Skip to main content
NCBI home page
Proceedings of the Royal Society B: Biological Sciences logoLink to Proceedings of the Royal Society B: Biological Sciences
. 2014 May 7;281(1782):20132927. doi: 10.1098/rspb.2013.2927

Improved heat tolerance in air drives the recurrent evolution of air-breathing

Folco Giomi 1,✉, Marco Fusi 2,3, Alberto Barausse 4, Bruce Mostert 5, Hans-Otto Pörtner 1, Stefano Cannicci 3
PMCID: PMC3973256  PMID: 24619438

Abstract

The transition to air-breathing by formerly aquatic species has occurred repeatedly and independently in fish, crabs and other animal phyla, but the proximate drivers of this key innovation remain a long-standing puzzle in evolutionary biology. Most studies attribute the onset of air-breathing to the repeated occurrence of aquatic hypoxia; however, this hypothesis leaves the current geographical distribution of the 300 genera of air-breathing crabs unexplained. Here, we show that their occurrence is mainly related to high environmental temperatures in the tropics. We also demonstrate in an amphibious crab that the reduced cost of oxygen supply in air extends aerobic performance to higher temperatures and thus widens the animal's thermal niche. These findings suggest that high water temperature as a driver consistently explains the numerous times air-breathing has evolved. The data also indicate a central role for oxygen- and capacity-limited thermal tolerance not only in shaping sensitivity to current climate change but also in underpinning the climate-dependent evolution of animals, in this case the evolution of air-breathing.

Keywords: air-breathing evolution, heat tolerance, oxygen limitation, terrestrial colonization

1. Introduction

The evolution of terrestrial forms from aquatic ancestors occurred independently in all animal phyla, leading to an enormous diversification of life forms that conquered the land [1–3]. While the exploitation of terrestrial niches and their resources represents the ultimate factor of success, the unifying reasons for the evolution of air-breathing have not been fully understood. It is generally assumed that air-breathing evolved in response to unfavourable conditions in the aquatic environment. Aquatic hypoxia, either perpetual or seasonal, may be involved, but in our view this explanation remains incomplete for two main reasons. First, a repeated and large-scale evolutionary process such as the transition to air implies a more widespread and persistent effect of a key environmental driver. Second, the transition to air is less favoured at higher latitudes, although hypoxia and even anoxia develop episodically even in shallow estuarine waters of temperate zones. Furthermore, the geographical distribution of extant amphibious ectotherms currently in transition to air is wide-ranging also in highly oxygenated coastal environments. While such range expansion may well have occurred after the evolution of air-breathing, both of these observations open the possibility that drivers other than severe hypoxia were effective. Earlier work suggested that temperature extremes constrain the aquatic–terrestrial transition [4–8]. Here, we provide contrasting evidence that temperature extremes and variability together with hypoxia may in fact synergistically enforce air-breathing. We studied this transition in the true crabs (Brachyura, Crustacea), which, owing to the high diversity of amphibious forms, are considered to be at the dawn of their transition to land [5,9,10].

We analysed the proximate reasons for the transition to air-breathing by applying the recent concept of oxygen and capacity limitation of thermal tolerance (OCLTT), proposed as a unifying principle to comprehend the sensitivity of aquatic and many terrestrial ectotherms to thermal stress [11,12]. According to this concept, the performance capacity of aquatic ectotherms and their thermal windows are constrained by a mismatch between oxygen supply and demand at both low and high edges of the thermal envelope. In general, the provision of oxygen to the peripheral circulation becomes a physiological constraint at thermal extremes [13]. As a consequence, the functional capacity of an organism declines progressively with the development of body fluid hypoxaemia and elevated costs of oxygen supply. At more extreme, critical temperatures, tissue anaerobiosis follows, compromising performance further, and thereby long-term survival. Thus, aerobic performance defines the thermal niche of marine and freshwater species [12]. The thermal limits identified in laboratory studies are those effective in the field [11,14–18], and these temperature thresholds are influenced by oxygen availability. Exposure of the Antarctic fish Pachycara brachycephalum to hyperoxia in fact shifted its thermal tolerance (TT) to higher temperatures by mitigating the increasing costs of oxygen supply upon warming [15].

An important corollary of the OCLTT concept is that breathing air instead of water may enhance TT owing to the higher concentration (30 times) and diffusivity (200 000 times at 20°C) of oxygen in air. Conversely, TT is reduced when animals undergo a shortage of available oxygen [19]. In general, applicability of the OCLTT principle to terrestrial species with convective and circulatory oxygen supply systems has been suggested [11,13,19]. By increasing oxygen availability, air-breathing may lead to the immediate benefit of enhanced heat tolerance through reduced maintenance cost and the extension of aerobic scope, promoting the exploitation of new habitats under the unpredictable temperature variations of the aquatic–terrestrial boundary. To exploit the aerial oxygen and to avoid collapse of respiratory epithelia in air they need to be reshaped, which in crustaceans occurs by gill sclerotization or the use of gill chambers as lungs, or formed de novo as in vertebrates. Further adaptations to terrestrial life include shifts in nitrogen excretion from ammonia to uric acid in crustaceans [5,9,10] or urea in vertebrates. Intermediate forms of terrestrial adaptation include intertidal species, active in air only at low tide. Truly terrestrial species no longer require regular immersion in water [1,2,5].

2. Results and discussion

The terrestrial invasion by true crabs is an evolutionary process that now involves 22 families of Brachyura and about 300 genera. The reconstruction of ancestral state of respiratory mode shows that the evolution of air-breathing occurred independently and repeatedly in phylogenetically unrelated families of both freshwater and marine terrestrial forms (figure 1). Indeed, two main pathways of terrestrialization are recognized, with some taxa clearly conquering land via freshwater systems and others from the marine intertidal environment. A survey and meta-analysis of existing literature on the distribution, biogeography and taxonomy of the world's air-breathing crabs identifies brachyuran terrestrial invasion as a mainly tropical phenomenon (figure 2). The higher richness of air-breathing crab genera in the tropics is not linked to the greater crab diversity occurring at low latitudes. Indeed, the geographical distribution of crabs, categorized by different respiratory modes (water-breathing and air-breathing), proves that the percentage of the air-breathing crab genera is inversely related to the latitude and peaks in the equatorial and tropical climatic regions (figure 2; when comparing the percentage of air-breathing genera with latitude across different climatic regions, Spearman's rank correlation coefficient rS = −1.00, p = 0.0167; n = 5). Moreover, the richness of air-breathing crabs, analysed at a magnified biogeographic scale, is consistently high in all tropical regions showing peaks of diversity in the Indian to west Pacific oceanic region (figure 3a). Inversely, only a small number of genera ranges from subtropical to warm-temperate zones and none in cooler regions. A strong positive relationship exists between the richness of air-breathing crabs of both freshwater and marine origin and the average habitat temperatures in the different biogeographic regions (figure 3b,c).

Figure 1.

Figure 1.

Open in a new tab

Ancestral state reconstruction of respiratory mode along the phylogenetic tree of brachyuran families (see text for details on tree reconstruction). At each node, the pie charts represent the relative likelihood support for reconstructed ancestors adopting a binary classification using either water-breather (white) or air-breather (black). Note the recurrent and independent evolution of air-breathing along the true crab radiation.

Figure 2.

Figure 2.

Open in a new tab

Proportion of crab genera, categorized on the basis of respiratory mode, in the five major climatic regions. The total number of crab genera is reported on the top of the bars, whereas the number of genera for each category is superimposed on the respective stack level. There is a strong, inverse correlation between the percentage of the air-breathing genera and the latitude of the climatic regions (Spearman's rank correlation coefficient rS = −1.00, p = 0.0167, n = 5).

Figure 3.

Figure 3.

Open in a new tab

(a) The richness of air-breathing crab genera from freshwater and coastal habitats is highest in the intertropical zone and particularly along the coast of Indian Ocean and in the North Australasian/Indo-Malayan ecozones. The box bordered by a dashed line circumscribes the Pacific Islands. Richness (G) is correlated with land and sea mean annual surface temperatures (T) for species of both (b) freshwater and (c) marine origin. Data were fitted to exponential growth curves (solid lines), G = 0.513e0.185•T for freshwater origin (r2 = 0.34, p < 0.01) and G = 0.219e0.174•T (r2 = 0.41, p < 0.001) for marine origin. Shaded areas indicate 95% CIs. (b) NA, non-tropical Asia; SAm, non-tropical South America; Me, Circum-Mediterranean regions; SAf, South Africa; SAu, non-tropical Australia; TAf, Tropical Africa; IS, India and Sri Lanka; TAm, Tropical America; MA, Malayan and Tropical Australasia ecozone. (c) SE, southern Europe; NEP, northeast Pacific Ocean; NE, Near East; NA, North Africa; NWA, northwest Atlantic Ocean; SAu, South Australia; SAf, South Africa; SEP, southeast Pacific Ocean; EAA, East Atlantic Africa; CG, Caribbean Gulf; SWA, southwest Atlantic Ocean; RS, Red Sea; WIO, West Indian Ocean; PI, Pacific Islands; EIO, East Indian Ocean and West Pacific.

We investigated the potential benefits of air-breathing for the thermal response of a model brachyuran, Pachygrapsus marmoratus (Fabricius, 1787), which is a dual breather and displays an amphibious mode of life. In animals exposed to progressive warming while breathing air or water, we analysed oxygen consumption as a measure of metabolic rate, as well as oxygen partial pressures in arterial and venous blood (haemolymph), as indicators of the level of oxygen supply to tissues (figure 4). Oxygen demand by P. marmoratus is strongly impacted by temperature in both milieus (F = 4.85, d.f. = 4, 76, p < 0.01). Moreover, in water oxygen demand follows a characteristic exponential function between 19 and 28°C. According to the OCLTT concept, a critical temperature is reached at 28°C in water, when respiration rate levels off and begins to decline, thereby indicating capacity limits to oxygen supply, onset of anaerobic metabolism and the associated metabolic depression [14–16]. The decline of arterial and especially venous haemolymph oxygen partial pressure (PO2) during warming, with the lowest values reached beyond the upper critical temperature (Tc), confirms the onset of hypoxaemia in body fluids at high temperatures [14]. A mismatch between oxygen supply and demand thus constrains the thermal niche realized by P. marmoratus in warming water.

Figure 4.

Figure 4.

Open in a new tab

Widening of the TT window in the dual breather crab Pachygrapsus marmoratus when in air (grey area) is indicated by (a) oxygen consumption (MO2) rising exponentially in warming water more steeply than in air. The exponential phase in MO2 in water (fitted by a solid grey line, y = 3.95 + 0.002·e0.29x, r2 = 0.92, p < 0.001) is limited by an upper critical temperature (Tc) of 28°C, where metabolic rate deviates from the exponential curve (dotted grey line). By contrast, oxygen consumption in air shows a markedly reduced temperature dependency indicating an upward shift of critical limit (black line, y = 3.13 + 0.002e0.24x, r2 = 0.87, p < 0.001) (n = 7–10 ± s.e.m.). (b) Arterial and venous haemolymph oxygen partial pressures (PO2) and (c) the arteriovenous differences in PO2 are significantly reduced when crabs are in warming water (beyond 25°C, p < 0.001), but remain unchanged during air-breathing. Venous PO2 values are generally lower in air, reflecting a trade-off between enhanced costs of posture and the reduction in circulatory blood flow in air (see text) (n = 6–9 ± s.e.m.).

When exposed to air, the amphibious crabs change their thermal response. While metabolic rate remains similar in both media at 19°C, oxygen uptake increases less in air than in water (F = 15.54, d.f. = 1, 75, p < 0.0001) and arterial PO2 remains higher over the entire temperature range (air versus water: F = 4.34, d.f. = 1, p < 0.05; interaction term Milieu × temperature: F = 2.162, d.f. = 2, 86, p = 0.12; figure 4). In conclusion, when warming in air P. marmoratus does not experience cost increments as strong as in water. The higher availability and diffusivity of oxygen in air probably facilitates gas exchange and alleviates the ventilatory and circulatory workload during warming such that animals cover their oxygen demand at a lower cost. These findings resemble the patterns observed in terrestrial arthropods [19], in fishes when exposed to hyperoxia [15] and in eurythermal crustaceans [18], confirming that excess oxygen supply alleviates the thermal burden. Similarly, early work on the green crab Carcinus maenas describes a behaviour adopted in response to acute heat rise in water. This aquatic species emerges in air and starts to ventilate the branchial chamber [20]. In line with the OCLTT principles, the enhanced oxygen content in air facilitates oxygen supply and causes a widening of the TT window, including sustenance of aerobic scope at higher temperatures in air than in water.

The processes involved in cost reductions in air require identification, considering that shifts in energy budget may also occur. In fact, cost increments may be reflected in enhanced oxygen removal from the venous sinus at the base of walking legs, leading to lower venous PO2 values and significantly larger arteriovenous differences in PO2 at all temperatures (F = 6.22, d.f. = 2, 86; p < 0.01) in air than in water (air versus water: F = 14.22, d.f. = 1, 43, p < 0.001; interaction term Milieu × temperature: F = 0.21, d.f. = 2, 86, p = 0.81; figure 4). In air, the cost of animal posture is evident from the extremely low value of venous oxygen content, which suggests that during emersion aquatic animals are subjected to amplified exercise. This is because of the eliminated buoyancy in air. Indeed, buoyancy during submergence counteracts 90% of the effective weight of grapsid crabs, and at the same time it increases the efficiency of their underwater kinematics [21,22]. However, even if the cost of sustaining the body weight in air may increase owing to greater vertical forces (i.e. gravity), this is not reflected in increased whole animal oxygen consumption in air over that in water. This potential cost may thus be negligible or compensated for by energy savings as in oxygen supply. At higher temperatures, whole animal oxygen demand in air even decreases below that in water while oxygen removal from venous blood remains enhanced. Therefore, cost savings probably include a reduction in blood flow and circulatory work owing to excess oxygen in air, again similar to findings under hyperoxia in fishes [15]. Such reduction in blood flow at constant or falling oxygen consumption would cause the observed decrease in venous PO2 and in metabolic rate.

In conclusion, the geographical distribution of extant genera of air-breathing crabs and the experimental evidence both support the hypothesis that the recurrent transition to land occurred for the sake of improved heat tolerance, provided by the reduced costs of oxygen supply in air. Both heat tolerance and energy savings probably constitute the proximate causal factors driving the evolution of air-breathing at tropical latitudes. This key innovation not only evolved in crabs, but also in euteleosts and neoteleosts, which show strikingly similar evolutionary and biogeographic features. As for crabs, in fact, air-breathing in fish evolved between 38 and 67 times at tropical latitudes [1,2,4]. With progressive warming, the decrease in water oxygen content, combined with a general trend for organismal metabolic rates to increase, may explain why air-breathing evolved. The more rapid transition to hypoxaemia would enhance sensitivity to warming and provide a stimulus to breathe air, stronger at lower than at higher (cooler) latitudes. This explains why, globally, the fraction of air-breathing crustaceans increases continually with decreasing latitude and increasing temperature.

The higher fraction of freshwater than marine species moving on land deserves further consideration. This finding contrasts intuitive expectations as at the same temperature oxygen solubility is higher in freshwater than in seawater. Assuming the same oxygen diffusivity, this higher solubility would slightly improve heat tolerance unless compensated for by higher costs of maintenance, a question that cannot be answered at present. However, freshwater species live in smaller water bodies which can periodically dry up or strongly reduce their water regime, a situation that strongly advantages the species undertaking the evolutionary path to air-breathing. Moreover, they may thus be exposed to larger temperature variability and extremes such that the heat stimulus to move into air may, on average, be stronger than in the marine environment.

These considerations thus do not exclude a role for aquatic hypoxia/anoxia in the evolution of air-breathing. Indeed, depletion of oxygen from ambient media exacerbates the mismatch between oxygen supply and demand, and causes a shift of temperature thresholds to lower values [12]. The seasonal occurrence of extreme hypoxia in certain tropical estuaries or in localized coastal zones would limit the heat tolerance of aquatic ectotherms further by narrowing the thermal envelope required for the maintenance of aerobic metabolism. In fact, the present hypothesis strengthens the view that oxygen limitation as provoked or exacerbated by extreme temperatures caused transition to air-breathing.

The evolutionary benefits projected here for air-breathing crabs and fish indicate that improved heat tolerance in air prepares them for more pronounced fluctuations of environmental temperature and enhanced heat exposure on land that, together with the risk of desiccation, may otherwise limit the terrestrialization of aquatic animals [6–8,16,17]. However, the water–air interface offers access to air while extreme air temperatures or sun exposure would still be buffered by the water. Both air-breathing amphibious fish and crabs also evolved effective thermoregulatory strategies, by cooling their body to several degrees below ambient temperature by means of transpiration [6,23,24]. The regular retreat into burrows, colonization of sheltered environments and nocturnal activity are universal behavioural strategies adopted during the evolutionary transition from aquatic to terrestrial life [4,5,6,8]. These strategies, together with the widened thermal window, probably supported the exploitation of amphibious habitat and the rapid diversification of terrestrial life.

Building on OCLTT principles, our hypothesis can explain the multiple and diverse evolutions of air-breathing and terrestrial life from both freshwater or marine ancestor fishes and crabs [1,4,5,25]. Heat constraints on aerobic metabolism and energy budget, and associated performance and fitness decrements, exert permanent evolutionary pressure in favour of air-breathing within the temperature range of tropical climates. OCLTT occurring at tropical latitudes is thus likely to constitute a central driving force for the impressive number of independent evolutionary transitions to air-breathing observed.

3. Material and methods

As a model aquatic ectotherm capable of facultative air-breathing, we chose the rocky shore crab Pachygrapsus marmoratus (Fabricius, 1787) belonging to the Grapsidae, a crab family widespread in intertidal habitats across the tropics. Specifically, the worldwide intertropical distribution of the majority of Pachigrapsus species indicates that the centre of origin of this genus meets the assumption of our working hypothesis [26]. Secondary extension of geographical range of the genus occurred, including the colonization of the Mediterranean basin by Pachygrapsus maurus, P. transversus and P. marmoratus. This last species is abundant in the intertidal belt of both Mediterranean and Atlantic European rocky shores, as well as in the Macaronesian region [26]. Pachygrapsus marmoratus represents an excellent bimodal species model as it displays, in air and in water, an equal oxygen consumption rate during moderate activity [21]. Indeed, it exhibits a truly semi-terrestrial lifestyle, moving freely between air and seawater, and being active on emerged rocks both during day and night [27].

Male crabs were hand-collected during spring of 2010 from the rocky shore of Calafuria, Ligurian Sea, Italy. The animals were held at the rearing facilities of the Department of Biology in Florence, in tanks of filtered, aerated re-circulating natural seawater at 19 ± 0.2°C, 38‰ salinity and a 12 h light cycle for at least four weeks before the beginning of the experiments.

(a). Oxygen consumption rate

Oxygen consumption rates were measured in an intermittent flow respirometer, equipped with five parallel darkened Perspex chambers and placed in a water bath to set experimental temperatures. An oxygen sensor (Sensor Type PSt3, PreSens, Regensburg, Germany) glued onto the wall of each chamber and connected to a single channel oxygen transmitter (Fibox 3, PreSens) through an optical SMA fibre was used to measure the partial pressure of oxygen in the water. Data were recorded by the use of FibSoft v. 1.0 software (Loligo Systems ApS). Prior to the measurements, sensors were calibrated in air-equilibrated seawater (100%) and saturated sodium hydrosulfite solution (0%). During trials, oxygen concentration was not allowed to fall below 60% saturation. Oxygen consumption rates were calculated by determining the decline in oxygen saturation from oxygen partial pressure and solubility over time. The movements of experimental specimens caused sufficient mixing of the water and a linear decline of oxygen content over time. An empty chamber (control) was run with each trial to account for background oxygen consumption, which was routinely less than 2–3% of the consumption recorded within the respiratory chambers in water, and negligible in air. Before applying the temperature ramp procedure, individuals were placed in the chambers and allowed to recover from handling stress overnight. Oxygen consumption rates were determined at assay temperatures of 19°C/22°C/25°C/28°C/31°C, which were changed in a stepwise procedure at an average rate of 1°C × h–1. Following each experiment, animal wet weight was measured and animal volume was calculated by immersing individuals into a graduated measuring cylinder to measure the volume of water displaced.

(b). Dissolved oxygen measurements

Oxygen partial pressure (PO2) in arterial and venous haemolymph was measured with fibre-optic oxygen microsensors (PreSens) connected to an oxygen meter (Microx-TX, PreSens) with integrated signal-processing software. The sensors were calibrated before and after each experiment using a two-point calibration in oxygen-free (addition of sodium hydrosulfite) and air-saturated (bubbled) seawater. Animals were acclimated overnight and the measurements were performed at 19°C/25°C/31°C, increasing the temperature as described earlier, in a temperature-controlled room where environmental temperature was maintained at an accuracy of ±1°C.

To measure the oxygen partial pressure of arterial blood, haemolymph was withdrawn from a hole (maximum width 0.2 mm) drilled in the carapace over the pericardial sinus, avoiding injury to the hypodermis, following the procedure described by Frederich & Pörtner [14]. Venous blood was withdrawn from the sinus below the arthrodial membrane at the base of the fourth or the fifth pereiopod. In both cases, small haemolymph samples (less than 20 µl) were drawn by capillary action into a manually sharpened Pasteur pipette in which the oxygen sensor was inserted and positioned close to the tip.

(c). Phylogenetic and ancestral state reconstructions

A composite phylogeny of brachyuran crabs at the level of family was elaborated from multiple literature sources. Updated general revisions on Brachyura, such the ones by Guinot et al. [28] and Schubart [29], were used as a basis for the construction of general phylogenetic reconstruction. Afterwards, the phylogenetic tree was implemented with information gathered from papers focusing on phylogenetic relationships within brachyuran groups. In particular, the most useful papers were the ones by Lai et al. [30–34]. In our analysis, we included almost all families (87 out of 96), with the exceptions being Aethridae, Acidopsidae, Atelecyclidae, Bythograeidae, Conleyidae, Crossotonotidae, Litocheiridae, Palicidae, Progeryonidae, because of lack of consensus about their phylogenetic relationships.

When the tree topology in the original studies was not sufficiently supported or when different studies presented contrasting phylogenetic reconstructions the uncertain branching was represented as polytomies.

Genus-specific information on respiratory mode was compiled from different sources [5,19] and by directly contacting specialists of those groups.

Ancestral state reconstruction of respiratory mode in this true crab ‘super tree’ was conducted using the maximum-likelihood computation in Mesquite v. 1.1 [35]. Considering that transition to air-breathing is very rarely reversible, the asymmetrical two-parameter Markov k-state model was adopted for the ancestral state reconstruction. This model assumes that reversible transition of character state is unlikely and that the probabilities for all character transitions are dissimilar (i.e. different rate of change for the directions of character transition).

(d). Worldwide richness and distribution of air-breathing crabs

The definition of the extant genera of the semi-terrestrial crabs of the world was done according to the comprehensive catalogue available [36]. Information on their biology and distribution was collected from studies reporting on the definition of new species of terrestrial crabs, their biology and distribution, as well as from review papers, checklists and web databases [26–28,30–34,36–46]. For the evaluation of distribution and richness, genera were preferred since they are more inter-independent taxonomical units than species.

In a first analysis, the distribution of air- and water-breathing crabs across the five major climatic regions [47] was assessed for 1020 genera. The remaining 280 (of which five are air-breathing) genera registered in the WoRMS [45] database were not employed because of the lack, or uncertainty, of confirmed distribution ranges on the OBIS database or other reliable biogeographic sources.

Subsequently, the analysis was refined solely to the air-breathing crabs. Biogeographic regions for species of marine origins were defined following the classification of major seawater basins [48] and considering the evolution and biogeography of brachyuran crabs. Following the theories on the centre of speciation of Sesarmidae and Grapsidae, and considering the present distribution of their genera, we divided the Western Indo-Pacific (WIP) region [48] into a Western Indian Ocean (WIO) region and Eastern Indian Ocean/Western Pacific (EIO/WP) region, merging the eastern part of WIP with the Central Indo-Pacific regions. For terrestrial crabs originating from freshwater, we adopted the biogeographic classification based on major terrestrial habitats within each ecoregion as developed by the World Wildlife Fund, USA [49,50]. To account for the particular evolutionary history of freshwater crabs distributed in southeast Asia, India and Sri Lanka, we split the Indo-Malayan region into two different regions (the biogeography and air-breathing genera datasets are available in the electronic supplementary material).

(e). Temperature data

We computed mean annual land surface air temperatures for each ecoregion using the climatological dataset CRU CL 2.0 [51], which contains mean monthly values of surface climate for global land areas on a 10 min latitude/longitude grid, averaged over the period 1961–1990. Similarly, we computed mean annual sea surface temperature for ecoregions using the climatological time-series NOAA Optimum Interpolation [52]. This dataset contains mean weekly values of sea surface climate at a resolution of 1°, averaged over the period 1980–2010.

(f). Statistical analysis

Given the small sample size, Spearman's rank correlation coefficient [53] was computed to test the presence of a monotonic relationship between the proportion of air-breathing crab genera and latitude across climatic regions.

As the groups of experimental crabs were different in the different trials, a full factorial permutational analysis of variance (PERMANOVA [54]) was used to test the null hypothesis of no differences between respiration rates of specimens across Milieu, defined as fixed and orthogonal. To test for statistical differences in oxygen consumption along the temperature ramp procedure, we used a paired-PERMANOVA design, as the specimens were the same along each experimental cycle, keeping the Milieu factor in the model, to disentangle its influence. Post hoc pairwise tests were performed when appropriate. All data are expressed as mean ± s.e. and the analyses were performed using the PERMANOVA+ routines for PRIMER 6 [55].

Acknowledgements

The authors thank Simone Babbini for laboratory assistance, Ivan Mičetić for graphical insights on figures and Christopher McQuaid for helpful comments and suggestions. S.C. thanks Richard Hartnoll for inspiration and Irene Ortolani for help in biogeographic data collection. M.F. is very grateful to Sara Borin and Daniele Daffonchio for the invaluable support and useful discussions on this manuscript.

Funding statement

F.G. was funded by the FP7-PEOPLE, IEF project ‘The weakest links’ (no. 221017), and both F.G. and H.-O.P. by the PACES program of the AWI. M.F. and S.C. were supported by MIUR funds and by the FP7-PEOPLE, IRSES Project ‘CREC’ (no. 247514). M.F. was also funded by MaCuMBA EU Project (FP7/2007–2013; no. 311975).

References

  • 1.Little C. 1983. The colonisation of land: origins and adaptations of terrestrial animals. Cambridge, UK: Cambridge University Press [Google Scholar]
  • 2.Little C. 1990. The terrestrial invasion: an ecophysiological approach to the origins of land animals. Cambridge, UK: Cambridge University Press [Google Scholar]
  • 3.Randall DJ, Burggren WW, Farrell AP, Haswell MS. 1981. The evolution of air breathing in vertebrates. Cambridge, UK: Cambridge University Press [Google Scholar]
  • 4.Graham JB. 1997. Air-breathing fishes: evolution, diversity, and adaptation. San Diego, CA: San Diego Academic Press [Google Scholar]
  • 5.Burggren WW, McMahon BR. 1988. Biology of the land crabs. Cambridge, UK: Cambridge University Press [Google Scholar]
  • 6.Sayer MDJ. 2005. Adaptations of amphibious fish for surviving life out of water. Fish Fish. 6, 186–211 (doi:10.1111/j.1467-2979.2005.00193.x) [Google Scholar]
  • 7.Dejours P. 1988. Respiration in water and air: adaptations–regulation–evolution. Amsterdam, The Netherlands: Elsevier [Google Scholar]
  • 8.Sayer MDJ, Davenport J. 1991. Amphibious fish: why do they leave water? Rev. Fish. Biol. Fish. 1, 159–181 (doi:10.1007/BF00157583) [Google Scholar]
  • 9.Bliss DE. 1968. Transition from water to land in decapod crustaceans. Am. Zool. 8, 355–392 (doi:10.1093/icb/8.3.355) [Google Scholar]
  • 10.Henry RP. 1994. Morphological, behavioral, and physiological characterization of bimodal breathing crustaceans. Am. Zool. 34, 205–215 [Google Scholar]
  • 11.Pörtner HO. 2001. Climate change and temperature-dependent biogeography: oxygen limitation of thermal tolerance in animals. Naturwissenschaften 88, 137–146 (doi:10.1007/s001140100216) [DOI] [PubMed] [Google Scholar]
  • 12.Pörtner HO. 2010. Oxygen- and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. J. Exp. Biol. 213, 881–893 (doi:10.1242/jeb.037523) [DOI] [PubMed] [Google Scholar]
  • 13.Pörtner HO. 2002. Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals. Comp. Biochem. Physiol. A 132, 739–761 (doi:10.1016/S1095-6433(02)00045-4) [DOI] [PubMed] [Google Scholar]
  • 14.Frederich M, Pörtner HO. 2000. Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance in spider crab, Maja squinado. Am. J. Physiol. 279, 1531–1538 [DOI] [PubMed] [Google Scholar]
  • 15.Mark FC, Bock C, Pörtner HO. 2002. Oxygen-limited thermal tolerance in Antarctic fish investigated by MRI and 31P-MRS. Am. J. Physiol. 283, 1254–1262 (doi:10.1152/ajpcell.00169.2002) [DOI] [PubMed] [Google Scholar]
  • 16.Melzner F, Bock C, Pörtner HO. 2006. Temperature-dependent oxygen extraction from the ventilatory current and the costs of ventilation in the cephalopod Sepia officinalis. J. Comp. Physiol. B 176, 607–621 (doi:10.1007/s00360-006-0084-9) [DOI] [PubMed] [Google Scholar]
  • 17.Wittmann AC, Schröer M, Bock C, Steeger HU, Paul RJ, Pörtner HO. 2008. Indicators of oxygen- and capacity-limited thermal tolerance in the lugworm Arenicola marina. Clim. Res. 37, 227–240 (doi:10.3354/cr00763) [Google Scholar]
  • 18.Giomi F, Pörtner HO. 2013. A role for haemolymph oxygen capacity in heat tolerance of eurythermal crabs. Front. Physiol. 4, 110 (doi:10.3389/fphys.2013.00110) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Stevens MM, Jackson S, Bester SA, Terblanche JS, Chown SL. 2010. Oxygen limitation and thermal tolerance in two terrestrial arthropod species. J. Exp. Biol. 213, 2209–2218 (doi:10.1242/jeb.040170) [DOI] [PubMed] [Google Scholar]
  • 20.Taylor EW, Wheatly MG. 1979. The behaviour and respiratory physiology of the shore crab, Carcinus maenas (L.) at moderately high temperatures. J. Comp. Physiol. B 130, 309–316 (doi:10.1007/BF00689848) [Google Scholar]
  • 21.Houlihan DF, Innes AJ. 1984. The cost of walking in crabs: aerial and aquatic oxygen consumption during activity of two species of intertidal crab. Comp. Biochem. Physiol. A 77, 325–334 (doi:10.1016/0300-9629(84)90069-0) [Google Scholar]
  • 22.Martinez MM. 2001. Running in the surf; hydrodynamics of the shore crab Grapsus tenuicrustatus. J. Exp. Biol. 204, 3097–3112 [DOI] [PubMed] [Google Scholar]
  • 23.Edney EB. 1962. Some aspects of the temperature relations of fiddler crabs (Uca spp.). In Biometeorology (ed. Tromp SW.), pp. 79–85 Oxford, UK: Pergamon Press [Google Scholar]
  • 24.Graham JB. 1973. Terrestrial life of the amphibious fish Mnierpes macrocephalus. Mar. Biol. 23, 83–91 (doi:10.1007/BF00394114) [Google Scholar]
  • 25.Graham JB, Rosenblatt RH, Gans C. 1978. Vertebrate air breathing arose in fresh waters and not in the oceans. Evolution 32, 459–463 (doi:10.2307/2407614) [DOI] [PubMed] [Google Scholar]
  • 26.Poupin J, Davie PJE, Cexus JC. 2005. A revision of the genus Pachygrapsus Randall, 1840 (Crustacea: Decapoda: Brachyura, Grapsidae), with special reference to the Southwest Pacific species. Zootaxa 1015, 1–66 [Google Scholar]
  • 27.Hartnoll RG. 1988. Evolution, systematics, and geographical distribution. In Biology of the land crabs (eds Burggren WW, McMahon BR.), pp. 6–54 Cambridge, UK: Cambridge University Press; (http://dx.doi.org/10.1017/CBO9780511753428.003) [Google Scholar]
  • 28.Guinot D, Tavares M, Castro P. 2013. Significance of the sexual openings and supplementary structures on the phylogeny of brachyuran crabs (Crustacea, Decapoda, Brachyura), with new nomina for higher-ranked podotreme taxa. Zootaxa 3665, 1–414 (doi:10.11646/zootaxa.3665.1.1) [DOI] [PubMed] [Google Scholar]
  • 29.Tsang LM, Schubart CD, Ahyong ST, Lai JCY, Au E, Chan TY, Ng PKL, Chu KH. In press Evolutionary history of true crabs (Crustacea: Decapoda: Brachyura) and the origins of freshwater crabs. Mol. Biol. Evol. (doi:10.1093/molbev/msu068) [DOI] [PubMed] [Google Scholar]
  • 30.Lai JCY, Thoma BP, Clark PF, Felder DL, Ng PKL. 2013. Phylogeny of eriphioid crabs (Brachyura, Eriphioidea) inferred from molecular and morphological studies. Zool. Scripta 43, 52–64 (doi:10.1111/zsc.12030) [Google Scholar]
  • 31.Lai JCY, Mendoza JCE, Guinot D, Clark PF, Ng PKL. 2011. Xanthidae MacLeay, 1838 (Decapoda: Brachyura: Xanthoidea) systematics: a multi-gene approach with support from adult and zoeal morphology. Zool. Anz. 250, 407–448 (doi:10.1016/j.jcz.2011.07.002) [Google Scholar]
  • 32.Klaus S, Yeo DCJ, Ahyong ST. 2011. Freshwater crab origins—laying Gondwana to rest. Zool. Anz. 250, 449–456 (doi:10.1016/j.jcz.2011.07.001) [Google Scholar]
  • 33.Kitaura J, Wada K, Nishida M. 1998. Molecular phylogeny and evolution of unique mud-using territorial behavior in ocypodid crabs (Crustacea: Brachyura: Ocypodidae). Mol. Biol. Evol. 15, 626–637 (doi:10.1093/oxfordjournals.molbev.a025966) [DOI] [PubMed] [Google Scholar]
  • 34.Kitaura J, Wada K, Nishida M. 2002. Molecular phylogeny of grapsoid and ocypodoid crabs with special reference to genera Metaplax and Macrophthalmus. J. Crust. Biol. 22, 682–693 [Google Scholar]
  • 35.Maddison WP, Maddison DR. 2006. Mesquite: a modular system for evolutionary analyses, version 1.1 See http://www.mesquiteproject.org
  • 36.Ng PKL, Guinot D, Davie PJF. 2008. Systema Brachyurorum: Part 1. An annotated checklist of extant brachyuran crabs of the world. Raffles. B. Zool. 17, 1–286 [Google Scholar]
  • 37.Cumberlidge N. 1999. The freshwater crabs of West Africa, Family Potamonautidae. Faune et Flore tropicales 35. Paris, France: IRD [Google Scholar]
  • 38.Cumberlidge N, Reed SK, Boyko CB. 2004. Distribution patterns of the Malagasy freshwater crabs (Crustacea: Decapoda: Brachyura). J. Nat. Hist. 38, 1133–1157 (doi:10.1080/0022293031000068419) [Google Scholar]
  • 39.Ng PKL. 1988. The freshwater crabs of peninsular Malaysia and Singapore. Singapore: Shinglee Press [Google Scholar]
  • 40.Pati SK, Roy MKD, Sharma RM. 2012. Checklist of Indian fauna—freshwater crabs See zsi.gov.in/checklist/freshwater_crabs.pdf
  • 41.Rathbun MJ. 1898. A contribution to a knowledge of the fresh-water crabs of America: the Pseudothelphusinae. Proc. US Nat. Mus. 21, 507–537 (doi:10.5479/si.00963801.21-1158.507) [Google Scholar]
  • 42.Rathbun MJ. 1900. The decapod Crustaceans of West Africa. Proc. US Nat. Mus. 22, 221–316 (doi:10.5479/si.00963801.22-1199.271) [Google Scholar]
  • 43.Rodriguez G. 1992. The freshwater crabs of America. Family Trichodactylidae. Paris, France: ORSTOM [Google Scholar]
  • 44.Bahir MM, Ng PKL, Crandall K, Pethiyagoda R. 2005. A conservation assessment of the freshwater crabs of Sri Lanka. Raffles B Zool. 12, 121–126 [Google Scholar]
  • 45.WoRMS Editorial Board 2014. World register of marine species See http://www.marinespecies.org at VLIZ. (consulted on 23 January 2014)
  • 46.OBIS 2014. Data from the Ocean Biogeographic Information System. Intergovernmental Oceanographic Commission of UNESCO Web See http://www.iobis.org (consulted on 23 January 2014)
  • 47.Aguado E, Burt JE. 2013. Understanding weather and climate, 6th edn, p. 576 Princeton, NJ: Prentice Hall [Google Scholar]
  • 48.Spalding MD, et al. 2007. Marine ecoregions of the world: a bioregionalization of coastal and shelf areas. Bioscience 57, 573–583 (doi:10.1641/B570707) [Google Scholar]
  • 49.Dinerstein E. 1995. A conservation assessment of the terrestrial ecoregions of Latin America and the Caribbean. Washington, DC: The World Bank [Google Scholar]
  • 50.Olson DM, Dinerstein E. 1998. The Global 200: a representation approach to conserving the Earth's most biologically valuable ecoregions. Conserv. Biol. 12, 502–515 (doi:10.1046/j.1523-1739.1998.012003502.x) [Google Scholar]
  • 51.New M, Lister D, Hulme M, Makin I. 2002. A high-resolution data set of surface climate over global land areas. Clim. Res. 21, 1–25 (doi:10.3354/cr021001) [Google Scholar]
  • 52.Reynolds RW, Rayner NA, Smith TM, Stokes DC, Wang W. 2002. An improved in situ and satellite SST analysis for climate. J. Climate 15, 1609–1625 (doi:10.1175/1520-0442(2002)015<1609:AIISAS>2.0.CO;2) [Google Scholar]
  • 53.Legendre P, Legendre L. 1998. Numerical ecology, 2nd English edn, p. 853 Amsterdam, The Netherlands: Elsevier [Google Scholar]
  • 54.Anderson MJ. 2001. A new method for non-parametric multivariate analysis of variance. Austral. Ecol. 26, 32–46 [Google Scholar]
  • 55.Anderson MJ, Gorley RN, Clarke KR. 2008. PERMANOVA+ for PRIMER: guide to software and statistical methods. Plymouth, UK: PRIMER-E Ltd. [Google Scholar]

Articles from Proceedings of the Royal Society B: Biological Sciences are provided here courtesy of The Royal Society

RESOURCES