Journal Pre-proof

Multiple stressor effects on alpha, beta and zeta diversity of

riverine fish

Tibor Erős, István Czeglédi, Rita Tóth, Dénes Schmera

PII: S0048-9697(20)34936-6
DOI: https://doi.org/10.1016/j.scitotenv.2020.141407
Reference: STOTEN 141407

To appear in: Science of the Total Environment

Received date: 15 April 2020

Revised date: 20 July 2020
Accepted date: 30 July 2020

Please cite this article as: T. Erős, I. Czeglédi, R. Tóth, et al., Multiple stressor effects on
alpha, beta and zeta diversity of riverine fish, Science of the Total Environment (2018),
https://doi.org/10.1016/j.scitotenv.2020.141407

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© 2018 Published by Elsevier.

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Multiple stressor effects on alpha, beta and zeta diversity of riverine fish

Tibor Erős1,2,*, István Czeglédi1, Rita Tóth3, and Dénes Schmera1,2

1
Centre for Ecological Research, Balaton Limnological Institute, Klebelsberg K. u. 3, H-8237

Tihany, Hungary
2
Centre for Ecological Research, GINOP Sustainable Ecosystems Group, Klebelsberg K. u. 3,

H-8237 Tihany, Hungary

3
University of Veterinary Medicine, Institute of Biology, Rottenbiller u. 50, H-1077

Budapest, Hungary

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*
Corresponding author: e-mail address: eros.tibor@okologia.mta.hu, Tel: +36 87448244,

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Fax.: +36 87448006 -p
Abstract
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We examined the effects of regional scale land use and local scale environmental and biotic

stressors on alpha, beta and zeta diversities of native fish communities in wadeable streams
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and non-wadeable rivers in the Danube basin, Hungary. Relationships among land use and
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local scale environmental and biotic stressors were weak both in streams and rivers,

suggesting that these stressors act relatively independently. Alpha diversity decreased strongly
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with increasing local scale environmental stressor intensity in rivers. On the contrary, its
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response to stressors was more obscure in streams, where the best-fit statistical model

indicated the importance of the interaction between land use, local scale environmental and

biotic stressors, while the secondly ranked model highlighted the negative impact of local

scale environmental stressors. Analysis of variance using distance matrices provided evidence

that stressors alone and in interactions explained compositional differences of pairs of study

sites (beta diversity). Considering the degree of overall degradation, both local (alpha) and

among-site (beta and zeta) diversity indices responded to increasing stressor intensity,

generally negatively. Riverine fish communities showed higher degrees of similarity (lower
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beta and higher zeta) than stream fish communities. They also showed increasing similarity

(i.e. homogenization) with increasing overall stressor intensity, unlike stream fish

communities, which showed no relationship with overall stressor intensity. Our results

suggest that the relationships between land use and local scale environmental and biotic

stressors can be complex and so do their effects on biodiversity. While stressor specific

indices can provide information on the role of specific stressors in some cases, the

examination of overall stressor effects is needed to assess realistically the effects of

anthropogenic disturbances on native fish diversity. Diversity indices that quantify among-site

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changes in species composition, such as measures of beta and zeta diversity, can be fruitful

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for better understanding the role of multiple stressors in structuring ecological communities.
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Keywords
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fish, streams and rivers, land use, invasive species, riparian degradation, habitat alteration

1. Introduction
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Freshwater biodiversity is declining at an alarming rate, which is consistently higher than that
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estimated for terrestrial ecosystems (Dudgeon et al., 2006; Reid et al., 2019). Understanding

the role of multiple human induced stressors in this decline is essential for evidence based
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environmental management. Difficulties arise, because most stressors do not act individually,
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and individual effects are not simply additive in a real world situation (e.g. Ormerod et al.,

2010; Göthe et al., 2019; but see Gieswein et al., 2017). Rather, a variety of stressors interact

in unexpected ways by either reducing (antagonism) or amplifying (synergism) the individual

effects of each stressor (Townsend et al., 2008; Jackson et al., 2016; Schäfer and Piggott,

2018). Therefore, studies on individual and complex stressor effects are needed for a more

mechanistic understanding of the changes in biodiversity patterns.

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Although theory and experiments strongly support the importance of multiple stressors in

shaping biodiversity, their effect has rarely been investigated at biogeographically relevant

spatial scales (Gieswein et al., 2017; Ryo et al., 2018). Ideally, such studies should address

local as well as regional or landscape level conditions using the same analytical framework

(e.g. data set, stressors, evaluation procedures) for a more accurate understanding of stressor

effects. In this regard, the exploration of within site (alpha) and between site (beta) diversity

changes in species composition has special relevance, since only their joint evaluation can

reveal unequivocally how anthropogenic stressors influence spatial patterns in biodiversity

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(Heino, 2011). However, studies which examine the effects of multiple stressors on a variety

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of diversity components (e.g. alpha and beta) in a single study are few (for review see
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Stendera et al., 2012; Heino, 2011, 2013; Jackson et al., 2016). In fact, most studies examined
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i) habitat dependent patterns in alpha, beta and gamma diversity without examining the role

of stressors (e.g. Erős, 2007), ii) the effects of stressors on local (alpha) diversity and
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community structure, without examining the response of other diversity components

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(Schmera et al., 2012), or iii) changes in compositional turnover (beta diversity) along

environmental degradation gradients (e.g. Bini et al., 2014). An exception, for example, is the
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study of Göthe et al. (2015), who tested the effects of multiple stressors on alpha, beta and
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gamma (or total) diversity components of stream macrophyte, macroinvertebrate and fish

communities in an intensively managed, agriculture dominated landscape. They found that

alpha diversity was generally lower in high- compared to low-impacted sites, whereas both

beta and gamma diversities varied more unpredictably among the differently impacted sites.

Overall, studies on freshwater organisms mirror a variety of responses to multiple stressors,

which can be due to biogeographic/taxonomic differences in stressor effects and/or

methodological differences in evaluating stressor-response relationships (Olaya-Marín et al.,

2012; Stendera et al., 2012; Jackson et al., 2016; Lange et al., 2018).
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Recently, a new concept and metric, called zeta diversity has been introduced for

characterizing spatial variation in species composition of multiple assemblages (Hui and

McGeoch, 2014; McGeoch et al., 2019). Zeta diversity allows to calculate how diversity is

structured across multiple spatial scales under the same framework. In brief, zeta diversity is

the mean number of species shared by i number of sites, with i referred to as the zeta order

(McGeoch et al., 2019). The mean number of species across all sites (i=1) gives alpha

diversity, while incidence-based pairwise beta diversity can be calculated based on the

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comparisons of compositional similarity of two sites (i=2). However, unlike pairwise beta

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diversity measures, zeta diversity partitioning quantifies the complete set of diversity
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components for a series of sites or assemblages (i>2), not just between two sites. It thus
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quantifies multiple site similarity in species composition in a scale dependent manner from

i=1 to i=n sites. Therefore, unlike incidence-based beta diversity metrics that are biased
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towards measuring the contribution of rare or satellite species to turnover in species

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composition, zeta diversity informs on the role of the full spectrum of rare, intermediate and

common species in driving compositional turnover (Hui et al., 2018; McGeoch et al., 2019).
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Although zeta diversity is a recent concept, it has already been successfully applied for a
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comprehensive exploration of regional scale patterns in biodiversity (Hui et al., 2018;

Latombe et al., 2019; Simons et al., 2019).

In this study, we examine the effect of multiple stressors on the diversity of fish assemblages

in streams and rivers. We explore the relationships among 1) regional or landscape scale

stressors, 2) local (or site scale) environmental stressors and 3) local scale biotic stressors, and

examine their individual and combined effects. To our knowledge this is the first study which

tests the effect of increasing stressor intensity on both within (alpha) and among-site (beta and
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zeta) diversity patterns of native fish assemblages using the same analytical framework for

streams (catchment size <1000 km2) and rivers (catchment size >1000 km2). Streams and

rivers may not only differ in their size, and therefore in well-known methodological

challenges in sampling their biota, but by the relative importance of individual stressors and

their interrelationships. In addition, streams are in more peripheral, while rivers are in more

central position in the riverine network for example, from the viewpoint of fish dispersal

(Erős et al., 2017) and receiving and integrating stressor effects (Hering et al., 2006; Hein et

al., 2019).

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We hypothesized decreasing alpha diversity with increasing stressor intensity, due either to
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local extirpation or emigration of species at the site level. We also predicted a decrease in
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among-site diversity with increasing stressor intensity, due to the extirpation (or emigration)

of species and consequently, increasing homogenization of assemblage composition. Note,

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that in this case zeta diversity should also increase because of the increasing dominance of
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common species in the samples with increasing zeta order (see Methods). However, we were

also interested in quantifying the relative differences between the two contrasting stream and
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river habitat types, since differences might appear due simply to differences in dispersal
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mechanisms between peripheral stream and more central river habitats. Beside alpha and beta

diversity, the quantification of zeta diversity may provide a novel way to gain information

about the response of variation in fish species composition to increasing stressor intensity.

2. Material and methods

2.1 Study sites

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The study area was located in the Pannon ecoregion, Hungary where all the streams and rivers

are tributaries of the River Danube, the second largest river in Europe (catchment area 796

250 km2; length 2847 km). The majority of the country’s 93,030 km2 are lowland areas (i.e.

situated below 300 m a.s.l.), with only a very small proportion being located in submontane

regions (highest mountain peak is only 1014 m). The dominant land use type in the

catchments is arable fields, with vineyards, orchards, pastures, and managed deciduous forest

forming a smaller proportion (Tóth et al., 2019).

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We used our institutional database of fish assemblages occurring in 429 stream and river
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localities (Fig. 1). We distinguished stream and river sites due to contrasting differences in
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their size and position in the riverine network (Erős et al., 2017). Submontane streams were

not considered due to their very small proportion in the data set (n = 17). Two very large or
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great rivers, the Danube and the Tisza were also disregarded for this study, due to their
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disproportionally large size, difficulties in sampling their fish assemblages, as well as

characterizing their stressor variables. Stream sites (n = 312) were wadeable and had a mean
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width of 2.8 ± 0.8 m, a mean depth of 34.5 ± 19.1 cm, and a catchment size <1000 km2. On
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the contrary, river sites (n = 100) were usually non-wadeable and had a mean width of 29.7 ±

32.2 m, a mean depth of 84.6 ± 54.3 cm, and a catchment size >1000 km2.

2.2 Individual stressors and overall degradation

We used three types of variable groups to characterize stressor effects. These were the

following: 1) land use stressors, 2) local scale environmental stressors and 3) local scale biotic
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stressors. We used the land use index (LUI) of Böhmer et al. (2004) for land use stress

indicator, which describes land use intensity and impact within a catchment along a gradient

from natural forest cover to agricultural and urban use. The index, which has been used in

other studies (e.g., Ligeiro et al., 2013; Erős et al., 2018), is calculated as follows:

LUI = % pasture + 2 × % arable land + 4 × % urban area

The index was scaled between 0 and 1. The higher is the index value, the higher is the land

use degradation.

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We used indicators of riparian degradation, water chemistry, and instream

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hydromorphological degradation for characterizing local scale environmental stressor effects.
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The habitat structure of the stream margin (i.e. along a ∼10m wide strip in both sides) was
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characterized by visually estimating the percentage coverage of vegetation (herbaceous and

arboreal) and concrete (Erős et al., 2017). Based on these surveys, riparian degradation
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(RIPD) was calculated using the following formula:

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RIPD = % arboreal + 2 × % herbaceous + 4 × % concrete coverage

Decreasing arboreal vegetation and increasing concrete cover can influence assemblage
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structure and integrity of the biota in streams and rivers (Dala‐ Corte et al., 2016; Tóth et al.,
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2019). Therefore, the higher is the index value, the higher is the riparian degradation.

For characterizing water chemistry, conductivity, dissolved oxygen content and pH were

measured in the field with an OAKTON Waterproof PCD 650 portable handheld meter. The

content of nitrate (the salicylate method), nitrite, ammonium (indophenol blue method) and

phosphate (molybdenate blue method), were obtained from the National Water Quality

Database. The national water authorities that provided the data have been accredited

according to the EN ISO/EC 17025 European standard (Istvánovics and Honti, 2012).
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Water chemistry parameters were transformed if necessary (using square-root, double square-

root or log-transformation), and analysed in a standardized Principal Component Analysis

(PCA). As the first principal component explained a remarkable amount of total variability

(51.1%), and because nitrogen forms and phosphate directed along the first principal

component (Suppl. Fig. 1), we used the first principal component as a measure of water

chemistry stressor indicator. Finally, we used the percentage coverage of instream concrete

surface for a simple measure of hydromorphological degradation. The higher is the proportion

of concrete surface, the higher is the within-stream habitat degradation.

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All three indices were scaled between 0 and 1 (the riparian degradation index was square-root
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transformed before scaling to increase its normality). Note that of these local scale
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environmental stressors, riparian degradation showed positive correlation with both chemical

(Pearson correlation value 0.276, P<0.001) and within-stream hydromorphological

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degradation (0.544, P<0.001), indicating a strong coupling between riparian and instream
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physical and chemical degradation. On the contrary, water chemistry did not show significant

relationship with instream hydromorphological degradation (0.052, P=0.296) ). The indices

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were then summed to get the local scale degradation index, which was also scaled between 0
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and 1. The higher is the index value, the higher is the local scale environmental degradation.

We used the relative abundance (%) of non-native fish species (double square-root

transformed) scaled between 0 and 1 for the indicator of biotic degradation. Invasion of non-

native species can influence the structure of native assemblages negatively, for example by

predation or competition for space and/or food (Cucherousset and Olden, 2011; Sagouis et al.,

2015). Therefore, the higher is the index value, the higher is the biotic degradation.
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The mean and minimum-maximum values of the index variables can be found in Suppl. Table

1. Finally, we also calculated the overall degradation (OD) of the sites, by summing land use,

and local scale environmental and biotic stressor index values. OD was also scaled between 0

and 1. We then divided the overall degradation gradient into three degradation categories:

low, moderate and high (Suppl. Table 2), which allowed us to examine the behaviour of

diversity indices (e.g. zeta diversity) to different levels of stressor intensity (Hui and

McGeoch, 2014; McGeoch et al., 2019; Simons et al., 2019).

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2.3 Fish sampling -p
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Fish were generally collected during the summer or early autumn base flow period between

2013 and 2018. Two types of electrofishing methods were used (Erős et al., 2017), which
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were fully consistent with generally used electrofishing protocols (see e.g. Oberdorff et al.,
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2001; Pont et al., 2006). For streams, a battery-powered electrofishing device was used (Hans-

Grassl IG 200/2B device, PDC). The crew sampled a 150 m long reach, slowly walking
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upstream and with single-pass fishing of the whole stream width. For non-wadeable rivers,
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boat electrofishing was applied with a generator driven device (Hans-Grassl EL64 II GI

device, max 7000 W, DC), slowly moving downstream and electrofishing 500 or 1000 m long

reaches in near shore areas of rivers and large rivers, respectively. This division in sampling

length between streams and rivers (and within rivers) was necessary to optimize sampling

effort and to sample fish assemblages representatively and proportionally to the size of the

water body (Erős, 2007). After identification and counting, fish were released into the water at

the site of capture.

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2.4 Data analyses

We calculated individual (land use, local scale environmental and biotic stressors) and overall

degradation (OD) indices for each study site. Principal component analysis (PCA) was used to

examine the relationship of individual stressors as well as to reveal how individual stressors

contribute to the separation of study sites assigned to low, moderate and high overall

degradation categories, separately for stream and river sites.

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Linear models were used to test whether and how individual stressors and their interactions
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shape alpha diversity (species richness) of native fish. We examined stream and river sites
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separately. The best-fit models were selected using Akaike Information Criterion (AIC). To

characterize the overall effects of stressors, richness of sites assigned to different levels of OD
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categories were compared using Analysis of Variance (ANOVA).

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Analysis of variance using distance matrices (called ADONIS in R) was used to test how
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individual stressors and their interactions influence community dissimilarity, which is a

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frequently used beta diversity measure (Anderson 2001). Note that the analysis is also

referred to as "permutational MANOVA" or "non-parametric MANOVA" (Anderson, 2001;

McArdle and Anderson, 2001) or "multivariate regression analysis of distance matrices"

(Zapala and Schork, 2006). As alpha diversity showed sensitivity to both habitat type and

degradation (see results), we searched for a dissimilarity measure (i.e. beta diversity) that is

relativized to a unit range. We selected the commonly used Jaccard dissimilarity (Jaccard,

1912) index, which varies between 0 (the species composition of the two study sites are
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identical) and 1 (the two study sites do not share common species) (Podani and Schmera,

2011).

To reveal the overall effects of stressors (i.e. OD) on beta diversity, we examined how habitat

type and the levels of OD influence beta diversity of fish. As beta diversities are not

independent from each other, we applied a two-way ANOVA test for unbalanced design with

permutation test developed by Pierre Legendre

(http://adn.biol.umontreal.ca/~numericalecology/labo/fonctions_r/anova.2way.unbalanced.R.z

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ip, access date: 19 January, 2020).

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Finally, rescaled zeta diversity was used to calculate diversity components across spatial
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scales under the same multi-site diversity partitioning framework (Hui and McGeoch, 2014;

McGeoch et al., 2019). We used rescaled zeta because it is insensitive to inherent richness
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differences between the habitat types. Consequently, we could directly compare differences in
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zeta diversity between streams and rivers within and between the degradation levels (Hui and

McGeoch, 2014; McGeoch et al., 2019). Statistical analyses were performed in R (R Core
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Team, 2019) using the MASS (Venables and Ripley, 2002), multcomp (Hothorn et al., 2008),
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MuMIn (Barton, 2019) and vegan (Oksanen et al., 2019) packages.

3. Results

3.1. Relationships between the stressor components

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The OD categories (low, moderate, high) separated well along the first two components of the

PCAs, which explained 76.6% and 78.2% of the variance for streams and rivers, respectively

(Fig. 2). The PCAs indicated that the stressor components (land use, local scale environmental

and biotic) determined the OD gradient relatively independently. For streams (Fig. 2a)

significant positive relationships were found between land use and local scale environmental

(r = 0.292, P < 0.001), land use and biotic (r = 0.172, P < 0.002) and local scale

environmental and biotic (r = 0.119, P < 0.036) stressors, albeit the relationships were weak (r

< 0.3). Similarly, for rivers (Fig. 2b) weak positive relationships were found between land use

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and local scale environmental (r= 0.318, P = 0.001), as well as between local scale

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environmental and biotic (r = 0.220, P = 0.027) stressors. However, the relationship proved to
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be insignificant between land use and biotic stressors (P>0.05).
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3.2. Alpha diversity

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Altogether 42 native fish species were recorded. Fish species richness varied between 1 and
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18 species (mean = 7.49) among the sites. The best-fit model showed that the species richness
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of stream fish was influenced by individual stressors (land use, local scale environmental and

biotic) as well as by the interaction of biotic and land use stressors (Table 1: model 1). Other,

but still very plausible statistical models indicated the negative effects of local scale

environmental stressors (Table 1: model 2) as well as the interactions between stressors

(Table 1: model 3). Other, less optimal models contained only individual effects (Table 1:

models 4 and 8) or both individual effects and interactions (Table 1: models 5 and7).
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Regarding rivers, the best-fit model showed that the species richness of fish was influenced

only by local scale environmental stressors (Table 2: model 1), while the second best-fit

model indicated the negative effect of local scale environmental stressors and, surprisingly, a

moderately positive effect of land use stressors (Table 2: model 2). Further models indicated

the negative effects of local scale environmental stressors.

ANOVA table showed that habitat type as well as the interaction of habitat type and OD

influenced the species richness of fish (Table 3). Richness in streams (mean: 6.19) was

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significantly smaller than that in rivers (mean: 11.52, Fig. 3). However, the effect of the levels

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of OD strongly depended on habitat type (Table 3: significant interaction effect). In streams,
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the richness of sites with high level of OD was significantly smaller than the richness of sites
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at moderate degradation level, while in rivers species richness decreased with increasing

levels of OD (Fig. 3).

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3.2 Beta diversity

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ADONIS showed that beta diversity of stream fish was influenced by individual stressors, as
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well as by their two- and three-way interactions (Table 4). However, these effects were very

weak and therefore predictors explained only a limited amount of variability (Table 4).

Regarding rivers, ADONIS showed that beta diversity of fish was influenced by individual

stressors, as well as by the interaction of the local scale environmental and land use stressors

(Table 5). However, similarly to streams, the variation explained by the statistical model was

limited.
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The permutation-based ANOVA showed that beta diversity of fish was significantly

influenced by habitat type and levels of OD, as well as by the interaction of habitat type and

OD (Table 6). Beta diversity was larger in streams (mean value: 0.76) than in rivers (mean

value: 0.64) and the effect of OD depended on the habitat type. Specifically, beta diversity

decreased with increasing level of OD in rivers, while in streams the highest beta diversity

was found at the moderate OD level (Fig. 4).

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3.3. Zeta diversity

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In general, rescaled zeta diversity decreased monotonically with increasing zeta order (Fig. 5).
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In streams, the 95% confidence intervals of the curves overlapped among the OD levels,

suggesting that the degree of OD had no effect on rescaled zeta diversity. On the contrary,
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sites with high level of OD showed higher rescaled zeta diversity values in rivers than sites
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with low and moderate OD. Moreover, the 95% confidence intervals of rescaled zeta diversity

observed at the highest level of OD did not overlap with the confidence intervals of the
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rescaled zeta diversity of sites under low or moderate level of degradation at zeta order larger
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than 7.

4. Discussion

There is a substantial lack of studies that have examined the effects of multiple stressors on

within and among-site components of native fish diversity. To fill this knowledge gap, we

characterized individual stressor intensity and fish communities at more than 400 running
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water sites in the Pannon ecoregion, Hungary, and evaluated the individual and combined

effects of different anthropogenic stressors. We found that both within-site (alpha) and

among-site (beta) diversity components responded to increasing stressor intensity, as well as

to the interaction of the studied stressors. However, our results showed that the effects of

multiple stressors on fish diversity were different when examining stream and riverine

communities. The application of a novel analytical framework (zeta diversity) revealed also

that the overall effects of stressors significantly homogenized riverine fish communities.

These findings suggest that stream and riverine fish communities suffer from the effects of

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multiple stressors, and underpin the need for more holistic approaches in conserving

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freshwater biodiversity. -p
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The effects of catchment level land-use transformation on local scale habitat alteration are

widely recognized in stream ecology (Allan, 2004). However, the strength of these effects
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varies largely, depending on the interrelationships between natural and anthropogenic factors,
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length of the environmental gradients, scale effects, and historical influences (Wang et al.,

2003; Allan, 2004; Leitao et al., 2017). Here, we found weak, but significant relationships
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between regional scale land use and local scale environmental stressors in both streams and
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rivers supporting the results of former studies. In addition, the relationships among biotic

degradation (here the abundance of non-natives fishes), land use, and local scale habitat

alteration were relatively weak, but significant in both streams and rivers, with the exception

of the relationship between land use and biotic degradation in rivers, which proved to be

insignificant. Former studies show that the abundance of non-native fishes is mostly related to

the distribution of fish ponds and water reservoirs in this and in other ecoregions (Marchetti et

al., 2004; Hermoso et al., 2011; Erős et al. 2012). These landscape level alterations may also

influence the local stream environment and facilitate the spread of non-natives species
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(Marchetti et al., 2004; Hermoso et al, 2011; Takács et al., 2017). Since fish ponds and

reservoirs are more common in streams than in rivers in this ecoregion, it is not surprising that

we found stronger coupling between biotic degradation and landscape and local scale habitat

alteration in streams than in rivers. In addition, streams have much smaller catchments, which

may also predetermine a stronger relationship between land use and local scale environmental

conditions. Nevertheless, our results suggest that land use, local scale environmental

degradation, and biotic stressors may affect stream and river communities relatively

independently, and therefore, all stressors should be taken into account in assessing the

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overall degradation of study sites in the Pannon ecoregion.

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Of the diversity components, alpha showed the most responsive changes to stressor intensity.
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Interestingly, the most clear response was found for rivers. Here, alpha diversity decreased

strongly with increasing level of overall stressor intensity (Fig 3), and this effect was related
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the most to local scale environmental alteration (Table 2). Although changes in fish
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assemblage structure or in fish based biotic indices have already been found (Schinegger et

al., 2018), to our knowledge, so contrasting changes in fish species richness to anthropogenic
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stressors have never been reported. The more responsive change in rivers compared with
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streams was probably due to their more homogenized habitat structure and fish communities

(see Fig. 4 in Erős, 2007). On the contrary, the response of alpha diversity to stressors was

more obscure in streams, where the best-fit candidate model indicated an interaction between

land use and biotic stressors, while the secondly ranked model highlighted the negative

influence of local scale environmental degradation (Table 1). In fact, we found more

interaction effects in streams than in rivers. Although the effect of stressor interactions is hard

to interpret, their importance has been widely recognized, especially in explaining the

diversity of stream macroinvertebrates (Jackson et al., 2016; Gieswein et al., 2017; Ryo et al.,
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2018). For stream fishes, interaction effects of stressors were found to be relatively

uncommon (Gieswein et al., 2017). Negative changes in alpha diversity were mostly related

to local, and especially to hydromorphic degradation (Hering et al., 2006; Zajicek et al.,

2018), albeit the lack of significant relationships was also common (Göthe et al., 2015). In

fact, the response of fish metrics to stress was generally low in multitaxa comparisons (Hering

et al., 2006; Dahm et al., 2013). In addition, unlike hydromorphic alterations, nutrient

enrichment has been mostly found to increase both the richness and abundance of species in

some streams to a certain degree (Johnson and Angeler, 2014; Takács et al, 2016). The

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observed hump shaped response of alpha diversity for stream fish communities may indicate a

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subsidy stress response (see e.g. Wagenhoff et al., 2011), albeit differences in richness do not
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seem to be biologically relevant. Overall, the differences we found in the patterns between
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streams and rivers, and the inconsistencies among studies suggest strong context dependency

in the response of alpha diversity of fish communities to multiple stressors. Further studies are
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thus necessary for a more mechanistic understanding of the role of stressors on alpha diversity
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(see also Alahuhta et al., 2018).

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The results of ADONIS provided clear evidence that the composition of fish communities was
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sensitive to the intensity of stressors acting individually or in interactions (Tables 4 and 5).

This sensitivity, however, was in general weak in both streams and rivers. Two or three way

interaction effects were more frequent in streams suggesting that multiple stressor effects are

somewhat more complex, and less predictable in streams. These differences may be related to

the more heterogeneous environmental features and spatial configuration of streams (Finn et

al., 2011; Erős et al., 2017), which, combined with the diversity of stressor effects, may

increase unpredictability for streams compared with rivers. Regarding the overall effects of

stressors, rivers showed the most responsive changes (Fig. 4). Beta diversity clearly decreased
 Journal Pre-proof

with increasing overall stressor intensity, which suggests a much stronger homogenization of

native communities in rivers compared with that in streams. Overall, these results suggest that

stream fish communities maybe less vulnerable to anthropogenic stressors than riverine ones,

since more heterogeneous communities may have a greater plasticity and resilience to stress,

at least at the regional scale (Johnson and Angeler, 2014).

Rescaled zeta diversity curves confirmed the pattern found for beta diversity. Specifically,

riverine communities showed less among-site compositional heterogeneity (lower beta

of
diversity and higher rescaled zeta diversity values) than stream fish communities, and river

ro
fish communities displayed also sensitivity to overall stressor intensity. This means that in
-p
sites with high level of overall degradation, riverine fish communities indicated a significantly
re
lower degree of among-site heterogeneity (higher rescaled zeta diversity) than in sites with

low or moderate level of overall degradation (lower rescaled zeta diversity). Contrary to these
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similarities in the possible inferences, beta and zeta diversities represent clearly different
na

facets of among-site community heterogeneity. While beta diversity, quantified by the Jaccard

dissimilarity, quantifies differences between site pairs exclusively (i.e. 2 sites), the rescaled
ur

zeta diversity inspects multiple sites; in our case a series of sites ranging from 1 (zeta order =
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1) to 9 (zeta order = 9). Consequently, the finding that rescaled zeta diversity of riverine fish

suffers from faunal homogenization at zeta orders above seven revealed a new and so far

undescribed response of native fish diversity to anthropogenic stressors. Based on these

findings we believe that the application of novel approaches, such as zeta diversity, can

significantly deepen our knowledge on patterns in freshwater fish biodiversity to multistressor

effects.
 Journal Pre-proof

Although we found relatively similar responses regarding alpha, beta and zeta diversities to

overall degradation effects (i.e. more responsive changes in rivers), we argue that these

approaches complement each other for a more holistic understanding of diversity patterns and

community responses (Simons et al., 2019). For example, it has been shown that while

pairwise beta diversity is insensitive to occupancy changes in common species, zeta diversity

is responsive to changes across the range of species occupancy classes (Hui and McGeoch,

2014). In fact, presently most bioassesment systems are based on local evaluation of

ecological integrity (Heino, 2013; Erős et al., 2019). Our results support other, recent studies

of
(Heino, 2013; Cid et al., 2020), which call the attention for the application of multisite

ro
approaches in bioassessment, especially for highly mobile organism, such as fish. In this
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regard, zeta diversity can be an especially good candidate, since it quantifies the
re
homogenization of communities across multiple sites considering the occupancy of rare and

common species (McGeoch et al., 2019; this study). The finding that riverine fish
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communities become less species rich and more homogenized regarding common species
na

with increasing stressor intensity underline the need for urgent conservation actions in this

ecoregion, which should involve the tributaries of the most altered rivers as well.
ur
Jo

A limitation of our study is that we could not incorporate the role of fragmentation in

evaluating stressor effects on fish diversity, due to the lack of detailed barrier data at the

national scale. However, several studies show that fragmentation can seriously influence fish

communities, and may explain some unrevealed portion of variance in diversity-stressor

relationships (Olaya-Marín et al., 2012; Perkin and Gido, 2012). Therefore, changes in fish

diversity to anthropogenic stressors can be even more detrimental than that characterized here.
 Journal Pre-proof

5. Conclusions

Our results justified that although the relationships among land use and local scale

environmental and biotic stressors were relatively weak, the effects of these stressors on

biodiversity were rather complex. Therefore, while stressor specific indices may be

informative on the possible impact of stressors, this knowledge seems to be ineffective in

assessing the significance of human alteration effects on native fish diversity. The evaluation

of overall stressor effects is needed for understanding the decline in different facets of

of
biodiversity. Our results showed that fish communities of streams and rivers differ in their

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response to individual and multiple stressor effects. In this regard, changes in the diversity of
-p
riverine fish communities proved to be more predictable. We suggest that diversity indices,
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which quantify among-site changes in species composition, such as measures of beta and zeta

diversity, can be fruitful for better understanding the role of multiple stressors in structuring
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ecological communities. The recently introduced concept of zeta diversity seems to be an

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especially ideal candidate for characterizing the response of biodiversity to increasing stressor

intensity at regional scales.

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Jo

Acknowledgement

This research was supported by the National Research, Development and Innovation Office

(NKFI K 128496), GINOP (2.3.3-15-2016-00019) and Ecology for Society (MTA KEP)

grants.
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Table 1: Best-fit models explaining the effect of stressors on stream fish richness (alpha diversity). The table contains individual effects

(individual biotic, local and land use) and two-, and three-way interactions. Cells show the slope of regressions.

Model individual individual individual interaction interaction interaction interaction df AICc delta weight R-squared RMSE

number biotic local land use biotic:local biotic:land use local:land use biotic: local: land use

f
1 0.85 -0.91 0.41 -1.75 6 621.9 0.00 0.165 0.060 0.643

o
2 -0.93 3 622.2 0.31 0.142 0.041 0.649

3 2.42 0.592 1.78 -4.39 -5.32 -3.59 9,66

r o9 622.4 0.50 0.128 0.078 0.637

p
4 -0.83 -0.33 4 622.7 0.76 0.113 0.046 0.648

5 0.81 -0.22 0.87 -1.72 -1.39

e - 7 623.0 1.02 0.099 0.064 0.641

r
6 -0.12 0.17 -1.44 5 623.6 1.67 0.072 0.049 0.647

P
7 0.77 -1.06 0.47 0.39 -1.88 7 623.8 1.87 0.065 0.061 0.642

8 -0.04 -0.92

a l 4 624.2 2.25 0.054 0.041 0.649

r n
o u
J
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Table 2: Best-fit models explaining the effect of stressors on river fish richness (alpha diversity). The table contains individual effects (individual

biotic, local environmental and land use) and two-, and three-way interactions. Cells show the slope of regressions.

Model individual individual individual interaction interaction interaction interaction df AICc delta weight R-squared RMSE

number biotic local land use biotic:local biotic:land use local:land use biotic: local: land use

f
1 -0.986 3 57.2 0.00 0.429 0.120 0.312

o
2 -1.03 0.13 4 59.1 1.89 0.167 0.123 0.312

3 -0.04 -0.97

r o4 59.3 2.11 0.149 0.121 0.312

p
4 -1.52 -0.08 1.01 5 61.1 3.96 0.059 0.124 0.311

5 -0.04 -1.02 0.14

e - 5 61.2 4.04 0.057 0.123 0.312

r
6 0.00 -0.896 -0.171 5 61.5 4.32 0.050 0.121 0.312

l P
n a
u r
J o
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Table 3: The ANOVA output explaining the species richness (alpha diversity) of fish

depending on the habitat type (stream vs river), overall degradation and their interaction.

Predictor Df SS MS F P

Habitat type 1 177.773 177.773 295.004 <0.001

Overall degradation 2 0.234 0.119 0.197 0.822

Interaction 2 15.284 7.642 12.681 <0.001

Residuals 406 244.660 0.603

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Table 4: Summary of the analyses of variance using distance matrices (ADONIS) testing the

effect of stressors (land use, local scale environmental and biotic) on the Jaccard dissimilarity

(beta diversity) of stream fish.

Stressor Df SS R2 F P

individual biotic 1 4.497 0.046 15.513 0.001

individual local 1 0.396 0.004 1.365 0.164

individual land use 1 1.691 0.017 5.834 0.001

intercation of biotic:local 1 0.861 0.008 2.971 0.002

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interaction of biotic:land use 1 0.739 0.008 2.550 0.007

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interaction of local: land use 1 0.929 0.009 3.205
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interaction of biotic: local:land use 1 0.707 0.007 2.439 0.010

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Residual 304 88.136 0.899

Total 311 97.958 1.000

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Table 5: Summary of the analyses of variance using distance matrices (ADONIS) testing the

effect of stressors (land use, local scale environmental and biotic) on the Jaccard dissimilarity

(beta diversity) of riverine fish.

Stressor Df SS R2 F P

individual biotic 1 1.717 0.079 9.036 0.001

individual local 1 1.014 0.046 5.338 0.001

individual land use 1 0.392 0.018 2.060 0.029

interaction of biotic:local 1 0.228 0.010 1.199 0.269

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interaction of biotic:land use 1 0.195 0.009 1.026 0.385

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interaction of local:land use 1 0.429 0.019 2.258 0.016
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interaction of biotic: local:land use 1 0.402 0.018 2.117 0.033
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Residual 92 17.481 0.802

Total 99 21.785 1.000

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Table 6: The output of permutation-based ANOVA explaining the Jaccard dissimilarity (beta

diversity) of fish.

Predictor Df MS F P

Habitat type 1 0.0004 205.409 0.001

Degradation 2 0.0001 24.870 0.001

Interactions 2 0.0001 11.769 0.001

Residuals 18482 0.0282

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Fig. 1: The map of the study area with the study sites in the Pannon ecoregion, Hungary (area:

93,030 km2). Red circles represent stream sites (n=312), while yellow triangles river sites
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(n=100).
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Fig. 2: Ordination plot of the results of principal component analysis (PCA) for (a) streams

and (b) rivers. Yellow squares represent sites with low, orange squares moderate, while red

squares high overall degradation categories based on land use, local scale environmental and

biotic stressors.
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Fig. 3: The effect of overall degradation on the species richness (alpha diversity) of fish in

streams and rivers. Bars indicate mean values, whiskers standard errors. Different letters in

the same habitat type indicate significant differences between degradation categories (Tukey

test, Bonferroni adjusted P value at P = 0.05).

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Fig. 4: The effect of overall degradation on the Jaccard dissimilarity (beta diversity) of fish in

streams and rivers. Bars indicate mean values, whiskers standard errors.
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Fig. 5: The effect of the levels of overall degradation on the rescaled zeta diversity of fish in
streams and rivers. Rescaled zeta diversity is the ratio of the average number of species shared
by a given number of randomly selected sites (i.e. the zeta order) to the total number of
species. The curves show the change in rescaled zeta diversity with increasing numbers of
sites included in the comparison. Solid lines show mean values, while dotted lines true 95%
confidence intervals.
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Declaration of competing interests

☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

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Author contributions
Tibor Erős: Conceptualization, Methodology, Visualization, Investigation, Supervision,
Writing- Original draft preparation, Writing- Reviewing and Editing. István Czeglédi, Rita
Tóth: Data curation, Methodology, Visualization, Investigation. Dénes Schmera:
Conceptualization, Methodology, Visualization, Investigation, Supervision, Writing- Original
draft preparation, Writing- Reviewing and Editing.

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Graphical abstract

Highlights

 We examined the response of fish diversity to land use, local and biotic stressors

 Intensity of stressors varied relatively independently in both streams and rivers

 Alpha and beta diversities were affected by both individual and interactive effects

 Zeta diversity indicated a homogenized fauna in the most degraded river sites

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Overall effect of stressors was inconsistent in streams and negative in rivers

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