Benthic communities: comparison among ecological indices
Concerning diatoms, the comparison between the two sampling campaigns is reported in Figure 4. From 2005 to 2020 we observed a slight increase in species richness and diversity, despite not being statistically relevant (Figure 4 a,b,c). Diatom indices also (Fig 4 d,e,f,g) depict a substantial unchanging situation, except for the Trophic Index TI, which is significantly higher in 2020 (Wilcoxon paired test V= 11.5, p<0.05). An increase in TI corresponds to a decrease in the environmental quality as this index increases along a gradient of human impact. The worsening in TI concerns, in particular, Valnontey, Orco, Noaschetta and Nampio.
Fig4
In both years, diatom communities were mainly composed of alcalobiontic and alcalophilous taxa, adapted to mean conductivity and very high oxygen saturation levels (99.5%). Concerning tolerance to organic matter and nutrients, both diatom communities were dominated by oligosaprobous taxa with a small proportion of mesosaprobous ones, which slightly increased in 2020; oligotrophic (more than 90%) and oligo-mesotrophic taxa. In terms of ecological guilds, diatom assemblages of 2005 were in mean dominated by low-profile species (76.5%), which are favored in nutrient-poor and high-disturbance habitats; 13.4% of the taxa belonged to the high-profile guild, developing in nutrient-rich sites, more lentic flows and low disturbance conditions; while only 1.9% was represented by motile species, mainly epipelic taxa favored by high nutrient content and able to move within periphyton and river bed toward suitable habitats. In comparison, samples from 2020 were characterized by a higher proportion of low-profile taxa (81.1%), which corresponded to a decrease in high-profile diatoms (10.3%). Motile species also increased, reaching 3%.
In both campaigns, the genus Achnanthidium  is by far the dominant one in all the sites, with the three species Achnanthidium lineareAchnanthidium pyrenaicum  and Achnanthidium minutissimum , very common in the Alpine rivers. Achnanthidium  species are low profile, resistant to current velocity thanks to physiological adaptation mechanisms which allow them to tightly adhere to the substrate. Some other interesting species reached consistent relative abundances, at least in some sites. This is the case of Fragilaria arcus , a rheophilic species, very sensitive to pollution and typical of low mineralized freshwaters, found in high percentages in Forzo and Orco in 2005, well distributed but with lower abundances in the 2020 campaign. Achnanthidium thienemannii  was found in Campiglia in 2020 with a 10.7% relative abundance. Epithemia goeppertiana,  a large motile species, usually epiphytic, was exclusively found in Nampio in 2020. Cymbella excisiformis , widely spread in headwater is the third species in Leviona in 2020, while Encyonema silesiacum,  a high-profile species typical of siliceous rocks, was abundant in Roc.
It is worth noting that many of the species sampled in the GPNP are included in the Red List of diatom species (Hofmann et al., 2018) as endangered at different levels. In this sense, the sites sheltering the highest number of endangered species were in 2005 Noaschetta and in 2020 Leviona and Vaudaletta. On average, 2005 samples hosted a lower abundance (39.5%) of individuals belonging to endangered species than 2020 ones (50.2%).
For macroinvertebrates we computed seven metrics, none of which showed a significant change between the two sampling years (Wilcoxon test p>0.05; boxplots in Figure 5). On average, macroinvertebrate community composition is characterized in both sampling campaigns by a relatively small number of well-adapted, orophilic and rheophilic taxa, with a mean number of taxa equal to 16, a minimum value of 9 in Savara (2005) and a maximum of 23 in Campiglia (2020). In all the sampling sites and for both years the stenoecious taxa account for at least 50% of the total taxa (average 64%, minimum 47% in Campiglia 2020 and maximum 87.5% in Gran Nomenon 2005). Indeed, these communities are mainly composed of orophilic and stenoxybiont organisms strictly adapted to cold and harsh environments, such as highly sensitive Diptera (namely Hapalotrix lugubris ) Ephemeroptera Heptageniidae (in particular, Epeorus alpicola ) and Plecoptera Systellognatha, such as Dictyogenus  alpinusPerlodes intricatus  and Siphonoperla montana .
No noticeable differences can be evidenced by comparing the two campaigns. For example, the occurrence of stenoecious Plecoptera remains constant, as in the case of Perlidae (58.3 % of stations on both occasions), Chloroperlidae (41.7 %) and Perlodidae (66.7 %in 2005, 58.3 in 2020) were found. The same pattern of occurrence is reported for Blephariceriidae, found in 75% of samples in 2005 and 83.3 % of samples in 2020.
We applied the Spearman correlation test to verify the association between HQA and biotic indices. HQA resulted as significantly and positively correlated with the Shannon diversity index of diatoms and macroinvertebrates (p<0.05) and with two macroinvertebrate indices: EPT and IBE (p<0.05 as well).