Hemoglobin,from microorganisms to man: a single structural motif,multiple functions

Haemoglobins from unicellular organisms, plants or animals, share a common structure, which results from the folding, around the heme group, of a polypeptide chain made from 6-8 helices. Nowadays, deciphering the genome of several species allows one to draw the evolutionary tree of this protein going back to 1800 millions of years, at a time when oxygen began to accumulate in the atmosphere. This permits to follow the evolution of the ancestral gene and of its product. It is likely that, only in complex multicellular species, transport and storage of oxygen became the main physiological function of this molecule. In addition, in unicellular organisms and small invertebrates, it is likely that the main function of this protein was to protect the organism from the toxic effect of O2, CO and NO*. The very high oxygen affinity of these molecules, leading them to act rather as a scavenger as an oxygen carrier, supports this hypothesis. Haemoglobins from microorganisms, which may probably be the closest vestiges to the ancestral molecules, are divided into three families. The first one is made from flavohaemoglobins, a group of chimerical proteins carrying a globin domain and an oxido-reduction FAD-dependant domain. The second corresponds to truncated haemoglobins, which are hexacoordinated with very high oxygen-affinity molecules, 20-40 residues shorter than classical haemoglobins. The third group is made from bacterial haemoglobins such as that of Vitreoscilla. Some specific structural arrangements in the region surrounding the heme are cause of their high oxygen affinity. In plants, two types of haemoglobins are present (non-symbiotic and symbiotic), that arose from duplication of an ancestral vegetal gene. Non-symbiotic haemoglobins, which are probably the oldest, are scarcely distributed within tissues having high energetic consumption. Conversely, symbiotic haemoglobins (also named leghaemoglobins) are present at a high concentration (mM) mostly in the rhizomes of legumes, where they are involved in nitrogen metabolism. In some species, haemoglobin was proposed to be an oxygen sensor bringing to the organism information to adjust metabolism or biosynthesis to the oxygen requirement. Elsewhere haemoglobin may act as final electron acceptors in oxido-reduction pathways. Evolution of haemoglobin in invertebrates followed a large variety of scenarios. Some surprising functions as sulphide acquisition in invertebrates living near hydrothermal vents, or a role in the phototrophism of worm need to be mentioned. In invertebrates, the size of haemoglobin varies from monomers to giant molecules associating up to 144 subunits, while in vertebrates it is always a tetramer. In some species, several haemoglobins, with completely different structure and function, may coexist. This demonstrates how hazardous may be to extrapolate the function of a protein from only structural data.     

Organic phosphates in the red blood cells of fish

Fish are dependent on aerobic metabolism. They respond to changes in oxygen availability by a wide spectrum of compensatory and respiratory adjustments to safeguard tissue oxygenation. Such adjustments are directed to facilitate both oxygen uptake at the gas exchange surfaces and oxygen unloading to tissues. The importance of erythrocytic organic phosphates as regards oxygen transfer has been recognised since 1967 when the 'dramatic' effect of 2,3DPG on human haemoglobin was first reported. The present review examines the appearance of all the major erythrocytic organic phosphates during the evolutionary radiation of fish. In addition, it provides examples illustrating qualitative and quantitative ontogenetic changes of organic phosphates in the red blood cell of several fish species and describes their effects on oxygen affinities. The interaction of the organic phosphates with haemoglobins and divalent cations are also examined. Of particular interest is the regulation of erythrocytic organic phosphates according to both environmental and physiological conditions.     

Physical, biochemical and functional characterization of haemoglobin from three strains of Artemia

The brine shrimp, Artemia, an inhabitant of coastal and inland salterns, encounter fluctuations in the salinity which in turn influences the oxygen availability of their habitat. Hence, experiments were performed to analyze variations in haemoglobin structure and patterns of three strains of Artemia from South India and also to reflect the effect of varying oxygen levels in their habitat. Haemoglobins were purified on a DEAE-Sephadex column and haemoglobin types were analyzed by comparing their relative mobility on a non-denaturing medium. Furthermore, their molecular masses were determined by gel filtration in Sepharose column and by dodecylsulfate polyacrylamide gel electrophoresis. Results clearly reveal the presence of three distinct extracellular haemoglobins Hb I, Hb II and Hb III in Tuticorin strain while the other strains displayed only trails or the complete absence of Hb III and Hb II. Estimated molecular masses of these haemoglobins are 235,000-250,000 Da. Denaturation of the reduced and alkylated haemoglobins revealed apparently one polypeptide chain with a molecular mass of 124,000 Da. Upon denaturing gel electrophoresis of native haemoglobin Hb II, it was found that the 124,000 Da, polypeptide was cleaved specifically into two unequally-sized fragments of 50,400 and 79,800 Da. With regard to oxygen affinity, Hb III has a very high affinity for oxygen, an almost negligible Bohr effect and a good physiological adaptation to temperature changes. By combining the three haemoglobins in different proportions Artemia strains must be able to withstand diverging environmental conditions. In particular, the absence of Hb III in Puthalam and its occurrence as a faint band in Thamaraikulam could be correlated to the oxygen levels of their habitats.     

Nonsymbiotic haemoglobins in plants.

General aspects regarding the presence of nonsymbiotic haemoglobin in plants are presented with the emphasis on those related to its function. As it becomes apparent that the nonsymbiotic haemoglobins are widespread across the plant kingdom and that they represent a more primitive and evolutionary older form of the plant globin genes, the question of their function becomes more attractive. While the physiological functions of the symbiotic haemoglobins in plants are well understood, almost nothing is known about their nonsymbiotic predecessors. Therefore, the known and hypothetical functions of haemoglobins in various systems are described along with information concerning properties and the regulation of expression of the nonsymbiotic haemoglobins. Interestingly, a number of nonsymbiotic haemoglobins have been shown to be hypoxia-inducible. The spatial and temporal pattern of this induction in barley may suggest that it is an integral part of the plants response to limiting oxygen stress.     

Evolutionary trace analysis of plant haemoglobins: implications for site-directed mutagenesis

Haemoglobins are found ubiquitously in eukaryotes and many bacteria. In plants, haemoglobins were first identified in species, which can fix nitrogen via symbiosis with bacteria. Recent findings suggest that another class of haemoglobins termed as nonsymbiotic haemoglobins are present through out the plant kingdom and are expressed differentially during plant development. Limited data available suggests that non-symbiotic haemoglobins are involved in hypoxic stress and oversupply of nutrients. Due to lack of information on structurally conserved, functionally important residues in non-symbiotic haemoglobins, further studies to elucidate the molecular mechanisms underlying the biological role are hampered. To determine functionally important residues in non-symbiotic haemoglobins, I have analyzed a number of sequences from plant haemoglobin family, in the context of the known crystal structures of plant by evolutionary trace method. Results indicate that the, evolutionary trace method like conventional phylogentic analysis, could resolve phylogentic relationships between plant haemoglobin family. Evolutionary trace analysis has identified candidate functional (trace) residues that uniquely characterize the heme-binding pocket, dimer interface and possible novel functional surfaces. Such residues from specific three-dimensional clusters might be of functional importance in nonsymbiotic haemoglobins. These data, together with our improved knowledge of possible functional residues, can be used in future structure-function analysis experiments.     

Physiological properties of eel haemoglobin: hypoxic acclimation, phosphate effects and multiplicity.

Unlike the whole body oxygen affinity, which adapts readily to environmental oxygen tensions, haemoglobins, prepared from normoxic- and hypoxic-accimated eels (Anguilla anguilla) show no adaptive changes in oxygenation properties or in multiplicity. Hypoxic acclimation is, howeveer, accompanied by a strong decrease in red cell nucleoside triphosphates, particularly guanosine triphospphate (GTP), which depresses oxygen affinity of the composite and component haemoglobins more strongly than does the concurring ATP. The effects of pH, temperature and salts on the oxygenation properties of the (isolated) haemoglobins are reported, discussed in relation to the varying environmetal conditions encountered by eels, and compared with data on American and Japanese eels (A. rostrata and A. juponica, respectively.     

Can stochastic geographical evolution re‐create macroecological richness–environment correlations?

Aim Richness gradients are frequently correlated with environmental characteristics at broad geographic scales. In particular, richness is often associated with energy and climate, while environmental heterogeneity is rarely its best correlate. These correlations have been interpreted as evidence in favour of environmental determinants of diversity gradients, particularly energy and climate. This interpretation assumes that the expected‐by‐random correlation between richness and environment is zero, and that this is equally true for all environmental characteristics. However, these expectations might be unrealistic. We investigated to what degree basic evolutionary/biogeographical processes occurring independently of environment could lead to richness gradients that correlate with environmental characteristics by chance alone. Location Africa, Australia, Eurasia and the New World. Methods We produced artificial richness gradients based on a stochastic simulation model of geographic diversification of clades. In these simulations, species speciate, go extinct and expand or shift their distributions independently of any environmental characteristic. One thousand two hundred repetitions of this model were run, and the resulting stochastic richness gradients were regressed against real‐world environmental variables. Stochastic species–environment relationships were then compared among continents and among three environmental characteristics: energy, environmental heterogeneity and climate seasonality. Results Simulations suggested that a significant degree of correlation between richness gradients and environment is expected even when clades diversify and species distribute stochastically. These correlations vary considerably in strength; but in the best cases, environment can spuriously account for almost 80% of variation in stochastic richness. Additionally, expected‐by‐chance relationships were different among continents and environmental characteristics, producing stronger spurious relationships with energy and climate than with heterogeneity. Main conclusions We conclude that some features of empirical species–environment relationships can be reproduced just by chance when taking into account evolutionary/biogeographical processes underlying the construction of species richness gradients. Future tests of environmental effects on richness should consider structure in richness–environment correlations that can be produced by simple evolutionary null models. Research should move away from the naive non‐biological null hypotheses that are implicit in traditional statistical tests.     

Exploring taxonomic filtering in urban environments

Question: Several mechanisms have been proposed that control the spatio‐temporal pattern of species coexistence. Among others, the species pool hypothesis states that the large‐scale species pool is an important factor in controlling small‐scale species richness through filtering of species that can persist within a species assemblage on the basis of their tolerance of the abiotic environment. Because of the process of environmental filtering, co‐occurring species that experience similar environmental conditions are likely to be more taxonomically similar than ecologically distant species. This is because, due to the conservatism of many species traits during evolutionary diversification, the ability of species to colonize the same ecological space is thought to depend at least partially on their taxonomic similarity. The question for this study is: Under the assumption of trait conservatism, does environmental filtering lead to nonrandom species assemblages with respect to their taxonomic structure? Methods: The significance of taxonomic filtering in regulating species coexistence is tested using data from 15 local species assemblages from the urban flora of Rome (Italy). To find out whether the taxonomic structure of the selected’ local’ species assemblages was significantly different from random, we used a Monte Carlo simulation in which for each local species assemblage, the actual taxonomic diversity was compared to the taxonomic diversity of 1000 virtual species lists of the same size extracted at random from a larger ‘regional’ species pool. Results: We found that in most cases the local species assemblages have a higher degree of taxonomic similarity than would be expected by chance showing a phenomenon of ‘species condensation’ in a small number of higher‐level taxa. Conclusions: Our observations support the species pool hypothesis and imply that environmental filtering is an important mechanism in shaping the taxonomic structure of species assemblages. Therefore, the incorporation of taxonomic diversity into landscape and community ecology may be beneficial for a better understanding of the processes that regulate species coexistence.     

Molecular modeling and small angle X-ray scattering studies of Hoplosternum littorale cathodic haemoglobin

Considerable interest is currently focused on fish haemoglobins in order to identify the structural basis for their diversity of functional behavior. Hoplosternum littorale is a catfish that presents bimodal gill (water)/gut (air)-breathing, which allows this species to survive in waters with low oxygen content. The hemolysate of this fish showed the presence of two main haemoglobins, cathodic and anodic. This work describes structural features analyzed here by integration of molecular modeling with small angle X-ray scattering. Here is described a molecular model for the cathodic haemoglobin in the unliganded and liganded states. The models were determined by molecular modeling based on the high-resolution crystal structure of fish haemoglobins. The structural models for both forms of H. littorale haemoglobin were compared to human haemoglobin.     

Intraspecific genotypic diversity in plants

Variations in the nuclear DNA, mainly as a result of quantitative modulations of DNA repeats belonging to different sequence families of satellite DNA and to the activity of transposable elements, have been assessed within several angiosperm species. These variations alter the amount and organization of the DNA and therefore the genotype, rather than the genome proper. They take place on an evolutionary time scale as the result of selection processes after the occurrence of uncontrolled events in the genome or may be due to direct responses of plant genomes to environmental stimuli that occur under plant-level control within a short developmental period of a single generation. These DNA changes are correlated to changes in the developmental dynamics and phenotypic characteristics of the plants, and the capability to carry out genotypic variation is an evolutionary trait that allows plant species to adapt to different environmental conditions, as well as to the variability of conditions in a given environment. The link between developmental and environmental stimuli and repetitive DNA that elicits the intraspecific diversity of plant genotypes may provide models of evolutionary change that extend beyond the conventional view of evolution by allelic substitution and take into account epigenetic effects of the genome structure.     

Environmental heterogeneity does not affect levels of phenotypic plasticity in natural populations of three Drosophila species

Adaptation of natural populations to variable environmental conditions may occur by changes in trait means and/or in the levels of plasticity. Theory predicts that environmental heterogeneity favors plasticity of adaptive traits. Here we investigated the performance in several traits of three sympatric Drosophila species freshly collected in two environments that differ in the heterogeneity of environmental conditions. Differences in trait means within species were found in several traits, indicating that populations differed in their evolutionary response to the environmental conditions of their origin. Different species showed distinct adaptation with a very different role of plasticity across species for coping with environmental changes. However, geographically distinct populations of the same species generally displayed the same levels of plasticity as induced by fluctuating thermal regimes. This indicates a weak and trait‐specific effect of environmental heterogeneity on plasticity. Furthermore, similar levels of plasticity were found in a laboratory‐adapted population of Drosophila melanogaster with a common geographic origin but adapted to the laboratory conditions for more than 100 generations. Thus, this study does not confirm theoretical predictions on the degree of adaptive plasticity among populations in relation to environmental heterogeneity but shows a very distinct role of species‐specific plasticity.     

Molecular evolution of haemoglobins of polar fishes

The Arctic and the Antarctic differ by age and isolation of the respective marine faunas. Antarctic fish are highly stenothermal, in response to stable water temperatures, whereas the Arctic ones are exposed to seasonal and latitudinal temperature variations. The knowledge of the mechanisms of phenotypic response to cold exposure in species of both polar habitats offers fundamental insights into the nature of environmental adaptation. In the process of cold adaptation, the evolutionary trend of Antarctic fish has led to unique specialisations, including modification of haematological characteristics, e.g. decreased amounts and multiplicity of haemoglobins.Unlike Antarctic Notothenioidei, Arctic teleosts have high haemoglobin multiplicity. Although the presence of functionally and structurally distinct haemoglobins is a plesiomorphic condition for many perciform-like fishes, it seems that the oxygen-transport system of teleost fish in the Arctic region has been adjusted to temperature differences and fluctuations of Arctic waters, much larger than in the Antarctic. The amino-acid sequences used to gain insight into the evolution history of α and β globins of polar fish have clearly shown that Antarctic and Arctic globins have different phylogenies, leading to the hypothesis that the selective pressure of environment stability allows the phylogenetic signal to be maintained in the Antarctic sequences, whereas environmental variability would tend to disrupt this signal in Arctic sequences.     

Opinion: Is gene mapping in wild populations useful for understanding and predicting adaptation to global change?

Changing environmental conditions will inevitably alter selection pressures. Over the long term, populations have to adapt to these altered conditions by evolutionary change to avoid extinction. Quantifying the ‘evolutionary potential’ of populations to predict whether they will be able to adapt fast enough to forecasted changes is crucial to fully assess the threat for biodiversity posed by climate change. Technological advances in sequencing and high‐throughput genotyping have now made genomic studies possible in a wide range of species. Such studies, in theory, allow an unprecedented understanding of the genomics of ecologically relevant traits and thereby a detailed assessment of the population's evolutionary potential. Aimed at a wider audience than only evolutionary geneticists, this paper gives an overview of how gene‐mapping studies have contributed to our understanding and prediction of evolutionary adaptations to climate change, identifies potential reasons why their contribution to understanding adaptation to climate change may remain limited, and highlights approaches to study and predict climate change adaptation that may be more promising, at least in the medium term.     

Alteration of functional properties associated with the change in quaternary structure in unliganded haemoglobin.

The preparation of a number of specifically modified haemoglobins lacking various C-terminal residues is described. These haemoglobins can be changed from the unliganded R (or oxy type) quaternary structure to the unliganded T (or deoxy type) on addition of inositol hexaphosphate. This paper shows that this transition is associated with a lowering of the oxygen affinity and an increase in the Hill's coefficient, n, except in the case of des-(Arg141α, Tyr140α) haemoglobin where addition of inositol hexaphosphate lowers the oxygen affinity but does not increase the Hill's coefficient, n. This shows that Tyr140α plays a more important role than Tyr145β in generating co-operativity. The transition between unliganded R and unliganded T is associated with a lowering of the reactivity of the sulphydryl group Cys93β; this is due both to the change in quaternary structure per se and to the formation of the salt bridge between His146β and Asp94β. The Bohr effect associated with the transition from the unliganded to liganded R structure was less than one-tenth of the normal Bohr effect.     

Interactions retain the co‐phylogenetic matching that communities lost

Both species and their interactions are affected by changes that occur at evolutionary time‐scales, and these changes shape both ecological communities and their phylogenetic structure. That said, extant ecological community structure is contingent upon random chance, environmental filters and local effects. It is therefore unclear how much ecological signal local communities should retain. Here we show that, in a host–parasite system where species interactions vary substantially over a continental gradient, the ecological significance of individual interactions is maintained across different scales. Notably, this occurs despite the fact that observed community variation at the local scale frequently tends to weaken or remove community‐wide phylogenetic signal. When considered in terms of the interplay between community ecology and coevolutionary theory, our results demonstrate that individual interactions are capable and indeed likely to show a consistent signature of past evolutionary history even when woven into communities that do not.     

Recurrent polymorphic mating type variation in Madagascan Bulbophyllum species (Orchidaceae) exemplifies a high incidence of auto‐pollination in tropical orchids

The transition from outcrossing to self‐fertilization is one of the most common evolutionary changes in angiosperms. The orchid family exemplifies this evolutionary trend but, because of a general lack of large‐scale surveys on auto‐pollination in orchid taxa, the incidence and modes of auto‐pollination among (sub)tropical orchids remain poorly known. In the present study, we assessed the frequency and mode of auto‐pollination within and among species of a largely monophyletic group of Madagascan Bulbophyllum. The capacity for autonomous fruit set was investigated by bagging experiments in the greenhouse and the field, complemented with detailed floral micromorphological studies of the gynostemium. Our survey comprises 393 accessions, representing at least 78 species, and thus approximately 37% of the species diversity of the genus in the Madagascan region. Our studies revealed that mating type is directly related to gynostemium structure, most often involving the presence or absence of a physical barrier termed ‘rostellum’. As a novel and unexpected finding, we identified eight species of a single lineage of Madagascan Bulbophyllum (termed ‘clade C’), in which auto‐pollinating morphs (selfers), either lacking a rostellum or (rarely) possessing a stigmatic rostellum, co‐exist with their pollinator‐dependent conspecifics (outcrossers). We hypothesize that auto‐pollination via rostellum abortion has a simple genetic basis, and probably evolved rapidly and recurrently by subtle changes in the timing of rostellum development (heterochrony). Thus, species of clade C may have an intrinsic genetic and developmental lability toward auto‐pollination, allowing rapid evolutionary response under environmental, perhaps human‐disturbed conditions favouring reproductive assurance. Overall, these findings should stimulate further research on the incidence, evolution, and maintenance of mating type variation in tropical orchids, as well as how they adapt(ed) to changing environmental conditions. © 2014 The Authors. Botanical Journal of the Linnean Society published by John Wiley & Sons Ltd on behalf of The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 242–258.     

Ecological differentiation between the Sardinian endemic Maniola nurag and the pan-European M. jurtina

Recently refined evolutionary theories have highlighted that ecological interactions and environmental gradients can play a major role in speciation. This paper reports on a 3‐year field study, in which the ecology of two congeneric butterfly species was used to explore and compare the environmental factors determining their spatial distribution. These data are discussed in the context of possible speciation scenarios between the Sardinian populations of Maniola nurag and M. jurtina. M. nurag is endemic to the island of Sardinia, while M. jurtina is widespread over Europe. In Sardinia, the two species are locally sympatric. Mark–release–recapture experiments were combined with measures of environmental variables in 15 1‐ha plots, established in areas of potential habitat for the butterflies. Constrained linear models were parameterized from mark–recapture data to estimate both individual (survival and capture probabilities) and population (population size and recruitment) parameters. The two species had similar demography, movement patterns, life history, and behaviour. Population sizes developed in a parabolic fashion from beginning to end of the flight season. Differences included local population size, adult phenology, and habitat requirements. Long‐distance movements larger than 1.5 km were observed, suggesting a substantial amount of gene‐flow between populations of the endemic as well as the widespread species. Multivariate analyses revealed four main environmental gradients responsible for the abundance of the butterflies in an area. Both species responded similarly to environmental variables. However, each species’s abundance was correlated with a different environmental gradient determined by vegetation cover and structure. When sympatric, the two species responded to subtle differences in microhabitat structure. This might originally have induced their divergence. This study is an example of how empirical field data on population dynamics, dispersal, and habitat characteristics of two sympatric congeners can further our understanding of how species differentiate despite existing gene‐flow. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89 , 561–574.     

Human‐modified habitats change patterns of population genetic structure and group relatedness in Peter's tent‐roosting bats

Although coloniality is widespread among mammals, it is still not clear what factors influence composition of social groups. As animals need to adapt to multiple habitat and environmental conditions throughout their range, variation in group composition should be influenced by adaptive adjustment to different ecological factors. Relevant to anthropogenic disturbance, increased habitat modification by humans can alter species’ presence, density, and population structure. Therefore, it is important to understand the consequences of changes to landscape composition, in particular how habitat modification affects social structure of group‐forming organisms. Here, we combine information on roosting associations with genetic structure of Peter's tent‐roosting bats, Uroderma bilobatum to address how different habitat characteristics at different scales affect structure of social groups. By dividing analyses by age and sex, we determined that genetic structure was greater for adult females than adult males or offspring. Habitat variables explained 80% of the variation in group relatedness (mainly influenced by female relatedness) with roost characteristics contributing the most explained variation. This suggests that females using roosts of specific characteristics exhibit higher relatedness and seem to be philopatric. These females mate with more males than do more labile female groups. Results describe ecological and microevolutionary processes, which affect relatedness and social structure; findings are highly relevant to species distributions in both natural and human‐modified environments.     

Multiple globins and haemoglobins in the bass, Dicentrarchus labrax (L.) (Serranidae: Teleostei)

The haemoglobins and globins of bass, Dicentrarchus labrax (L.) have been studied by starch and polyacrylamide gel electrophoresis. Five haemoglobin components were found. These haemoglobins appear to result from the combination of four different globin monomers. The molecular weight of the pooled haemoglobin is about 54 400, confirming its tetrameric form. The evolutionary significance of multiple haemoglobins is discussed.