*2.1. Study Area*

The Venice Lagoon is a shallow water body of about 550 km2, located in the north-eastern part of Italy (Figure 1). It has an average depth of approximately 1.1 m, and it is characterized by a variety of aquatic and terrestrial habitats, such as canals, shoals, salt-marshes and islands [16]. According to the WFD, the Venice Lagoon is a large coastal microtidal lagoon, divided into 11 natural WBs and 3 heavily modified WBs [10]. In 2011, during the first cycle of the WFD Operational Monitoring, 114 stations, spread within the 11 natural WBs of the Venice Lagoon, were sampled to assess the ecological status

by macrophyte assemblages (Figure 1). The dataset analyzed during the current study is available online at Environmental prevention and protection agency of Veneto Region (ARPAV) web portal [17]. For the classification of the BQE macrophytes, the MaQI [9] was used.

**Figure 1.** The Venice Lagoon map with its morphological and hydrological characteristics, and the WFD (Water Framework Directive) MaQI (Macrophyte Quality Index [8]) classification in 2011. Names of water bodies (WBs) are indicated in uppercase bold characters; place names are in lowercase. Final assessment of each WB is labeled like for stations: blue for high, green for good, yellow for moderate, orange for poor, red for bad status classes.

### *2.2. Reliability of Classification at Water Body Scale*

The classification of the macrophyte assemblage at WB scale was obtained by averaging the MaQI EQRs of all stations within each WB (j).

First, the confidence interval (*L*) was also calculated for each WB (j) by the following formula:

$$L\_j = t\_{\mathcal{N}\_j - 1, \mathbf{s}' \mathbf{t}} \frac{\mathcal{S}\_j}{\sqrt{\mathcal{N}\_j}} \tag{1}$$

where *tNj*−1,α/2 is the critical value of the *t*-student distribution for the confidence level 1−<sup>α</sup> (two tiled distribution); *Sj* is the standard deviation of the EQRs within each WB; *Nj* is the number of stations within each WB. In this study, the confidence intervals were stated at the 95% confidence level (α = 0.05).

The *L* value provides an overall view of the confidence of the mean EQRs, but it does not take into account the closeness of the face value to the class boundaries, being independent from the WFD classification system. Therefore, the reliability of the classification was assessed in terms of probabilities that the observed ecological status classification (sample mean) lies within the right class. The cumulative probability that the actual (population) mean value of MaQI fell in each status class was assessed by *t*-student distribution. Data normality was tested by the Kolmogorov–Smirnov test whilst the absence of autocorrelations was verified by variogram analyses and Moran's test of autocorrelation. Classes were identified by the WFD boundaries reported by the Italian Ministry decree 260/2010 (high/good = 0.8, good/moderate = 0.6, moderate/poor = 0.4, poor/bad = 0.2). The confidences related to the critical boundary good/moderate were also calculated by the sum of probability of classes being lower/higher than good.

Statistics were carried out using the R software [18].

### *2.3. Optimization of Sampling E*ff*ort*
