Anthony V. Signore

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.

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.

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.

Interordinal gene capture, the phylogenetic position of Steller's sea cow based on molecular and morphological data, and the macroevolutionary history of Sirenia.

The recently extinct (ca. 1768) Stellers sea cow (Hydrodamalis gigas) was a large, edentulous North Pacific sirenian. The phylogenetic affinities of this taxon to other members of this clade, living and extinct, are uncertain based on previous morphological and molecular studies. We employed hybridization capture methods and second generation sequencing technology to obtain >30kb of exon sequences from 26 nuclear genes for both H. gigas and Dugong dugon. We also obtained complete coding sequences for the tooth-related enamelin (ENAM) gene. Hybridization probes designed using dugong and manatee sequences were both highly effective in retrieving sequences from H. gigas (mean=98.8% coverage), as were more divergent probes for regions of ENAM (99.0% coverage) that were designed exclusively from a proboscidean (African elephant) and a hyracoid (Cape hyrax). New sequences were combined with available sequences for representatives of all other afrotherian orders. We also expanded a previously published morphological matrix for living and fossil Sirenia by adding both new taxa and nine new postcranial characters. Maximum likelihood and parsimony analyses of the molecular data provide robust support for an association of H. gigas and D. dugon to the exclusion of living trichechids (manatees). Parsimony analyses of the morphological data also support the inclusion of H. gigas in Dugongidae with D. dugon and fossil dugongids. Timetree analyses based on calibration density approaches with hard- and soft-bounded constraints suggest that H. gigas and D. dugon diverged in the Oligocene and that crown sirenians last shared a common ancestor in the Eocene. The coding sequence for the ENAM gene in H. gigas does not contain frameshift mutations or stop codons, but there is a transversion mutation (AG to CG) in the acceptor splice site of intron 2. This disruption in the edentulous Stellers sea cow is consistent with previous studies that have documented inactivating mutations in tooth-specific loci of a variety of edentulous and enamelless vertebrates including birds, turtles, aardvarks, pangolins, xenarthrans, and baleen whales. Further, branch-site dN/dS analyses provide evidence for positive selection in ENAM on the stem dugongid branch where extensive tooth reduction occurred, followed by neutral evolution on the Hydrodamalis branch. Finally, we present a synthetic evolutionary tree for living and fossil sirenians showing several key innovations in the history of this clade including character state changes that parallel those that occurred in the evolutionary history of cetaceans.

Inactivation of thermogenic UCP1 as a historical contingency in multiple placental mammal clades

Inactivation of uncoupling protein 1 is linked to shifts in metabolic rate, body size, and species richness of eight mammalian lineages. Mitochondrial uncoupling protein 1 (UCP1) is essential for nonshivering thermogenesis in brown adipose tissue and is widely accepted to have played a key thermoregulatory role in small-bodied and neonatal placental mammals that enabled the exploitation of cold environments. We map ucp1 sequences from 133 mammals onto a species tree constructed from a ~51-kb sequence alignment and show that inactivating mutations have occurred in at least 8 of the 18 traditional placental orders, thereby challenging the physiological importance of UCP1 across Placentalia. Selection and timetree analyses further reveal that ucp1 inactivations temporally correspond with strong secondary reductions in metabolic intensity in xenarthrans and pangolins, or in six other lineages coincided with a ~30 million–year episode of global cooling in the Paleogene that promoted sharp increases in body mass and cladogenesis evident in the fossil record. Our findings also demonstrate that members of various lineages (for example, cetaceans, horses, woolly mammoths, Steller’s sea cows) evolved extreme cold hardiness in the absence of UCP1-mediated thermogenesis. Finally, we identify ucp1 inactivation as a historical contingency that is linked to the current low species diversity of clades lacking functional UCP1, thus providing the first evidence for species selection related to the presence or absence of a single gene product.

Origin and mechanism of thermal insensitivity in mole hemoglobins: a test of the 'additional' chloride binding site hypothesis.

SUMMARY The structural and evolutionary origins underlying the effect of temperature on the O2 binding properties of mammalian hemoglobins (Hbs) are poorly understood, despite their potential physiological importance. Previous work has shown that the O2 affinities of the blood of the coast mole (Scapanus orarius) and the eastern mole (Scalopus aquaticus) are significantly less sensitive to temperature changes than that of the star-nosed mole (Condylura cristata). It was suggested that this difference may arise from the binding of ‘additional’ chloride ions within a cationic pocket between residues 8His, 76Lys and 77Asn on the β-like δ-globin chains of coast and eastern mole Hbs. To test this hypothesis, we deduced the primary sequences of star-nosed mole and American shrew mole (Neurotrichus gibbsii) Hb, measured the sensitivity of these respiratory proteins to allosteric effector molecules and temperature, and calculated their overall oxygenation enthalpies (ΔH′). Here we show that the variability in ΔH′ seen among mole Hbs cannot be attributed to differential Cl– binding at δ8, δ76 and δ77, as the Cl– sensitivity of mole Hbs is unaffected by amino acid changes at this site (i.e. the proposed ‘additional’ Cl– binding site is not operational in mole Hbs). Rather, we demonstrate that the numerically low ΔH′ of coast and eastern mole Hbs results from heightened proton binding relative to other mole Hbs. Comparative sequence analysis and molecular modelling moreover suggest that this attribute evolved in a common ancestor of these two fossorial lineages and arises from the development of a salt bridge between a pair of amino acid residues (δ125His and α34Glu/Asp) that are not present in other mole Hbs.

Divergent and parallel routes of biochemical adaptation in high-altitude passerine birds from the Qinghai-Tibet Plateau

Significance Mountain ranges and highland plateaus in different parts of the world provide an opportunity to investigate the extent to which native species have followed similar or different routes of adaptation to the challenges of life at high altitude. Here we demonstrate that high-altitude songbirds from the Qinghai-Tibet Plateau independently evolved derived increases in hemoglobin–O2 affinity in comparison with their closest lowland relatives in East Asia. In comparisons that also included more distantly related high-altitude avian taxa, site-directed mutagenesis experiments revealed two cases in which convergent increases in hemoglobin–O2 affinity were caused by identical amino acid substitutions at the same sites. However, most adaptive convergence in protein function was attributable to different amino acid substitutions in different species. When different species experience similar selection pressures, the probability of evolving similar adaptive solutions may be influenced by legacies of evolutionary history, such as lineage-specific changes in genetic background. Here we test for adaptive convergence in hemoglobin (Hb) function among high-altitude passerine birds that are native to the Qinghai-Tibet Plateau, and we examine whether convergent increases in Hb–O2 affinity have a similar molecular basis in different species. We documented that high-altitude parid and aegithalid species from the Qinghai-Tibet Plateau have evolved derived increases in Hb–O2 affinity in comparison with their closest lowland relatives in East Asia. However, convergent increases in Hb–O2 affinity and convergence in underlying functional mechanisms were seldom attributable to the same amino acid substitutions in different species. Using ancestral protein resurrection and site-directed mutagenesis, we experimentally confirmed two cases in which parallel substitutions contributed to convergent increases in Hb–O2 affinity in codistributed high-altitude species. In one case involving the ground tit (Parus humilis) and gray-crested tit (Lophophanes dichrous), parallel amino acid replacements with affinity-enhancing effects were attributable to nonsynonymous substitutions at a CpG dinucleotide, suggesting a possible role for mutation bias in promoting recurrent changes at the same site. Overall, most altitude-related changes in Hb function were caused by divergent amino acid substitutions, and a select few were caused by parallel substitutions that produced similar phenotypic effects on the divergent genetic backgrounds of different species.