Acentuada diferenciação regional numa espécie de caranguejo norte americana ao longo da sua distribuição de origem e em estuários europeus: análise filogeográfica e considerações ecológicas

Rhithropanopeus harrisii (Gould 1841) tem uma distribuição nativa desde o Canadá até ao México e é considerada uma espécie invasora nos rios e estuários da costa noroeste e sudeste do continente americano e na costa Europeia, onde foi descoberto antes de 1874, na Holanda. Neste estudo uma parte do gene citocrómio oxidase subunidade I foi analisada em populações dos EUA e da Europa, de modo a obter a informação sobre a diversidade genética das populações e sobre uma potencial população de origem. Análises morfométricas foram efetuadas através de medições da carapaça. Um gargalo genético deverá ter ocorrido aquando da colonização Europeia e Nova Jersey é a população que mais se assemelha geneticamente à potencial população de origem. A heterogeneidade genética ao longo da costa este dos EUA e da costa Europeia tomou-se evidente. Pela análise morfométrica não foi possível discriminar as populações. Verifica-se que esta espécie ainda se encontra em expansão e que pode usar vários vetores de invasão. /ABSTRACT - Rhithropanopeus harrisii (Gould 1841) has a native distribution from Canada to Mexico and is considered an invasive species in northwestem American, southeastem American and European estuaries and rivers. In Europe it was observed for the first time before 1874, in The Netherlands. In this study we analyzed a part of cytochrome oxidase subunit I gene of populations from the east coast of United States of America and from Europe, in order to assess to a the genetic diversity of the populations and to determine a potential founder population. Morphometric analyses were also performed using measurements of the carapace. A genetic bottleneck might have occurred during the European colonization. New Jersey is the most similar population to the potential founding population and there is evidence of genetic heterogeneity along the North American east coast and European coast. It was not possible to discriminate all populations morphometrically. R. harrisii is still in expansion and might use several invasion mechanisms

Epistasis Constrains Mutational Pathways of Hemoglobin Adaptation in High-Altitude Pikas

A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. This question can be addressed most directly by using site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here, we evaluate how pleiotropic trade-offs and epistatic interactions influence the accessibility of alternative mutational pathways during the adaptive evolution of hemoglobin (Hb) function in high-altitude pikas (Mammalia: Lagomorpha). By combining ancestral protein resurrection with a combinatorial protein-engineering approach, we examined the functional effects of sequential mutational steps in all possible pathways that produced an increased Hb–O2 affinity. These experiments revealed that the effects of mutations on Hb–O2affinity are highly dependent on the temporal order in which they occur: Each of three -β chain substitutions produced a significant increase in Hb–O2 affinity on the ancestral genetic background, but two of these substitutions produced opposite effects when they occurred as later steps in the pathway. The experiments revealed pervasive epistasis for Hb–O2 affinity, but affinity-altering mutations produced no significant pleiotropic trade-offs. These results provide insights into the properties of adaptive substitutions in naturally evolved proteins and suggest that the accessibility of alternative mutational pathways may be more strongly constrained by sign epistasis for positively selected biochemical phenotypes than by antagonistic pleiotropy

Integrating Evolutionary and Functional Tests of Adaptive Hypotheses: A Case Study of Altitudinal Differentiation in Hemoglobin Function in an Andean Sparrow, \u3ci\u3eZonotrichia capensis\u3c/i\u3e

In air-breathing vertebrates, the physiologically optimal blood-O2 affinity is jointly determined by the prevailing partial pressure of atmospheric O2, the efficacy of pulmonary O2 transfer, and internal metabolic demands. Consequently, genetic variation in the oxygenation properties of hemoglobin (Hb) may be subject to spatially varying selection in species with broad elevational distributions. Here we report the results of a combined functional and evolutionary analysis of Hb polymorphism in the rufouscollared sparrow (Zonotrichia capensis), a species that is continuously distributed across a steep elevational gradient on the Pacific slope of the Peruvian Andes. We integrated a population genomic analysis that included all postnatally expressed Hb genes with functional studies of naturally occurring Hb variants, as well as recombinant Hb (rHb) mutants that were engineered through site-directed mutagenesis. We identified three clinally varying amino acid polymorphisms: Two in the αA-globin gene, which encodes the α-chain subunits of the major HbA isoform, and one in the αD-globin gene, which encodes the α-chain subunits of the minor HbD isoform. We then constructed and experimentally tested single- and double-mutant rHbs representing each of the alternative αA-globin genotypes that predominate at different elevations. Although the locusspecific patterns of altitudinal differentiation suggested a history of spatially varying selection acting on Hb polymorphism, the experimental tests demonstrated that the observed amino acid mutations have no discernible effect on respiratory properties of the HbA or HbD isoforms. These results highlight the importance of experimentally validating the hypothesized effects of genetic changes in protein function to avoid the pitfalls of adaptive storytelling

Epistasis Constrains Mutational Pathways of Hemoglobin Adaptation in High-Altitude Pikas

A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. This question can be addressed most directly by using site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here, we evaluate how pleiotropic trade-offs and epistatic interactions influence the accessibility of alternative mutational pathways during the adaptive evolution of hemoglobin (Hb) function in high-altitude pikas (Mammalia: Lagomorpha). By combining ancestral protein resurrection with a combinatorial protein-engineering approach, we examined the functional effects of sequential mutational steps in all possible pathways that produced an increased Hb–O2 affinity. These experiments revealed that the effects of mutations on Hb–O2affinity are highly dependent on the temporal order in which they occur: Each of three -β chain substitutions produced a significant increase in Hb–O2 affinity on the ancestral genetic background, but two of these substitutions produced opposite effects when they occurred as later steps in the pathway. The experiments revealed pervasive epistasis for Hb–O2 affinity, but affinity-altering mutations produced no significant pleiotropic trade-offs. These results provide insights into the properties of adaptive substitutions in naturally evolved proteins and suggest that the accessibility of alternative mutational pathways may be more strongly constrained by sign epistasis for positively selected biochemical phenotypes than by antagonistic pleiotropy

Evolution des adaptations respiratoires chez les Polynoidae (Polychaeta) des sources hydrothermales

Hydrothermal vents are deep-sea ecosystems that are characterized by widely changing chemical and physical characteristics that are the result of the chaotic mixing of the hydrothermal fluid, rich in toxic compounds, such as sulfide and heavy metals, with low pH, high temperature and no oxygen; with the deep-sea water that contains no heavy metals, has a pH of ~7.8, low temperature and normal oxygen concentrations. Among others constraints, hypoxia and anoxia pose a serious problem for the fauna colonizing the hydrothermal ecosystems.Scale-worms (Polynoidea) living at hydrothermal vents are abundant, diverse and widely distributed in the range of chemical and thermal conditions. To obtain the benefits of living there (abundant local primary production), they need to cope with the harsh conditions that characterize these ecosystems. Respiratory pigments play a central function in the adaptation to hypoxia (uptake, transport and storage of the oxygen). Hydrothermal vent polynoids possess hemoglobins, a feature that clearly sets them aside from shallow water relatives. These hemoglobins are unique among annelids and their evolutionary history was the interest of this work. There are two main types of hemoglobins in hydrothermal vent polynoids: single- and tetra-domain globins. Although they are extracellular, both globins types are more closely related to intracellular globins that to the typical annelid extracellular ones. This indicates a distinct origin for these hemoglobins. We first studied the evolutionary history of the multi-domain gene hemoglobin and found out that it originates from the tandem duplication of a myoglobin/like ancestral. Both globin types possess residues in the heme pocket that have been shown to be responsible for the high oxygen affinity in the nematode Ascaris hemoglobin. These amino acids are also found in the globin from Harmothea, a non-vent-endemic species. This indicates that the adaptative value of these hemoglobins most likely resides in their expression at high levels in the body and did not require specific adaptations in the heme pocket in the lineage to give rise to all the vent species. In addition, some amino acid sites were shown to be under positive selection in some lineages. Some of these amino acids are located in the heme pocket where they will likely affect the functional properties and potentially provide protection against sulfide. The other amino acids under positive selection are located in areas where they will probably affect interactions between subunits for the single-domain globins, and between domains for the tetra-domain globins.Les sources hydrothermales sont des écosystèmes profonds qui sont caractérisés par une large et variable caractéristiques chimiques et physiques, résultat du mélange chaotique du fluide hydrothermal, riche en composés toxiques, tels que le sulfide et les métaux lourds, avec un pH bas, une température élevée et pas d'oxygène; avec une eau profonde ne contenant pas de métaux lourds, qui a un pH ~7.8, une basse température et une concentration en oxygène normale. Parmi d'autres contraintes, l'hypoxie et l'anoxie posent un sérieux problème à la faune colonisant les écosystèmes hydrothermales.Les annélides (Polynoidea), vivant près des sources hydrothermales, sont abondants, variés et largement répandu dans la variété des conditions chimiques et thermiques. Pour obtenir les avantages de vivre dans ce lieu (abondante production primaire locale), ils doivent surmonter les conditions difficiles qui caractérisent ces écosystèmes. Les pigments respiratoires jouent un rôle central dans l'adaptation à l'hypoxie (assimilation, transport et stockage de l'oxygène). Les Polynoidea des sources hydrothermales ont des hémoglobines, une particularité qui les distingue clairement de leurs proches en eaux peu profondes. Ces hémoglobines sont uniques parmi les annélides, leur évolution était l'objet de ce travail.Il y a deux principaux types d'hémoglobines chez les polynoids des sources hydrothermales : les globines "classiques" et les globines tétradomaines. Bien qu'elles soient extracellulaires, ces deux types de globines sont plus proches des globines intracellulaires que celles extracellulaires d'un annélide typique. Cela indique une origine distinct entre ces hémoglobines. Nous avons d'abord étudié l'évolution de ces gènes hémoglobines tétradomaines et nous avons découvert qu'elles étaient à l'origine d'une duplication en tandem entre une myoglobine-genre ancestral.Toutes deux possèdent des résidus dans la poche de l'hème qui ont indiqué être responsable de l'affinité forte pour l'oxygène dans l’hémoglobine du nématode Ascaris. Ces acides aminés sont aussi découverts dans les globines de Harmothea, une espèce non endémique aux sources hydrothermales. Cela indique que la valeur adaptative de ces hémoglobines résident sans doute dans l'expression à des niveaux élevés dans le corps et n'exigent pas d'adaptations spécifiques dans la poche de l'hème à l'origine de la lignée des espèces hydrothermales. De plus, certaines de ces acides aminés sont présents sous sélection positive dans certaines lignées. Certains de ces acides aminés sont localisés dans la poche de l'hème où ils sont probablement affectés par les propriétés fonctionnelles et potentiellement protégés contre le sulfide. Les autres acides aminés sous sélection positive sont localisés dans des zones où ils interagissent probablement entre les sous-unités des globines classiques et entre les domaines des globines tétradomaines

Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds

The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O2 affinity in the presence of allosteric effectors such as organic phosphates and Cl− ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O2 affinity stems primarily from changes in the O2 association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene that is shared with the embryonic α-like globin gene. Includes supplemental tables & references

Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different altitudinal ranges

Semi-fossorial ground squirrels face challenges to respiratory gas transport associated with the chronic hypoxia and hypercapnia of underground burrows, and such challenges are compounded in species that are native to high altitude. During hibernation, such species must also contend with vicissitudes of blood gas concentrations and plasma pH caused by episodic breathing. Here, we report an analysis of hemoglobin (Hb) function in six species of marmotine ground squirrels with different altitudinal distributions. Regardless of their native altitude, all species have high Hb–O2 affinities, mainly due to suppressed sensitivities to allosteric effectors [2,3-diphosphoglycerate (DPG) and chloride ions]. This suppressed anion sensitivity is surprising given that all canonical anion-binding sites are conserved. Two sciurid species, the golden-mantled and thirteen-lined ground squirrel, have Hb–O2 affinities that are characterized by high pH sensitivity and low thermal sensitivity relative to the Hbs of humans and other mammals. The pronounced Bohr effect is surprising in light of highly unusual amino acid substitutions at the C-termini that are known to abolish the Bohr effect in human HbA. Taken together, the high O2 affinity of sciurid Hbs suggests an enhanced capacity for pulmonary O2 loading under hypoxic and hypercapnic conditions, while the large Bohr effect should help to ensure efficient O2 unloading in tissue capillaries. In spite of the relatively low thermal sensitivities of the sciurid Hbs, our results indicate that the effect of hypothermia on Hb oxygenation is the main factor contributing to the increased blood–O2 affinity in hibernating ground squirrels

Repeated elevational transitions in hemoglobin function during the evolution of Andean hummingbirds

Animals that sustain high levels of aerobic activity under hypoxic conditions (e.g., birds that fly at high altitude) face the physiological challenge of jointly optimizing blood-O2 affinity for O2 loading in the pulmonary circulation and O2 unloading in the systemic circulation. At high altitude, this challenge is especially acute for small endotherms like hummingbirds that have exceedingly high mass-specific metabolic rates. Here we report an experimental analysis of hemoglobin (Hb) function in South American hummingbirds that revealed a positive correlation between Hb-O2 affinity and native elevation. Protein engineering experiments and ancestral- state reconstructions revealed that this correlation is attributable to derived increases in Hb-O2 affinity in highland lineages, as well as derived reductions in Hb-O2 affinity in lowland lineages. Site-directed mutagenesis experiments demonstrated that repeated evolutionary transitions in biochemical phenotype are mainly attributable to repeated amino acid replacements at two epistatically interacting sites that alter the allosteric regulation of Hb-O2 affinity. These results demonstrate that repeated changes in biochemical phenotype involve parallelism at the molecular level, and that mutations with indirect, second-order effects on Hb allostery play key roles in biochemical adaptation. Includes supplementary materials

Integrating Evolutionary and Functional Tests of Adaptive Hypotheses: A Case Study of Altitudinal Differentiation in Hemoglobin Function in an Andean Sparrow, \u3ci\u3eZonotrichia capensis\u3c/i\u3e

In air-breathing vertebrates, the physiologically optimal blood-O2 affinity is jointly determined by the prevailing partial pressure of atmospheric O2, the efficacy of pulmonary O2 transfer, and internal metabolic demands. Consequently, genetic variation in the oxygenation properties of hemoglobin (Hb) may be subject to spatially varying selection in species with broad elevational distributions. Here we report the results of a combined functional and evolutionary analysis of Hb polymorphism in the rufouscollared sparrow (Zonotrichia capensis), a species that is continuously distributed across a steep elevational gradient on the Pacific slope of the Peruvian Andes. We integrated a population genomic analysis that included all postnatally expressed Hb genes with functional studies of naturally occurring Hb variants, as well as recombinant Hb (rHb) mutants that were engineered through site-directed mutagenesis. We identified three clinally varying amino acid polymorphisms: Two in the αA-globin gene, which encodes the α-chain subunits of the major HbA isoform, and one in the αD-globin gene, which encodes the α-chain subunits of the minor HbD isoform. We then constructed and experimentally tested single- and double-mutant rHbs representing each of the alternative αA-globin genotypes that predominate at different elevations. Although the locusspecific patterns of altitudinal differentiation suggested a history of spatially varying selection acting on Hb polymorphism, the experimental tests demonstrated that the observed amino acid mutations have no discernible effect on respiratory properties of the HbA or HbD isoforms. These results highlight the importance of experimentally validating the hypothesized effects of genetic changes in protein function to avoid the pitfalls of adaptive storytelling