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Adaptation to simultaneous warming and acidification carries a thermal tolerance cost in a marine copepod

James A. deMayo

James A. deMayo

Department of Marine Sciences, University of Connecticut, Groton, CT 06340-6048, USA

[email protected]

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Amanda Girod

Amanda Girod

Department of Molecular Biology and Biochemistry, Middlebury College, Middlebury, VT 05753, USA

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Matthew C. Sasaki

Matthew C. Sasaki

Department of Marine Sciences, University of Connecticut, Groton, CT 06340-6048, USA

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Hans G. Dam

Hans G. Dam

Department of Marine Sciences, University of Connecticut, Groton, CT 06340-6048, USA

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    The ocean is undergoing warming and acidification. Thermal tolerance is affected both by evolutionary adaptation and developmental plasticity. Yet, thermal tolerance in animals adapted to simultaneous warming and acidification is unknown. We experimentally evolved the ubiquitous copepod Acartia tonsa to future combined ocean warming and acidification conditions (OWA approx. 22°C, 2000 µatm CO2) and then compared its thermal tolerance relative to ambient conditions (AM approx. 18°C, 400 µatm CO2). The OWA and AM treatments were reciprocally transplanted after 65 generations to assess effects of developmental conditions on thermal tolerance and potential costs of adaptation. Treatments transplanted from OWA to AM conditions were assessed at the F1 and F9 generations following transplant. Adaptation to warming and acidification, paradoxically, reduces both thermal tolerance and phenotypic plasticity. These costs of adaptation to combined warming and acidification may limit future population resilience.

    1. Introduction

    The unprecedented rate of global climate change is expected to yield atmospheric CO2 levels of 1000 µatm by the end of 2100 [1], and 2000 µatm by 2300 [2] generating temperature increases of 3–5°C and pH levels between 7.8–7.6 [1]. Evolutionary adaptation provides a mechanism to mitigate deleterious effects of climate change [3]. Yet, adaptation may incur costs. Here, we are concerned with adaptation costs arising from reduced phenotypic plasticity—the ability for phenotypes to respond to environmental change without changes to the genome—which ultimately may reduce fitness [4,5]. These adaptation costs can be demonstrated through reciprocal transplant experiments, which can identify potential fitness penalties in non-native environments [68]. Selection can limit phenotypic plasticity [912], either by genetic assimilation [1315] or other mechanisms, but can also support the maintenance of phenotypic plasticity by phenotypic or genetic accommodation [1618].

    Thermal tolerance—the temperature that represents an organism's ability to handle thermal stress (i.e. CTmax, CTmin, LD50 [19])—should reflect environmental temperature [20,21]. The projected combination of warming and acidification, however, may result in novel environments for marine species [22,23]. Expanding studies into multi-generational, multi-stressor experiments that provide a more comprehensive view of future conditions is necessary to assess climate change adaptation and its costs [24]. Copepods, likely the most abundant metazoans [25,26], provide a critical link in marine food webs between primary producers and upper trophic level consumers, contributing greatly to biogeochemical cycles [27]. They also have short generation times, making them excellent candidates for evolutionary adaptation studies [28]. Despite their experimental potential, most copepod studies separately evaluate evolutionary or plastic effects of temperature [11,2931] and acidification [3237]. Previous studies addressing both effects on metazoans are rare, limited to few generations, or have not considered potential costs of adaptation [24,38,39].

    We took advantage of a separate study to examine transgenerational response of a copepod species (25 generations) to combined ocean warming and acidification (OWA) conditions [39,40]. Here, we extended the study to 65 generations (approx. 2 years) and evaluated thermal performance (the thermal reaction norm for survival across a temperature range [41]) relative to cultures raised for the same calendar time frame under average ambient, present-day conditions (AM). We then reciprocally transplanted both treatments and re-evaluated thermal performance to assess costs of adaptation to OWA and explore effects on developmental phenotypic plasticity. Treatments that were transplanted from OWA to AM, denoted as OWAAM, were evaluated twice: once at the initial transplant generation (F1T), and again after nine generations in the AM treatment (F9T) to evaluate transgenerational effects and their potential for evolutionary rescue [3,42]. We previously identified costs to OWA adaptation with a loss of plasticity after three transplant generations and measured life-history traits to estimate overall population fitness [39]. Here, we repeated the transplant but extended it to nine generations to identify if costs were sustained past three generations and to evaluate costs separate from conventional life-history traits.

    2. Material and methods

    (a) Study organism and culturing

    Acartia tonsa (Copepoda: Calanoida) is an estuarine and coastal foundational species [25,26], used in previous experimental evolution studies [28,33,40]. Acartia tonsa was collected June 2016 from Long Island Sound CT USA (41.3 N, −72.0 W). Copepods were cultured in the laboratory for at least three generations prior to the start of the transgenerational experiment to minimize the influence of parental effects and acclimation to field conditions on observed patterns [43]. Copepods were fed every 48–72 h at food-replete concentrations of carbon (greater than 800 µg C l−1) on a mixed prey phytoplankton diet [44]. Parental lineages were acclimated to one of two experimental conditions: (i) AM—ambient temperature (18°C), ambient CO2 (400 µatm CO2, pH ∼ 8.2), (ii) OWA—high temperature (22°C), high CO2 (2000 µatm CO2, pH ∼ 7.5). About 400 females and 200 males were used to seed each treatment, yielding approximately 15 000 eggs and newly hatched nauplii to start the F1 generation. During the first 30 generations, copepods for each treatment were separated into four replicate 10 l culture containers kept in separate temperature-controlled incubators per treatment. After generation 30, cultures were moved to a different location for long-term maintenance. Culture volume was increased to 18 l with containers kept in one of two large water baths. Temperatures in this new location were controlled with aquarium heaters (JBJ Aquariums, CA, USA) and temperature chillers (Aqua Logic, San Diego, CA, USA). Elevated CO2 levels for all 65 generations were maintained with two-gas proportioners (Cole-Parmer, Vernon Hills, IL, USA) combining air with 100% bone dry CO2 that was delivered continuously to the bottom of each replicate culture for the entire experiment. Target pH values were monitored daily using a pH probe (Orion Ross Ultra pH/ATC Triode with Orion Star A121pH Portable Meter; Thermo FisherScientific®, Waltham, MA, USA). Continuous bubbling maintained dissolved oxygen levels at greater than 8 mg l−1. Copepod water was changed every generation (approx. 10–12 days for OWA, approx. 14–16 days for AM) for the first 30 generations, and every two weeks thereafter. Media were replaced with pre-acidified, temperature-acclimated water for respective cultures.

    (b) Transplants

    For transplants, eggs and newly hatched nauplii representing half of each culture were moved into new developmental environments at the start of a new generation. Each transplant contained greater than 1500 individuals per replicate, minimizing chances of genetic drift or bottlenecks on performance [45]. Transplant between AM and OWA conditions yielded four replicates for each of two new treatments: AM in OWA (AMOWA) and OWA in AM (OWAAM). A schematic of the experiment and transplant is highlighted in electronic supplementary material, figure S1. Copepods were fed and media replaced as described above.

    (c) Heat stress experiments

    Acute heat stress experiments were done at 10 temperatures between 18 and 38°C at 2°C intervals following previous protocols [30,46,47]. Individual copepods were placed in 1.5 ml microcentrifuge tubes with 1.5 ml of 0.22 µm filtered Long Island Sound seawater. Copepods in OWA developmental environments were evaluated with pre-acidified water in a custom plexiglass enclosure with elevated atmospheric CO2 to maintain concentrations of 2000 µatm CO2 during the 24 h experiment. Copepods in AM developmental environments were evaluated at ambient pCO2. For temperatures ranging between 22 and 32°C, 14 individuals (seven females and seven males) per replicate culture (N = 4) per treatment were evaluated. For temperatures ranging from 18 to 20°C and 34 to 38°C, 14 individuals (seven females and seven males) from two of the four replicates per culture were evaluated. The different number of individuals at different stress temperatures allowed us to use more temperatures across a larger range. OWAAM transplants were evaluated twice: at the initial transplant generation (F1T) and after nine generations (F9T). The number of copepods evaluated totalled 2224 individuals. Survivorship was recorded under a dissection microscope as the response to physical stimuli or visible gut-passage movement and scored as binary data (1, survival; 0, mortality).

    (d) Analyses

    All analyses were performed in R (v. 3.6.1). For survival, we constructed generalized linear mixed-effects models using the lme4 package [48] to test for effects of lineage, developmental environment, generation, sex and stress temperature on survivorship (survivorship∼lineage × dev.treatment × generation × sex × temp) with culture replicates included as a random factor. We created secondary models of specific data subsets based on lineage or developmental environment including the same fixed and random effects mentioned previously. We used post hoc ANOVAs on these secondary models to evaluate the effects of an environment on a lineage, or to compare survival across different lineages. Generational effects were tested in the OWA lineage-specific model. Comparisons across lineages (i.e. OWA to AM) tested for effects of adaptation. Comparisons within lineages across developmental environments (i.e. OWAAM to OWAOWA) tested for effects of developmental plasticity. Generalized linear models were constructed for each treatment/developmental environment/generation combination to calculate the LD50 (temperature at which there is 50% mortality) and maximum survivorship (i.e. the highest survivorship at any temperature). ‘Thermal tolerance’ was estimated as LD50 with accompanying standard error, as calculated using the MASS package [49].

    3. Results

    (a) Costs linked to adaptation

    Copepods adapted to OWA conditions (OWAOWA; figure 1a), surprisingly, displayed a thermal performance curve shifted towards colder temperatures, which was significantly lower than AMAM due to lowered high-temperature performance (figure 1a) (p < 0.01, d.f. = 1, χ2 = 16.53). This is also reflected in the thermal tolerance of OWAOWA, which is the lowest of all treatments (OWAOWA LD50 = 27.32 ± 0.31°C; AMAM LD50 = 29.34 ± 0.35°C; AMOWA LD50 = 28.13 ± 0.53°C; OWAAM F1T LD50 = 28.12 ± 0.42°C; OWAAM F9T LD50 = 29.84 ± 0.62°C; figures 1b and 2a). Thermal performance of copepods from ambient conditions (AMAM), by contrast, was shifted furthest towards warmer temperatures out of any treatment (OWAOWA: p < 0.01, d.f. = 1, χ2 = 16.53; OWAAM: p = 0.01, d.f. = 1, χ2 = 6.38; AMOWA: p < 0.01, d.f. = 1, χ2 = 10.21; figure 1a) and experiences the highest thermal tolerance of any treatment (figure 2a).

    Figure 1.

    Figure 1. Thermal performance curves. (a) Thermal performance of AM (blue) and OWA (red) lineages among developmental environments during the F1 generation. AMAM (blue solid line) is significantly different from all other F1 treatments (OWAOWA (red dashed line): p < 0.01, d.f. = 1, χ2 = 16.53; OWAAM (red solid line): p = 0.01, d.f. = 1, χ2 = 6.38; AMOWA (blue dashed line): p < 0.01, d.f. = 1, χ2 = 10.21). (b) Thermal performance comparing F1T (yellow) and F9T (grey) OWAAM transplant treatments. Model for F9T is significantly different from OWAOWA and F1T OWAAM (p < 0.03, d.f. = 1, χ2 = 5.08).

    Figure 2.

    Figure 2. Thermal tolerance and maximum survivorship for AM and OWA treatments at F1. (a) LD50 stress temperatures evaluated for AM and OWA lineages in the respective treatments and reciprocally transplanted treatments. (b) Maximum survivorship across all temperatures for AM and OWA lineages. Colours represent lineage. Error bars represent standard error of the model. The low OWAOWA thermal tolerance reflects the lowest thermal performance of any treatment (p = 0.01, d.f. = 1, χ2 = 6.26), while AMAM represents the highest (p = 0.01, d.f. = 1, χ2 = 6.62).

    (b) Transgenerational effects

    Thermal tolerance (LD50) for the OWA treatment was partially recovered after development in AM conditions for multiple generations (figure 2b). After one generation, OWAAM (figure 1a) copepods experienced no significant change in thermal performance relative to OWAOWA (figure 1a; p = 0.11, d.f. = 1, χ2 = 2.54). However, after nine generations, we observed a significant increase in high-temperature thermal performance relative to that of the OWA lineage (OWAOWA and OWAAM) or the AMOWA transplant (p < 0.03, d.f. = 1, χ2 = 5.08) but like that of AMAM (p = 0.49, d.f. = 1, χ2 = 0.46). Additionally, a significant stress temperature × lineage interaction effect for OWAAM F9T is observed relative to AMAM (p < 0.002, d.f. = 9, χ2 = 26.23), and a significant stress temperature × generation effect relative to OWAAM (p < 0.0001, d.f. = 1, χ2 = 26.82). This suggests a change in the shape of the survivorship curve, resulting from increased mortality at low temperatures.

    (c) Developmental phenotypic plasticity

    The developmental environment effect for a lineage tests for developmental phenotypic plasticity. Copepods in the AM lineage showed developmental phenotypic plasticity—AMOWA thermal performance was lower than AMAM (p < 0.01, d.f. = 1, χ2 = 10.21; figure 1a). Copepods from the OWA lineage did not—OWAAM and OWAOWA had similar thermal performance (p = 0.11, d.f. = 1, χ2 = 2.54; figure 1a). Both transplant treatments (AMOWA and OWAAM) had the same thermal performance (p > 0.1, d.f. = 1, χ2 = 0.16), with values significantly lower than AMAM (p < 0.005, d.f. = 1, χ2 = 8.01), but not significantly different from OWAOWA (figure 2a). Moreover, AMOWA showed lowered maximum survivorship relative to AMAM (p < 0.01, d.f. = 75.49, t = 3.16; figure 2b), but the maximum survivorship for OWAAM at the F1T generation was not significantly different from OWAOWA (figure 2b).

    (d) Sex effects

    We observed no sex effects on the thermal performance of A. tonsa.

    4. Discussion

    (a) Costs of adaptation

    Copepods adapted to elevated temperatures and CO2 should perform better at higher temperatures, as has been reported for populations adapted to warmer temperatures [50]. A decrease in thermal performance of these copepods relative to those in ancestral conditions (cooler temperature, ambient CO2) reveals a cost to adaptation [7]. High-temperature thermal performance of copepods under OWA conditions decreased relative to AMAM in all cases. We previously observed that copepods raised under warming conditions increase thermal performance [30,50]. Here, exposure to acidification and warming decreases thermal performance, likely due to increased energetic costs of acid–base regulation to maintain homeostasis in a lower pH environment [51]. Across the stress temperatures evaluated, copepods adapted to OWA conditions exhibit a decrease in maximum survivorship in addition to a shift towards cooler temperatures. The reduced thermal tolerance for OWA copepods could also be explained by antagonistic interactions of warming and acidification, which we observed after 25 generations of adaptation [40]. Copepods from OWA lineages (OWAOWA and OWAAM) and those that developed in OWA conditions (AMOWA) show significantly lower high-temperature thermal performance relative to AMAM, with the OWAOWA treatment exhibiting the lowest LD50 (figure 2a). Meanwhile, the AMAM performance curve was the furthest towards warmer temperatures, which reflects a typical thermal tolerance for A. tonsa collected from Connecticut raised at 18°C [30].

    (b) Transgenerational effects

    Transgenerational effects for OWA copepods returning to ambient conditions are evident via changes between F1T and F9T (figure 1b). F9T cultures show an increase in thermal tolerance relative to the F1T cultures and resemble the AMAM thermal tolerance values. Moreover, comparing models for F1T and F9T reveals a significant temperature × generation effect, suggesting differential thermal performance across time with increased high-temperature performance at F9T. This suggests a relatively fast recovery after returning to ambient conditions, consistent with some degree of evolutionary rescue. Previously, we observed signals of selection for OWAAM transplants over multiple generations consistent with genetic changes rather than plasticity [39], which could explain the thermal performance patterns we observe here. Further analysis of F1T and F9T performance curves suggests lower maximum performance for F9T cultures than for F1T (figure 1b), which is consistent with a trade-off: LD50 improves at the expense of maximum survivorship. Thus, while tolerance of warm temperatures post-transplantation may increase after nine generations of habitation, tolerance of cold temperatures decreases, which represents costs associated with OWA adaptation.

    (c) Loss of plasticity

    Increased environmental temperature can increase thermal performance via both adaptation and plasticity [20,21]. Transplant experiments that include a return to ancestral conditions can be used to test for local adaptation [68], or maintenance of plasticity [52]. Comparisons among transplant treatments can also be used to assess for additional adaptation costs [7]. First, we consider how well populations maintained developmental phenotypic plasticity by comparing thermal performance between developmental conditions for each lineage. In our experiment, thermal performance changed only for copepods from the AM lineage exposed to OWA conditions. We have previously observed AM lineages maintain transcriptional plasticity when transplanted to OWA conditions [39]. This plastic response is also reflected here in the similar thermal tolerance and lowered maximum survivorship for the AMOWA treatment relative to OWAOWA (figure 2).

    In contrast with the AM lineage, we observe no change in thermal performance for copepods from the OWA lineage. Other work has shown that as populations adapt to environments closer to their thermal limit, the potential for plasticity decreases [911,30,50]. If plasticity were sustained and thermal performance reflected developmental temperature, we would expect thermal performance of OWAAM to decrease relative to OWAOWA. Our results suggest that warming and acidification combined lead to both lower plasticity and lower thermal tolerance. These results are also consistent with genetic assimilation [1315] because copepods from the OWA lineage do not change thermal performance with developmental environment [912,50]. Consequently, the thermal regime alone does not determine thermal tolerance or developmental plasticity in this species when CO2 is elevated. Forecasting changes in thermal performance in a warmer, more acidic environment should account for this interaction between stressors; predictions based solely on temperature changes may overestimate a population's capacity to respond via both adaptation and plasticity. Future work should evaluate whether adaptation to alternate multiple stressors exacerbates this loss of plasticity.

    5. Conclusion

    We provide clear evidence that thermal performance in this species is affected by multiple environmental factors, not just temperature regimes. In this study, adaptation to warming and acidification reduces thermal tolerance. All lineages that were raised in OWA conditions exhibit lowered thermal tolerance relative to their counterparts in ambient conditions. Further, consistent with previous experiments, the OWA lineage loses phenotypic plasticity [39] and expresses constitutive performance consistent with genetic assimilation [1315]. After nine generations in ambient conditions, the OWA lineage thermal performance is partially recovered, but this results in a trade-off of reduced survival at lower temperatures and a lower maximum survivorship. Thus, adaptation to OWA conditions is costly. We present evidence that (i) adaptation to warming and acidification incurs costs in the form of reduced phenotypic plasticity and thermal performance, (ii) developmental temperature does not predict thermal performance in the presence of acidified conditions, (iii) climate change conditions may, hence, limit evolutionary rescue. With future climate scenarios approaching rapidly, adaptation to simultaneous warming and acidification may limit population resilience.

    Data accessibility

    All data and code for analysing and visualizing the data are deposited into a Zenodo repository located at: [53].

    Authors' contributions

    J.A.d. designed the study, analysed the data, created figures, drafted and critically revised the manuscript. A.G. performed the experiments and revised the manuscript. M.C.S. designed the study and critically revised the manuscript. H.G.D. conceived the study, co-drafted the manuscript and critically revised the manuscript. All authors gave final approval for publication and agree to be held accountable for the work performed therein.

    Competing interests

    The authors declare no competing interests.


    This work was supported by NSF OCE-1559180, NSF (REU) 1658742, Connecticut Sea Grant R/LR_25, and the UConn Crandall Cordero Graduate Fellowship.


    The authors thank Drs Michael Finiguerra and Tracy Romano for leading the Mystic Aquarium REU Site that allowed A.G. to be a part of this study.


    Electronic supplementary material is available online at

    Published by the Royal Society. All rights reserved.