==== Front Ecol Evol Ecol Evol 10.1002/(ISSN)2045-7758 ECE3 Ecology and Evolution 2045-7758 John Wiley and Sons Inc. Hoboken 10.1002/ece3.6909 ECE36909 Original Research Original Research Loss and resiliency of social amoeba symbiosis under simulated warming SHU et al.Shu Longfei https://orcid.org/0000-0001-9683-906X 1 2 shulf@mail.sysu.edu.cn Qian Xinye 2 Brock Debra A. 2 Geist Katherine S. 2 Queller David C. 2 Strassmann Joan E. 2 1 Environmental Microbiomics Research Center School of Environmental Science and Engineering Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai) Sun Yat‐sen University Guangzhou China 2 Department of Biology Washington University in St. Louis St. Louis MO USA * Correspondence Longfei Shu, Sun Yat‐sen University, No. 132 East Circle, University Town, Guangzhou 510006, China. Email: shulf@mail.sysu.edu.cn 20 10 2020 12 2020 10 23 10.1002/ece3.v10.2313182 13189 02 6 2020 21 8 2020 17 9 2020 © 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Anthropogenic global change is increasingly raising concerns about collapses of symbiotic interactions worldwide. Therefore, understanding how climate change affects symbioses remains a challenge and demands more study. Here, we look at how simulated warming affects the social ameba Dictyostelium discoideum and its relationship with its facultative bacterial symbionts, Paraburkholderia hayleyella and Paraburkholderia agricolaris. We cured and cross‐infected ameba hosts with different symbionts. We found that warming significantly decreased D. discoideum's fitness, and we found no sign of local adaptation in two wild populations. Experimental warming had complex effects on these symbioses with responses determined by both symbiont and host. Neither of these facultative symbionts increases its hosts’ thermal tolerance. The nearly obligate symbiont with a reduced genome, P. hayleyella, actually decreases D. discoideum's thermal tolerance and even causes symbiosis breakdown. Our study shows how facultative symbioses may have complex responses to global change. Experimental warming had complex effects on the social amoeba symbioses with responses determined by both symbiont and host. The less facultative symbiont, P. hayleyella, actually decreases D. discoideum's thermal tolerance and even causes symbiosis breakdown. bacterial symbiontsDictyostelium discoideumglobal warmingParaburkholderiasymbiosisNational Natural Science Foundation of China 10.13039/5011000018093197038441907021Fundamental Research Funds for the Central Universities19lgpy162Hundred Talents Program through Sun Yat‐sen University38000‐18841205National Science FoundationNSF IOS‐1656756NSF DEB‐1753743John Templeton Foundation 10.13039/10000092543667 source-schema-version-number2.0cover-dateDecember 2020details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:5.9.5 mode:remove_FC converted:03.12.2020 Shu L , Qian X , Brock DA , Geist KS , Queller DC , Strassmann JE . Loss and resiliency of social amoeba symbiosis under simulated warming . Ecol Evol . 2020 ;10 :13182 –13189 . 10.1002/ece3.6909 Funding information This material is based upon work supported by the National Natural Science Foundation of China (31970384 and 41907021), the Fundamental Research Funds for the Central Universities (19lgpy162), the Hundred Talents Program through Sun Yat‐sen University (38000‐18841205), the National Science Foundation (NSF IOS‐1656756 and NSF DEB‐1753743), and the John Templeton Foundation (43667). ==== Body 1 INTRODUCTION Global warming causes biodiversity crises, which impact organisms not only directly but also indirectly through other organisms with which they interact (Bellard et al., 2012; Blois et al., 2013; Harley, 2011; Penuelas et al., 2013; Ullah et al., 2018). Symbiosis is important for global biodiversity, ecosystem services, and agriculture (Soka & Ritchie, 2015; Wernegreen, 2012; Werner et al., 2018). In recent years, the possibility that elevated temperatures resulting from global warming may substantially affect biodiversity through disrupting mutualistic associations such as the coral–dinoflagellate symbiosis (Hoegh‐Guldberg et al., 2007; Pandolfi et al., 2011), insect–bacteria symbioses (Kikuchi et al., 2016; Wernegreen, 2012), and plant–pollinator interactions (Eckert et al., 2010; Hegland et al., 2009) has been highlighted. The coral–dinoflagellate model, which is an obligate symbiotic relationship, showed that thermal stress could lead to coral bleaching (corals’ loss of zooxanthellae that provide up to 90% of host nutritional requirements) (Baker et al., 2018; Ferrier‐Pages et al., 2018). The stable, long‐term mutualistic relationship between insects and their carried symbionts is also vulnerable to thermal stress (Kiers et al., 2010). However, empirical investigations of facultative mutualism under global warming have been scarce and mostly focus on insects (Burke et al., 2010; Wernegreen, 2012). Research on facultative symbiosis is needed. The symbiosis between social amebae and certain Paraburkholderia bacterial species is a promising system for gaining insight into how facultative mutualisms respond to global warming. The soil‐dwelling ameba Dictyostelium discoideum is a good model to address eukaryote–microbe interactions because of its dynamic relationship with bacteria. In a nutrient‐rich environment, D. discoideum lives as independent haploid amebae that reproduce by binary fission. When food is scarce, cAMP‐mediated aggregation occurs, leading to the formation of multicellular slugs that move to a favorable location to develop into fruiting bodies. In these fruiting bodies, approximately 20% of the cells die to form a long thin stalk that supports a spherical structure called the sorus, while the remaining 80% ascend into the sorus and turn into spores (Kessin, 2001). D. discoideum is a predator of bacteria and a popular model for studying biological phenomena, including multicellularity, chemical signaling, and social phenomena (Chen et al., 2016; DiSalvo et al., 2015; Ho et al., 2013; Kessin, 2001; Shu et al., 2018; Strassmann & Queller, 2011; Zhang et al., 2016). In addition to eating bacteria, D. discoideum can also form symbiotic associations with some bacterial species (Brock et al., 2011; DiSalvo et al., 2015; Strassmann & Shu, 2017). About one‐third of wild‐collected clones of D. discoideum, which are referred to as “primitive farmers,” have stable associations with their symbiotic bacteria throughout their life cycle (Brock et al., 2011). These farmer clones can carry bacteria during spore dispersal and seed them as new food sources (Figure 1). Later studies found that farming status is induced by symbiotic bacteria belonging to the genus Paraburkholderia (DiSalvo et al., 2015; Haselkorn et al., 2019; Shu, et al., 2018) (named P. agricolaris, P. hayleyella, and P. bonniea (Brock et al., 2018)). These Paraburkholderia are not edible themselves, but they facilitate further carriage of food bacteria that on their own would be digested. The inedible symbionts actively find their ameba hosts through chemotaxis, reside within food vacuoles, and form very stable associations (Figure 1) (Shu et al., 2015; Haselkorn et al., 2019; Shu, et al., 2018; Shu, et al., 2018). Therefore, we also define their association as “bacterial carriage” by social ameba. FIGURE 1 Scheme summarizing the social ameba–Paraburkholderia symbiosis. Figure courtesy of Susanne DiSalvo Both D. discoideum and their Paraburkholderia symbionts can live independently, making them facultative symbioses. However, P. hayleyella shows three indications of being more obligate than P. agricolaris. First, it is a sister species comprising a very long branch in the phylogeny, suggesting that it has been associated with amebas for a long time (Brock et al., 2018; Haselkorn et al., 2019). Second, consistent with greater dependence on the host, it grows slowly on its own under laboratory conditions compared to P. agricolaris. P. hayleyella also has greatly reduced carbon usage compared to P. agricolaris (Brock et al., 2020). Finally, it shows the genome size reduced by over one half compared to close relatives (Brock et al., 2018). This system gives us an opportunity to investigate how increased temperatures associated with global warming could potentially affect facultative symbioses. Facultative symbioses could be more vulnerable to global warming compared to obligate symbioses because their relationships are less stable. Alternatively, facultative symbioses may be more resilient to global warming because both partners can live on their own and therefore may be more resilient to environmental changes. We will test whether these facultative symbionts help or harm their hosts under warming, and also whether the symbiosis is less or more resilient with the more facultative species P. agricolaris versus the more obligate species P. hayleyella. We first tested the thermal tolerance of social amebas using common garden experiments. Then, we mixed and matched social ameba hosts with different Paraburkholderia symbionts (Figure 2a) to investigate how different combinations respond to simulated global warming. FIGURE 2 (a) Diagram of symbiosis experimental design. The experiment explores how thermal stress affects D. discoideum–Paraburkholderia symbiosis by mixing and matching D. discoideum with two facultative symbionts P. agricolaris and P. hayleyella. (b) Spore count (mean ± 95% CI) of three D. discoideum clones under different temperatures ranging from 21 to 30°C. QS9, naïve host; QS11, native host carrying P. hayleyella B2qs11, and QS70, native host carrying P. agricolaris B1qs11; (c) Spore count (mean ± 95% CI) of two D. discoideum populations (Texas and Virginia) under two temperature treatments (27.5 and 21°C). All tested Texas and Virginia clones are naïve host which do not carry any Paraburkholderia symbionts 2 MATERIALS AND METHODS 2.1 D. discoideum clones and culture conditions We used wild D. discoideum isolates (Table 1) collected at Mountain Lake Biological Station in Virginia (N37°21′, W80°31′), Houston Arboretum and Nature Center in Texas (N29°77′, W95°45′) and Little Butt's Gap in North Carolina (35°46′ N, 82°20′ W). These clones were uninfected (called naïve hosts in this paper) or infected with either P. agricolaris or P. hayleyella (called native hosts in this paper). We grew D. discoideum from previously frozen spores on SM/5 agar plates (2 g glucose, 2 g BactoPeptone (Oxoid), 2 g yeast extract (Oxoid), 0.2 g MgCl2, 1.9 g KH2PO4, 1 g K2HPO4, and 15 g agar per liter) with food bacterium Klebsiella pneumoniae (obtained from the Dicty Stock Center) at room temperature (21°C). TABLE 1 List of wild D. discoideum clones and Paraburkholderia isolates used in this study. Checkmarks indicate specific clones in each test Clones Location Host types Symbionts Choosing test temperature Amebae under warming Symbioses under warming QS177 Texas Naïve host √ QS198 Texas Naïve host √ QS323 Texas Naïve host √ QS325 Texas Naïve host √ QS600 Texas Naïve host √ QS68 Texas Naïve host √ QS71 Texas Naïve host √ QS74 Texas Naïve host √ √ QS76 Texas Naïve host √ QS78 Texas Naïve host √ QS1010 Virginia Naïve host √ QS1041 Virginia Naïve host √ QS1068 Virginia Naïve host √ QS1072 Virginia Naïve host √ QS1080 Virginia Naïve host √ QS17 Virginia Naïve host √ QS18 Virginia Naïve host √ QS4 Virginia Naïve host √ QS6 Virginia Naïve host √ QS9 Virginia Naïve host √ √ √ QS1 Virginia Naïve host √ QS70 Virginia Native host P. agricolaris B1qs70 √ √ QS159 Virginia Native host P. agricolaris B1qs159 √ NC21 North Carolina Native host P. agricolaris B1nc21 √ QS11 Virginia Native host P. hayleyella B2qs11 √ √ QS21 Virginia Native host P. hayleyella B2qs21 √ NC28 North Carolina Native host P. hayleyella B2nc28 √ QS70C Virginia Cured native host √ QS159C Virginia Cured native host √ NC21C North Carolina Cured native host √ QS11C Virginia Cured native host √ QS21C Virginia Cured native host √ NC28C North Carolina Cured native host √ John Wiley & Sons, Ltd2.2 Symbionts We used D. discoideum‐associated Paraburkholderia symbionts isolated and described in previous studies (Brock et al., 2011; DiSalvo et al., 2015; Haselkorn et al., 2019; Shu, et al., 2018). P. agricolaris strains were isolated from QS70, QS159, and NC21 D. discoideum hosts, while P. hayleyella strains were isolated from QS11, QS21, and NC28 D. discoideum hosts, respectively. Specific isolates used in this study are listed in Table 1. 2.3 Choosing experimental temperature for simulating warming We wanted to choose an experimental temperature that is stressful to social amebae but does not cause complete death. We tested growth conditions of D. discoideum (three clones: QS11, QS70, and QS9) under different temperatures ranging from 21 to 30°C. We found that almost no clone can survive above 28°C, while there were drastic changes between 27 and 28°C (Figure 2b). Therefore, we chose 27.5°C as the thermal stress temperature for this experiment. We want to test how extreme warming event (from D. discoideum ameba's perspective) affects the social ameba symbiosis and whether its bacterial symbionts could help. 2.4 Effects of thermal stress on two wild D. discoideum populations We used two D. discoideum populations from geographic and climate divergent locations Texas (N29°46′, W95°27′; elevation, 11 m; annual temperatures: 5.7–34.7°C; average temperatures: 20.6°C) and Virginia (N37°21′, W80°31′; elevation, 1,160 m; annual temperatures: −15–25°C; average temperatures: 5.2°C) to investigate how D. discoideum responds to simulated thermal stress and whether they could locally adapt to it. We randomly chose 10 Texas clones and 10 Virginia clones of wild D. discoideum and plated those (2 × 105 spores) in association with K. pneumoniae (200 µl, OD1.5) on SM/5 plates. We incubated these clones at room temperature 21°C (control) and 27.5°C (thermal stress treatment), respectively. We harvested spores from each plate after one week. We flooded the plate with 10 ml KK2 + 0.1%NP‐40 and collected spores into 15 ml falcon tubes. We counted spores on a hemocytometer using a light microscope. This design resulted in a total of 2 (populations) × 10 (clones) × 2 (temperatures) × 3 (replicates) =  120 experimental units. The mean of three replicates was used for further statistical analyses. 2.5 Effects of thermal stress on D. discoideum–Paraburkholderia symbiosis We generated symbiont‐free native host clones (QS70C, QS159C, NC21C, QS11C, QS21C, and NC28C) by curing them of their bacteria with tetracycline, or by ampicillin–streptomycin treatment as previously described (Brock et al., 2011; DiSalvo et al., 2015; Shu, et al., 2018). We confirmed the loss of infection status by plating them out on bacteria‐free plates and confirming that the social amebae could not proliferate, a test we call a spot test (Brock et al., 2011). We mixed and matched (Figure 2) D. discoideum (naïve hosts: QS1, QS9, and QS74; native hosts: QS70C, QS159C, NC21C, QS11C, QS21C, and NC28C) with two facultative symbionts P. agricolaris (B1qs70, B1qs159, and B1nc21) and P. hayleyella (B2qs11, B2qs21, and B2nc28) to investigate how thermal stress affects their symbiotic relationships. We tested four combinations under two temperature treatments (21 and 27.5°C): native hosts—P. agricolaris, naïve hosts—P. agricolaris, native hosts—P. hayleyella and naïve hosts—P. hayleyella with three replicates. To set up each experiment, we plated 2 × 105 spores in association with K. pneumoniae (200 µl, OD1.5) on SM/5 plates. For experiments adding Paraburkholderia, we mixed the specified Paraburkholderia (OD1.5) clones at 3% (6 µl) and K. pneumoniae at 97% (194 µl) vol and plated D. discoideum spores (2 × 105) with 200 µl of the bacterial mixture on SM/5 plates. We incubated these clones at room temperature 21°C (control) and 27.5°C (thermal stress treatment), respectively. We harvested spores from each plate after one week and flooded the plate with 10 ml KK2 + 0.1%NP‐40 and collected spores into 15 ml falcon tubes. We counted spores on a hemocytometer using a light microscope. 2.6 Statistical analyses 2.6.1 Effects of thermal stress on two D. discoideum populations We analyzed the data (N = 40) with a general linear mixed model in IBM SPSS Statistics 24. In these analyses, population (two levels: Texas and Virginia), temperature (two levels: 21 and 27.5°C), and their interactions were used as fixed factors. D. discoideum clone was nested within population and used as a random factor. The data passed the normality test (Kolmogorov–Smirnov test) and tested for homogeneity of variance (Levene's test). We analyzed spore production (outcome variable) as a measure of ameba fitness. A significant temperature main effect would indicate thermal stress affects D. discoideum's fitness, a significant population type main effect would indicate that populations differ in their fitness, and a significant population × temperature interaction would indicate adaptive divergence in thermal tolerance in two populations. 2.6.2 Effects of thermal stress on D. discoideum–Paraburkholderia symbiosis We analyzed and plotted four combinations separately (native hosts—P. agricolaris, Figure 3a; naïve hosts—P. agricolaris, Figure 3b; native hosts—P. hayleyella, Figure 3c and naïve hosts—P. hayleyella, Figure 3d). Native hosts—P. agricolaris (N = 12), naïve hosts—P. agricolaris (N = 24), and native hosts—P. hayleyella (N = 12) data were log‐transformed to improve normality. Transformed data passed the normality test (Kolmogorov–Smirnov test) and tested for homogeneity of variance (Levene's test). We analyzed these data with general linear models. Naïve P. hayleyella data (N = 24) were analyzed with a generalized linear model (GLM) with Negative binomial distribution in IBM SPSS Statistics 24. FIGURE 3 Spore counts (mean ± 95% CI) of D. discoideum hosts (with and without P. agricolaris and P. hayleyella) under two temperature treatments (27.5 and 21°C). (a) P. agricolaris with their native hosts (hosts which they are isolated: QS70, QS159, NC21); (b) P. agricolaris with naïve hosts (non‐farmer clones: QS1, QS9, QS74); (c) P. hayleyella with their native hosts (hosts which they are isolated: QS11, QS21, NC28); (d) P. hayleyella with naïve hosts (non‐farmer clones: QS1, QS9, QS74) We used spore production as a measure of ameba fitness. A significant temperature main effect indicates that thermal stress can affect D. discoideum fitness. A significant symbiont main effect indicates that the presence of a symbiont can affect D. discoideum fitness. A significant temperature × symbiont interaction will indicate that the presence of symbiont can affect D. discoideum fitness under thermal stress. 3 RESULTS 3.1 The pattern of local adaptation to thermal stress in D. discoideum Increased temperature decreased the fitness of both the Texas clones and the Virginia clones (Figure 2c), as indicated by the significant temperature main effect (GLM, F 1,18 = 351.25, P < .001). Virginia clones outperformed Texas clones at both temperatures (Figure 2c). However, we found no variation in thermal tolerances of Texas and Virginia populations, as shown by the nonsignificant population × temperature interaction (GLM, F 1,18 = 2.141, P = .161). These results suggest that increased temperature significantly decreases D. discoideum's fitness. We did not find adaptive divergence to thermal stress in two wild populations of D. discoideum from locations that differed in ambient temperature. 3.2 The complex effects of simulated warming on D. discoideum–Paraburkholderia symbioses 3.2.1 P. agricolaris had no effect on D. discoideum's thermal tolerance When P. agricolaris clones were mixed with their native hosts, thermal stress decreased D. discoideum's fitness, as indicated by the significant temperature main effect (GLM, F 1,20 = 20.188, p < .001, Figure 3a). However, adding P. agricolaris made no difference to host fitness (GLM, F 1,20 = 2.406, p = .137, Figure 3a). The effect of thermal stress did not change with the addition of P. agricolaris, as indicated by the nonsignificant temperature*symbiont interaction (GLM, F 1,20 = 0.427, p = .521, Figure 3a). When P. agricolaris clones (n = 3) were mixed with naïve hosts (n = 3), the pattern is the same (Figure 3b). Thermal stress decreased D. discoideum's fitness (General linear model, F 1,8 = 82.087, p < .001, Figure 3b), while adding P. agricolaris made no difference to host's fitness (GLM, F 1,8 = 1.803, p = .216, Figure 3b). Also, there was no significant temperature*symbiont interaction (General linear model, F 1,8 = 0.004, p = .953, Figure 3b), indicating that the effect of thermal stress did not change with the addition of P. agricolaris. Overall, these results suggest that the more facultative P. agricolaris neither helps nor harms D. discoideum under thermal stress. In addition, there is no difference between native and naïve hosts. 3.2.2 P. hayleyella decreased D. discoideum's thermal tolerance and caused a symbiosis breakdown when mixed with naïve hosts When P. hayleyella clones (n = 3) were mixed with their native hosts (n = 3), thermal stress decreased D. discoideum's fitness, as indicated by the significant temperature main effect (GLM, F 1,8 = 44.747, p < .001, Figure 3c). We also found that adding P. hayleyella decreased host fitness (GLM, F 1,8 = 17.287, p = .003, Figure 3c). There was no significant temperature*symbiont interaction (GLM, F 1,8 = 2.624, p = .144, Figure 3c), indicating that adding P. hayleyella did not further decrease the native host's fitness under thermal stress (Figure 3c). When P. hayleyella clones (n = 3) were mixed with naïve hosts (n = 3), both adding P. hayleyella (Negative binomial GLM, χ2 = 6.73, p = .009) and thermal stress (Negative binomial GLM, χ2 = 8.471, p = .004) decreased D. discoideum's fitness (Figure 3d). There was also a significant temperature*symbiont interaction (Negative binomial GLM, χ2 = 4.958, p = .026, Figure 3d), indicating that adding P. hayleyella further decreased naïve host's fitness under thermal stress. In addition, 2 out of 3 tested naïve hosts showed zero growth under thermal stress when mixed with P. hayleyella, indicating symbiosis breakdown, while this did not happen in any of the native hosts. Taken together, these results suggest that adding P. hayleyella, like thermal stress, can decrease D. discoideum's fitness. In addition, it further decreases host fitness under thermal stress. We also found evidence of symbiosis breakdown when P. hayleyella was mixed with naïve hosts, while this does not happen in the native hosts. This indicates potential partner adaptation between P. hayleyella and their native hosts. 4 DISCUSSION Overall, we show that increased temperature affects symbiotic interactions. Increased temperature can significantly decrease D. discoideum's fitness. We found no adaptive divergence to thermal stress in two wild populations. Neither symbiont increased its hosts’ thermal tolerance. Our study shows that facultative symbioses can also have complex responses to warming. Previous studies found that facultative symbionts provide greater flexibility in response to temperature change compared to obligate symbioses (Burke et al., 2010; Renoz et al., 2019). For example, facultative bacterial symbionts benefit aphids under heat stress (Montllor et al., 2002) and may protect both host and obligate symbiont from thermal stress (Burke et al., 2010). However, in the social ameba symbiosis system, we find no evidence that facultative Paraburkholderia symbionts increase D. discoideum hosts’ thermal tolerance. We find that different symbionts behave differently within the same host under simulated warming, and we also find evidence of host adaptation. Of the two symbionts, the more facultative P. agricolaris has no effects on the thermal tolerance of either native or naïve D. discoideum hosts. On the other hand, the more obligate P. hayleyella induces a significant difference to the host's thermal tolerance, imposing a higher cost to D. discoideum. Our study shows that the addition of P. hayleyella to its native host decreases host fitness at both temperatures indicating that native hosts suffer a fitness cost when they carry P. hayleyella. In addition, P. hayleyella harms and even kills naïve hosts exposed to thermal stress, disrupting the symbiosis. The more severe fitness costs exerted by P. hayleyella colonization in naïve hosts compared to native hosts suggest potential host adaptation between P. hayleyella and their native host clones. One potential drawback of this study is that we did not monitor the population dynamics of K. pneumoniae and Paraburkholderia symbionts under different temperatures. Simulated warming can directly affect the interactions between food bacteria and symbionts, which in turn affects the growth of amebae. Indeed, a recent study reported that the optimal growth temperature for both Paraburkholderia symbionts is 30°C, and P. agricolaris grows faster than P. hayleyella (Brock et al., 2020). Therefore, in this study, both food bacterium Klebsiella pneumoniae and Paraburkholderia symbionts grow faster under warming conditions. However, we argue that their interactions may have little effect on host fitness. First, K. pneumoniae grows much faster than symbionts, and their starting proportion is very high (97%) compared to symbionts (3%). Second, the faster‐growing symbiont, P. agricolaris, did not change host's fitness in both temperatures, indicating its frequency has little effect on host fitness. Moreover, P. hayleyella grows much slower than P. agricolaris. Therefore, despite their faster growth under warmer temperatures, the major conclusion of this study still holds. Still, it will be useful to have such information in future studies. Taken together, our results provide insight into facultative symbioses under extreme warming. For the ameba–Paraburkholderia symbiotic relationship, the effects of adding different Paraburkholderia can be complex. The responses of social ameba symbioses to warming depend on both symbiont types and host types. Our study also shows that facultative symbionts are not necessarily more resilient to global change. In this system, the less facultative, more obligate symbiont has the less resilient symbiosis. Different symbioses may develop different evolutionary trajectories leading to unpredictable symbiosis resiliency with global warming. CONFLICT OF INTEREST The authors declare no conflicts of interest. AUTHOR CONTRIBUTION Longfei Shu: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Visualization (lead); Writing‐original draft (lead). Xinye Qian: Data curation (equal); Formal analysis (equal); Visualization (equal); Writing‐original draft (equal). Debra A. Brock: Data curation (equal); Formal analysis (equal); Writing‐review & editing (equal). Katherine S. Geist: Data curation (equal); Formal analysis (equal); Visualization (equal); Writing‐review & editing (equal). David C. Queller: Conceptualization (lead); Data curation (equal); Formal analysis (equal); Funding acquisition (lead); Supervision (lead); Writing‐review & editing (equal). Joan E. Strassmann: Conceptualization (lead); Data curation (equal); Formal analysis (equal); Funding acquisition (lead); Supervision (lead); Writing‐original draft (equal); Writing‐review & editing (equal). ACKNOWLEDGMENTS Many thanks to Mountain Lake Biological Station of the University of Virginia where we collected the samples. DATA AVAILABILITY STATEMENT All data are available from the Mendeley Data: http://dx.doi.org/10.17632/fjj9mbm6hw.1 ==== Refs REFERENCES Baker , D. M. , Freeman , C. J. , Wong , J. C. Y. , Fogel , M. L. , & Knowlton , N. (2018 ). Climate change promotes parasitism in a coral symbiosis . The ISME Journal , 12 (3 ), 921 –930 . 10.1038/s41396-018-0046-8 29379177 Bellard , C. , Bertelsmeier , C. , Leadley , P. , Thuiller , W. , & Courchamp , F. (2012 ). Impacts of climate change on the future of biodiversity . Ecology Letters , 15 (4 ), 365 –377 . 10.1111/j.1461-0248.2011.01736.x 22257223 Blois , J. L. , Zarnetske , P. L. , Fitzpatrick , M. C. , & Finnegan , S. (2013 ). Climate change and the past, present, and future of biotic interactions . Science , 341 (6145 ), 499 –504 . 10.1126/science.1237184 23908227 Brock , D. A. , Douglas , T. E. , Queller , D. C. , & Strassmann , J. E. (2011 ). Primitive agriculture in a social amoeba . Nature , 469 (7330 ), 393 –396 . 10.1038/Nature09668 21248849 Brock , D. A. , Hubert , A. M. , Noh , S. , DiSalvo , S. , Geist , K. S. , Haselkorn , T. S. , Queller , D. , & Strassmann , J. E. (2018 ). Endosymbiotic adaptations in three new bacterial species associated with Dictyostelium discoideum: Burkholderia agricolaris sp. nov., Burkholderia hayleyella sp. nov., and Burkholderia bonniea sp. nov . bioRxiv. 10.1101/304352 Brock , D. A. , Noh , S. , Hubert , A. N. M. , Haselkorn , T. S. , DiSalvo , S. , Suess , M. K. , Bradley , A. S. , Tavakoli‐Nezhad , M. , Geist , K. S. , Queller , D. C. & Strassmann , J. E. (2020 ) Endosymbiotic adaptations in three new bacterial species associated with Dictyostelium discoideum: Paraburkholderia agricolaris sp. nov., Paraburkholderia hayleyella sp. nov., and Paraburkholderia bonniea sp. nov . Peerj 8 , e9151 10.7717/peerj.9151 32509456 Burke , G. , Fiehn , O. , & Moran , N. (2010 ). Effects of facultative symbionts and heat stress on the metabolome of pea aphids . The ISME Journal , 4 (2 ), 242 –252 . 10.1038/ismej.2009.114 19907504 Chen , X. , Kollner , T. G. , Jia , Q. , Norris , A. , Santhanam , B. , Rabe , P. , Dickschat , J. S. , Shaulsky , G. , Gershenzon , J. , & Chen , F. (2016 ). Terpene synthase genes in eukaryotes beyond plants and fungi: Occurrence in social amoebae . Proceedings of the National Academy of Sciences of the United States of America , 113 (43 ), 12132 –12137 . 10.1073/pnas.1610379113 27790999 DiSalvo , S. , Haselkorn , T. S. , Bashir , U. , Jimenez , D. , Brock , D. A. , Queller , D. C. , & Strassmann , J. E. (2015 ). Burkholderia bacteria infectiously induce the proto‐farming symbiosis of Dictyostelium amoebae and food bacteria . Proceedings of the National Academy of Sciences of the United States of America , 112 (36 ), 5029 –5037 . 10.1073/pnas.1511878112 25848034 Eckert , C. G. , Kalisz , S. , Geber , M. A. , Sargent , R. , Elle , E. , Cheptou , P. O. , Goodwillie , C. , Johnston , M. O. , Kelly , J. K. , Moeller , D. A. , Porcher , E. , Ree , R. H. , Vallejo‐Marín , M. , & Winn , A. A. (2010 ). Plant mating systems in a changing world . Trends in Ecology & Evolution , 25 (1 ), 35 –43 . 10.1016/j.tree.2009.06.013 19683360 Ferrier‐Pages , C. , Sauzeat , L. , & Balter , V. (2018 ). Coral bleaching is linked to the capacity of the animal host to supply essential metals to the symbionts . Global Change Biol , 24 (7 ), 3145 –3157 . 10.1111/gcb.14141 Harley , C. D. G. (2011 ). Climate change, keystone predation, and biodiversity loss . Science , 334 (6059 ), 1124 –1127 . 10.1126/science.1210199 22116885 Haselkorn , T. S. , DiSalvo , S. , Miller , J. W. , Bashir , U. , Brock , D. A. , Queller , D. C. , & Strassmann , J. E. (2019 ). The specificity of Burkholderia symbionts in the social amoeba farming symbiosis: Prevalence, species, genetic and phenotypic diversity . Molecular Ecology , 28 (4 ), 847 –862 . 10.1111/mec.14982 30575161 Hegland , S. J. , Nielsen , A. , Lazaro , A. , Bjerknes , A. L. , & Totland , O. (2009 ). How does climate warming affect plant‐pollinator interactions? Ecology Letters , 12 (2 ), 184 –195 . 10.1111/j.1461-0248.2008.01269.x 19049509 Ho , H. I. , Hirose , S. , Kuspa , A. , & Shaulsky , G. (2013 ). Kin Recognition protects cooperators against cheaters . Current Biology , 23 (16 ), 1590 –1595 . 10.1016/j.cub.2013.06.049 23910661 Hoegh‐Guldberg , O. , Mumby , P. J. , Hooten , A. J. , Steneck , R. S. , Greenfield , P. , Gomez , E. , Harvell , C. D. , Sale , P. F. , Edwards , A. J. , Caldeira , K. , Knowlton , N. , Eakin , C. M. , Iglesias‐Prieto , R. , Muthiga , N. , Bradbury , R. H. , Dubi , A. , & Hatziolos , M. E. (2007 ). Coral reefs under rapid climate change and ocean acidification . Science , 318 (5857 ), 1737 –1742 . 10.1126/science.1152509 18079392 Kessin , R. H. (2001 ). Dictyostelium: Evolution, cell biology, and the development of multicellularity . Cambridge University Press . Kiers , E. T. , Palmer , T. M. , Ives , A. R. , Bruno , J. F. , & Bronstein , J. L. (2010 ). Mutualisms in a changing world: An evolutionary perspective . Ecology Letters , 13 (12 ), 1459 –1474 . 10.1111/j.1461-0248.2010.01538.x 20955506 Kikuchi , Y. , Tada , A. , Musolin , D. L. , Hari , N. , Hosokawa , T. , Fujisaki , K. , & Fukatsu , T. (2016 ). Collapse of insect gut symbiosis under simulated climate change . Mbio , 7 (5 ), e01578‐16 10.1128/mBio.01578-16 27703075 Montllor , C. B. , Maxmen , A. , & Purcell , A. H. (2002 ). Facultative bacterial endosymbionts benefit pea aphids Acyrthosiphon pisum under heat stress . Ecological Entomology , 27 (2 ), 189 –195 . 10.1046/j.1365-2311.2002.00393.x Pandolfi , J. M. , Connolly , S. R. , Marshall , D. J. , & Cohen , A. L. (2011 ). Projecting coral reef futures under global warming and ocean acidification . Science , 333 (6041 ), 418 –422 . 10.1126/science.1204794 21778392 Penuelas , J. , Sardans , J. , Estiarte , M. , Ogaya , R. , Carnicer , J. , Coll , M. , Barbeta , A. , Rivas‐Ubach , A. , Llusia , J. , Garbulsky , M. , Filella , I. , & Jump , A. S. (2013 ). Evidence of current impact of climate change on life: A walk from genes to the biosphere . Global Change Biol , 19 (8 ), 2303 –2338 . 10.1111/gcb.12143 Renoz , F. , Pons , I. , & Hance , T. (2019 ). Evolutionary responses of mutualistic insect‐bacterial symbioses in a world of fluctuating temperatures . Current Opinion in Insect Science , 35 , 20 –26 . 10.1016/j.cois.2019.06.006 31302355 Shu , L. , Brock , D. A. , Geist , K. S. , Miller , J. W. , Queller , D. C. , Strassmann , J. E. , & DiSalvo , S. (2018 ). Symbiont location, host fitness, and possible coadaptation in a symbiosis between social amoebae and bacteria . Elife , 7 , e42660 10.7554/eLife.42660 30596477 Shu , L. , Zhang , B. , Queller , D. C. , & Strassmann , J. E. (2018 ). Burkholderia bacteria use chemotaxis to find social amoeba Dictyostelium discoideum hosts . The ISME Journal , 12 (8 ), 1977 –1993 . 10.1038/s41396-018-0147-4 29795447 Soka , G. , & Ritchie , M. (2015 ). Arbuscular mycorrhizal symbiosis, ecosystem processes and environmental changes in tropical soils . Applied Ecology and Environmental Research , 13 (1 ), 229 –245 . Strassmann , J. E. , & Queller , D. C. (2011 ). Evolution of cooperation and control of cheating in a social microbe . Proceedings of the National Academy of Sciences of the United States of America , 108 , 10855 –10862 . 10.1073/pnas.1102451108 21690338 Strassmann , J. E. , & Shu , L. (2017 ). Ancient bacteria‐amoeba relationships and pathogenic animal bacteria . PLoS Biology , 15 (5 ), e2002460 10.1371/journal.pbio.2002460 28463965 Ullah , H. , Nagelkerken , I. , Goldenberg , S. U. , & Fordham , D. A. (2018 ). Climate change could drive marine food web collapse through altered trophic flows and cyanobacterial proliferation . PLoS Biology , 16 (1 ), e2003446 10.1371/journal.pbio.2003446 29315309 Wernegreen , J. J. (2012 ). Mutualism meltdown in insects: Bacteria constrain thermal adaptation . Current Opinion in Microbiology , 15 (3 ), 255 –262 . 10.1016/j.mib.2012.02.001 22381679 Wernegreen , J. J. (2012 ). Endosymbiosis . Current Biology , 22 (14 ), 555 –561 . 10.1016/j.cub.2012.06.010 Werner , G. D. A. , Cornelissen , J. H. C. , Cornwell , W. K. , Soudzilovskaia , N. A. , Kattge , J. , West , S. A. , & Kiers , E. T. (2018 ). Symbiont switching and alternative resource acquisition strategies drive mutualism breakdown . Proceedings of the National Academy of Sciences of the United States of America , 115 (20 ), 5229 –5234 . 10.1073/pnas.1721629115 29712857 Zhang , X. , Zhuchenko , O. , Kuspa , A. , & Soldati , T. (2016 ). Social amoebae trap and kill bacteria by casting DNA nets . Nature Communications , 7 , 10938 10.1038/ncomms10938