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.

Sexual Dichromatism Drives Diversification within a Major Radiation of African Amphibians.

Theory predicts that sexually dimorphic traits under strong sexual selection, particularly those involved with intersexual signaling, can accelerate speciation and produce bursts of diversification. Sexual dichromatism (sexual dimorphism in color) is widely used as a proxy for sexual selection and is associated with rapid diversification in several animal groups, yet studies using phylogenetic comparative methods to explicitly test for an association between sexual dichromatism and diversification have produced conflicting results. Sexual dichromatism is rare in frogs, but it is both striking and prevalent in African reed frogs, a major component of the diverse frog radiation termed Afrobatrachia. In contrast to most other vertebrates, reed frogs display female-biased dichromatism in which females undergo color transformation, often resulting in more ornate coloration in females than in males. We produce a robust phylogeny of Afrobatrachia to investigate the evolutionary origins of sexual dichromatism in this radiation and examine whether the presence of dichromatism is associated with increased rates of net diversification. We find that sexual dichromatism evolved once within hyperoliids and was followed by numerous independent reversals to monochromatism. We detect significant diversification rate heterogeneity in Afrobatrachia and find that sexually dichromatic lineages have double the average net diversification rate of monochromatic lineages. By conducting trait simulations on our empirical phylogeny, we demonstrate that our inference of trait-dependent diversification is robust. Although sexual dichromatism in hyperoliid frogs is linked to their rapid diversification and supports macroevolutionary predictions of speciation by sexual selection, the function of dichromatism in reed frogs remains unclear. We propose that reed frogs are a compelling system for studying the roles of natural and sexual selection on the evolution of sexual dichromatism across micro- and macroevolutionary timescales.

Land flatworms (Platyhelminthes, Geoplanidae) of São Tomé: a first account on their diversity, with the description of five new species.

The present contribution provides the first faunistic and taxonomic account of six species of land flatworm from the island of São Tomé, including five new species of the genus Othelosoma Gray, 1869 and the introduced Bipalium kewense Moseley, 1878. One of the new species represents the first African land flatworm that has specks on its dorsal body surface, instead of stripes or a more or less uniform colouration. At least two of the new species were observed to prey on snails. The study details the fourth record of a sclerotic spermatophore in a species of land flatworm, and discusses the definition and homology of double female genital canals in African and Indian species of the genus Othelosoma.

Reed frog diversification in the Gulf of Guinea: overseas dispersal, the progression rule, and in situ speciation.

Oceanic islands accumulate endemic species when new colonists diverge from source populations or by in situ diversification of resident island endemics. The relative importance of dispersal versus in situ speciation in generating diversity on islands varies with a number of archipelago characteristics including island size, age, and remoteness. Here, we characterize interisland dispersal and in situ speciation in frogs endemic to the Gulf of Guinea islands. Using mitochondrial sequence and genome-wide single-nucleotide polymorphism data, we demonstrate that dispersal proceeded from the younger island (São Tomé) to the older island (Príncipe) indicating that for organisms that disperse overseas on rafts, dispersal between islands may be determined by ocean currents and not island age. We find that dispersal between the islands is not ongoing, resulting in genotypically distinct but phenotypically similar lineages on the two islands. Finally, we demonstrate that in situ diversification on São Tomé Island likely proceeded in allopatry due to the geographic separation of breeding sites, resulting in phenotypically distinct species. We find evidence of hybridization between the species where their ranges are sympatric and the hybrid zone coincides with a transition from agricultural land to primary forest, indicating that anthropogenic development may have facilitated secondary contact between previously allopatric species.

Population genetics of the São Tomé caecilian (Gymnophiona: Dermophiidae: Schistometopum thomense) reveals strong geographic structuring.

Islands provide exciting opportunities for exploring ecological and evolutionary mechanisms. The oceanic island of São Tomé in the Gulf of Guinea exhibits high diversity of fauna including the endemic caecilian amphibian, Schistometopum thomense. Variation in pigmentation, morphology and size of this taxon over its c. 45 km island range is extreme, motivating a number of taxonomic, ecological, and evolutionary hypotheses to explain the observed diversity. We conducted a population genetic study of S. thomense using partial sequences of two mitochondrial DNA genes (ND4 and 16S), together with morphological examination, to address competing hypotheses of taxonomic or clinal variation. Using Bayesian phylogenetic analysis and Spatial Analysis of Molecular Variance, we found evidence of four geographic clades, whose range and approximated age (c. 253 Kya-27 Kya) are consistent with the spread and age of recent volcanic flows. These clades explained 90% of variation in ND4 (φCT = 0.892), and diverged by 4.3% minimum pairwise distance at the deepest node. Most notably, using Mismatch Distributions and Mantel Tests, we identified a zone of population admixture that dissected the island. In the northern clade, we found evidence of recent population expansion (Fu's Fs = -13.08 and Tajima's D = -1.80) and limited dispersal (Mantel correlation coefficient = 0.36, p = 0.01). Color assignment to clades was not absolute. Paired with multinomial regression of chromatic data, our analyses suggested that the genetic groups and a latitudinal gradient together describe variation in color of S. thomense. We propose that volcanism and limited dispersal ability are the likely proximal causes of the observed genetic structure. This is the first population genetic study of any caecilian and demonstrates that these animals have deep genetic divisions over very small areas in accordance with previous speculations of low dispersal abilities.

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 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.

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.

Evolutionary implications of the distribution and variation of the skeletal muscles of the anuran lymphatic system.

Lymphatic return to the circulation in anurans is dependent upon the interaction of a number of skeletal muscles and lung deflation. We define character states and describe variation of these putative lymphatic skeletal muscles: the M. cutaneus pectoris (CP), M. cutaneus dorsi (CD), M. piriformis (P), M. sphincter ani cloacalis (SAC), and the complex of the M. gracilis minor/M. abdominal crenator (GM/AC). We include examination of over 400 specimens of 377 species belonging to 40 of the 42 currently recognized anuran families. Some muscles show limited variation (P) or are clearly linked to phylogeny (CP; CD) and thus have limited value in the determination of form and function. However, the GM/AC and SAC show a high degree of structural variation that appears in taxa across the phylogenetic spectrum. This allows us to make phylogenetically independent determinations of form and function. We define an ancestral state of the GM and conclude that evolution of the GM/AC and SAC has progressed in two directions from this ancestral state: toward either elaboration or reduction. Where present, the character states of both of these muscle groups were observed in all species examined and the number of states correlated within each family as well. The degree of development of the GM/AC and SAC compliance pump system is strongly correlated with previously determined lymph flux rates in a three species test. Our data suggest there may be a relationship between greater elaboration of the GM/AC and SAC system and terrestriality among the Anura.

Lymphatic regulation in nonmammalian vertebrates.

All vertebrate animals share in common the production of lymph through net capillary filtration from their closed circulatory system into their tissues. The balance of forces responsible for net capillary filtration and lymph formation is described by the Starling equation, but additional factors such as vascular and interstitial compliance, which vary markedly among vertebrates, also have a significant impact on rates of lymph formation. Why vertebrates show extreme variability in rates of lymph formation and how nonmammalian vertebrates maintain plasma volume homeostasis is unclear. This gap hampers our understanding of the evolution of the lymphatic system and its interaction with the cardiovascular system. The evolutionary origin of the vertebrate lymphatic system is not clear, but recent advances suggest common developmental factors for lymphangiogenesis in teleost fishes, amphibians, and mammals with some significant changes in the water-land transition. The lymphatic system of anuran amphibians is characterized by large lymphatic sacs and two pairs of lymph hearts that return lymph into the venous circulation but no lymph vessels per se. The lymphatic systems of reptiles and some birds have lymph hearts, and both groups have extensive lymph vessels, but their functional role in both lymph movement and plasma volume homeostasis is almost completely unknown. The purpose of this review is to present an evolutionary perspective in how different vertebrates have solved the common problem of the inevitable formation of lymph from their closed circulatory systems and to point out the many gaps in our knowledge of this evolutionary progression.

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.

Interspecific comparisons of lymph volume and lymphatic fluxes: do lymph reserves and lymph mobilization capacities vary in anurans from different environments?

The femoral lymph sac volumes and lymph mobilization capacity were compared in three anuran species that span a range of environments, dehydration tolerance, ability to maintain blood volume with dehydration, and degrees of development of skeletal muscles putatively involved in moving lymph vertically to the posterior lymph hearts. The femoral lymph sac volume determined by Evans blue injection and dilution in the femoral lymph sac varied interspecifically. The semiaquatic species, Lithobates catesbeianus, had the greatest apparent lymph volume expressed either as 18.7 mL kg body mass⁻¹ or 94 mL kg thigh mass⁻¹, compared with both the terrestrial and aquatic species, Rhinella marina (7.3 mL kg body mass⁻¹ and 57 mL kg thigh mass⁻¹) and Xenopus laevis (6.5 mL kg body mass⁻¹ and 40 mL kg thigh mass⁻¹, respectively. Injections of Evans blue into the subvertebral lymph sac, which communicates with both pairs of lymph hearts, yielded the highest rates of lymph return to the circulation in all three species. The most terrestrial species had a greater rate of lymphatic return from the subvertebral lymph sac, compared with the other two species. The rate of lymph flux from the femoral sac varied interspecifically and was correlated with the number and development of skeletal muscles involved in lymph movement. The results indicated that the three species differ in both the volume of lymph present and the capacity to return lymph. Lymph flux was correlated with habitat and the ability to maintain blood volume when challenged by dehydration or hemorrhage, whereas femoral lymph volume was not correlated with these factors.

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.

Lung ventilation contributes to vertical lymph movement in anurans.

Anurans (frogs and toads) generate lymphatic fluid at 10 times the rate in mammals, largely as a consequence of their very 'leaky' vasculature and high interstitial compliance. Lymph is ultimately pumped into the venous system by paired, dorsally located lymph hearts. At present, it is unclear how lymphatic fluid that accumulates in central body subcutaneous lymph sacs is moved to the anterior and posterior lymph hearts in the axillary regions and how lymph is moved, against gravity, to the dorsally located lymph hearts. In this study, we tested the hypothesis that lung ventilation, through its consequent effects on lymph sac pressure, contributes to the vertical movement of lymphatic fluid in the cane toad (Chaunus marinus) and the North American bullfrog (Lithobates catesbeiana). We measured pressure in the dorsal, lateral and subvertebral lymph sacs of anesthetized cane toads and bullfrogs during artificial lung inflation and deflation. We also measured pressure in the subvertebral lymph sac, which adheres to the dorsal surface of the lungs, simultaneously with brachial (forelimb) and pubic (posterior) sac pressure during ventilation in freely behaving animals. There were highly significant (P<0.001) relationships between lung pressure and lymph sac pressures (r(2)=0.19-0.72), indicating that pulmonary pressure is transmitted to the highly compliant lymph sacs that surround the lungs. Subvertebral sac pressure of resting animals was not significantly different between L. catesbeiana (518+/-282 Pa) and C. marinus (459+/-111 Pa). Brachial sac compliance (ml kPa(-1) kg(-1)) also did not differ between the two species (33.6+/-5.0 in L. catesbeiana and 37.0+/-9.4 in C. marinus). During expiration (lung deflation), reductions in expanding subvertebral sac pressure are communicated to the brachial lymph sac. Changes in brachial and pubic lymph sac pressures were correlated almost entirely during expiration rather than inspiration. The change in brachial sac pressure during expiration was 235+/-43 Pa for C. marinus and 215+/-50 Pa for L. catesbeiana, which is of sufficient magnitude to move lymph the estimated 0.5-1.0 cm vertical distance from the forelimb to the vicinity of the anterior lymph hearts. We suggest that lymph is moved during expiration to the subvertebral sac from anterior and posterior lymph sacs. During lung inflation, increased lymph sac pressure moves lymph to axillary regions, where lymph hearts can return lymph to the vascular space. Consequently, pulmonary ventilation has an important role for lymph movement and, hence, blood volume regulation in anurans.

Unique role of skeletal muscle contraction in vertical lymph movement in anurans.

Electromyographic (EMG) activity of skeletal muscles that either insert on the skin or are associated with the margins of subcutaneous lymph sacs was monitored for two species of anurans, Chaunus marinus and Lithobates catesbeiana (formerly Bufo marinus and Rana catesbeiana). Our hypothesis was that contraction of these muscles varies the volume, and hence pressure, within these lymph sacs, and that this pressure is responsible for moving lymph from ventral, gravitationally dependent reaches of the body to dorsally located lymph hearts. EMG activity of M. piriformis, M. gracilis minor, M. abdominal crenator, M. tensor fasciae latae, M. sphincter ani cloacalis, M. cutaneous pectoris and M. cutaneous dorsi was synchronous with pressure changes in their associated lymph sacs. These muscles contracted synchronously, and the pressures generated within the lymph sacs were sufficient to move lymph vertically against gravity to the lymph hearts. The pressure relationships were complex; both negative and positive pressures were recorded during a contractile event, a pattern consistent with the addition and loss of lymphatic fluid to the lymph sacs. Severing the tendons of some of the muscles led to lymph pooling in gravitationally dependent lymph sacs. These data are the first to: (1) describe a function for many of these skeletal muscles; (2) document the role of skeletal muscles in vertical lymph movement in anurans; and (3) reinterpret the role of the urostyle, a bony element of the anuran pelvic girdle.

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.

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.

A molecular phylogenetic analysis of the family Rhacophoridae with an emphasis on the Asian and African genera.

Using characters from mitochondrial DNA to construct maximum parsimony and maximum likelihood trees, we performed a phylogenetic analysis on representative species of 14 genera: 12 that belong to the treefrog family Rhacophoridae and two, Amolops and Rana, that are not rhacophorids. Our results support a phylogenetic hypothesis that depicts a monophyletic family Rhacophoridae. In this family, the Malagasy genera Aglyptodactylus, Boophis, Mantella, and Mantidactylus form a well-supported sister clade to all other rhacophorid genera, and Mantella is the sister taxon to Mantidactylus. Within the Asian/African genera, the genus Buergeria forms a well-supported clade of four species. The genera, except for Chirixalus, are generally monophyletic. An exception to this is that Polypedates dennysii clusters with species of Rhacophorus, suggesting that the taxonomy of the rhacophorids should be revised to reflect this relationship. Chirixalus is not monophyletic. Unexpectedly, there is strong support for Chirixalus doriae from Southeast Asia forming a clade with species of the African genus Chiromantis, suggesting that Chiromantis dispersed to Africa from Asia. Also, there is strong support for the sister taxon relationship of Chirixalus eiffingeri and Chirixalus idiootocus apart from other congeners.