Trait‐based diatom functional diversity as an appropriate tool for understanding the effects of environmental changes in soda pans

Abstract Saline lakes, among the most seriously endangered ecosystems, are threatened due to climate change and human activities. One valuable feature of these environments is that they constitute areas of high biodiversity. Ecologists are, therefore, under great pressure to improve their understanding of the effects of natural and anthropogenic disturbances on the biodiversity of saline lakes. In this study, a total of 257 samples from 32 soda pans in Central Europe between 2006 and 2015 were examined. The effects of environmental variables and of geographical and limnoecological factors on functional diversity were analyzed. Furthermore, the explanatory power of the trait‐based approach was assessed, and the applicability of the indices for biomonitoring purposes was determined. It was found that low habitat heterogeneity and harsh environments lead to the selection of a small number of suitable traits, and consequently, to a naturally low level of functional diversity. Anthropogenic activities enhance diversity at functional level due to the shift toward freshwater characteristics. On the regional scale, the effects of the region and status (natural, degraded, reconstructed) on diatom functional diversity were significant and more pronounced than that of the environmental and other limnoecological factors. The degree of variance found in functional diversity ascribed to environmental variables is five times greater in the case of the application of a trait‐based approach, than when a taxonomic one is employed in the literature. Each of the tested functional diversity indices was sensitive to the most important environmental variables. Furthermore, these were type‐specific and proved to be more complex indicators than taxonomic metrics. It is possible to suggest four functional diversity indices (FGR, FRic, FDis, and FDiv) which emphasize their independence from substrate and seasonal variations for ecological status assessment and conservation planning.


| INTRODUC TI ON
In recent decades, biodiversity research has focused mostly on species richness and diversity metrics based on taxa as taxonomic units (e.g., Robinson, Rushforth, & Minshall, 1994;Tews et al., 2004).
These diversity metrics have been applied as common indicators of environmental impacts (He, Jiang, Tang, & Cai, 2015), in which the species correctly identified under the microscope have served as a basis for the analyses (Korponai et al., 2019). Nowadays, a new generation method, DNA metabarcoding, has established the conditions for the identification of operational taxonomic units (OTU) in many hundreds of samples simultaneously (Taberlet, Bonin, Zinger, & Coissac, 2018). This method seems likely to broaden our knowledge of biodiversity and with phylogenetic estimation of OTU ecological profiles it will move closer to functional biomonitoring (Keck, Vasselon, Rimet, Bouchez, & Kahlert, 2018).
Recently, trait-based approaches using functional trait units have drawn attention to the ecological and biological importance of the species (Schneider et al., 2017). In this sense, improved or more accurate predictions of ecosystem functions may be expected than were available using the taxonomic approach (Thompson, Davies, & Gonzalez, 2015). It was for this reason that the usefulness of this approach has been rapidly recognized and applied by ecologists. This recognition initiated an intensive search to discover the nature of the relationship between traits and habitat properties (Schneider et al., 2017) via the identification of the drivers of the diversity patterns. However, functional diversity metrics (He et al., 2015) have rarely been used recently, even though they promise to improve our knowledge of community and ecosystem responses to environmental changes at different scales (Péru & Dolédec, 2010). Furthermore, functional diversity can be a good indicator of ecosystem stability (Schneider et al., 2017) and can be strongly correlated with DNAbased phylogenetic diversity (Li et al., 2019) through ecological traits as phylogenetic signals (Keck, Rimet, Franc, & Bouchez, 2016;Keck et al., 2018;Winter, Devictor, & Schweiger, 2013). Consequently, functional diversity can play an effective role in conservation management using phylogenetic tools (Webb, Ackerly, McPeek, & Donoghue, 2002).
Functional approaches require simpler data than do traditional taxonomic approaches, and at first glance, this may appear to reduce ecological information. Nonetheless, this approach is capable of increasing the variance which can be explained in a community by the environmental variables . This is because of their sensitivity and consistent response to distinct ecological drivers (Tolonen, Leinonen, Marttila, Erkinaro, & Heino, 2017). Moreover, complementary functional diversity indices are available, which are capable of indicating different aspects of ecosystem functioning and environmental changes (e.g., Mouchet, Villéger, Mason, & Mouillot, 2010;Schmera, Erős, & Podani, 2009).
Trait-based approaches can provide an easier, faster, and more general understanding (Flynn, Mirotchnick, Jain, Palmer, & Naeem, 2011) of community organization than traditional taxonomical methods.
The application of functional traits and diversity indices as indicators of stressors of aquatic organisms is scarce (Ding et al., 2017).
Only a few studies connecting structural patterns to the primary production are to be found (Niyogi, Lewis, & McKnight, 2002;Rowe, Sánchez-España, Hallberg, & Johnson, 2007), and especially in the case of phytoplankton Török et al., 2016) and benthic algal communities (B.- Béres et al., 2019;Cibils, Principe, Márquez, Gari, & Albariño, 2015). However, diatoms are one of the most understudied groups of biota from this point of view (Alahuta et al., 2018), despite the possibility that diatom trait diversity (e.g., thickness of the valves, size, morphology or life strategies, and linking ability) may have a crucial role in environmental processes such as the ocean carbon pump (Tréguer et al., 2018).
Saline lakes are among the most vulnerable types of aquatic ecosystems due to the environmental threat generated by diverse human impacts (e.g., drainage and immoderate pumping of ground water) and climate change (Williams, 2002). The maintenance of the natural hydrological cycles and natural characteristics of these endorheic shallow lakes is key ecological and conservation tasks (Stenger-Kovács et al., 2014). In contrast to typical saline waters, which are often permanent and characterized mainly by chloride ions, astatic soda pans are mostly dominated by bicarbonate (Boros & Kolpakova, 2018) and are to be found across Africa, Europe, Asia, Australia, and America. The various aquatic communities (such as benthic and planktic algae, zooplankton and macroinvertebrates) of these ecosystems are exposed to extreme physical and chemical stress (strongly alkaline pans with high conductivity, nutrient concentration, turbidity, and diurnal temperature variation) (Boros, 2013;Stenger-Kovács et al., 2014), all of which may play a decisive role in selection of a given species  able to survive under such circumstances . This strong environmental filter causes a low degree of α-diversity in alkaline lakes, not only in the case of benthic communities (Stenger-Kovács, Hajnal, Lengyel, Buczkó, & Padisák, 2016), but also in planktic communities (Nkambo et al., 2015;Vidaković et al., 2019;Vignatti, Paggi, Cabrera, & Echaniz, 2012). However, the degree of β-diversity found in these communities, primarily determined by the environmental variables, is high due to species turnover (Szabó, Lengyel, Padisák, Vass, & Stenger-Kovács, 2018); this is true even in sodic anthropogenic, bomb crater ponds (Vad et al., 2017). The conservation of saline lakes is essential if the loss of biodiversity and the disappearance of these unique habitats are to be limited (Williams, 2002).
The main aim of this study was to assess the effects of environmental variables (conductivity, pH, dissolved oxygen, temperature, nutrients [P and N forms], HCO 3 -, CO 3 2-, SO 4 2-, Cl -) and compare these with the individual effect of the geographical (regions) and limnoecological factors (watercolor, substrate, status, hydrological phase, and season) on benthic diatom diversity patterns in soda pans. In order to achieve this, a functional, trait-based approach has been adopted. In this way, the applicability of functional diversity as an element of ecological status assessment and conservation planning is evaluated, along with the degree to which factors such as adequate sampling time and substrate selection can modify the final results of a status assessment. Two hypotheses were adopted: (a) functional diversity will be an effective indicator of the most characteristic environmental variables, and consequently, of the ecological/conservational status of soda pans, and (b) the individual effects of spatial as well as limnoecological factors on diatom functional diversity will be less pronounced than that of extreme environmental constraints.

| Sample collection and background variables
A total of 257 diatom and water samples were collected from 32 soda pans (Table 1)  Degraded pans (n = 6) were excluded from the analyses of the status effect because of their underrepresentation.
The choice of substrate (mud/macrophyte) and sampling sites followed the recommendations of King, Clarke, Bennion, Kelly, and Yallop (2006). Samples were taken at a water depth of 5-10 cm close to the shorelines of the pans. Diatoms were collected from the macrophytes using toothbrush and were collected from mud by pipetting ~10 cm 3 of the superficial layer of the pan sediments (Cochero, Romaní, & Gómez, 2013).

| Laboratory analyses
Diatom samples were preserved in ethanol and were kept at pH ~7-8 by cc. HCl, thereby avoiding the dissolution of the silica walls.
A hot hydrogen peroxide treatment was applied to oxidize the protoplasms (CEN, 2003

| RE SULTS
Using the six functional diversity metrics, variation partitioning showed that environmental variables, region, watercolor, and ecological status had considerable and significant explanatory power with regard to the variations in functional diversity (Figure 2a,b,d). Only one index, functional evenness, was not sensitive to variation by region (Figure 3).
In the case of the watercolor, the responses of the indices were more varied (Figure 4). The values of RaoQ and FDis differed significantly depending on watercolor. RaoQ and FDis had the lowest values in colored soda pans, while in the transitional pans, they had the highest. FDiv was significantly lower both in the colored and turbid pans.
FRic was lower in colored waters and showed no significant variation in value between the transitional and turbid ones. In the values of FEve and FGR, no significant differences were observed (Figure 4).
Five indices (FDiv, FDis, RaoQ, FGR, and FDis) differed significantly between natural and reconstructed areas, with lower diversity values indicating the natural status of the soda pans ( Figure 5).
The FEve values were similar in soda pans with different statuses ( Figure 5).
The individual indices were not sensitive to the seasons, except for FEve, which was significantly different in summer and winter ( Figure 6).
Significant effects of the environmental variables on the functional diversity indices were found in the course of the RDA analysis ( Figure 7). On the first axis, 89% of the total constrained variance of the functional diversity indices was explained by the environmental variables. DIN, temperature, pH, conductivity, and DO were the main constraints that determined functional diversity to a great extent. After the reduction of the full models containing ten environmental variables, FRic was determined by eight, FDiv, RaoQ, and FGR by seven, and FDis was determined by six variables (  indicating the harsh (high conductivity and turbidity, temporary drying out) environment . The motility allows the species to change their position to find the "best place" under these unfavorable conditions. The small cell size and this elongated shape further facilitate their movement among the inorganic sediment particles and their ability to hide in the mud. Small size has also been highlighted in planktic communities (Alfonso, Zunino, & Piccolo, 2017;Somogyi et al., 2014) as well as the motile feature (Földi et al., 2018) in other saline lake ecosystems, where species reduce their cell and pore size due to the osmotic stress (Leterme et al., 2010).

| D ISCUSS I ON
Functional diversity metrics displayed significantly lower values in natural soda pans, indicating their pristine features. The diversity values of the degraded pans did not differ either from the natural ones F I G U R E 4 Results of the Kruskal-Wallis test of the six applied functional diversity indices for different watercolor types (groups with the same letters are not distinct, whereas groups with different letters differ significantly) or from those of reconstructed pans, which had significantly higher functional diversity than natural lakes. Disturbed hydrological cycles (e.g., by water abstraction or resupply) can modify limnological variables (e.g., lower conductivity and pH) , potentially leading to less extreme features characteristic of fresh water, and therefore resulting in higher diversity. This result calls attention to anthropogenic activities (Alfonso et al., 2017), including even those undertaken for conservation purposes, which have considerable impacts on biodiversity both on the taxonomic (Heino, 2005;Stenger-Kovács et al., 2016) and at the functional levels.
As forest shading of streams reduces functional diversity intensity, high levels of humic materials can modify the spectral composition of the incoming light (Kirk, 1994;V.-Balogh, Németh, & Vörös, 2009). In contrast to other aquatic ecosystems where the light intensity is high and different growth forms can coexist (Passy & Larson, 2011), here only those species with adequate traits can survive, and this results in a low degree of functional diversity.
One possible adaptation strategy, besides the chromatic adaptation of algae, might be size as a key trait, since the surface area of small cells is relatively large in proportion to their volume/size, an advantage in the competition for light (Somogyi & Vörös, 2004).

F I G U R E 6
Results of the Kruskal-Wallis test of the six applied functional diversity indices for soda pans in different seasons (groups with the same letters are not distinct, whereas groups with different letters differ significantly) In these ecosystems, seasons had a less pronounced effect, and the related hydrological cycle, as well as the substrate type, had no significant effect on functional diversity, in contrast to the case of freshwater, where high water periods support the appearance of a number of periphytic algal species with different traits, thus resulting in a high degree of functional diversity (Dunck, Algarte, Cianciaruso, & Rodrigues, 2016;Dunck, Rodrigues, & Bicudo, 2015).
At the taxonomic level, seasonal effects  can also be detected in the benthic diatom, as well as in the planktic communities of saline lakes (Alfonso et al., 2017). However, microhabitat preference (such as substrate type) is negligible at taxonomic levels as a consequence of the extreme environmental conditions (Cejudo-Figueiras, Álvarez-Blanco, Bécares, & Blanco, 2011;Lengyel et al., 2016). This result further emphasizes the primarily role of local factors (Bichoff et al., 2018) and of the strong environmental filters on the structure and function of the communities, (Ding et al., 2017;Soininen, 2012) even in saline ecosystems .
Of the environmental variables, DIN, temperature, pH, conductivity, and DO were the main determinants of the functional diversity metrics as revealed by the RDA analyses. This stands in contrast to the taxonomic diatom assemblages that were chiefly determined by conductivity, bicarbonate, and sulfate concentration (Stenger-Kovács et al., 2014) in natural ponds, while, salinity, pH, and turbidity dominated in artificial saline ones (Földi et al., 2018). All specific functional diversity indices were sensitive to the most important environmental variables of soda pans-conductivity and pH-as has also been found in subarctic ponds (Teittinen et al., 2018  Abbreviations: temp, temperature; cond, conductivity; DO, dissolved oxygen; TP, total phosphorus; DIN, dissolved inorganic nitrogen. *p < .05; **p < 0. 01; ***p < .001. distinctiveness) , the functional diversity indices were more complex indicators since they integrated the effects of more environmental variables (from three to five, instead of two or three), and this plays a crucial role in the indication of environmental changes. On the basis of the strong correlation between these key variables, they are very effective and informative metrics (for macroinvertebrates, see He et al., 2015). Of the functional diversity metrics studied, FGR, FRic, FDis, and FDiv proved to be the most useful for assessing the ecological status and conservation value of soda pans. FEve was not related to changes in the environment, as has been shown in the case of diatoms in tropical headwater streams (Taniwaki et al., 2019), and over the long term by phytoplankton communities in a large river .

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interests to declare.