Thermal specialization across large geographical scales predicts the resilience of mangrove crab populations to global warming

The broad prediction that ectotherms will be more vulnerable to climate change in the tropics than in temperate regions includes assumptions about centre/edge population effects that can only be tested by withinspecies comparisons across wide latitudinal gradients. Here, we investigated the thermal vulnerability of two mangrove crab species, comparing populations at the centre (Kenya) and edge (South Africa) of their distributions. At the same time, we investigated the role of respiratory mode (watervs air-breathing) in determining the thermal tolerance in amphibious organisms. To do this, we compared the vulnerability to acute temperature fluctuations of two sympatric species with two different lifestyle adaptations: the free living Perisesarma guttatum and the burrowing Uca urvillei, both pivotal to the ecosystem functioning of mangroves. The results revealed the air-breathing U. urvillei to be a thermal generalist with much higher thermal tolerances than P. guttatum. Importantly, however, we found that, while U. urvillei showed little difference between edge and centre populations, P. guttatum showed adaptation to local conditions. Equatorial populations had elevated tolerances to acute heat stress and mechanisms of partial thermoregulation, which make them less vulnerable to global warming than temperate co-specifics. The results reveal both the importance of respiratory mode on thermal tolerance and the unexpected potential for low latitude populations/species to endure a warming climate and furthermore contribute to build a conceptual model on the latitudinal Oikos

The broad prediction that ectotherms will be more vulnerable to climate change in the tropics than in temperate regions includes assumptions about centre/edge population effects that can only be tested by withinspecies comparisons across wide latitudinal gradients. Here, we investigated the thermal vulnerability of two mangrove crab species, comparing populations at the centre (Kenya) and edge (South Africa) of their distributions. At the same time, we investigated the role of respiratory mode (water-vs air-breathing) in determining the thermal tolerance in amphibious organisms. To do this, we compared the vulnerability to acute temperature fluctuations of two sympatric species with two different lifestyle adaptations: the free living Perisesarma guttatum and the burrowing Uca urvillei, both pivotal to the ecosystem functioning of mangroves. The results revealed the air-breathing U. urvillei to be a thermal generalist with much higher thermal tolerances than P. guttatum. Importantly, however, we found that, while U. urvillei showed little difference between edge and centre populations, P. guttatum showed adaptation to local conditions. Equatorial populations had elevated tolerances to acute heat stress and mechanisms of partial thermoregulation, which make them less vulnerable to global warming than temperate co-specifics. The results reveal both the importance of respiratory mode on thermal tolerance and the unexpected potential for low latitude populations/species to endure a warming climate and furthermore contribute to build a conceptual model on the latitudinal The current trend of global warming has severe implications for the structure and function of 2 ecosystems (Walther et al. 2002;Rockström et al. 2009;Byrne 2011, Borja, 2014. At large 3 geographical scales, increasing temperatures cause shifts in species distributions and 4 community composition (Sagarin et al. 1999;Deutsch et al. 2008;Beaugrand 2009;5 Bozinovic, Calosi & Spicer 2011;Sunday, Bates & Dulvy 2011, 2012Rius et al., 2014), 6 while at smaller scales temperature anomalies may cause local extinction of vulnerable 7 species often resulting in the contraction of a species range (Helmuth et al. 2002(Helmuth et al. , 20068 Pörtner & Farrell 2008). Global and local warming have direct effects on the functions of 9 organisms, population size and growth rate and consequently drive shifts in distribution 10 ranges which can destabilise community structure and dynamics (Helmuth et al. 2002;11 Somero 2012; Sunday, Bates & Dulvy 2012). Ecologically, such shifts mediate and most 12 likely alter intra-and inter-specific dynamics such as competition for resources and prey- stenotherm species and on their specific potential to cope with anomalous climatic events.

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With these premises, the present work aims to address the current projection of a greater 18 vulnerability to warming of low latitude populations, incorporating an accurate comparison 19 between stenotherm and eurytherm model species with special reference to their respiratory 20 strategies. Intertidal organisms are ideal candidates for studies of thermal adaptation as they 21 live at the interface of marine and terrestrial systems, may rely on different sources of oxygen 22 (i.e. aerial or dissolved) and are susceptible to larger climate anomalies, since they have to 23 cope daily with abrupt and extreme heat stress (Helmuth et al. 2002(Helmuth et al. , 2006Kelly et al. 2012 muddy open spaces to graze, filter sediment and build burrows (Hartnoll 1975;Litulo 2005).   Tidal time series data were retrieved using Wtide software version 3.1.7 (www.wtide.com), in 22 order to link the tidal level in each site with the time-temperature data described below. humidity. These were attached to branches under the canopy about 4 m above ground to avoid 5 submersion and protected by an umbrella-like transparent plastic cover. All loggers were set 6 to record at 5 min intervals for 15 days, after which data were retrieved and the i-buttons re-7 deployed. Data were downloaded with Cold Chain ThermoDymanimcs software (version 4.9 -8 Fairbridge Technologies) and temperature and humidity were averaged at an hourly scale. Adult males of each species were collected at each site during the warmer season 2011 12 (average air temperature 27°C at both sites). Animals were maintained in tanks for two days 13 with fresh mud and aerated seawater at 27 ± 0.5°C, 35‰ salinity before experimentation.
14 Crabs were fed with commercial cat food pellets and exposed to a natural 12 h light cycle.  measurements, sensors were calibrated in air-equilibrated seawater (100% saturation) and in 1 sodium dithionite saturated solution in seawater (0%). During trials, oxygen concentration 2 was not allowed to fall below 60% in order to avoid exposing the animals to severe hypoxic 3 conditions (Schurmann & Steffensen 1992). The limited movement of individuals was 4 adequate to ensure mixing of the water and MO 2 was determined by measuring the linear 5 decline in oxygen saturation. An empty chamber was run as a control with each trial to 6 account for background oxygen depletion, which was less than 2% of crab consumption in 7 water and negligible in air. Before applying the temperature ramp, individuals were placed in 8 the chambers and allowed to recover from handling stress 8 hours at 27 ± 0.5°C. MO 2 was 9 determined every two degrees of temperature. Following each experiment, animals were  software. Sensors were calibrated before each experiment using a two-point calibration in 20 oxygen-free (addition of sodium dithionite) and air-saturated seawater. Animals were 21 acclimated overnight at 27°C and PO 2 was estimated between 27 and 38°C (accuracy ± 1°C), 22 using the protocol described for the MO 2 experiment.

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To measure arterial PO 2 , haemolymph small haemolymph samples (less than 20 µl) were 24 collected through capillary action, using a manually sharpened Pasteur pipette in which the To measure the body temperature of animals, ten similar-sized adult male U. urvillei and P. 10 guttatum were selected (carapace width: 20 mm and 15 mm respectively). We used a two-11 input digital thermometer (Omega©, HH504) with two micro-K-type thermocouples 12 (Omega©) to record body and environmental temperature simultaneously. The day before 13 experimentation, a hole was drilled through the carapace to the gill chamber. Crabs were then 14 kept in aerated sea water to recover overnight at 27°C (Frederich & Pörtner 2000). The 15 following day, the micro-thermocouple registering the body temperature was carefully 16 introduced into the gill chamber and fixed to the carapace using cyanoacrylate glue, while the 17 other one was fixed to the wire of the other sensor close by the crab to detect the exact 18 external temperature experienced by the animal. Specimens were released in the field during 19 low tide, allowed 30 min of field/handling acclimation and then followed for one hour, during 20 which body and environmental temperatures were recorded at 5 min intervals. Since the wire 21 of the thermocouple was really thin and light the animal behaved normally, not disturbed or 22 was not impeded. The position of the animal (e.g. in a hole or a crevice, in sun or shade, in 23 water) was also recorded. South Africa) and Medium (water, air) the categorical explanatory variables.

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The same test was applied to detect statistical significance of PO 2 , considering it as the 6 response variable and the temperature as explanatory variable, while Species (P. guttatum, U. In Kenya, the tidal range is 0 to 4 m and 1 to 2.5 m during spring and neap tides, respectively.

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In South Africa, the tidal range is 0 and 2 m and 0.5 to 1.5 during spring and neap tides, 4 respectively. Tidal levels during the observation periods differed between the two regions 5 (Fig. 2 Thermal responses in MO 2 were clearly different between the two species across two 1 geographic regions and medium (Fig. 3, Tab. S1). In both regions, P. guttatum showed a 2 pronounced increase in MO 2 during the thermal ramp that was steeper than for U. urvillei. The

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Kenyan population of P. guttatum had a higher metabolic rate in air than in water (ANCOVA; 4 F= 605.6, df=1,1162; p < 0.0001), while in South Africa the opposite was true, with lower 5 MO 2 in air. Surprisingly, the South African population showed significant indications of a 6 critical thermal threshold above 33°C in both air and water (Fig.3), when the temperature 7 induced rise in MO 2 levelled off in air and inverted in water. In contrast, the temperature  showed body temperatures as much as 2 o C lower than environmental temperatures, especially  Overall, Uca urvillei consistently increased its oxygen consumption, suggesting that its 10 respiration was not impaired by heat during aerial respiration. In addition, this species, 11 displays a large difference between arterial and venous PO 2 when relies on aerial oxygen. This  Consequently, the fact that U. urvillei is essentially an air breather can explain its pronounced 20 general heat tolerance, lack of thermoregulation and ability to sustain activity at high 21 temperatures (Fusi, unpublished data). This interpretation is supported by the dramatic 22 hypoxemia that this species develops when submerged, which is a sign of extremely 23 inefficient oxygen extraction from water. These findings indicate that U. urvillei is essentially 24 an air-breathing species, poorly adapted to underwater respiration, even though it inhabits plugging of the hole by carving a mud disc adjacent to the entrance of the burrow, as 4 described for other fiddler crab species (de la Iglesia, 1994). Once plugged, crabs carefully 5 seal the disc from inside the hole to avoid water leakage and to trap air inside the burrow.  avoid long periods of exposure to higher temperatures (Fusi, unpublished data), which would 21 require excessive energy expenditure to sustain metabolic demands (Fig. 4).

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The results of this study reveal unexpected climate induced response of tropical ectotherms to 24 intertidal environments, supporting the need to adopt a multilevel integrated approach when 2008). In particular, the two studied species showed different mechanisms of adaptation to 2 temperature regimes and a diverse degree of resilience to regional warming. U. urvillei, 3 through a high temperature tolerance and a consistent metabolic response across a wide range 4 of temperatures, exhibits a single level of thermal response in different environments and can 5 be described as a thermal generalist, highly tolerant to a broad range of temperatures across its 6 entire geographical distribution. In contrast, the specialist, P. guttatum, showed distinct 7 tolerance ranges across the geographical gradient and appeared to be adapted to local climatic   Based on the present results, we would propose a conceptual model to draft the differences in 16 thermal vulnerability between thermal specialist and thermal generalist species (Fig. 6). Since 17 such model is based on the comparisons between two populations at two latitudes future 18 studies are needed to validate and extend the proposed rationales.

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Across its entire geographical distribution, a thermal generalist will maintain a wide window In contrast, the thermal specialist is 2 more sensitive to temperature variations and locally adapted to regional climate. Thus, while 3 stenothermal populations at low latitudes are able to withstand increased heat stress, as stated 4 for high-latitude species by Sunday et al. (2012), those specialised to a subtropical climate 5 appear vulnerable to a long term warming. Further, we should also take into account that low 6 latitudes in this study were also the centre of distribution for these species, implying that other 7 factors must contribute to the well-being of the tropical populations. Perhaps populations at 8 the edge of their distribution have to cope with increased habitat degradation and therefore 9 genetic isolation compared to their tropical counterparts (Pearson et al. 2009).

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In conclusion, a detailed appreciation of thermal induced response, extended at population 11 level, appears to be crucial to predicting the resilience to climate anomalies in species with  We thank the anonymous reviewer to help us to deeply improve the overall manuscript. MF 10 thanks Laura Sbaragli for invaluable help during the Kenyan fieldwork, Francesco Gori,

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Fabrizio Bartolini and Tommaso Guerra for essential support.      to the sun (c, d, i, j), in shade but always on the ground (e, f, k, l). Grey area represents 95% 1 confidence interval, the bisect represents the isotherm line.      Temperature-dependent performances (curves depict responses to acute temperature fluctuation) are maintained within the entire climatic range (horizontal bars) and maximised around the optimum which generally represents the preferred temperature for activity. Acute heat events (vertical bars) beyond the range of environmental temperatures affect organismal fitness limiting the capability to sustain performance. Thermal generalists undergo pronounced temperature fluctuations both on a daily and seasonal basis and manifest a pronounced eurythermy throughout their distribution. This category shows a low thermal vulnerability, even to anomalous warming, and a single pattern of responses to temperature fluctuation along the geographic gradient (further supported by wide ranging gene flow among populations).
Thermal specialists manifest distinctive tolerance ranges among different regions as a result of climate induced response to local climatic signatures and the genetic differentiation of phylogeographic clades. Tropical stenotherms are particularly adapted to a constantly hot climate and may endure irregular increases of environmental temperatures. In contrast, temperate-adapted populations have a broader thermal tolerance, likely due to the larger fluctuation of environmental temperature, but are more vulnerable to the effects of global warming. 68x58mm (300 x 300 DPI) Temperature-dependent performances (curves depict responses to acute temperature fluctuation) are maintained within the entire climatic range (horizontal bars) and maximised around the optimum which generally represents the preferred temperature for activity. Acute heat events (vertical bars) beyond the range of environmental temperatures affect organismal fitness limiting the capability to sustain performance. Thermal generalists undergo pronounced temperature fluctuations both on a daily and seasonal basis and manifest a pronounced eurythermy throughout their distribution. This category shows a low thermal vulnerability, even to anomalous warming, and a single pattern of responses to temperature fluctuation along the geographic gradient (further supported by wide ranging gene flow among populations).
Thermal specialists manifest distinctive tolerance ranges among different regions as a result of climate induced response to local climatic signatures and the genetic differentiation of phylogeographic clades. Tropical stenotherms are particularly adapted to a constantly hot climate and may endure irregular increases of environmental temperatures. In contrast, temperate-adapted populations have a broader thermal tolerance, likely due to the larger fluctuation of environmental temperature, but are more vulnerable to the effects of global warming. 144x122mm (300 x 300 DPI)