1. Introduction
Phytobenthos and macrophytes are primary producers and, thus, at the base of the food web in rivers. Phytobenthos is a crucial element of the periphytic community that colonises different submerged substrates in the photic zone of the majority of aquatic ecosystems, including rivers. The diversity of the phytobenthos community is directly related to the heterogeneity of the habitats in the water body [
1,
2]. Their occurrence and abundance also depend on other factors, such as the availability of light and nutrients, water temperature, changes in water levels, and water velocity [
3,
4]. Human activity can significantly alter these parameters, which can consequently affect the structure and function of phytobenthic communities [
5]. After disturbance, the colonization of the substrate with diatoms, which are important elements of phytobenthos, occurs relatively quickly, while the complex community develops only over a longer period [
6,
7]. Phytobenthos has multiple functions. It stabilises the substrate and presents a habitat for other organisms. In many rivers, the contribution of organic carbon by phytobenthos photosynthesis is considerable. The short life cycle of phytobenthos enables a quick response to environmental changes; therefore, phytobenthos is a good indicator for assessing the ecological status of surface water bodies [
8,
9,
10,
11,
12].
Macrophytes also present an important element of an aquatic ecosystem community. The presence and abundance of macrophytes in running waters depend primarily on the water quality, flow velocity, and substrate characteristics [
13,
14]. Moreover, they are involved in the energy flow, nutrient cycling, and sedimentation processes. When present in abundance, they affect ecosystems significantly [
15]. They provide habitats, food, breeding locations, and refugia for aquatic invertebrates, fish, and a range of other organisms [
16,
17,
18]. The degradation of macrophyte communities results in a degradation of the communities of dwelling organisms [
19]. Macrophytes affect the water quality, and concurrently, they reflect the conditions in the water, in the riverbed, and in the interstitial water and sediment [
20], which makes them valuable indicators of the water and sediment quality [
21,
22,
23]. Macrophytes absorb nutrients through the root system from the sediment [
24,
25,
26] and from the water column throughout their surface [
27]. The proportion of uptake by the subterranean or aboveground parts of the plant depends on the specific adaptations of each species [
28,
29,
30]. The knowledge about the structure of the macrophyte community is of great importance in assessing the ecological status of rivers [
31].
Macrophytes and phytobenthos respond differently to nutrients and other stressors, since phytobenthos is more sensitive to nutrient enrichment while macrophytes better reflect the secondary effects of this enrichment [
32]. However, the presence and abundance of representatives of the two groups reflect the trophic condition of the river, which is a measure of the autotrophic and heterotrophic bioproduction rates in the water body. The trophic status is one of the most important indicators of water quality [
33,
34,
35]. Different trophic statuses are used as a stressor gradient in the development of indices for the assessment of the ecological status of waters in accordance with the Water Framework Directive [
31,
36,
37]. Different studies have suggested that phosphorus and nitrogen are the most important nutrients regulating the trophic status in rivers [
38]; however, the results are not consistent. Francoeur [
39], who performed 237 nutrient enrichments on benthic algae in streams, found out that the algae in 16.5% of cases positively responded to N, 18.1% to P, 23.2% required N and P, 5% showed N or P inhibition, and 43% had no response to N or P.
For the evaluation of the ecological status of rivers in Slovenia on the basis of the biological element phytobenthos and macrophytes, we used the River Macrophytes Index (RMI), developed in 2007 [
22,
40] and supplemented in 2012, for intercalibration purposes at the European Union level [
41] and the phytobenthic diatoms-based Trophic Index (TI) and Saprobic Index (SI), adapted for the ecological status assessment of Slovenian rivers from 2006 to 2008 [
42,
43] and supplemented in 2012 for intercalibration purposes at the European Union level [
44]. Based on the RMI index, we primarily aimed to evaluate the trophic status and partly the general degradation of the river [
22,
40], as was the case in most of the methods evaluating the ecological status [
32]. The phytobenthic diatoms-based TI index presumably reflects nutrients in the water (trophic state), whereas the SI index reflects the watercourse load with dissolved organic matter (saprobic state) [
44,
45,
46,
47]. Phytobenthos and macrophytes communities may respond differently to trophic conditions in the river. The response of macrophytes is relatively slow, while the phytobenthos respond quickly to changes of the trophic status. Therefore, both communities might reflect different aspects of the trophic status of the river ecosystem. However, rarely has it been tested whether the trophic stressor gradient length impacts the relationship between environmental variables and phytobenthos and macrophyte assemblage-based indices. The relations between trophic indices and nutrients are usually made on the basis of a few annual measurements of the nutrients, while the nutrient content in the water can vary greatly during the year [
48]. The empirical research revealed relatively strong correlations between the nutrients and trophic indices based on phytobenthos; however, the correlations between nutrients and trophic indices based on macrophytes are rather weak [
20,
44,
46,
47]. This is possibly related to the length of the life cycle and growth form of both groups of primary producers. For macrophytes with a long life cycle and complex growth forms, the instantaneous chemical status of water could not be directly translated to the water quality during the macrophyte vegetation season. In addition, macrophytes are a very variable group regarding their growth forms, which also affects their response to various environmental factors [
49,
50,
51], including water chemistry [
52,
53].
The aims of this study were (i) to examine the relationships between the nutrient variables and values of the River Macrophyte Index (RMI) and Trophic index (TI) using varied stressor gradient lengths and (ii) to explain the variability of the macrophyte and diatom communities with different stressors, namely nutrients and land cover variables and their combinations. We hypothesized (i) that TI will be related to nutrient concentrations in the water, while this will not be the case for RMI, (ii) that the relationships between stressor gradients and biotic indices will be affected by the length of the considered stressor gradients, and (iii) that a composite stressor gradient will explain a similar share of the variability of the macrophyte and phytobenthic communities.
4. Discussion
Nutrients are crucial for river biocenoses, supporting primary production, on one hand, while, on the other hand, their high concentrations compromise species relations within the community [
65] and, thus, ecological status of the rivers. The response of macrophyte communities to nutrients differed significantly from that of diatom communities and so did the indices based on these two groups of organisms. This is possibly a consequence of the sampling time, since the macrophytes data used in our study were obtained at the peak growing season. The majority of studies relate to nutrients with macrophyte abundance only; however, we took into account species presence and abundance. For example, the study of O’Hare [
66] revealed the increase of macrophyte biomass with P availability as a soluble reactive phosphorus and total phosphorus in water. The study of D’Aiuto [
67] showed a significant correlation between stream nutrient enrichment (total phosphorus, soluble reactive phosphorus, and nitrate nitrogen) and macrophyte production. On the contrary, with epiphytic algae that obtains nutrients from water, an important aspect defining the response of the majority of macrophyte species to nutrients is also root uptake from the sediment. Carr [
26] showed that, in streams, the macrophyte abundance increased linearly with increasing the sediment P concentration in water, while the study of Mebane [
68] revealed that both the nitrogen contents in sediment and water were positively correlated to a macrophyte biomass. Thus, the response of the macrophyte community largely reflects the trophic conditions during their entire growth cycle and on the long-term accumulation of nutrients in the sediment, as shown by Clarke [
28]. The absence of significant relationships of the RMI with the majority of the nutrient parameters is also a consequence of specific traits of species and communities, namely species life cycle, their growth dynamics, and their modes of nutrient uptake [
69,
70,
71]. In the case of perennials with different underground storage organs, the impact of nutrients can be even more delayed and observed only over a long period [
72]. An additional reason for the lack of correlation between the RMI with total phosphorus in our study could be the consequence of the relatively high total phosphorus concentrations in Slovenian watercourses, since, in our dataset, only 11% of the samples contained concentrations below 0.05 mg/L. The research of Fabris [
73] applying the macrophyte Trophic Index (TIM) as a measure of the macrophyte community status revealed significant changes at relatively low concentrations of total phosphorus (up to 0.05 mg/L).
For the TI, we determined weak-to-moderate statistically significant correlations for the majority of the studied nutrient parameters. The significance of the correlations for the TI was expected due to the expansion mode of phytobenthos growth [
11,
74,
75] and their quick response to nutrients [
39].
The strength of the relationships between the RMI and TI and nutrients strongly depended on the length of the stressor gradient. The findings based on shorter stressor gradients with lower values (up to 0.3 mg/L of the TP mean) differed significantly from those based on longer ones. These inconsistent responses of the RMI relationships to nutrients may be a consequence of macrophytes that can function as a sink and a source of nutrients [
76,
77]. It was shown that the bulk of nutrients can be released from macrophyte beds during decay processes after disturbances and at the end of the vegetation period [
78]. Besides that, macrophytes can also affect the nutrient balance by trapping fine organic and inorganic particles, enhancing the mineralization of organic matter through oxidation of the sediments, and altering the local environment, thus enabling P release due to the reducing conditions [
79]. For the TI, statistically significant relationships with an annual mean total phosphorous value were obtained at gradient lengths ≥ 0.3 mg/L of the TP mean. For the variable maximum value of ammonium, the strength of the relationship also varied with the length of the gradient in the case of the RMI, but it showed an increase with the length in the case of the TI. Although some studies indicate a strong correlation between the stressor gradient and biotic indices/communities [
80], the weak correlations obtained in our study were not against our expectations [
61]. Weak correlations between the groups of stressor gradients (e.g., eutrophication) or individual stressor gradient (e.g., TP stressor gradient) and biotic indices confirmed that it is difficult to distinguish among the effects of individual stressor gradients or a group of stressor gradients on biotic communities in a multiple-stressor ecosystem. Moreover, different responses of biotic communities to the same stressor gradients are also possible [
81]. Even in a case when we primarily assess the individual stressor gradient, there are always other events or influences that could change the conditions. For instance, removing riparian vegetation, which is a hydro-morphological element, affects the water temperature, nutrient cycling, and consequently, the nutrient uptake and trophic conditions for biotic communities [
82,
83]. In addition, different environmental factors may act at multiple scales [
84].
The explained variability of macrophytic and phytobenthic assemblage structures by different stressor gradient groups was similar for macrophytes and phytobenthic diatoms. The greatest share of variability was explained by the land use and nutrients (LandNut) stressor group and the least by the phosphorus variables and nitrogen variables. Thus, the unexplained portion of variability was still prevalent. Different studies revealed that, even with indices that focus on individual stressors (e.g., trophic status), authors rarely explain >50% of the variability of an index [
85]. However, in all these cases, one does not evaluate only the trophic status but also the general degradation of the riverine ecosystem. To comprise these complex effects, we built a composite gradient using a PCA. Along the first and second axes of the PCA plot, we obtained different land cover gradients. The relationship with the land cover variables was expected, since many studies showed that the land cover presented a key factor affecting the aquatic community [
15,
22,
86]. The first axis represented mainly a gradient of intensive agriculture, while the second axis a gradient of natural areas and urban areas. The gradient vectors of natural areas and urban areas pointed in opposite directions. The relationships between the RMI EQR values, TI EQR values, and the PCA1 and PCA2 varied and were dependent on the gradient lengths. In the case of the RMI EQR values, stronger relationships were observed for shorter gradients of lower stressor values and decreased as the gradient extended towards higher values. We obtained a significant relationship with PCA1 at low mean annual values of the total phosphorus, which was related to intensive agriculture in the subcatchment. With the TI EQR values, no significant relationships were determined. This may be due to the long-term response of macrophytes to their habitat conditions, including those related to land use changes, which may result in continuous sediment accumulation and nutrient input [
87], while phytobenthos mainly responded to short-term changes of the nutrients in the water [
1,
8].
The observed similarities in the explained variability of the macrophytic and phytobenthic assemblage structures by different stressor gradient groups (nutrients and land use) and differences in the indices performances might indicate the individual assessment index limitations. Each index was developed with a specific dataset and tested against selected stressor gradient data. Usually, the stressor gradient applied in studies is for necessity, a simplification of the true stressor gradient to which biota are exposed. Despite all the limitations, biological assemblages offer more stable information about the ecological status of a given ecosystem than abiotic variables, although we cannot always perfectly relate them. Macrophytes and phytobenthos assemblages reflect varied faces of the same river ecosystems.