*3.5. Relationships between Prokaryotes, Meiofauna, Organic Matter, and Environmental Characteristics*

The results of the linear regression analyses indicate a significant and positive relationship between the BPC sedimentary contents and prokaryotic abundance (p < 0.05, R2 0.622; Figure 5A) and biomass (p < 0.01, R2 0.611; Figure 5B), as well as between the chlorophyll-a sedimentary contents and meiofaunal abundance (p < 0.05, R<sup>2</sup> 0.590; Figure 5C) and biomass (p < 0.05, R2 0.372; Figure 5D).

The results of the multivariate multiple regression analysis (DISTLM forward; Table 3), conducted on the composition of prokaryotic OTUs and meiofaunal assemblages, reveal that, when pooling together all data irrespective of season and station, the sub-set of variables that explained significant variations in the prokaryotic OTU composition explained a total of ca. 85% of variance and included all of the considered variables, with the exception of the protein to carbohydrate ratio.

**Figure 5.** Relationships between biopolymeric C contents and prokaryotic abundance (**A**) and biomass (**B**), as well as between chlorophyll-a and meiofauna abundance (**C**) and biomass (**D**) in the sediments.

**Table 3.** Results of the multivariate multiple regression analysis carried out to ascertain the effect of the quantity and nutritional quality of sedimentary organic matter on the prokaryotic OTU composition (**A**) and meiofaunal taxonomic composition irrespective of the season (**B**), in summer, (**C**) and in winter (**D**). % Variance = percentage of explained variance (SS = sum of squares; F = F statistic; P = probability level; \*\*\* = P < 0.001; \*\* = P < 0.01; \* = P < 0.05; ns = not significant).



**Table 3.** *Cont.*

The multivariate multiple regression analysis also reveals that, when pooling together all data irrespective of season and station, a total of 75% of variation in the meiofaunal community composition is significantly explained by the phaeopigment, salinity, chlorophyll-a to biopolymeric C, protein, carbohydrate, protein to carbohydrate ratio, chlorophyll-a, and temperature.

The DISTLM forward analysis carried out separately for the two seasons revealed that two different sub-sets of variables significantly explained the observed variations (ca. 91% and 90% in summer and winter, respectively). In summer, the most important variables explaining the observed variations in the meiofaunal taxonomic composition were chlorophyll-a and temperature, whereas in winter, they were phaeopigment and salinity.

#### **4. Discussion**

The rapid increase of human activities has substantially altered the biogeochemical cycles of carbon, nitrogen, and phosphorous, thus becoming, in the last decades, a major issue for most of the freshwater and coastal marine ecosystems worldwide [63,64].

For many years, the North Adriatic Sea has been the most productive region of the whole Mediterranean Sea, with high marine production at all trophic levels, from phytoplankton to fish. Nevertheless, during the last decades, this basin has experienced severe eutrophication, beside hypoxic/anoxic crises and mucilage spreads [47,65]. These events have caused the mass mortality of pelagic and benthic organisms, as well as a deep degradation of the benthic compartment [66]. In the last two decades, the combination of reduced nutrient loads (due to recent regulations limiting detergent use and to a continuously diminished runoff due to climate change [67]) has caused an overall trend of oligotrophication in the basin [68], although some authors recently highlighted that the continental loads of nutrients are still high [63,69].

According to previous studies [3,70], the sedimentary trophic status can be assessed through rankings based on the quantity, biochemical composition, and nutritional quality of organic matter, and their combinations. Such studies have proposed a marine benthic trophic status classification based on the sedimentary contents of protein, carbohydrate, and biopolymeric C and the algal fraction of biopolymeric C [3,70]. In this study, applying the classifications proposed above, all of the investigated sediments can be ranked as meso-eutrophic. Specifically, we observed that, during our study, either the marine or lagoon sediments can be ranked only partially as eutrophic. Our results, when compared with early studies carried out in the same area [3,9,16,33,38,71], pinpoint the decrease of the benthic trophic status, and confirm the "regime shift" of the Adriatic Sea towards progressively more oligotrophic conditions [68,72–74].

Extending the classification proposed by Dell'Anno et al. (2002) [70] and Pusceddu et al. (2009) [3] to the river sediments, the Po and its tributary sediments would be meso-eutrophic. Unfortunately, the lack of similar data from previous study periods in Po River sediments does not allow any inference on temporal changes in the benthic trophic status of the riverine station. Nevertheless, the overall BPC contents in the Po River sediments during our study were in the lowest range observed in other rivers, although in largely different ecological contexts and latitudes [75]. This would suggest that, most likely due to the increased use of inner freshwaters for human usage [76], the sediments of the Po

River could also have recently experienced a decrease in the benthic trophic status. However, most recent studies have demonstrated that excessive simplification of the landscape due to the removal of buffer strips and riparian wetlands has accelerated the nutrient transfer to water bodies [37,63].

The results of our study indicate that, in both seasons, the lagoon sediments (i.e., Gorino, Giralda, and Delta stations) were characterized by the highest sedimentary organic loads and OM nutritional quality. This result is in agreement with the general view of Mediterranean coastal lagoons as transitional water sites of high production [77], where the accumulation of organic matter turns these ecosystems into 'detritus traps' [16,78], and they are net heterotrophic [79,80].

Moreover, according to previous studies [16,20], both the lagoon and marine sites under scrutiny, showing the highest sedimentary BPC concentrations, were characterized by the highest values of prokaryotic abundance, biomass, and diversity. This result confirms the presence of an overwhelming positive effect of OM quantity and bioavailability on benthic prokaryotes [19], but it can also be interpreted on the basis of concurrent physical-chemical gradients along the investigated transect. Indeed, a strong difference in the prokaryote assemblage's composition was observed along the salinity gradient across the river-lagoon-sea transect and was accompanied by an increasing dominance of exclusive taxa in each of the investigated systems. This result corroborates previous investigations showing either an increasing presence of freshwater taxa at stations more influenced by the river discharge or a core microbiome present across all study areas [81]. Unfortunately, as our results do not allow us to provide information on the prokaryotic taxonomic identity (due to the limitations of the ARISA molecular technique, which does not allow DNA sequencing), we cannot make inferences about the specific ecological role of the different prokaryotic groups in each investigated system. Despite this, we observed the highest values of extracellular enzymatic activities in the lagoon sediments, where the highest prokaryotic biomass and diversity were also observed. These results let us hypothesize that lagoons not only behave as 'detritus traps' (sensu [78]), but also as OM degradation hot spots, such as deltas and estuaries, already identified as metabolic reactors for OM and nutrients [63]. The rates of enzymatic OM degradation have been repeatedly used as proxies of benthic ecosystem functioning [12,13]. We report here a clear coupling between OM degradation and prokaryotic and meiofaunal biomass and diversity, so we can infer that the investigated lagoon sediments are hotspots of ecosystem functioning, and that this, at least partially, is promoted by either prokaryotes [19] or meiofauna.

Our results also provided evidence of a concurrent positive relationship between sedimentary OM and meiofaunal abundance and biomass. Meiofauna are considered to be highly sensitive to environmental changes, so they can provide useful information about the benthic component response to ecosystem "regime shifts" in a variety of aquatic ecosystems [16,82–84]. Previous studies carried out in typically oligotrophic conditions (like the coastal W Mediterranean Sea) reported an evident decrease of the abundance, biomass, and richness of higher meiofaunal taxa exposed to high (excess) organic loads [82]. In contrast, our results, obtained in even more eutrophic conditions, show that the higher the organic loads, the higher the values of meiofaunal abundance and biomass. This result suggests that the apparent positive relationship between sedimentary OM and meiofaunal abundance and biomass observed across the river-lagoon-sea transect is more the result of a pattern of prokaryotic and meiofaunal communities specifically adapted to different habitats, rather than an actually 'positive' response to OM enrichment. Accordingly, despite no differences being observed in the richness of prokaryotic OTUs and meiofaunal higher taxa among stations or between seasons, the multivariate analysis revealed that different stations in the two seasons were characterized by very different prokaryotic and meiofaunal assemblages. In this regard, SIMPER analyses also revealed a high % of dissimilarity among systems (particularly between river and lagoon/sea systems) and seasons in each system. These results suggest that meiofaunal assemblages retrieved in the different investigated environments appear well-adapted to the specific trophic and environmental characteristics they face in each environment. Indeed, when the data are presence/absence transformed, different sets of taxa

are responsible for the observed dissimilarity (mostly rare taxa, i.e., accounting for <1% of the total assemblage, each, [16]).

In this regard, the results of the multiple multi-regression analysis (DISTLM forward) indicated that both the compositions of prokaryotic OTUs and meiofaunal assemblages were significantly affected by environmental settings (temperature and salinity, cumulatively explaining ca. 24% and 19% of the observed variance in prokaryote and meiofauna assemblages, respectively), the trophic resource quantity (cumulatively 46% and 43%, respectively), and the nutritional quality (cumulatively 16% and 13%, respectively).

## **5. Conclusions**

Our results allow us to reject the null hypothesis that the OM quantity, biochemical composition and degradation rates, prokaryotic and meiofaunal biodiversity, and ecosystem functioning do not vary among different ecosystems along a strong salinity gradient in different periods of the year.

Overall, the results of this study also allow us to conclude that the synoptic analysis of prokaryotes and meiofauna can provide useful information on the relative effects of organic enrichment and environmental settings across gradients of the environmental continuum. Our results also pinpoint that transitional water systems, including rivers, lagoons, and marine coastal ecosystems, represent a sort of end-of-pipe of the watershed continuum connecting the terrestrial and coastal domains, and acting as either a filter or source for nutrients and contaminants [64].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/12/5/189/s1, Table S1. Results of the one-way PERMANOVA testing for differences in the concentration of biochemical compounds and composition (A), descriptors of nutritional quality (B) of organic matter in the sediment between sampling stations and seasons. Table S2. Results of multivariate PERMANOVA and pair wise test on prokaryotic (A) and meiofaunal (B) abundance, biomass, richness of meiofaunal taxa and taxonomic composition in the sediment among stations and sampling seasons.

**Author Contributions:** Conceptualization, P.V. and A.P.; methodology, S.B., D.N., M.B., P.V., E.R., and A.P.; writing, S.B. and A.P., revision, S.B., D.N., M.B., P.V., E.R., and A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study has been carried out in the frame of the project "Origin, composition and fate of organic nitrogen loads in the NW Adriatic: from the Po River to the sea", funded by the Italian Ministry of University and Research under the PRIN 2008 call.

**Acknowledgments:** The authors are indebted to M. Lo Martire for the precious help during sampling activities.

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

#### **References**


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*Article*
