately 232 kg dw m<sup>−</sup><sup>2</sup> y<sup>−</sup>1. On the contrary, extremely low values were found at stations 8 and 17, where aquatic angiosperms had widely taken root.

**Figure 7.** Values of salinity, total chlorophyll-*a* (Chl-*a* tot), and settled particulate matter (SPM) collected by sedimentation traps).

Two other important parameters that marked the differences between the stations were the percentage of sediment fraction <63 μm (fines) and the amount of dry sediment per volume unit (dry density) of surface sediments. Grain-size was >70% in all the stations (Figure 8), but with a different degree of compactness. Dry density values were overall very low (0.43 ± 0.14 g dw cm<sup>−</sup>3), but the lowest were recorded at station 5 (0.16 g dw cm<sup>−</sup>3) and station 1 (0.32 g dw cm<sup>−</sup>3), where aquatic angiosperms had not taken root.

**Figure 8.** Percentage of fines (fraction < 63 μm) and dry density of surface sediments.

The non-parametric Spearman's coefficients calculated using 38 physico-chemical parameters, seven macrophyte variables, and three indices of ecological status in the eight stations showed that freshwaters coming from the river Siloncello affected the salinity of the water column seriously, deteriorating the ecological conditions and hindering the angiosperm rooting (Table 1). In fact, sod diameter (cm), sod surface (m2), and sod growth (cm day−1) were positively correlated to salinity, whereas rhizome survival (% yr<sup>−</sup>1) was positively correlated to light availability at the bottom (Light-B), and inversely to SPM and the concentration of organic phosphorus (OP) in surface sediments.

Moreover, rhizome growth was counteracted by the concentrations of Si, RP, and ammonium (NH4+) in the water column by TP and OP in surface sediments and SPM. Macroalgal cover (M-cover) was also inversely and significantly correlated to angiosperm growth. The accumulation of tionitrophilic macroalgae, especially Ulvaceae and Gracilariaceae, on sod and rhizome transplants prevented their growth. These macroalgae have a much higher growth rate [18] than angiosperms [19]. The same inverse correlations were also found between sod growth and TP, OP in surface sediments, and OP in SPM. The indices of ecological status: MaQI, M-AMBI, and HFBI showed that the correlations with the physico-chemical parameters in the water column had a similar trend, whereas in surface sediments the correlations were not always clear, especially when M-AMBI was applied.

The principal component analysis (PCA) applied to the whole set of parameters and variables, after the exclusion of redundancies, confirmed the above relationships (Figure 9). About 66% of the total variance was explained by the first two components that clearly separate the variables of the aquatic angiosperms and most of the environmental parameters into two major groups. In the two minor groups, the variables of the first two components were intermediate. In group 1, both sod and rhizome rooting were associated with salinity, sediment density, sediment total carbon, sediment and water pH, and light availability at the bottom. The ecological quality ratio (EQR) of the indices of ecological status of the biological elements, macrophytes (MaQI) and fish fauna (HFBI), were also associated to aquatic angiosperm variables and the same environmental parameters.

In group 2, most of the nutrient concentrations in the water column, surface sediments, and SPM were plotted together with sediment moisture and porosity, water temperature, the %DO, Chl*-a*, FPM, and M-AMBI. Groups 3 and 4 highlighted some parameters which were intermediate between groups 1 and 2.

**Figure 9.** Principal component analysis (PCA) between physico-chemical parameters and macrophyte variables. Legend: Rhiz = rhizomes, Surv = survived, Diam = diameter, surf = surface, Temp = water temperature, Transp = water transparency, %DO = percentage of dissolved oxygen, w = water, sed = sediment, dens = density; moist = moisture, Light-S = light at a depth of −5 cm, Light-B = light at bottom, FPM = filtered particulate matter, Si = silicates, RP = reactive phosphorus, NH4<sup>+</sup> = ammonium, NO2− = nitrites, NO3<sup>−</sup>, = nitrates, DIN = dissolved inorganic nitrogen, M-Biom = macroalgal biomass, M-cov = macroalgal cover, Chl*-a* T = total chlorophyll-*<sup>a</sup>*, TP = total phosphorus, OP = organic phosphorus, TN = total nitrogen, TC = total carbon, OC = organic carbon, SPM = settled particulate matter, part = particulate.
