Genética quantitativa

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Biol. Rev. (2007), 82, pp. 551–572. 551
doi:10.1111/j.1469-185X.2007.00024.x
Conceptual bases for quantifying the role
of the environment on gene evolution:
the participation of positive selection
and neutral evolution
Anthony Levasseur1*, Ludovic Orlando2, Xavier Bailly3, Michel C. Milinkovitch4,
Etienne G. J. Danchin5 and Pierre Pontarotti1*
1 Phylogenomics Laboratory, EA 3781 Evolution Biologique Universit�e de Provence, Case 19, Pl. V. Hugo, 13331 Marseille Cedex 03, France
2 Pal�eog�en�etique et Evolution mol�eculaire, Ecole Normale Sup�erieure de Lyon, Universit�e de Lyon, Institut de G�enomique Fonctionnelle de Lyon,
CNRS UMR 5262 - INRA, 46 All�ee d’Italie, 69364 Lyon Cedex 07, France
3 Station Biologique de Roscoff, Place Georges Teissier 29680 Roscoff, France
4 Laboratory of Evolutionary Genetics, Institute for Molecular Biology & Medicine, Universit�e Libre de Bruxelles (ULB), 12 rue Jeener &
Brachet, 6041 Gosselies, Belgium
5Glycogenomics and Biomedical Structural Biology, AFMB UMR 6098 - CNRS - Aix-Marseille I and II, 163 Av. de Luminy, Case 932,
13288 Marseille Cedex 09, France
(Received 30 August 2006; revised 6 July 2007; accepted 9 July 2007)
ABSTRACT
To demonstrate that a given change in the environment has contributed to the emergence of a given genotypic
and phenotypic shift during the course of evolution, one should ask to what extent such shifts would have
occurred without environmental change. Of course, such tests are rarely practical but phenotypic novelties can
still be correlated to genomic shifts in response to environmental changes if enough information is available. We
surveyed and re-evaluated the published data in order to estimate the role of environmental changes on the
course of species and genomic evolution. Only a few published examples clearly demonstrate a causal link
between a given environmental change and the fixation of a genomic variant resulting in functional modification
(gain, loss or alteration of function). Many others suggested a link between a given phenotypic shift and a given
environmental change but failed to identify the underlying genomic determinant(s) and/or the associated
functional consequence(s).
The proportion of genotypic and phenotypic variation that is fixed concomitantly with environmental changes
is often considered adaptive and hence, the result of positive selection, even though alternative causes, such as
genetic drift, are rarely investigated. Therefore, the second aim herein is to review evidence for the mechanisms
leading to fixation.
Key words: genome evolution, environmental changes, positive selection, adaptation, evolutionary shift.
CONTENTS
I. Introduction ...................................................................................................................................... 553
(1) Defining biological function ....................................................................................................... 553
(2) The molecular mechanisms of gene co-option .......................................................................... 553
(a ) Co-option without shift of the original function .................................................................. 553
(b ) Co-option with shift of the original function ....................................................................... 553
* Address for correspondence: Tel: ]33 491 106 489; E-mail: Anthony.Levasseur@up.univ-mrs.fr; Pierre.Pontarotti@up.univ-mrs.fr
Biological Reviews 82 (2007) 551–572 � 2007 The Authors Journal compilation � 2007 Cambridge Philosophical Society
II. Functional losses ............................................................................................................................... 554
(1) Convergent loss with a suggested relationship between gene function and environmental
change ......................................................................................................................................... 554
(a ) Gene losses that occurred faster than under neutrality (positive selection) ........................ 554
(i ) FR1 ................................................................................................................................. 554
(ii ) CCR5 .............................................................................................................................. 555
(iii ) G6PD .............................................................................................................................. 555
(iv ) Riftia pachyptila haemoglobin ........................................................................................... 555
(b ) When positive selection cannot be detected ........................................................................ 556
(i ) Sws1 ................................................................................................................................. 556
(ii ) Ectodysplasin (EDA) ....................................................................................................... 556
( c ) Mass gene losses .................................................................................................................... 556
(d ) Loss not directly linked to an environmental change but to the acquisition of
a new function ....................................................................................................................... 556
(2) Suggested relationship between gene function and environmental change but absence of
convergent losses ......................................................................................................................... 556
(a ) Cases with positive selection ................................................................................................. 556
(i ) The selfing locus in Arabidopsis thaliana ........................................................................... 556
(ii ) The Duffy blood group locus (FY) ................................................................................. 556
(iii ) CASP12 ........................................................................................................................... 556
(b ) No positive selection detected ............................................................................................... 557
(i ) Olfactory receptors in Stenella coeruleoalba ....................................................................... 557
(ii ) Human bitter taste receptor genes ................................................................................. 557
(3) Convergent loss with no clear relationship between gene function and environment ............. 557
(a ) Galactose pathway ................................................................................................................ 557
(b ) Class I vomeronasal receptor ............................................................................................... 557
(4) No convergence and no clear relationship between gene function and environment ............. 557
III. Functional gains ................................................................................................................................ 557
(1) Cases in which all criteria are met ............................................................................................ 558
(a ) Artificial cases ........................................................................................................................ 558
(i ) Insecticides ...................................................................................................................... 558
(ii ) Antibiotics ....................................................................................................................... 558
(b ) Natural cases .........................................................................................................................558
(i ) Somatic evolution ........................................................................................................... 558
(ii ) RNAseI from ruminants and colobine monkeys ........................................................... 558
(2) Cases where only some criteria are met .................................................................................... 559
(a ) Case A: All criteria are met except that positive selection is not shown to occur for sites
involved in the function ........................................................................................................ 559
(i ) Lysosyme ......................................................................................................................... 559
(ii ) Major histocompatibility complex .................................................................................. 560
(b ) Case B: convergence, with no demonstrated positive selection for sites involved
in the function, and no clear correlation with the environment ......................................... 560
(i ) Aldehyde oxidase (AOX) and xanthine dehydrogenase (XDH) .................................... 560
(ii ) Semenogelin .................................................................................................................... 560
( c ) Case C: convergent evolution at the molecular level, with demonstrated positive
selection, but no demonstrated functional shift or relation with the environment ............ 561
(d ) Case D: convergent evolution at the molecular and functional levels, existence of
correlation with an environmental change, but no detection of positive selection ............ 561
(i ) Evolution of orthologous genes ...................................................................................... 561
(a ) Vertebrate rhodopsins .............................................................................................. 561
(b ) TTX-resistant sodium channel ................................................................................ 562
(ii ) Evolution of non-orthologous genes ............................................................................... 562
(a ) Antifreeze glycoproteins (AFGP) ............................................................................. 562
(b ) Crystallins ................................................................................................................. 562
(iii ) Evolution of genomic repertoire .................................................................................... 562
( e ) Case E: demonstrated positive selection on sites involved in the functional shift,
with functional convergence ................................................................................................. 562
( f ) Case F: demonstrated positive selection on sites involved in the functional shift,
but with no observed convergence among lineages ............................................................. 563
(i ) Probable link with an environmental change ................................................................ 563
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(a ) Proteorhodopsin ....................................................................................................... 563
(b ) TRIM5a ................................................................................................................... 563
(g ) ECP and EDN ......................................................................................................... 563
(d ) Lipase/feruloyl esterase A ....................................................................................... 563
(ii ) No clear link with an environmental shift ..................................................................... 564
(a ) Glutamate dehydrogenase 2 (GLUD2) ................................................................... 564
(b ) Glutathione transferases (GSTs) .............................................................................. 564
(g ) Case G: functional shift linked to an environmental change but with no evidence
for positive selection or convergence .................................................................................... 564
(i ) Iota crystallin .................................................................................................................. 564
(ii ) Melanocortin-1 receptor (Mc1r) ..................................................................................... 564
(h ) Case H: functional shift but no evidence for positive selection .......................................... 564
( i ) Case I: demonstrated positive selection but with no evidence for functional shift ............ 564
IV. Co-option through transcriptional shifts .......................................................................................... 565
(1) IL4 ............................................................................................................................................... 565
(2) Coagulation factor VII ............................................................................................................... 565
(3) MMP3 ......................................................................................................................................... 566
(4) LCT ............................................................................................................................................. 566
V. Subcellular localisation shift driven by environmental change ....................................................... 566
VI. Evidence for a role of environment on phenotypic shifts without information at the
genomic level ..................................................................................................................................... 567
VII. Conclusions ....................................................................................................................................... 567
VIII. Acknowledgments ............................................................................................................................. 568
IX. References ......................................................................................................................................... 568
I. INTRODUCTION
(1) Defining biological function
During the evolutionary history of species, genomic events
become fixed, first at the population then at the species level
due to selection or to genetic drift. These changes can have
different impacts at different functional levels (e.g. mod-
ifications of protein-coding or regulatory sequences).
We first need to clarify the notions of gene and protein
function. Indeed, depending on the author, the word
‘‘function’’ refers to either (i) the general biochemical
activity of a given gene product, or (ii) the cellular process in
which the gene product is involved, (iii) the detailed
mechanisms of catalysis or recognition, or (iv) a generalized
phenotype (e.g. ‘‘olfaction’’). The term ‘‘function’’ refers to
the specific results of specific experiments, and for that
reason, a ‘‘function’’ can be defined at different organiza-
tional levels of organisms.
The first level describes molecular functions such as
catalytic or binding activities. As two proteins with similar
molecular activities can generate different phenotypes
according to their subcellular locations, a second level
refers to the cell compartments where the molecular activity
takes place. The next functional level is the cell as a whole
and refers to cellular pathways, cascades or processes in
which a given gene is involved. In multicellular organisms,
functions can go beyond cellular functions: tissue distribu-
tion of biological processes, cellular interactions and
communications, etc. These notionscould even be further
developed towards yet higher levels of integration such as
population or social levels.
The function of a given gene and its product(s) can
change due to mutations that alter coding or regulatory
sequences resulting in a shift at the biochemical level,
subcellular localisation and/or transcriptional level which,
in turn, may lead to functional shifts at higher levels of
organization. Novelty in evolution is mainly the result of
functional shifts, also called ‘‘gene co-option’’ (Ganfornina &
Sanchez, 1999) whereas truly new genes (gene occurring
via overprinting for example, see Vernet et al., 1993) have
not been identified (Long et al., 2003). We will describe in
the following paragraph reported cases of co-option.
(2) The molecular mechanisms of gene
co-option
(a ) Co-option without shift of the original function
As demonstrated by the complexity of the immune system
in vertebrates, a given enzymatic product (e.g. the products
of the proteasome holoenzyme) may be recruited to a new
pathway (e.g. antigen presentation to the major histocom-
patibility complex) without any change in the basic
biochemical function of the enzymes (Danchin et al.,
2004). Thus, a mutation is not always necessary for a novel
biochemical function to appear.
(b ) Co-option with shift of the original function
Different types of mutations can generate co-option: (1)
Micromutations (substitutions, small indels). If located in
the coding sequence, such mutations can lead to bio-
chemical or sub-cellular localisation shifts whereas they can
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lead to transcriptional shifts when they affect promoters.
(2) Gene shuffling between coding sequences or between
a coding sequence and promoter. (3) Gene duplications by
regional or whole-genome duplications (i.e. polyploidiza-
tion) followed by micromutations and gene shuffling.
All these events alter the genomic context, as illustrated
by single gene (promoter plus coding sequence) duplication:
either the two copies are subfunctionalized at the bio-
chemical or transcriptional level (for reviews see Prince &
Picket, 2002, and van Hoof, 2005) or neofunctionalized, i.e.
one of the duplicates maintains the ancestral function while
the other accumulates substitutions and evolves towards
a new function (e.g. see Rodriguez-Trelles, Tarrio & Ayala,
2001, and Bos, 2005). Neofunctionalization can be
associated with the asymmetric distribution of type I
substitution sites (conserved in one subfamily but not in
the other) indicating relaxed selection followed by positive
selection (not detectable if the event is ancient due to
saturation of synonymous substitutions).
Van Hoof (2005) designed a test to discriminate between
neo- and subfunctionalization. He analysed a pair of
duplicates in yeast that evolved in an asymmetric manner
and the non-duplicated corresponding orthologue in the
closest related species. It appeared that the non-duplicated
orthologue was able to complement both copies, while the
copies were not able to complement each other, suggesting
that both copies were not neofunctionalized but subfunc-
tionalized. Another possibility to explain maintenance of
duplicates under neutrality is to consider that the pre-
duplication gene was once expressed under two conditions
that were distributed between the two copies after
duplication. Several lines of experimental evidence support
this model. For instance, the en1 gene is expressed in the
pectoral appendage bud and in some neurones in mice and
chicks, while in zebrafish Brachydario rerio two paralogues are
found, one being expressed in the pectoral appendage bud
and a second in neurons (Force et al., 1999).
The best examples of co-option are found among the
crystallin genes. The ocular lens in vertebrates and some
invertebrates is a transparent cellular tissue involved in light
refraction. The necessary refraction index is achieved by the
accumulation of soluble proteins: the crystallins. The genes
coding for crystallins have been recruited in a recurrent
manner from genes having non-lens functions (see Section
III.2.d). Because in some organisms the crystallin function
and the enzymatic function are encoded by two related but
distinct genes, Piatigorsky & Wistow (1989) have suggested
that the acquisition of the new, additional, molecular or
transcriptional behaviour occurred at first, then was
followed by a duplication event, and finally each duplicate
lost a part of its ancestral ‘‘dual-behaviour’’. In the text we
do not distinguish direct co-option followed by gene
duplication from gene duplication followed by co-option.
Shifts in molecular, transcriptional, and subcellular local-
ization parameters could have an impact at different
functional levels of the organism. For example, neo expression
of a ‘‘master’’ regulator gene can modify the expression of
several genes from the same cascade in a new cellular
environment. One of the most famous examples is that of the
Dll gene and butterfly eyespots (True & Carroll, 2002).
The main questions addressed herein are: how many co-
option events have been fixed in response to environmental
changes and what is the role of positive selection in this
process? Positive selection is indeed implicated in many
studies but this conclusion is often based on observed
sequence changes without evidence for a possible link
between these evolutionary shifts and functional shifts or
even for the existence of an environmental change. These
events are rarely related to a precise environmental shift.
We will focus here mainly on genomic changes that have
easily detectable functional impacts, such as functional
losses or biochemical, transcriptional, and cellular localiza-
tion shifts. Many phenotypes have been suggested being
selected due to environmental changes but the genomic loci
involved have generally not been characterized. They will
be briefly discussed in the final part of this review.
Extensive reviews explaining how to detect positive
selection at the population, species or sequence levels are
already available (see, for example, Nielsen, 2005; Ponting &
Lunter, 2006; Yang & Bielawski, 2000).
II. FUNCTIONAL LOSSES
Functional loss can be seen as an extreme case of co-option
and can occur at the level of functional sites (subfunction-
alization), genes (pseudogenization) or of whole cascades.
Under selective constraints, the role of the environment on
genomic evolution is not always obvious unless the same
functional loss has occurred independently several times in
similar environments (convergence). Consider a species
subjected to a particular environmental change, some of its
original functions are no longer essential for survival and
may be lost (together with the associated genes) through
genetic drift. Here, genomic evolution would have been
driven by environmental change despite the lack of any
form of selection (or because it allowed relaxation of the
selective pressure). Therefore, loss of function can either be
fixed by positive selection (if the maintenance of the
function is deleterious) or by genetic drift (if the loss/
maintenance of function is neutral) in response to
environmental change. Ideally, statistical tests should be
applied to determine how often a given functional loss has
occurred for a given environmental shift.
Table 1 provides examples from the literature of losses
possibly associated with environmental changes, classified
according to the presence or absence of evidence for
convergence, positive selection or a functional link.
(1) Convergent loss with a suggested
relationship between gene function and
environmental change
(a ) Gene losses that occurred faster than under neutrality (positive
selection)
( i ) FR1The FRIGIDA (FR1) gene has been shown to be a major
determinant of flowering time in Arabidopsis thaliana. A
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majority of early-flowering ecotypes shows one or two
deletions that generate a frameshift in the FR1 open reading
frame (ORF), suggesting that this phenotype has arisen at
least twice. Le Corre, Roux & Reboud (2002) performed
a population analysis on different ecotypes and confirmed
that the loss of function mutations was associated with an
early-flowering phenotype, these gene inactivations system-
atically evolved in a non-neutral fashion. Moreover, they
confirmed that the gene inactivation was phenotypically
linked to an early flowering ecotype adaptated to cold
environments (Johanson et al., 2000; Le Corre et al., 2002).
This represents a strong indication that environmental
change has driven this genetic change.
( ii ) CCR5
This primate transmembrane receptor is a cellular gateway
for the entry of HIV-1 and all strains of SIV. Human
homozygotes for the CCR5 null allele which has a 32 base
pair (bp) deletion are highly resistant to HIV-1 infection.
Another null allele (24 bp deletion) of CCR5 has
convergently evolved in sooty mangabeys (Cercocebus atys),
a natural host of SIV. The occurrence of the mangabey null
allele at an appreciable frequency (around 4%) could be
explained by positive selection; null homozygotes are
protected from SIV infection because the encoded protein
is not transported to the cell surface (Palacios et al., 1998).
The null allele has been shown to be positively selected in
humans (Galvani & Novembre, 2005). However, the exact
nature of the selective pressure involved in the origin of the
CCR5-{delta} 32 allele and its high prevalence in
European populations (approximately 10%) is unclear as
the HIV epidemic in humans is much more recent than the
age of the null allele (about 700 years). However, both HIV
and poxviruses enter leukocytes using chemokine receptors;
it is plausible that the loss of the CCR5 chemokine receptor
originally conferred resistance against smallpox. This
hypothesis is supported by a correlation between historical
smallpox epidemics and allele geographic distribution
(Galvani & Slatkin, 2003).
( iii ) G6PD
The frequencies of the low-activity coding alleles of glucose-
6-phosphate dehydrogenase (G6PD) in humans are highly
correlated with the prevalence of malaria. The low activity
coding alleles are thought to reduce the risk of infection by
Plasmodium falciparum and are maintained at high frequencies
despite the haemopathologies they cause (anaemia). Hap-
lotype analysis of the low-activity coding alleles (8–20% for
the G6PDA- and < 5% for the G6PDMed) indicated that the
mutations at this locus evolved independently (Tishkoff
et al., 2001) and their frequencies have increased at a rate
too rapid to be explained by genetic drift (Tishkoff et al.,
2001; Sabeti et al., 2002). Therefore, though a functional
link between low activity of G6PD and protection from
Plasmodium falciparum has not been established, the parasite
could have driven genome evolution at this locus. Such
a mechanism might also be involved in the sickle cell
haemoglobin allele (given as a classical example of positive
selection driven by the environment by Haldane, 1949),
although new data are needed to attest formally for fixation
under positive selection (Pagnier et al., 1984; Currat et al.,
2002).
(iv) Riftia pachyptila haemoglobin
Haemoglobin of the deep-sea hydrothermal vent vestimen-
tiferan Riftia pachyptila (Annelida) is able to bind hydrogen
sulphide (H2S) to free-cysteine residues and transport it to
fuel endosymbiotic sulphide-oxidizing bacteria. Cysteine
residues are key amino acids (aa) conserved in annelid
globins living in sulphide-rich environments, but not in
globins from annelids living in sulphide-free environments.
Synonymous and non-synonymous substitutions analyses
from two different sets of orthologous annelid globin genes
revealed that free-cysteine residues in annelids living in
sulphide-free environments were lost during the course of
evolution due to positive selection (free cysteines are
disadvantageous in H2S-free environments because they
interact with blood components disturbing homeostasis and
reducing fitness; Bailly et al., 2003). The ability to bind
hydrogen sulphide has been lost in several worms living in
H2S-free environments. These worms form polyphyletic
groups (McHugh, 1997), suggesting according to Bailly et al.
(2003), that binding to H2S could represent the ancestral
state, and that loss of binding capacity occurred indepen-
dently many times. However, the positive selection has been
shown to occur in only one case; it would be interesting to
investigate other annelids living in H2S-free environments.
Table 1. Functional losses of genes classified according to the presence of convergence, positive selection or a functional link
FR1 CCR5 G6PD
Haemoglobin Trichromacy
SWS1
EDA
SCR FY
CASP12
OR
TAS2R
Galactose
pathway V1RL
TRP2
V1R
Functional explanation for selective
environmental advantage associated with gene loss
] ] ] [ [
New environment should lead to relaxed
functional constraint
NA ] NA ] NA NA
Positive selection ] [ ] [ [ [
Convergence : same gene loss in similar
environmental changes
] ] [ [ ] [
NA, not applicable.
–: criteria not fulfilled or not tested.
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Biological Reviews 82 (2007) 551–572 � 2007 The Authors Journal compilation � 2007 Cambridge Philosophical Society
(b ) When positive selection cannot be detected
( i ) Sws1
The sws1 (short wavelength sensitive type I pigment group)
gene mediates ultraviolet (UV) and violet vision in
vertebrates except dolphins and coelacanths Latimeria
chalumnae. Since sister species of both dolphins and
coelacanths have functional sws1, it has been deduced that
the sws1 gene became independently non-functional in
these two groups. Such loss of function is unsurprising for
coelacanths which live at depths exceeding 80 m, where UV
and violet light are not available, but is less straightforward
for dolphins which spend a significant portion of their time
at the surface. It is possible that UV and violet vision may
have been replaced by other communication means in these
species (Shi & Yokoyama, 2003).
( ii ) Ectodysplasin (EDA)
Morphological characters of several groups of teleost fish
changed following the colonization of new freshwater
environments; many shows a reduction of the bony armour
found in their oceanic ancestors (Bell & Foster, 1994).
Marine and freshwater stickleback populations have been
studied with reference to the presence (‘‘complete’’ morph)
or absence (‘‘low’’ morph) of armour plates. Mapping,
sequencing, and transgenic studies demonstrated that
ectodysplasin (EDA) (a member of the tumour necrosis
family of secreted signalling molecules) played a key role in
these evolutionary changes in natural populations and that
parallel evolution of freshwater stickleback low-plated
phenotypes has occurred repeatedly by selection of Eda
alleles derived from an ancestral haplotype (Colosimo, et al.,
2005). The selective advantage of armour plate reduction
after freshwater colonization could be due to increased
body flexibility and manoeuvrability, changes in swimming
performance and predation regime in the freshwater
environment.
(c ) Mass gene losses
In the endosymbiotic bacterium Buchnera aphidicola the
genome contains 580 genes whereas the closely related
species Escherichia coli has 4,300 genes. It seems that
B. aphidicola lost 85 % of its genes 220–250 million years
ago, during adaptation to an endosymbiotic life-style. Many
other examples of multiple gene losses are foundamong
bacteria symbionts (Moran, 2003) or eukaryotic intracellu-
lar parasites (e.g. the microsporidian Encephalitozoon cunil;
Katinka et al., 2001). Such genome shrinkage could be
explained by the fact that host tissues supply many
metabolic intermediates and cofactors making these
symbiont genes redundant. Furthermore, as host-associated
bacteria have small genetic population sizes relative to free-
living relatives (Funk, Wernegreen & Moran, 2001), genetic
drift would accelerate the loss of non-essential genes.
Pathways for the synthesis of vitamins and amino acids
present in Bacteria, Archea, fungi, plants, etc., but absent in
Metazoa, provide good examples of this class of gene losses
(Danchin, Gouret & Pontarotti, 2006). Using data from
complete animal genomes, Friedman & Hughes (2004)
showed that the same gene families have been lost
independently in different lineages and that this has
occurred more often than expected if gene loss occurred
randomly.
(d ) Loss not directly linked to an environmental change but to the
acquisition of a new function
The acquisition of obligate trichromacy occurred indepen-
dently in the Catharrhini (apes, old-world-monkeys and
a new-world monkey (the howler monkey Alouatta seniculus).
In these trichromatic groups, a significantly higher pro-
portion of olfactory receptor pseudogenes is found com-
pared with closest relatives, suggesting that the deterioration
of the olfactory repertoire occurred concomitantly with the
acquisition of full trichromatic colour vision in primates
(Gilad et al., 2004). However, a link with environmental
conditions is difficult to demonstrate formally.
(2) Suggested relationship between gene
function and environmental change but absence
of convergent losses
(a ) Cases with positive selection
( i ) The selfing locus in Arabidopsis thaliana
Shimizu et al. (2004) provided very strong evidence that
inactivation of the SCR gene, which encodes a pollen coat
protein required for reproductive self-incompatibility, has
been selected during the evolution of A. thaliana. This may
have been a key event enabling A. thaliana to expand its
range from glacial regions into Eurasia post-Pleistocene.
This phenomenon has not been observed for other species
under the same environmental shift.
( ii ) The Duffy blood group locus (FY)
A single cis-regulatory single nucleotide mutation has been
demonstrated to shut down the expression of the FY locus
in humans and confers resistance to malaria. This null
expression allele is over-represented in sub-Saharan Africa
whereas it is present at very low frequencies in other
populations. Moreover, Fixation index (FST) values based on
that locus are higher than those based on surrounding
sequences or 10 non-coding and non-functional regions
scattered throughout the genome. There is thus good
evidence for local adaptation of human subpopulations to
the malarial selective agent through selection on this
mutation despite the absence of convergence (Hamblin &
Di Rienzo, 2000).
( iii ) CASP12
The gene encoding for the CASPASE12 protein is non-
functional in the human lineage. This null allele appeared
shortly before modern humans migrated out of Africa
(Wang, Grus & Zhang, 2006). Wang et al. (2006) show that
nearly complete fixation of the null allele has been driven by
positive selection, and propose that the functional loss was
a consequence of exposure to new antigens during
colonization of new areas. Indeed, CASPASE12 is involved
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in the regulation of inflammatory and immune responses to
endotoxins and protections against severe sepsis. In this
case, the role and nature of environmental change is not
clear and it remains to be tested whether this gene has also
been convergently lost in other lineages although initial
analysis shows that the null allele is rare in other mammals
(Saleh et al., 2004). However, in contrast with the CCR5
and Duffy genes, whose null alleles are specifically present
in limited geographic areas (Europe and Africa, respec-
tively), the loss of CASPASE 12 is characteristic of the
whole human lineage.
(b ) No positive selection detected
( i ) Olfactory receptors in Stenella coeruleoalba
The olfactory receptor repertoire of the dolphin S. coeruleoalba
consists only of non-functional class II olfactory receptor
genes (pseudogenes; Freitag et al., 1998). Class II olfactory
receptors, which recognize volatile odorant molecules, were
duplicated en masse when tetrapods colonized land. The
terrestrial ancestor of dolphins would therefore have had
a set of class II genes like most mammals for which sequences
are available. As these class II receptors presumably did not
function in an aquatic environment, pseudogenization pre-
sumably was not counter-selected. It would be interesting to
examine convergent gene losses in other aquatic mammals
that colonized the aquatic environment independently.
( ii ) Human bitter taste receptor genes
Bitter taste perception prevents mammals from ingesting
poisonous substances because many toxins taste bitter.
Wang, Thomas & Zhang (2004) hypothesized that selective
constraints on human bitter taste receptor (TAS2R) genes
might have been relaxed because of changes in diet, use of
fire and reliance on other means of toxin avoidance that
emerged during human evolution. They looked at intra-
specific variations of all 25 genes of the human TAS2R
repertoire and found hallmarks of neutral evolution
including : (1) similar rates of synonymous (dS) and non-
synonymous (dN) nucleotide changes among rare poly-
morphisms, (2) no variation in dN/dS among functional
domains; and (3) segregation of pseudogene alleles within
species and fixation of loss-of-function mutations.
(3) Convergent loss with no clear relationship
between gene function and environment
In most studies investigating functional losses or gains,
a clear causal relationship between an environmental
change and a genotypic/phenotypic shift has not been
demonstrated. However, establishing such links is extremely
difficult since the effect of the environment is possibly
indirect and palaeontological and palaeoenvironmental
data are often incomplete.
(a ) Galactose pathway
Repeated losses of functionally linked genes have been
described for seven genes involved in the galactose pathway
(Hittinger, Rokas & Carroll, 2004). These genes were lost at
least three times independently in the yeasts Eremothecium
gossypii, Candida glabrata and Saccharomyces kudriavzvii and
Hittinger et al. (2004) linked this to a change in ecological
niche. An alternative view is that loss of these genes
prevented the collapse of the gene network, rather than
representing an adaptation to new environmental param-
eters (Silwa & Korona, 2005).
(b ) Class I vomeronasal receptor
Another example of recurrent gene losses can be found for
the class I vomeronasal receptor-like genes (V1RL) (Mundy &
Cook, 2003) which were lost seven times independently. No
obvious environmental shift can be linked to these gene losses.
(4) No convergence and no clear relationship
between gene function and environment
Genes encoding the TRP2 ion channel and V1R pheromone
receptors are two components of the vomeronasal phero-
mone transduction pathway and have been pseudogenized
during the evolution of old world monkeys (OWM) (Zhang &
Webb, 2003). It is however difficult to link this loss to
a particular environmental change. Phylogenetic distribution
of vomeronasal pheromone insensitivity is concordant with
conspicuous female sexual swelling and male trichromatic
colour vision, suggesting that vision-based signaling may
have replaced a vomeronasal mediated chemical-based
system in hominoids and OWM. Trichromacy may have
arisen or becomefixed because aided detection of young
leaves and ripe fruits against dappled foliage. Once acquired,
trichromatic vision allowed perception of subtle colour
changes which may have provided the selective force for
sexual swelling. However, Webb, Cortes-Ortiz & Zhang
(2004) showed that pheromone perception and full tri-
chromatic vision coexist in howler monkeys. Consequently, it
is difficult to determine the validity of this scenario.
III. FUNCTIONAL GAINS
Like functional losses, functional gains can be positively
selected or not. To show that a gain of function has
occurred due to an environmental change, the following
criteria should be met: (1) there is a clear correlation
between the environmental change and the functional gain.
(2) The sequence (or part of the sequence) has undergone
substitutions characterized by positive selection. (3) The
corresponding protein has undergone a functional shift (co-
option) and the shift is associated with sites that have been
shown to be under positive selection. (4) There is evidence
for convergence at the functional and, possibly, at the
molecular level (substitutions at relevant amino-acid sites
occurring in several lineages, and a statistical test demon-
strating significant convergent or parallel substitutions). (5)
The functional gain is correlated with an environmental
shift. (6) The novelty confers a selective advantage in the
new environment.
Role of the environment on gene evolution 557
Biological Reviews 82 (2007) 551–572 � 2007 The Authors Journal compilation � 2007 Cambridge Philosophical Society
Alternatively a gain of function could be driven by an
environmental change without positive selection, i.e. by
genetic drift. Note that an apparent lack of positive selection
can be due to low statistical power preventing detection; in
this case, the role of the environment can still be detected in
the presence of convergent gains. Many studies have
focused on specific genes where functional shift can be
detected but adaptation to new environments could involve
a combination of several genes producing a particular
phenotypic trait.
(1) Cases in which all criteria are met
(a ) Artificial cases
To our knowledge, most cases reported in the literature that
fulfill all the above criteria are associated with artificial
selection imposed by humans (most notably, insecticide,
herbicide and antibiotic-resistance genes: Palumbi, 2001;
Wright et al., 2005).
( i ) Insecticides
Three main types of resistance mechanisms have been
described to date, two involving enhanced insecticide
detoxification and one rendering the target site for the
insecticide insensitive to its effects. This latter case has been
found repeatedly for a large range of species and types of
insecticide in proteins including acetylcholinesterase,
g-aminobutyric acid (GABA) receptors and voltage-gated
Na] channels which are the targets of organophosphate,
cyclodiene and synthetic pyrethroids, like DDT, respectively.
One of the remarkable aspects of insecticide resistance is
the recurrence of exactly the same amino acid changes in
orthologous proteins across different species (Ffrench-
Constant, Daborn & Le Goff, 2004; Hartley et al., 2006). In
the GABA receptor case, insecticide resistance is associated
with replacement of alanine at position 302 with either
a serine or a glycine residue. Alanine 302 is thought to lie in
the narrowest part of the chloride ion channel and
replacement of this crucial residue plays a dual role both in
reducing insecticide-binding and in destabilizing the insecti-
cide-bound conformation of the receptor. Population studies
have documented increases in resistant allele frequencies in
response to insecticide application and have shown that the
corresponding loci are under positive selection (see reviews by
Roush & Mckenzie, 1987; Guillemaud et al., 1998; and Scott,
Diwell & McKenzie, 2000).
Resistance to organophosphates is particularly interest-
ing: molecular analysis of preserved specimens collected
indicate that insecticide-resistant haplotypes of the esterase 3
gene present in two Australasian sibling blowfly species
(Lucilia cuprina and Lucilia sericata) spread during the
resistance outbreak associated with the first use of these
insecticides in Australia (around 1955) (Hartley et al., 2006).
( ii ) Antibiotics
ß-lactam antibiotics, including penicillin, ampicillin, ceph-
alosporins and monobactams (and their derivatives) account
for 50% of global antibiotic consumption. The integrity of
the ß-lactam ring is necessary for the activity of the
antibiotic which exerts its effect through inactivation of
transpeptidases that catalyse key cross-linking reactions in
peptidoglycan synthesis. Resistance to ß-lactams is the result
of expression of ß-lactamases, enzymes that degrade and
inactivate ß-lactams. The most common ß-lactamases are
the TEM ß-lactamases encoded by the TEM-1 gene and its
relatives. TEM-1 is taxonomically widely distributed
(Meideiros, 1997) and exists at high frequencies in diverse
antibiotic-resistant bacterial species (Chanal et al., 2000; Yan
et al., 2000). Antibiotic resistance in bacteria can occur by
many diverse mechanisms including nucleotide changes in
the gene coding for the antibiotic target. In the case of
TEM b-lactamase resistance, nine amino acid substitutions
have been demonstrated to have occurred more than once;
most of the observed substitutions are non-synonymous
suggesting that positive selection is occuring (Barlow & Hall,
2002).
Similar examples can be found for herbicide resistance
(see, for example, Tranel & Wright, 2002).
(b ) Natural cases
The only natural cases where all the above criteria are met
have been described in somatic evolution concerning
antibodies and for the pancreatic ribonuclease in leaf-
eating monkeys.
( i ) Somatic evolution
Concerning antibodies, it has been shown that B cells
undergo positive selection for mutations that increase their
affinity for the antigen. The mutations that occur during the
process of somatic recombination have been shown to be
functional (by promoting antigen binding shifts; see, for
example, Wellmann et al., 2005) and under positive selection
(Manser, 1989). Parallel evolution has also been described
(Wysocki, Gefter & Margolies, 1990).
( ii ) RNAseI from ruminants and colobine monkeys
The ancestral function of RNAseI is the degradation of
double-stranded RNA. A new biochemical function (i.e. the
ability to digest single-stranded RNAs at low pH) appeared
independently in both colobines (primates) and ruminants
(artiodactyls) in relation to their capacity to perform
fermentation in the pre-stomach. Recently, Zhang (2006)
showed that the gene encoding RNaseI was independently
duplicated in Asian and African leaf-eating monkeys and
that those new genes acquired enhanced digestive efficien-
cies through parallel amino acid replacements driven by
positive selection.
In ruminants, positive selection has not been demon-
strated for RNAseI (Golding & Dean, 1998). Furthermore
the amino acids involved in the functional shift are different
for ruminant and colobine RNAseI: five amino acid
substitutions in ruminant RNAseI that are known to affect
its catalytic activity against double-strand RNA (Jermann
et al., 1995) are not present in colobines.
The relationship between digestive behaviour and
environment is not straightforward. However, according
Anthony Levasseur and others558
Biological Reviews 82 (2007) 551–572 � 2007 The Authors Journal compilation � 2007 Cambridge Philosophical Society
to palaeomolecular work (Jermann et al., 1995), the co-
option events in ruminants occurred around 35 million
years (Myr) ago which is also the age of the earliest known
ruminant fossil. Therefore it appears that the biochemical
functional shift occurred in response to acquisition of
herbivory. The Oligoceneglobal cooling that followed
allowed tough grasses to expand and possibly gave
a selective advantage to tough-grass eaters. For colobines
monkeys, fossil evidence suggests that a shift to foregut
fermentation occurred at least 10 Myr ago, predating the
duplications of RNaseI (Delsen, 1994). Therefore a func-
tional shift in the optimal pH of RNaseI was not necessary
for the change in diet but rather provided a selective
pressure for enhanced performance of digestive RNases in
acidified environments. To our knowledge, no other
example in this category has been described.
(2) Cases where only some criteria are met
Other natural cases reported in the literature do not fulfill
all the criteria required to demonstrate a role of the
environment on gene evolution via positive selection. These
different cases are listed in Table 2 and shown in a simplified
overview in Fig. 1.
(a ) Case A: All criteria are met except that positive selection is not
shown to occur for sites involved in the function
( i ) Lysosyme
Lysosyme is an enzyme which disrupts bacterial peptido-
glycans and in tetrapods is normally expressed in tear
macrophages, saliva, avian egg white and mammalian milk;
however, lysosyme has been recruited independently in the
foregut of ruminants, colobine monkeys, and the hoatzins
Opisthocomus hoazin (a leaf-eating bird). These stomach
lysosymes have similar convergent biochemical properties:
lytic activity is clearly optimal approximately at pH 5 for
ruminant, colobine, and hoatzin lysosymes compared to pH
5–7 in other species. Ruminant and colobine digestive
lysosymes also show increased resistance to inactivation by
pepsin compared to other lysosymes. Positive selection has
been detected in primate lysosyme (Messier & Stewart,
1997) by inference of ancestral sequences and dN/dS
analysis on each branch of the tree. Most adaptive
substitutions in lysosyme seem to have occurred at the
origin of the colobine group. The same result was obtained
by Yang & Nielsen (2002) using codon-substitution models
at individual sites along specific lineages. Stomach lysozyme
from ruminants also underwent positive selection (Jolles
et al., 1990; Yu & Irwin, 1996; Zhang & Kumar, 1997): sites
75, 87 underwent parallel evolution, however, it is necessary
to test functionally these two positions by site-directed
mutagenesis for a definitive demonstration of their involve-
ment in the gain of function.
In the hoatzin, the lack of available sequence prevented
testing the possible occurrence of positive selection.
The relationship between digestive behaviour and
environment changes in ruminants remains hypothetical.
However, according to ancestral sequence reconstruction T
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Role of the environment on gene evolution 559
Biological Reviews 82 (2007) 551–572 � 2007 The Authors Journal compilation � 2007 Cambridge Philosophical Society
(Messier & Stewart, 1997), the co-option events seem to
have occurred, as in the case of ruminant digestive RNAseI,
around 35 million years ago. Additional palaeontological
data and phylogenetic analyses are necessary to confirm the
nature of the environmental shift responsible for lysozyme
recruitment in the stomach.
( ii ) Major histocompatibility complex
In the major histocompatibility complex (MHC) adaptive
evolution has promoted diversity of the antigen recognition
site (Hughes & Nei, 1988). Further analyses identified that
the majority of residues located in the antigen recognition
site are involved in antigen-binding (Yang & Swanson,
2002) and are under positive selection. However no
functional study has been undertaken yet to test whether
positively selected residues are indeed directly involved in
the peptide-binding shift. Convergent changes in the MHC
have been demonstrated (Andersson et al., 1991; Kriener
et al., 2000).
(b ) Case B: convergence, with no demonstrated positive selection for
sites involved in the function, and no clear correlation with the
environment
( i ) Aldehyde oxidase (AOX) and xanthine dehydrogenase (XDH)
Aldehyde oxidase (AOX) and xanthine dehydrogenase
(XDH) encode two members of the xanthine oxidase family
of molibdo-flavoenzymes with different functions. AOX and
XDH are homodimers (290 kDa) but each monomer acts
independently in catalysis. XDH is involved in catabolism
of purines by oxidizing hypoxanthine into xanthine, and
xanthine into uric acid, whereas AOX catalyses the
oxidation of aldehydes into acids and does not show
reactivity with hypoxanthine. AOX and XDH originated
from duplication events and thus provide an interesting case
of neofunctionalization. Rodriguez-Trelles et al. (2001)
demonstratedthat Aox evolved independently twice from
two different Xdh paralogues whose duplicates were
subjected to positive selection after each round of
duplication. Moreover, in both cases, the same amino acids
(located in the flavin adenine dinucleotide and substrate-
binding pockets) have been positively selected. Although the
link with an environmental change is difficult to demon-
strate, convergence at functional and molecular levels
strongly argues in favour of an adaptive event.
( ii ) Semenogelin
In primates, semenogelin is the main protein of the seminal
fluid produced by seminal vesicles. After ejaculation,
semenogelin undergoes covalent cross-linking to become
the principal structural component of semen coagulum
in the reproductive tract of the recipient female. Over
time, the coagulum is liquefied through the cleavage of
semenogelin and this process leads to the release of sperm
from the coagulum. This process is crucial in preventing
fertilization of a recently inseminated female by rival males
in subsequent copulations and thus is subject to different
selective regimes under different mating systems. Dorus et al.
(2004) showed a correlation between the rate of evolution of
SEMG2, and three behavioural and physiological repro-
ductive parameters in primates: mean number of male
partners per female periovulatory period; the residual testis
size within the species; and semen coagulation rates.
Occurrence of convergent or parallel evolution at the
molecular level would strengthen the case for selection
although ultimate demonstration requires functional tests
Fig. 1. Simplified overview of cases of functional gain. Cases are classified according to the presence (blue arrows) or absence (red
crosses) of simplified criteria (left box). Cases are defined and discussed in the text, section numbers are as in Table 2.
Anthony Levasseur and others560
Biological Reviews 82 (2007) 551–572 � 2007 The Authors Journal compilation � 2007 Cambridge Philosophical Society
(using site-directed mutagenesis to quantify the impact of
the incriminated substitutions on semen coagulation). The
link between an environmental change and this behavioural
shift remains unclear.
(c ) Case C: convergent evolution at the molecular level, with
demonstrated positive selection, but no demonstrated functional
shift or relation with the environment
FOXP2 (forkhead box P2) is a gene probably involved in
cerebral and cognitive processes such as speech acquisition.
Positive selection in humans has been shown at the
population level (Zhang, Webb & Podlaha, 2002; Enard,
2002) but neither a functional shift nor a correlation with an
environmental change has been demonstrated. Convergent
or parallel evolution at the molecular level has been
documented between humans and carnivores although
statistically significant positive selection was not detected in
carnivores.
(d ) Case D: convergent evolution at the molecular and functional
levels, existence of correlation with an environmental change, but
no detection of positive selection
When positive selection is not detected, gain of function
could also be the consequence of a relaxed functional
constraint allowing the new function to appear under
neutrality. Moreover, many instances of selection are
probably not detectable by any currently available method.
The presence of convergence provides significant proof of
a role of the environment on the evolutionary change.
Therefore evidence of positive selection is not strictly
required to substantiate an environmental influence.
( i ) Evolution of orthologous genes
(a) Vertebrate rhodopsins. Retinal photoreceptors consist
of a light-absorbing component (the chromophore) and
a protein moiety (the visual pigment or opsin Wald,
1968). In vertebrates, two types of chromophore coexist,
11-cis-retinal and 11-cis-3,4 dehydroretinal and five types
of opsins. There are five evolutionary groups of opsins,
one in rods and four in cones. The rod opsin (rhodopsin
1, RH1) facilitates formation of black and white images in
dim light whereas the cone opsins mediate colour vision
in bright light. The four colour opsins differ in their light
sensitivity: short-wavelength ultraviolet (UV)-sensitive 1
(SWS1, maximum absorbance lmax ¼ 360–430 nm),
short-wavelength sensitive 2 (SWS2, lmax ¼ 440–460 nm),
rhodopsin–like 2 (RH2, lmax ¼ 470–510 nm) and middle
(green) and long (red) wavelength sensitive (MWS/LWS,
lmax ¼ 510–560 nm). The light sensitivity of a visual pig-
ment is determined by the chromophore and its interac-
tion with the amino acid residues lining the pocket of the
opsin in which chromophore is embedded.
RH1 Most RH1 pigments tested so far have lmax values
around 500 nm; however, marine conger eel Conger myriaster,
bottlenose Tursiops truncates and saddleback dolphins
Delphinus delphis, and coelacanths Latimeria chalumnae have
RH1 pigments that show a 10–20 nm blue-shift in lmax
value (Yokoyama, 2000; Yokoyama, personal communica-
tion) possibly because they inhabit aquatic environments
dominated by blue light. Although several pigments are
known to have a lmax, the molecular basis of this shift
has been analysed only for coelacanths (Yokoyama et al.,
1999). While positive selection could not be detected
with statistical significance, their analysis suggested that
the same amino acid substitution occurred several times
independently.
In squirrel fish rhodopsins (Yokoyama & Takenaka,
2004), RH1 lmax ranges from 481nm to 501nm. Phyloge-
netic and mutagenesis analyses suggest that the common
ancestor of these pigments had a lmax value of 493 nm and
that extant values were generated largely by three amino
acid replacements: E122M, F261Y and A292S. The
probability of simultaneous substitution of these three
amino acids occurring by chance is only 2.5 � 10[9. The
close correlation between the lmax values of these pigments
and the wavelengths of light available to these species
suggests that this functional shift can be associated with an
identified environmental change.
LWS/MWS Yokayama & Yokoyama (1990) reported that
red pigments in humans and fish independently evolved
from green pigments by identical amino acid substitutions
at key functional positions. Indeed, three amino acid sites
underwent parallel substitutions in bony fish and primate
opsins, generating a similar change in lmax. Zhang (2003)
using the statistical test developed by Zhang & Kumar
(1997) rejected the null model of parallel substitutions due
to chance alone. The same parallel substitutions were later
found to have occurred in other species, and in all cases, the
events were correlated with changes in lmax (Boissinot et al.,
1998; Yokoyama & Radlwimmer, 2001). The environmen-
tal changes that selected for these functional shifts are not
yet known.
SWS1/SWS2 Shi & Yokoyama (2003) showed that
reconstructed SWS pigments of the ancestor of bony
vertebrates had a lmax of 360 nm, enabling UV vision. A
shift towards the violet spectrum occurred independently in
different vertebrate lineages, partly due to a single amino-
acid substitution. Shi & Yokoyama (2003) identified amino-
acid substitutions that affected the lmax of SWS1 opsins. As
noted by Zhang (2003), from such information, it should be
possible to predict the lmax of any SWS1 opsin simply from
its sequence. Odeen & Hastad (2003) obtained the SWS1
sequence for 41 birds and predicted that UV vision was
regained several times independently.
The difference in peak sensitivity between the UV and
violet spectrum (at least 23 nm) is quite dramatic and
changes not only the perception of objects that reflect light
solely in the UV or violet ranges but also the perception of
objects reflecting longer wavelengths. All of the six raptors
examined by Odeen & Hastad (2003) possessed violet-
sensitive SWS1, unlike many of their Passeriform prey. This
could mean that preybirds use colours for signaling that are
conspicuous to members of their own species but cryptic to
raptors (Odeen & Hastad, 2003). Selection should favour
stronger signals in the wavelengths to which predators
(including human hunters) are insensitive: either higher
plumage reflectance in the SWS1 and SWS2 ranges or
Role of the environment on gene evolution 561
Biological Reviews 82 (2007) 551–572 � 2007 The Authors Journal compilation � 2007 Cambridge Philosophical Society
higher sensitivity to those parts of the spectrum (Odeen &
Hastad, 2003).
(b) TTX-resistant sodium channel. Coevolution between
the garter snake Thamnophilis sirtalis and its toxic prey, the
newt Tarisha granulosa, has resulted in geographic variabil-
ity in a physiological trait in this snake: resistance to tetro-
dotoxin (TTX) (Brodie, 2002). Tetrodotoxin causes
paralysis and death by binding to the outer pore of
voltage-gated Na] channels [tsNa(V)1.4] blocking nerve
and muscle fibre activity. Some populations of T. granulosa
have high levels of TTX in their skin, providing a defence
against predators. Sequence analyses of the TTX Na]
channel gene showed that TTX resistance evolved at least
twice during the radiation of T. sirtalis and in vitro experi-
ments revealed the amino acid substitutions involved in
the functional shift (Geffeney et al., 2005). One substitu-
tion may represent parallel evolution at that site among
resistant populations.
( ii ) Evolution of non-orthologous genes
The above examples involve orthologous genes driven by
the environment towards a similar new function, but
convergence can occur among paralogues or even non-
homologous sequences. Two examples are found: the
antifreeze glycoproteins (AFGP) and the crystallins.
(a) Antifreeze glycoproteins (AFGP). Antarctic notothenioid
fish and Arctic cod (Chen, DeVries & Cheng, 1997) show
similar AFGP gene structures that did not arise by descent
from a common progenitor, but from the tendency for
short repetitive sequences to undergo expansion through
slippage replication and unequal crossing-over that gave
rise to similar mature glycotripeptide gene products capa-
ble of ice binding. The underlying environmental change
(glaciation) can be linked to the same physiological adap-
tation to life at low temperatures in these two groups
althought a test for positive selection is difficult here since
we need to identify the sequence of the protein before it
was recruited to become an antifreeze.
(b) Crystallins. The ocular lens in vertebrates and some
invertebrates is a transparent cellular tissue whose princi-
pal function is light refraction. The refraction index is
achieved by the accumulation of soluble proteins: the crys-
tallins. The genes coding for crystallins were recruited
repeatedly from genes with non-lens functions. The oldest
event involved a crystallin that was co-opted in the com-
mon ancestor of vertebrates from small heat shock pro-
teins (Ingolia & Craig, 1982).
Genes co-opted to form crystallins are often easily
identifiable as many of their products still function as
enzymes in tissues outside the lens. Wistow (1993) suggested
that these co-option events played a major role in the rise
and radiation of land vertebrates. The vertebrate eye
evolved in an optically dense medium (water) requiring
a high refractive index. g crystallins have a high refractive
index and predominate in both fish and mammalian lenses.
During the tetrapods radiation in the less optically dense
medium of air, the eye lens refractive index was reduced to
allow accommodation and focusing at large distances. This
reduction occurred via either the elimination of g crystallins
or their dilution with other, unrelated, crystallin proteins.
Birds with high diurnal visual acuity have completely
replaced g crystallins with crystallins of low refractive index,
co-opted from the argininosuccinate lyase (ASL), lactate
dehydrogenase B (LDHB), and enolase enzymes, giving rise
to d, 3, and t crytallins, respectively.
Co-option events in mammals can be understood given
their evolutionary history: the few mammals that survived
the Cretaceous-Tertiary extinctions are thought to have
been nocturnal. During this nocturnal episode in mamma-
lian evolution, g crystallin genes underwent amplification
and any lens-softening crystallins were lost. The explosive
radiation of diurnal mammals in the tertiary led to the loss
of some g crystallins and the independent recruitment of
several enzymes to reduce the refractive index once more.
Less well-studied crystallins found in invertebrate eye lenses
have been similarly co-opted from a variety of genes (see
Tomarev & Piatigorsky, 1996).
Therefore, shifts in lens refraction indices can be linked
to environmental changes requiring aquatic versus aerial and
nocturnal versus diurnal vision causing shifts in the crystallin
composition in relation to the co-option of different
proteins. However, there is no indication that the co-opted
genes were fixed more rapidly than under neutrality.
Analyses of crystallin from amphibious species, such as
hippopotami, pinnipeds, etc., might shed light on this.
( iii ) Evolution of genomic repertoire
The genomic repertoire of a community is specific to its
biotope, suggesting that genomic architecture is shaped by the
environment. For instance, distributional patterns of genes in
microbial planktonic communities between the surface of the
ocean and the sea floor allowed the identification of depth-
variable trends in gene contents and metabolic pathway
components (De Long et al., 2006). Likewise, Rocap et al.
(2003) compared the genome of two Prochlorococcus ecotypes
(MED4 and MIT9313) that exhibit different light sensitivity
for growth. Although displaying 1350 genes in common,
a significant number (;1300 non orthologous genes between
both ecotypes) were not shared but were either differentially
retained from a common ancestor or acquired through lateral
transfer. Some of these genes are likely to help determine the
relative fitness of the ecotypes in response to key environ-
mental variables and hence directly participate to their
distribution and reproductive success in oceans.
(e ) Case E: demonstrated positive selection on sites involved in the
functional shift, with functional convergence
The great star coral Montastrea cavernosa possesses several
genes coding for fluorescent GFP-like proteins with cyan,
shortwave green, longwave green, and red emission colours
(Ugalde, Chang & Matz, 2004). Phylogenetic analysis
suggested convergent evolution within this gene family.
Field et al. (2006) suggested that the increase in fluorescent
colour diversity is adaptive and hypothesized that multi-
coloured fluorescent proteins could have evolved as part of
a mechanism regulating the relationship between the coral
and its algal endosymbionts (zooxanthellae).
Anthony Levasseur and others562
Biological Reviews 82 (2007) 551–572 � 2007 The Authors Journal compilation � 2007 Cambridge Philosophical Society
Field et al. (2006) recreated the ancestral proteins to
establish where in the evolutionary lineages the phenotypic
transition happened, they searched for and found evidence
of episodic positive selection in these lineages, and used
mutagenesis of extant and ancestral proteins to confirm that
the predicted positively selected mutations were involved in
the colour change. Mutagenesis experiments showed that
positively selected sites were both essential and sufficient to
generate cyan colour from ancestral green. However,
mutagenesis proved that positively selected sites were
essential but not sufficient for the phenotypic change in
the case of the red colour raising the possibility of a role of
neutral evolution in addition to positive selection (Field
et al., 2006).
( f ) Case F: demonstrated positive selection on sites involved in the
functionalshift, but with no observed convergence among lineages
In this category two cases in which there is no molecular
convergence can be defined: basic examples concerning
different genes involved in the same functional gain, also
examples where different sites in an individual gene are
linked to the gain in function.
( i ) Probable link with an environmental change
(a) Proteorhodopsin. Bielawski et al. (2004) detected posi-
tively selected amino acid sites in proteorhodopsin, a
retinal-binding membrane protein in marine bacteria that
functions as a light-driven pump. Site-directed mutagene-
sis (Man-Aharonovich et al., 2004) showed that two out of
four positively selected amino acids sites could account for
the spectral difference between the two major proteorho-
dopsin families found in marine bacteria populations.
Members of the two related proteorhodopsin families
absorb light with different lmax (525 nm, green; 490 nm,
blue) and their distribution in the water column was
shown to be stratified according the available wave-
lengths.
(b) TRIM5a. The primate genome encodes a variety
of genes involved in immune strategies against retrovi-
ruses. One of these gene products, TRIM5a, probably
involved in an antagonistic conflict with proteins from the
viral capsid, can restrict diverse retroviruses in a species-
specific manner (Sawyer et al., 2005): whereas rhesus
monkey’s TRIM5a can strongly restrict HIV-1, human
TRIM5a exhibits only weak HIV-1 restriction. Sawyer
et al. (2005) found strong evidence for ancient positive selec-
tion of TRIM5a in the primate lineage and suggested that
TRIM5a evolution was driven by antagonistic interactions
with a wide variety of viruses that pre-dated the origin of
primate lentiviruses. A 13 amino-acid patch in the B30.2
functional domain bears multiple positively selected resi-
dues, potentially acting at the viral interface. Experiments
with recombinant proteins later have shown that this patch
is generally essential for retroviral restriction.
The antiquity of the detected positive selection rules out
the emergence of primate lentiviruses (like HIV-1) as the
major cause. However, TRIM5a from humans and old
world monkeys are active against murine leukaemia virus
(a gamma-retrovirus closely related to human endogenous
retroviruses) that has episodically invaded primate genomes
and still continues to be active. This suggests that TRIM5a
evolution may have been strongly influenced by episodes of
endogenous retrovirus infection and subsequent retroposi-
tion events. HIV-1 and other primate lentiviruses are likely
to be newcomers in this conflict, with the TRIM5a
restriction against HIV-1 in old world monkeys being just
an evolutionary coincidence.
(g) ECP and EDN. The eosinophil cationic protein
(ECP/RNase3) and eosinophil-derived neurotoxin (EDN/
RNAse2) are paralogues that emerged from a duplication
event around 31 million years ago in the old world mon-
keys lineage; the orthologues found among new world
monkeys and prosimians are named EDNs by convention
(Zhang & Rosenberg, 2002; Bielawski & Yang, 2004). In
humans, EDN and ECP proteins are found in large gran-
ules in eosinophilic leukocytes. In vitro studies showed that
human EDN reduces the infectivity of certain RNA viruses
through an RNAse-dependent process. This antiviral activ-
ity is also found in old world monkeys EDN. Human ECP
however shows only a weak antiviral activity (even at rela-
tively high concentrations) but exhibits a cell membrane
disruptive function that is probably responsible for its toxic-
ity against bacteria and parasites. New world monkey
EDNs lack antibacterial and antiviral activities. As (i) both
EDN and ECP can digest RNA and (ii) EDN RNases from
old world monkeys are catalytically more efficient than
both ECP and new world monkey EDNs, significant
enhancement of RNAse activity most probably occurred
in the EDN lineage after gene duplication. Zhang &
Rosenberg, (2002) were able to determine that after dupli-
cation nine amino acid substitutions occurred in the EDN
of the hominoid ancestor. Site-directed mutagenesis analysis
shows that two of these substitutions, located at two inter-
acting sites (positions 64 and 132) resulted in a 13-fold
enhancement of EDN ribonucleolytic activity. Since the
temporal order of these substitutions is unknown, two sce-
narios are possible: R64S replacement first decreased
RNAse activity by 46% then T132R substitution raised
RNAse activity 24-fold. Alternatively, T132R substitution
occurred first, reducing the RNAse activity by 21%, with
the second substitution (R64S) allowing a 17-fold increase.
(d) Lipase/feruloyl esterase A. Despite strong structural
and sequence similarities, two distinct enzymatic activities,
i.e. lipase and type-A feruloyl esterase (FAEA), are
encoded by different members of this fungal gene family.
Evolutionary analyses suggested that the lipase function
was co-opted after gene duplication, leading to subsequent
enzymatic novelty (FAEA) involved in the lignocellulolysis
of plant cell wall. This functional shift was detected, and
the corresponding positively selected amino acids were
identified, using the branch-site model for testing positive
selection on individual codons along specific lineages. Fur-
thermore, site-directed mutagenesis experiments clearly
confirmed that three of the amino acids under positive
selection were involved in the functional shift. It could be
argued that environmental changes such as colonization
by terrestrial plants might have driven adaptation by func-
tional diversification (Levasseur et al., 2006).
Role of the environment on gene evolution 563
Biological Reviews 82 (2007) 551–572 � 2007 The Authors Journal compilation � 2007 Cambridge Philosophical Society
( ii ) No clear link with an environmental shift
(a) Glutamate dehydrogenase 2 (GLUD2). The ancestral
GLUD gene was duplicated in the hominoid lineage, after
the split with old world monkeys, giving rise to two
paralogues: GLUD1 and GLUD2. GLUD1 is an impor-
tant house-keeping gene and is expressed in many tissues,
whereas GLUD2 is specifically and highly expressed in
nerve tissues (brain and retina) and in testis. Burki &
Kaessmann (2004) have shown that the amino acid
changes responsible for GLUD2 brain-specificity occurred
during a period of positive selection after the duplication
event. Maximum likelihood analyses that test for selection
at certain sites on the phylogenetic tree identified a subset
of sites with a dN/dS value significantly greater than 1:
only two among them (443 and 456) explain high brain-
specificity activity. Even if the origin and adaptive phase
of GLUD2 are approximately concomitant with a period
of increased size and structural and functional complexity
of the brain, it is difficult to identify an environmental
change that could have driven this evolution. We do not
pretend that such an environmental change must have
existed: conceptually, a new function can emerge even in
a stable environment.
(b) Glutathione transferases (GSTs). Ivarsson et al. (2003)
identified positively selected amino acid residues in GSTs
that are multifunctional enzymes providing cellular
defence against toxic electrophiles of both exogenous and
endogenous origins. Site-directed mutagenesis confirmed
that those substitutions might drive functional diversifica-
tion in substrate specificities. It is however probably
impossible to design experiments that could test if muta-
tions have been fixed in response to the emergence of
a specific xenobiotic or a new catalytic pathway.
(g ) Case G: functional shift linked to an environmental change but
with no evidence for positive selection or convergence
( i ) Iota crystallin
The crystallins are not only involved in modifying the
refraction index of the eye lens (see Section III.2.dii). They
also, in association with a chromophore,