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Convergent evolution of increased urine concentrating ability in desert mammals

One of the most celebrated textbook examples of physiological adaptations to desert environments is the unique ability that desert mammals have to produce hyperosmotic urine. Commonly perceived as an adaptation mainly observed in small rodents, the extent to which urine concentrating ability has independently evolved in distinct lineages, including medium-sized and large desert mammals, has not previously been assessed using modern phylogenetic approaches. Here, we explicitly test the general hypothesis that desert-dwelling mammals have evolved increased ability to concentrate urine compared to non-desert species, controlling for body mass and other covariates. Phylogenetic generalized least-squares models show that the mean aridity index of a species distribution range largely predicts its urine concentrating ability, even when accounting for body mass differences and phylogenetic correlations. In contrast, we find much weaker correlations between mass-adjusted basal metabolic rate and environmental variables.


Background 38 39
Deserts are defined as regions with aridity index, a ratio of annual precipitation and 40 potential evapo-transpiration, below 0.20 [1]. Desert species independently evolved striking 41 adaptations to cope with the water scarcity and extreme climatic and physical conditions that 42 characterize such habitats [2][3][4][5][6][7]. For the last fifty years, this has captured the attention of many 43 eco-physiologists who turned to desert biology to study the physiological mechanisms that help 44 maintaining body temperature and retain water. Remarkably, most of the classic works on 45 mammalian desert physiology, pioneered by Schmidt-Nielsen et al [8][9][10][11], have survived the test 46 of time, as researchers have applied new perspectives and tools to test specific hypothesis 47 regarding the evolution of adaptive traits [12,13]. 48 Among the classical findings of physiological adaptations that minimize water loss, is the 49 ability of some desert mammals to produce highly concentrated urine [14]. This phenotype is 50 mostly associated with desert 'evaders', such as the Australian Hopping mouse, which has the 51 record for highest hyperosmotic urine (above 9000 mOsm/kg) [3,15]. Evaders, part of a 52 classification system proposed by Willmer et al. (2000), are small body-sized animals that are able 53 to evade extreme conditions through behavior [5]. By contrast, large-sized mammals unable to 54 shelter from extreme climates that are forced to withstand heat, are called 'endurers', and 55 medium-sized mammals unable to evade nor withstand extremes as efficiently as evaders and 56 endurers are called 'evaporators' [5]. Because mammalian urine concentrating ability (mOsm/Kg) 57 is negatively correlated with body mass [16], desert evaders stand out in this capacity [15]. 58 However, when correcting for differences in body mass, the capacity to highly concentrate urine 59 seems to have independently evolved in deserts. For example, evaporators such as the fennec fox 60 can concentrate urine up to 4022 mOsm/Kg, which is much more concentrated than in similar-61 sized non-desert counterparts and, when mass-adjusted, nearly as impressive as the 5500 62 mOsm/Kg of a lesser Egyptian gerbil [5,17,18].

63
Classical comparative physiology studies have provided evidence for convergent adaptive 64 evolution of ecologically relevant phenotypes in multiple desert mammals. A textbook example 65 includes basal metabolic rate (BMR), which is positively correlated with zoogeographical zones, 66 even when accounting for phylogeny and body mass [19,20]. Desert mammals evolved lower 67 production of metabolic heat (low BMR) to maintain body temperatures and avoid water loss by 68 evaporative cooling [19][20][21][22][23]. Surprisingly, and to the best of our knowledge, no study to date has 69 attempted to identify continuous climatic or environmental variables that help explain variation in 70 urine concentrating ability, one of the most celebrated examples of mammalian desert adaptation. 71 Moreover, and in contrast to BMR, previous studies concerning urine concentrating ability have 72 ignored phylogenetic correlations when analyzing associations between this trait and the 73 environment [16,24]. 74 Here, we explicitly test the hypothesis that the aridity index of a species range predicts 75 maximum urine concentrating ability, even when accounting for body mass and phylogenetic 76 relationship, and provide statistical evidence that the ability to avoid water loss by producing 77 hyperosmotic urine has evolved independently in multiple phylogenetic lineages of desert 78 mammals. Additionally, we use similar models to re-analyze mass adjusted BMR for species in 79 which urine osmolality was obtained, and test aridity index and temperature variables as 80 predictors for metabolism in deserts. Phenotypes were obtained by extensive literature search and by taking advantage of large 85 datasets from previously published revision studies [16,19,24,25]. A total of 108 mammalian 86 species' maximum urine concentrating abilities came from two previous studies by Beuchat 87 [16,24], which recorded the specific condition (factor defined as C) under which urine osmolality 88 (hereafter mOsm) was measured, including: dehydrated (D), given salt (S) or protein (P) loading, 89 combinations of these conditions (DP, DS, SP, DSP), or treatment not specified (X), as well as the 90 method used to measure urine mOsm (see 15,23 for more information). We also recorded the 91 highest urine osmolality values for 13 additional species from other studies [18,26-30], and also 92 noted the experimental conditions (C) the study-cases were subjected to, the method (M) used to 93 measure urine mOsm, and the study-species diet (Di), following the same notation as Beuchat (see 94 15,23 for more information). For species with more than one record of urine mOsm, due to 95 different treatments/studies, we opted to keep values taken from captive study-subjects under 96 controlled/experimentally induced dehydration, as opposed to measurements of unknown 97 condition taken from the field. Ultimately, only one individual was used per species.

98
As mOsm is a plastic trait, the experimental conditions under which this parameter is 99 measured can greatly affect estimates. In particular, the degree of hydration of the study animals 100 naturally affects urine osmolality. As such, we curated two datasets: 1) a larger dataset including 101 all 121 reported measurements of mOsm from Beuchat [16,24] and our search [18,26-30]; 2) a 102 subset of the larger dataset only containing 87 observations in which the study-subject, in 103 captivity, was dehydrated (D) and not given food or any other treatment. However, we note that 104 there is still some residual variation in the number of days under water deprivation for an animal 105 to be considered fully dehydrated and variation in the method used for measuring mOsm. Also, 106 for the 121 species dataset, 24 records came from studies that did not report the procedures used 107 to measure mOsm. A full detail of the conditions and studies can be found in electronic 108 Supplementary Material TableS1. Downstream analyses were performed on both data sets. 109 The of variance-covariance matrix in a Brownian evolution model with a multiplicative factor, λ, such 131 that when λ = 0 a star phylogeny is obtained representing no phylogenetic signal, while when λ = 132 1 a standard Brownian on the reference tree is obtained. As residuals of maximum urine osmolality 133 are not normally distributed ( Figure S1), we performed PGLS analyses on log10(maximum mOsm) 134 using log10(body mass) as covariate. Mean annual aridity index (hereafter mean AI) and 135 uncorrelated mean temperature variables were used in PGLS models as potential predictors of 136 urine concentrating ability. Condition (C), method (M) and diet (Di) were also included as 137 covariates. For the dataset including only dehydrated individuals, the same PGLS models were 138 tested with AI converted to a categorical variable using the following binning defined in [33]: 139 humid for AI > 0.65, dry sub-humid for 0.5< AI < 0.65, semi-arid for 0.2 < AI < 0.5, and arid for AI 140 < 0.2. To test for phylogenetic signal for specific traits, we calculated Pagel's λ on urine 141 concentrating ability, body mass and AI using the function 'phylosig' as implemented in 'phytools'. 142

Results 143 144
We analyzed a dataset with information on maximum urine concentrating abilities, mean 145 annual aridity index and WorldClim temperatures for a total of 121 species of mammals, with 146 available DNA sequence information, spanning 6 g to 479 kg of body mass. Using the classification 147 of [33] the dataset contained 32, 35, 15, and 39 species with distribution ranges classified as arid, 148 semi-arid, dry sub-humid, and humid, respectively. Ancestral state reconstruction across our 149 mammalian dataset's phylogeny shows that the ability to produce hyperosmotic urine has evolved 150 multiple times in species with distribution ranges characterized by low mean AI (Figure 1). 151 When testing for phylogenetic signal using Pagel's λ we found highly significant 152 phylogenetic signal for most variables, validating the relevance of adjusting the linear models for 153 phylogenetic covariance (Table 1). The mean AI correlates with all temperature variables, except 154 mean annual temperature range ( Figure S3). To avoid collinearity, all WorldClim temperatures but 155 the latter were excluded from PGLS analysis with mean AI (Table S2). 156 All PGLS models tested in this study point to mean annual aridity index as a highly 157 significant and important predictor of mammalian maximum urine concentration ability (Table 2,  158 TableS2). The lower the mean AI, the more efficient mammals are able to concentrate urine 159 (Figure 2, FigureS2), even when accounting for the differences in body mass, diet, experimental 160 condition, methodology and phylogeny (Table 2). Overall, species inhabiting arid and semi-arid 161 environments can maximize urine concentration well above the levels of dry sub-humid and humid 162 counterparts (Figure 2). In congruence with past studies, our results also show that log-163 transformed body mass is also negatively correlated with log-transformed maximum urine 164 osmolality [16], though not to the same extent as mean aridity index (Table 2, Figure S2). Diet is 165 also a significant predictor of maximum urine osmolality in fructivorous mammals, which 166 significantly concentrate more urine than strictly carnivore species (Table 2). Overall, no strong 167 correlation was found between urine concentrating ability and temperature variables ( Figure S4,  168 TableS2).

169
When the experimental condition was used as co-variate in the PGLS analysis, its role in 170 predicting maximum urine osmolality was not significant ( Table 2, Table S2). Nonetheless, we also 171 tested similar PGLS models on a subset of the data that only included individuals fulfilling the 172 dehydrated criterion (see Methods section), resulting in a smaller dataset including 87 species (18 173 arid, 26 semi-arid, 13 dry sub-humid, and 30 humid). The results from this analysis are similar to 174 those of the larger dataset conditions (Table 2). When using AI as a discrete variable we found 175 that urine concentrating ability is significantly higher in species from arid environments than in 176 species from semi-arid, dry sub-humid and humid regions (Table S3). In all analyses, the aridity 177 index of the species distribution is a stronger predictor of maximum urine osmolality than body 178 mass and diet. Notably, experimental condition and methodology are non-significant. 179 We also obtained mass-adjusted basal metabolic rates (BMR) for 84 of the 121 species 180 from our dataset. BMR is weakly correlated with bioclimatic variables (Figure S5), and differences 181 in this parameter are better explained by log10 body mass (TableS4). 182 183 4. Discussion 184 185 The extent to which different mammalian species from desert environments have 186 significantly higher urine concentrating abilities, a phenotype to better retain water in extreme 187 conditions of water-deprivation and extreme temperature ranges, has not previously been subject 188 to rigorous statistical analyses that account for phylogeny. Here we show that increased ability to 189 concentrate urine, measured as maximum urine osmolality, has convergently evolved in many 190 desert mammals (Figure 1).

191
PGLS analysis on a multi-species dataset revealed that desert-dwelling species effectively 192 concentrate more urine than non-desert species, even when accounting for both body mass and 193 ancestry, and furthermore, that differences in mammalian maximum urine concentrating ability 194 can be predicted, to some degree, by the aridity of species' range. A contentious debate in the fields of ecological and evolutionary physiology is whether 209 phenotypic differences between species and populations inhabiting contrasting environments are 210 due to genetic (evolutionary) adaptations or due to plastic responses, and difference in the ability 211 to concentrate urine is no exception. Although all mammals, when water-deprived, are able to 212 increase urine concentration in relation to their optimal hydrated states [14,39], our results for a 213 dataset in which species were all captive and water-deprived, provide strong evidence that 214 increased maximal urine concentration is an adaptation to high aridity and has evolved multiple 215 times in different phylogenetic lineages. The hypothesis that higher maximum urine concentrating 216 ability in mammals is an adaptive trait is also supported by an observed increase in renal medullary 217 thickness in desert mammals [3,16].

218
Our PGLS analyses also suggest that, among the many physical and climatic challenges 219 faced by desert species, aridity, more than temperature, has been one of the main selective 220 pressures increasing maximum urine concentrating ability, and driving its repeated evolution in 221 different desert mammalian lineages.