Evolution of an extreme hemoglobin phenotype contributed to the sub-Arctic specialization of extinct Steller's sea cows.

The extinct Steller's sea cow (Hydrodamalis gigas; †1768) was a whale-sized marine mammal that manifested profound morphological specializations to exploit the harsh coastal climate of the North Pacific. Yet despite first-hand accounts of their biology, little is known regarding the physiological adjustments underlying their evolution to this environment. Here, the adult-expressed hemoglobin (Hb; α2ß/δ2) of this sirenian is shown to harbor a fixed amino acid replacement at an otherwise invariant position (ß/δ82Lys→Asn) that alters multiple aspects of Hb function. First, our functional characterization of recombinant sirenian Hb proteins demonstrates that the Hb-O2 affinity of this sub-Arctic species was less affected by temperature than those of living (sub)tropical sea cows. This phenotype presumably safeguarded O2 delivery to cool peripheral tissues and largely arises from a reduced intrinsic temperature sensitivity of the H. gigas protein. Additional experiments on H. gigas ß/δ82Asn→Lys mutant Hb further reveal this exchange renders Steller's sea cow Hb unresponsive to the potent intraerythrocytic allosteric effector 2,3-diphosphoglycerate, a radical modification that is the first documented example of this phenotype among mammals. Notably, ß/δ82Lys→Asn moreover underlies the secondary evolution of a reduced blood-O2 affinity phenotype that would have promoted heightened tissue and maternal/fetal O2 delivery. This conclusion is bolstered by analyses of two Steller's sea cow prenatal Hb proteins (Hb Gower I; ζ2ε2 and HbF; α2γ2) that suggest an exclusive embryonic stage expression pattern, and reveal uncommon replacements in H. gigas HbF (γ38Thr→Ile and γ101Glu→Asp) that increased Hb-O2 affinity relative to dugong HbF. Finally, the ß/δ82Lys→Asn replacement of the adult/fetal protein is shown to increase protein solubility, which may have elevated red blood cell Hb content within both the adult and fetal circulations and contributed to meeting the elevated metabolic (thermoregulatory) requirements and fetal growth rates associated with this species cold adaptation.

In 1741, shipwrecked naturalist Georg Wilhelm Steller made detailed observations of large marine mammals grazing on seaweed in the shallow waters surrounding a remote island in the North Pacific Ocean. Within thirty years, these 'Steller's sea cows' had been hunted to extinction. Unlike their remaining tropical relatives ­ dugongs and manatees ­ Steller's sea cows were specialized to cold, sub-Arctic environments. Measuring up to 10 meters long, they were much larger than other sea cow species. This, along with having very thick skin, helped them to reduce heat loss. Previous work showed that the hemoglobin protein ­ which binds to and carries oxygen around mammalian bodies ­ of Steller's sea cows had a decreased affinity for oxygen, resulting in greater delivery of oxygen to organs and tissues. It was thought that this could be an adaptation to fuel heightened metabolic heat production in cold conditions. Studies of ancient DNA also identified the substitution of a single building block in the Steller's sea cow hemoglobin protein that is not present in other mammals and was suspected to underlie this modification. To determine how this unique substitution affects Steller's sea cow hemoglobin function ­ and whether it contributed to their ability to live in cold environments ­ Signore et al. generated hemoglobin proteins of Steller's sea cows, dugongs and Florida manatees. Testing their biochemical properties showed that this single exchange profoundly alters multiple aspects of how the Steller's sea cow hemoglobin works. Alongside reducing hemoglobin's oxygen affinity, the Steller's sea cow substitution also makes the protein more soluble, potentially increasing the level of hemoglobin within red blood cells. Additionally, it eliminates hemoglobin sensitivity to a molecule involved in oxygen binding ­ known as DPG ­ saving energy by no longer requiring production of this molecule. Furthermore, the same substitution makes hemoglobin less sensitive to changes in temperature, which would have helped to safeguard the delivery of oxygen to cool limbs and other extremities, reducing costly heat loss. Together, these changes in hemoglobin would have helped the Steller's sea cow to more efficiently transport oxygen around the body. Importantly, generating and testing Steller's sea cow pre-natal hemoglobins suggested this substitution may have also helped to enhance the fetal growth rate of these immense marine mammals by improving gas exchange between the mother and fetus. Signore et al. have revealed how a mutated form of hemoglobin allowed an extinct mammal to adapt to an extreme environment. Similar methods could be used to understand the physiological attributes of other extinct animals. In the future, this increased understanding of hemoglobin mutations could aid the development of human hemoglobin substitutes for therapeutic uses.

Thermoconforming rays of the star-nosed mole.

The star-nosed mole (Condylura cristata) is renowned for its densely innervated 22 appendage star-like rostrum ('star') specialized for tactile sensation. As a northerly distributed insectivorous mammal exploiting aquatic and terrestrial habitats, these vascularized nasal rays are regularly exposed to cold water and thermally conductive soil, leading us to ask whether the star surface temperature, a proxy for blood flow, conforms to the local ambient temperature to conserve body heat. Alternatively, given the exquisite sensory nature of the star, we posited that the uninsulated rays may be kept warm when foraging to maintain high mechanosensory function. To test these hypotheses, we remotely monitored surface temperatures in wild-caught star-nosed moles. Although the tail acted as a thermal window exhibiting clear vasoconstriction/vasodilation, the star varied passively in surface temperature, with little evidence for thermoregulatory vasomotion. This thermoconforming response may have evolved to minimize conductive heat loss to the water or wet soils when foraging.

Dive Performance and Aquatic Thermoregulation of the World's Smallest Mammalian Diver, the American Water Shrew (Sorex palustris).

Allometry predicts that the 12-17-g American water shrew (Sorex palustris)-the world's smallest mammalian diver-will have the highest diving metabolic rate coupled with the lowest total body oxygen storage capacity, skeletal muscle buffering capacity, and glycolytic potential of any endothermic diver. Consistent with expectations, and potentially owing to their low thermal inertia, water shrews had a significantly higher diving metabolic rate in 10°C water (8.77 mL O2 g-1 h-1) compared with 30°C water (6.57 mL O2 g-1 h-1). Unlike larger-bodied divers, muscle myoglobin contributed minimally (7.7%-12.4%) to total onboard O2 stores of juvenile and adult water shrews, respectively, but was offset by high blood O2 carrying capacities (26.4%-26.9% v/v). Diving was predominantly aerobic, as only 1.2%-2.3% of dives in 10°C and 30°C water, respectively, exceeded the calculated aerobic dive limits at these temperatures (10.8-14.4 s). The mean voluntary dive time of water shrews during 20-min trials in 3°C-30°C water was 5.0±0.1 s (N=25, n=1,628), with a mean maximum dive time of 10.1±0.4 s. However, the average dive duration (6.9±0.2 s, n=257) of radio-telemetered shrews exclusively foraging in a simulated riparian environment (3°C water) for 12-28 h suggests that mean (but not maximum) dive times of water shrews in the wild may be longer. Mean dive duration, duration of the longest dive, and total time in water all decreased significantly as water temperature declined, suggesting that shrews employed behavioral thermoregulation to defend against immersion hypothermia. Additionally, free-diving shrews in the 24-h trials consistently elevated core body temperature by ∼1°C immediately before initiating aquatic foraging bouts and ended these bouts when body temperature was still at or above normal resting levels (∼37.8°C). We suggest that this observed predive hyperthermia aids to heighten the impressive somatosensory physiology, and hence foraging efficiency, of this diminutive predator while submerged.

Burrowing star-nosed moles (Condylura cristata) are not hypoxia tolerant.

Star-nosed moles (Condylura cristata) have an impressive diving performance and burrowing lifestyle, yet no ventilatory data are available for this or any other talpid mole species. We predicted that, like many other semi-aquatic and fossorial small mammals, star-nosed moles would exhibit: (i) a blunted (i.e. delayed or reduced) hypoxic ventilatory response, (ii) a reduced metabolic rate and (iii) a lowered body temperature (Tb) in hypoxia. We thus non-invasively measured these variables from wild-caught star-nosed moles exposed to normoxia (21% O2) or acute graded hypoxia (21-6% O2). Surprisingly, star-nosed moles did not exhibit a blunted HVR or decreased Tb in hypoxia, and only manifested a significant, albeit small (<8%), depression of metabolic rate at 6% O2 relative to normoxic controls. Unlike small rodents inhabiting similar niches, star-nosed moles are thus intolerant to hypoxia, which may reflect an evolutionary trade-off favouring the extreme sensory biology of this unusual insectivore.

Myoglobin primary structure reveals multiple convergent transitions to semi-aquatic life in the world's smallest mammalian divers.

The speciose mammalian order Eulipotyphla (moles, shrews, hedgehogs, solenodons) combines an unusual diversity of semi-aquatic, semi-fossorial, and fossorial forms that arose from terrestrial forbearers. However, our understanding of the ecomorphological pathways leading to these lifestyles has been confounded by a fragmentary fossil record, unresolved phylogenetic relationships, and potential morphological convergence, calling for novel approaches. The net surface charge of the oxygen-storing muscle protein myoglobin (ZMb), which can be readily determined from its primary structure, provides an objective target to address this question due to mechanistic linkages with myoglobin concentration. Here, we generate a comprehensive 71 species molecular phylogeny that resolves previously intractable intra-family relationships and then ancestrally reconstruct ZMb evolution to identify ancient lifestyle transitions based on protein sequence alone. Our phylogenetically informed analyses confidently resolve fossorial habits having evolved twice in talpid moles and reveal five independent secondary aquatic transitions in the order housing the world's smallest endothermic divers.

The shrews, moles and hedgehogs that surround us all belong to the same large group of insect-eating mammals. While most members in this 'Eulipotyphla order' trot on land, some, like moles, have evolved to hunt their prey underground. A few species, such as the water shrews, have even ventured to adopt a semi-aquatic lifestyle, diving into ponds and streams to retrieve insects. These underwater foragers share unique challenges, burning a lot of energy and losing heat at a high rate while not being able to store much oxygen. It is still unclear how these semi-aquatic habits have come to be: the fossil record is fragmented and several species tend to display the same adaptations even though they have evolved separately. This makes it difficult to identify when and how many times the Eulipotyphla species started to inhabit water. The protein myoglobin, which gives muscles their red color, could help in this effort. This molecule helps muscles to capture oxygen from blood, a necessary step for cells to obtain energy. Penguins, seals and whales, which dive to get their food, often have much higher concentration of myoglobin so they can spend extended amount of time without having to surface for air. In addition, previous work has shown that eight groups of mammalian divers carry genetic changes that help newly synthetized myoglobin proteins to not stick to each other. This means that these animals can store more of the molecule in their muscles, increasing their oxygen intake and delivery. He et al. therefore speculated that all semi-aquatic Eulipotyphla species would carry genetic changes that made their myoglobin less likely to clump together; underground species, which also benefit from absorbing more oxygen, would display intermediate alterations. In addition, reconstructing the myoglobin sequences from the ancestors of living species would help to spot when the transition to aquatic life took place. A variety of approaches were harnessed to obtain myoglobin and other sequences from 55 eulipotyphlan mammals, which then were used to construct a strongly supported family tree for this group. The myoglobin results revealed that from terrestrial to subterranean to semi-aquatic species, genetic changes took place that would diminish the ability for the proteins to stick to each other. This pattern also showed that semi-aquatic lifestyles have independently evolved five separate times ­ twice in moles, three times in shrews. By retracing the evolutionary history of specific myoglobin properties, He et al. shed light on how one of the largest orders of mammals has come to be fantastically diverse.

The Rapid Response to the COVID-19 Pandemic by the Arthroplasty Divisions at Two Academic Referral Centers.

The COVID-19 pandemic has created widespread changes across all of health care. As a result, the impacts on the delivery of orthopedic services have been challenged. To ensure and provide adequate health care resources in terms of hospital capacity and personnel and personal protective equipment, service lines such as adult reconstruction and lower limb arthroplasty have stopped or substantially limited elective surgeries and have been forced to re-engineer care processes for a high volume of patients. Herein, we summarize the similar approaches by two arthroplasty divisions in high-volume academic referral centers in (1) the cessation of elective surgeries, (2) workforce restructuring, (3) phased delivery of outpatient and inpatient care, and (4) educational restructuring.

Body condition impacts blood and muscle oxygen storage capacity of free-living beluga whales (Delphinapterus leucas).

Arctic marine ecosystems are currently undergoing rapid environmental changes. Over the past 20 years, individual growth rates of beluga whales (Delphinapterus leucas) have declined, which may be a response to climate change; however, the scarcity of physiological data makes it difficult to gauge the adaptive capacity and resilience of the species. We explored relationships between body condition and physiological parameters pertaining to oxygen (O2) storage capacity in 77 beluga whales in the eastern Beaufort Sea. Muscle myoglobin concentrations averaged 77.9 mg g-1, one of the highest values reported among mammals. Importantly, blood haematocrit, haemoglobin and muscle myoglobin concentrations correlated positively to indices of body condition, including maximum half-girth to length ratios. Thus, a whale with the lowest body condition index would have ∼27% lower blood (26.0 versus 35.7 ml kg-1) and 12% lower muscle (15.6 versus 17.7 ml kg-1) O2 stores than a whale of equivalent mass with the highest body condition index; with the conservative assumption that underwater O2 consumption rates are unaffected by body condition, this equates to a >3 min difference in maximal aerobic dive time between the two extremes (14.3 versus 17.4 min). Consequently, environmental changes that negatively impact body condition may hinder the ability of whales to reach preferred prey sources, evade predators and escape ice entrapments. The relationship between body condition and O2 storage capacity may represent a vicious cycle, in which environmental changes resulting in decreased body condition impair foraging, leading to further reductions in condition through diminished prey acquisition and/or increased foraging efforts.

Emergence of a Chimeric Globin Pseudogene and Increased Hemoglobin Oxygen Affinity Underlie the Evolution of Aquatic Specializations in Sirenia.

As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller's sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb-O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb-O2 affinity in (sub)Arctic Steller's sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.

Evolution of UCP1.

Brown adipose tissue (BAT), the specialized heat-producing organ found in many placental mammals including humans, may be accessible for clinical drug intervention to help combat metabolic diseases. Understanding the biology of BAT and its thermogenic uncoupling protein 1 (UCP1) will benefit from an assessment of its evolution, answering where UCP1 originated and how it has been modified and integrated into cellular energy metabolism. Here, we review topical insights regarding the molecular evolution of UCP1-also reconstructing the proximate and ultimate factors selecting for brown fat thermogenesis in placental mammals. This new thinking on "old" events will assist our understanding of how thermogenic mitochondrial uncoupling was integrated into the physiology of the brown adipocyte. Recent comparative studies examining the occurrence of UCP1 in vertebrates not only identified the ancient (pre-mammal) rise of UCP1 but also its repeated downfall during mammalian evolution as evidenced by multiple independent gene loss and/or inactivation events. Together with the comparative physiology of various species, we may be able to find conditions that favor UCP1 thermogenesis and, learning from these insights, identify molecular networks that will be useful to pharmacologically stimulate the tissue.

Altered hemoglobin co-factor sensitivity does not underlie the evolution of derived fossorial specializations in the family Talpidae.

The high O2 affinity of European mole (Talpa europaea) blood is postulated to largely arise from the presence of two ß-globin chain residues (ß4 Ser and ß5 Gly) that weaken the interaction of its hemoglobin (Hb) with the red cell organophosphate 2,3-diphosphoglycerate (DPG). This latter trait is generally accepted to be an 'adaptation to subterranean life', despite the fact that no data are available for more basal mole lineages that have no evolutionary history of fossoriality (i.e. the ambulatory, high-elevation shrew-like moles and the semi-aquatic desmans), and which may similarly benefit from an elevated blood O2 affinity. To test whether evolution of a low DPG sensitivity phenotype is linked to derived fossorial lifestyles or represents an ancestral trait for the family, we determined the globin gene sequences and measured the intrinsic O2 affinity and co-factor sensitivity of the major Hb component of the gracile shrew-like mole (Uropsilus gracilis) and the Pyrenean desman (Galemys pyrenaicus). Our results unequivocally demonstrate that the presence of ß4 Ser and ß5 Gly, together with a low DPG sensitivity Hb phenotype, predates the radiation of the family Talpidae, and hence did not evolve as a specific adaptation to fossorial life. By contrast, our comparative analyses suggest that variations in whole blood O2 affinity among members of this family predominantly arose from amino acid substitutions that increase or decrease the intrinsic O2 affinity of the protein.

Evolution of UCP1 Transcriptional Regulatory Elements Across the Mammalian Phylogeny.

Uncoupling protein 1 (UCP1) permits non-shivering thermogenesis (NST) when highly expressed in brown adipose tissue (BAT) mitochondria. Exclusive to placental mammals, BAT has commonly been regarded to be advantageous for thermoregulation in hibernators, small-bodied species, and the neonates of larger species. While numerous regulatory control motifs associated with UCP1 transcription have been proposed for murid rodents, it remains unclear whether these are conserved across the eutherian mammal phylogeny and hence essential for UCP1 expression. To address this shortcoming, we conducted a broad comparative survey of putative UCP1 transcriptional regulatory elements in 139 mammals (135 eutherians). We find no evidence for presence of a UCP1 enhancer in monotremes and marsupials, supporting the hypothesis that this control region evolved in a stem eutherian ancestor. We additionally reveal that several putative promoter elements (e.g., CRE-4, CCAAT) identified in murid rodents are not conserved among BAT-expressing eutherians, and together with the putative regulatory region (PRR) and CpG island do not appear to be crucial for UCP1 expression. The specificity and importance of the upTRE, dnTRE, URE1, CRE-2, RARE-2, NBRE, BRE-1, and BRE-2 enhancer elements first described from rats and mice are moreover uncertain as these motifs differ substantially-but generally remain highly conserved-in other BAT-expressing eutherians. Other UCP1 enhancer motifs (CRE-3, PPRE, and RARE-3) as well as the TATA box are also highly conserved in nearly all eutherian lineages with an intact UCP1. While these transcriptional regulatory motifs are generally also maintained in species where this gene is pseudogenized, the loss or degeneration of key basal promoter (e.g., TATA box) and enhancer elements in other UCP1-lacking lineages make it unlikely that the enhancer region is pleiotropic (i.e., co-regulates additional genes). Importantly, differential losses of (or mutations within) putative regulatory elements among the eutherian lineages with an intact UCP1 suggests that the transcriptional control of gene expression is not highly conserved in this mammalian clade.

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

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.

Talpid Mole Phylogeny Unites Shrew Moles and Illuminates Overlooked Cryptic Species Diversity.

The mammalian family Talpidae (moles, shrew moles, desmans) is characterized by diverse ecomorphologies associated with terrestrial, semi-aquatic, semi-fossorial, fossorial, and aquatic-fossorial lifestyles. Prominent specializations involved with these different lifestyles, and the transitions between them, pose outstanding questions regarding the evolutionary history within the family, not only for living but also for fossil taxa. Here, we investigate the phylogenetic relationships, divergence times, and biogeographic history of the family using 19 nuclear and 2 mitochondrial genes (∼16 kb) from ∼60% of described species representing all 17 genera. Our phylogenetic analyses help settle classical questions in the evolution of moles, identify an ancient (mid-Miocene) split within the monotypic genus Scaptonyx, and indicate that talpid species richness may be nearly 30% higher than previously recognized. Our results also uniformly support the monophyly of long-tailed moles with the two shrew mole tribes and confirm that the Gansu mole is the sole living Asian member of an otherwise North American radiation. Finally, we provide evidence that aquatic specializations within the tribes Condylurini and Desmanini evolved along different morphological trajectories, though we were unable to statistically reject monophyly of the strictly fossorial tribes Talpini and Scalopini.

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) Steller's 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 Steller's 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.

The globin gene repertoire of lampreys: convergent evolution of hemoglobin and myoglobin in jawed and jawless vertebrates.

Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus, thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O2 transport in the blood and O2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O2-transport and O2-storage proteins in agnathans and gnathostomes involved the convergent co-option of different precursor proteins in the ancestral globin repertoire of vertebrates.

Enthalpic partitioning of the reduced temperature sensitivity of O2 binding in bovine hemoglobin.

The oxygenation enthalpy of the heme groups of hemoglobin (Hb) is inherently exothermic, resulting in decreased Hb-O2 affinity with rising temperature. However, oxygenation is coupled with endothermic dissociation of allosteric effectors (e.g. protons, chloride ions and organic phosphates) from the protein, which reduces the overall oxygenation enthalpy. The evolution of Hbs with reduced temperature sensitivity ostensibly safeguards O2 unloading in cold extremities of regionally-heterothermic vertebrates permitting energy-saving reductions in heat loss. Ungulate (e.g. bovine) Hbs have long served as a model system in this regard in that they exhibit numerically low oxygenation enthalpies that are thought to correlate with the presence of an additional Cl(-) binding site (compared to human Hb) comprised of three cationic residues at positions 8, 76 and 77 of the ß-chains of Hb. However, ungulate Hbs also exhibit distinctive amino acid exchanges at the N-termini of the ß-chains that stabilize the low-affinity deoxystructure of the Hb, mimicking the action of organic phosphates. In order to assess the relative contributions from these two effects, we measured the temperature sensitivity of Hb-O2 affinity in bovine and human Hbs in the absence and presence of Cl(-) ions under strictly controlled pH conditions. The data indicate that Cl(-)-binding accounts for a minority (~30%) of the total reduction in the oxygenation enthalpy manifested in bovine compared to human Hb, whereas the majority of this reduction is ascribable to structural differences, including increased ß-chain hydrophobicity that would increase the heat of oxygenation-linked conformational change in bovine Hb.

Repeated evolution of chimeric fusion genes in the ß-globin gene family of laurasiatherian mammals.

The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the ß-globin gene family of placental mammals, the two postnatally expressed δ- and ß-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian ß-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the ß-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20-100%) of ß-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion.

Catch the wave: prairie dogs assess neighbours' awareness using contagious displays.

The jump-yip display of black-tailed prairie dogs (Cynomys ludovicianus) is contagious, spreading through a prairie dog town as 'the wave' through a stadium. Because contagious communication in primates serves to assess conspecific social awareness, we investigated whether instigators of jump-yip bouts adjusted their behaviour relative to the response of conspecifics recruited to display bouts. Increased responsiveness of neighbouring town members resulted in bout initiators devoting a significantly greater proportion of time to active foraging. Contagious jump-yips thus function to assess neighbours' alertness, soliciting social information to assess effective conspecific group size in real time and reveal active probing of conspecific awareness consistent with theory of mind in these group-living rodents.

Multilocus phylogeny of talpine moles (Talpini, Talpidae, Eulipotyphla) and its implications for systematics.

The tribe Talpini is a group of strictly subterranean moles distributed across the Eurasian Continent whose phylogenetic relationships and taxonomy remain unresolved. Here we report a multi-locus nuclear-mitochondrial DNA dataset (9468 bp) from 11 talpine species encompassing all five recognized genera, together with analyses of their divergence times and evolutionary affinities inferred from maximum likelihood and Bayesian approaches. Our results finely resolved all relationships except the root of the four recognized Asian genera, which was placed sister to the genus Talpa. With respect to the Asian clade, we moreover provide the first molecular support for a sister-taxon relationship between Parascaptor and Scaptochirus and confirm that the genus Euroscaptor is paraphyletic. Further, and despite a relatively small sample size (22 specimens), our species delimitation analyses support the existence of at least two genetically distinct, and hence potentially cryptic species. Taken together, these findings argue that generic status should be given to E. mizura and illustrate that the taxonomic diversity of the tribe Talpini in mountainous regions of southwestern China and Southeast Asia is underestimated. Finally, results of our divergence time analyses support a rapid radiation of the endemic Asian genera in the late-Miocene, which temporally corresponds with enhanced aridity and cooling arising from a significant uplift of the Himalayan-Tibetan plateau.