Integrative Approaches to Understanding Organismal Responses to Aquatic Deoxygenation.

AbstractOxygen bioavailability is declining in aquatic systems worldwide as a result of climate change and other anthropogenic stressors. For aquatic organisms, the consequences are poorly known but are likely to reflect both direct effects of declining oxygen bioavailability and interactions between oxygen and other stressors, including two-warming and acidification-that have received substantial attention in recent decades and that typically accompany oxygen changes. Drawing on the collected papers in this symposium volume ("An Oxygen Perspective on Climate Change"), we outline the causes and consequences of declining oxygen bioavailability. First, we discuss the scope of natural and predicted anthropogenic changes in aquatic oxygen levels. Although modern organisms are the result of long evolutionary histories during which they were exposed to natural oxygen regimes, anthropogenic change is now exposing them to more extreme conditions and novel combinations of low oxygen with other stressors. Second, we identify behavioral and physiological mechanisms that underlie the interactive effects of oxygen with other stressors, and we assess the range of potential organismal responses to oxygen limitation that occur across levels of biological organization and over multiple timescales. We argue that metabolism and energetics provide a powerful and unifying framework for understanding organism-oxygen interactions. Third, we conclude by outlining a set of approaches for maximizing the effectiveness of future work, including focusing on long-term experiments using biologically realistic variation in experimental factors and taking truly cross-disciplinary and integrative approaches to understanding and predicting future effects.

Controversial Roles of Oxygen in Organismal Responses to Climate Warming.

AbstractDespite the global ecological importance of climate change, controversy surrounds how oxygen affects the fate of aquatic ectotherms under warming. Disagreements extend to the nature of oxygen bioavailability and whether oxygen usually limits growth under warming, explaining smaller adult size. These controversies affect two influential hypotheses: gill oxygen limitation and oxygen- and capacity-limited thermal tolerance. Here, we promote deeper integration of physiological and evolutionary mechanisms. We first clarify the nature of oxygen bioavailability in water, developing a new mass-transfer model that can be adapted to compare warming impacts on organisms with different respiratory systems and flow regimes. By distinguishing aerobic energy costs of moving oxygen from environment to tissues from costs of all other functions, we predict a decline in energy-dependent fitness during hypoxia despite approximately constant total metabolic rate before reaching critically low environmental oxygen. A new measure of oxygen bioavailability that keeps costs of generating water convection constant predicts a higher thermal sensitivity of oxygen uptake in an amphipod model than do previous oxygen supply indices. More importantly, by incorporating size- and temperature-dependent costs of generating water flow, we propose that oxygen limitation at different body sizes and temperatures can be modeled mechanistically. We then report little evidence for oxygen limitation of growth and adult size under benign warming. Yet occasional oxygen limitation, we argue, may, along with other selective pressures, help maintain adaptive plastic responses to warming. Finally, we discuss how to overcome flaws in a commonly used growth model that undermine predictions of warming impacts.

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 role of myoglobin in the evolution of mammalian diving capacity - The August Krogh principle applied in molecular and evolutionary physiology.

After the Devonian tetrapod land invasion, groups of terrestrial air-breathing and endothermic mammals repeatedly went back to live in the sea, relying on air intake at the surface for extended breath-hold dives to forage underwater, often at great depths and even in the coldest oceans. Studies on the physiological mechanisms behind prolonged breath-hold diving have a long history, including August Krogh's estimates of the maximal dive duration of the blue whale. Yet the molecular underpinnings of such extreme physiological adaptations are only beginning to be understood. The present review focuses on the molecular properties of the respiratory protein myoglobin that has repeatedly evolved an elevated net positive surface charge in several distantly related groups of diving mammals. This has enabled substantial increases of maximal myoglobin concentration in muscle cells, and hence muscle oxygen storage capacity and maximal dive duration. Using myoglobin net surface charge as a marker has allowed unprecedented insights into the evolution of mammal diving capacity and into the general mechanisms of adaptive protein evolution. From these findings it is argued, in an extension of the August Krogh principle, that for a large number of problems in molecular and evolutionary physiology there will be some protein of choice, or a few such proteins, on which it can be most conveniently studied.

Retinal oxygen supply shaped the functional evolution of the vertebrate eye.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

ATP-induced reversed thermal sensitivity of O2 binding in both major haemoglobin polymorphs of the non-endothermic Atlantic cod, Gadus morhua.

Atlantic cod is a species that is affected by climate change, with some populations being exposed to higher temperatures than others. The two polymorphs of its major haemoglobin type (HbI) show an inverse change in frequency along a latitudinal temperature cline in the North East Atlantic, which has been associated with differences in population performance and behavioural traits. An earlier study at the northern distribution limit of the species reported differential temperature sensitivities of red blood cell oxygen (O2) affinity between the northern cold-water HbI-2 polymorph and its southern, warm-water HbI-1 counter-part, which has since widely been held as adaptive for the species across its distributional range. The present study critically re-examined this hypothesis by comparing the thermal sensitivity of O2 binding in both purified HbI polymorphs from the southern, high-temperature distribution limit of the species under controlled conditions of allosteric modifiers of Hb function. Contrary to the prevailing view, the O2 affinity of the major HbI polymorphs did not differ from each other under any of the tested conditions. Depending on pH and ATP concentration, the temperature-sensitive and temperature-insensitive Hb-O2 affinity phenotypes - previously exclusively ascribed to HbI-1 and HbI-2, respectively - could be induced in both HbI polymorphs. These results are the first to establish a molecular mechanism behind a reversed temperature dependence of red blood cell O2 affinity in a non-endotherm fish and lay the basis for future studies on alternative mechanisms behind the differences in distribution, performance and behavioural traits associated with the different HbI polymorphs of Atlantic cod.

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.

Regulation of erythrocyte function: Multiple evolutionary solutions for respiratory gas transport and its regulation in fish.

Gas transport concepts in vertebrates have naturally been formulated based on human blood. However, the first vertebrates were aquatic, and fish and tetrapods diverged hundreds of millions years ago. Water-breathing vertebrates live in an environment with low and variable O2 levels, making environmental O2 an important evolutionary selection pressure in fishes, and various features of their gas transport differ from humans. Erythrocyte function in fish is of current interest, because current environmental changes affect gas transport, and because especially zebrafish is used as a model in biomedical studies, making it important to understand the differences in gas transport between fish and mammals to be able to carry out meaningful studies. Of the close to thirty thousand fish species, teleosts are the most species-numerous group. However, two additional radiations are discussed: agnathans and elasmobranchs. The gas transport by elasmobranchs may be closest to the ancestors of tetrapods. The major difference in their haemoglobin (Hb) function to humans is their high urea tolerance. Agnathans differ from other vertebrates by having Hbs, where cooperativity is achieved by monomer-oligomer equilibria. Their erythrocytes also lack the anion exchange pathway with profound effects on CO2 transport. Teleosts are characterized by highly pH sensitive Hbs, which can fail to become fully O2 -saturated at low pH. An adrenergically stimulated Na+ /H+ exchanger has evolved in their erythrocyte membrane, and plasma-accessible carbonic anhydrase can be differentially distributed among their tissues. Together, and differing from other vertebrates, these features can maximize O2 unloading in muscle while ensuring O2 loading in gills.

Extreme anoxia tolerance in crucian carp and goldfish through neofunctionalization of duplicated genes creating a new ethanol-producing pyruvate decarboxylase pathway.

Without oxygen, most vertebrates die within minutes as they cannot meet cellular energy demands with anaerobic metabolism. However, fish of the genus Carassius (crucian carp and goldfish) have evolved a specialized metabolic system that allows them to survive prolonged periods without oxygen by producing ethanol as their metabolic end-product. Here we show that this has been made possible by the evolution of a pyruvate decarboxylase, analogous to that in brewer's yeast and the first described in vertebrates, in addition to a specialized alcohol dehydrogenase. Whole-genome duplication events have provided additional gene copies of the pyruvate dehydrogenase multienzyme complex that have evolved into a pyruvate decarboxylase, while other copies retained the essential function of the parent enzymes. We reveal the key molecular substitution in duplicated pyruvate dehydrogenase genes that underpins one of the most extreme hypoxic survival strategies among vertebrates and that is highly deleterious in humans.

Life on the edge: O2 binding in Atlantic cod red blood cells near their southern distribution limit is not sensitive to temperature or haemoglobin genotype.

Atlantic cod are a commercially important species believed to be threatened by warming seas near their southern, equatorward upper thermal edge of distribution. Limitations to circulatory O2 transport, in particular cardiac output, and the geographic distribution of functionally different haemoglobin (Hb) genotypes have separately been suggested to play a role in setting thermal tolerance in this species. The present study assessed the thermal sensitivity of O2 binding in Atlantic cod red blood cells with different Hb genotypes near their upper thermal distribution limit and modelled its consequences for the arterio-venous O2 saturation difference, Sa-vO2 , another major determinant of circulatory O2 supply rate. The results showed statistically indistinguishable red blood cell O2 binding between the three HbI genotypes in wild-caught Atlantic cod from the Irish Sea (53° N). Red blood cells had an unusually low O2 affinity, with reduced or even reversed thermal sensitivity between pH 7.4 and 7.9, and 5.0 and 20.0°C. This was paired with strongly pH-dependent affinity and cooperativity of red blood cell O2 binding (Bohr and Root effects). Modelling of Sa-vO2  at physiological pH, temperature and O2 partial pressures revealed a substantial capacity for increases in Sa-vO2  to meet rising tissue O2 demands at 5.0 and 12.5°C, but not at 20°C. Furthermore, there was no evidence for an increase of maximal Sa-vO2  with temperature. It is suggested that Atlantic cod at such high temperatures may solely depend on increases in cardiac output and blood O2 capacity, or thermal acclimatisation of metabolic rate, for matching circulatory O2 supply to tissue demand.

Gene turnover in the avian globin gene families and evolutionary changes in hemoglobin isoform expression.

The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and ß-type subunits of hemoglobin (Hb), the tetrameric α2ß2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and ß-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the α(D)-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the α(A)-globin gene), recurrent losses of α(D)-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa.

Pronounced in vivo hemoglobin polymerization in red blood cells of Gulf toadfish: a general role for hemoglobin aggregation in vertebrate hemoparasite defense?

Two human hemoglobin (Hb) variants, Hb C and Hb S, are known to protect against Plasmodium falciparum malaria and have evolved repeatedly in malaria endemic areas. Both aggregate to insoluble crystals (Hb C) or polymers (Hb S) under certain physiological conditions, impair parasite growth, and may facilitate retention of infected red blood cells (RBCs) in the spleen. Given the profound effects of parasites on host evolution in general, and that RBC Hb concentration is often close to its solubility limit throughout vertebrates, similar mechanisms may operate in nonhuman vertebrates. Here we show exercise-induced, profound in vivo Hb polymerization in RBCs of the Gulf toadfish. Hb aggregation was closely associated with the extent of plasma acidosis, fully reversible, and without any signs of hemolysis or anemia. Our literature analysis suggests that aggregation prone Hbs may be relatively old, evolved multiple times in nonhuman vertebrates, show enhanced aggregation during hemoparasite infections, and can be uncovered in vivo by splenectomy. We discuss the working hypothesis that widespread Hb aggregation within several vertebrate groups may be the result of ongoing or past selection pressure against RBC parasites. Further comparative studies of these evolutionary old systems may provide valuable insights into hemoparasite susceptibility and reservoir potential of livestock and companion animals but also into human malaria and sickle cell disease.

Evolution of mammalian diving capacity traced by myoglobin net surface charge.

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.

Nitrite regulates hypoxic vasodilation via myoglobin-dependent nitric oxide generation.

BACKGROUND: Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for >100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO·) and thereby contribute to cardiomyocyte NO· signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO·. METHODS AND RESULTS: We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO· from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO·, and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin. Hypoxic vasodilation studies in myoglobin and endothelial and inducible NO synthase knockout models suggest that only myoglobin contributes to systemic hypoxic vasodilatory responses in mice. CONCLUSIONS: Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO· via the heme globin myoglobin enhances blood flow and matches O(2) supply to increased metabolic demands under hypoxic conditions.

Functional differentiation of myoglobin isoforms in hypoxia-tolerant carp indicates tissue-specific protective roles.

Because of a recent whole genome duplication, the hypoxia-tolerant common carp and goldfish are the only vertebrates known to possess two myoglobin (Mb) paralogs. One of these, Mb1, occurs in oxidative muscle but also in several other tissues, including capillary endothelial cells, whereas the other, Mb2, is a unique isoform specific to brain neurons. To help understand the functional roles of these diverged isoforms in the tolerance to severe hypoxia in the carp, we have compared their O(2) equilibria, carbon monoxide (CO) and O(2) binding kinetics, thiol S-nitrosation, nitrite reductase activities, and peroxidase activities. Mb1 has O(2) affinity and nitrite reductase activity comparable to most vertebrate muscle Mbs, consistent with established roles for Mbs in O(2) storage/delivery and in maintaining nitric oxide (NO) homeostasis during hypoxia. Both Mb1 and Mb2 can be S-nitrosated to similar extent, but without oxygenation-linked allosteric control. When compared with Mb1, Mb2 displays faster O(2) and CO kinetics, a lower O(2) affinity, and is slower at converting nitrite into NO. Mb2 is therefore unlikely to be primarily involved in either O(2) supply to mitochondria or the generation of NO from nitrite during hypoxia. However, Mb2 proved to be significantly faster at eliminating H(2)O(2,) a major in vivo reactive oxygen species (ROS), suggesting that this diverged Mb isoform may have a specific protective role against H(2)O(2) in the carp brain. This property might be of particular significance during reoxygenation following extended periods of hypoxia, when production of H(2)O(2) and other ROS is highest.

Diverse cell-specific expression of myoglobin isoforms in brain, kidney, gill and liver of the hypoxia-tolerant carp and zebrafish.

Myoglobin (Mb) is famous as a muscle-specific protein--yet the common carp expresses the gene (cMb1) encoding this protein in a range of non-muscle tissues and also expresses a novel isoform (cMb2) in the brain. Using a homologous antibody and riboprobes, we have established the relative amounts and cellular sites of non-muscle Mb expression in different tissues. The amounts of carp myoglobin (cMb) in supernatants of different tissues were just 0.4-0.7% relative to that of heart supernatants and were upregulated by two-to-four fold in liver, gill and brain following 5 days of hypoxic treatment. Brain exhibited both cMb proteins in western analysis, whereas all other tissues had only cMb1. We have also identified cells expressing cMb protein and cMb mRNA using immunohistology and RNA in situ hybridisation (RNA-ISH), respectively. Mb was strongly expressed throughout all cardiac myocytes and a subset of skeletal muscle fibres, whereas it was restricted to a small range of specific cell types in each of the non-muscle tissues. These include pillar and epithelial cells in secondary gill lamellae, hepatocytes, some neurones, and tubular epithelial cells in the kidney. Capillaries and small blood vessels in all tissues exhibited Mb expression within vascular endothelial cells. The cMb2 riboprobe located expression to a subset of neurones but not to endothelial cells. In zebrafish, which possesses only one Mb gene, a similar expression pattern of Mb protein and mRNA was observed. This establishes a surprisingly cell-specific distribution of Mb within non-muscle tissues in both carp and zebrafish, where it probably plays an important role in the regulation of microvascular, renal and brain function.

Ancient and modern duplication events and the evolution of stearoyl-CoA desaturases in teleost fishes.

Stearoyl-CoA desaturases (SCDs) are key enzymes of fatty acid biosynthesis whose regulation underpins responses to dietary, thermal, and hormonal treatment. Although two isoforms are known to exist in the common carp and human and four in mouse, there is no coherent view on how this gene family evolved to generate functionally diverse members. Here we identify numerous new SCD homologs in teleost fishes, using sequence data from expressed sequence tag (EST) and cDNA collections and genomic model species. Phylogenetic analyses of the deduced coding sequences produced only partially resolved molecular trees. The multiple SCD isoforms were, however, consistent with having arisen by an ancient gene duplication event in teleost fishes together with a more recent duplication in the tetraploid carp and possibly also salmonid lineages. Critical support for this interpretation comes from comparison across all vertebrate groups of the gene order in the genomic environments of the SCD isoforms. Using syntenically aligned chromosomal fragments from large-insert clones of common carp and grass carp together with those from genomically sequenced model species, we show that the ancient and modern SCD duplication events in the carp lineage were each associated with large chromosomal segment duplications, both possibly linked to whole genome duplications. By contrast, the four mouse isoforms likely arose by tandem duplications. Each duplication in the carp lineage gave rise to differentially expressed SCD isoforms, either induced by cold or diet as previously shown for the recent duplicated carp isoforms or tissue specific as demonstrated here for the ancient duplicate zebrafish isoforms.