the evolution of multiple haemoglobins in fishes

Reviews in Fish Biology and Fisheries, 5,304-319 (1995)

The evolution of multiple haemoglobins in fishes

J U L I O P I ~ R E Z 1, K E N T R Y L A N D E R 2 * a n d M A U R O N I R C H I O 3

l lnstituto Oceanografico de Venezuela, Universidad de Oriente, Apartado de Correos 245, Cumana, Venezuela 2Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, USA 3 Escuela de C~encias Aplicadas del Mar, Universidad de Oriente, Isla de Margarita, Venezuela

Contents

Introduction The Bohr and Root effects in teleost haemoglobins Evolution of protein polymorphism

The selectionist hypothesis and haemoglobin multiplicity Functional heterogeneity of haemoglobin components in fishes Haemoglobin multiplicity and environment Haemoglobin multiplicity and levels of activity Changes in haemoglobin multiplicity due to temperature variation

The neutralist hypothesis and amino acid substitution Constancy of evolutionary rate Functional constraints of evolutionary rate DNA sequence data

Concluding remarks and summary Acknowledgements References

page 304 305 306

314 315 316

Introduction

The great diversity of haemoglobins in fishes (much greater than in other vertebrate groups) is indicated by the higher frequency of multiple haemoglobins and haemoglobin polymorphism (reviews: Brittain, 1991; Di Prisco and Tamburrini, 1992). It has been suggested that this phenomenon reflects the diversity of ways fishes adapt to their environments, a point of view supported by studies showing more haemoglobins in fishes from thermolabile than from thermostable environments (Sullivan, 1977). While this explanation may be adequate for fishes under some environmental conditions, it may be insufficient to explain the high level of haemoglobin diversity in all fishes. For example, P6rez and Rylander (1985) found that the mean number of haemoglobins and the incidence of polymorphism was almost as high in tropical fishes living in a relatively thermostable environment as in temperate species.

If multiple haemoglobins are the result of selective pressures, then their physiological properties must be different as well as be correlated with specific environmental parameters. However, the mechanisms enabling fishes to adapt to their environment

*Author to whom correspondence should be addressed.

0960-3166 (~) 1995 Chapman & Hall

Multiple haemoglobins in fishes 305

may not be simple, For example, an increase in water temperature may reduce available oxygen by increasing the biological activity of bacteria, plankton, etc., decreasing the solubility of oxygen, decreasing the affinity of haemoglobin for oxygen, and increasing the fish's own oxygen requirements.

Fishes may respond to reduced available oxygen behaviourally by migrating to more favourable habitats. They may respond physiologically by (1) increasing ventilation volume, heart rate, haematocrit, haemoglobin and the density of erythrocytes; (2) increasing haemoglobin affinity by changing intracellular levels of altosteric modifiers; or (3) evolving haemoglobins the affinities of which are unaffected by temperature changes.

An interesting strategy for responding to variable oxygen availability is exhibited by a toadfish (Thalasophryne maculosa, Batrachoididae). This species is polymorphic with respect to haemoglobin affinity (P6rez, 1986), and the heterozygote, being intermediate in its haemoglobin affinity, appears best adapted for both oxygen-rich and oxygen-poor waters. It has a higher haemoglobin affinity than the homozygote with the lowest affinity; yet it has a greater efficiency of oxygen release in its tissues than the homozygote having the highest affinity.

The above examples illustrate the diversity of the physiological mechanisms in fishes for supplying tissues with sufficient concentrations of oxygen. In this review we will summarize the occurrence of multiple haemoglobins in fishes and discuss their evolution from a selectionist and from a neutralist point of view.

The Bohr and Root effects in teleost haemoglobins

Among the numerous haemoglobins in teleosts which appear to offer no selective advantages (see discussion of neutralist hypothesis below), two properties characteristic of some haemoglobins, the Bohr effect and the Root effect, do appear to be subjected to evolutionary pressures. The Bohr and Root effects refer to the effect of pH (concentration of protons) on haemoglobin oxygen affinity. In teleosts, an increase in pH above about pH 6.5 results in increased oxygen affinity (Bohr effect, or alkaline Bohr effect), and a decrease in pH below about pH 6.5 results in decreased oxygen affinity (acid Bohr effect), to the point that in some cases haemoglobin cannot be saturated even at high pressures of pure oxygen (Root effect). Although it has been postulated that multiple haemoglobins may have evolved in fishes to compensate for oxygen deficiencies due to the Root effect, currently there are no data to support such a relationship (Farmer et al., 1979).

Perutz and Brunori (1982) suggest that the constellation of polar residues needed to produce the Root effect and the accompanying large Bohr effect in teleosts seems to consist of SerF9b, LysEF6b, GluFGlb, ArgH21b and HisHC3b; and that the Root effect is caused mainly by a single amino acid replacement (cysteine F9b in mammals and serine in fishes) (Perutz, 1983). However, there could be many more replacements. As Brittain (1987) indicates, the assignment of all residues responsible for the alkaline Bohr effect is far from universal, with measurements at various ionic strengths, suggesting that as many as 10 or even 28 different residues may be involved.

For many years it was believed that the Root effect was related solely to the existence of a swim bladder (Binotti et al., 1971), its assumed function being to

306 Pdrez et al.

facilitate the secretion of oxygen into the swim bladder. However, since many teleosts without swim bladders have a Root effect (Farmer et al., 1979), Root effect and the presence of a swim bladder are not inevitably linked.

The Root effect may have arisen from the need to oxygenate the poorly vascularized retina of many fishes (Brittain, 1987). In Antarctic fishes which lack a swim bladder, the presence of Root-effect haemoglobins is correlated with the presence of the oxygen-secreting choroid rete mirabile, and the high oxygen tension present in the eyes of many fishes may be produced by a secretory mechanism involving Root-effect haemoglobins.

Evolution of protein polymorphism

Two mechanisms have been proposed to explain the occurrence of widespread protein polymorphism in natural populations: (1) the selectionist hypothesis, which holds that polymorphism is the result of natural selection, and that geographic variation in gene frequencies reflects different fitness values; and (2) the neutralist hypothesis, which maintains that polymorphism is the product of the interaction between selective neutral mutations and genetic drift. According to the latter hypothesis, genetic variants may be functionally equivalent.

THE SELECTIONIST HYPOTHESIS AND HAEMOGLOBIN MULTIPLICITY

Four types of evidence may be considered in support of the selectionist hypothesis. If multiple haemoglobins have been fixed by selection, we would expect (1) evidence for functional heterogeneity in the different haemoglobin components; (2) a higher multiplicity in fishes that live in variable environments (Fyhn et al., 1979); (3) a correlation between multiplicity and activity level in fishes; and (4) a change in the concentrations of specific haemoglobin fractions during the thermoacclimatory process.

Functional heterogeneity o f haemoglobin components in fishes

Fishes may be divided into two groups according to their oxygen affinities and the modifications of this affinity with changes in temperature, salinity and pH. Functional heterogeneity among fishes may be marked or slight or absent.

Group i, members of which have marked functional heterogeneity in their haemoglobins, contains several species with two functionally distinct components and includes the chum salmon (Oncorhynchus keta, Salmonidae) (Hashimoto et al., 1960); a cichlid (Oreochromis mossambicus, Cichlidae) (P6rez and Maclean, 1976); a catfish (Hoplo- sternum littorale, Callichthyidae) (Garlick et al., 1979b; P6rez, 1980); a characid (Mylossoma sp., Characidae) (Martin et al., 1979a); an eel (AnguiUa japonica, Anguillidae) (Yamaguchi et al., 1966; Yoshioka et al., 1966; however, Shimada et al., 1980, detected six haemoglobin components in this species); another eel (A. anguilla) (Breespol et al., 1981); and a third species of eel (A. rostrata) (Poluhowich, 1972).

In A. rostrata, Gillen and Riggs (1973b) found either two or three components in the specimens they examined. In specimens having two components, both functional properties were identical; in those having three components, two were the same and


the other was different. A similar finding was reported by Wilhem and Weber (1983a) for a catfish (Callichthys callichthys, Callichthyidae), which is a close relative of Hoplosternum littorale. C. callichthys had five haemoglobins, four of which had similar functional properties.

In the following species there are four or more haemoglobin components but they always can be divided into just two groups having different functional properties: the rainbow trout (Oncorhynchus mykiss, Salmonidae) (Binotti et al., 1971); the sockeye salmon (Oncorhynchus nerka) (Sauer and Harrington, 1988); a catostomid (Catostornus clarkii, Catostomidae) (Powers, 1974); the tench (Tinca tinca, Cyprinidae) (Jensen and Weber, 1982); an anostomid (Leporinus friderice, Anostomidae) (Peterson et al., 1989); a catfish (Pterygoplichthys pardalis, Loricariidae) (Brunori et al., 1979); the dogfish (Squalus acanthias, Squalidae) (Weber et al., 1983); and a species of sea chub ( Girella tricuspidata, Kyphosidae) (Wells et al., 1984).

In general, the electrophoretic anodic ('fast') haemoglobin component(s) in these fishes show low oxygen affinities, a well-defined Bohr effect, and temperature and salt effects. In contrast, the cathodic ('slow') component(s) possess high oxygen affinities, a low or reverse Bohr effect, and reduced temperature and salt sensitivities.

Group ii, which lack (or have very slight) functional heterogeneity in their haemoglobins, includes several species with two components, among them the arapaima ( Arapaima gigas, Osteoglossidae) (Galdames-Portus et al., 1979; however, Fyhn et al., 1979, reported only one component from this species). Other representatives of this group have three components, such as the carp (Cyprinus carpio, Cyprinidae) (Gillen and Riggs, 1972); the Rio Grande cichlid (Cichlasoma (= Herichthys) cyanoguttatum, Cichlidae) (Gillen and Riggs, 1973a); and a catfish (Pimelodus maculatus, Pime- lodidae) (Reischl, 1977). In the last species, only the two most concentrated haemoglobins were studied.

The following species in Group II have more than three haemoglobins: the plaice (Pleuronectes platessa, Pleuronectidae) and the flounder (Platichthys flesus, Pleuronec- tidae) (Weber and Wilde, 1976); the killifish (Fundulus heteroclitus, Fundulidae) (Mied and Powers, 1978); a catfish (Catostomus insignis, Catostomidae) (Powers, 1974); the cichlids Crenicichla lepidota, Aequidens portalegrensis and Geophagus brasiliensis (Cichlidae) (Wilheim and Weber, 1983b); the tilapia (Tilapia grahami, Cichlidae) (Lykkeboe et al., 1975); and the bowfin (Amia calvia, Amiidae) (Weber et al., 1976).

In fishes of group i, which have haemoglobins that substantially differ in their functional properties, one or more components are affected in their oxygen affinity by changes in pH. However, these haemoglobins also have components that are unaffected (or very little affected) by changes in pH. In fishes of group ii, which have haemoglobins that do not differ in function, all isohaemoglobins are affected by changes in pH.

Haemoglobins in fishes with only one component are also affected by change in pH. Examples include the spot (Leiostomus xanthurus, Sciaenidae) (Bonaventura et al., 1976); a squirrelfish (Myripristis berndti, Holocentridae) (Pennelly et al., 1978); a knifefish (Sternopygus macrurus, Sternopygidae) (Garlick et al., 1979a); a catfish (Brachyplatystoma sp., Pimelodidae) (Martin et al., 1979b); an osteoglossid (Osteo- glossum bicirrhosum, Osteoglossidae) (Galdames-Portus et al., 1979); the South American lungfish (Lepidosiren paradoxa, Lepidosirenidae) (Phelps et al., 1979); an Antarctic dragonfish (Bathydraco marri, Bathydraconidae) (Kunzmann et al., 1991),

308 Pdrez et al.

and two species of cod icefish (Dissostichus mawsoni, Nototheniidae (Qvist et al., 1977) and Aethodaxis mitopteryx (D'Avino et al., 1992)).

The only exception to the above correlations seems to be a cold-adapted Antarctic dragonfish (Gymnodraco acuticeps, Bathydraconidae). This species has a single haemoglobin that has been found not to be modulated by pH and aUosteric effects (Tambur- rini et al., 1992). Although G. acuticeps lives in a stable environment and is a slow predator on marine organisms, it does not need a large oxygen turnover. The absence of a Bohr effect is balanced by the low oxygen affinity of the haemoglobin, which facilitates oxygen release to the tissues in conditions of acidosis (Tamburrini et al., 1992).

Powers (1972) found that two species of catfish (Catostomus insignis and C. clarkii) possess multiple haemoglobins that have approximately the same molecular weight. C. insignis has seven haemoglobin components, all pH dependent, whereas C. clarkii has nine components, including a cathodic fraction of about 20%, the oxygen equilibria of which are independent of pH. Since the Bohr effect is normally a beneficial phenomenon, the maintenance of some haemoglobin without a Bohr effect evidently provides a physiological advantage that is habitat specific if natural selection is important. Both species are sympatric, but C. insignis belongs to the subgenus Pantosteus (which prefer fast-moving parts of the stream) and C. clarkii belongs to the subgenus Catostomus (which prefers pools or sluggish water). Fish in fast-water habitats generally have haemoglobins with low oxygen affinities and a large Bohr effect. Although a large Bohr effect is beneficial in releasing oxygen at the cellular level, it can suppress oxygen binding at the gills when the blood pH is sufficiently low. Furthermore, the presence of cathodal haemoglobins in several catfish that inhabit fast-flowing waters suggests that this haemoglobin provides an emergency system for oxygen transport during periods of activity when decreased blood pH values unload the pH-sensitive haemoglobin component.

Haemoglobin multiplicity and environment

To identify trends in haemoglobin diversity in fishes, we assembled published data on 408 species that included type of gel employed, the taxonomic position of the species, and whether the species was marine, estuarine or freshwater (Tables 1 and 2). Orders containing marine, estuarine and freshwater species are especially useful for our analysis because they permit a comparison of species that live in the more variable estuarine habitat with related species found in the more stable marine and freshwater habitats. We used data derived only from polyacrylamide and starch gel electrophoresis for which there was an adequate sample. No attempt was made to include polymorphism and we have included only data from adult fish to avoid possible ontogenetic changes. Also, in analyses such as this one, one must always consider the possibility that some haemoglobin multiplicity reported may represent artefacts caused by the assembly of hybrid tetramers (Brittain, 1991).

Data from Tables 1 and 2 support the selectionist hypothesis if they indicate an increase in the number of haemoglobins in the direction of marine/freshwater/estuarine species, since the variability of the environment increases in this direction. Such support is clearly provided by the orders Salmoniformes and Batrachoidiformes (in PAA), and partially provided by the Atheriniformes and Perciformes. Within the

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Perciformes, the families Gerreidae, Haemulidae, Lutjanidae, and to some extent the family Sciaenidae, show the tendency for more haemoglobin components to be found in the more variable environments.

Bonaventura et al. (1975) pointed out that deep sea species such as rattails (Macro- urus sp., Macrouridae) have simple one-banded or two-banded haemoglobin electrophoretic patterns that may be correlated with the relatively thermostable environment in which they live. They also described a single haemoglobin for the coelacanth (Latimeria chalumnae, Latimeridae), although Weber et al. (1976) described six haemoglobin components for this species. Gorr et al. (1991) sequenced the haemoglobin of the coelacanth and found only one component. Bonaventura et al. (1975) also compared two closely related species that are often collected together, the spot and the Atlantic croaker (Micropogon undulatus, Sciaenidae). They found a single haemoglobin component in the former species and five to ten highly polymorphic haemoglobins in the latter.

The relatively stable Antarctic waters would be expected to support fishes having simple haemoglobins (unless it is argued that weak selective pressures enhance the possibility of mutations to persist). This seems to be the case for fishes in the largely endemic perciform suborder Nototheriodei (families Channichthyidae, Nototheniidae, Bathydraconidae, Harpagiferidae), which have a reduced number of erythrocytes as well as a reduction in haemoglobin (Kunzmann et al., 1991). The chaenichthyids lack haemoglobin altogether and the families Nototheniidae and Bathydraconidae have only a single haemoglobin (Hbl). In these last two families, a second component was identified in a very few cases. It was in low concentration, invariable, and functionally indistinguishable from Hbl (Di Prisco et al., 1981). However, there is an exception in at least one species of cod icefish (Trematomus newnesi, Nototheniidae), which has two functionally distinct major heamoglobins: Hbl (70%) and HbC (25%), and one minor Hb2 component (5%). T. newnesi is cryopelagic (other species in the family are mostly sedentary bottom feeders), very active and feeds underneath the pack and fast ice. In contrast to the other Nototheriodei, its behaviour and lifestyle more closely resemble that of the trout, which also has haemoglobins with different functional properties.

This simple haemoglobin composition - a single major haemoglobin, often accom- panied by a second minor component (approximately 5% of the total) - is not found in three Antarctic species of the family Zoarcidae (of the non-endemic suborder Zoarcoi- dei), which shows a high multiplicity with four to five functionally distinct major components (Di Prisco et al., 1981).

Tables 1 and 2 and the above discussion do not appear to either strongly support or refute the selectionist hypothesis that haemoglobin heterogeneity is adaptive for unstable environments, even though it is reasonable to expect that some species may have evolved haemoglobins in response to instability in the environment. The assump- tion here is that different haemoglobins are adapted to specific stressful elements that occur in variable environments.

It is also possible that species with fewer haemoglobins may survive in a variable environment by compensating in other ways. For example, natural selection could favour a single haemoglobin with multiple capabilities that matched the diversity exhibited by several haemoglobins having single capabilities. Also, even if a species in a variable environment had a haemoglobin with limited capabilities, the fish could migrate to more favourable areas during stressful periods. Finally, a species with

312 P~rez et al.

limited haemoglobin capabilities might simply function more efficiently during periods that are critical for reproductive success, such as the breeding season.

Haemoglobin multiplicity and levels of activity

In a study of haemolysate complexity, including haemoglobin heterogeneity, in 31 species of marine, estuarine and freshwater fishes, Prrez et al. (1986) found no correlations between activity level and oxygen affinity, Root effect, pH of the blood, and number of haemoglobins. However, the values for haemoglobin concentration, haematocrit and erythrocyte concentration were higher in highly active fishes (branchial respiration) than in species having low activity levels (aero-branchial respiration).

Changes in haemoglobin multiplicity due to temperature variation

The selectionist hypothesis is supported by studies indicating that some species selectively alter the concentration of specific haemoglobin fractions during the thermoacclimatory process. This response has been reported in the goldfish (Carassius auratus, Cyprinidae) and rainbow trout (Houston and Cyr, 1974); pumpkinseed (Lepomis gibbosus, Centrarchidae); white sucker (Catostomus commersoni, Catostomidae); carp (Cyprinus carpio, Cyprinidae), and carp-goldfish hybrids (Houston et al., 1976). Moreover, Houston and Rupert (1976) observed in goldfish acclimated to 3 °C and 23 °C the existence of two and three haemoglobin components, respectively. When fish acclimated at 23 °C were abruptly transferred to 3 °C, and vice versa, the minor component appeared and disappeared, respectively, within 3 h. Cooling and warming the whole blood and haemolysate samples in vitro indicated that modification of the haemoglobin system occurs under cell-free as well as cell-intact conditions (Houston and Rupert, 1976). These observations suggest that the observed variations may be the result, at least partially, of aggregation of pre-existing subunits in combinations not possible, or not stable, at other temperatures, rather than de novo haemoglobin synthesis that would require longer intervals of times and demand erythrocyte integrity. Houston and Rupert (1976) indicate that this finding raises the possibility that teleos- tean haemoglobin systems may possess a capacity for rapid, adaptive reorganization after environmental temperature variation. However, Vaccaro et al. (1975) found a single haemoglobin in this species, which does not change during acclimatization at 4, 21 and 37 °C.

Qualitative changes (i.e. the appearance of new haemoglobins in the same indi- viduals through time) reported for the goldfish (Houston and Rupert, 1976; Houston et al., 1976) support the concept of an adaptive response through selective modification of the haemoglobin system; however, the mechanisms involved are not dear. Along these lines, Van Vuren and Hattingh (1978) found that the proportions of multiple haemoglobins change seasonally in the carp, yeUowfish (Barbus holubi, Cyprinidae) and two species of mudfish (Labeo umbratus and L. capensis, Cyprinidae).

THE NEUTRALIST HYPOTHESIS AND AMINO ACID SUBSTITUTION The neutralist theory (Kimura, 1968), in contrast to the Darwinian theory of evolution by natural selection, claims that the great majority of evolutionary changes at the molecular level are caused by random fixation (genetic drift) of selectively neutral or


very nearly neutral mutants under continued mutation pressure. The neutralist theory does not deny the role of natural selection in determining the course of evolution, but it assumes that only a small fraction of DNA changes are adaptive (Kimura, 1989). It regards protein and DNA polymorphism as a transient phase of molecular evolution and rejects the notion that the majority of such polymorphs are adaptive and maintained by some form of molecular evolution.

The strongest support for the neutralist theory in this case may well be our inability to find sufficient functional correlates for the different haemoglobins found in fishes. For example, because cave environments, like deep-sea or Antarctic waters, are greatly stable and uniform both spatially and temporally, cave fishes would be expected to have a single Hb modulated by pH and organic phosphates. However, one species of catfish (Trichomycterus sp., Trichomycteridae), which lives in the Guacharo Cave in Venezuela, has three haemoglobins for which no adaptive values can be determined (Prrez and Moodie, 1993). Therefore, the neutralist hypothesis seems to be appropriate to explain haemoglobin multiplicity at least in this species.

On the other hand, indirect support for the neutralist theory is suggested by (1) the constancy of evolutionary rate of amino acid substitution; (2) the occurrence of functionally indistinct properties in different haemoglobins; and (3) DNA sequence data.

Constancy of evolutionary rate The rate of evolution in terms of amino acid substitution per site per year is approximately constant for each protein or gene region in haemoglobin and is about 10 -9 substitutions per amino acid site per year. Kimura (1989) pointed out the possibility that haemoglobins and other molecules of 'living fossils' may have under- gone as many amino acid (and therefore DNA base) substitutions as corresponding molecules (genes) in more rapidly evolving species.

Kimura (1989) used as evidence the similar divergence between c~ and fl-globins of humans and ol and fl-globins of the Port Jackson shark (Heterodontus portusjacksoni, Heterodontidae), which is considered a relict survivor from the early Jurassic (Maisey, 1984). We also find confirmation for this idea in the data of Gorr et al. 1991) for Latimeria, a survivor of the Devonian, some 380 million years ago. In both comparisons it is clear that genes coding for the c~ and fl-chains of the haemoglobins have diverged to roughly the same extent (or slightly more) as in the corresponding two genes in humans. By comparing the amino acid differences between cr and fl-chains of human, shark and coelacanth haemoglobins, we find the following (listed in order of human-shark-coelacanth): no change, 62-50-52; one change, 55-56-50; two changes, 21-32-35; three changes, 0-1-2 ; gaps, 9-11-8. The total number of sites in humans was 147; in the shark, 150; and in the coelacanth, 147.

Functional constraints of evolutionary rate The second feature that distinguishes molecular evolution from phenotypic evolution is that molecules or portions of molecules that are subject to fewer functional constraints evolve faster (in terms of mutant substitutions) than those that are subject to stronger constraints. Thus, mutant substitutions that cause less drastic changes in the existing structure and function of a molecule occur more frequently than those which cause

314 P~rez et al.

more drastic ones. As an example, the surface of the haemoglobin molecule is less important for maintaining the structure and function of the molecule than the vitally important haem pocket. It was shown that in both alpha and beta haemoglobins the surface portion evolves about 10 times as fast as the haem pocket (Kimura, 1989).

One species of cod icefish (Notothenia coriiceps, Nototheniidae) was found to contain two haemoglobins which are functionally indistinct. Both display a large Bohr effect and a Root effect. Beta chains (146 residues) were common to both haemoglobins but a~ chains (142 residues) were different. They differed in 51 residues, but amino acid residues known to be invariant in all vertebrates were essentially unchanged and most of the replacements in the sequences appeared to be localized in domains where the structural requirements for the biological functions are less stringent (Di Prisco et al., 1981). A similar situation is found in an Antarctic dragonfish (Cygnodraco mawsoni, Bathydraconidae) (Caruso et al., 1991), which also possesses two haemoglobins (Hbl = 95%, Hb2 = 5%). These two haemoglobins have identical functional properties (large Bohr and Root effects) and the same a~ chains, but they have different fl chains. The reasoning here is that if different haemoglobins can have the same function, then natural selection would not be causing the evolution of specific molecular configurations. On the other hand, convergent evolution could also have oc- curred.

D N A sequence data

Other evidence which, according to Kimura (1989), supports the neutralist theory, emerged as a consequence of DNA sequence data during the last decade. First, it was shown that the 'synonymous' changes (nucleotide substitutions within codons that do not cause amino acid changes) occur at a much higher rate than 'non-synonymous' changes - those that alter the amino acid sequences. Second, support for the neutralist theory is provided by the rapid evolutionary changes demonstrated in pseudogenes, sometimes called 'dead genes'. A pseudogene is a sequence found in the genome of eukaryotes that closely resembles a known coding sequence, but which differs in that transcription is rendered impossible by the insertion of numerous stop codons, or sometimes by more substantial additions or deletions (Maclean, 1987). Comparisons of DNA reveal that base substitutions occur at very high rates in the pseudogenes after they are created by gene duplications. In the carp it has been found that chromosomal DNA contains seven different aMike genes. Genes number 1 and 5 are probably pseudogenes (Hirono et al., 1991).

Concluding remarks and summary

Although there are few supporting studies, it is possible that when fishes possess two or more haemoglobin components that are affected by pH, their a~ and r-chains are very similar (Di Prisco et al., 1981). The same applies to a~ and r-chains in haemoglobins not affected by pH changes. For example, Bossa et al. (1982) compared the structure of the four haemoglobins present in a South American catfish (Pterygophichthys pardalis, Loricariidae). The estimated percentage sequence differences were very low (1.1 to 8.1%) between Hb ii, Hb m and Hb iv but high (46.2 to 51.3%) between Hb i and the other three haemoglobins. Components n, in and ~v, which together comprise approxi-


mately half of the haemoglobin (the other half being Hb 0, are functionally similar to each other and affected by pH changes.

If the multiplicity of haemoglobin components in fishes is best explained by the neutralist hypothesis, one would expect a higher number of components in species with a very restricted distribution (one or several small populations), such as in Tricho- mycterus.

What selective advantages could genes for multiple haemoglobins provide? In fast-swimming fishes like trout or Catostomus clarkii, the pH at the level of the gills may drop too low for an efficient oxygen uptake by haemoglobins that exhibit the Root effect. In order to ensure a continued oxygen supply, it would be an advantage to possess haemoglobins that do not respond to heterotrophic ligands. However, the haemoglobins that do respond to changes in pH assure that the supply of oxygen to the eye and to the swim bladder is maintained.

However, in other cases the presence of multiple haemoglobins does not seem to be adapative, as in fishes where multiple haemoglobins are equivalent in function.

At least sometimes the presence of multiple haemoglobins might allow a higher total haemoglobin concentration to be maintained in the erythrocytes than if there were only a single haemoglobin, as was suggested by Bunn (cited in Perutz, 1983).

One very important question raised by Perutz (1983) is whether adaptation is due to the gradual accumulation of minor mutations, each producing a small shift in chemical affinity, or to a few amino acid substitutions in key positions. Perutz (1983) indicated that the second possibility seems to have a sound basis. For example, the haemoglobin of the Port Jackson shark has only a weak alkaline Bohr effect and has alanine in position F9. It seems that the Root effect in teleosts has evolved primarily by the substitution of alanine by serine. As another example, Perutz (1983) points out that only five amino acid substitutions probably are needed to inhibit, in trout haemoglobin, all of the heterotrophic interactions exhibited by haemoglobin iv.

On the other hand, it seems that most amino acid substitutions between haemoglobins of different species are functionally equivalent and that such replacements have little, if any, influence on the functional properties of haemoglobins. To illustrate this point, Perutz (1983) mentions two cases: (1) human and horse haemoglobins differ in sequence in 42 of the 287 positions, yet their functional properties are the same; and (2) only a minority of the approximately 400 different amino acid replacements found in abnormal haemoglobins affect any of the haemoglobin functions. It has been shown that mutations that merely change surface charges do not have a significant effect on the respiratory functions of haemoglobins.

Therefore, the structural evidence suggests that most amino acid replacements between species are neutral or nearly so, and are caused by random drift of selectively equivalent mutant genes; and that adaptive mechanisms generally operate by a few replacements in key positions. Here again the evidence supports Kimura's view that "adaptive mutations are much less frequent than selectively neutral substitutions caused by random drift" (Kimura, 1968).

Acknowledgements

We would like to thank two anonymous reviewers for the valuable criticism they offered on the first version of the manuscript.

316 P~rez et al.

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Accepted 13 September 1994

the evolution of multiple haemoglobins in fishes

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