Kevin L. Campbell

Evolution of Mammalian Diving Capacity Traced by Myoglobin Net Surface Charge

Introduction Evolution of extended breath-hold endurance enables the exploitation of the aquatic niche by numerous mammalian lineages and is accomplished by elevated body oxygen stores and morphological and physiological adaptations that promote their economical use. High muscle myoglobin concentrations in particular are mechanistically linked with an extended dive capacity phenotype, yet little is known regarding the molecular and biochemical underpinnings of this key specialization. We modeled the evolutionary history of this respiratory pigment over 200 million years of mammalian evolution to elucidate the development of maximal diving capacity during the major mammalian land-to-water transitions. Evolutionary reconstruction of myoglobin net surface charge in terrestrial and aquatic mammals. The figure reveals a molecular signature of elevated myoglobin net surface charge in all lineages of living elite mammalian divers with an extended aquatic history (upper silhouettes). This signature is used here to infer the diving capacity of extinct species representing stages during mammalian land-to-water transitions (†). Methods We first determined the relationship between maximum myoglobin concentration and its sequence-derived net surface charge across living mammalian taxa. By using ancestral sequence reconstruction we then traced myoglobin net surface charge across a 130-species phylogeny to infer ancestral myoglobin muscle concentrations. Last, we estimated maximum dive time in extinct transitional species on the basis of the relationship of this variable with muscle myoglobin concentration and body mass in extant diving mammals. Results We reveal an adaptive molecular signature of elevated myoglobin net surface charge in all lineages of mammalian divers with an extended aquatic history—from 16-g water shrews to 80,000-kg whales—that correlates with exponential increases in muscle myoglobin concentrations. Integration of this data with body mass predicts 82% of maximal dive-time variation across all degrees of diving ability in living mammals. Discussion We suggest that the convergent evolution of high myoglobin net surface charge in mammalian divers increases intermolecular electrostatic repulsion, permitting higher muscle oxygen storage capacities without potentially deleterious self-association of the protein. Together with fossil body-mass estimates, our evolutionary reconstruction permits detailed assessments of maximal submergence times and potential foraging ecologies of early transitional ancestors of cetaceans, pinnipeds, and sea cows. Our findings support amphibious ancestries for echidnas, talpid moles, hyraxes, and elephants, thereby not only establishing the earliest land-to-water transition among placental mammals but also providing a new perspective on the evolution of myoglobin, arguably the best-known protein. Holding Your Breath Hemoglobin and myoglobin are widely responsible for oxygen transport and storage (see the Perspective by Rezende). The ability of diving mammals to obtain enough oxygen to support extended dives and foraging is largely dependent on muscle myoglobin (Mb) content. Mirceta et al. (p. 1234192) found that in mammalian lineages with an aquatic or semiaquatic lifestyle, Mb net charge increases, which may represent an adaptation to inhibit self-association of Mb at high intracellular concentrations. Epistasis results from nonadditive genetic interactions and can affect phenotypic evolution. Natarajan et al. (p. 1324) found that epistatic interactions were able to explain the increased hemoglobin oxygen-binding affinity observed in deer mice populations at high altitude. In mammals, the offloading of oxygen from hemoglobin is facilitated by a reduction in the bloods pH, driven by metabolically produced CO2. However, in fish, a reduction in blood pH reduces oxygen carrying capacity of hemoglobin. Rummer et al. (p. 1327) implanted fiber optic oxygen sensors within the muscles of rainbow trout and found that elevated CO2 levels in the water led to acidosis and elevated oxygen tensions. Increasing the number of charged amino acids allows for higher myoglobin concentrations in the muscles of diving mammals. [Also see Perspective by Rezende] Extended breath-hold endurance enables the exploitation of the aquatic niche by numerous mammalian lineages and is accomplished by elevated body oxygen stores and adaptations that promote their economical use. However, little is known regarding the molecular and evolutionary underpinnings of the high muscle myoglobin concentration phenotype of divers. We used ancestral sequence reconstruction to trace the evolution of this oxygen-storing protein across a 130-species mammalian phylogeny and reveal an adaptive molecular signature of elevated myoglobin net surface charge in diving species that is mechanistically linked with maximal myoglobin concentration. This observation provides insights into the tempo and routes to enhanced dive capacity evolution within the ancestors of each major mammalian aquatic lineage and infers amphibious ancestries of echidnas, moles, hyraxes, and elephants, offering a fresh perspective on the evolution of this iconic respiratory pigment.

Molecular phylogenetic relationships of moles, shrew moles, and desmans from the new and old worlds.

A Rich variety of anatomical and physiological specializations has enabled members of the family Talpidae (moles, shrew moles, and desmans) to exploit a diverse range of habitats: terrestrial, semi-aquatic, aquatic/fossorial, semi-fossorial, and fossorial. While numerous morphological and biochemical studies pertaining to the origin and radiation of the Talpidae have been completed, phylogenetic hypotheses remain controversial. To address this shortcoming we sequenced the mitochondrial DNA cytochrome b gene (1140bp) from 29 individuals spanning 12 talpid species. Phylogenetic trees incorporating 12 New and Old World genera (18 species; all 3 extant subfamilies) were then constructed using NJ, MP, ML, and NJ-ML (NJ with ML parameters) methods. Our results provide molecular support for a mononphyletic Talpidae, and suggest that the 12 genera are clustered into seven major clades; (1) Asiatic shrew-like moles (Uropsilus), (2) North American aquatic/fossorial moles (Condylura), (3) North American fossorial moles (Parascalops, Scalopus, and Scapanus), (4) North American semi-fossorial shrew moles (Neurotrichus), (5) Japanese semi-fossorial shrew moles (Dymecodon and Urotrichus), (6) European semi-aquatic desmans (Desmana), and (7) Eurasian fossorial moles (Euroscaptor, Mogera, and Talpa). None of these groupings comprised mole species from both continents. In fact, North American moles and shrew moles do not appear to have specific affinities with Asian moles and shrew moles, respectively. Although low bootstrap support was generally found for evolutionary nodes uniting the major talpid clades, all gene trees constructed identified fossorial North American and Eurasian mole lineages as nonmonophyletic groups, suggesting subterranean specializations arose independently at least twice during the evolution of the Talpidae. Additionally, our data set provides molecular support for a basal divergence and long independent history of Uropsilus from the main talpid line, and refutes the traditional taxonomic status and secondarily basal phylogenetic placement of the subfamily Desmaninae within the Talpidae.

Substitutions in woolly mammoth hemoglobin confer biochemical properties adaptive for cold tolerance

We have genetically retrieved, resurrected and performed detailed structure-function analyses on authentic woolly mammoth hemoglobin to reveal for the first time both the evolutionary origins and the structural underpinnings of a key adaptive physiochemical trait in an extinct species. Hemoglobin binds and carries O2; however, its ability to offload O2 to respiring cells is hampered at low temperatures, as heme deoxygenation is inherently endothermic (that is, hemoglobin-O2 affinity increases as temperature decreases). We identify amino acid substitutions with large phenotypic effect on the chimeric β/δ-globin subunit of mammoth hemoglobin that provide a unique solution to this problem and thereby minimize energetically costly heat loss. This biochemical specialization may have been involved in the exploitation of high-latitude environments by this African-derived elephantid lineage during the Pleistocene period. This powerful new approach to directly analyze the genetic and structural basis of physiological adaptations in an extinct species adds an important new dimension to the study of natural selection.

Water shrews detect movement, shape, and smell to find prey underwater

American water shrews (Sorex palustris) are aggressive predators that feed on a variety of terrestrial and aquatic prey. They often forage at night, diving into streams and ponds in search of food. We investigated how shrews locate submerged prey using high-speed videography, infrared lighting, and stimuli designed to mimic prey. Shrews attacked brief water movements, indicating motion is an important cue used to detect active or escaping prey. They also bit, retrieved, and attempted to eat model fish made of silicone in preference to other silicone objects showing that tactile cues are important in the absence of movement. In addition, water shrews preferentially sniffed model prey fish and crickets underwater by exhaling and reinhaling air through the nostrils, suggesting olfaction plays an important role in aquatic foraging. The possibility of echolocation, sonar, or electroreception was investigated by testing for ultrasonic and audible calls above and below water and by presenting electric fields to foraging shrews. We found no evidence for these abilities. We conclude that water shrews detect motion, shape, and smell to find prey underwater. The short latency of attacks to water movements suggests shrews may use a flush-pursuit strategy to capture some prey.

Evolution and Biogeography of Talpid Moles from Continental East Asia and the Japanese Islands Inferred from Mitochondrial and Nuclear Gene Sequences

Abstract We sequenced the cytochrome b gene from two little-studied mammal species from the highlands of Southwest China, the long-tailed mole Scaptonyx fusicaudus and the gracile shrew-like mole Uropsilus gracilis. This data was used to examine the phylogenetic relationships among 19 talpid species within the family Talpidae (Mammalia: Eulipotyphla). Cytochrome b gene trees supported a basal placement of shrew-like moles (Uropsilus) within the Talpidae, and suggested that fossorial specializations arose twice during talpid evolution. To assess the evolutionary relationships of moles endemic to this region, we additionally sequenced the 12S rRNA gene and the nuclear recombination-activating gene-1 from eight and ten East Asian taxa, respectively. Analyses of these single and concatenated data sets suggested that East Asian shrew moles diverged prior to the evolution of fossorial Eurasian moles. However, we were unable to determine whether semi-fossorial shrew moles are monophyletic. In contrast, fossorial Eurasian genera (Talpa, Mogera and Euroscaptor) were consistently found to form a monophyletic clade, with Mogera and Euroscaptor representing sister taxa. Furthermore, this fossorial clade grouped with the semi-aquatic Desmana, although with fairly low (35–62%) bootstrap support. Mogera imaizumii was found to be more closely related to M. wogura than to M. tokudae. This implies that the ancestors of these three species entered Japan from the Asian continent in this order via a series of migration events, suggesting that the Japanese Islands have played an important role in preserving mole lineages from ancient to recent times.

Temperature dependence of haemoglobin-oxygen affinity in heterothermic vertebrates: mechanisms and biological significance

As demonstrated by August Krogh et al. a century ago, the oxygen‐binding reaction of vertebrate haemoglobin is cooperative (described by sigmoid O2 equilibrium curves) and modulated by CO2 and protons (lowered pH) that – in conjunction with later discovered allosteric effectors (chloride, lactate and organic phosphate anions) – enhance O2 unloading from blood in relatively acidic and oxygen‐poor tissues. Based on the exothermic nature of the oxygenation of the haem groups, haemoglobin–O2 affinity also decreases with rising temperature. This thermal sensitivity favours oxygen unloading in warm working muscles, but may become detrimental in regionally heterothermic animals, for example in cold‐tolerant birds and mammals and warm‐bodied fish, where it may perturb the balance between O2 unloading and O2 requirement in organs with substantially different temperatures than at the respiratory organs and thus commonly is reduced or obliterated. Given that the oxygenation of haemoglobin is linked with the endothermic release of allosteric effectors, increased effector interaction is an effective strategy that is widely exploited to achieve adaptive reductions in the temperature dependence of blood‐O2 affinity. The molecular mechanisms implicated in heterothermic vertebrates from different taxonomic groups reveal remarkable variability, both as regards the effectors implicated (protons in tunas, organic phosphates in sharks and billfish, chloride ions in ruminants and chloride and phosphate anions in the extinct woolly mammoth, etc.) and binding sites for the same effectors, indicating multiple evolutionary origins, but convergent physiological functionality (reductions in temperature dependence of O2‐binding affinity that safeguard tissue O2 supply).

Heat increment of feeding and its thermoregulatory benefit in the muskrat (Ondatra zibethicus)

The calorigenic effect of feeding and its potential benefit in defraying thermoregulatory costs and attenuating immersion hypothermia of adult muskrats were investigated. A single session of feeding on aquatic vegetation was sufficient to raise the metabolic rate of muskrats for a period of at least 5 h. The peak postprandial rate of oxygen consumption averaged 1.42 times the level established for fasted animals, and the heat increment of feeding accounted for about 40% of the metabolizable energy intake of muskrats. There was no evidence of a postprandial rise in oxygen consumption of muskrats that entered water at 18–19°C after feeding. In aquatic trials, average and minimum steady-state oxygen consumption rates of fed muskrats were similar to, or even lower than values recorded from fasted animals, implying substitution of heat increment of feeding for thermoregulatory heat production. Our data did not support the hypothesis that heat increment of feeding retards body cooling in water. Net body temperature decline in water was actually higher in fed animals than in fasted controls. However, since previously fed muskrats also entered water at an elevated body temperature, the final body temperature (at 30 min immersion) was similar in all groups. These findings suggest that metabolic heat generated incidental to preimmersion feeding could provide a thermoregulatory benefit to muskrats by reducing the need for active thermogenesis in water.

Molecular basis of a novel adaptation to hypoxic-hypercapnia in a strictly fossorial mole

BackgroundElevated blood O2 affinity enhances survival at low O2 pressures, and is perhaps the best known and most broadly accepted evolutionary adjustment of terrestrial vertebrates to environmental hypoxia. This phenotype arises by increasing the intrinsic O2 affinity of the hemoglobin (Hb) molecule, by decreasing the intracellular concentration of allosteric effectors (e.g., 2,3-diphosphoglycerate; DPG), or by suppressing the sensitivity of Hb to these physiological cofactors.ResultsHere we report that strictly fossorial eastern moles (Scalopus aquaticus) have evolved a low O2 affinity, DPG-insensitive Hb - contrary to expectations for a mammalian species that is adapted to the chronic hypoxia and hypercapnia of subterranean burrow systems. Molecular modelling indicates that this functional shift is principally attributable to a single charge altering amino acid substitution in the β-type δ-globin chain (δ136Gly→Glu) of this species that perturbs electrostatic interactions between the dimer subunits via formation of an intra-chain salt-bridge with δ82Lys. However, this replacement also abolishes key binding sites for the red blood cell effectors Cl-, lactate and DPG (the latter of which is virtually absent from the red cells of this species) at δ82Lys, thereby markedly reducing competition for carbamate formation (CO2 binding) at the δ-chain N-termini.ConclusionsWe propose this Hb phenotype illustrates a novel mechanism for adaptively elevating the CO2 carrying capacity of eastern mole blood during burst tunnelling activities associated with subterranean habitation.

Body oxygen stores, aerobic dive limits, and the diving abilities of juvenile and adult muskrats (Ondatra zibethicus).

Intraspecific variability in body oxygen reserves, muscle buffering capacity, diving metabolic rate, and diving behavior were examined in recently captured juvenile and adult muskrats. Allometric scaling exponents for lung ( \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape