Environ Earth Sci (2016) 75:627

DOI 10.1007/s12665-016-5442-7

ORIGINAL ARTICLE

Testate amoebae as indicators of water quality and contamination

in shallow lakes of the Middle and Lower Yangtze Plain
Yangmin Qin1,2 • Richard Payne3,4 • Xiangdong Yang5 • Min Yao5,6 •

Jiantao Xue1,2 • Yansheng Gu2 • Shucheng Xie1,2

Received: 29 August 2015 / Accepted: 6 February 2016

Ó Springer-Verlag Berlin Heidelberg 2016

Abstract Testate amoebae are micro-organisms charac- links between the environmental variables and testate
terized by an agglutinated or autogenous shell enclosing amoeba composition. Results showed that testate amoebae
the cytoplasm. Testate amoebae have been widely pro- are widely distributed in most of the lakes despite often
posed as valuable bioindicators in a range of ecosystems high levels of pollution. Our data highlight some links
(such as soils, peatlands and lakes). The use of testate between water quality variables and testate amoeba com-
amoebae as bioindicators of water quality in aquatic munities. In particular, our results suggest that heavy
ecosystems is much less developed than for other metals have a role in shaping testate amoeba assemblage
microorganisms and previous research is geographically structure, although correlations are comparatively weak,
restricted. We investigated a large range of environmental perhaps due to lagged responses. Our results support pre-
variables and their relations to testate amoeba communities vious suggestions that testate amoebae may be useful
from 37 shallow lakes in the middle and lower Yangtze bioindicators in aquatic ecosystems but emphasize the need
River Plain of China. Multiple factor analysis, redundancy for an improved mechanistic understanding.
analysis (RDA) and a forward-selection approach were
used to explore the overall community structure and the Keywords Testate amoebae  Lakes  Indicator  Middle
and Lower Yangtze Plain

Electronic supplementary material The online version of this

article (doi:10.1007/s12665-016-5442-7) contains supplementary Introduction
material, which is available to authorized users.

& Yangmin Qin The middle and lower Yangtze reaches of southern China
qinyangmin2005@163.com are known as the ‘country of a thousand lakes’ (Jin et al.
1
1990). This is one of only 17 regions recognized as both a
Department of Geography, School of Earth Science, China
University of Geosciences, 430074 Wuhan, China
Global Biodiversity Hotspot and a Global 200 Priority
2
Ecoregion (Mittermeier and Mittermeier 1997; Olson and
State Key Laboratory of Biogeology and Environmental
Geology, China University of Geosciences, 430074 Wuhan,
Dinerstein 1998). However, this region is also one of the
China most developed regions of China. With the development of
3
Environment, University of York,
extensive agriculture, fishing, farming and urbanization
Heslington York YO105DD, UK during recent decades, most lakes in this area have
4
Department of Zoology and Ecology, Penza State University,
undergone eutrophication (Shu et al. 1996; Cheng and Li.
Krasnaya str. 40, 440026 Penza, Russia 2006; Yang et al. 2005). The deterioration of drinking
5 water due to cyanobacterial blooms in many lakes has
State Key Laboratory of Lake Science and Environment,
Nanjing Institute of Geography and Limnology, Chinese become a serious socio-economic problem (Qin et al. 2004;
Academy of Sciences, Nanjing, China Dong et al. 2008; Wang et al. 2012; Shi et al. 2015). A
6
School of Environment and Planning, Liaocheng University, large amount of wastewater has been released into many of
252059 Liaocheng, China the lakes from industrial activities, residential areas and

123
627 Page 2 of 11 Environ Earth Sci (2016) 75:627

agriculture. In addition, the building of dikes and dams Materials and methods
since late twentieth century has isolated the lakes from the
Yangtze River, changed the natural hydrological conditions Study sites and sampling
and reduced the lake areas (Qin et al. 2009). Such human
activities have seriously damaged the aquatic ecosystems This study forms part of an ongoing research theme
leading to a decline in biodiversity, and threats to local investigating the ecology and paleoecology of lakes in the
human health (Dearing et al. 2012; Wang et al. 2012; Shi middle and lower Yangtze River plain (Dong et al. 2008;
et al. 2015). Limnological investigations are essential to aid Yang et al. 2008; Yao et al. 2011). The lakes were
future long-term watershed management and sustainable selected beside the Yangtze River and sampled beginning
development (Dong et al. 2008; Yang et al. 2008; Wang at Shanghai City in the estuary of Yangtze River to
et al. 2012; Chen et al. 2014). Wuhan, the middle reach of Yangtze River. In total we
An increasing body of evidence suggests that microor- sampled 37 shallow lakes, with one sample from nearly
ganisms such as protozoa and diatoms are sensitive to a the middle or the deepest point of each lake (Fig. 1). The
wide variety of environmental changes (Charman et al. lakes span a broad range of physical and chemical gra-
1998; Bobrov et al. 1999; Booth 2001; Beyens and Meis- dients. The lakes span more than 800 km and range in
terfeld 2001; Mitchell et al. 2008; Mazei et al. 2012; Payne depth from 1.2 to 6.55 m. Samples from four of these
2013; Lamentowicz et al. 2015; Li et al. 2015; Song et al. lakes were analyzed for testate amoebae, but do not have a
2014). High species diversity, rapid responses to environ- complete set of environmental data so these are only
mental change and cosmopolitan distribution mean that briefly discussed.
microorganisms can be useful bioindicators in ecological Water samples were taken seasonally from July 2007 to
and palaeoecological studies (Qin et al. 2013; Payne 2013). July 2008 and analyzed for water chemistry within 1 week
Recent research has suggested that a group of protozoa of collection. Sediment samples were also collected on
with particular potential as bioindicators foidentifications these sampling occasions but proved insufficiently large for
were made with the aid of Penardr water quality is the metal and testate amoeba analysis. Additional sediment
testate amoebae. Testate amoebae have been suggested to samples were therefore collected from each lake in October
respond to anthropogenic eutrophication, acidification and 2010 and the upper 0.5 cm sub-sampled for analysis. These
metal inputs in a number of studies worldwide (Asioli sediment samples are likely to integrate conditions in the
et al.1996; Mitchell et al. 1999; Patterson and Kumar 2000; lake over several years so the 2-year gap between water
Kauppila et al. 2006; Escobar et al.2008; Neville et al. and sediment sampling is unlikely to have a large influence
2010; Kihlman and Kauppila 2012; Ju et al. 2014; on our results.
Macumber et al. 2014; Roe and Patterson 2014).
Testate amoebae are micro-organisms characterized by an Water and sediment chemistry
agglutinated or autogenous shell enclosing the cytoplasm
(Mitchell et al. 2008). Their high abundance and responses to Results of water quality analyses have been previously
environmental stress which are both large-magnitude and published (Yang et al. 2008; Yao et al. 2011). Chemical
distinctive, suggest considerable potential to indicate water variables including total phosphorus (TP), total nitrogen
conditions. Excellent preservation of tests in lacustrine (TN), nitrate (NO3-N), nitrite (NO2-N), ammonia
sediments also shows potential for the reconstruction of past (NH4-N), chlorophyll-a (Chl-a), total iron, chemical oxy-
trajectories of change in water quality (Patterson et al. 2002; gen demand (COD), SiO2, dissolved inorganic phosphorus,
Tsugeki et al. 2003; Escobar et al. 2008; Roe et al. 2010; Qin CO3-, and HCO- were determined using standard tech-
et al. 2011). It is reasonable to expect that testate amoebae niques (Institute of Hydrogeography and Engineering
will respond to aquatic pollution by metals or nutrients but Geology, MGMR 1990; Jin et al. 1990). K? and Na?
previous studies have been restricted in terms of their geo- concentrations in water were measured by inductively
graphic location (particularly in North America) and the coupled plasma-atomic emission spectrometry (ICP-AES;
types of lakes and pollutants considered. Leeman-Labs Profile, Hudson, NH, USA.. Concentrations
This study focuses on the water quality of lakes within of Ca2?, Mg2?, and SO42- were analyzed using EDTA
the middle and lower Yangtze River plain and the potential titrimetric methods. The argentometric method (AgNO3)
role of testate amoebae as bioindicators. The aims are to was used for Cl- determination (Greenberg et al. 1992).
investigate the testate amoeba community structure, test Water pH and conductivity were measured in the field
the physico-chemical controls on amoeba communities and using a HI-214 conductivity meter and Hanna EC-214 pH
to understand how the testate amoeba community responds meter (HANNA instruments, Canada). Data used in our
to human activities in contaminated shallow lakes. analyses are means of the four seasonal samples.

123
Environ Earth Sci (2016) 75:627 Page 3 of 11 627

Fig. 1 Map showing the

location of sample sites in the
middle and lower reaches of
Yangtze River. See Table 1 for
names of the lakes

Metal concentrations in sediments were determined in procedure of Patterson and Kumar (2000): sediment sam-
the State Key Laboratory of Geological Processes and ples were washed through 500 and 35 lm sieves into pertri
mineral Resource, China University of Geosciences dishes to retain the testate amoeba shells. However, we
(Wuhan). Sediment samples were dried, ground and passed note that sieve sizes used to retain testate amoeba shells
through 180 lm mesh. Sieved 20 mg samples were acid have varied considerably, including sieve sizes larger and
digested and analyzed by ICP-MS to determine heavy smaller than that used in this study and that the smallest
metal concentrations in sediments (Supplementary tests may pass through the mesh (Charman et al. 1998;
Table 2). Metal oxide weight percentages were normalized Patterson and Kumar 2000; Patterson et al. 2002; Payne
to 100 % and corrected by the ablation yield correction and Mitchell 2009; Avel and Pensa 2013). As material and
factor (AYCF) (Moor et al. 2001; Liu et al. 2008). test concentration were limited we analyzed all tests in the
dishes. Slides were prepared and examined using a stere-
Testate amoeba analysis omicroscope (Olympus SZX16) at 70–2009 magnification
for species identification. In counting we aimed for at least
Wet subsamples of about 5 g of benthic sediment were 100 shells, however, the abundance of testate amoebae was
analyzed for testate amoebae. Isolation of testate amoeba very low in some samples, with as few as 32 individuals.
from the sediment samples using a modified version of the We acknowledge that these relatively low total are sub-

123
627 Page 4 of 11 Environ Earth Sci (2016) 75:627

Table 1 Summary details for lakes studied fications were made with the aid of Penard (1902), Ogden
# Site name Depth Richness Shannon H Count total
(1983), Shen (1983), Kumar and Dalby (1998).

1 Qinglan 2.55 9 1.73 54 Numerical analyses

2 Baidang 1.7 15 2.49 78
3 Fengsha 1.325 12 2.06 155 We compiled the testate amoeba data and converted to
4 Niuya 1.9 11 2.00 56 percentages of the total count. To quantify sample diversity
5 Jinxi 2.075 7 1.77 41 we determined the Shannon diversity index (SDI), calcu-
6 Yaohu 1.45 12 2.15 52 lated as follows:
7 Qingshanhu 1.265 11 2.30 88 X S    
Xi Xi
8 Banghu 1.9 9 1.58 132 S:D:I ¼   ln
i¼1
N i N i
9 Shahu 2.325 12 2.03 148
10 Junshan 2.3 11 2.02 52 where Xi is the abundance of each taxon in a sample, Ni is the
11 Poyang 3.75 12 2.24 135 total abundance of the sample, and S is the species richness of
12 Huangbo 1.225 14 2.20 66 the sample (Shannon and Weaver 1949). The Shannon
13 Huangni 1.8 13 2.28 106 diversity index assumes that all individuals are represented
14 Pogang 1.525 10 1.68 112 in the sample and are randomly sampled from an ‘‘infinitely
15 Shengjin 1.775 14 2.06 141 large’’ population (Shannon and Weaver 1949).
16 Taipo 2.425 17 2.47 129 To explore the overall structure of the environmental
17 Huayuan 2.3 5 1.02 87 and testate amoeba data we first conducted non-metric
18 Tuohu 2.35 5 1.32 56 multi-dimensional scaling ordinations of both testate
19 Nvshan 6.55 5 1.05 34 amoebae and environmental datasets (Clarke 1993). The
20 Qili 3.2 4 1.11 32 species data was Hellinger transformed prior to all analyses
21 Luoma 3.8 12 2.22 70 (Legendre and Gallagher 2001). To identify groupings of
22 Hongze 3.9 9 2.02 37 samples with similar environmental or species character-
23 Baima 1.325 7 1.62 81 istics we used cluster analysis based on UPGMA (Un-
24 Jinhu 2.6 8 1.52 68 weighted Pair Group Method with Arithmetic Mean). We
25 Gaoyou 3.15 7 1.70 44 used IndVal (Dufrêne and Legendre 1997; Podani and
26 Shaobo 2.55 10 1.75 97 Csányi 2010) to identify indicator species for sample
27 Shanghu 3.175 14 2.56 42 groupings based on the first splits of the cluster analysis of
28 Tuanjiu 1.2 9 1.78 62 environmental data.
29 Dongjiu 1.35 11 1.76 131 To examine the links between species composition
30 Caohu 3.35 10 2.05 78 and environment we first simultaneously ordinated both
31 Kuncheng 2.15 7 1.16 98 the species and environmental data using multiple factor
32 Ezhendang 3 10 1.82 131 analysis (MFA: Escofier and Pages 1994). To quantita-
33 Shijiu 2.05 4 0.99 70 tively test the explanatory power of environmental
34 Yanghu 2.35 7 1.16 103 variables we used redundancy analysis (RDA; Legendre
35 Nanhu 2.85 10 1.71 67 and Legendre 2012). The environmental data consisted
36 Huangmaotan 1.875 13 2.36 75
of 55 distinct environmental variables, compared to 33
37 Xiujiu 1.85 7 1.61 17
sites and 23 taxa. To reduce the variables included in
the redundancy analysis to a manageable number we
Numbers correspond to locations shown on Fig. 1 used a forward-selection approach in which variables
Sites shown in italics have incomplete environmental data and are not were successively added according to their explanatory
included in statistical analyses
power while accounting for variables already selected
(999 Monte Carlo permutations, cut-off at P \ 0.05).
optimal, but given the lack of previous research believe This approach allowed us to objectively select a mini-
that our results are still of value. Simulation studies suggest mum suite of explanatory variables but it is important to
that even low totals can give valuable data on community note that selection does not imply causation as many
structure (Payne and Mitchell 2009). Our results should be variables are strongly correlated (Supplementary
interpreted in the light of the count totals. Species identi- Table 1).

123
Environ Earth Sci (2016) 75:627 Page 5 of 11 627

Results a mean taxon richness of 9.8. Shannon diversity varied

from 0.99 to 2.56 supporting the low diversity of the
Water quality community when considering evenness as well as richness.
The most abundant taxa were Centropyxis ecornis (mean
All the studied lakes are quite shallow with depth ranges abundance 21.4 %), Centropyxis cassis (15.3 %), Difflugia
from 1.2 to 6.55 m, and weakly alkaline, with pH values oblonga (13.5 %) and Centropyxis aculeata (12.7 %).
varying between 7.6 and 8.3. Across the 37 studied lakes, pH These are all taxa which are relatively frequently found in
ranged from 7.55 to 8.27, Ca2? from 4.78 to 41.56 lg/L, lakes and other wet habitats (Booth 2001; Warner et al.
Mg2? from 2.45 to 15.57 lg/L, concentrations of TP ranged 2007; Payne et al. 2008; Qin et al. 2009; Swindles et al.
from 10 to 390 lg/L, TN ranged from 320 to 3320 lg/L, 2009; Roe et al. 2010; Sullivan and Booth 2011; Patterson
conductivity from 67.5 to 700.5 lS/cm2 and chlorophyll a et al. 2013a; Turner et al. 2013; Lamentowicz et al. 2015).
from 1.25 to 38.53 lg/L (Supplementary Table 2). No taxa were present in all samples and some species were
Many of the environmental variables are strongly corre- notably abundant in a few samples but comparatively rare
lated. Correlations are particularly strong between many of overall (e.g., Centropyxis platystoma and Pontigualsia
the heavier elements with atomic mass above 60 (Cu-Pb). In compressa). Overall the community composition is
this range most correlations are significant (P \ 0.05) and notable for the dominance of taxa with xenosome tests, and
many very strong (Supplementary Table 1). Strong and Difflugia species in particular (14 of 23 taxa).
significant correlations are also apparent between many
variables associated with nutrient status such as TP, TN, Multiple factor analysis and ordination
NO3-N, NH3-N, etc. (Supplementary Table 1). These cor-
relations most likely represent commonality of source. The Non-metric Multidimensional Scaling (NMDS) ordi-
nation plot for environmental variables (Fig. 2a) shows
Testate amoebae species composition strong clustering of around half the sites but with others
strongly scattered along the two axes. Lakes Kuncheng,
The testate amoeba community overall was comparatively Caohu, Ezhendang and Qingshanhu have high scores on
species-poor with only 23 taxa identified (Tables 1, 2) and axis one and are distinguished from the other sites in the

Table 2 Details of testate

# Taxon N Minimum % Maximum % Mean %
amoeba communities of the
sites 1 Arcella discoides 14.0 0.0 21.6 2.9
2 Centropyxis aculeata 20.0 0.0 67.1 12.7
3 Centropyxis cassis 29.0 0.0 50.0 15.3
4 Centropyxis ecornis 29.0 0.0 64.7 21.4
5 Centropyxis platystoma 11.0 0.0 41.1 2.6
6 Cucurbitella tricuspis 16.0 0.0 27.3 4.5
7 Difflugia amphoralis 2.0 0.0 2.3 0.1
8 Difflugia acuminata 13.0 0.0 7.3 1.2
9 Difflugia acutissima 19.0 0.0 16.1 2.6
10 Difflugia bicornis 4.0 0.0 3.9 0.2
11 Difflugia biwae 1.0 0.0 5.9 0.2
12 Mediolus (Difflugia) corona 1.0 0.0 14.8 0.8
13 Difflugia elegans 17.0 0.0 25.6 4.1
14 Difflugia lanceolata 11.0 0.0 24.3 1.4
15 Difflugia mammillaris 6.0 0.0 7.7 0.8
16 Difflugia oblonga 32.0 0.0 37.0 13.5
17 Difflugia pulex 18.0 0.0 27.3 4.3
18 Difflugia urceolata 20.0 0.0 8.2 1.7
19 Difflugia smilion 15.0 0.0 12.8 1.9
20 Difflugia sp.1 1.0 0.0 2.9 0.1
21 Lesquereusia modesta 4.0 0.0 5.4 0.3
22 Pontigulasia compressa 19.0 0.0 34.2 3.7
23 Pontigulasia incisa 17.0 0.0 17.3 3.3

123
627 Page 6 of 11 Environ Earth Sci (2016) 75:627

Fig. 2 Non-metric multidimensional scaling plots for a Environmen- A omits some site names where points are tightly clustered. Note
tal variables and b Hellinger transformed species data. Differences in difference in stress between the two plots
colour represent groupings based on a UPGMA cluster analysis. Plot

first split of the cluster analysis. These sites are noted by

high abundance of several heavy metals including Zn (up
to 789 ppm) and Cr (up to 319 ppm) and TN (up to
3.3 mg/L, all in Kuncheng Lake). The NMDS results
highlight Kuncheng Lake as a clear extreme outlier in
terms of these variables and this site is assigned its own
grouping in further cluster analysis splits. On axis 2, sites
Tuanjiu, Dongjiu, Nanhu, Luoma and Tuohu have notably
high scores and Tuanjiu and Dongjiu form a distinct group
in the cluster analysis. This gradient is weaker but appears
to be associated with base richness with these sites typi-
cally having higher values for variables such as Mg2?,
Ca2? and Na?. The NMDS plot for species (Fig. 2b) fits
the data less well but suggests less extreme scattering of
sites. Cluster analysis first separates species with low and
high scores on axis 1 and then splits samples with higher
axis 1 scores along axis 2. There is little obvious simi-
larity between the two NMDS plots. The sites which have
notably high scores on particularly axis one in Fig. 2a
(e.g., Kuncheng Lake) are not distinguished on Fig. 2b. Fig. 3 Multiple factor analysis plot of testate amoeba species (blue)
We found no significant IndVal indicator species and environmental data (red). Only variables with more extreme axis
(P \ 0.05) for UPGMA clusters based on environmental scores are labelled
data.
The Multiple Factor Analysis (MFA) plot (Fig. 3) sug- The first selected variable in RDA forward selection is
gests some possible links between species and environ- Praseodymium (Pr), a rare earth metal explaining 7.3 % of
mental variables. Axis 1 is positively correlated with variability. This illustrates the problem of forward selection
environmental variables including TN, conductivity and when environmental data includes numerous correlated
some metals, and negatively correlated with some moder- variables. Pr is a very rare element and found at low
ately abundant taxa such as Difflugia pulex and Pontigu- concentrations in our samples (mean 10.6 ppm) but is
lasia incisa. Two of the most abundant taxa C. aculeata strongly correlated with many other heavy metals (Sup-
and C. ecornis are negatively correlated with axis 2; many plementary Table 1). Our result clearly indicates a rela-
of the lanthanide metals are positively correlated with this tionship between the testate amoeba community and a suite
axis. of heavy metals rather than Pr per se. Other variables

123
Environ Earth Sci (2016) 75:627 Page 7 of 11 627

Table 3 Variables identified in RDA forward selection and Discussion

explanatory power as sole environmental variable, listed in order of
selection
Water quality
Variable Variance explained (%) P
According to the OECD (1982) nutrient classification
Pr 7.3 0.03
system (Ryding and Rast 1989), 20 of these lakes can be
Conductivity 5.8 0.08
classed as eutrophic (TP: 35–100 lg/L) and 13 hypereu-
Chlorophyll a 5.0 0.12
trophic (TP [ 100 lg/L). Three lakes are mesotrophic
NO3-N 5.8 0.08
((TP \ 35 lg/L)) while only one lake (Lake Baidang) has
SiO2 6.3 0.06
TP less than 10 lg/L. The concentrations of heavy metals
Mg 3.6 0.3
are relatively high indicating that the water in most lakes is
All above variables: 36.4
heavily polluted. These indications of high pollutant
Note that not all variables are significant at P \ 0.05 by this test loadings can be attributed to high inputs from adjacent
(these shown in italics) industry since the early twentieth century (Hu et al. 2012)
with levels of heavy metals sufficient to be toxic to aquatic
organisms (Yang et al. 2011). Previous limnological and
palaeolimnological investigations in the lakes of this region
suggest that the increasing concentrations of heavy metals
and nutrients including TP are primarily linked to the rapid
increase in intensity of human activities since the early to
middle 20th century (Dong et al. 2008; Yang et al. 2008;
Qin et al. 2009; Dearing et al. 2012; Wang et al. 2012).
This has resulted in serious eutrophication and loss of
biodiversity. The reduction in water quality is acknowl-
edged to threaten the security of drinking-water resources
and the stability of ecosystems and is an acknowledged
issue for environmental management and policy in the
region (Qin et al. 2004; Shi et al. 2015).

Testate amoeba community composition

Shannon diversity of these samples was low, falling

between 0.99 and 2.56 (although note the low counts used
to derive these figures). Previous aquatic testate amoeba
studies have suggested that ‘healthy’ lakes have SDI values
Fig. 4 RDA results based on forward selection of environmental [2.5 (Patterson and Kumar 2000; Roe and Patterson 2006;
variables. Environmental vectors exaggerated 59 for clarity and some Patterson et al. 2012). Our results suggest that these lakes
taxa with short vectors unlabelled
have impoverished faunas, most likely due to the impacts
of pollution.
In previous testate amoeba studies from lakes in China,
identified as important by forward selection include (in several unusual taxa such as Diffilugia biwae, D. tuber-
order of selection): conductivity, chlorophyll a, NO3-N, spinifera, D. mulanensis and Pentagonia zhangduensis
SiO2 and Mg. A total of 36.4 % of variability is explained have been identified which are endemic to eastern Asia
by these variables, although all but Pr are non-significant (Tsugeki et al. 2003; Yang et al. 2004, 2005; Qin et al.
when tested independently (Table 3). Our results therefore 2008, 2011; Gomaa et al. 2015). The community of these
imply that the testate amoeba community is affected by samples, however, is primarily composed of ubiquitous
heavy metals and also by nutrient availability (albeit species which are abundant in lakes around the world
weakly). The ordination plot (Fig. 4) suggests that heavy (Reinhardt et al. 1998; Patterson et al. 2002; Escobar
metals (typified by Pr) are positively correlated with three et al.2008; Neville et al. 2010; Roe et al. 2010; Patterson
comparatively rare taxa: D. bicornis, Mediolus (Difflugia) et al. 2013b). D. biwae was firstly detected in the sediments
corona and P. compressa and negatively correlated with D. of Lake Biwa in an early phase when the water was olig-
acuminata and L. modesta. C. tricuspis is positively cor- otrophic (Kawamura 1918). Palaeoecological evidence
related with SiO2 and NO2-N. shows that the abundance of D. biwae decreased rapidly

123
627 Page 8 of 11 Environ Earth Sci (2016) 75:627

due to declining water quality after the middle 1960s dominated by both Difflugia and Centropyxis taxa with no
(Tsugeki et al. 2003). A similar decline was also found in clear shift in dominance between these groups along pol-
Lake Zhangdu where the abundance of D. biwae declined lution gradients. Given the limited previous research on the
with increasing eutrophication and hydrological modifica- influence of heavy metals on testate amoebae the mecha-
tion since 1970s (Qin et al. 2009). D. biwae can thus be nisms underlying species responses are difficult to explain
considered as an indicator of oligotrophic conditions. In the and are likely to be complex.
37 lakes of this study, D. biwae was only found in Lake
Poyang, the largest freshwater lake in China connecting to Potential for bioindication
the Yangtze River and a lake with relatively good water
quality. It seems probable that all of the other lakes we Overall our results highlight how little is known of testate
consider have become too eutrophic for D.biwae. Other amoeba ecology in aquatic systems in general, and par-
regional endemics (D. tuberspinifera, D. mulanensis and ticularly in highly polluted lakes. Our environmental
Pentagonia zhangduensis) are also believed to be present dataset includes a very large number of variables, including
primarily in oligotrophic conditions and were not detected most that would be expected to be important determinants
in our samples. It is therefore probable that pollution has of testate amoeba community composition. However, the
caused a loss of these species; palaeolimnological studies proportion of variance explained by these variables is
would be valuable to confirm this. It is possible that pol- unexpectedly low. Our strongest evidence is for impacts of
lution may be a systemic threat to the endemic testate heavy metals, but the results also provide some suggestion
amoeba species of China. of links to nutrient status. Further work will be required to
confirm the potential of testate amoebae as bioindicators in
Response of testate amoebae to pollution these systems. These lakes have unique histories of human
impact, with some impacted by early industrial develop-
The Multiple Factor Analysis (MFA) plot (Fig. 3) suggests ment while others have been comparatively undisturbed
axis 1 is more related to taxa such as Difflugia mammil- until recently. The sediment samples we analyse are likely
laris, D. pulex and Pontigulasia incisa while axis 2 is to integrate testate amoebae over a comparatively long
negatively correlated with two of the most abundant taxa, period of time. It may be that the relatively weak correla-
C. aculeata and C. ecornis. Axis 1 is correlated with many tions we detect are because the testate amoeba community
general water quality indicators, including TN and con- is still adapting to a changed environment. It is also pos-
ductivity, Ca, Cu, Cr, Cl and Na, while axis 2 is primarily sible that the gap between water and sediment sampling or
correlated with many lanthanide series metals, suggesting other, unmeasured, variables are also important. The
specific responses of different species to differing aspects comparatively low count totals may also have impaired our
of pollution. Pr is the most significant correlate to testate ability to detect significant correlations. As a group of
amoeba community composition in redundancy analysis. protists which are abundant and perform important func-
Pr is a trace element but widely used in several industrial tional roles in aquatic ecosystems the potential of testate
fields like petrochemicals and metallurgy, including the amoebae as bioindicators in highly polluted lakes deserves
manufacture of electric motors. Our results cannot show further detailed attention.
that Pr per se has an influence on testate amoebae but do
imply that heavy metal concentrations have an influence on
assemblage structure. Conclusion
In previous studies on testate amoebae ecology in lakes
in the world it has been considered that most Difflugia and The use of testate amoebae as bioindicators of pollution in
Pontigulasia taxa are more abundant in mesotrophic to aquatic ecosystems is much less developed than for other
eutrophic conditions or with high pH (Asioli et al. 1996; micoorganisms, particularly diatoms. Previous studies
Patterson et al. 1996; Beyens and Meisterfeld 2001; Kihl- demonstrate considerable potential for testate amoebae to
man and Kauppila 2012; Macumber et al. 2014; Roe and indicate various aspects of anthropogenic impact on lakes
Patterson 2014). While most Centropyxis taxa are oppor- but these studies have been geographically restricted. Our
tunistic and capable of existing in oligotrophic water, low study considers an unprecedentedly large range of envi-
organic content, high concentration metal contaminated ronmental variables and their relations to testate amoebae
waters (Patterson et al. 1996; Kauppila et al. 2006; Kihl- communities from lakes in China. Results show that these
man and Kauppila 2009, 2012). However, they also lakes—many of which are highly disturbed—do, never-
recently reported can also tolerate to mesotrophic and theless, host testate amoebae but that communities are
highly eutrophic conditions (Patterson et al. 2013b). In this species poor and lack characteristic regional endemics. We
study the community of these polluted lakes was found links between testate amoebae and their environment

123
Environ Earth Sci (2016) 75:627 Page 9 of 11 627

but disentangling precise environmental controls is com- Escobar J, Brenner M, Whitmore TJ, Kenney WF, Curtis JH (2008)
plicated by correlations between many variables. Our data Ecology of testate amoebae (thecamoebians) in subtropical
Florida lakes. J Paleolimnol 40:715–731
provides the strongest support for impacts of heavy metals Escofier B, Pages J (1994) Multiple factor analysis (AFMULT
but also suggest a role for nutrient status in shaping testate package). Comput Stat Data An 18:121–140
amoeba assemblages. Our results support previous sug- Gomaa F, Yang J, Mitchell EAD, Zhang WJ, Yu Z, Todorov M, Lara
gestions that testate amoebae may be useful bioindicators E (2015) Morphological and molecular diversification of Asian
endemic Difflugia tuberspinifera (Amoebozoa, Arcellinida): a
but also show that controls may be complex and involve case of fast morphological evolution in protists? Protist
many pathways of impact. Clearly further research will be 166(1):122–130
required. Greenberg AE, Clesceri LS, Eaton AD (1992) Standard methods for
the examination of water and wastewater. American Public
Acknowledgments This work was supported by 973 program Health Association, Washington, DC, pp 3/34–4/138
(2011CB808800), the National Natural Science Foundation of China Hu Y, Qi S, Wu C, Ke Y, Chen J, Chen W, Gong X (2012)
(No. 41530753, 41330103, 41502167), and the 111 project (B08030) Preliminary assessment of heavy metal contamination in surface
and. RJP was supported by the Russian Scientific Fund, Grant 14-14- water and sediments from Honghu Lake, East Central China.
00891. Front Earth Sci 6:39–47
Institute of Hydrogeography and Engineering Geology, MGMR
(1990) Water quality analysis. Geology Press, Beijing (in
Chinese)
Jin X, Liu H, Tu Q, Zhang Z, Zhu X (1990) Lake eutrophication in
References China. China Environment Science Press, Beijing (in Chinese)
Ju LH, Yang J, Liu LM, Wilkinson DM (2014) Diversity and
Asioli A, Medioli FS, Patterson RT (1996) Thecamoebians as a tool distribution of freshwater testate amoebae (Protozoa) along
for reconstruction of paleoenvironments in some Italian lakes in latitudinal and trophic gradients in China. Microb Ecol
the foothills of the southern Alps (Orta, Varese and Candia). 68:657–670
J Foraminifer Res 26:248–263 Kauppila T, Kihlman S, Mäkinen J (2006) Distribution of arcel-
Avel E, Pensa M (2013) Preparation of testate amoebae sa mples af- laceans (Testate Amoebae) in the sediments of a mine water
fects water table depth reconstructions in peatland palaeoeco- impacted Bay of Lake Retunen, Finland. Water Air Soil Poll
logical studies. Est J Earth Sci 62:113–119 172:337–358
Beyens L, Meisterfeld R (2001) Protozoa: Testate amoebae. In: Smol Kawamura T (1918) Japanese Freshwater Biology. Syoukabou,
JP, Birks HJB, Last WM (eds) Tracking Environmental Change Tokyo, Japan: 113–115 (in Japanese)
Using Lake Sediments. Kluwer Academic Publishers, Dordrecht, Kihlman S, Kauppila T (2009) Mine water-induced gradients in
pp 121–153 sediment metals and arcellacean assemblages in a boreal
Bobrov AA, Charman DJ, Warner BG (1999) Ecology of testate freshwater bay (Petkellahti, Finland). J Paleolimnol 42:533–550
amoebae (Protozoa: Rhizopoda) on peatlands in western Russia Kihlman S, Kauppila T (2012) Effects of mining on testate amoebae
with special attention to niche separation in closely related taxa. in a Finnish lake. J Paleolimnol 47:1–15
Protist 150(2):125–136 Kumar A, Dalby AP (1998) Identification key for Holocene lacustrine
Booth RK (2001) Ecology of testate amoebae (protozoa) in two lake Arcellacean (Thecamoebian) taxa. Paleontol Soc 1:1–36
superior coastal wetlands: implications for palaeoecology and Lamentowicz M, Gałka M, Lamentowicz Ł, Obremska M, Kühl N,
environmental monitoring. Wetlands 21:564–576 Lücke A, Jassey VEJ (2015) Climate change over the last
Charman DJ, Roe HM, Gehrels WR (1998) The use of testate 4000 years in a Baltic bog in northern Poland revealed by a trait-
amoebae in studies of sea-level change: a case study from the based approach, biotic proxies, and stable isotopes. Palaeogeogr
Taf Estuary, south Wales, UK. Holocene 8:209–218 Palaeocl 418:261–277
Chen X, Qin Y, Stevenson MA, McGowan S (2014) Diatom Legendre P, Gallagher ED (2001) Ecologically meaningful transfor-
communities along pH and hydrological gradients in three mations for ordination of species data. Oecologia 129:271–280
montane mires, central China. Ecol Indic 45:123–129 Legendre P, Legendre L (2012) Numerical Ecology. Elsevier,
Cheng X, Li S (2006) An analysis on the evolvement processes of Amsterdam
lake eutrophication and their characteristics of the typical lakes Li H, Wang S, Zhao H, Wang M (2015) A testate amoebae transfer
in the middle and lower reaches of Yangtze River. Chinese Sci function from Sphagnum -dominated peatlands in the Lesser
Bull 51:1603–1613 Khingan Mountains, NE China. J Paleolimnol
Clarke KR (1993) Non-parametric multivariate analyses of change in Liu Y, Hu Z, Gao S, Günther D, Xu J, Gao C, Chen H (2008) In situ
community structure. Aust J Ecol 18:117–143 analysis of major and trace elements of anhydrous minerals by
Dearing JA, Yang X, Dong X, Zhang E, Chen X, Langdon PG, Zhang LA-ICP-MS without applying an internal standard. Chem Geol
K, Zhang W, Dawson RP (2012) Extending the timescale and 257:34–43
range of ecosystem services through paleoenvironmental anal- Macumber AL, Patterson RT, Roe HM, Reinhardt EG, Neville LA,
yses, exemplified in the lower Yangtze basin. Proc Natl Acad Sci Swindles GT (2014) Autoecological approaches to resolve
109(18):E1111–E1120 subjective taxonomic divisions within Arcellacea. Protist
Dong X, Bennion H, Battarbee R, Yang X, Yang H, Liu E (2008) 65(3):305–316
Tracking eutrophication in Taihu Lake using the diatom record: Mazei YA, Marfina OV, Chernyshov VA (2012) Distribution of soil-
potential and problems. J Paleolimnol 40:413–429 inhabiting testate amoebae along a mountain slope (Baikal Lake
Dufrêne M, Legendre P (1997) Species assemblages and indicator region, Khamar-Daban ridge, Cherskii peak). Biol Bull
species: the need for a flexible asymmetrical approach. Ecol 39(10):800–804
Monogr 67:345–366 Mitchell EAD, Buttler AJ, Warner BG, Gobat JM (1999) Ecology of
testate amoebae (Protozoa: Rhizopoda) in Sphagnum peatlands

123
627 Page 10 of 11 Environ Earth Sci (2016) 75:627

in the Jura Mountains, Switzerland and France. Ecoscience Qin Y, Booth RK, Gu Y, Wang Y, Xie S (2009) Testate amoebae as
6:565–576 indicators of 20th century eutrophication in Lake Zhangdu,
Mitchell EAD, Charman DJ, Warner BG (2008) Testate amoebae China. Fund Appl Limnol 175(1):29–38
analysis in ecological and paleoecological studies of wetlands: Qin Y, Xie S, Smith HG, Swindles GT, Gu Y (2011) Diversity,
past, present and future. Biodivers Conserv 17(9):2115–2137 distribution and biogeography of testate amoebae in China:
Mittermeier RA, Mittermeier CG (1997) Megadiversity: Earth’s implications for ecological studies in Asia. Eur J Protistol 47:1–9
biological wealthiest nations. Graphic Arts Center Publishing Qin Y, Mitchell EAD, Lamentowicz M, Payne RJ, Lara E, Gu Y,
Company, Portland Huang X, Wang H (2013) Ecology of testate amoebae in
Moor C, Lymberopoulou T, Dietrich V (2001) Determination of peatlands of central China and development of a transfer
heavy metals in soils. Sediments and geological materials by function for palaeohydrological reconstruction. J Paleolimnol
ICP-AES and ICP-MS. Microchim Acta 136(3–40):123–128 50(3):319–330
Neville LA, Christie DG, McCarthy FMG, MacKinnon MD (2010) Reinhardt EG, Dalby AP, Kumar A, Patterson RT (1998) Arcel-
Biogeographic variation in Thecamoebian (Testate amoeba) laceans as pollution indicators in mine tailing contaminated lakes
assemblages in lakes within various vegetation zones of Alberta, near Cobalt, Ontario, Canada. Micropaleontol 44:131–148
Canada. Int J Biodivers Conserv 2:215–224 Roe HM, Patterson RT (2006) Distribution of thecamoebians (testate
Ogden CG (1983) Observations on the systematics of the genus amoebae) in small lakes and ponds, Barbados, West Indies.
Difflugia in Britain (Rhizopoda, Protozoa). Bull Br Mus Nat Hist J Foraminifer Res 36:116–134
(Zool.) 44:1–73 Roe HM, Patterson RT (2014) Arcellacea (Testate Amoebae) as bio-
Olson DM, Dinerstein E (1998) The global 200: a representation indicators of road salt contamination in lakes. Microbial Ecol
approach to conserving the Earth’s most biologically valuable 68:299–313
ecoregions. Conserv Biol 12:502–515 Roe HM, Patterson RT, Swindles GT (2010) Controls on the
Organisation for Economic Co-operation and Development, OECD contemporary distribution of lake thecamoebians (testate amoe-
(1982) Eutrophication of waters: monitoring, Assessment and bae) within the Greater Toronto Area and their potential as water
Control. OECD, Paris quality indicators. J Paleolimnol 43:955–975
Patterson RT, Kumar A (2000) Assessment of arcellacea (thecamoe- Ryding SO, Rast W (1989) The control of eutrophication of lakes and
bian) assemblages, species and strains as contaminant indicators reservoirs. The United Nations Educational Scientific and
in variably contaminated James Lake, north eastern Ontario. Cultural Organization, Paris, pp 37–43
J Foraminiferal Res 30:310–320 Shannon CE, Weaver W (1949) The mathematical theory of
Patterson RT, Barker T, Burbridge SM (1996) Arcellaceans (the- communication. University of Illinois Press, Urbana
camoebians) as proxies of arsenic and mercury contamination in Shen Y (1983) Protozoa of the Teibetan Plateau. In: Jiang XZ, Shen
Northeastern Ontario lakes. J Foraminifer Res 26:172–183 YF, Gong XJ (eds) Aquatic invertebrates of the Teibetan Plateau.
Patterson RT, Dalby A, Kumar A, Henderson LA, Boudreau REA Science press, Beijing, pp 48–100 (in Chinese)
(2002) Arcellaceans (thecamoebians) as indicators of land-use Shi K, Zhang Y, Zhu G, Liu X, Zhou Y, Xu H, Qin B, Liu G, Li Y
change: settlement history of the Swan Lake area, Ontario as a (2015) Long-term remote monitoring of total suspended matter
case study. J Paleolimnol 28(3):297–316 concentration in Lake Taihu using 250 m MODIS-Aqua data.
Patterson RT, Roe HM, Swindles GT (2012) Development of an Remote Sens Environ 164:43–56
Arcellacean (testate lobose amoebae) based transfer function for Shu J, Huang W, Wu Y (1996) Studies on the classification of trophic
sedimentary phosphorous in lakes. Palaeogeogr Palaeocl types of China’s lakes. Lake Sci 8:193–200 (in Chinese)
348–349:32–44 Song L, Li H, Wang K, Wu D (2014) Ecology of testate amoebae and
Patterson RT, Chang AS, Prokoph A, Roe HM, Swindles GT (2013a) their potential use as palaeohydrologic indicators from peatland
Influence of the Pacific Decadal Oscillation, El Niño-Southern in Sanjiang Plain, Northeast China. Front Earth Sci 8:564–572
Oscillation and solar forcing on climate and primary productivity Sullivan ME, Booth RK (2011) The potential influence of short-term
changes in the northeast Pacific. Quat Int 310:124–139 environmental variability on the composition of testate amoeba
Patterson RT, Lamoureux EDR, Neville LA, Macumber AL (2013b) communities in Sphagnum peatlands. Microbial Ecol 62:80–93
Arcellaceans (testate lobose amoebae) as pH indicators in a Swindles GT, Charman DJ, Roe HM, Sansum PA (2009) Environ-
pyrite mine acidified lake, northeastern Ontario, Canada. mental controls on peatland testate amoebae (Protozoa: Rhi-
Microbial Ecol 65(3):541–554 zopoda) in the North of Ireland: Implications for Holocene
Payne RJ (2013) Seven reasons why protists make useful bioindica- palaeoclimate studies. J Paleolimnol 42:123–140
tors. Acta Protozool 52:105–113 Tsugeki N, Oda H, Urabe J (2003) Fluctuation of the zooplankton
Payne R, Mitchell EA (2009) How many is enough? Determining community in Lake Biwae during the 20th century: a paleolim-
optimal count totals for ecological and palaeoecological studies nological analysis. Limnology 4:101–107
of testate amoebae. J Paleolimnol 42(4):483–495 Turner TE, Swindles GT, Charman DJ, Blundell A (2013) Comparing
Payne R, Charman DJ, Matthews S, Eastwood W (2008) Testate regional and supra-regional transfer functions for palaeohydro-
amoebae as palaeoclimate proxies in Sürmene Ağaçbaşi Yaylasi logical reconstruction from Holocene peatlands. Palaeogeogr
peatland (Northeast Turkey). Wetlands 28:311–323 Palaeocl 369(1):395–408
Penard E (1902) Faune rhizopodique du bassin du Léman. Kündig.v, Wang R, Dearing JA, Langdon PG, Zhang E, Yang X, Dakos V,
Genève Scheffer M (2012) Flickering gives early warning signals of a
Podani J, Csányi B (2010) Detecting indicator species: Some critical transition to a eutrophic lake state. Nature
extensions of the IndVal measure. Ecol Ind 10:1119–1124 492(7429):419–422
Qin B, Hu W, Guang G, Luo L, Zhang J (2004) Dynamics of Warner BG, Asada T, Quinn NP (2007) Seasonal influences on the
sediment resuspension and the conceptual schema of nutrient ecology of testate amoebae (Protozoa) in a small Sphagnum
release in the large shallow Lake Taihu, China. Chinese Sci Bull peatland in southern Ontario, Canada. Microbial Ecol
49:54–64 54(1):91–100
Qin Y, Xie S, Swindles GT, Gu Y (2008) Pentagonia zhangduensis Yang J, Beyens L, Shen Y, Feng W (2004) Redescription of Difflugia
nov. spec. (Lobosea, Arcellinida), a new freshwater species from tuberspinifera Hu, Shen, Gu et Gong, 1997 (Protozoa:
China. Eur J Protistol 44(4):287–290

123
Environ Earth Sci (2016) 75:627 Page 11 of 11 627

Rhizopoda: Arcellinida: Difflugiidae) from China. Acta Proto- implications for assessing long-term eutrophication. Freshwater
zool 43:281–289 Biol 53:1273–1290
Yang X, Dong X, Gao G, Pan H, Wu J (2005) Relationships between Yang Z, Wang Z, Zhang Z (2011) Biomonitoring of testate amoebae
surface sediment diatoms and summer water quality in shallow (protozoa) as toxic metals absorbed in aquatic bryophytes from
lakes of the middle and lower reaches of Yangtze River. J Integr the Hg-Tl mineralized area (China). Environ Monit Assess
Plant Biol 47(2):153–164 176:321–329
Yang X, Jone A, Dong X, Shen J (2008) Surface sediment diatom Yao M, Li Y, Yang X, Liu Q (2011) Three-year changes in planktonic
assemblages and epilimnetic total phosphorus in large, shallow diatom communities in a eutrophic lake in Nanjing, Jiangsu
lakes of the Yangtze floodplain: their relationships and Province. China. J Freshwater Ecol 26(1):133–141

123