Acta Oecologica 99 (2019) 103443

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Acta Oecologica
journal homepage: www.elsevier.com/locate/actoec

Exploring the relationships between aquatic macrophyte functional traits T

and anthropogenic pressures in freshwater lakes
Dimitrios Zervasa,b,∗, Vasiliki Tsiaoussib, Athanasios S. Kallimanisc, Panayotis Dimopoulosd,
Ioannis Tsiripidisa
a
Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-54124, Greece
b
The Goulandris Natural History Museum, Greek Biotope, Wetland Centre, 14th km Thessaloniki-Mihaniona, P.O. Box 60394, Thermi, 57001, Thessaloniki, Greece
c
Department of Ecology, School of Biology, Aristotle University of Thessaloniki, GR-54124, Greece
d
Department of Plant Biology, School of Biology, University of Patras, GR-54124, Greece

A R T I C LE I N FO A B S T R A C T

Keywords: Modern water resource management requires biomonitoring of the structure and functioning of freshwater
Aquatic plant ecosystems, which may be better illuminated by functional trait distribution patterns and responses across
Trait pattern human-induced pressure gradients. In this study, we applied the RLQ, the fourth-corner and their novel com-
RLQ fourth-corner bination methods, in order to assess the relationship between the distribution of 30 aquatic macrophyte func-
Biomonitoring
tional traits and 14 indicators of anthropogenic pressures across 16 freshwater lakes. Our findings showed that
Eutrophication
there is a statistically significant relationship between the distribution of specific functional traits and anthro-
Greece
pogenic pressures. Eutrophication was the dominant pressure and the shift from a submerged-macrophyte
dominated vegetation community to an emergent one was the most important functional response. Aerial re-
production and dispersal traits were found to replace water-related ones under higher nutrient concentrations.
Trophic and light preferences of macrophytic species increased, while their leaf morphology was found to
change from tubular/capillary leaf types with low leaf area values to entire leaf types with greater leaf area.
These results provide hints on the changes in ecosystem functioning occurring as a response to human-induced
drivers. Therefore, this assessment approach could provide important support to the tasks of biomonitoring,
conservation and management planning in freshwater ecosystems.

1. Introduction methods based on a number of different groups of organisms as biolo-

Freshwater lakes are ecosystems of high biodiversity and their
sustainable management is a conservation priority worldwide (Coops
et al., 2004; Weisner and Ekstam, 1993), after being in decline during
the last century due to anthropogenic activities (Johnson et al., 2001;
Sala et al., 2000). Identifying the anthropogenic driving forces of this
decline and the pressure-impact-response relationships pertaining to
ecosystem quality are important for conservation and restoration
management (Kerr et al., 2007). This necessity has been acknowledged
by modern water resource management, and biological indicators have
been incorporated, in order to assess the quality status of freshwater
ecosystems (e.g. Clean Water Act, U.S. Congress, 2002). Water Frame-
work Directive (WFD; European Commission, 2000), the European
water legislation currently in force, which established the rules for
monitoring and assessment of the ecological status of surface waters
within the EU, required the development of ecological assessment
gical indicators. Aquatic macrophytes are one of these biological in-
dicators, considered as a key component of freshwater ecosystems, in-
fluencing a great number of other organisms (Carpenter and Lodge,
1986; Jeppesen et al., 1997; Scheffer, 1998) and highlighting changes
in lake ecosystem functioning (Penning et al., 2008; Schaumburg et al.,
2004; Søndergaard et al., 2010).
Since the release of the Millennium Ecosystem Assessment (MA,
2005), there has been an expanding interest on ecosystem functions and
services (Carpenter et al., 2009; Fisher et al., 2009; Naeem et al., 2012).
Conservation and restoration ecology have paid extra attention to the
development of appropriate tools that assess the relationships between
environmental change, biological community composition and ecolo-
gical functions, in order to guide management planning (Benayas et al.,
2009; Cortina et al., 2006; Hobbs and Cramer, 2008). WFD also re-
quired the assessment of the ecological status of surface water ecosys-
tems as an expression of the quality of their structure and functioning

∗
Corresponding author. Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-54124, Greece.
E-mail address: dzervas@ekby.gr (D. Zervas).

https://doi.org/10.1016/j.actao.2019.103443
Received 14 November 2018; Received in revised form 6 May 2019; Accepted 19 June 2019
Available online 27 June 2019
1146-609X/ © 2019 Elsevier Masson SAS. All rights reserved.
D. Zervas, et al. Acta Oecologica 99 (2019) 103443

(European Commission, 2000). A number of such developed tools (e.g. Table 1

functional diversity indices, Mouchet et al., 2010; Petchey and Gaston, Sampling period and number of transects established for the investigation of the
2006) attempted to assess the ecosystem functions on the basis of or- macrophytic vegetation, in the 16 lakes of the Greek National Monitoring
ganismal functional traits (Grime, 2006; Hooper et al., 2005). Func- Network. GR-DNL type are warm monomictic, deep natural lakes with mean
depth > 9 m, while GR-SNL type are polymictic, shallow natural lakes with
tional trait is any measurable morphological, physiological or pheno-
mean depth 3–9 m. Transboundary lakes that only the part of their surface area
logical property of an individual that affects its performance, response
in Greece was surveyed, are marked with asterisks.
or biotic fitness directly or indirectly (Cadotte et al., 2011; McGill et al.,
2006; Violle et al., 2007). Trait patterns can depict the response of a No Lake Abbreviation Type No of Sampling
Transects Period
biological community and its constituent species to ecosystem processes
and simultaneously provide a good predictor of ecosystem functioning 01 Volvi VOL13 GR-DNL 20 September
(Mouchet et al., 2010; Petchey and Gaston, 2006; Tilman et al., 2001). 2013
Therefore, trait-based bioassessment tools are considered to have clear 02 Doirani* DOI13 GR-SNL 10 August 2013
potential in environmental monitoring by (i) allowing the under- 03 Vegoritida VEG13 GR-DNL 20 August 2013
05 Kastoria KAS14 GR-SNL 20 August 2014
standing of cause-effect relationships between species and their en- 06 Megali Prespa* MEP15 GR-DNL 12 August 2015
vironment, (ii) enabling evaluation and prediction of anthropogenic 07 Mikri Prespa* MIP15 GR-SNL 15 August 2015
impacts at different spatial and temporal scales, and (iii) revealing the 08 Pamvotida PAM13 GR-SNL 20 September
links between organisms and ecosystem services (Doledec and Statzner, 2013
09 Amvrakia AMV14 GR-DNL 20 June 2014
2008; Reyjol et al., 2014). However, a common issue when attempting
10 Ozeros OZE14 GR-SNL 20 June 2014
to apply a trait-based method is the methodological decisions needed to 11 Lysimachia LYS14 GR-SNL 20 June 2014
be taken such as: a) how many and which traits contain the appropriate 12 Trichonida TRI15 GR-DNL 20 July 2015
functional information and which are irrelevant, b) how they should be 13 Paralimni PAR14 GR-SNL 19 July 2014
weighted according to their relative functional importance, and c) 14 Yliki YLI14 GR-DNL 20 July 2014
15 Feneos FEN14 GR-DNL 10 August 2014
which mathematical characteristics more efficiently predict variation in 16 Kourna KOU14 GR-DNL 14 May 2014
ecosystem processes (Leps et al., 2006; Petchey and Gaston, 2006; 01 Volvi VOL16 GR-DNL 20 August 2016
Schleuter et al., 2010). The use of different functional traits produces 02 Doirani* DOI16 GR-SNL 10 August 2016
diverse spatial functional diversity patterns for the same biological 03 Vegoritida VEG16 GR-DNL 20 June 2016
04 Zazari ZAZ16 GR-SNL 12 July 2016
community (Tsianou and Kallimanis, 2016). Moreover, the requirement
of lower number of traits than the number of the studied taxa per Total 16 Lakes 19 Lake-Years 2 Types 322 2013–2016
sampling site for the calculation of some functional diversity indices
(e.g. Functional Richness, Villeger et al., 2008; Functional Volume,
Cornwell et al., 2006) further increases the need for a strong filtering of functional bio-assessment tools, with the ultimate goal to guide con-
functional traits used in assessments (Schleuter et al., 2010; Villeger servation and management planning.
et al., 2008).
Aquatic macrophyte functional trait patterns have been examined in
a number of studies (e.g. Baattrup-Pedersen et al., 2015, 2016; Bornette 2. Materials and methods
and Puijalon, 2011; Cavalli et al., 2014; de Bello et al., 2010; Fu et al.,
2014a, 2014b; 2015; Jänes et al., 2017; Mouton et al., 2019; Mueller 2.1. Aquatic macrophytes sampling
et al., 2013; Pan et al., 2017). Despite their differences in objectives and
methodologies, these studies advocated the use of functional trait The data of macrophyte communities in freshwater lakes were ac-
composition of aquatic plants as a helpful tool in understanding the quired during the first five years of operation of the Greek National
relationship between aquatic plant diversity and environmental para- Monitoring Program (2012–2016; Zervas et al., 2018). In total, 16 lakes
meters in water ecosystem functioning. Although numerous studies belonging to two national types (type GR-DNL and type GR-SNL) have
relate functional traits with environmental gradients, we were able to been extensively sampled for aquatic macrophytes during 2013–2015
find only four (Baattrup-Pedersen et al., 2015, 2016; Mouton et al., and three of them were revisited during 2016, resulting in a dataset of
2019, for a number of lowland streams; Pan et al., 2017, for a eutrophic 19 lake-years (Table 1). All 16 lakes belong to Mediterranean lake types
lake), that statistically explored the response of aquatic plant functional (ETC/IC, 2015), they are distributed throughout Greece (Zervas et al.,
traits to human-induced pressures. To our knowledge, studies that 2018) and they cover a wide range of nutrient loading pressures and
quantify and statistically test the relationship between functional trait catchment area land use (Mavromati et al., 2018).
distributions of aquatic plants and gradients of anthropogenic pressures Surveys of aquatic macrophytes followed the belt transect-mapping
in differentially impacted freshwater lakes are scarce in the literature. method described in Zervas et al. (2018). A number of transects per-
The new statistical methods summarized in Dray et al. (2014) offer the pendicular to the shoreline were established in each lake according to
possibility for an in-depth analysis of these relationships, in order to their riparian heterogeneity (Table 1). Sampling plots were positioned
identify the most important linkages between human-induced pressures along the increasing depth gradient and the abundance of each aquatic
and aquatic vegetation responses in aquatic ecosystems. macrophyte taxon (angiosperms, pteridophytes, bryophytes, char-
The aim of this study was to explore the relationships between ophytes and filamentous macroalgae) was estimated using the semi-
several macrophytes’ functional traits and anthropogenic pressure in- quantitative five-point DAFOR scale (Palmer et al., 1992).
dicators reflecting the ecological status of freshwater lakes. In parti- For the calculation of relative species abundance at the transect
cular, our first objective was to test if there is a statistically significant level, the ordinal values of DAFOR scale were transformed to percen-
correlation between aquatic plant functional traits and anthropogenic tage cover values on the basis of the following correspondence:
pressures present in freshwater lakes. Secondly, we attempted to de- Dominant = 87.5%, Abundant = 50%, Frequent = 17.5%,
termine which functional traits, separately or jointly, are positively or Occasional = 5.5% and Rare = 0.5% and the mean percentage cover of
negatively associated with each pressure indicator, providing insights each taxon in all transect's plots were divided by the total percentage
into the underlying mechanisms of species assembly patterns due to cover of all taxa in the transect. Afterwards, relative species abundance
anthropogenic factors. These results may provide a basis for the selec- at lake level was calculated as the mean relative species abundance in
tion of appropriate functional traits in the functional evaluation of the all its transects. Finally, the resulting percentage cover values were
ecological status of freshwater lakes and in the development of raised to the power 0.2, to avoid a high weight of dominant taxa in the

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D. Zervas, et al. Acta Oecologica 99 (2019) 103443

Table 2
The 30 functional traits of aquatic macrophytes selected in order to explore their relations with anthropogenic pressures in freshwater lakes. Plant life aspects
(Category), abbreviations for graphs (Code) and type of allocated values (Value) are listed.
Functional Trait Category Code Value

Free-floating, surface Life form frflsr 0 (Absent); 1 (Occasional); 2 (Present)

Free-floating, submerged ‘‘ frflsb ‘‘
Anchored, floating leaves ‘‘ anflle ‘‘
Anchored, submerged leaves ‘‘ ansule ‘‘
Anchored, emergent leaves ‘‘ anemle ‘‘
Anchored, heterophylly ‘‘ anh ‘‘
Meristem growth point Growth morphology growth api (Apical); bas (Basal)
Morphology Index (Height class + lateral extension of the ‘‘ MI 1 (< 1 cm); 2 (1–10 cm); 3 (10–40 cm); 4 (40–100 cm); 5 (> 100 cm) plus
canopy class)/2 in between classes
Leaf type ‘‘ LT Ent (Entire); cap (Capillary); tub (Tubular)
Leaf area ‘‘ LA 1 (< 1 cm2); 2 (1–20 cm2); 3 (20–100 cm2); 4 (> 100 cm2) plus in between
classes
Rooting at nodes ‘‘ nodal 0 (Absent); 1 (Occasional); 2 (Present)
High below-ground/above-ground biomass fraction ‘‘ root ‘‘
Perennation Survival Peren 1 (Annual); 2 (Perennial)
Overwintering leaves ‘‘ winter 0 (Absent); 1 (Occasional); 2 (Present)
Amphibiousness ‘‘ amph ‘‘
Water level stability Ecological preference wls flu (Fluctuating water regime); sta (Stable water regime; all (All water
regimes)
Water depth ‘‘ wdep 1 (always shallow < 2 m); 2 (reaching deep > 2 m)
Water trophic level ‘‘ troph 1 (Mostly oligotrophic); 2 (Mostly mesotrophic); 3 (Mostly eutrophic)
Ellenberg index value for Light ‘‘ ELL 1-9 (Sciophilous to Photophilous)
Ellenberg index value for Moisture ‘‘ ELM 1-12 (Xerophilous to Hydrophilous)
Ellenberg index value for Reaction of soil ‘‘ ELR 1-9 (Acidophilous to Baseophiloous)
Ellenberg index value for Nutrients ‘‘ ELN 1-9 (Oligonutrient to Hypernutrient)
Rhizome Reproduction and dispersal rhiz 0 (Absent); 1 (Occasional); 2 (Present)
Fragmentation ‘‘ fragm ‘‘
Budding and turions ‘‘ budd ‘‘
Stolons ‘‘ stol ‘‘
Vector of gamete dispersion ‘‘ gamvec win (Anemogamous); wat (Hydrogamous); ins (Entomogamous); sel
(Autogamous); com (Combinations)
Vector of propagule dispersion ‘‘ provec win (Anemochorous); wat (Hydrochorous); oth (Other)
Fruit type ‘‘ FT fle (Fleshy); nfl (Non-fleshy); spo (Spore)
Seed size ‘‘ SZ 1 (< 1 mm); 2 (1–3 mm); 3 (> 3 mm)

calculations. summer seasons (March to August), respectively. Both WLFy and WLFs
were based on monthly water level measurements for each lake (12 and
6, respectively) taken by means of a Nikon-AC-2S topographic equip-
2.2. Pressure indicators
ment.
Land use data for the catchment area of each lake, were analyzed
A number of environmental data indicating anthropogenic pressures
using the Corine Land Cover (CLC) 2012, Version 18.5.1 (Copernicus
were also collected for the 16 lakes during the Greek National
Service - Pan-European Component, 2012). Three parameters were
Monitoring Program operations. Fourteen parameters were selected for
derived from these data, artificial land use (ALU), intensive agriculture
further analysis, grouped into four categories: water chemistry, water
(IA) and natural and semi-natural land use (NASN). ALU was the cover
level fluctuation, land use and population density in the catchment
of all Class 1 CLC categories (Urban areas continuous and dis-
area, and eutrophication indicators for each lake.
continuous, industrial and commercial zones, communication infra-
Regarding water chemistry, mean annual total phosphorus con-
structures and networks, mines, etc.). IA was the cover of CLC cate-
centration (TP), mean summer (June–August) Chlorophyll-a con-
gories corresponding to a high potential impact from agricultural
centration (CHLA), mean summer water transparency (SD), mean
activities (arable and irrigated land, permanent and annual crops, vi-
summer pH values (PH) and mean summer electrical conductivity (EC)
neyards, orchards, olive groves, complex cultivation patterns; CLC
values were measured. Mean summer values were based on three
codes 2.1, 2.2, 2.4.1, 2.4.2). NASN was the cover of forest and natural
monthly samples, while mean annual TP values were based on at least
areas, wetlands and water bodies (CLC codes 3.1.1, 3.1.2, 3.1.3, 3.2,
one sample from each of four seasons for each lake. Transparency was
3.3, 4 and 5). Population data were acquired by the 2011 Population-
measured by means of a Secchi disk. Water samples for water chemistry
Housing Census (Hellenic Statistical Authority, 2011) and population
measurements were taken at the deepest part of the lakes, from the
density (PD) was calculated as inhabitants per square kilometer (h/
euphotic zone (2.5 × SD depth) by using either an integrated type or a
km2) in the catchment area.
Nansen type sampler (de Hoyos et al., 2014). Analyses of TP were
Finally, the Hellenic Lake Macrophyte assessment method values
carried out in unfiltered water using ascorbic method following per-
(HeLM) and its two-constituent metrics, Trophic Index for HeLM
sulfate digestion (Rice et al., 2012). CHLA measurements were carried
(TIHeLM) and Maximum Colonization Depth (Cmax), were used as
out in filtrated water through Whatman GF/F glass-fiber filters, by
eutrophication indicators for each lake (Zervas et al., 2018). TIHeLM is
using 90% acetone and applying the trichromatic equation (Jeffrey and
a trophic index metric based on taxon-specific trophic (phosphorus
Humphrey, 1975; Rice et al., 2012). PH and EC were measured with
optima) scores, weighted by their relative abundance in sampling sites,
portable meters from ThermoFisher Scientific.
therefore it grades lakes according to their eutrophication level as ex-
Two water level fluctuation parameters were used, the annual
pressed by vegetation's taxonomic composition (low TIHeLM va-
(WLFy) and the seasonal (WLFs) maximum water level fluctuation,
lues = low eutrophication levels). Cmax metric grades lakes by their
calculated as the difference between maximum and minimum water
aquatic vegetation expansion levels by simply expressing the maximum
levels in each lake during the whole year or during the spring and

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Table 3
Overview of mean, minimum – maximum values, standard deviation and variation coefficient of the environmental variables used as pressure indicators in the
studied lakes. See text (2.2 Methods – Pressure indicators) for delineation of variables. Total macrophyte coverage of the sampling area in the studied lakes is also
given.
Variable Code Mean Standard Deviation Variation Coefficient Min Max

Total Phosphorus (μg/L) TP 46,83 50,92 1,09 9,75 182,43

Chlorophyll-a (μg/L) CHLA 12,61 14,33 1,14 0,42 60,75
Water transparency (m) SD 3,51 3,14 0,89 0,58 10,57
pH PH 8,60 0,36 0,04 7,98 9,31
Electrical Conductivity (μS/cm) EC 600,28 451,10 0,75 174,45 2014,00
Water Level Fluctuation intra-annual (m) WLFy 1,20 0,87 0,73 0,29 4,20
Water Level Fluctuation intra-seasonal (m) WLFs 0,67 0,55 0,82 0,19 2,17
Artificial Land Use (%) ALU 2,34 2,44 1,04 0,00 9,33
Intensive Agriculture (%) IA 24,89 12,40 0,50 0,00 42,89
Natural and Semi Natural areas (%) NASNA 64,00 16,15 0,25 47,70 97,30
Population Density (h/km2) PD 45,76 53,27 1,16 0,00 220,85
Trophic Index HeLM TIHeLM 7,78 0,51 0,07 7,14 9,07
Maximum Colonization depth (m) Cmax 5,88 3,39 0,58 0,53 13,20
HeLM final values HeLM 0,70 0,19 0,28 0,20 1,00

Macrophyte coverage (%) 42,91 13,94 0.32 3,53 63,93

observed depth where submerged rooted macrophytes are present and
ranges from zero meters for hyper-eutrophic lakes to many meters for
oligotrophic lakes. HeLM values are the arithmetic combination of
these two aquatic vegetation metrics, assigning lakes to an ecological
status class (low HeLM values = low ecological quality) (Zervas et al.,
2018).
2.3. Functional traits
A total of 30 functional traits were selected for the analysis. The
selection of traits was based on their potential relationship to anthro-
pogenic pressures in lakes, as well as on their availability in literature
and online databases. A number of publications (e.g. Casper & Krausch,
1980–1981; Correll and Correll, 1972; Ellenberg et al., 1992; Krause,
1997; Pignatti et al., 2005; Tutin et al., 1968–1980, 1993; Willby et al.,
2000; Wood and Imahori, 1964, 1965) and online databases (Guiry and
Guiry, 2017; Julve, 1998; Kleyer et al., 2008; Klotz et al., 2002) were
consulted in order to collect all values needed. These traits cover a
number of different aspects of aquatic plant life, such as life forms,
growth morphology, survival characteristics, ecological preferences,
reproduction and dispersal attributes (Table 2).
2.4. Statistical analyses
In order to assess the relationships between functional trait com-
position and anthropogenic pressures, RLQ (Doledec et al., 1996) and
fourth-corner analysis (Legendre et al., 1997) were used. The data were
organized in three different tables for these analyses: a table R with the
environmental variables measured in each sampling locality, a table L
with the abundances of species found in each locality and a table Q with
the functional trait values for each species. In our data, each lake
sampled in different year represents a sampling locality. RLQ analyzes
the co-inertia found in all three tables, assigns scores to environmental
variables, species and functional traits and performs a simultaneous
ordination for all three types of variables (Dray et al., 2003). Fourth-
corner approach uses the species abundance data as a link to combine
each functional trait with each environmental variable into a matrix
and statistically evaluate the associations between them (Legendre
et al., 1997). One of the reasons for selecting these analyses for this
study is that both RLQ and fourth-corner are not affected by inter-
correlation of explanatory variables because the former is based on the
calculations of uncorrelated ordination axes, and the latter examines
bivariate associations between traits and explanatory variables. Fur-
thermore, these two analyses represent the most integrated methods to
analyze trait-environment relationships to date (Dray et al., 2014;
Kleyer et al., 2012).
The first step for RLQ analysis is to perform ordinations on each
table R, L and Q separately. The environmental table R, contained only
quantitative data, thus we applied a Principal Component analysis
(PCA). Correspondence analysis (CA) was performed on species data in
table L. Functional trait table Q contained both quantitative and cate-
gorical variables, therefore we used a Hill and Smith analysis for mixed
data (Hill and Smith, 1976). Afterwards, RLQ analysis was applied by
using the site and species scores from the CA of table L as row weights
in the PCA of table R and in the Hill and Smith of table Q, respectively.
The resulting graphs and reports show the maximum covariance be-
tween functional traits and environmental variables mediated by spe-
cies abundances (Dray et al., 2014). A Monte-Carlo randomization test
of the total inertia with 99999 permutations of model 6 (combination of
models 2 and 4, Dray and Legendre, 2008) to correct type I errors, was
performed in order to assess the statistical significance of the re-
lationship between the environmental variables and the functional
traits. Finally, in order to assess the relative importance of each func-
tional trait and environmental variable separately and identify the
prime factors affecting the trait-environment covariance, we used the
contribution to total inertia (Wesuls et al., 2012).
The fourth-corner method was applied to test the statistical sig-
nificance of all possible bivariate associations between individual
functional traits and environmental variables. Both stats “D2” (Pearson
correlation coefficient r) and “G” (Global statistic F) were used to
quantify the strength of the associations between each individual trait
variable separately and between categorical groups of traits respec-
tively (Legendre et al., 1997). Due to the number of traits and variables
used in the analysis, 99999 permutations of model 6 (Dray & Legendre,
2008) to correct type I errors were performed, using false discovery rate
method for adjusting P values to control the overall error rate (Dray
et al., 2014).
Finally, we took into consideration that RLQ analysis does not
provide a significance test so as to identify which combination of en-
vironmental variables acts on which combination of functional traits.
Additionally, the fourth-corner approach tests only the association be-
tween one trait and one environmental variable at a time and does not
take into consideration the covariation among traits and environmental
variables. Therefore, we applied the combined method proposed by
Dray et al. (2014). RLQ scores from the first two RLQ axes were used to
position functional traits and environmental variables on the RLQ fac-
torial map and fourth-corner statistics were used to detect associations
between environmental and trait syndromes. The significance of the
associations was tested again by 99999 permutations of model 6 (Dray
& Legendre, 2008) to correct type I errors, using false discovery rate

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D. Zervas, et al. Acta Oecologica 99 (2019) 103443

Fig. 1. Results of the first two axes of RLQ analysis. Environmental coefficients (a), trait coefficients (b), species scores (c) and eigenvalues of the analysis with the
first two axes in black (d) are presented. Grid size values are given (“d” values) for each graph for comparability. See Table 3 for the pressure and Table 2 for the trait
abbreviations. Species codes can be found at Supplementary Information S1.

method for adjusting P values to control the overall error rate (Dray
et al., 2014).
All analyses were performed by using ade4 package library (Dray
and Dufour, 2007) in R environment version 3.4.2 (R Core Team,
2017).
3. Results
Pressure variables among the studied lakes showed great variability
(Table 3), indicating that studied lakes cover a wide range of the an-
thropogenic pressure gradient.
The RLQ analysis explained fairly well the cross-covariance between
species functional traits and environmental variables. First two RLQ
axes alone explained 91.47% of the total inertia (1st axis = 80.32%;
2nd axis = 11.15%; Fig. 1d; Table 4). First axis (horizontal) differ-
entiated sites with low eutrophication impact, indicated by high HeLM,
Cmax and SD values, from sites with high eutrophication impact, in-
dicated by high TIHeLM, TP, CHLA and PD values (Fig. 1a; Table 4).
Second axis (vertical) correlated with PH and IA (Fig. 1a; Table 4), both
indicating anthropogenic pressures in the catchment area of the lakes.
Hydrological indicators WLFy and WLFs were correlated to both axes.
With regard to species functional traits, first axis was positively corre-
lated with the anchored-submerged leaves life form, tubular or capil-
lary leaf types, hydrogamous or autogamous gamete vectors and spore
fruit type, and negatively correlated with the anchored-emergent leaves
life form, high trophic and leaf area values, entire leaf types, anemo-
gamous or entomogamous gamete vectors and other than spores fruit
types (Fig. 1b; Table 4). Second axis was positively correlated also with
high trophic values, high Ellenberg Nutrient and Moisture index values,
hydrochorous propagule vector, great seed size and water stability
preference, and negatively correlated with anchored with emergent
leaves life form, high Ellenberg Light index values, other than hydro-
chorous propagule vectors, and fluctuating water preference (Fig. 1b;
Table 4). Species were also discriminated according these two axes
(Fig. 1c; see Supplementary Information S1 for species codes). Char-
ophytes were positioned at the right part of the first axis, elodeids were
positioned at the middle, while lemnids and nymphaeids were dis-
tributed on the left part of the first axis, following the eutrophication
gradient. Ceratophyllids were positioned at the upper part of the second
axis, while helophytes were distributed on the bottom left corner of the

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D. Zervas, et al. Acta Oecologica 99 (2019) 103443

Table 4
Summary of RLQ analysis results. For each pressure and trait variable, their relative contributions to the first two axes of the analysis and to the total inertia of the
analysis, are given. Pressure and trait variables are sorted (ordered) according to their relative contribution to total inertia of the analysis. Lower left corner
summarizes the variable contribution to the first two axes of the analysis. See Table 3 for the pressure and Table 2 for the trait abbreviations.
Order Pressure variables Inertia Contribution (%) Order Trait variables Inertia Contribution (%)

Axis 1 Axis 2 Total Axis 1 Axis 2 Total

01 TIHeLM 15.20 0.53 12.77 01 ansule 12.87 1.56 10.78

02 HeLM 15.32 0.02 12.65 02 gamvec 9.63 1.73 8.86
03 TP 12.84 15.52 12.44 03 troph 8.44 10.15 8.06
04 Cmax 13.35 5.24 11.80 04 FT 7.15 6.70 7.16
05 PD 11.62 3.15 10.14 05 LA 7.91 1.69 6.73
06 SD 6.96 5.10 6.72 06 LT 7.06 1.07 6.58
07 EC 4.76 4.14 5.38 07 provec 5.54 9.74 6.12
08 NASN 4.49 14.03 5.33 08 SZ 5.70 8.97 5.83
09 CHLA 4.40 0.44 4.66 09 ELL 5.80 4.84 5.39
10 IA 2.45 21.08 4.49 10 anemle 3.74 10.98 4.25
11 WLFy 3.28 3.36 4.05 11 Peren 4.40 0.41 3.74
12 ALU 3.49 2.46 3.66 12 amph 3.04 4.61 3.06
13 PH 0.03 23.52 3.34 13 wls 2.10 7.80 2.97
14 WLFs 1.81 1.41 2.57 14 MI 2.27 1.04 2.22
15 growth 2.36 0.59 2.10
16 frflsr 1.30 4.25 2.01
17 ELN 1.41 4.14 1.94

Summary Axis 1 Axis 2 Total 18 winter 2.17 0.29 1.91

Eigenvalues 3.05 0.42 3.80 19 stol 2.13 0.17 1.90

Total inertia projected (%) 80.32 11.15 100 20 fragm 1.94 0.01 1.83
21 ELM 0.49 5.41 1.13
Covariance 1.75 0.65 1.95 22 root 0.45 4.07 1.03
Correlation 0.32 0.23 0.62 23 frflsb 0.67 2.87 0.94
Environmental inertia projected cumulative ratio (%) 91.28 92.54 100 24 budd 0.36 1.65 0.74
25 anflle 0.14 0.40 0.56
26 wdep 0.37 0.04 0.55
Trait inertia projected cumulative ratio (%) 64.69 76.56 100 27 rhiz 0.30 1.44 0.54
28 ELR 0.01 2.81 0.39
29 anh 0.23 0.02 0.35
30 nodal 0.03 0.56 0.33

graph (Fig. 1c).
The statistical relationship between all pressure variables and spe-
cies functional traits was found highly significant (Monte-Carlo ran-
domization test; p-value = 0.00169). Among pressure variables, eu-
trophication indicators TIHeLM, HeLM and Cmax, as well as TP from
water chemistry and PD from catchment pressures were the ones that
contributed the most to RLQ analysis, adding up to 59.8% of total in-
ertia (Table 4). Land use indicators ALU, IA, NASN and water level
fluctuation variables WLFy and WLFs, contributed 20.1% of the total
inertia. With regard to functional traits, anchored-submerged leaves life
form had the largest contribution to total inertia with 10.78%, followed
by gamete vector categorical variable (8.86%), water trophic ecological
preference (8.06%), fruit type categorical variable (7.16%), leaf area
(6.73%) and leaf type categorical variable (6.58%) (Table 4). The traits
with the smallest contribution (less than 1% each) were nodal rooting,
anchored-heterophylly, anchored-floating leaves and free-floating sub-
merged life forms, budding and rhizome asexual reproduction, water
depth and Ellenberg soil reaction ecological preferences.
The fourth-corner method showed some significant associations
between pressure variables and species functional traits (Fig. 2). In
total, nine out of the 30 functional traits were found to associate sig-
nificantly (p > 0.05) with six out of the 14 different pressure variables,
in 15 out of 420 possible (30 × 14) bivariate associations (without
applying a p-adjustment method, the number of statistically significant
bivariate associations would be 53/420, see Supplementary Informa-
tion S2). Eutrophication indicators TIHeLM, Cmax and HeLM were in-
volved in most bivariate associations (10/15), followed by TP from
water chemistry, and NASN and PD from land use and catchment
pressures. Remarkably, neither of hydrological variables WLFy and
WLFs showed statistically significant association with any of the
functional traits. Anchored with submerged leaves life form was found
to be significantly positively associated with HeLM and negatively with
TIHeLM and TP, while anchored with emergent leaves life form was
positively associated with TP. Leaf area and water trophic preference
index were both negatively associated with HeLM and positively with
TIHeLM, while trophic preference index was also negatively associated
with Cmax and NASN. Ellenberg indicator light index was positively
associated with TP and seed size was negatively associated with Cmax.
Gamete vector, propagule vector and fruit type categorical variables
were found to be positively associated with PD, TIHeLM and Cmax,
respectively. Checking categorical trait variables separately (D2 stat,
see Supplementary Information S2), PD was positively associated with
entomogamous and negatively with hydrogamous gamete vectors, TI-
HeLM was positively associated with anemochorous propagule vectors
and Cmax was positively associated with spore fruit types and nega-
tively with non-fleshy fruit types.
Finally, the application of the combined RLQ and fourth-corner
methodologies was able to detect bivariate associations between pres-
sure and trait syndromes. The overall relationship between functional
trait and pressure syndromes was found statistically significant (Monte-
Carlo randomization tests; Model 2 p-value = 0.00018; Model 4 p-
value = 0.00173). First environmental RLQ axis (AxcR1) was found to
significantly associate with three out of the 30 functional traits
(Fig. 3a), positively with anchored-submerged leaves life form and
negatively with leaf area and lake trophic preference. No significant
association was found between traits and the second environmental
RLQ axis (AxcR2). First trait RLQ axis (AxcQ1) was found to sig-
nificantly associate with six out of the 14 pressure variables (Fig. 3b),
positively with SD, Cmax and HeLM (all indicators of clearer water
conditions), and negatively with TP, PD and TIHeLM, (indicators of

6
D. Zervas, et al.
Fig. 2. Results of the fourth-corner method (model 6, stat “G”, p adjustment
“fdr”, 99999 permutations) testing the bivariate associations between func-
tional trait characteristics (rows) and pressure variables (columns). Significant
(p < 0.05) positive and negative associations are represented by black and
grey cells, respectively. Non-significant associations are represented by white.
See Table 3 for the pressure and Table 2 for the trait abbreviations.
eutrophic conditions in lakes). No significant association was found
between the second trait RLQ axis (AxcQ2) and pressure variables.
4. Discussion
Our results showed that there are statistically significant relation-
ships between aquatic plant functional traits and various pressure in-
dicators that reflect anthropogenic alteration in lake ecosystems.
Moreover, we managed to associate specific functional traits as re-
sponses to specific human-induced pressures. These results indicate that
human pressures, which affect either directly a lake or indirectly
through its catchment area, can strongly influence the species selection
and assembly processes taking place in the aquatic plant communities,
by affecting specific functional species groups. The findings of our re-
search as to the total significance of macrophyte functional traits/
human-induced pressures relationship, as well as to the significance of
specific trait responses to anthropogenic pressures, are in line with the
work of Baattrup-Pedersen et al. (2016) in lowland streams and Pan
et al. (2017) in a eutrophic lake in China.
Life form trait “anchored plants with submerged leaves” was found
consistently among the traits responding strongly to pressure gradients
in all three methodological approaches applied in our data (RLQ,
fourth-corner analysis and their combination). Another life form trait
that explained a great ratio of the trait-pressure covariance and showed
significant association with TP was “anchored plants with emergent
leaves”. Pan et al. (2017) also found submerged/emergent life form
traits to be significantly associated with a number of environmental
parameters depicting lake eutrophication. Both these traits are con-
nected with the shift from a submerged macrophytic vegetation to an
emergent one, commonly observed in lakes under eutrophication
pressure (Kolada, 2014; Squires et al., 2002; van den Berg et al., 2003).
7
Acta Oecologica 99 (2019) 103443
Fig. 3. Results of the combination of RLQ and fourth-corner methodologies for
testing the associations between functional traits and pressure syndromes
(model 6, stat “G”, p adjustment “fdr”, 99999 permutations). (a) Fourth-corner
tests between functional traits (rows) and the two environmental axes from RLQ
analysis (AxcR1 and AxcR2). (b) Fourth-corner tests between pressure variables
(columns) and the two functional trait axes from RLQ analysis (AxcQ1 and
AxcQ2). Significant (p < 0.05) positive and negative associations are re-
presented by black and grey cells, respectively. Non-significant associations are
represented by white. See Table 3 for the pressure and Table 2 for the trait
abbreviations.
This shift has been found to be strongly related with a change from a
clear water state to a turbid one, thus reflecting a water deterioration
gradient (Hilt et al., 2010; Kolada, 2014; Søndergaard et al., 2010).
Nutrient loading promotes substantial phytoplankton growth in lakes,
leading to a decrease in light penetration and availability to submerged
macrophytes, resulting in their deterioration and their gradual re-
placement by emergent macrophytes (Blindow et al., 2006; Hilt et al.,
2006; Kolada, 2014).
Leaf area was also a trait found in our study to be significantly af-
fected by eutrophication. Interspecific variation of leaf traits has been
shown to represent the leaf investment strategies of plant species,
rendering them as useful indicators of plant community responses to
abiotic conditions (Lavorel and Garnier, 2002; Wright et al., 2004). Leaf
habit traits specifically have been found as useful tools in locating plant
species preferences on nutrient gradients (Garnier et al., 2001; Shipley
and Almeida-Cortez, 2003; Wilson et al., 1999). Interspecific leaf area
D. Zervas, et al.
traits follow a positive correlation with nutrient availability (Garnier
et al., 2001; Shipley and Almeida-Cortez, 2003; Wright et al., 2001) and
primary productivity (Asner et al., 2003; Garnier et al., 2001; Reich
et al., 1992), therefore species with higher values of leaf area are ex-
pected to outcompete those with lower ones under eutrophic condi-
tions. Leaf type is also a trait tightly connected with macrophyte life
forms and leaf area (Fu et al., 2014a, 2014b; Sand-Jensen, 2003). The
shift from a submerged macrophytic vegetation to an emergent one
inevitably leads to a shift from capillary leaf types to entire leaf ones.
As expected, water trophic-level preference was one more trait that
correlated strongly with eutrophication pressure indicators. On the
other hand, Ellenberg indicator values (ELL, ELM, ELR and ELN) did not
perform well. Light indicator was the only one that gave a statistically
significant association with TP (fourth-corner method), following the
dominance of photophilous rush communities observed under eu-
trophication pressures, while all Ellenberg indicator values had very
low contributions to the total trait-pressure inertia (RLQ method). We
expected that Ellenberg indicator for nutrients (ELN) would be asso-
ciated with water trophic-level but our analysis did not support this
expectation, a similar result with those presented by Kolada et al.
(2014). The reason may be that Ellenberg N values grades the pre-
ference of plant species to soil nitrogen status, while water trophic-level
preference, grades plant species according to phosphorus optima and
tolerance values within the water body (Ellenberg et al., 1992; Schaffers
and Sykora, 2000; Willby et al., 2000). While emergent macrophytes
(helophytes) have been found to respond more directly to soil char-
acteristics, submerged macrophytes are able to absorb nutrients from
both sediment and overlying water (Kolada, 2016; Partanen et al.,
2009; Raun et al., 2010). Taking into consideration the processes as-
sociated with sediment-water nutrient exchange, such as sediment iron-
bound phosphorus retention or sediment resuspension and phosphate
release (Fan et al., 2001; Song and Burgin, 2017), direct nutrient
availability or limitations in the water seem to be better indicated by
water trophic optima values than Ellenberg N values (Kolada et al.,
2014; Lyche-Solheim et al., 2013; Schneider, 2007).
Finally, reproduction and dispersal functional traits, gamete vectors,
propagule vectors, fruit type and seed size, were all found to be asso-
ciated significantly with eutrophication pressures. The main reason
behind this correlation is the species turnover observed along the eu-
trophication gradient (Egertson et al., 2004; Kolada, 2014; Sand-Jensen
et al., 2008; Zhong et al., 2017). The shift from a submerged macro-
phytic vegetation to an emergent one leads also to a shift in re-
production and dispersal mechanisms from those adapted to aquatic life
to those adapted to aerial life (Barrat-Segretain, 1996; Kautsky, 1988).
Therefore, as eutrophication pressure increases, aquatic vegetation in
lakes shifts from hydrogamous and hydrochorous plant species in oli-
gotrophic-mesotrophic conditions to entomogamous and anemochorous
ones in mesotrophic-eutrophic conditions. Most common fruit types
change from spores, a characteristic of charophytes which is a group of
submerged aquatic macrophytes indicative of oligotrophic or less de-
graded conditions (Kolada, 2014; Penning et al., 2008; Toth et al.,
2008), to achene and caryopsis types, characteristics of helophytes
which is a group of emergent aquatic macrophytes indicative of eu-
trophic or more degraded conditions (Kolada, 2014; Penning et al.,
2008; Toth et al., 2008). Alongside the fruit type, increased seed size is
also a characteristic of plant species adapted to emergent or amphibious
life strategies (Barrat-Segretain, 1996; Kautsky, 1988).
All significant associations between functional trait patterns and
anthropogenic pressures found in our study, involved direct or indirect
eutrophication indicators. Nutrient loading and eutrophication are
considered to be the most common direct stressors to freshwater eco-
systems, following widespread land cover changes driven by intensive
agriculture and urbanization in water catchment areas (Foley et al.,
2005; Vörösmarty et al., 2010). At the same time though, intensive
agriculture and urbanization raise water demand, either for irrigation
or drinking water supply purposes, leading to declining water tables in
8
Acta Oecologica 99 (2019) 103443
catchment areas and water withdrawal from surface waterbodies
(Alcamo et al., 2008; Foley et al., 2005). Therefore, hydro-morpholo-
gical pressures are also increasingly found to play an important role in
aquatic ecosystem alterations (Coops et al., 2003; Lyche-Solheim et al.,
2013; Manel and Cantonati, 2008). The extent, frequency and duration
of water level fluctuations along with lake-morphometric character-
istics can be important drivers of ecosystem processes in littoral areas
(Coops et al., 2003; Manel and Cantonati, 2008). In our study though,
no significant association was found between any of the functional
traits and the two hydrological parameters used, inter-annual water
level fluctuation (WLFy) and intra-monthly water level fluctuation
during growing season (WLFs). We suggest there may be three main
reasons behind this result, which call for extra study on these re-
lationships. Firstly, aquatic macrophytes respond differently to water
level changes. Hydrophytes that colonize the deepest parts of the lakes
are far less sensitive to water level fluctuations than helophyte com-
munities found at the vegetated littoral edges (Coops et al., 2003;
Dienst et al., 2004). Therefore, the identification and selection of sen-
sitive taxa is an important procedure when trying to connect macro-
phyte response to water level fluctuations (Hellsten, 2001; Mjelde et al.,
2012). Secondly, water level fluctuations are basically natural phe-
nomena on which many plant species depend for their survival and
reproduction. This means that there are response thresholds for aquatic
vegetation, with no apparent distribution changes if fluctuations are not
extreme enough to surpass the physiological limits of the plants (Coops
et al., 2004; Hill et al., 1998; Wantzen et al., 2008). Finally, vegetation
responses to water level fluctuations may vary greatly under simulta-
neous influence of a number of different parameters such as lake
morphometry, catchment characteristics, climate and water manage-
ment, all dependent by time-scale and spatial amplitude (Coops et al.,
2003; Manel and Cantonati, 2008; Wantzen et al., 2008).
This study was based on a limited dataset of 19 lake-years which
may be considered that sets certain limitations in the robustness of the
analyses applied in the study. However, the pressure variables used in
the analyses cover most of the relevant gradient of anthropogenic
pressures (Table 3) and may be considered sufficient to detect biolo-
gical responses in the 322 transects comprising the vegetation dataset.
Moreover, the nature of the analyses (i.e. multiple randomization per-
mutations) allows the detection and validation of statistically important
relationships in small data sets (Martens and Dardenne, 1998; Suresh,
2011). Taking into consideration that the current study is of explorative
nature, examining functional traits-anthropogenic pressures relation-
ships in a number of different lacustrine ecosystems, additional re-
search with larger datasets representing wider geographical areas may
add new insights concerning the above-mentioned relationships.
In order to provide a basis for the selection of appropriate functional
traits relevant to eutrophication and general degradation pressures in
freshwater lakes, we summarize our findings in Fig. 4. These findings
give insights into the underlying mechanisms of aquatic plant species
assembly patterns as influenced by the most common anthropogenic
pressures in freshwater ecosystems. These functional traits and their
responses could be taken under consideration in future studies ex-
ploring the functional diversity patterns or assessments of ecosystem
services under the influence of human-induced pressures in freshwater
lakes. Besides, biomonitoring of both structure and function of surface
water ecosystems, in order to express their ecological status and in-
crease understanding of the ecological characteristics necessary for
their conservation or restoration, is required for the implementation of
modern water resource management legislation. Under WFD, a number
of EU Member States developed biomonitoring systems that contain
some functional traits (more commonly life forms), in order to assess
the quality of their aquatic ecosystems (Poikane et al., 2015), but the
majority of them focused on monitoring individual structural para-
meters on the assumption that good quality of those elements equals to
good ecosystem functioning (Solimini et al., 2009; Voulvoulis et al.,
2017). Functional trait approaches and subsequent functional diversity
D. Zervas, et al.
Fig. 4. A graphical representation of the study's findings. Functional trait patterns (+increasing/- decreasing) are depicted in relation to human-induced eu-
trophication and general degradation pressure patterns (+increasing/- decreasing).
estimations may serve as the necessary tools towards the integration of
more effective assessment strategies, which will provide a holistic ap-
proach on monitoring and management of surface water ecosystems
(Reyol et al., 2014; Solimini et al., 2009; Vlachopoulou et al., 2014).
5. Conclusions
The relationship between the functional trait patterns of aquatic
macrophytes and the human-induced pressures and impacts in a
number of freshwater lakes was statistically assessed and found sig-
nificant. Eutrophication pressure was found to strongly influence spe-
cific functional trait responses of aquatic vegetation. The shift from a
submerged-macrophyte dominated vegetation community to an emer-
gent one was the most important functional response to high levels of
eutrophication in lakes. Submerged/emergent life forms and aquatic/
aerial reproduction and dispersal functional traits were among the
changes observed in the functional distribution patterns. Trophic and
light preferences also raised along the eutrophication gradient, while
leaf morphology traits were also highly influenced by eutrophication
levels. These findings are a glance on the functional changes occurring
in aquatic macrophyte communities over eutrophication and general
degradation gradients in freshwater lakes; in this respect they provide
an insight into functional diversity research, into the connection be-
tween ecological status and lake ecosystem functioning, and the de-
velopment of bio-assessment tools for monitoring and management
purposes in freshwater lakes.
Acknowledgements
The present study was executed in the frame of the Greek National
Water Monitoring Network, according to the JMD 140384/2011, im-
plemented by The Goulandris Natural History Museum, Greek Biotope/
Wetland Centre (EKBY). The study was co-financed by the European
Union Cohesion Fund (Partnership Agreement 2014–2020, MIS
5001204). The Network is supervised by the Directorate for the
Protection and Management of Water Resources of the Special
Secretariat for Waters of the Ministry of Environment and Energy. The
data used in this report come from Acts MIS 371010, 371138, 371140,
371144, 371145 of the Operational Program “Environment and
Sustainable Development” financed by the European Regional
Development Fund. Special thanks must be given to G. Poulis, for his
contribution to aquatic macrophyte samplings and identification and to
9
Acta Oecologica 99 (2019) 103443
E. Tsakiri for the identification of bryophytes. EKBY's personnel con-
ducted monitoring samplings and analysis of pressure data.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.actao.2019.103443.
Author contributions statement
D.Z. designed the study, carried out the macrophyte surveys, the
trait data collection and the statistical analyses, wrote the manuscript
and prepared the tables and figures, V.T. supervised macrophyte sur-
veys and collected the pressure data, A.S.K. and P.D. supervised the
statistical analyses, I.T. supervised the trait data collection, the statis-
tical analyses and manuscript writing; all authors reviewed the manu-
script.
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