Control of blood volume following hypovolemic challenge in vertebrates: Transcapillary versus lymphatic mechanisms.

Anurans have an exceptional capacity for maintaining vascular volume compared with other groups of vertebrates. They can mobilize interstitial fluids via lymphatic return at rates that are ten-fold higher than mammals. This extraordinary capacity is the result of coordination of specialized skeletal muscles and pulmonary ventilation that vary volume and pressure of subcutaneous lymph sacs, thus moving lymph to dorsally located lymph hearts that return lymph to the vascular space. Variation in the capacity to mobilize lymph within anurans varies with the degree of terrestriality, development of skeletal muscles, lung volume and lung compliance, and lymph heart pressure development. This ability enable anurans, which have the highest rates of evaporative water loss among terrestrial vertebrates, to withstand levels of dehydration far exceeding that of other vertebrates, and to successfully occupy virtually all terrestrial environments during their evolution. Maintenance of vascular fluid volume for all vertebrates can be achieved primarily by moving fluid from the interstitial space to the vascular space by transcapillary uptake and mobilization of interstitial (lymphatic) fluid. Transcapillary fluid uptake at the capillary level has been analyzed historically by Krogh and others from a Starling perspective and involves a balance of hydrostatic and oncotic forces. A complete evaluation of blood volume homeostasis also incorporates pressures and compliances of the vascular and interstitial spaces, but has been applied to only a few species. In this review we outline the current understanding of how anurans and other vertebrates maintain blood volume during hypovolemic challenges such as dehydration and hemorrhage which is crucial for maintaining cardiac output.

Anuran amphibians as comparative models for understanding extreme dehydration tolerance: a unique negative feedback lymphatic mechanism for blood volume regulation.

Anurans are the most terrestrial order of amphibians. Couple the high driving forces for evaporative loss in terrestrial environments and their low resistance to evaporation, dehydration is an inevitable stress on their water balance. Anurans have the greatest tolerances for dehydration of any vertebrate group. Some species can tolerate evaporative losses up to 45% of their standard body mass. Anurans have remarkable capacities to regulate blood volume with hemorrhage and dehydration compared with mammals. Stabilization of blood volume is central to extending dehydration tolerance, since it avoids both the hypovolemic and hyperviscosity stresses on cardiac output and its consequential effects on aerobic capacity. Anurans, in contrast to mammals, seem incapable of generating a sufficient pressure difference, either oncotically or via interstitial compliance, to move fluid from the interstitium into the capillaries. Couple this inability to generate a sufficient pressure difference for transvascular uptake to a circulatory system with high filtration coefficients and a high rate of plasma turnover is the consequence. The novel lymphatic system of anurans is critical to a remarkable capacity for blood volume regulation. This review summarizes what is known about the anatomical and physiological specializations that are involved in explaining differential blood volume regulation and dehydration tolerance involving a true centrally mediated negative feedback of lymphatic function involving baroreceptors as sensors and lymph hearts, arginine vasotocin, pulmonary ventilation and specialized skeletal muscles as effectors.

A metabolic hypothesis for the evolution of temperature effects on the arterial PCO2 and pH of vertebrate ectotherms.

Body temperature increases in ectothermic vertebrates characteristically lead to both increases in arterial PCO2  (PaCO2 ) and declines in resting arterial pH (pHa) of about 0.017 pH units per 1°C increase in temperature. This 'alphastat' pH pattern has previously been interpreted as being evolutionarily driven by the maintenance of a constant protonation state on the imidazole moiety of histidine protein residues, hence stabilizing protein structure-function. Analysis of the existing data for interclass responses of ectothermic vertebrates shows different degrees of PaCO2  increases and pH declines with temperature between the classes, with reptiles>amphibians>fish. The PaCO2  at the temperature where maximal aerobic metabolism (V̇O2,max) is achieved is significantly and positively correlated with temperature for all vertebrate classes. For ectotherms, the PaCO2  where V̇O2,max is greatest is also correlated with V̇O2,max, indicating there is an increased driving force for CO2 efflux that is lowest in fish, intermediate in amphibians and highest in reptiles. The pattern of increased PaCO2  and the resultant reduction of pHa in response to increased body temperature would serve to increase CO2 efflux, O2 delivery and blood buffering capacity and maintain ventilatory scope. This represents a new hypothesis for the selective advantage of arterial pH regulation from a systems physiology perspective in addition to the advantages of maintenance of protein structure-function.

A meta-analysis of in vivo vertebrate cardiac performance: implications for cardiovascular support in the evolution of endothermy.

Endothermy in birds and mammals is associated with high body temperatures, and high rates of metabolism that are aerobically supported by elevated rates of cardiovascular O2 transport. The purpose of this meta-analysis was to examine cardiovascular data from ectothermic and endothermic vertebrates, at rest and during exercise, with the goal of identifying key variables that may have contributed to the role of the cardiovascular system in supporting high rates of O2 transport associated with endothermy. Vascular conductance, cardiac power and stroke work were summarized and calculated from a variety of studies at rest and during exercise for five classes of vertebrates where data were available. Conductance and cardiac power were linearly related to cardiac output from rest to exercise and also interspecifically. Exercise cardiac power and stroke work were greater in the endothermic species, owing to increased flow resulting from increased heart rate and increased pressure. Increased relative ventricle mass (RVM) was related to increased stroke volume in both groups. However, the increased RVM of endotherms was related to the increased pressure, as stroke work per gram of ventricle during exercise was equivalent between the groups. Cardiac power was linearly related to aerobic metabolic power, with 158 mW aerobic power output achieved per mW of cardiac power input. This analysis indicates that the greatly increased heart rate and cardiac stroke work leading to increased blood flow rate and blood pressure was necessary to support the metabolic requirements of endothermy.

Net cardiac shunts in anuran amphibians: physiology or physics?

Amphibians have a single ventricle and common conus arteriosus that produces an equal pressure to the parallel pulmocutaneous and systemic vascular circuits. The distribution of blood flows between the pulmocutaneous (Qpul) and systemic (Qsys) circuits (net cardiac shunt) varies with a number of environmental conditions and behaviours; although autonomic regulation of pulmonary vascular resistance conductance has been emphasized, little attention has been paid to the possible contribution of the passive physical characteristics of the two circuits to pressure changes associated with variation in cardiac output. In this study, we re-analysed three recent studies that recorded net cardiac shunts in the cane toad (Rhinella marina) under a variety of conditions and treatments. In all three studies, Qpul and Qsys were linearly related to cardiac output (Qtot), but the slope was threefold higher for Qpul compared with Qsys as predicted by relative conductance increases associated with increases in pressure from perfused preparations where autonomic regulation and humoral control were eliminated. Our analysis indicates that the net cardiac shunt in the cane toad is predicted primarily by the physical, rather than physiological, characteristics of the parallel pulmonary and systemic vascular circuits.

Physiological vagility and its relationship to dispersal and neutral genetic heterogeneity in vertebrates.

Vagility is the inherent power of movement by individuals. Vagility and the available duration of movement determine the dispersal distance individuals can move to interbreed, which affects the fine-scale genetic structure of vertebrate populations. Vagility and variation in population genetic structure are normally explained by geographic variation and not by the inherent power of movement by individuals. We present a new, quantitative definition for physiological vagility that incorporates aerobic capacity, body size, body temperature and the metabolic cost of transport, variables that are independent of the physical environment. Physiological vagility is the speed at which an animal can move sustainably based on these parameters. This meta-analysis tests whether this definition of physiological vagility correlates with empirical data for maximal dispersal distances and measured microsatellite genetic differentiation with distance {[F(ST)/[1-F(ST))]/ln distance} for amphibians, reptiles, birds and mammals utilizing three locomotor modes (running, flying, swimming). Maximal dispersal distance and physiological vagility increased with body mass for amphibians, reptiles and mammals utilizing terrestrial movement. The relative slopes of these relationships indicate that larger individuals require longer movement durations to achieve maximal dispersal distances. Both physiological vagility and maximal dispersal distance were independent of body mass for flying vertebrates. Genetic differentiation with distance was greatest for terrestrial locomotion, with amphibians showing the greatest mean and variance in differentiation. Flying birds, flying mammals and swimming marine mammals showed the least differentiation. Mean physiological vagility of different groups (class and locomotor mode) accounted for 98% of the mean variation in genetic differentiation with distance in each group. Genetic differentiation with distance was not related to body mass. The physiological capacity for movement (physiological vagility) quantitatively predicts genetic isolation by distance in the vertebrates examined.

Pulmonary compliance and lung volume varies with ecomorphology in anuran amphibians: implications for ventilatory-assisted lymph flux.

Vertical movement of lymph from ventral regions to the dorsally located lymph hearts in anurans is accomplished by specialized skeletal muscles working in concert with lung ventilation. We hypothesize that more terrestrial species with greater lymph mobilization capacities and higher lymph flux rates will have larger lung volumes and higher pulmonary compliance than more semi-aquatic or aquatic species. We measured in situ mean and maximal compliance (Δvolume/Δpressure), distensibility (%Δvolume/Δpressure) and lung volume over a range of physiological pressures (1.0 to 4.0 cmH(2)O) for nine species of anurans representing three families (Bufonide, Ranidae and Pipidae) that span a range of body masses and habitats from terrestrial to aquatic. We further examined the relationship between these pulmonary variables and lymph flux for a semi-terrestrial bufonid (Rhinella marina), a semi-aquatic ranid (Lithobates catesbeianus) and an aquatic pipid (Xenopus laevis). Allometric scaling of pulmonary compliance and lung volume with body mass showed significant differences at the family level, with scaling exponents ranging from ∼0.75 in Bufonidae to ∼1.3 in Pipidae. Consistent with our hypothesis, the terrestrial Bufonidae species had significantly greater pulmonary compliance and greater lung volumes compared with semi-aquatic Ranidae and aquatic Pipidae species. Pulmonary distensibility ranged from ∼20 to 35% cmH(2)O(-1) for the three families but did not correlate with ecomorphology. For the three species for which lymph flux data are available, R. marina had a significantly higher (P<0.001) maximal compliance (84.9±2.7 ml cmH(2)O(-1) kg(-1)) and lung volume (242.1±5.5 ml kg(-1)) compared with L. catesbeianus (54.5±0.12 ml cmH(2)O(-1) kg(-1) and 139.3±0.5 ml kg(-1)) and X. laevis (30.8±0.7 ml cmH(2)O(-1) kg(-1) and 61.3±2.5 ml kg(-1)). Lymph flux rates were also highest for R. marina, lowest for X. laevis and intermediate in L. catesbeianus. Thus, there is a strong correlation between pulmonary compliance, lung volume and lymph flux rates, which suggests that lymph mobilization capacity may explain some of the variation in pulmonary compliance and lung volume in anurans.

Posterior lymph heart function in two species of anurans: analysis based on both in vivo pressure-volume relationships by conductance manometry and ultrasound.

Rhinella marina and Lithobates catesbeianus have known differences in the capacity to mobilize lymph to stabilize blood volume following dehydration and hemorrhage. The purpose of these experiments was to assess whether there are interspecific differences in basic lymph heart functions. The end diastolic volumes of posterior lymph hearts averaged 10.8 µl kg⁻¹ in R. marina and 7.9-10.8 µl kg⁻¹ in L. catesbeianus by conductance manometry, and 9-32 µl kg⁻¹ in R. marina by ultrasound techniques, which correlated with body mass. Stroke volumes were approximately 20% of end diastolic volumes in both species. Peak systolic pressures and stroke work were correlated with the index of contractility (dP/dt(max)) in both species. Stroke volume was correlated to stroke work but not peak systolic pressure, end diastolic volume or end diastolic pressure indicating the preload variables do not seem to determine stroke volume as would be predicted from Starling considerations of the blood heart. Renal portal elastance (end systolic pressure/stroke volume) an afterload index did not differ interspecifically, and was equivalent to values for systemic flow indices from mice of equivalent ventricular volume. These data, taken together with predictions derived from mammalian models on the effect of high resistance indicate afterload (renal portal pressure), may be important determinants of posterior lymph heart stroke volume. The shape of the pressure-volume loop is different from an idealized version previously reported, and is influenced by end diastolic volume. Our data indicate that increasing end diastolic pressure and volume can influence the loop shape but not the stroke volume. This indicates that lymph hearts do not behave in a Starling Law manner with increased preload volume.

Lymph flux rates from various lymph sacs in the cane toad Rhinella marina: an experimental evaluation of the roles of compliance, skeletal muscles and the lungs in the movement of lymph.

A new method for quantitatively determining lymph flux from various lymphatic sacs of an anuran, the cane toad, was developed. This method used the dye dilution principle of C(i)V(i)=C(f)V(f) following injection of Evans Blue into specific lymph sacs and measuring its appearance in the venous circulation. The apparent lymph volume was 57 ml kg(-1). The greatest rate of lymph return (0.5-0.8 ml kg(-1) min(-1)) and best linear fit of Evans Blue appearance in the circulation with time followed injections into the subvertebral lymph sac, which has direct connections to both the anterior and posterior pairs of lymphatic hearts. Rate of lymph flux from the pair of posterior lymph hearts was three times greater than the anterior pair. Rates of lymph flux were only influenced by injection volume in the crural lymph sacs, implicating lymph sac compliance as the source of the pressure for lymph movement from these sacs. Femoral lymph sac fluxes were decreased by 60% following ablation of the tendons of the sphincter ani cloacalis, abdominal crenators and piriformis. This supports a role for these muscles in generating the pressure for vertical lymph movement. Femoral lymph sac fluxes were also decreased by 70% by the insertion of a coil in the subvertebral lymph sac, preventing normal compression and expansion of this sac by the lungs. This supports a role for lung ventilation in generating the pressure for vertical movement of lymph. Contrary to previous hypotheses, fluxes from the brachial sac were not influenced by insertion of the coil into the subvertebral sac. A haemorrhage equivalent to 50% of the blood volume did not change lymph flux rates from the femoral lymph sacs. These data provide the first experimental evidence that actual lymph fluxes in the cane toad Rhinella marina depend on lymph sac compliance, contraction of specific skeletal muscles and lung ventilation to move lymph laterally and vertically to the dorsally located lymphatic hearts.

Commentary on: "Vascular distensibilities have minor effects on intracardiac shunt patterns in reptiles" by Filogonio et al. (2017).

The recent study by Filogonio et al. (2017) suggested that net cardiac shunt patterns in two species of reptiles (Trachemys scripta and Crotalus durissus) were not significantly influenced by the vascular distensibilities of the systemic and pulmonary vasculatures. This is in contrast to a previously published study (Hillman et al., 2014) in the toad (Rhinella marina) in which net cardiac shunts were predicted primarily by the physical properties of vascular distensibility rather than physiological control of resistance of the systemic and pulmonary vasculature. We analyze the data and conclusions reached by Filogonio et al. (2017) regarding the role of vascular distensibilities in determining net cardiac shunt patterns in reptiles in comparison with toads. In our view, the conclusions reached by Filogonio et al. (2017) are not supported by the data primarily because vascular distensibilities were not measured in the reptiles analyzed in their study.

Osmotically absorbed water preferentially enters the cutaneous capillaries of the pelvic patch in the toad Bufo marinus.

Cutaneously absorbed water in anurans has two potential routes of movement from the skin interstitium into the body fluids, via the cutaneous capillaries and/or the lymphatic system. We investigated the lymphatic route at the pelvic patch skin under the influence of isoproterenol and arginine vasotocin in Bufo marinus by continuously aspirating lymph from the lymph sacs draining the pelvic patch while the animals absorbed water. Changes in body mass, lymph mass, and lymph osmolality were measured. If absorbed water entered the lymph space directly, we expected, relative to controls, (1) no difference in change in body mass, (2) lymph mass to be greater, and (3) lymph osmolality to be lower. None of these predictions were confirmed. We also tested the possibility that absorbed water was stored in the skin interstitium by measuring the surface density of pelvic skin immediately after it absorbed water. If water was stored, we expected the surface density of this skin to be greater than that of control skin. No difference in surface density was found. These results provide strong evidence that absorbed water does not directly enter the lymphatic system and is not stored in the skin. Consequently, osmotically absorbed water must enter via a transcapillary route.

Functional roles for the compartmentalization of the subcutaneous lymphatic sacs in anuran amphibians.

Compliance of the subcutaneous lymph sacs of the hindlimbs increases from distal to proximal, as does limb segment mass (and presumably rate of lymph formation), for the semiaquatic bullfrog Rana catesbeiana and the cane toad Bufo marinus but not the aquatic clawed toad Xenopus laevis. Subcutaneous lymph-sac compliances vary interspecifically. The distal-to-proximal increase in lymph-sac compliance and estimates of lymph formation rate in the various hindlimb segments indicate that partitioning of hindlimb subcutaneous lymphatic sacs establishes a differential decrease in the intra-lymph-sac pressure for R. catesbeiana and B. marinus. These pressure differentials constitute a "compliance pump" that drives distal-to-proximal intersac lymph flow. The compliance pump alone explains lymphatic return for the aquatic frog X. laevis but does not explain how lymph would reach the dorsally located lymph hearts for terrestrial anurans, so we hypothesize that skeletal muscle pumps return lymph from the femoral and pubic lymph sacs to the lymph heart. This is a fundamentally different role of the subcutaneous lymph-sac system than has been previously proposed. We suggest that the more proximal subcutaneous lymph sacs are important for fluid storage because they have a relatively high compliance.

Metabolism at the Max: How Vertebrate Organisms Respond to Physical Activity.

Activity metabolism is supported by phosphorylated reserves (adenosine triphosphate, creatine phosphate), glycolytic, and aerobic metabolism. Because there is no apparent variation between vertebrate groups in phosphorylated reserves or glycolytic potential of skeletal muscle, variation in maximal metabolic rate between major vertebrate groups represents selection operating on aerobic mechanisms. Maximal rates of oxygen consumption in vertebrates are supported by increased conductive and diffusive fluxes of oxygen from the environment to the mitochondria. Maximal CO2 efflux from the mitochondria to the environment must be matched to oxygen flux, or imbalances in pH will occur. Among vertebrates, there are a variety of modes of locomotion and vastly different rates of metabolism supported by a variety of cardiorespiratory architectures. However, interclass comparisons strongly implicate systemic oxygen transport as the rate-limiting step to maximal oxygen consumption for all vertebrate groups. The key evolutionary step that accounts for the approximately 10-fold increase in maximal oxygen flux in endotherms versus ectotherms appears to be maximal heart rate. Other variables such as ventilation, pulmonary/gill, and tissue diffusing capacity, have excess capacity and thus are not limiting to maximal oxygen consumption. During maximal activity, the ratio of ventilation to respiratory system blood flow is remarkably similar among vertebrates, and CO2 extraction efficiency increases while oxygen extraction efficiency decreases, suggesting that the respiratory system provides the largest resistance to maximal CO2 flux. Despite the large variation in modes of activity and rates of metabolism, maximal rates of oxygen and CO2 flux appear to be limited by the cardiovascular and respiratory systems, respectively.

General function and endocrine control of the posterior lymph hearts in Bufo marinus and Rana catesbeiana.

The effects of hypervolemia and graded increases in arginine vasotocin (AVT), angiotensin II (ANGII), and atrial natriuretic peptide (ANP) on lymph heart pressure (P(lh)) and rate (f(lh)) were examined in Bufo marinus and Rana catesbeiana. The P(lh) and f(lh) for normally hydrated B. marinus at rest were 1.45+/-0.01 kPa and 52.8+/-0.38 beats min(-1). The P(lh) and f(lh) were significantly lower in R. catesbeiana, 1.05+/-0.01 kPa and 48.4+/-0.35 beats min(-1). Hypervolemia, induced by intravenous infusion of isotonic saline, stopped the lymph hearts at volumes of 0.48%+/-0.06% and 0.32%+/-0.04% body mass in B. marinus and R. catesbeiana, respectively, equivalent to an 8% increase of their respective plasma volumes. ANP had no effect on P(lh) or f(lh) at any of the dosages tested. ANGII decreased f(lh) in both species, approximating the physiological range of concentrations. AVT, at physiological concentrations, increased P(lh) 48% in B. marinus and 38% in R. catesbeiana without changing f(lh) in either species. At higher than physiological dosages, P(lh) and f(lh) in both species declined. The results suggest that AVT, normally released during hemorrhage and dehydration, would increase lymph heart output and help compensate for the hypovolemia. This is a contrary result to previous work using supraphysiologic doses of AVT.

Lymph pools in the basement, sump pumps in the attic: the anuran dilemma for lymph movement.

Amphibians are a vertebrate group transitional between aquatic and terrestrial environments. Consequently, both increases and decreases in blood volume are a natural biological stress associated with aquatic and terrestrial environments. In comparison with other vertebrate classes, anuran amphibians have the most rapid compensation and greatest capacity to compensate for changes in blood volume and survive dehydration. Unlike in mammals, a Starling transcapillary uptake mechanism does not account for this fluid mobilization because lymph flow is a substantial and important additional factor. The role of the lymphatic system in flux of fluids back into the circulation varies interspecifically in anurans and is an order of magnitude greater in anurans than in mammals. Current models of lymph movement in anurans are centered on the role of lymph hearts, but we suggest that these models are untenable. We present a new hypothesis for lymph movement involving (1) pressure differences created by compartmentalization of the hind limb lymph spaces into sacs of serially graded compliance to move lymph horizontally and (2) both negative and positive pressure differences created by contraction of skeletal muscles to move lymph vertically. The primary function of some of these skeletal muscles may be solely for lymph movement, but some may also be involved with other functions such as pulmonary ventilation.

Cardiovascular oxygen transport limitations to thermal niche expansion and the role of environmental Po2 in Antarctic notothenioid fishes.

The notothenioid fishes of the Southern Ocean possess some of the lowest upper thermal thresholds of any species and display a range of cardiovascular features that distinguish them from other fishes. Some species lack hemoglobin, and it has been posited that the inability to deliver sufficient oxygen at elevated temperature may in part determine upper thermal thresholds. Here, we provide an analysis of systemic O2 transport based on circulatory resistance, cardiac outputs, and cardiac power for three species of Antarctic fishes, including species that possess hemoglobin (Trematomus bernacchii, Pagothenia borchgrevinki) and a species lacking hemoglobin (Chaenocephalus aceratus) and that differ in their cardiovascular characteristics. This analysis supports the hypothesis that the mutation resulting in the lack of hemoglobin would be metabolically prohibitive at elevated temperatures. The analysis also suggests that such a mutation would be least detrimental to species with greater cardiac power outputs and lower total peripheral resistance. Decreased environmental Po2 has the greatest detrimental effect on the metabolic capacity in the species without hemoglobin. These data indicate that differences in cardiovascular characteristics of the notothenioid fishes place varying limits on thermal niche expansion in these species, but any significant increase in environmental temperature or decrease in environmental Po2 will prohibit maintenance of cardiovascular systemic O2 transport in all species. These data also suggest an evolutionary sequence of events such that a reduction in hematocrit, to reduce blood viscosity and resistance, was a first step in the invasion of low-temperature habitats and loss of hemoglobin was followed by increased cardiac power output to achieve sustainable metabolic rates.

Physiological vagility: correlations with dispersal and population genetic structure of amphibians.

Physiological vagility represents the capacity to move sustainably and is central to fully explaining the processes involved in creating fine-scale genetic structure of amphibian populations, because movement (vagility) and the duration of movement determine the dispersal distance individuals can move to interbreed. The tendency for amphibians to maintain genetic differentiation over relatively short distances (isolation by distance) has been attributed to their limited dispersal capacity (low vagility) compared with other vertebrates. Earlier studies analyzing genetic isolation and population differentiation with distance treat all amphibians as equally vagile and attempt to explain genetic differentiation only in terms of physical environmental characteristics. We introduce a new quantitative metric for vagility that incorporates aerobic capacity, body size, body temperature, and the cost of transport and is independent of the physical characteristics of the environment. We test our metric for vagility with data for dispersal distance and body mass in amphibians and correlate vagility with data for genetic differentiation (F'(ST)). Both dispersal distance and vagility increase with body size. Differentiation (F'(ST)) of neutral microsatellite markers with distance was inversely and significantly (R2=0.61) related to ln vagility. Genetic differentiation with distance was not significantly related to body mass alone. Generalized observations are validated with several specific amphibian studies. These results suggest that interspecific differences in physiological capacity for movement (vagility) can contribute to genetic differentiation and metapopulation structure in amphibians.

Pulmonary compliance and lung volume are related to terrestriality in anuran amphibians.

Dehydration tolerance of anuran amphibians is directly related to their ability to mobilize lymphatic reserves, with more terrestrial species having more effective lymph mobilization dependent on specialized skeletal muscles acting directly on the lymph sacs and via pulmonary ventilation. Consequently, we tested the hypothesis that pulmonary compliance, lung volume, and femoral lymphatic sac volume were related to terrestriality-and, hence, lymph mobilization-for 18 species of aquatic, semiaquatic, or terrestrial anuran amphibians. Lung compliance and volume were significantly related to body mass, but there was no significant phylogenetic pattern. There were significant habitat-related patterns for mass-corrected and phylogenetically corrected residuals for these pulmonary variables. Femoral lymph volume was significantly related to body mass, with no significant phylogenetic pattern, and there was only a weak correlation for habitat with mass-corrected and phylogenetically corrected residuals. These results suggest that pulmonary volume and compliance are strongly related to terrestriality in anuran amphibians and are under significant selection pressure to enhance lymph mobilization, but lymph sac volume does not appear to have a major role in adaptation to terrestriality.