's University, Biology experiments, landscape … aquatic community structure (Jeziorski et al....

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Calcium decline reduces population growth rates of

zooplankton in field mesocosms

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2016-0105.R1

Manuscript Type: Article

Date Submitted by the Author: 19-Dec-2016

Complete List of Authors: Arnott, Shelley; Queen\'s University, Biology Azan, Shakira; Queen\'s University, Biology Ross, Alex; Queen\'s University, Biology

Keyword: calcium decline, growth rate, ZOOPLANKTON < Taxon, mesocosm, experiments, landscape stressors

https://mc06.manuscriptcentral.com/cjz-pubs

Canadian Journal of Zoology

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Calcium decline reduces population growth rates of zooplankton in field mesocosms* 1

2

3

S. E. Arnott1, S. S. E Azan

2, and A. J. Ross

3 4

5

6

7

8

Department of Biology, Queen’s University, Kingston, ON, K7L 3N6 9

1Corresponding author: S. E. Arnott, Department of Biology, Queen’s University, Kingston, ON, 10

K7L 3N6, phone: 613-533-6384, fax: 613-533-6617, email: [email protected] 11

[email protected] 12

[email protected] 13

*This article is one of a series of invited papers arising from the symposium “Large, landscape-14

level ecological disturbances” that was co-sponsored by the Canadian Society of Zoologists and 15

Canadian Science Publishing and held during the Annual Meeting of the Canadian Society of 16

Zoologists at the University of Calgary, Calgary, Alberta, 25–29 May 2015.17

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Calcium decline reduces population growth rates of zooplankton 18

Abstract 19

Regional calcium decline, a legacy of acid deposition and logging, is a potential threat to aquatic 20

organisms. Lake surveys and laboratory studies indicate that Ca-rich daphniids are likely most 21

susceptible, allowing for competitive release of other taxa with low Ca demand. Indeed, dramatic 22

shifts in zooplankton community structure have been documented in lakes where Ca has declined, 23

amid multiple other stressors. Given the perceived threat of this large-scale stressor, manipulative 24

studies are needed to evaluate causal relationships between calcium decline and zooplankton 25

community structure. We analysed per capita growth rates of zooplankton from three independent 26

mesocosm experiments where we manipulated aqueous Ca concentrations to reflect current and 27

future Ca concentrations. In two experiments where Ca was reduced to 0.6 or 0.9 mg Ca/L, we 28

observed reduced growth rates for several taxa, including daphniids, bosminids, and copepods. No 29

effect of Ca was detected in the experiment where Ca ranged from 1.2 to 2.5 mg Ca/L, a gradient 30

representing 68% of lakes in south-central Ontario. These results suggest that future Ca decline in 31

softwater Shield lakes may be accompanied by shifts in community structure and overall declines in 32

zooplankton production. 33

Keywords: calcium decline, growth rates, crustacean zooplankton, mesocosm experiments, 34

landscape stressors 35

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Introduction 37

Soft water lakes across the Canadian Shield are facing multiple environmental changes, 38

including climate change, the spread of invasive species, altered nutrient loading, and acidification 39

(Schindler et al. 1998; Paterson et al. 2008; Yan et al. 2008). Recently, a new threat to aquatic food 40

webs has been identified; calcium (Ca) concentration has declined in hundreds of lakes in the eastern 41

regions of the Canadian Shield (Keller et al. 2001; Jeziorski et al. 2008; Reid and Watmough 2016). 42

Calcium decline is a growing concern because the potential loss of key Ca-rich herbivores, including 43

daphniids (Jeziorski et al. 2008) and crayfish (Edwards et al. 2009; 2015), has implications for 44

aquatic community structure (Jeziorski et al. 2015) and ecosystem function (Korosi et al. 2011). 45

The Ca concentration of lake water is determined primarily by the export of Ca from 46

terrestrial watersheds and thus depends on the amount of exchangeable base cation reserves in the 47

soil. To a lesser extent, atmospheric inputs associated with reductions in diffuse and point-source 48

emissions of particulates have also contributed to declining aqueous Ca concentrations (Hedin et al. 49

1994). For the most part, however, Ca decline in soft water lakes is largely a legacy of historical 50

regional acid deposition that enhanced the release of Ca from the soil, as well as past logging and 51

subsequent uptake of Ca from soils by growing forests (Likens et al. 1998; Watmough et al. 2003). 52

The decline of aqueous Ca is part of a global trend, with regions in North America (Ontario, 53

Adirondacks, Quebec, Vermont, Atlantic Canada, Maine) and Europe (Scandinavia, United 54

Kingston, East Central Europe), experiencing reduced base cation concentrations (Ca and Mg) 55

ranging from 10 to 27% when compared to concentrations in the 1990s (Garmo et al. 2014). Further 56

declines of 10-40% compared to 1997 Ca concentrations are expected in Canada (Watmough and 57

Aherne 2008). 58

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Aqueous Ca concentration is an important determinant of crustacean zooplankton community 59

composition and richness (Wærvågen et al. 2002; Strecker et al. 2008), generally influencing the size 60

distribution of daphniids (Tessier and Horwitz 1990) and the relative abundance of daphniids, 61

bosminids, (Jeziorski et al. 2008; DeSellas et al. 2011), and Holopedium spp. (Hessen et al. 1995; 62

Jeziorski et al. 2015). Species shifts are likely driven by physiological tolerances associated with 63

species-specific Ca influx and efflux rates (Tan and Wang 2010), as well as shifts in competitive 64

interactions that favour Ca-poor taxa (Hessen et al. 2000) and possibly changes in predator-prey 65

interactions driven by impaired anti-predator defense in low Ca environments (Riessen et al. 2012). 66

Daphniids have been a primary research focus because they are thought to be more sensitive to low 67

Ca environments than other taxa because their exoskeletons are rich in Ca (Cowgill 1976; Jeziorski 68

and Yan 2006). The Ca content of daphniids is generally higher (1-7 %DW) than other zooplankton 69

taxa, which range from 0.04 to 2.32 %DW (Azan et al. 2015). Furthermore, Ca-demand is high 70

throughout their entire life cycle because they continually shed their Ca-rich exoskeleton, losing 71

>90% of their body Ca with each moult (Alstad et al. 1999). The availability of aqueous Ca is crucial 72

because it is the primary source of Ca used to regenerate the carapace, with a negligible amount of 73

Ca coming from food sources (Cowgill et al. 1986; Alstad et al. 1999). Laboratory studies using 74

Daphnia pulex Leydig 1960 have identified 1.5 mg Ca/L as an important ecological threshold for 75

reproduction (Ashforth and Yan 2008) and the induction of anti-predator defenses (Riessen et al. 76

2012). 77

Our knowledge of species-effects for the wider zooplankton community is limited because 78

controlled laboratory trials across a range of environmentally-relevant Ca concentrations have been 79

conducted on only a few species; mainly daphniids (e.g., Daphnia magna Straus 1820; D. pulex, 80

Daphnia galeata G.O. Sars 1864; Daphnia tenebrosa G.O. Sars 1898; Daphnia longispina O. F. 81

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Mueller 1785; Alstad et al. 1999; Hessen et al. 2000; Hessen and Rukke 2000a; Rukke 2002; 82

Ashforth and Yan 2008; Riessen et al. 2012; Jiang et al. 2014; Jesus et al. 2014) and a few littoral 83

taxa (Tan and Wang 2010). In general, aqueous Ca concentrations below 0.5 mg Ca/L are lethal to 84

Daphnia in laboratory studies (reviewed in Cairns and Yan 2009). Field thresholds, based on species 85

occurrence data, range from 0.5 to 1.69 mg Ca/L for most daphniids (Azan et al. 2015). Reduced 86

body size, delayed maturity (Cairns and Yan 2009), and possible increased energy expenditure 87

(Hessen and Rukke 2000a) are expected to result in reduced population growth rates for daphniids 88

experiencing low Ca, while birth rates and population growth rates of small cladocerans and 89

copepods are hypothesized to be unaffected by low Ca (Azan et al. 2015). However, the paucity of 90

species-specific tolerances limits our ability to reliably predict how declining Ca will influence 91

diverse species assemblages and how differential sensitivities may play out in the context of complex 92

communities where individual growth rates are influenced by species interactions. 93

Despite the perceived threat of this large-scale stressor, that has the potential to impact 94

thousands of lakes on the Canadian Shield, few manipulative studies have been carried out that can 95

evaluate causal relationships between Ca decline and zooplankton community structure, particularly 96

at the low concentrations that many Canadian Shield lakes are likely to attain (Reid and Watmough 97

2016). Despite correlations between [Ca] and cladoceran community structure in some studies (e.g., 98

Jeziorski et al. 2008), others have detected minor and sometimes inconsistent community change 99

through time (e.g., Jeziorski et al. 2012a; 2012b). Some of this may be attributed to inadequate 100

taxonomic resolution of cladoceran fossils in sediments, particularly the lumping of Ca-poor and Ca-101

rich daphniid species. In addition, it can be difficult to differentiate community response to Ca from 102

other regional changes such as acidification (but see Barrow et al. 2014) using field survey data. 103

Lakes with low concentrations of Ca have poor buffering capacity and therefore are more susceptible 104

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to regional acidification (e.g., pH is correlated with Ca in 382 lakes in the Muskoka River 105

Watershed; R2=0.53, P<0.001, unpublished analysis). As with any correlative study, uncertainty 106

exists because multiple drivers, including unknown/unmeasured factors, could be influencing species 107

distributions. Interpretation of survey results is further complicated by the potential for intraspecific 108

variation in Ca response (Rukke 2002; Wærvågen et al. 2002). 109

To control for some of the inherent problems in regional surveys (discussed above) and to 110

account for the natural ecological complexity not captured in laboratory studies, we assessed the 111

influence of declining Ca on zooplankton population growth rates of crustacean zooplankton using 112

three independent, manipulative field experiments (Azan and Arnott 2016; Azan 2016; Ross 2015). 113

Each experiment was designed to address the individual and interactive effect of [Ca] and an 114

additional factor (e.g., Bythotrephes, pH, and historical [Ca]) on zooplankton community structure, 115

but for this comparative study we only considered Ca treatments. In each experiment, Ca 116

concentration was manipulated to reflect future scenarios and to bracket threshold concentrations 117

from laboratory and field studies. Diverse zooplankton communities from multiple lakes were 118

inoculated in experimental mesocosms to incorporate possible intra-specific variation in tolerances. 119

We hypothesized that population growth rates of Ca-rich taxa, especially daphniids, would be 120

reduced in our low Ca conditions compared to our high Ca treatments. Conversely, we expected that 121

taxa with low Ca content would either not be influenced by our Ca treatments or would have higher 122

growth at low Ca because of decreased competition with species sensitive to low Ca. Here we 123

summarize the effect of Ca on population growth rates for crustacean zooplankton communities, 124

comparing responses among the three independent mesocosm studies. Detailed assessment of 125

individual species and functional group responses for each experiment can be found in Azan and 126

Arnott (2016), Azan (2016), and Ross (2015). 127

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Methods: 128

To assess the potential impact of declining Ca on crustacean zooplankton communities in 129

Canadian Shield lakes, we calculated population growth rates over a six-week period for diverse taxa 130

in each of the three mesocosm experiments. Two studies (Havelock and Dorset) were conducted in 131

the Muskoka-Haliburton region of Ontario, a lake district that is experiencing multiple 132

environmental changes, including Ca decline (Palmer et al. 2013). Lake Ca concentration in the 133

Muskoka-Haliburton region has, on average, declined by 30% since the 1980s and currently 28% and 134

5% of the lakes are below 1.5 and 1.0 mg Ca/L, respectively (Reid and Watmough 2016). The third 135

experiment (Lumsden) was conducted in Killarney Provincial Park, Ontario, where the combination 136

of geology and historic acid deposition from nearby metal smelters (Gunn and Sandoy 2003) resulted 137

in diminished Ca concentrations with 50% and 18% of the lakes having [Ca] below 1.5 and 1.0 mg 138

Ca/L, respectively (SE Arnott 2011, unpublished data for 45 lakes). Water chemistry (Ca, pH, TP, 139

DOC) and the location of lakes used in each experiment are presented in Table 1. 140

We used a gradient design to test for the effect of Ca on zooplankton per capita growth rates 141

for the Havelock experiment (Azan and Arnott 2016). Calcium concentration was manipulated in 142

fourteen enclosures to create a gradient from 1.2 to 2.5 mg Ca/L. This gradient represents [Ca] for 143

68% of the lakes in the Muskoka-Haliburton region (Ontario Ministry of Environment and Climate 144

Change). For the Dorset experiment we established two gradients; aqueous [Ca] ranged from 0.5 to 145

2.5 mg Ca/L and was crossed with 8 zooplankton source lakes that ranged from 1.8 to 24.8 mg Ca/L. 146

Each treatment combination was replicated 2 times for a total of 64 mesocosms (Azan 2016). The 147

Lumsden experiment had two Ca treatments; 0.9 and 2.3 mg Ca/L, each replicated 5 times (Ross 148

2015). 149

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The Havelock and Lumsden experiments were conducted during summer 2013 in 1-m 150

diameter polyethylene bags (Filmtech Plastics, Brampton Canada) that were closed at the bottom, 151

open to the atmosphere, and suspended from wooden frames that floated on the lake surface. The 152

mesocosms in Havelock Lake were 10 m deep, whereas in Lumsden Lake, they were 3.2 m deep. 153

The Dorset experiment was conducted during summer 2014 using 800L aquaculture tanks (Fish 154

Farm Supply, Guelph, Canada) that were 0.94 m diameter, 1.2 m high, and located in an open field 155

on the Dorset Environmental Science Centre (DESC) site in Dorset, Ontario. To reduce solar 156

heating, we shaded each tank in the Dorset experiment with black plastic that was wrapped around 157

its perimeter, leaving an air space between the plastic and the tank. In each of the three experiments, 158

we used mesh coverings made from window screening for shade and to prevent aerial colonization 159

by insects and other organisms. 160

We filled mesocosms with lake water that was filtered through 80-µm mesh to remove 161

zooplankton but allow most phytoplankton to pass through. The lowest Ca treatment in the Havelock 162

and Lumsden experiments was equal to the ambient [Ca] of the lake where the enclosures were 163

deployed (Table 1). For the Dorset experiment, we extended the minimum [Ca] by passing lake 164

water through a deionization filter system (High Purity Water Services, Inc., Mississauga, Ontario). 165

Water from Plastic Lake (Table 1) was trucked to DESC where each mesocosm was filled with 350 166

L of Plastic Lake water and 350 L of deionized Paint Lake water (the water source for DESC). This 167

reduced [Ca] in each mesocosm to ~0.6 mg/L. To compensate for the loss of nutrients with 168

deionization, we added 7.38 mg of monopotassium phosphate (KH2PO4; ACS, Fisher Scientific), 169

197.52 mg of sodium nitrate (NaNO3; 99.0% Sigma-Aldrich), and 124.3 mg of ammonium chloride 170

(NH4Cl; USP/FCC, Fisher Scientific) to each enclosure. We added 0.5M sodium hydroxide (0.5 N 171

NaOH; N/2 certified, Fisher Scientific) to each mesocosm in Lumsden Lake to attain pH 6.3, which 172

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is above the threshold value of pH 6, where community composition is impacted (Holt et al. 2003). 173

This wasn’t necessary for the Havelock and Dorset experiments as pH of water in the mesocosms 174

was above 6. Calcium gradients/treatments for each experiment were achieved by adding calcium 175

sulfate dehydrate (CaSO4(H2O)2; ACS reagent, Fisher Scientific. Water samples were analysed at 176

least 3 times (e.g., beginning, middle, and end of experiment) for each study by the Ontario Ministry 177

of Environment and Climate Change, using atomic absorption spectrophotometry (Ontario Ministry 178

of Environment, 1981; Appendix 1). 179

We added a diverse assemblage of zooplankton from lakes with relatively high [Ca] to each 180

enclosure. Zooplankton were collected from nearby lakes with [Ca]>2.5 mg/L for Havelock, [Ca] of 181

the source lakes ranged from 1.8 to 24.8 mg Ca/L for the Dorset experiment, and from 1.4-1.8 mg 182

Ca/L for the Lumsden experiment (see Table 1 for list of lakes). For the Havelock and Lumsden 183

experiments, zooplankton from all source lakes were pooled and aliquots were evenly distributed 184

among enclosures resulting in a final density that approximated the mean ambient density of all 185

colonist source lakes, based on volume of water filtered through nets during collection. We stocked 186

mesocosms for the Dorset experiment with zooplankton at ambient density from 8 lakes so we could 187

assess how zooplankton population growth rate varied a long a gradient of source lake [Ca] (Azan 188

2016). 189

We sampled zooplankton in each mesocosm to assess species abundance at the beginning 190

(Week 0) and end of experiment (Week 6). After a period greater than 6 weeks, periphyton growth 191

and colonization by littoral zooplankton species changes the communities from those typical of 192

pelagic regions to those more typical of littoral regions (i.e., ‘enclosure effects’ begin to appear). For 193

the Havelock and Lumsden experiments, we sampled zooplankton using 0.15 m diameter, 80-um 194

mesh nets that were pulled to the top of the water column starting ~0.2 m from the bottom of the 195

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enclosure, sampling ~7% of total enclosure volume. We collected Week 0 zooplankton from the 196

Dorset mesocosms by combining 3 discrete samples that were taken from the bottom, middle, and 197

top of the water column using a 12-L Schindler trap. This methodology was altered to include 198

mixing mesocosm contents before sampling for the Week 6 samples. These samples were 199

approximately 5% of the total volume of the mesocosm. 200

Crustacean zooplankton samples from the Havelock and Lumsden experiments were 201

enumerated using a subsample of a known volume and counting all individuals within the subsample 202

until three subsamples in a row contained no new species (usually >8 subsamples were counted for 203

each sample). For the Dorset experiment, entire samples were processed. Crustacean zooplankton 204

were identified to species and counted on a Leica MZ12.5 dissecting microscope (Leica 205

Microsystems; Solms, Germany). Bosmina freyi DeMelo and Hebert 1994 and Bosmina liederi 206

DeMelo and Hebert 1994 were grouped as “Bosmina spp.” and D. pulex and Daphnia pulicaria 207

Forbes 1893 as “Daphnia pulex/pulicaria” due to difficulties in distinguishing these species based on 208

morphology. Immature copepods (nauplii and copepodids) were identified to order. 209

Statistical analyses 210

For each experiment, we estimated daily per capita growth rate (individuals 211

produced/individual/day) for each taxon in every mesocosm as loge(final abundance/initial 212

abundance)/days. To compensate for the change in sampling methodology between Week 0 and 6 for 213

the Dorset experiment, we calculated the initial abundance (before treatments were applied) as the 214

mean abundance of the 8 mesocosms associated with each lake; final abundance was estimated from 215

individual mesocosms. 216

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We occasionally failed to detect individual taxa in the initial or final sample of a mesocosm. 217

Zeros could result from colonization events (0/L in Week 0), extirpation events (0/L in Week 6), or 218

abundances below detection limits. To calculate per capita growth for these cases, we replaced 0s 219

with a density equivalent to the occurrence of one individual in the total volume of the sample 220

(minimum detectable concentration). 221

To visualize the community growth performance for each mesocosm, we conducted a 222

principle component analysis (PCA) using a covariance matrix based on growth rate for each taxon. 223

Because species X site matrices cannot contain missing growth values for PCA, we combined some 224

species that were not present in every enclosure (e.g., Eubosmina longispina and Eubosmina tubicen; 225

Leptodiaptomus sicilis and Leptodiaptomus minutus) and removed from the analyses taxa that were 226

absent from two or more mesocosms for each experiment. Three taxa (of 20) were 227

removed/combined for the Havelock PCA, two taxa (of 18) were removed/combined for the Dorset 228

PCA, and three taxa (of 19) were removed/combined for the Lumsden PCA. Where a species was 229

absent from only one enclosure, the enclosure was removed from the analyses. D. pulex/pulicaria 230

was absent from one enclosure in both the Lumsden and Havelock experiments and Skistodiaptomus 231

oregonensis (Lilljeborg in Guerne and Richard 1889) was absent from one enclosure in the Havelock 232

experiment. 233

To examine the effect of [Ca] on community growth performance we used permutational 234

MANOVA (PERMANOVA) (Anderson et al. 2001) on the distance between all pairs of mesocosm 235

communities in ordination space for the Lumsden and Dorset experiments. For the Havelock 236

experiment, we investigated the linear relationship between PC1, PC2 and [Ca] using lm in R. PCAs 237

and PERMANOVAs were performed in R (R Development Core Team, 2014) using the vegan 238

package (Oksanen et al. 2013). 239

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Results: 240

Havelock Experiment 241

The average per capita growth rate of the 20 taxa that were present in the Havelock 242

experiment was 0.012 and ranged from -0.069 to 0.114 ind/ind/day (Figure 1a). Bosminids 243

experienced the highest per capita growth rates across all treatments, whereas most copepods 244

declined throughout the experiment. Daphniids tended to have low, but generally positive average 245

per capita growth rates. The per capita growth rate of the total crustacean community was not related 246

to aqueous calcium concentration (P=0.985, R2=-0.08, df=1, 2; Figure 1b). Similarly, there was no 247

detectable relationship between [Ca] and community growth performance in the Havelock 248

experiment (Regression PC1 vs Ca, F=0.174, P=0.683, df=1, 12; Regression PC2 vs Ca, F=0.011, 249

P=0.918, df=1, 12; Figure 2a). For an in-depth analysis of the relationship between [Ca] and species 250

and functional group abundances, see Azan and Arnott (2016). 251

Lumsden Experiment 252

Average per capita growth rate of the 19 taxa present in the Lumsden experiment was 0.025 253

ind/ind/day in the high Ca treatment, compared to 0.009 ind/ind/day in the low Ca treatment (Fig 3). 254

E. tubicen, Diaphanosoma birgei Korinek, 1981, D. pulex/pulicaria, Orthocyclops modestus 255

(Herrick 1883), and E. longispina had the highest per capita growth rates, whereas Holopedium 256

glacialis (Zaddach 1855), Diacyclops thomasi (S.A. Forbes 1882), Epischura lacustris S.A. Forbes 257

1882, Daphnia retrocurva Forbes 1882, and Acanthocyclops vernalis (Fischer 1853) had the lowest 258

per capita growth rates, declining in abundance in both treatments through time. Although the range 259

of per capita growth rates was similar among treatments (Figure 3), there were marked community-260

level differences in per capita growth rates between treatments (PERMANOVA, F=2.76, R2=0.26, 261

P=0.007, df =1,9; Figure 2b). Most taxa had higher per capita growth rates in the high Ca compared 262

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to the low Ca treatment, although only Bosmina spp., D. birgei, Leptodiaptomus minutus (Lilljeborg 263

in Guerne and Richard 1889), and Mesocyclops edax (S. A. Forbes 1890) had confidence intervals 264

that did not overlap zero (Figure 4). 265

Dorset Experiment 266

Zooplankton per capita growth rates were positive for most taxa in the high Ca treatment in 267

the Dorset experiment, with an average of 0.034 ind/ind/day compared to an average of 0.012 268

ind/ind/day in the lowest Ca treatment (Figure 5). Of the 18 taxa, only H. glacialis and D. 269

pulex/pulicaria experienced negative mean per capita growth rates when provided ~2.4 mg Ca/L; -270

0.41 and -0.32 ind/ind/day, respectively. As [Ca] decreased, mean per capita growth rates also 271

decreased (Figure 5). Taxa with the highest per capita growth rates (e.g., Eubosminids) continued to 272

maintain high per capita growth rates as [Ca] declined, but 11 of the 18 taxa experienced decreased 273

growth rates at low Ca (~0.6 mg/L) compared to high Ca (2.4 mg/L) (Figure 4). Community growth 274

performance was strongly effected by [Ca] (PERMANOVA, F=5.49, R2= 0.081, P<0.001, df=1, 63). 275

Performance was highest in the 2.4 mg Ca/L treatment and decreased as [Ca] was reduced to 0.6 mg 276

Ca/L (Figure 2c). 277

Discussion: 278

Our study, which is the first to experimentally assess the effect of low [Ca] on growth rates of 279

crustacean zooplankton communities, provides evidence that declining aqueous [Ca] is associated 280

with reduced growth rates of a variety of taxa, in addition to Ca-rich daphniids that are frequently 281

considered the most vulnerable. In two of our experiments, cyclopoid copepods (M. edax, A. 282

vernalis, Tropocyclops extensis (Kiefer 1931)), diaptomid copepods, and small cladocerans (D. 283

birgei, Bosmina spp.) had reduced population growth rates in low [Ca] treatments (0.6 and 0.9 mg 284

Ca/L) when compared to high [Ca] treatments. Based on steady state water chemistry models (Reid 285

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and Watmough 2016), 37% and 8% of lakes in our study region are expected to reach [Ca] less than 286

1.5 and 1 mg Ca/L, respectively, suggesting that the most severe impacts on zooplankton 287

communities have yet to be seen. In contrast, the lack of a strong Ca effect on zooplankton 288

population growth rates in our Havelock experiment, where [Ca] ranged from 1.2 to 2.5 mg Ca/L, 289

suggests that Ca declines within this range are unlikely to have a large influence on crustacean 290

community growth performance. 291

Differences among experiments: 292

We were surprised that population growth rates, particularly of daphniids, were not 293

influenced by [Ca] in our Havelock experiment where [Ca] ranged from 1.2 to 2.5 mg Ca/L. It is 294

possible that our decision to use 2.5 mg Ca/L as an upper concentration for our experiments limited 295

our ability to detect Ca effects, particularly for Ca-rich daphniids, where Ca-uptake in laboratory 296

studies appears to saturate (e.g., Tan and Wang 2010) and population growth rates decline at lower-297

limit concentrations well above 2.5 mg Ca/L (Jiang et al. 2014; Jesus et al. 2014). A lake survey in 298

south-central Ontario revealed that cladoceran community structure changed along a [Ca] gradient 299

from 1.0 to 24 mg Ca/L, but no association between [Ca] and cladoceran community structure was 300

detected for lakes along a gradient from 1.0 to 3.1 mg Ca/L (Jeziorski et al. 2012a). This may suggest 301

that current community composition in Canadian Shield lakes has already been altered by Ca 302

decline, and indeed dramatic changes in the relative abundance of Ca-rich daphniids and Ca-poor 303

cladocera, such as bosminids and H. glacialis, over the past several decades support this conclusion 304

(Jeziorski et al. 2008). Despite this, there is considerable evidence from laboratory studies and field 305

surveys to suggest that reproduction or survival thresholds for daphniids occur at low [Ca], within 306

the range used in our experiments (Cairns and Yan 2009; Azan et al. 2015). In North America, a 307

field survey identified survival thresholds between 1.26 and 1.69 mg Ca/L for several species of Ca-308

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rich daphniids (Cairns 2010). Lower lethal and reproductive thresholds have yet to be identified for 309

non-daphniid species (Azan et al. 2015). Based on this, we believe that our contrasting results for the 310

Havelock experiment compared to the Lumsden and Dorset experiment results indicate that a lower 311

threshold, between 0.9 and 1.2 mg Ca/L, exists for crustacean zooplankton community growth 312

performance. The most severe changes in community structure may only be apparent as lakes with 313

the lowest Ca concentrations decline below threshold concentrations. 314

It is also possible that other factors are interacting with Ca and thus obscuring a relationship 315

between [Ca] and population growth in the Havelock experiment. Several studies have indicated that 316

food quantity and quality can influence daphniid response to low [Ca] (e.g., Jiang et al. 2014; Prater 317

et al. 2015) and other ions (Brown and Yan 2015). In combination, low P-content of food has a 318

larger effect on D. pulex growth than [Ca] (Prater et al. 2015). Similarly, [Ca] had a stronger effect 319

on D. magna individual growth rates, reproduction, and survival at high food concentration (0.4-0.8 320

mg C/L) than at low food concentration (0.02-0.05 mg C/L), likely because carbon limitation 321

overrides the potential effects of low Ca (Hessen et al. 2000). In contrast, Ashforth and Yan (2008) 322

found that the effect of low Ca on population growth rate of D. pulex was much greater at low food 323

(3 ug Chl a/L) compared to high food (30 ug Chl a/L). It is likely that food was limiting growth in all 324

of our experiments, as chlorophyll a (Chl a) concentrations in our mesocosms were low (Havelock: 325

Chl a =4.32 ± 7.3 µg/L; Dorset: Chl a=2.19 ± 1.22 µg/L; Lumsden: Chl a=1.40 ± 0.75 µg/L). Likely 326

because of low food availability, per capita growth rates in our experiments (Figure 4) were lower 327

than published laboratory values for a diversity of cladoceran taxa (including Bosmina sp. and 328

daphniids) which ranged from 0.14 to 0.58 /day at temperatures ~20°C (Lynch 1980). The 329

importance of food quantity and quality on zooplankton community response to Ca merits further 330

investigation. 331

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Inter-population variation in sensitivity to low Ca conditions could have contributed to 332

differences in Ca-response among our experiments. Although daphniids exhibit inter-population 333

variation in response to many stressors, including metals (DeMille et al. 2016; Baird et al. 1990) and 334

predators (DeMeester 1996; Boeing et al. 2006), only one study has compared Ca thresholds between 335

populations of a single species (Rukke 2002). Despite observing a different Ca threshold for D. 336

galeata from a lake with high and low [Ca] (Rukke 2002), we cautiously assert that inter-population 337

differences in tolerance to low [Ca] were unlikely associated with differences in results among our 338

experiments. In each of our experiments we used zooplankton from several populations (3 lakes for 339

Lumsden; 5 for Havelock, and 8 for Dorset), thus reducing the likelihood of responses being driven 340

by aberrant Ca tolerances in individual populations. In addition, we found no evidence in the Dorset 341

experiment that zooplankton from lakes with high [Ca] had different sensitivity to low Ca than 342

zooplankton from lakes with low [Ca] (Azan 2016). However, inter-population variation in tolerance 343

to low Ca conditions merit further investigation, particularly among regions that differ in bedrock 344

geology and, therefore, soil Ca (Wærvågen et al. 2002). 345

Which species were affected? 346

Our results contribute to the growing evidence that Ca body content is not a reliable predictor 347

of tolerance to low Ca conditions (Tan and Wang 2010; Azan et al. 2015). We found that reductions 348

in growth rate in low [Ca] occurred in taxa with high and low Ca content (Figure 5). Much of the 349

research investigating the effects of low Ca has focused on daphniids because they have relative high 350

Ca content and expected vulnerability to Ca decline. Based on results from lake surveys and fossil 351

records in lake sediment cores (e.g., Hessen et al. 2000; Jeziorski et al. 2008) we expected to observe 352

reduced growth rates of Ca-rich taxa and no effect or compensatory increases in other taxa with low 353

Ca body content (Azan et al. 2015). Instead, we found that several species, including non-daphniid 354

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cladocerans and copepods, contributed to the shift in community growth performance in the 355

Lumsden and Dorset experiments (Figure 2). As expected, daphniids in our study were sensitive to 356

low Ca, but we did not detect a clear distinction between high-Ca and low-Ca species, as defined by 357

Jeziorski et al. (2015). Daphnia catawba Coker, 1926 and Daphnia ambigua Scourfield, 1947, two 358

species considered less susceptible to Ca decline (Jeziorski et al. 2015), tended to have lower growth 359

rates in low Ca compared to high Ca treatments in the Lumsden and Dorset experiments (Figure 4). 360

Similarly, Bosmina spp., a species that appears to thrive in low Ca environments (DeSellas et al. 361

2011; Jeziorski et al. 2015), experienced reduced growth rates in our low Ca treatments. In contrast, 362

the closely related genus, Eubosmina, showed little effect of reduced [Ca]. It is not clear whether 363

direct effects of [Ca] or species interactions are driving these changes in growth rates; there have 364

been no studies examining physiological tolerances of bosminids to [Ca] gradients. 365

In addition, several copepod species, which are generally considered less susceptible to 366

declining Ca due to their low Ca body content, had lower per capita growth rates in treatments with 367

reduced [Ca]. Although species-level tolerances have not been assessed in laboratory studies, 368

copepod distribution does not appear to be associated with [Ca] gradients (Wærvågen et al. 2002). 369

Copepod sensitivity to low Ca has received little attention in the literature, particularly in 370

paleolimnological studies, because their body parts do not preserve well in lake sediments and they 371

are thought to be tolerant of low [Ca]. However, one study did find that the Ca content of M. edax 372

varied depending on lake [Ca] (Jeziorski and Yan 2006), suggesting low [Ca] may impose 373

physiological constraints on this species. Our observation of reduced growth rates for M. edax, A. 374

vernalis, L. minutus, Leptodiaptomus sicilis (S. A. Forbes 1882), and juvenile copepods in low [Ca] 375

environments (Figure 4) emphasizes the need to explore the effects of declining [Ca] on a broader 376

range of taxa. 377

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Holopedium glacialis, a jelly-coated species with low Ca body content (0.1 to 0.5% DW; 378

Azan et al. 2015), has increased in abundance over the past few decades in Canadian Shield lakes 379

(Jeziorski et al. 2015). This increase has likely arisen from changes in species interactions that were 380

driven by declining [Ca], particularly the reduction of daphniid competitors (Hessen et al. 1995; 381

Jeziorski et al. 2015). We expected that H. glacialis would thrive in our low Ca enclosures, because 382

its distribution includes low-Ca lakes and the loss of daphniids would release it from competition. 383

Instead, H. glacialis experienced negative growth rates in all enclosures for the Lumsden and 384

Havelock experiments, suggesting that other factors were limiting their survival in these 385

experiments. Holopedium glacialis growth rates were variable in the Dorset experiment (negative in 386

33 of 64 mesocosms) but there was no relationship with [Ca] (S. Azan, unpublished data). Overall, 387

the poor performance of H. glacialis in our experimental mesocosms limits our ability to predict how 388

future Ca declines may impact this species. 389

The strength of our experiments is that we were able to directly manipulate [Ca] while 390

ensuring that other potentially confounding variables were similar across treatments. One of the 391

difficulties associated with assessing the impact of Ca on zooplankton communities using lake 392

surveys is that [Ca] is often correlated with other variables, such as pH, making it challenging to 393

attribute community shifts to a single variable. In our experiments, we ensured that pH was above 6, 394

a recognized threshold for zooplankton communities (Holt et al. 2003), in each treatment. Although 395

we are confident that [Ca] is the driver of differences in growth rates among treatments, we do not 396

know whether they were direct effects of physiological intolerances or indirect effects caused by 397

changes in species interactions. Cladocerans in our experiments would have produced several 398

generations at the temperatures they experienced in our mesocosms, allowing for indirect effects of 399

competition, as well as physiological effects on birth and death rates. Experiments to assess 400

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physiological tolerance to Ca and mechanistic studies to assess the influence of Ca on species 401

interactions are needed to tease apart direct and indirect effects of low [Ca] on aquatic food webs. 402

Our assembled communities represent simplified food webs because we excluded vertebrate 403

predators and stocked low abundances of invertebrate predators. Our communities were collected 404

from lakes during the day when common invertebrate predators, such as Chaoborus spp., are deep in 405

the water column and less likely to be captured. Consequently, our results do not include potential 406

synergistic interactions between predators and [Ca]. Riessen et al. (2012) demonstrated that anti-407

predator defenses in D. pulex are reduced at low Ca, suggesting that the effect of low Ca on 408

zooplankton could be greater with the inclusion of predators. 409

Similarly, interactions with other regional stressors, including changes in ultraviolet radiation 410

(associated with changes in dissolved organic carbon), nutrient loading, and climate change (Hessen 411

and Rukke 2000b; Ashforth and Yan 2008; Prater et al. 2015) could have interactive effects with Ca 412

decline. The results presented in this study consider the effects of [Ca] in isolation of other potential 413

environmental changes. Going forward, there is a clear need to assess interactive effects of multiple 414

stressors, as an increasingly large fraction of lakes on the Canadian Shield are impacted by human 415

activities that have changed nutrient loading, caused declines in calcium concentrations, and 416

increased the spread of non-native predators (Palmer et al. 2011), with resultant changes in food web 417

structure (Paterson et al. 2008; Yan et al. 2008). To fully understand how Ca decline will influence 418

aquatic food webs, we will need to quantify its interaction with additional stressors that co-occur in 419

Canadian Shield lakes. 420

Conclusion 421

The combined results of our three experiments suggest that crustacean zooplankton 422

communities will be dramatically impacted by on-going declines in aqueous calcium concentrations. 423

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In our two experiments where Ca treatments were as low as 0.6 and 0.9 mg Ca/L, we observed 424

declines in population growth rates of a diversity of taxa, including daphniids, bosminids, and 425

several species of copepods. This suggests that predicted future [Ca] (38% of lakes <1.5 and 8% of 426

lakes <1.0 mg Ca/L; Reid and Watmough 2016) are likely to result in lower zooplankton 427

productivity with potential effects on other food web components. 428

429

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Acknowledgements: 430

Funding was provided by NSERC-Discovery and NSERC-Strategic Network (CAISN) 431

grants. Logistical support was provided by Killarney Provincial Park, the Ontario Ministries of 432

Natural Resources and Forestry, and Environment and Climate Change. Water chemistry was 433

provided by the Dorset Environmental Science Centre, Dorset, ON. Field assistance was provided 434

by Milly Corrigan, Laura Redmond, James Sinclair, Phillip Anderson, Matthew Laird, Bill Nelson, 435

Adam Sprott, Sarah Lamb, and Sarah Hasnain. Norman Yan, Howard Riessen, Jim Rusak, Keith 436

Somers, Brian Cumming, and Lonnie Aarssen provided valuable input during the design and 437

execution of the experiments. 438

439

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833. 577

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582

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Table 1: Location and water chemistry of lakes used in experiments. Water chemistry analyzed by 583

Ontario Ministry of Environment and Climate Change Dorset Environmental Science Centre, based 584

on a single mid-summer sample. * indicates lakes where experiments were conducted or where water 585

was used for experiments. Other lakes provided zooplankton for experiments. 586

587

588

589

590

Lake name Location Experiment Ca

(mg/L) Mg

(mg/L)

pH TP

(µg/L)

DOC

(mg/L)

Havelock* 45.29, -78.63 Havelock 1.38 0.36 6.4 4.2 3.6

McFadden 45.20, -78.51 Havelock 2.8 1.08 6.9 3.3 3.1

Big Brother 45.08, -78.46 Havelock 3.3 0.62 6.7 6.6 5.9

Beech 45.40, -78.41 Havelock 6.72 1.83 7.1 5.8 3.7

Moose 45.09, -78.27 Havelock 7.22 1.86 7.2 4.5 4.1

Percy 45.21, -78.36 Havelock 7.31 1.04 7.3 4.9 5.6

Plastic* 45.11, -78.50 Dorset 1.1 0.32 6.0 4.2 3.0

Ridout 45.10, -78.59 Dorset 1.78 0.54 6.4 3.9 4.3

Fifteen Mile 45.35, -78.96 Dorset 1.84 0.62 6.7 4.6 3.4

Red Chalk 45.18, -78.93 Dorset 1.84 0.62 6.5 3.1 3.7

Blue Chalk 45.18, -78.93 Dorset 2.26 0.63 6.8 2.4 2.3

Big Glamour 44.96, -78.37 Dorset 13.8 - 8.0 16.0 4.7

Glen 45.13, -79.50 Dorset 24.8 - 7.5 10.6 3.8

Grandview 45.20, -79.05 Dorset 4.19 1.78 6.4 6.9 2.7

Long Line 45.25, -78.98 Dorset 3.27 1.22 5.2 5.2 3.8

Lumsden* 46.03, -81.43 Lumsden 0.93 0.36 5.7 2.2 2.3

AY Jackson 46.02, -81.40 Lumsden 1.44 0.62 6.1 6.1 3.8

Bell 46.13, -81.21 Lumsden 1.82 0.64 6.3 5.1 5.8

Johnnie 46.09, -81.23 Lumsden 1.52 0.54 6.2 5.4 4.2

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Figure Legends: 591

Figure 1: a) Distribution of mean per capita growth rates (ind/ind/day) for each taxon across all 592

enclosures. Vertical dashed line represents the mean growth rate of all taxa. Black bars represent 593

daphniids, gray bars represent non-daphniid cladocera, and white bars represent copepods. b) Mean 594

growth rate of crustacean zooplankton in each enclosure in relation to Ca concentration in 595

mesocosms in Havelock Lake experiment. 596

Figure 2: Principle component analysis on growth rates (ind/ind/day) of species found in individual 597

enclosures for a) Havelock, b) Lumsden, and c) Dorset experiments. Grayscale associated with 598

points represent Ca concentrations of mesocosms (white = low Ca to black = high Ca). 599

Figure 3: Mean growth rates (ind/ind/day) of taxa in a) low Ca (0.9 mgCa/L) and b) high Ca (2.3 600

mgCa/L) enclosures in Lumsden Lake experiment. Black bars represent daphniids, gray bars 601

represent non-daphniid cladocera, and white bars represent copepods. Vertical dashed line represents 602

the mean. 603

Figure 4: Difference in growth rates of individual taxa between the highest and lowest calcium 604

treatments in the Lumsden (grey triangle) and Dorset (black circle) experiments. Error bars are 605

standard error of the mean. Taxa are ordered based on Ca body content from the literature (Azan et 606

al. 2015) and grouped based on taxonomic similarity. Numbers in brackets give the range of 607

measured Ca body content (%DW) from Azan et al. (2015). 608

Figure 5: Mean growth rates (ind/ind/day) of taxa in a) 0.6 mgCa/L, b) 1.0 mgCa/L, c) 1.4 mgCa/L, 609

and d) 2.4 mgCa/L treatments in the Dorset Experiment. Black bars represent daphniids, gray bars 610

represent non-daphniid cladocera, and white bars represent copepods. Vertical dashed line represents 611

the mean. 612

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613

Figure 1 614

615

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616 Figure 2 617

D.pul

Eub

Bos

Lepto

M.ed

A.ver

HolD.cat

D.men

a.

1.2 2.5

b.

Eub

D.pulCal.cp

L.m in

BosM.ed

D.cat

cal.np

cyc.np

A.ver

c.

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618

Figure 3 619

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620

Figure 4 621

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622

Figure 5 623

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