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First published online June 11, 2007
Journal of Experimental Biology 210, 2170-2180 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.004861
Exceptional longevity in songbirds is associated with high rates of evolution of cytochrome b, suggesting selection for reduced generation of free radicals
New Hope Biomedical R&D, 23 W. Bridge Street, New Hope, PA 18938, USA
e-mail: rotteh{at}hotmail.com
Accepted 17 April 2007
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Key words: lifespan, songbirds, cytochrome b, energy expenditure, reactive oxygen species
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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;
Sohal and Weindruch, 1996;
Beckman and Ames, 1998;
Barja, 2004;
Sanz et al., 2006). Because
the rate of generation of reactive oxygen species (ROS) is largely a function
of metabolic rates (Ku and Sohal,
1993) this postulate is compatible with the fact that, in animals,
longevity (L) is dependent on mass-specific basal metabolic rates
(BMRw). L is inversely related to BMRw, obeying
by the power law:
 | (1) |
 | (2) |
 | (3) |
Since L is a function of BMRw, L is also a function of
M, obeying the power law:
 | (4) |
From Eqn 1 and Eqn 2 it follows that e=a–(bc) and that the scaling
exponent f=(bd) (for a review, see
Speakman, 2005).
The `rate of living' theory postulated that the mass-specific lifetime
expenditure of energy (i.e. LxBMRw) is nearly
constant in all animals (Pearl,
1928; Speakman,
2005). If L was a reciprocal function of BMRw,
i.e. b=1, then f=d and the lifetime expenditure of energy would be constant,
since in this case
LxBMRw=(eMd)(cM–d)=ec=k.
In fact, both in birds and mammals, b<<1 and d>f
(Speakman, 2005), and the
lifetime expenditure of energy should be inversely related to body mass, since
from Eqn 4 and Eqn 2:
 |
 | (5) |
The relationships between longevity and body mass (power law 4), longevity
and mass-specific basal metabolic rates (power law 1), and lifetime
expenditure of energy and body mass (power law 5) could be explained very well
by the free radical theory of aging if we accept that the ratio between the
rate of generation of reactive oxygen species
(
ROS) and the rate of
oxygen consumption (and hence BMRw) is not necessarily the same in all
animals. Thus, the fact that L is not a reciprocal function of BMRw,
as well as the commonly observed large deviations (residuals) from the power
laws 1, 4 and 5, could be accommodated by the theory if these were the result
of the evolution of taxon-specific modulations of the relationship between
metabolic rates and ROS.
Indeed, increasing evidence suggest that longevity correlates negatively with
ROS, and that this
correlation is stronger than the negative correlation with BMRw
(Ku et al., 1993;
Perez-Campo et al., 1998;
Herrero and Barja, 1998).
Recently, we showed that in a large clade of placental mammals the rate of
evolution of cytochrome b, and most other mtDNA-coded proteins, is
positively correlated with longevity
(Rottenberg, 2006). Moreover,
we showed later that, in the same clade the longevity residuals from power
laws 1 and 4 are also positively correlated with the rate of evolution of
cytochrome b, suggesting that longevity dependence on the rate of
evolution of mtDNA-coded proteins is independent of body mass or mass-specific
basal metabolic rates (Rottenberg,
2007). Since ROS production is largely a byproduct of
mitochondrial electron transport, and cytochrome b and other
components of the mitochondrial electron transport complexes, are coded by
mtDNA, we suggested that the evolution of mtDNA-coded proteins in placental
mammals is driven by adaptive selection of mutations that reduce
ROS. Anthropoid primates,
as well as elephants, whales, dolphins and bats exhibit exceptionally high
rates of evolution of cytochrome b and other mtDNA-coded proteins,
and in addition to exceptional longevity (i.e. positive longevity residuals
from power laws 1 and 4), exhibit also exceptionally high BMRw values (i.e.
positive BMRw residuals from power law 2). These observations suggested that
adaptive selection of mutations in cytochrome b and other mtDNA-coded
proteins reduced ROS, in
part, by increasing mitochondrial proton leak
(Rottenberg, 2007).
Mitochondrial proton leak reduces the mitochondrial proton electrochemical
potential difference and thus accelerates basal electron transport rate,
thereby reducing ROS
(Korshunov et al., 1997).
Therefore, the free radical theory of aging predicts that an increase of
mitochondrial proton leak should increase lifespan
(Brand, 2000). We suggested
that the observed association between positive L residuals from Eqn 1
and Eqn 4 with positive BMRw residuals from Eqn 2, that appear to be
incompatible with the free radical theory of aging, actually reflects a lower
degree of mitochondrial coupling that is associated with increased basal
respiration rate, and consequently lower
ROS/BMRw ratio. A lower
mitochondrial degree of coupling could accounts for the positive residuals
from the power laws 1, 2, 4 and 5 observed in these taxa. Interestingly, most
of these taxa also share exceptional sociality and cognitive abilities, and
therefore it is not clear whether the selection for reduced
ROS is driven by pressure
to increase lifespan or sociality and cognitive abilities (see
Discussion).
It is well-established that the power laws that describe the relationships
between longevity, body mass and metabolic rates in birds and mammals are
different: in the power laws 1, 2, 4 and 5 the coefficients a, c, e and g are
larger in birds than in mammals, i.e. at equal body mass birds have higher
mass-specific basal metabolic rates than mammals, and yet live longer than
mammals of equal body mass, and their lifetime expenditure of energy is also
larger (Holmes et al., 2001;
Speakman, 2005). Birds and
mammals share endothermic metabolism, but they evolved independently, and it
should not be surprising that these coefficients, which are influenced by the
anatomy, physiology and metabolism of these classes of animals are different.
Moreover, there is direct evidence that birds live longer than mammals of
equal mass or BMRw because the ratio between
ROS and oxygen consumption
is smaller in birds than in mammals (Ku
and Sohal, 1993; Barja et al.,
1994; Barja, 1998).
Similarly, the large deviations of some species from the birds' power laws
most likely result from modulation of the relationships between BMRw and ROS
generation in these species. We can therefore ask: do these modulations also
result from adaptive selection in the evolution of mtDNA-coded proteins as
observed in placental mammals?
In mammals the relative rate of evolution of most mtDNA-coded proteins is
very different in different taxa, and this fact and other independent
evidence, suggest that in mammalian taxa the evolution of mtDNA-coded proteins
is driven by adaptive (positive) selection
(Gissi et al., 2000). There is
also evidence that adaptive selection drives the evolution of mtDNA in most
other classes of animals (Bazin et al.,
2006). However, there are no studies of the relative rates of
evolution of mtDNA-coded proteins in birds, and there are relatively few
complete sequences of mtDNA of birds that are available for such studies. The
exception is cytochrome b, the core peptide of complex III, which has
been sequenced extensively for phylogenetic studies of birds. Cytochrome
b is a major source of ROS (Demin
et al., 1998), and its rate of evolution correlates strongly with
longevity in placental mammals
(Rottenberg, 2006;
Rottenberg, 2007). It was also
suggested that complex III (together with complex I) is responsible for the
low ROS in birds
(Herrero and Barja, 1997;
Herrero and Barja, 1998).
Therefore, in this study, we determined the relative rate of cytochrome
b evolution in modern birds and investigated the relationships
between the rate of cytochrome b evolution and longevity, basal
metabolic rates and lifetime expenditure of energy.
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Materials and methods |
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)]
to obtain the longevity (L, years) and body mass (M; g) of
809 species of birds belonging to 287 genera. We also collected all the
available values of mass-specific basal metabolic rates (BMRw; mW
g–1) for these species from the AnAge databank. Data
collection was terminated in February 2006. We computed the genera averages of
L, M and BMRw, and these average values were used for the
analysis (Table S1 in supplementary material). Using the Entrez/protein search
engine
(http://www.ncbi.nlm.nih.gov/entrez/)
we searched for complete cytochrome b sequences for the 287 genera.
If more than one complete sequence was found for a genus we used the sequence
of the species with life history traits that was closer to the genus average.
In a few cases, where several complete sequences were available for a genus,
we aligned them and used the one that was closest to the genus consensus. In
most cases there was no significant difference between the values of
substitution per site calculated for species of the same genus (see below).
Analysis of the data at the genera level rather than at the species level
provides several advantages. Firstly, it allowed us to increase the size of
our database since very few species that have BMRw value in the
AnAge databank also have a complete cytochrome b sequence. Secondly,
the error in estimating species L values is quite large, and the
genus average is therefore more reliable. Thirdly, a few genera are
represented by a large number of species in the AnAge databank, whereas most
other genera are represented by a few, often a single, species; therefore,
analysis at the species level would give unduly large weight to a few
genera.
The computation of cytochrome b substitution per site
The complete cytochrome b sequences of 122 genera of birds were
aligned using the CLUSTALX program
(http://bips.u-strasb.fr/fr/Documentation/ClustalX)
and submitted for genetic distance determination by the PRODIST program at the
phylogeny site PHYLIP
(http://bioweb.pasteur.fr/seqanal/interfaces/prodist.html).
The Dayhoff PAM matrix distance model with the default setting was used. The
distance matrix of the 122 cytochrome b sequences was used to compute
the relative rates of cytochrome b evolution in modern birds. The
detailed phylogeny of extant bird species has not been fully elucidated as
yet. Nevertheless, it is accepted that the clade of Neognathae (modern birds)
that contains most extant bird species diverged from the Paleognathae clade
early on and later split into the Neoaves and the Galloanserae branches
(Cracraft et al., 2004).
Therefore, we use the three sequences of the Paleognathae clade as an outgroup
to calculate the relative rate of evolution (i.e. substitution per site) of
the 119 genera of modern birds from the node of divergence of the neoaves and
the Galloanserae branches. To compute the substitution per site for a species
i (Si) belonging to the Neoaves branch we used the formula:
Si=(Di,p+Di,g–Dp,g)/2
where Di,p is the distance from species i to p, a species
that belongs to the Paleognathae branch; Di,g is the
distance from i to g, a species that belongs to the Galloanserae branch; and
Dp,g is the distance from p to g. For each species we
computed Si with the three different Paleognathae species (Table S1 in
supplementary material) and three different Galloanserae species (Gallus
gallus, Anser albifrons, Ortalis vetula), in all possible combinations
(3x3), and averaged the nine values. To compute the substitution per
site for species that belong to the Galloanserae branch we used the formula
Si=(Di,p+Di,n–Dp,n)/2
where Di,n is the distance between species i and n, a
species that belong to the Neoaves branch, and Dp,n is the
distance from p to n. For each species we computed Si with the three different
Paleognathae species and three Neoaves species belonging to different orders,
in all possible combination, and averaged the nine values. The results of
these computations are listed in Table S1 in supplementary material.
Statistical analysis (e.g. linear regression, multiple linear regression, Student's t-test) was carried out with the SigmaStat3.1 Statistical analysis package (Systat Software inc.)
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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), suggesting
adaptive selection of mutations in taxa that exhibit accelerated evolution of
cytochrome b. The computed value of substitution per site, Scytb, is a sum of
both neutral mutations and selected mutations. Assuming that the rate of
neutral mutations is the same in all species [the Molecular Clock assumption
(Page and Holmes, 1998)],
genera with exceptionally low value of Scytb represent genera with relatively
few positively selected mutations. By contrast, genera with exceptionally high
values of Scytb, represent genera where most of the mutations were selected.
Of the 119 genera of Neognathae that have a complete cytochrome b
sequence, only 59 also have BMRw values listed in the AnAge
database (Table S2 in supplementary material). To find out if there are
correlations between life history traits and selection of cytochrome
b mutations we compared the relationship between body mass
(M), basal metabolic rates (BMR), mass-specific basal metabolic rates
(BMRw), and longevity (L) in a group of genera with
exceptionally high Scytb (the upper quadrant of the rates distribution,
Scytb>0.1345, N=15) and a group with exceptionally low Scytb (the
lower quadrant, Scytb<0.069, N=15, see Table S2 in supplementary
material).
Fig. 1A (solid line) shows
the linear regression of power law (3) that describe BMR as a function of body
mass in the 59 Neognathae genera (Table S2 in supplementary material):
BMR=3.76*M0.663. The scaling exponent 0.663±0.014
is similar to previously reported value for birds, 0.671
(Speakman, 2005). However,
when plotted separately there was a significant difference between the scaling
exponent of the high Scytb group, that is composed entirely of small birds
(empty symbols), and the scaling exponent of the whole group
(0.826±0.075 compared to 0.663±0.014; d.f.=70, t=2.09,
P=0.02). By contrast, the scaling exponent of the low Scytb group
(0.666±0.0458) was not significantly different from the exponent of the
entire Neognathae group. This difference between the high and low Scytb groups
is shown more clearly in Fig.
1B, in which the dependence of BMRw on M
(power law 2) is shown. In the Neognathae clade
BMRw=3.712M–0.337 (solid line) the
scaling exponent of the low Scytb group (–0.334±0.0458) is nearly
identical to the scaling exponent of the whole clade (–0.337) but the
scaling exponent of the high Scytb is only half as much
(–0.174±0.0753); the coefficient of determination was also much
higher in the low Scytb group than in the high Scytb group
(r2=0.804 and r2=0.291, respectively),
indicating a much weaker dependence of BMRw on M in the
high Scytb group. These results suggest that accelerated evolution of
cytochrome b is associated with taxon-specific modulation of
BMRw.
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There is also a large difference between the two groups in the dependence of L, on BMRw (power law 1), or on M (power law 4). Fig. 1C shows that within the low Scytb group L increases with M with a slope that is similar to the dependence in the whole clade. However, within the high Scytb group, L, which is highly variable, is not dependent on M at all. Fig. 1D shows that within the low Scytb group L decreases with BMRw, with a slope that is similar to the dependence in the whole clade, but within the high Scytb group L is not dependent on BMRw at all (Fig. 1D).
If longevity in the high Scytb group, a group of taxa, where most of the cytochrome b mutations were selected, is not strongly dependent on either M or BMRw does it depend on Scytb? To answer this question we computed the longevity residuals from power laws 1 and 4, to obtain the extent to which the L value of each genus deviates from the prediction of the birds' power laws, and plotted the residuals of the two groups against theirs Scytb values (Fig. 2A). Within the low Scytb group there was no significant correlation between Scytb and the L residuals, but within the high Scytb group there was a very strong positive correlation between the L residuals and Scytb. These results indicate that Scytb does not have strong effect on longevity in the low Scytb group, in which few mutations were selected and L depend strongly on the magnitude of BMRw or M (Fig. 1C,D). By contrast, in the high Scytb group, in which most of the mutations were selected, and BMRw (or M) have little effect on L; the L residuals are strongly dependent on the value of Scytb. These results suggest that the selection of mutations in cytochrome b increased longevity independently of M or BMRw.
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An alternative measure of the effect of BMRw on L is the lifetime expenditure of energy, LxBMRw. In general LxBMRw decrease with increasing M (power law 5), and this holds true for the low Scytb group; however, in the high Scytb group LxBMRw varies greatly but shows no significant dependence on M (Fig. 2B). By contrast in the high Scytb group, LxBMRw depends strongly on Scytb, but this is not true for the low Scytb group (Fig. 2C). Thus, the selected mutations in cytochrome b increased the lifetime expenditure of energy. The genera with the highest values of Scytb (i.e. Serinus and Fringilla, Table S2 in supplementary material), also exhibit the highest values of LxBMRw (Fig. 2C) and the highest L residuals (Fig. 2A).
The averaged value of Scytb is also different in taxonomic groups of higher order. The average Scytb for all neognath genera is 0.096±0.034, but for oscines (song birds) a branch of the order Passeriformes (passerines, or perching birds) the average Scytb value (0.134±0.011) is nearly twice as large as that of all other neognaths (0.073+0.019, t=19.6, P<0.001). The phylogeny of neognaths is not fully resolved yet, but the average Scytb of each non-oscine family (and therefore any ordinal level clade of non-oscine birds) is significantly lower than that of songbirds. All the genera in the high Scytb group (Scytb>0.1345) are oscine birds; but the low Scytb group (Scytb<0.069) includes genera from several families that belong to the Neoaves branch, and from the Galliformes order that belong to the Galloanserae branch (Table S2 in supplementary material). Therefore, the differences between the high and low Scytb groups, described above (Figs 1, 2), are to a considerable extent the differences between oscine birds and other orders of modern birds.
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We, therefore, examined the relationships between Scytb and life history traits in oscine birds, and particularly in the Fringillidae family, in comparison with all other neognaths. Within the Fringillidae family BMRw was rather a weak function of M (Fig. 3B), and the residuals of BMRw of the family genera (from the Neognathae power law BMRw=3.712M–0.337) were positively correlated with Scytb: residuals. lnBMRw(M)= –1.244+8.74Scytb (N=16, r2=0.406, P=0.008). In fact in this family both M and Scytb are independent variables that determine the value of BMRw as shown by the multiple linear regression: lnBMRw=2.304–0.157xlnM+6.37Scytb (N=16, r2=0.706, P<0.001; Fig. 4A). Therefore, in this family, cytochrome b mutations were apparently selected to increase basal respiration and thus the increased BMRw. As a result, within the true finches family L is not dependent significantly on either M or BMRw but is dependent on Scyb: L=–40+368Scytb (N=16, r2=0.459, P=0.004).
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If the accelerated evolution of cytochrome b in birds involved
positive selection of mutations, the sites and nature of these mutations
should relate to their effect on the protein function. The results presented
in Figs 3 and
4 suggest that in the
Fringillidae family these mutations increase both BMRw and
L (hence also LxBMRw), most likely by
increasing mitochondrial proton leak, which accelerates metabolic rate
(Porter and Brand, 1993;
Rolfe et al., 1999), and
inhibits ROS
(Korshunov et al., 1997). The
Fringillidae genus Serinus has the highest values of Scytb (0.163),
LxBMRw (415),
Lo/Lp(M) (2.55) and
Lo/Lp(BMRw) (2.48) of all
birds (Table S2 in supplementary material), which suggests that in this genus
the modulation of cytochrome b function is the most extensive. Indeed
it has been shown that in canaries (Serinus canaria) the ratio
between ROS and oxygen
consumption is very low and involves inhibition of ROS generation at both
complex I and cytochrome b
(Herrero and Barja, 1998).
Fig. 5 shows the sites of the
cytochrome b substitutions in this genus, in comparison to the
sequence of the Tyrannidae family (suboscine) genus Empidonax
(Scytb=0.083). It is apparent that the substitutions are not distributed
randomly in the protein. Most of the substitutions are on the mitochondrial
matrix side of the protein and many of these are in the vicinity of the
Qi ubiquinone binding site. There are large domains of the protein
with no substitutions at all whereas in others, e.g. helices A and E, which
flank the Qi binding site, approximately half of the residues have
been substituted. Moreover, the substituted residues in Serinus are,
on average, smaller and less hydrophobic than in Empidonax. This
pattern supports the suggestion that most of these mutations were selected to
accelerate the reduction of ubiquinone at the Qi site (see
Discussion).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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ROS depends on
BMRw. If ROS was
the same fraction of the rate of oxygen consumption in all genera of birds we
would expect that L=K/BMRw or
LxBMRw=K. In fact, in birds, the linear
regression of L as a function of BMRw exhibits a slope
that is approximately half of that expected for reciprocal relationship (cf.
Fig. 1D) and consequently
LxBMRw is not constant but is inversely related to
body mass (power law 5 and Fig.
3C). The free radical theory of aging could accommodate this fact
if the ratio
ROS/BMRw in
birds with high BMRw values was lower than the ratio in birds with
low BMRw values. Since BMRw is inversely related to body mass
(power law 2), this is equivalent to the requirement that the ratio
ROS/BMRw would
be lower in small birds than in large birds. In fact, it was reported recently
that liver mitochondria from small birds have increased proton conductance,
and are therefore less coupled, than liver mitochondria from large birds
(Brand et al., 2003). A
similar finding was reported earlier for mammalian liver mitochondria
(Porter and Brand, 1993).
Thus, to the extent that the degree of coupling of liver mitochondria
represents mitochondrial coupling in all tissues, and that the small number of
studied species represents these classes of animals, these data support the
suggestion that in birds and mammals mitochondria of large animals are better
coupled than mitochondria from small animals. All the small birds in the study
of Brand et al. (Brand et al.,
2003) were passerines, which as we show here, share exceptionally
high rate of cytochrome b evolution, and it is therefore possible
that the modulation of coupling in mtDNA-coded proteins contribute to the
observed difference between small and large birds. However, it is quite likely
that the pressure to reduce the mitochondrial degree of coupling or
ROS resulted in selective
adaptation of many other proteins, as well as changes of the mitochondrial
membrane properties (Porter and Brand,
1993; Brand et al.,
2003).
Additional support for the conclusion that the mitochondrial degree of
coupling in small animals is lower than in large animals, could be obtained
from studies of the relationships between metabolic rates (MR) and body mass.
The scaling exponent ß, for the power law MR=cMß
is larger for maximal metabolic rates, MMR, than for basal metabolic rates,
BMR (
0.9 and 0.7, respectively)
(Bishop, 1999;
Weibel et al., 2004). Since
MMR is always higher than BMR [i.e. c(MMR) is larger than c(BMR)], the higher
scaling exponent of MMR means that the ratio MMR/BMR is higher in large
animals compared with small animals. The ratio MMR/BMR reflects to a large
extent the ratio between oxygen consumption during intense exercise, when the
rate of ATP synthesis (and consumption) is very high, and oxygen consumption
during rest where the rate of ATP synthesis is minimal. This ratio therefore
may reflect the mitochondrial respiratory control ratio, which is a classic
indicator of the mitochondrial degree of coupling.
We, therefore, suggest that the fact that the scaling exponent b of power law 1 is less than 1 reflects the body mass dependence of the mitochondrial degree of coupling. Consequently the body mass dependence of LxBMRw (power law 5) also results from the body mass dependence of the mitochondrial degree of coupling.
The dependence of LxBMRw on M accounts for some, but not all, of the variability in LxBMRw in birds (Fig. 3C). The residuals of LxBMRw could result from taxon-specific modulation of mitochondrial coupling as demonstrated by the case of the Frangillidae family. In this family of small birds, LxBMRw varies from 84 to 415, a fivefold range, but it is not dependent on M at all. In contrast, LxBMRw depends on Scytb (Fig. 4C). Since mitochondrial uncoupling can increase both L and BMRw, and in this family, BMRw, L and LxBMRw all depend on Scytb (Fig. 4A–C), it is likely that the increase of LxBMRw within this family is due to a decrease in the mitochondrial degree of coupling as a result of the evolution of cytochrome b (and probably other mtDNA-coded proteins).
The results of this study suggest that BMRw, and therefore also
BMR, are not only a function of body mass but also a function of the
mitochondrial degree of coupling. To the extent that mitochondrial coupling
itself depends on body mass this dependence is already embedded in the power
laws 2 and 3. However, mitochondrial coupling is not dependent only on body
mass and there are large taxon-specific deviations from this dependence (cf.
Dobson, 2003). The best
example in birds is provided by the Fringillidae family in which the value of
BMRw (and hence BMR), and presumably mitochondrial coupling, depend strongly
on the rate of cytochrome b evolution (and presumably other
mtDNA-coded proteins), independently of body mass
(Fig. 4A). As a result, the
scaling exponent of power law 3 in the Fringillidae family is 0.857, compared
with 0.648 in all birds (Fig.
3A). Similar effects of the accelerated evolution of cytochrome
b on the scaling exponent of power law 3 were observed in mammals
(Rottenberg, 2007).
There is a longstanding controversy regarding the `true' value of the
scaling exponent of power law 3 and its biological significance
(West et al., 2002;
Darveau et al., 2002;
White and Seymour, 2003). Our
analysis suggest that in addition to many other factors
(Darveau et al., 2002) the
observed value is influenced by the body-mass dependence, as well as the
taxon-specific evolution, of the mitochondrial degree of coupling.
A possible modulation of cytochrome b function in Serinus
It has been demonstrated previously that the exceptionally low rate of ROS
generation in Serinus canaria resulted, in part, from inhibition of
ROS generation in the bc1 complex (Herrero
and Barja, 1998). In coupled mitochondria, the proton
electrochemical gradient inhibits the oxidation of ubihydroquinine at the
Qo site by inhibiting the transfer of the second electron from the
bL heme, to the bH heme, and the
subsequent reduction of ubiquinone at the Qi site
(Crofts, 2004). The inhibition
of this segment of the Q cycle increases the steady state concentration of the
radical semiquinone at the Qo site, which interacts with oxygen to
generate superoxide (Demin et al.,
1998). Therefore, the rate of ROS generation could be inhibited,
in coupled mitochondria, by accelerating the reduction of ubiquinone at the
Qi site. A modulation of the dielectric environment surrounding
Qi, bL and bH may result in
acceleration of this segment of the Q cycle. Acceleration may also result from
increasing internal proton leak. If protons can find a leak path from the
outer surface (where the proton electrochemical potential is high) to the
inner surface the proton electrochemical gradient will decrease and the
reduction of the quinone at the Qi site will be accelerated. The
distribution of substitutions in Serinus cytochrome b, and the fact
that the substituted residues are, on average, smaller and less hydrophobic,
is compatible with increased proton leak and/or an increase of the dielectric
constant at the protein core (Fig.
5).
Adaptive selection and the evolution of cytochrome b in birds
The results of this study suggest that, similar to placental mammals, the
evolution of the mtDNA-coded protein cytochrome b in birds is driven
by adaptive selection. It is likely that, similar to mammals, the evolution of
other mtDNA-coded proteins is driven by the same forces. However, there are
significant differences between the evolution of cytochrome b in
birds and placental mammals. The positive correlation between the residuals
from power laws 1 and 4 and the rate of evolution of cytochrome b was
observed in a large clade of placental mammals that included all ordinal level
taxa except rodents (Rottenberg,
2007). In birds, this positive correlation was observed only in
songbirds, a suborder of the order Passeriformes. However, one should point
out that the songbirds (oscines) group is a very large and diversified clade,
containing nearly half of extant birds species. In mammals, the positive
correlations that are observed between exceptional longevity and the rate of
evolution of cytochrome b, and other mtDNA-coded proteins, at the species
level are stronger at the genera level, become stronger when analyzed at the
averaged family level values, and even stronger when analyzed at the ordinal
level (Rottenberg, 2007).
However, there were no significant correlations between longevity residuals
and the rate of cytochrome b evolution within most families. This
suggests that the selection for reduced ROS generation begun shortly after the
divergence of placental mammals ordinal taxa, coinciding with the explosive
radiation of mammals, about 65 million years ago.
By contrast, in songbirds, the correlations between the rate of cytochrome
b evolution and exceptional longevity are positive and significant at
the averaged genera level but not at the averaged families level. However, the
correlations are strong within the families. This is true not only within the
Fringillidae family (Figs 3,
4), but also within the
Muscicapidae family where the correlation between the longevity residuals from
power law 4 and Scytb is very strong:
Lo/Lp(M)=–2.25+26.2Scytb
(Lo, observed longevity; Lp, predicted
longevity; N=5, r2=0.865, P=0.024). This
observation suggests that the selection for reduced
ROS is relatively recent,
and begun only after the divergence of the oscines families. There is some
evidence that this selection continued after the divergence of species, at
least in some genera. For example, in the genus Carduelis (golden
finches), the longevity of Carduelis carduelis (European golden
finch) is 27 years compared to an average of 12.7 years in nine species of
this genus. However, Carduelis carduelis body mass, 16 g, is the same
as the genus average (15.5 g). That means that this species has exceptional
longevity, not only relatively to all other birds, but also relatively to its
sister species. The cytochrome b sequence of Carduelis
carduelis has 10 nonconservative substitutions from the consensus
sequence of the genus. No other Cardulelis species show more than two
or three substitutions from the consensus (results not shown).
Outside the oscines clade, there are taxa that evolved to reduce ROS by a
different mechanism. For example, the Psittacidae family exhibits an average
Scytb that is only slightly higher that most other non-oscine Neognathae
families, but exhibits positive L(M) residuals for all its
genera. However, the BMRw(M) residuals of all the genera
of this order are all negative (results not shown). Thus, reduced
ROS appears to result from
reduction of BMRw and not from mitochondrial uncoupling. Indeed, it
was found that a species of this order has reduced
ROS, which is the result of
a slow rate of oxygen consumption and not reduced
ROS/BMRw ratio
(Herrero and Barja, 1998).
If the selection for reduced
ROS is relatively recent,
what was the driving force for cytochrome b evolution before the
divergence to families? It appears that the unexpected answer to this question
is that, in modern birds, in general, early evolution of cytochrome b
selected mutations that increased ROS production. The longevity residuals in
neognaths, when oscines are excluded, are inversely correlated with the rate
of cytochrome b evolution (Fig.
4B). Unlike the positive correlations in oscines, these negative
correlations, in other neognaths, are stronger on the family average level,
but they are not significant within families (results not shown). This
suggests that, early, but slow, evolution of cytochrome b involved
adaptive selection that increased
ROS. Most likely the
selection was for increased mitochondrial coupling, perhaps to allow birds to
travel longer distances on a limited amount of stored fat.
We therefore suggest the following narrative for the evolution of
mtDNA-coded proteins and the mitochondrial degree of coupling in birds: as
birds evolved from reptiles and developed endothermic metabolism they
increased their metabolic rates to increase heat production by various means
including a reduction of their mitochondrial degree of coupling. The latter
apparently did not happened in the endothermic evolution of mammals hence the
generally higher metabolic rates and longevity in birds compared to mammals.
Later, during the early evolution of modern birds, they perfected their flight
capabilities and consequently many bird species selected mtDNA mutations that
slightly increased their mitochondrial degree of coupling in order to improve
their metabolic efficiency during flight. However, more recently, during the
evolution of oscine birds, and particularly the small size birds of the
Fringillidae family, many species reverse this trend, and selected mtDNA
mutations that decreased the mitochondrial degree of coupling further, most
likely to increase heat production (which was necessary when they decrease
their body size), and possibly also to reduce
ROS in order to increase
longevity and/or to enable improved cognitive functions. Larger birds, which
did not need to increase heat production (e.g. Psittacidae), reduced ROS
production to increase longevity and/or cognitive function by lowering their
metabolic rates.
Long-term memory, learning and ROS generation
A prerequisite for exceptional cognitive abilities is a large capacity for
long-term memory (Fagot and Cook,
2006). Although the mechanism of the formation and maintenance of
long-term memory is not yet clearly understood there is enough evidence to
suggest that excess ROS production interferes with the formation and
maintenance of long-term memory (Auerbach
and Segal, 1997). There is also evidence that the age-dependent
decline in cognitive functions in humans and non-human animals result from the
increased damage to proteins, DNA and membrane lipids, from mitochondrial ROS
(Beckman and Ames, 1998;
Barja, 2004). It is therefore
likely that ROS generation by mitochondria constrains the development of
exceptional cognitive abilities. Although exceptional cognitive abilities
appear to have evolved independently in several mammalian and bird taxa there
is often convergence in aspects of brain anatomy, physiology and specific
protein functions in these taxa (Emery and
Clayton, 2004; Scharff and
Haesler, 2005; Bolhuis and
Gahr, 2006).
Anthropoid primates, elephants, whales and dolphins share exceptional
cognitive abilities, and exceptional sociality. They also share exceptional
longevity (i.e. positive longevity residuals from power laws 1 and 4),
exceptionally high mass-specific basal metabolic rates (i.e. positive
residuals from power law 2), and accelerated evolution of mtDNA-coded proteins
(Rottenberg, 2007). The latter
association suggests that they also share low
ROS.
In birds, the Psittacidae family (parrots) combines exceptional cognitive
functions with exceptional longevity (i.e. positive residuals from power law
4, data not shown), and low rate of ROS generation
(Herrero and Barja, 1998). The
Corvidae family, which belongs to the oscines branch of the passerines, also
combine exceptional longevity (positive residuals from power laws 1 and 4),
and exceptionally high lifetime expenditure of energy, with exceptional
cognitive ability (Emery and Clayton,
2004). Songbirds that exhibit exceptional capacity to memorize
songs (Bolhuis and Gahar, 2006), e.g. Serinus canaria, exhibit
exceptional longevity and low rate of ROS generation
(Herrero and Barja, 1998),
which are also associated with exceptionally high mass-specific basal
metabolic rates and accelerated evolution of cytochrome b (this
study). Therefore, we suggest that, in mammals and birds, there is a
convergence in the evolution of mitochondria with reduced rates of ROS
production in taxa with high cognitive abilities.
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List of abbreviations |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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