1

Catchment properties controls photosynthetic trait composition of 1

freshwater plant communities 2

Short title: Catchments rules aquatic plant traits 3

One sentence summary: The widespread photosynthetic trait of freshwater plants, bicarbonate 4

use, has a global biogeography controlled by catchment characteristics. 5

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Author list: 7

Iversen#*, L. L.1, Winkel*, A.1, Baastrup-Spohr*, L.1, Hinke, A. B.1, Alahuhta, J.2, Baattrup-8

Pedersen, A.3, Birk, S.4, Brodersen, P.1, Chambers, P A.5, Ecke, F.6, Feldmann, T.7, Gebler, D.8, 9

Heino, J.9, Jespersen, T S.1, Moe, S J.10, Riis, T.3, Sass, L.11, Vestergaard, O.12, Maberly§, S C.13, 10

Sand-Jensen§, K.1, Pedersen#§, O.1 11

12

* contributed equally 13

§ contributed equally 14

# Corresponding author (s) 15

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1 University of Copenhagen; 2 University of Oulu; 3 Aarhus University; 4 Universität Duisburg 17

Essen; 5 Environment Canada; 6 Swedish University of Agricultural Sciences; 7 Estonian 18

University of Life Sciences; 8 Poznán University of Life Sciences; 9 Finnish Environment 19

Institute; 10 Norwegian Institute for Water Research; 11 University of Illinois; 12 United 20

Nations Environmental Program; 13 Centre for Ecology & Hydrology 21

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Abstract: 28

Unlike land plants, the photosynthesis of many aquatic plants replies on bicarbonate in addition 29

to CO2 to compensate for the low diffusivity and potential depletion of CO2 in water. 30

Concentrations of bicarbonate and CO2 vary greatly with catchment geology, however, it is 31

unclear whether there is a link between these concentrations and the frequency of freshwater 32

plants possessing the bicarbonate-use trait. Here we show, globally, that the frequency of plants 33

with this trait increases with bicarbonate concentration. Regionally however, the frequency of 34

bicarbonate-use is reduced at sites that are supersaturated with CO2, such as many streams, 35

consistent with this trait being an adaptation to carbon limitation. Future anthropogenic changes 36

of bicarbonate and CO2 concentration may alter the species composition of freshwater plants. 37

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Main Text: 39

The biogeography of terrestrial plants is controlled largely by climatic factors; primarily air 40

temperature and precipitation (1). Furthermore, the distribution of biochemical traits such as the 41

two terrestrial CO2 concentrating mechanisms, C4 photosynthesis and Crassulacean Acid 42

Metabolism, are linked to temperature and water availability (2). Freshwater angiosperms 43

evolved from terrestrial ancestors (3), but unlike terrestrial plants do not face water-supply 44

problems. Instead, their growth is controlled by light, nutrients and inorganic carbon (4). 45

3

Inorganic carbon potentially limits photosynthesis in aquatic systems, because the diffusion of 46

CO2 is 104-fold lower in water than in air. Consequently, the CO2 concentration needed to 47

saturate photosynthesis is up to 12 times the air equilibrium concentration (5). Moreover, rapid 48

photosynthesis can reduce CO2 in water substantially below air saturation (4). 49

50

In response to carbon limitation, a few aquatic angiosperms evolved the same CO2 concentrating 51

mechanisms found in their terrestrial ancestors, but the most frequent mechanism, found in about 52

half of studied submerged freshwater plants, is the use of bicarbonate (HCO3-; 4,6), exploiting 53

bicarbonate derived from mineral weathering in soils and rocks in the catchment. Bicarbonate is 54

the dominant form of inorganic carbon in fresh waters when pH is between ~6.3 and ~10.2, and 55

concentrations often exceeds that of CO2 by 10- to 100-fold (6). The ability to use bicarbonate 56

appears to have evolved independently in most groups of cyanobacteria, algae and vascular 57

aquatic plants. This shows the fundamental importance of bicarbonate-use to plant fitness (6) and 58

an increase of photosynthesis, growth and primary productivity at higher bicarbonate 59

concentrations has been documented (7-9). However, bicarbonate-use is not ubiquitous because 60

its use involves costs as well as benefits. Costs include energy: it is an active process (10) and 61

permeability: rates of photosynthesis at limiting concentrations of CO2 are greater in CO2-users 62

than in bicarbonate-users (5,11). Thus, where CO2 concentrations are substantially above air 63

saturation, as is often the case in streams, the benefit of bicarbonate-use will be low (12). 64

Furthermore, obligate CO2-users can exploit alternative CO2 sources in the air, lake sediment or 65

in the water overlying the sediment (13), allowing continued photosynthesis without the need to 66

invest in mechanisms required for bicarbonate-use. 67

68

4

We hypothesized that since limitation of photosynthesis by inorganic carbon supply is 69

widespread in freshwater plants, the relative concentration of bicarbonate and CO2 at a particular 70

site should determine the proportion of plants that are obligate CO2-users vs bicarbonate-users. 71

Since geochemical catchment characteristics determine bicarbonate concentration, there should 72

be broad biogeographical patterns in the proportion of freshwater plants able to use bicarbonate 73

while at a smaller scale, both the CO2 and bicarbonate concentrations in lakes and streams might 74

structure the functional group composition. 75

76

To test these hypotheses, we generated a database for freshwater angiosperms and their ability to 77

use bicarbonate as an inorganic carbon source, based on data found in the literature. These were 78

complemented with new data we gathered on 27 species from mainly tropical regions where few 79

prior data existed (Extended Data Table 1 and Methods). Of the 131 species for which the carbon 80

source preference was known or measured as part of this investigation, 58 (44%) could use 81

bicarbonate. In order to quantify the distribution of bicarbonate-users vs CO2-users, we used: i) 82

approximately 1 million georeferenced plant records; ii) global plant ecoregion species lists; and 83

iii) 963 site-specific plant compositions from northern hemisphere lakes and streams (Extended 84

Data Fig. 1). In each of the investigated 963 sites plant composition was related to measured, 85

concentration of CO2 and bicarbonate. The georeferenced plant records and ecoregion species 86

lists were linked to local bicarbonate concentrations derived from a constructed global map of 87

bicarbonate concentration (Methods; Extended Data Fig. 2). 88

89

Concentrations of both bicarbonate and CO2 affected the occurrence of obligate CO2-users vs 90

bicarbonate-users, but differently within and between lakes and streams (Fig. 1, and Extended 91

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Data Fig. 3). The chance of observing a bicarbonate-user in lakes and streams correlated directly 92

with concentrations of bicarbonate and CO2 (∆Habitat = -0.82 [-1.64; 0.01] (mean [95% 93

confidence intervals], ∆ represents the difference between streams and lakes in parameter 94

estimates), Fig. 1A). However, with increasing bicarbonate concentrations, the likelihood of 95

observing a bicarbonate-user increased in lakes, but not in streams (∆βBicarbonate = -0.82 [-1.10; -96

0.54], Fig. 1B). Moreover, with an increase in CO2, the chance of observing a bicarbonate-user 97

decreased in both habitat types (∆βCO2= -0.04 [-0.22; 0.13], Fig. 1C). This is the first time that 98

the concentration of bicarbonate has been shown to have a different effect on the proportion of 99

bicarbonate-users in lakes vs streams. Unlike in lakes, no relationship between bicarbonate 100

availability and bicarbonate-users was found in streams. This upholds our hypothesis that where 101

concentrations of CO2 are high, the competitive advantage of using bicarbonate as a carbon 102

source for photosynthesis will be reduced. 103

104

Across global plant regions (14), the shifting proportions of bicarbonate-users vs obligate CO2-105

users showed distinct spatial patterns (Fig. 2A). Compared to the overall mean, a higher 106

proportion of bicarbonate-users was observed in Africa, temperate Asia, and the northern part of 107

North America (Fig. 2A). Globally, greater proportions of species utilizing bicarbonate were 108

found in areas with higher bicarbonate concentrations (bicarbonate-users - CO2-users = 0.16 109

[0.02; 0.30]; Fig. 2C; see Fig. 3 for a local example). The proportion of bicarbonate-users also 110

increased with bicarbonate concentrations within ecoregions (β = 0.14 [0.05; 0.24], (mean [95% 111

confidence limits]), Fig. 2B). Because catchment geology and geological history shape the 112

distribution of lakes and rivers, as well as the bicarbonate concentrations in freshwater 113

ecosystems (15-16), they are the chief determinants of plant distribution in freshwaters (17). CO2 114

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concentrations are largely regulated by local CO2 supersaturated inflow (18) and ecosystem 115

metabolism, making modeling difficult at large spatial scales (16,19). Thus, future models of 116

freshwater CO2 concentrations may improve the prediction of plant distributions even further. 117

Although global lake and river data exist to some extent as annual means (20) given the temporal 118

variability in CO2 concentration, the appropriate concentration would be that during the growing 119

season at the specific site (21). 120

121

Anthropogenic changes as a consequence of deforestation, cultivation of land, application of 122

nitrate fertilizers and reduced atmospheric acid deposition (22) are causing large-scale increases 123

in bicarbonate concentrations (23-24). The observed increasing bicarbonate concentrations are 124

expected to cause a severe impact on bicarbonate-poor lakes, because higher bicarbonate 125

concentrations will markedly change species composition (17) by allowing tall, fast-growing 126

bicarbonate-users to colonize and suppress smaller species adapted to the use of CO2 alone in or 127

near the sediment (25). Due to eutrophication, many small sediment-CO2 users are already 128

declining globally or regionally (26). In contrast, increasing atmospheric CO2 concentrations, 129

even if they influence dissolved CO2, will have little effect on the abundance of bicarbonate-130

users, since increases in CO2 are small relative to bicarbonate concentrations and will have little 131

effect on their photosynthesis rate. 132

133

Our study shows that bicarbonate-use by aquatic angiosperms is widespread in fresh waters 134

around the globe, and that the proportion of obligate CO2-users to bicarbonate-users is 135

significantly related to the bicarbonate concentration. Among terrestrial plants, the evolution of 136

leaf traits and different photosynthetic pathways that enable rapid carbon assimilation and 137

7

improved water economy (27) has resulted in global biogeographical patterns that are linked to 138

variations in climate (28-29). In contrast, for freshwater plants, we show that biogeographical 139

patterns of bicarbonate-use exist and that these are caused by catchment properties that 140

determine the concentration of bicarbonate and CO2. This insight will help evaluate the 141

repercussions of future changes in concentration of bicarbonate and CO2 on the biodiversity and 142

ecosystem function for fresh waters. 143

144

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Fig. 1: Bicarbonate-use in 145 submerged freshwater plant 146 communities. (A) likelihood of 147 observing a bicarbonate-user vs a 148 CO2-user in streams (n=172, red) and 149 lakes (n=791, blue); (B and C), 150 modeled odds of observing a 151 bicarbonate-user vs a CO2-user as a 152 function of bicarbonate (B) and CO2 153 (C) concentrations. Values > 1 154 indicate a higher likelihood (A) or 155 increase in likelihood (B and C) of 156 observing a bicarbonate-user vs a 157 CO2-user. The dotted vertical lines 158 show mean estimates and shaded 159 areas the 95% confidence limits 160 around the mean. 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177

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178 179 Fig. 2: Global relationship between bicarbonate and the proportion of bicarbonate-users in 180 freshwater plants. (A) Proportion of bicarbonate-users across 52 plant ecoregions. Grey areas 181 indicate regions where information on bicarbonate-use in local plants is not available. (B) 182 Relationship between mean bicarbonate concentration in plant regions and frequency of 183 bicarbonate-users. The solid line represents the mean proportion of bicarbonate-users. (C) 184 Density plots of bicarbonate preferences for bicarbonate-users (n = 57) and obligate CO2-users (n 185 = 72). The central horizontal black line represents the mean and the boxes indicate the 95% 186 confidence intervals around the mean. 187 188 189 190

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191 Fig. 3: Steep gradients in bicarbonate concentrations and spatial separation in species 192 distribution in the British Isles. Distribution of two pondweed species with contrasting 193 bicarbonate-use in the British Isles. Potamogeton polygonifolius (obligate CO2-user, black 194 triangles) is found in areas with lower bicarbonate concentrations compared to Potamogeton 195 crispus (bicarbonate-user, white circles). The top left insert shows the density distribution of the 196 two species across bicarbonate concentrations. 197 198

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Supplementary information is available 280

Materials and Methods 281

Figure S1 - Site-specific observations of bicarbonate use 282

Figure S2 - Global bicarbonate map 283

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Figure S3 - The probability of observing bicarbonate use in a species at 963 study sites 284

Figure S4 - Overview of in situ lake bicarbonate measures 285

Table S1 - List of freshwater angiosperms and their trait of inorganic carbon use 286

Table S2 - Source data 287

288

Acknowledgements 289

L.L.I was funded by the Carlsberg Foundation (grant 0604-02230B and CF17-0155). L.B.-S. was 290

funded by the Aage V. Jensen Foundation. K.S.-J. was funded by the Carlsberg Foundation 291

(grant CF14-0136). 292

Author contributions 293

L.L.I., A.W., L.B-S., S.C.M., K. S.-J. and O. P. designed the study, framed the research 294

questions, and wrote the manuscript, with input from the working group (A.B.H., J.A., A.B-P., 295

P.B., P.A.C., F.E., T.F., J.H., T.S.J, S.J.M., T.R., L.S. and O.V.). L.L.I. analyzed the data and 296

prepared the figures. A.B.H and O.P. performed the pH-drift experiments and together with 297

A.W. searched the literature for bicarbonate uptake in aquatic plants. A.W., L.L.I., and L.B-S. 298

assembled the data for the global analysis. 299

F.E., L.B-S, L.S., S.C.M., S.J.M, J.A., and T.F. assembled the site-specific lake data and the site-300

specific stream data was assembled by A. B.-P., P.B., P.A.C., D.G., K.S.-J., T.R, T.S.J., and 301

O.V., A.W., L.L.I., and L. B-S. prepared the site-specific data for further analysis. 302

Additional information 303

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The authors declare no competing financial interests. Supplementary information accompanies 304

this paper. Correspondence and requests for materials should be addressed to L.L. Iversen or O. 305

Pedersen. 306