One experiment to rule them all? Testing multiple drivers of the temperature-size rule with nonlinear temperature increase

The temperature-size rule (TSR) describes the inverse relationship between organism size and environmental temperature in uni- and multicellular species. Despite the TSR being widespread, the mechanisms for shrinking body size with warming remain elusive. Here, we experimentally test three hypotheses (differential development and growth [DDG], maintain aerobic scope and regulate oxygen supply [MASROS] and the supply-demand hypothesis [SD]) potentially explaining the TSR using the aquatic protist Colpidium striatum in three gradually changing and one constant temperature environment crossed with three different nutrient levels. We find that the constant and slowly warming environments show similar responses in terms of population dynamics, whereas populations with linear and fast warming quickly decline and show a stronger temperature-size response. Our analyses suggest that acclimation may have played a role in observing these differences among treatments. The SD hypothesis is most parsimonious with the data, however, neither the DDG nor the MASROS hypothesis can be firmly dismissed. We conclude that the TSR is driven by multiple ecological and acclimatory responses and hence multicausal. Author statement A.T. designed and led the experiment, and A.T. and A.G. performed the sampling. A.T. and F.P. analyzed and interpreted the data. All authors contributed to the writing of the manuscript.


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Temperature and body size jointly influence biological rates from the individual to the ecosys-22 tem level (Brown et al. 2004). But temperature and size are not independent. About 80% of 23 ectotherms are smaller when grown in warmer environments (Atkinson 1994). This pattern is 24 so widespread that is has been named the temperature-size rule (TSR), describing the negative 25 correlation between body size and environmental temperature observed both in multi-and uni-26 cellular organisms (Atkinson 1994, Atkinson et al. 2003, Forster and Hirst 2012 2013). Despite the TSR being widespread and having important consequences for the structure 28 and functioning of communities, the mechanistic drivers of the TSR remain elusive. 29 Multiple hypotheses have been proposed and tested to explain the TSR ( Figure 1A). The 30 differential development and growth (DDG) hypothesis states that differences in thermal sensitiv-31 ities of growth and development rates of organismal ontogeny explain the TSR, i.e. temperature 32 changes the growth/reproduction trade-off (Forster et al. 2011). The DDG hypothesis assumes that 33 the rates of cell division and differentiation are more sensitive to temperature than cell growth and 34 hence increased temperature would result in smaller organisms (van der Have and de Jong 1996). 35 If, however, cell growth would be more strongly affected than development, larger sized individu- only occur during acclimation to novel temperature regimes and indicated by a temporary change 41 in the ratio of mother and daughter cell sizes (Forster et al. 2013). 42 Despite direct temperature effects on the physiology of individuals, it is also possible that 43 temperature affects size indirectly, that is, mediated by another environmental factor that is in-44 fluenced by temperature. The MASROS (i.e. maintain aerobic scope and regulate oxygen supply) 45 hypothesis proposes that organisms change their body size to regulate the oxygen demand of 46 their tissues (Atkinson et al. 2006) (Figure 1A). At higher temperatures organisms have increased 47 expect to see various responses such as vegetative enlargement to increase viability without induc-98 ing sexual reproduction (Atkinson et al. 2003), whereas in the optimal thermal range an inverse 99 relationship between size and temperature is expected. We predict a decoupling of cell mother 100 and daughter cell size ratios in all but the constant temperature treatment. We also predict an 101 increased growth rate with temperature increase, whereas resource supply is expected to remain  2015) with two autoclaved wheat seeds added for the slow release of carbon and nutrients.

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A mix of bacteria (Bacillus subtilis, Serratia fonticola and Brevibacillus brevis) forms the 109 basal food supply of the bacterivorous protist Colpidium striatum. We inoculated bacteria in the 110 medium 2 days prior to the experiment. We then added 1 mL Colpidium drawn from stock cultures 111 kept at 15 • C, to obtain an initial concentration of 3 individuals per mL and randomly assigned 112 microcosms to temperature treatments.

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The experiment has a full-factorial design of four levels of temperature treatment (constant 114 15 • C, three rising temperature between 15-30 • C, see Figure 1C) and three levels of nutrient con-115 centration (low = 0.28, medium = 0.56, and high = 1.12 g Protist Pellet medium per liter). We where µ is the growth rate, K is the carrying capacity and k is a constant which determines   In order to understand the relationship between cell size and population abundance, temper-166 ature, time and nutrients, we used a general linear model: where CS is the cell size measured as the median cell volume (MCV), t is time (days), P is 168 the cell density (cells mL −1 ), T is temperature ( • C) and N is the nutrient concentration (three  (Table S3). Cell shape also varied with time ( Figure S2). The changes in cell shape were generally 219 negatively correlated with the cell size over time (see Table S5 in Supplementary Information). In   Table 1). Variation in cell size was best explained in the constant and 235 slow warming treatments. Across treatments, variation in oxygen content of the medium was best 236 explained (R 2 : 0.56 -0.63), whereas abundance of bacteria had the lowest explanatory power (R 2 : 237 0.08 -0.11).

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The SEM indicated that cell size was impacted directly and indirectly by temperature (see 239 Figure 4 and Table S4). The direct effect of temperature on cell size (MCV) was negative in the fast   protist, support was found for the DDG hypothesis the during acclimation phase, which took 70h 274 and 120h for mother and daughter cells respectively (Forster et al. 2013). However, organisms were exposed to different constant temperatures, unlike the gradually changing environmental temper-276 atures as in our study. It is possible that acclimation to gradually changing temperature masked 277 the pronounced decoupling visible when cells were exposed immediately to higher temperatures.

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Nevertheless, we would have expected to see a decoupling at least in the linear and fast changing 279 temperature treatments. Therefore, we cannot fully exclude or support the DDG hypothesis. we did not reach in this experiment. Therefore, we cannot fully exclude or support the MASROS 294 hypothesis.

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The supply-demand model predicts changes in cell size as a function of temperature-dependent 296 demand and supply (DeLong 2012). Higher temperature will generally lead to higher metabolic 297 rates and hence demand. Temperature may also affect the supply of resources. We found that 298 temperature had a negative effect on growth rate, i.e. quickly increasing temperatures decreased 299 growth rates more than linearly or slowly increasing temperatures. Regarding supply, temperature a new population showed higher cell size at low density, and then a decrease in size until reaching 316 the carrying capacity. Therefore it is possible that the mechanisms explaining the TSR act at 317 different temporal scales, depending on the supply constraints: during the growth phase, when 318 resources are not limiting, the increased metabolic rate (and growth rate) may be most important.

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In contrast, when the population reaches carrying capacity (i.e., a density-dependent parameter 320 which balances the density, the cell size and the resource use), the SD model seems to fit the  where cells were immediately exposed to higher temperatures and the response compared to cell 329 size in control temperatures to which cells were adapted (see examples in Table S1). Our design 330 allows us to investigate the dynamic acclimation process as we exposed cells to gradually changing   (1) The differential development and growth (DDG) hypothesis states that in warm environment an organism has less time to grow, because warming accelerates the rate of development (++) faster than that of growth (+), which results in a smaller cell size. However, a reversed TSR has been observed in some cases, which might indicate that growth has higher sensitivity relative to development.