1 2 3 Larvae of Caribbean Echinoids Have Small Warming Tolerances for Chronic Stress in 4 Panama 5 6 7 Valentina Perricone1,2 and Rachel Collin1,3 8 9 10 11 1 Smithsonian Tropical Research Institute, Apartado Postal 0843-03092, Balboa Ancon, Panama. 12 13 2 Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 14 Villa Comunale, I-80121 Napoli, Italy 15 16 17 3Corresponding author: e-mail: [email protected]; (202) 633-4700 x28766. Address for 18 correspondence: STRI, Unit 9100, Box 0948, DPO AA 34002, USA. 19 20 Biological Bulletin 21 22 Keywords: , , thermal tolerance, coral reefs, Bocas del Toro, seagrass, 23 24 Short Title: Warming tolerance of echinoid larvae 25 26 October, 2018 27 28 Abbreviations: Eucidaris tribuloides: Et; : Ev; : El; 29 Lytechinus williamsi: Lw; Tripneustes ventricosus: Tv; Clypeaster rosaceus: Cr; Clypeaster 30 subdepressus: Cs

Perricone and Collin, Page 1 of 30 31 Abstract 32 In with complex life cycles, early developmental stages are often less thermally 33 tolerant than adults, suggesting they are key to predicting organismal response to environmental 34 warming. Here we document the optimal and lethal temperatures of larval sea urchins and use 35 those to calculate the warming tolerance and the thermal safety margin of early larval stages of 7 36 tropical species. Larvae of Echinometra viridis, Echinometra lucunter, Lytechinus williamsi, 37 Eucidaris tribuloides, Tripneustes ventricosus, Clypeaster rosaceus and Clypeaster 38 subdepressus, were reared at 26°C, 28°C, 30°C, 32°C and 34°C for 6 days. The temperatures at 39 which statistically significant reductions in larval performance are evident are generally the same 40 temperatures at which statistically significant reductions in larval survival were detected, 41 showing that the optimal temperature is very close to the lethal temperature. The two 42 Echinometra species had significantly higher thermal tolerance than the other species, with some 43 surviving culture temperatures of 34°C and showing minimal impacts on growth and survival at 44 32°C. In the other species, larval growth and survival were depressed at and above 30°C or 32°C. 45 Overall these larvae have lower warming tolerances (1°C to 5°C) and smaller thermal safety 46 margins (-3°C to 3°C) than adults. Survival differences among treatments were evident by the 47 first sampling on day 2, and survival at the highest temperatures increased when embryos were 48 exposed to warming after spending the first 24 hours at ambient temperature. This suggests that 49 the first days of development are more sensitive to thermal stress than are later larval stages. 50

Perricone and Collin, Page 2 of 30 51 Introduction 52 Environmental warming is one of the greatest current threats to biodiversity. Many 53 studies of thermal limits and thermal performance curves have been conducted on adult 54 ectotherms (Sunday et al., 2011; 2012; Nguyen et al., 2011). However, the majority of metazoan 55 have complex life cycles and, in general, early developmental stages are considered to 56 be more susceptible to environmental stressors (Byrne, 2012; Keshavmurthy et al., 2014). For 57 example, studies of crustaceans and echinoids have found that larval stages have lower thermal 58 tolerance than adults (e.g., Miller et al., 2013; Schiffer et al., 2014; Collin and Chan 2016) and 59 late larval stages show less tolerance to thermal stress than do early larval stages (Storch et al., 60 2011; Fitzgibbon and Battaglene, 2012). However, a few species show the opposite pattern, with 61 embryos and early life stages showing a higher tolerance to thermal stress than adults (Diederich 62 and Pechenik, 2013; Tangwancharoen and Burton, 2014). In general, the thermal tolerances of 63 embryonic and larval stages of marine invertebrates remain poorly documented, especially in the 64 tropics. 65 To fully understand the vulnerability of different life stages to warming, comparisons of 66 thermal tolerances need to be placed in an environmental context that accounts for the changes in 67 habitat between larval and adult stages (e.g., Lu et al., 2016). Two metrics have commonly been 68 used to do this. Warming tolerances (WT) are the differences between the environmental 69 temperature and the lethal temperature and thermal safety margins (TSM) are the differences 70 between the optimal temperature and the environmental temperature. Sufficient data on 71 terrestrial animals and marine fishes exist to form the basis of meta-analyses of these metrics. 72 They show that tropical marine organisms are more vulnerable to warming than are temperate 73 organisms (e.g., Deutsch et al. 2008; Comte and Olden 2017). This has been primarily based on 74 estimates of warming tolerances, which are generally around 5°C for tropical marine fishes and > 75 10°C for temperate marine fishes (Comte and Olden 2017). 76 Unfortunately, similar comparative data are not available for other groups of marine 77 organisms. This is especially true for information on optimal temperatures and the TSM, which 78 are vital to understanding the negative impacts of environmental warming on populations prior to 79 evidence of a lethal effect. Generally experimental studies of thermal tolerance detect non-lethal 80 reductions in performance at temperatures lower than those at which survival is impaired. These 81 negative effects can become apparent at temperatures much lower than the lethal temperature.

Perricone and Collin, Page 3 of 30 82 For example, larvae of Pollicipes elegans barnacles show a reduction in swimming activity at 5- 83 10 °C below the lethal temperature (Walther et al., 2013) and larval spiny lobsters growth rate 84 decreases above 21°C, while reduced survival is not evident until temperatures exceed 25°C 85 (Fitzgibbon et al., 2017). Although large difference between the optimal and lethal temperatures 86 have been documented for temperate organisms, this may not be the case for tropical organisms, 87 where the more extreme left skew of the thermal performance curve may reduce the difference 88 between the optimal and lethal temperatures. If this difference is very small in tropical animals, 89 the more easily measured lethal temperatures and WT could be used in place of more 90 complicated measures of performance. 91 The objective of the present study was to document the optimal and lethal temperatures 92 of larval sea urchins and to use those in combination with environmental temperatures to 93 calculate the warming tolerance and the thermal safety margin of early larval stages of 7 tropical 94 species. To do this we documented the impact of rearing temperature on larval growth and 95 survival over the first 6 days of larval life and used this to answer the following primary 96 questions: As temperature increases, does performance decrease before reduced survival can be 97 detected and how different is the optimal temperature from the lethal temperature (i.e, how 98 different are WT and TSM)? In addition, we conducted a follow-up short-term rearing 99 experiment to determine if upper limits of thermal tolerance are altered when thermal stress is 100 imposed after the first day of development. 101 We focused on tropical sea urchins for several reasons. Sea urchins are well-known eco- 102 system engineers that play important roles as herbivores in both reef and seagrass ecosystems 103 and can be important for erosion and bioturbation (Birkeland, 1989; Valentine and Heck, 1991; 104 Heck and Valentine, 1995; Perkins et al., 2015; Ling et al., 2015; Davidson and Grupe, 2015; 105 Lessios, 2016; Kuempel and Altieri, 2017). There is a significant body of literature on the 106 thermal tolerance of the developmental stages of temperate sea urchins (e.g., Fujisawa, 1995; 107 Sewell and Young, 1999; Byrne et al., 2009; 2011; 2013; Pecorino et al., 2013; Delorme and 108 Sewell, 2013; 2014; Gianguzza et al., 2014; Karelitz et al., 2017) upon which to base 109 comparisons, and the thermal tolerance of the adults of our focal species has previously been 110 documented (Collin et al. 2018). Finally, sea urchins are abundant charismatic megafauna in 111 shallow water tropical environments and their transcriptomic and cellular responses to 112 environmental stressors are relatively well-understood, for marine invertebrates, making them

Perricone and Collin, Page 4 of 30 113 potential candidates to serve as easily surveyed sentinel species (e.g., Bonacci et al., 2007; 114 Pinsino and Matranga, 2015). 115 116 Materials and Methods 117 We studied larvae of 7 species of common sea urchins from the Caribbean (the cidaroid 118 Eucidaris tribuloides; the echinometrid euechinoids Echinometra viridis and E. lucunter, the 119 toxopneustid euechinoids Lytechinus williamsi and Tripneustes ventricosus and the clypeasteroid 120 euechinoids Clypeaster rosaceus and C. subdepressus). These all develop through an obligatory 121 planktotrophic larval stage, except for C. rosaceus, which produces larger eggs that develop into 122 facultative feeding larvae (Schroeder, 1981; Emlet, 1986; Sewell and Young, 1999; Wolcott and 123 Messing, 2005; Vellutini and Migotto, 2010; McAlister and Moran, 2013). There is either little 124 or no published information on larval thermal tolerance for any of these species except for E. 125 lucunter (Sewell and Young, 1999), although some information on the thermal tolerance of 126 earlier developmental stages is available for 3 others (Cameron et al., 1985). 127 Adult sea urchins were collected from seagrass meadows and fringing reefs in the 128 shallow waters around Isla Colon and Isla Solarte, in Bocas del Toro, Panama. The 129 environmental conditions in Bocas del Toro have been well-documented over the last 10 years 130 (D’Croz et al., 2005; Kaufmann and Thompson, 2005; Collin et al., 2009; Neal et al., 2014; 131 Seemann et al., 2014). Adults were collected by hand from the same locations as described in 132 Collin et al. (2018) while snorkelling to a depth of less than 5 meters. Animals were maintained 133 submerged in buckets while they were transported to the Smithsonian Tropical Research 134 Institute's Bocas del Toro Research Station. They were maintained, unfed, outside in 250L tanks 135 of aerated running seawater at environmental temperature (28.5 ± 0.5 °C). 136 Animals were spawned no more than 7 days after collection following standard methods 137 (Strathmann, 1987). Adults were induced to spawn by injecting 2-5 ml 0.55M KCl into the 138 perivisceral cavity of each . For each cross, we pooled the gametes of 3 females and 3 139 males to produce a mixed population of larvae, with up to 12 alleles at any one genetic locus. 140 The eggs were washed gently and diluted with filtered sea water to bring the final volume to 141 500ml. Then, 0.3 ml of diluted sperm from each of 3 males were added to each beaker of eggs. 142 After 15 minutes, embryos with fertilization envelopes were counted into culture vessels and 143 placed in waterbaths to gradually come to the treatment temperatures.

Perricone and Collin, Page 5 of 30 144 145 Experiment 1: Chronic Thermal Stress During Development 146 To understand the tolerance of larvae to chronic elevated temperatures, we reared larvae 147 for 6 days at six different temperatures: 26, 28, 30, 32, 34, 36°C. These temperatures were 148 selected as studies of adult thermal tolerance show that some Caribbean sea urchin species can 149 survive short-term exposures to temperatures as high as 36°C (Collin et al. 2018) and that 150 fertilization may occur up to 37°C (Sewell and Young, 1999). Large water baths were made 151 using 40L aquaria with heaters, and bubblers, to mix the water. Temperature was measured twice 152 daily to ensure that the experimental temperatures were maintained throughout the trial. For each 153 trial, we added 50 cleaving embryos to 150ml of 0.45 m-filtered seawater with 20 g/ml 154 ampicillin in a 250 ml Erlenmeyer flask. 15 such flasks were placed in each water bath on day 0. 155 Water was changed every 2 days (i.e., on days 2 and 4) by gently concentrating the larvae into a 156 volume of ~10-20ml using reverse filtration. The larvae were poured into a clean flask with new 157 filtered sea water. The larvae were fed by adding 1250 cells/ml of Rhodomonas sp. and 1250 158 cells/ml of Dunaliella sp. 12 hours after fertilization, and every day thereafter. 159 Starting on Day 2 (i.e., 48 hours post-fertilization) and every day thereafter, 3 flasks were 160 removed from each treatment. The number of surviving larvae in each flask was counted and, 161 when present, 20 individuals were photographed under a compound microscope for subsequent 162 measurement. The total body length, the stomach length and the average length of the postoral 163 arms from the posterior margin of the larva to the arm tip were measured using ImageJ. Previous 164 studies of echinoplueti have shown these measurements to reflect growth of larvae and differing 165 ratios of these measures to reflect allometries induced by differing food rations (Bertram and 166 Strathmann, 1998; Sewell et al. 2004). In this way, we obtained estimates of growth and survival 167 over 5 days (Days 2-6). We conducted two trials of this experiment for each species, using 6 168 different parents for each trial. The trials were conducted on different dates at least a month 169 apart. Unfortunately we could only complete one trial for C. subdepressus. Measurements were 170 taken from the first survival trial for all the species, and we also measured larvae in the second 171 trial for E. viridis (Table 1). 172 173 Experiment 2: Survival After Early Development

Perricone and Collin, Page 6 of 30 174 Because the first experiment showed that most of the survival differences between 175 temperature treatments were evident by Day 2 (see results section), we designed a follow-up 176 experiment to determine if early development is more sensitive to elevated temperatures than are 177 the hatched larvae. Such a difference has been previously reported for E. lucunter (Sewell and 178 Young, 1999). We followed the same spawning and rearing protocols as described above, with 179 the following modification. We held the bulk cultures from each female at 28°C (ambient 180 environmental temperature) for 24 hours before allocating the embryos into the 250ml flasks and 181 rearing them at 28, 30, 32, 34, and 36°C. Larvae were counted after 48-hour exposure to the 182 experimental temperature. This exposure means that the larvae were kept at the experimental 183 temperatures for the same duration as the Day 2 sample in Experiment 1, but that we counted 184 them at the same age as those counted on Day 3 in Experiment 1. Higher relative survival of 185 larvae exposed to stressfully warm conditions in this experiment compared to Experiment 1 186 would indicate that the first 24 hours of development are particularly susceptible to thermal 187 stress. We assessed this by calculating the survival of the larvae raised at 30°C, 32°C and 34°C 188 after 48 hours relative to larvae from the same experiment raised at 28°C. If the timing of 189 thermal stress is important these ratios should differ more at higher temperatures than at cooler, 190 non-stressful temperatures. 191 192 Statistical Analyses 193 Analyses of variance were used to determine how the fully factorial combination of 194 temperature treatment and day affected survival and growth. Survival data are counts of the 195 number of larvae found in the flasks on each day and were analysed as such rather than being 196 transformed into proportions. These data were analysed in JMP version 12, using a REML 197 (Restricted Maximum Likelihood) ANOVA, the preferred method for model fitting, which uses 198 maximum likelihood estimation of a restricted likelihood function that is independent of fixed- 199 effect parameter (Patterson and Thompson 1974; Searle et al. 1992). The REML ANOVA 200 model included a fully factorial combination of temperature and day, and trial was included as a 201 random effect to account for variation due to differences between the 2 trials. The distributions 202 of the residuals were checked for normality and data were transformed if necessary. Only the 203 survival data for E. viridis could not be adequately transformed in this way, but it should be 204 noted that ANOVA analyses are generally robust to the violation of the normality assumption

Perricone and Collin, Page 7 of 30 205 with similar group sizes (Glass et al. 1972 ; Schmider et al. 2010). Larval measurements were 206 analysed with the same fixed factors, and included jar as a random effect nested within day and 207 temperature, to avoid over-inflated degrees of freedom because we measured multiple larvae 208 from each jar. Post-hoc Tukey HSD comparisons were used to determine which treatments 209 differed significantly from each other. Repeated measures ANOVA was not used as each jar was 210 independent and only counted and/or measured once. 211 212 Results 213 Survival in Experiment 1 - Larvae of each species survived in cultures at 26-32°C through the 6 214 days of the experiment. In addition, some larvae of both Echinometra species also survived at 215 34°C (Table 1 and Figure 1). No larvae of the Echinometra species survived to Day 2 at 36°C 216 and no larvae of the other species survived to Day 2 at 34°C or at 36°C. 217 For all of the species ANOVA analysis showed significant effects of temperature on 218 larval survival (Table 2; Figure 2). In all but 2 species (T. ventricosus and C. subdepressus) there 219 was a significant effect of day on survival (Table 2; Figure 2). This slow reduction in the number 220 of larvae across all of the treatments is most likely the result of gradual larval mortality and 221 possibly larval loss during water changes. The rate of reduction was independent of temperature 222 treatment in all but E. lucunter where a significant interaction between day and temperature was 223 detected (Table 2). Differences between trails was due to different rates of initial survival 224 between the trials. 225 Post-hoc Tukey HSD tests for the 6 species for which we did not detect significant 226 interactions show that for all of the species except for C. subdepressus survival did not differ 227 between the 26°C and 28°C treatments (Figure 2). In 3 of these species survival at 30°C was 228 significantly lower than survival at 26°C (Table 1). In L. williamsi survival was significantly 229 reduced at 32°C but not at 30°C. In E. viridis survival was significantly reduced at 34°C 230 compared to 26-32°C. The significant interaction between day and temperature makes 231 interpretation of the results for E. lucunter slightly more complicated (Figure 2). However post- 232 hoc tests conducted on each day show that survival at 34°C was significantly lower than survival 233 at 28°C on every day (Figure 2). Survival at 32°C was significantly lower than survival at 26°C 234 on Days 3 of the 5 days. In all of the species the significant reduction of survival at the higher 235 temperatures was already evident on Day 2 (Figure 1). In addition, in all of the species except for

Perricone and Collin, Page 8 of 30 236 E. lucunter, there was no indication of differential mortality rates across the temperature 237 treatments as would be evidenced by a significant interaction between treatment and day. 238 239 Growth in Experiment 1 – Some aspects of larval size was significantly affected by day, 240 temperature and the interaction between day and temperature for all of the species examined here 241 (Table 3). In general, the statistical effect of replicate beakers accounted for 5-30% of the 242 variance in the data. All species showed sustained growth in all 3 aspects of morphology during 243 the 6 days of the experiment, except for C. rosaceus in which growth more or less ceased after 244 Day 4, after which some reduction in size was evident (Figures 3 & 4). This species is a 245 facultative planktotroph which can metamorphose after 5-7 days (Emlet, 1986) and therefore a 246 reduction in arm length and larval body size was not unexpected. 247 The impact of rearing temperature was evident from visual inspection of the growth 248 trajectories (Figure 3 & 4). Post-hoc Tukey HSD tests comparing the size measurements from 249 each temperature treatment on day 6 showed how the treatments differ for each species (Table 250 1). In the 2 Echinometra species postoral arm length, stomach length and body length showed no 251 significant differences at 26°C, 28°C, 30°C and 32°C. In E. viridis larvae raised at 34°C were 252 significantly smaller in all 3 measures. So few larvae of E. lucunter survived at 34°C that it was 253 not possible to compare larval size on Day 6. But the few that could be measured on Days 2 and 254 5 were small and misshapen. Among the other species, L. williamsi had reduce body length and 255 stomach length at 32°C compared to 28-30°C. Eucidaris tribuloides, T. ventricosus, and C. 256 rosaceus had reduced arm length and body length at 32°C. Similar to the results for survival, the 257 irregular sea urchin C. subdepressus showed significantly longer bodies and stomachs at 26°C 258 than at 28 °C, while these lengths did not differ significantly at 30°C and 32°C (Table 1; Figure 3 259 & 4). 260 261 Experiment 2 – Embryos raised for 24 hours at ambient sea temperatures (28°C) prior to a 48- 262 hour exposure to one of the experimental temperatures show higher survival after exposures of 263 the same duration at stressfully warm temperatures (Figure 5; Tables 4&5) than those that were 264 placed in the temperature treatments immediately after fertilization (i.e., Day 2 larvae). For all 5 265 species, there was a significant effect of temperature on survival, and for all of them survival was 266 significantly reduced at 32°C compared to 28°C and 30°C. In addition, survival of T. ventricosus

Perricone and Collin, Page 9 of 30 267 larvae had reduced survival at 30°C compared to 28°C. Larvae had greater survival at higher 268 temperatures relative to 28°C when exposed to heat stress later in development in this 269 experiment compared to Experiment 1. In 3 species some larvae survived in a warmer treatment 270 in Experiment 2 than they did in Experiment 1 (Figures 1 & 5). A few larvae of L. williamsi 271 survived at 34°C in Experiment 2, but no larvae survived at 34°C in Experiment 1. A similar 272 pattern was evident in both species of Echinometra. In Experiment 2 some larvae survived a 48- 273 hour exposure to 36°C, while no larvae survived a 48-hour exposure to 36°C in Experiment 1. In 274 both Echinometra species, the relative survival at 34°C compared to 28°C was much higher in 275 Experiment 2 (~30%) than in Experiment 1 (~5%) (Table 5). Likewise, in T. ventricosus the 276 relative survival at 32°C compared to 28°C was much higher in Experiment 2 (~50%) than in 277 Experiment 1 (~5%) (Table 5). 278 279 Discussion 280 Larval WT and TSM 281 Calculations of warming tolerance and thermal safety margins following Deutsch et al. 282 (2008) show that warming tolerance and thermal safety margins for these tropical sea urchin 283 larvae are minimal (Table 6). We calculated warming tolerance as the difference between the 284 average habitat temperature using 29°C, the grand average of the temperatures at the 2 Bocas del 285 Toro reefs over 10 years reported in Collin & Chan (2016), and the temperature treatment at 286 which our larval cultures experienced >50% mortality relative to the temperature with the

287 highest survival. This is similar to a LT50 generated by acute warming experiments, which is 288 often used as an estimate of the critical temperature. We calculated the thermal safety margin, 289 the difference between the optimal temperature and the habitat temperature in two ways, using 290 estimates of optimal temperatures based on growth or on survival. Because temperature-specific 291 growth and temperature-specific survival were often statistically indistinguishable across several 292 experimental temperatures and because curve fitting approaches failed in many cases to estimate 293 an optimum (results not shown and Collin et al. 2018) we used the warmest temperature at which 294 (1) body length or (2) survival were statistically indistinguishable from the value in the treatment 295 with the highest values. When several temperatures resulted in the same values, we chose the 296 warmest temperature because performance curves are expected to be left skewed with a rapid 297 reduction in performance once the optimum has been exceeded. This approach gives estimates

Perricone and Collin, Page 10 of 30 298 of warming tolerances ranging from 5°C for the warm tolerant Echinometra viridis to 1°C for the 299 least tolerant C. subdepressus, and thermal safety margins ranging from 3°C for E. viridis to - 300 3°C for C. subdepressus (Table 6). 301 These estimates need to be interpreted with these caveats in mind. Since early larval 302 development is relatively rapid and since it appears that the earliest few hours of development 303 are the most sensitive to thermal stress (see below and Sewell and Young, 1999; Kapsenberg and 304 Hofmann, 2014; Collin and Chan, 2016), average annual temperatures may not represent the 305 typical thermal experience of early development. This may be particularly true if reproductive 306 effort varies seasonally. Several of the species studied here reproduce throughout the year in the 307 San Blas Islands of Panama (Lessios, 1985), while E. viridis, L. williamsi and C. rosaceus reduce 308 reproductive effort during the coolest times of the year (Lessios, 1985). Likewise, the 2 309 Echinometra species have been shown to focus reproductive effort only during the two warmest 310 months of the year in Puerto Rico (Cameron et al., 1985). If anything, this suggests that 311 development may be taking place at temperatures above the annual average, in Panama, and 312 raises the possibility that our WT and TSMs may be underestimated. The most extreme result for 313 C. subdepressus, that it is already experiencing negative thermal safety margins in Bocas del 314 Toro, is unfortunately the least well-supported result in this study. We have found this species to 315 be particularly difficult to spawn successfully and repeated attempts resulted in only a single 316 successful rearing trial, from which the larvae had low overall survival at all temperatures 317 compared to the other species. This species may be difficult to spawn precisely because it is 318 already living under stressfully warm conditions in Bocas del Toro. However, the possibility 319 remains that the low thermal tolerance measured in this study could be due to low gamete quality 320 resulting from some other stressor that resulted in artificially low estimates of thermal tolerance 321 in this species. 322 Regardless of these caveats, it is clear that these tropical sea urchins have very small 323 warming tolerances and thermal safety margins for early development in Bocas del Toro. This is 324 likely to put them at risk from even moderate environmental warming. Their warming tolerances 325 are lower than any reported in a review of latitudinal patterns in thermal tolerances of insects 326 (Deutsch et al., 2008), but are similar to WTs reported for larval frogs from warm subtropical 327 environments (Duarte et al., 2012). Other comparable data from sea urchins is sparse and 328 variation in methods make them not strictly comparable, but the available data do suggest a

Perricone and Collin, Page 11 of 30 329 latitudinal trend similar to that observed in other groups (e.g., Deutsch et al. 2008; Comte and 330 Olden 2017). In the Antarctic Sterechinus neumayeri developmental stages have an WT of at 331 least 20°C for acute 2 h exposures (Kapsenberg and Hofmann, 2014), and larvae of the temperate 332 Strongylocentrotus purpuratus also show an acute WT of approximately 20°C along the west 333 coast of the USA (Hammond and Hofmann, 2010). In the subtropics, adult Arbacia stellata 334 studied in Baja California, Mexico had an estimated WT of 14.5-16.4°C but a much smaller 335 TSM of 3-4.3°C (Díaz et al., 2017). In the tropics, TSM for adult is 5.7°C 336 and for adult E. lucunter is 3.7°C in the Cayman Islands (Sherman, 2015), slightly higher than 337 those obtained from chronic thermal stressors experienced during development for our species in 338 Panama. 339 340 How Do Larvae and Adults Compare? 341 Despite limited interspecific variation in upper thermal tolerance, the rank order of larval 342 thermal tolerance is similar in some respects to the rank order of adult thermal tolerances. The 343 warmest acute (2 hour) temperature exposures survived by adult sea urchins breaks the species 344 into 3 groups. The two Echinometra species can survive at the highest temperatures (37°C), 345 followed by E. tribuloides and C. rosaceus which can survive up to 36°C, with L. williamsi, T. 346 ventricosus and C. subdepressus all surviving only to 35°C (Collin et al., 2018 Figures 3&4). 347 The rankings of the maximum temperatures survived are as follows: 348 Adult Survival: Ev, El > Et, Cr > Lw, Tv, Cs 349 Larval Survival: Ev, El > Et, Cr, Lw, Tv, Cs 350 Ranking adults with respect to righting performance gives a similar ranking to that based 351 on survival. Unfortunately, neither of the Clypeaster species can right, so this metric could not be 352 measure those species (see Collin et al., 2018 Figure 5). 353 Adult Righting Performance: Ev, El > Et > Tv > Lw 354 Larval Growth Performance: Ev, El > Tv, Et, Lw 355 The larval and adult rankings are similar in that the two Echinometra species clearly have higher 356 warming tolerance, and that the tolerances of the other species are all very similar to each other. 357 The fact that adult survival was determined to a precision of 1°C, while larval survival was 358 determined with a precision of 2°C probably explains why adult tolerance could be more 359 effectively ranked.

Perricone and Collin, Page 12 of 30 360 Comparisons of adult and larval WT and TSM show that larvae are more sensitive to 361 warming than adults (Table 6). In general, larval WTs are 3-5°C smaller than adult warming 362 tolerances and larval TSMs are 0-4°C smaller than adult thermal safety margins. However, it is 363 important to emphasize that these assays used different exposure times. Assays of adults were 364 conducted after a 2-hour exposure, while our differences in larval survival were assayed after a 365 minimum of a 2-day exposure. Although lethal thermal limit often decreases with extended 366 exposures, possibly biasing this comparison towards finding lower larval tolerances, two lines of 367 evidence suggest that this cannot account entirely for this result. First, a comparison of 2-hour 368 exposures of embryonic development, 4-day old larvae and adults for ,

369 another sea urchin species from Bocas del Toro, showed that the LT50 for embryonic 370 development was 3°C lower than it was for adults and 2°C lower than it was for larvae (Collin 371 and Chan 2016), a result roughly similar to our comparisons of 2 hour adult and 2 day larval 372 exposures. Secondly, preliminary trials of adult thermal tolerance after 12-hour exposures gave 373 largely similar results to those of 2-hour exposures (Collin et al., 2018) for E. lucunter and E.

374 viridis, and very slightly lower (<1°C) estimates of LT50 for L. williamsi (unpublished data). 375 Despite these caveats, a major result is that distantly related genera and families of echinoids 376 with similar geographic ranges do not differ from each other by more than ~2-3°C in their acute 377 thermal tolerance as adults or in their growth under chronic stress as larvae, and that larvae or 378 early developmental stages do appear to be more sensitive to warming than are adults. 379 380 How different is the thermal optimum from the lethal thermal limit? 381 Studies of thermal tolerance often detect non-lethal reductions in performance at 382 temperatures just lower than those at which survival is impaired. This can include reductions in 383 ecologically relevant organismal function like sprint speed, or physiological function like cardiac 384 performance. For example, larvae of Pollicipes elegans barnacles show a reduction in swimming

385 activity at 5-10 °C below the lethal temperature (as measured by LT50), followed by a decrease in 386 oxygen consumption 0-7 °C below the lethal temperature (Walther et al., 2013). This is 387 explained by the general energy-limited tolerance to stress (Sokolova 2013) and the more widely 388 known but controversial special case, the oxygen and capacity limited thermal tolerance 389 (OCLTT) model which was developed primarily with data from arctic and temperate ectothermic 390 organisms (Pörtner and Knust, 2007; Pörtner, 2010; Jutfelt et al. 2018). These models posit that

Perricone and Collin, Page 13 of 30 391 the pattern of reduced performance followed by death as temperatures increase is due to a 392 reduction in aerobic scope due to rising temperatures and increased stress. The OCLTT posits 393 that this is due to increased metabolic rate which cannot be matched by the ability of the 394 organism to deliver oxygen to the tissues (Pörtner and Knust, 2007; Pörtner, 2010). This general 395 pattern fits a large body of empirical data showing reduced measures of ecological and 396 physiological performance at temperatures below the lethal temperature and is consistent with 397 the somewhat left-skewed thermal performance curve (Pörtner and Knust, 2007). However, 398 recent studies have found that cardiorespiratory capacity and aerobic scope can be maintained up 399 to the critical thermal limit in some tropical species (e.g., Ern et al., 2013; 2015; Fitzgibbon et 400 al., 2017). Even in these species where aerobic scope continues to increase until the critical limit 401 is reached, the optimal temperature for other aspects of performance often still occurs well below 402 that critical limit (Clark et al., 2013). For example, in Sagmariasus verreauxi metabolic rates and 403 aerobic scope increase through 25°C, and no mortality is evident at 25°C, yet the optimal growth 404 rate is around 21°C (Fitzgibbon et al., 2017). Our data, however do not fit this common pattern 405 of reduced performance at temperatures below the lethal temperature. The temperatures at which 406 statistically significant reductions in larval size are evident, are generally the same temperatures 407 at which statistically significant reductions in larval survival were detected (Table 1). In several 408 species survival was impaired at a lower temperature than at which we observed reduced growth. 409 The similarity between the temperature at which performance declines and the lethal temperature 410 suggest that WT could be used as an estimate of TSM. 411 412 Is the timing of exposure to elevated temperatures important? 413 Previous work with echinoid embryos suggest that the timing and duration of exposures 414 to elevated temperatures can impact estimates of upper thermal limits (Sewell and Young, 1999; 415 Kaspenberg and Hofmann, 2014; Collin and Chan, 2016). For two tropical species E. lucunter 416 and Lytechinus variegatus the acute thermal tolerance of fertilization and of larvae exceed the 417 tolerance of early developmental stages (cleavage-gastrulation). Likewise, the early embryos of 418 the Antarctic sea urchin Sterechinus neumayeri appear to be more susceptible to stress during 419 early embryological development (Kaspenberg and Hofmann, 2014). This suggests that the 420 pattern that most of the mortality in our rearing experiments occurred prior to the first survival 421 counts on Day 2 is due to a more pronounced impact of rearing temperature earlier in

Perricone and Collin, Page 14 of 30 422 development. Our follow-up experiment which maintained embryos at ambient (28°C) for the 423 first 24 hours followed by a 2-day exposure to elevated temperatures showed an increase in 424 tolerance of extreme temperatures. This provides further evidence that early developmental 425 stages may be one of the stages most susceptible to thermal stress and should be the focus of 426 future acute tolerance studies. It is important to note, however, that 2-hour acute exposures to 427 thermal stress show that early hatchlings (blastula and beyond) can survive short exposures of 428 temperatures 4-5°C warmer than their upper limit for normal development based on continuous 429 exposures during the first 2 days of development (Sewell and Young, 1999), suggesting that tests 430 using very short exposures may over-estimate the upper thermal tolerance limits by ecologically 431 meaningful amounts. 432 433 Conclusion 434 Results of this and previous studies concur that early developmental stages of echinoids 435 prior to the late 4-armed larval stage may be more susceptible to thermal stress than are later 436 stages. Larvae of 7 species of sea urchins representing 5 genera from 3 major clades which co- 437 occur in the Caribbean show very similar thermal tolerances, with the 2 Echinometra species 438 being consistently more tolerant of warmer conditions than the other species, but little difference 439 between the 5 others. Comparisons with environmental conditions show that warming tolerance 440 and thermal safety margins of these larvae are similar to or smaller than those reported for 441 tropical terrestrial organisms. 442 443 Acknowledgements 444 We thank Sean Murphy, Adriana Rebolledo, and Valerie Goodwin and especially Emily 445 Smith for vital help with laboratory trials, and the staff of the Bocas del Toro Research Station 446 for logistic support. This work was supported by a Competitive Grant from the Smithsonian 447 Institution, and was performed with permission from the Autoridad de Recursos Acuáticos de 448 Panamá in 2015, and the Ministerio de Ambiente de Panamá in 2016. Part of this work was 449 completed by VP as part of a Masters in Marine Biology at the University of Bologna, Italy. 450 Two anonymous reviewers helped improve the manuscript. 451

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Perricone and Collin, Page 21 of 30 698 Table 1: Summary of the results of Experiment 1 showing the temperatures at which larval 699 growth and survival are significantly reduced based on Tukey HSD Post-hoc tests from the 700 ANOVA analyses. 701 Species 100% Reduced Reduced Reduced Reduced702 Lethal by survival# PO arm stomach larval Day 2 length* size* length* Echinometra viridis 36 °C 34°C 34°C 34°C 34°C Echinometra lucunter 36 °C 32, 34 °C 34°C 34°C 34°C Lytechinus williamsi 34, 36 °C 32 °C 26°C 26, 32°C 26, 32°C Tripneustes ventricosus 34, 36 °C 32 °C 32°C 26, 32°C 32°C Cidaroidea Eucidaris tribuloides 34, 36 °C 30, 32 °C 32°C none 32°C

Clypeasteroida Clypeaster rosaceus 34, 36 °C 30, 32 °C 32°C NA 32°C Clypeaster subdepressus 34, 36 °C 32 °C none 28°C 28, 32°C 703 704 # Significant differences based on Tukey post-hoc tests, compared to 28°C. 705 * Significant differences based on Tukey post-hoc tests, compared to 28°C on Day 6 for all species except for C. 706 subdepressus where the largest size was observed at 26°C. 707 NA = not applicable; Stomach is not clearly visible. 708 709

Perricone and Collin, Page 22 of 30 710 Table 2: ANOVA tables from analysis of larval survival in Experiment 1. 6 out of 7 species had 711 multiple trials which were included in the model as a random effect. Survival was counted on 712 days 2-6. Bold highlights statistically significant results. 713 Factor df F Ratio p

Echinometra lucunter R2 = 0.82; N = 117 Temperature 4 67.12 <0.0001 Day 4 10.43 <0.0001 Temperature X Day 16 2.36 0.0056 Trial [Random] -2LogLikelihood = 688.07 4.5% of total variance explained

Echinometra viridis R2 = 0.80; N = 150 Temperature 4 51.65 <0.0001 Day 4 2.88 0.03 Temperature X Day 16 1.19 0.28 Trial [Random] -2LogLikelihood = 979.17 77.6% of total variance explained

Lytechinus williamsi R2 = 0.73; N =120 Temperature 3 28.98 <0.0001 Day 4 36.43 <0.0001 Temperature X Day 12 1.53 0.13 Trial [Random] -2LogLikelihood = 109.08 21.1% of total variance explained

Eucidaris tribuloides R2 = 0.63; N = 119 Temperature 3 16.64 <0.0001 Day 4 2.85 0.03 Temperature X Day 12 0.53 0.53 Trial [Random] -2LogLikelihood = 803.05 62.4% of total variance explained

Tripneustes ventricosus R2 = 0.80; N = 117 Temperature 3 119.63 <0.0001 Day 4 2.12 0.08 Temperature X Day 12 0.60 0.83 Trial [Random] -2LogLikelihood = 750.26 16.0% of total variance explained

Clypeaster subdepressus r2 = 0.54; N = 60 Temperature 3 12.36 <0.0001 Day 4 0.64 0.63 Temperature X Day 12 0.66 0.78

Clypeaster rosaceus

Perricone and Collin, Page 23 of 30 r2 = 0.82; N = 120 Temperature 3 127.16 <0.0001 Day 4 5.61 <0.0004 Temperature X Day 12 1.42 0.17 Trial [Random] -2LogLikelihood = 752.46 20.1% of total variance explained 714 715

Perricone and Collin, Page 24 of 30 716 Table 3: Nested ANOVA results of 3 morphological measurements from Experiment 1. 717 Replicate beakers were treated as nested random effects and generally account for little of the 718 overall variance. Fixed factors with a significant effect are highlighted in bold. 719 Factor df F-Ratio P-value F-Ratio P-value F-Ratio P-value arms arms stomach stomach body body

Echinometra lucunter N=1049 Temperature 3 4.85 0.0062 4.16 0.012 4.18 0.013 Day 4 130.83 <0.0001 70.65 <0.0001 133.13 <0.0001 Temperature X Day 12 1.56 0.15 2.30 0.027 2.09 0.047 Jar [Day, -2 loglike. = 11929.22 -2 loglike. = 9624.92 -2 loglike. = 10721.06 Temperature] Random Var. explained =15.51% Var. explained = 9.17% Var. explained = 11.36%

Echinometra viridis 1 N=1296 Temperature 4 62.60 <0.0001 76.68 <0.0001 134.08 <0.0001 Day 4 26.73 <0.0001 17.40 <0.0001 18.18 <0.0001 Temperature X Day 16 2.09 0.026 1.18 0.32 0.47 0.47 Jar [Day, Var. explained =35.24% Var. explained = 15.45% Var. explained = 15.39% Temperature] Random -2 loglike. = 13662.57 -2 loglike. = 10911.52 -2 loglike. = 12643.03

Echinometra viridis 2 N=739 Temperature 4 265.83 <0.0001 140.35 <0.0001 206.90 <0.0001 Day 4 30.15 <0.0001 31.94 <0.0001 41.12 <0.0001 Temperature X Day 16 2.35 0.037 5.45 <0.0001 4.08 0.0008 Jar [Day, Var. explained =4.47% Var. explained = 1.11% Var. explained = 4.13% Temperature] Random -2 loglike. = 9207.25 -2 loglike. = 5664.88 -2 loglike. = 7212.49

Lytechinus williamsi N = 1116 Temperature 3 55.05 <0.0001 72.23 <0.0001 42.72 <0.0001 Day 4 216.98 <0.0001 202.46 <0.0001 281.60 <0.0001 Temperature X Day 12 6.33 <0.0001 4.02 0.0004 4.62 0.0001 Jar [Day, -2 loglike. =12021.16 -2 loglike. = 9202.96 -2 loglike. = 10972.16 Temperature] Random Var. explained = 5.90% Var. explained = 6.49% Var. explained = 8.88%

Eucidaris tribuloides N = 1395 Temperature 3 27.03 <0.0001 24.09 <0.0001 19.59 <0.0001 Day 4 (3) 72.02 <0.0001 56.65 <0.0001 28.29 <0.0001 Temperature X Day 12 2.78 0.015 1.58 0.14 2.11 0.039 (9) Jar [Day, Var. explained = 18.45 Var. explained = 10.51% Var. explained = 17.21% Temperature] Random -2 loglike. = 8254.93 -2 loglike. = 7982.38 -2 loglike. =9931.80

Tripneustes ventricosus* N= 758 Temperature 3 25.25 <0.0001 97.72 <0.0001 35.35 <0.0001

Perricone and Collin, Page 25 of 30 Day 3 208.66 <0.0001 64.30 <0.0001 45.80 <0.0001 Temperature X Day 9 11.44 0.0014 7.42 <0.0001 3.56 0.0031 Jar [Day, -2 loglike. =6388.11 -2 loglike. =7517.52 -2 loglike. = 7034.85 Temperature] Random Var explained = 8.01% Var explained =14.23% Var. explained =15.51%

Clypeaster subdepressus N = 664 Temperature 3 16.19 <0.0001 14.05 <0.0001 17.15 <0.0001 Day 4 47.72 <0.0001 19.56 <0.0001 47.00 <0.0001 Temperature X Day 12 1.84 0.081 2.90 0.0060 2.06 0.049 Jar [Day, --2 loglike. =7677.37 -2 loglike. = 3196.43 --2 loglike. = 5914.64 Temperature] Random Var. explained =18.65% Var. explained =18.76% Var. explained = 18.03%

Clypeaster rosaceus N= 970 Temperature 3 12.80 <0.0001 - - 18.09 <0.0001 Day 4 31.43 <0.0001 - - 41.01 <0.0001 Temperature X Day 12 4.77 <0.0001 - - 6.17 <0.0001 Jar [Day, -2 logLike. = 9677.74 - -2 logLike. = 10227.49 Temperature] Random Var. explained =17.36% - Var. explained =16.14% 720 721 * Only for Days 3-6, as insufficient larvae survived at 32°C on day 2 to calculate the interaction 722 effect. 723

Perricone and Collin, Page 26 of 30 724 Table 4: One-way ANOVA results from Experiment 2, showing the effect of a 48-hour 725 temperature exposure after the first 24 hours of development were spent at 28°C. 726 Species df F-Ratio p Echinometra viridis R2 = 0.97; N = 20 Temperature 4 105.13 <0.0001 Echinometra lucunter R2 = 0.97; N = 20 Temperature 4 127.07 <0.0001 Lytechinus williamsi R2 = 0. 94; N = 16 Temperature 3 65.85 <0.0001 Tripneustes ventricosus R2 = 0.89; N = 12 Temperature 2 37.39 <0.0001 Clypeaster rosaceus R2 = 0.96; N = 12 Temperature 2 110.02 <0.0001 727 728 729

Perricone and Collin, Page 27 of 30 730 731 Table 5: Comparisons of survival after a 2-day exposure to experimental temperatures in 732 Experiments 1 and 2 calculated as the ratio of survival at the experimental temperature relative to 733 survival at 28°C in the same experiment. Letters indicate Tukey HSD post-hoc tests for 734 significant differences in survival at the different experimental temperatures, where temperatures 735 linked with the same letter do not differ significantly from each other. Larvae in Experiment 2 736 were 3-days old but larvae from both experiments had experienced the experimental 737 texmperatures for 2 days. 738 Species 28°C 30°C 32°C 34°C Echinometra viridis Experiment 2 A 0.88 (A) 0.53 (B) 0.34 (C) Experiment 1 Day 2 A 0.90 (A) 0.84 (A) 0.08 (B) Echinometra lucunter Experiment 2 A 0.88 (A) 0.51 (B) 0.30 (C) Experiment 1 Day 2 A 0.89 (A) 0.46 (B) 0.03 (C) Lytechinus williamsi Experiment 2 A 0.83 (A) 0.48 (B) 0.07 (C) Experiment 1 Day 2 A 0.87 (AB) 0.71 (B) - Tripneustes ventricosus Experiment 2 A 0.77 (B) 0.46 (C) - Experiment 1 Day 2 A 0.89 (A) 0.06 (B) - Clypeaster rosaceus Experiment 2 A 0.93 (A) 0.28 (B) - Experiment 1 Day 2 A 0.80 (A) 0.16 (B) - 739 740

Perricone and Collin, Page 28 of 30 741 Table 6: Warming Tolerance (WT) and Thermal Safety Margin (TSM) for each species, using 742 habitat temperature of 29°C. Optimal temperature as the warmest temperature in Experiment 1 743 with the longest body length on day 6 or the warmest temperature with the highest survival. 744 Warming Tolerance Thermal Safety Margin (T50 – Thab) (Topt-Thab)

Species Larval Adult* Larval Larval Adult* Body Survival Length Echinometra viridis 5°C 8.0°C 3°C 3°C 5°C Echinometra lucunter 3°C 7.5°C 3°C 1°C 7°C Lytechinus williamsi 3°C 6.0°C 1°C 1°C 1°C Eucidaris tribuloides 3°C 7.6°C 1°C 1°C 5°C Tripneustes ventricosus 3°C 5.9°C 1°C 1°C 3°C Clypeaster rosaceus 3°C 7.0°C 1°C -1°C - Clypeaster subdepressus 1°C 6.1°C 1°C -3°C - 745 746 *From Collin et al. 2018; Post-hoc Tukey test results indicated in Figure 4, indicating the 747 warmest temperature where righting was not significantly different from the optimal 748 temperature.

Perricone and Collin, Page 29 of 30 749 Figure 1: Bargraph of larval survival for the 7 species of sea urchins raised for 6 days over the 750 array of 5 experimental temperatures and pooled across the 2 trials. Error bars show standard 751 errors. 752 753 Figure 2: Bargraph of larval survival represented as the least square means of survival on each 754 day and at each temperature generated by the ANOVA. Letters above the bars show the results 755 of the Tuckey post-hoc test and significant differences are indicated when bars do not share a 756 letter. There was no significant interaction between day and temperature except for E. lucunter, 757 for which we show the Tuckey post-hoc test for the full factorial combination of temperatures 758 and days. 759 760 Figure 3: Larval growth trajectories of body length, stomach length and average postoral arms 761 for 4 species of sea urchins raised over the array of experimental temperatures. Error bars show 762 standard errors which are often too small to be visible. 763 764 Figure 4: Larval growth trajectories of body length, stomach length and average postoral arms 765 for 3 species of sea urchins raised over the array of experimental temperatures. Error bars show 766 standard errors which are often too small to be visible. The stomach is not clearly visible in C. 767 rosaceus and was not measured. 768 769 Figure 5: Bargraph showing the survival of 3-day-old larvae of 5 species of sea urchins raised 770 for 2 days at experimental temperatures after a day at 28°C in Experiment 2. Letters represent the 771 results of the Tukey’s HSD post-hoc test conducted separately for each species. Error bars show 772 standard errors. 773

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