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Ecology Letters, (2007) 10: 63–76
REVIEW AND
SYNTHESIS
Ary A. Hoffmann* and Phillip J.
Daborn
Department of Genetics, Centre
of Environmental Stress and
Adaptation Research, The
University of Melbourne,
Melbourne, Vic. 3010, Australia
*Correspondence: E-mail:
ary@unimelb.edu.au
doi: 10.1111/j.1461-0248.2006.00985.x
Towards genetic markers in animal populations as
biomonitors for human-induced environmental
change
Abstract
Genetic markers provide potentially sensitive indicators of changes in environmental
conditions because the genetic constitution of populations is normally altered well before
populations become extinct. Genetic indicators in populations include overall genetic
diversity, genetic changes in traits measured at the phenotypic level, and evolution at
specific loci under selection. While overall genetic diversity has rarely been successfully
related to environmental conditions, genetically based changes in traits have now been
linked to the presence of toxins and both local and global temperature shifts. Candidate
loci for monitoring stressors are emerging from information on how specific genes
influence traits, and from screens of random loci across environmental gradients.
Drosophila research suggests that chromosomal regions under recent intense selection can
be identified from patterns of molecular variation and a high frequency of transposable
element insertions. Allele frequency changes at candidate loci have been linked to
pesticides, pollutants and climate change. Nevertheless, there are challenges in
interpreting allele frequencies in populations, particularly when a large number of loci
control a trait and when interactions between alleles influence trait expression. To meet
these challenges, population samples should be collected for longitudinal studies, and
experimental programmes should be undertaken to link variation at candidate genes to
ecological processes.
Keywords
Adaptation, allele frequency, biomarker, climate change, environmental stress, genetics,
selection signature, toxin.
Ecology Letters (2007) 10: 63–76
INTRODUCTION: RAPID ADAPTATION AND
GENETIC MARKERS
Evolutionary changes in animal populations can be rapid,
particularly as a consequence of human-induced environmental disturbances (Hoffmann & Parsons 1997; Kinnison
& Hendry 2001). These include the evolution of resistance
to chemicals applied to control weeds, pests and diseases,
adaptation in plants and invertebrates to heavy metals,
adaptation to chemical and thermal emissions from
factories, responses to salinity, evolutionary responses to
overfishing, and adaptation to global temperature changes
(Hoffmann & Parsons 1997; Kinnison & Hendry 2001;
Stockwell et al. 2003).
Large phenotypic shifts can evolve in populations over a
short time period. For example, large and rapid evolutionary
changes are evident from population responses to pollutants
and chemical stresses. Strains of insects resistant and
susceptible to pesticides can differ in the lethal dose by
several hundred-fold (McKenzie 1996), and the survival of
adapted invertebrate populations can be unaffected by
contaminants even when all non-adapted populations die
(Klerks & Levinton 1989). Evolved life-history responses to
climatic stresses can also translate into large-fitness
differences between populations (Mitrovski & Hoffmann
2001; Bradshaw et al. 2004). A measure of the rate of
evolution in populations is the haldane, representing
standard deviations of change per generation and defined
as [(x2/sp) ) (x1/sp)]/g, where sp is the pooled standard
deviation of trait values of the population (x) and g is the
number of generations since separation (Hendry &
Kinnison 1999). Whilst rates of evolutionary change
! 2006 Blackwell Publishing Ltd/CNRS
64 A. A. Hoffmann and P. J. Daborn
correspond to a median of 0.0058 haldanes, rates can exceed
0.20 haldanes over short periods (Kinnison & Hendry
2001). Therefore, traits have the potential to evolve several
phenotypic standard deviations over a few generations.
Underlying these evolutionary changes are shifts in allele
frequencies at loci. Allele frequency changes have long been
considered as having potential for monitoring environmental stress (Luoma 1977). As populations adapt, alleles
that are initially rare in populations increase to a high
frequency. Studies of insecticide resistance in particular
show how the evolution of resistance in response to
pesticides in the environment involves allelic changes at a
limited number of loci (Raymond et al. 2001). Although
insecticide-resistance research has focused on understanding
the mechanisms of resistance and on the development of
strategies to minimize its evolution, it has also produced
markers for monitoring resistance development in populations, as in the case of resistance to a pyrethroid insecticide
in field populations of the Colorado potato beetle (Kim et al.
2005) and to Bacillus thuringiensis (Bt) toxins in the pink
bollworm (Morin et al. 2004).
Genes involved in rapid evolution to a range of
environmental stresses are likely to be identified over the
next few years. Whereas previously researchers have focused
on testing whether an evolutionary response had a genetic
basis, they have now moved to dissecting responses at the
physiological and molecular levels. By analogy, genetic
research in the health area a few decades ago centred on the
issue of whether illnesses such as heart disease or breast
cancer had a genetic basis. Large quantitative genetic
analyses were completed to test if a genetic basis was
present. Now research has moved on to identifying specific
marker genes that underlie these genetic variants (Kleyn &
Vesell 1998). Banks of markers are emerging that can be
used in the treatment of human diseases, indicating the likely
vulnerability of individuals to different diseases and aiding in
drug applications (Hiratsuka et al. 2006). An understanding
of this genetic variability among individuals is providing a
revolution in healthcare delivery, and a similar breakthrough
seems possible in the environmental area.
There is abundant evidence that genetic changes occur in
response to anthropogenic stresses, and that these can be
rapid and involve a variety of physiological, morphological
and life-history traits. Can evolutionary changes and the
genes underlying them be used to understand and detect
environmental changes?
LINKING EVOLUTION TO ENVIRONMENTAL
CHANGE – THE PHENOTYPIC LEVEL
Evolutionary shifts are traditionally detected at the phenotypic level. Even the evolution of chemical resistance in
pests is normally first detected by screening organisms for
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Review and Synthesis
their level of susceptibility. Typically a sensitive strain
provides a baseline and resistance is identified by comparing
field individuals or their offspring to this strain (e.g. Shelton
et al. 1993). Resistance can also occur in organisms that are
not the targets of insecticide applications, suggesting that
the particular chemical is having non-specific biological
effects. For instance, Drosophila melanogaster has evolved
resistance to several chemicals despite never being targeted
directly as a pest species (Wilson 2005). The presence of
resistance may be indicative of population-level effects of
the chemical. Phenotypic studies indicate that resistance has
also developed in animal populations to other chemical
changes including heavy metals (Klerks & Levinton 1989),
polychlorinated biphenyl (PCB) (Nacci et al. 2002) and
acidification (Merilä et al. 2004).
Evolutionary shifts have also been detected in response
to thermal changes in the environment. Populations of
copepods living in warm water produced by power plants
are locally adapted to thermal stress (Bradley 1978) and
there is evidence of local adaptation to thermal stress in
Drosophila populations (Hoffmann et al. 2003). On a wider
geographical scale, Bradshaw & Holzapfel (2001) and
Bradshaw et al. (2004) have documented evolution in
diapause induction in the pitcher plant mosquito, Wyeomyia
smithii, as a consequence of warmer conditions over the last
few decades. Diapause induction shows a latitudinal cline
along the east coast of the USA, with southern populations
from warmer areas entering a diapause at shorter daylengths
than northern populations from colder regions. This allows
southern populations to take advantage of the extended
favourable conditions in their environment. The cline has
shifted towards a more southern shorter daylength form
over the last 30 years, particularly in populations at the
northern end of the distribution of this species (Bradshaw &
Holzapfel 2001). This clinal shift is likely to reflect the largefitness benefit arising from entering diapause at the
appropriate stage. For instance, when a southern population
was exposed to mid-latitude daylengths, the southern
populations entered diapause too late and suffered a 74%
decline in fitness, while the northern populations entered
diapause too early and experienced an 88% decline
(Bradshaw et al. 2004). The rapid shift in this cline suggests
strong selection allowing populations to take advantage of
changing climatic conditions (Bradshaw & Holzapfel 2001).
Body size evolution has been related to climate change in
several organisms. Smith et al. (1998) followed changes in
pellet size (a measure of body size) in middens of woodrats
over a period of 25 000 years. Pellet size decreased as
temperatures increased during a series of temperature
fluctuations. Size changes have now also been linked to
climate change over a period of decades. Yom-Tov et al.
(2006) tested the prediction that global warming was
associated with decreases in body weight in 14 species of
Review and Synthesis
passerine birds at two localities in England: they found
linear decreases in size in four species, nonlinear decreases
in two other species, but an increase in size in one species
that may have reflected changes in food supply. Therefore,
predictions were met in the majority of species, although
these were phenotypic changes and a genetic basis for the
changes can be inferred only indirectly.
Genetic changes in size and other characteristics have
been linked to recent climate change in Darwin’s finches
from the Galapagos. By estimating genetic parameters in
finch populations, including interactions among traits, Grant
& Grant (1995) accurately predicted changes in body size
and beak characteristics as a consequence of a shifting food
supply during drought conditions. As the incidence of
drought events increased, selection was expected to favour a
decrease in body size and blunter beaks. These shifts were
documented over 30 years in one species of finch, although
they were not found in a second species (Grant & Grant
2002).
One problem in predicting changes in body size due to
shifts in environmental conditions is that size is influenced
by other selective factors, including competitive interactions,
mating success and resource acquisition. Most animals
follow Bergmann’s rule, where the size increases away from
the equator. In mammals and other warm-blooded animals,
this rule is thought to be associated with thermoregulation
of internal body temperature, because of changes in
surface/volume ratio. However, there are exceptions to
Bergmann’s rule. For instance, body size in Alaskan shrews
shows the opposite pattern to Bergmann’s rule, with size
increasing with latitude (Yom-Tov & Yom-Tov 2005).
Moreover, size has increased in these shrews in the last half
of the 20th century, despite temperatures increasing. This
pattern may be related to food availability; as more food
becomes available, the shrews are likely to have sufficient
resources to develop a larger body size. While body size
changes can therefore occur rapidly in populations, they
represent an indirect response to climate change.
Changes in morphological traits other than size might
signal climate change. In barn swallows, Møller & Szep
(2005) have found changes in the length of the outermost
tail feathers that provide a mating advantage to males. In
male birds from a Danish population length had increased
more than 1 SD over a 20-year period. This increase was
associated with selection on survival and breeding date of
the birds. The swallows in Denmark originate from Algeria,
where deterioration in the vegetation has occurred as a
result of unfavourable climatic conditions. Perhaps, males
with relatively longer outer tail feathers have an advantage
under unfavourable conditions for vegetation growth,
although this connection remains to be established.
In several species of birds migratory behaviour seems to
have evolved due to climate change. The heritability of traits
Genetic markers and environmental change 65
associated with migration in several bird species is high
(Pulido & Berthold 2004), reflecting the potential for rapid
evolution. In blackcaps, common garden experiments
indicate that there has been a heritable decrease in migratory
activity over 13 years, reflecting a change of more than 1 SD
(Pulido & Berthold 2004). Changes in the spring arrival
dates of barn swallows also appear to have evolved
suggesting a shift in migration patterns (Møller & Merilä
2004).
Finally, rapid evolutionary changes can occur because of
direct human biotic effects. Evolution can take place when
humans act as predators and exploit resources. For instance,
Atlantic cod off Newfoundland and Labrador declined
precipitously in the 1970s and collapsed by the 1990s.
Exploitation of mature fish was expected to select for early
maturation, and Olsen et al. (2005) found evidence for an
evolved phenotypic change in maturation time. They
showed that reaction norms in populations had shifted
towards maturation at earlier ages and at smaller sizes at the
time the fishery collapsed. This shift to early maturation was
likely to have been a consequence of selection imposed by
fishing, particularly as maturation rate in fish has a genetic
basis (Conover et al. 2005). Human activities can also
indirectly generate evolution by altering natural environments, such as through the introduction of predators that in
turn select for changes in morphology and physiology of
prey species (Phillips et al. 2004).
These examples illustrate that phenotypic studies can be
used to detect rapid evolution in response to human
activities, including the effects of environmental contaminants and climate change. However, trait changes are not
necessarily easily related to environmental factors, as
illustrated by problems in making links between body size
and climate change. Genetic changes in populations can be
inferred only when familial relationships are established in
the field or controlled breeding experiments are undertaken
in the laboratory, which is not possible for many groups.
Nevertheless, longitudinal monitoring of traits at the
phenotypic level might serve as a way of identifying the
biological impact of some environmental stressors. We
return to this issue below.
LINKING EVOLUTION TO ENVIRONMENTAL
CHANGE – ALLELE MARKERS
The notion that genetic markers can be used to monitor
environmental changes has been around for a number of
years and several types of markers have been associated with
environmental changes (Table 1). Early studies in aquatic
environments compared frequencies of genes in polluted
and unpolluted environments to isolate genetic markers
underlying tolerance variation. Attempts to link changes in
genes encoding enzyme polymorphisms (allozymes) with
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66 A. A. Hoffmann and P. J. Daborn
Review and Synthesis
Table 1 Types of genetic markers available for investigating environmental change associations
Markers
Advantage
Limitations
Example
Allozymes
Protein products provide targets
under selection
Limited number of loci,
requires well-preserved tissue
for protein isolation
Chromosome inversions
Rapid responses to environmental
change due to number of loci
in disequilibrium with inversions,
can be scored cytologically or by
polymerase chain reaction
Large numbers of microsatellites
can be identified from all species
Only described for limited
number of insects, different
gene combinations may be
involved
Variation in GPI (glucose
phosphate isomerase)
and mercury pollution
in mosquitofish
(Tatara et al. 2002)
Associations between
inversion polymorphism and
climatic warming in several
Drosophila species (Umina
et al. 2005; Etges et al. 2006)
Drosophila surveys linking
microsatellite markers with
environmental gradients
(Kennington et al. 2006)
Associations between
transposable elements and
insecticide resistance
(Daborn et al. 2002)
Associations between markers
and insecticide resistance
(Morin et al. 2004)
Microsatellites
Transposable elements
Scattered throughout genome, can
provide signatures of selection at
genomic levels
Other DNA
polymorphisms
EPICS (exon primed intron crossing), single nucleotide polymorphisms, and others
Heterozygotes readily detected
Clones can be identified with any
type of genetic marker, rapid
changes in clonal composition
expected due to strong selection
on entire genome
Clonal organisms (can be
distinguished by any of
the above marker systems)
pollutants were successful in some cases (Nevo et al. 1978,
1984) but not in others. Various studies have also tested if
overall levels of genetic diversity are reduced under
pollution stress (genetic erosion) using randomly selected
sets of markers. However, these have produced unequivocal
results because factors other than selection influence overall
genetic diversity (van Straalen & Timmermans 2002) and
this approach is not considered further here.
The evolution of insecticide resistance provides the bestknown examples where specific genetic changes have been
linked to environmental toxins. Precise changes at the DNA
and protein levels have been identified in several cases. In
D. melanogaster, resistance to the once commonly used
cyclodiene insecticide, dieldrin, is due to a single mutation in
the Rdl gene (ffrench-Constant et al. 1993). Rdl encodes the
molecular target of dieldrin, a receptor for the c-aminobutyric acid (GABA) neurotransmitter (ffrench-Constant et al.
1991). The point mutation in Rdl results in an amino acid
substitution in the GABA receptor, reducing the ability of
dieldrin to bind to the receptor, disrupt neurotransmission,
and thereby kill the insect. The same amino acid substitution
has now been found in natural populations of several insect
species that are resistant to dieldrin (Thompson et al. 1993).
! 2006 Blackwell Publishing Ltd/CNRS
Markers are often neutral, can
only be related to selection
through identified disequilibrium with other loci
Depends on elements being
identified for loci under
selection
No limit to number of markers
that can be identified
Depends on clonal organisms
being present across
environmental gradient
Daphnia clones associated with
thermal conditions (Lopes
et al. 2004)
Another example is resistance to pyrethroid insecticides
associated with the kdr gene that encodes a protein involved
in movement of compounds across membranes. A particular mutation in kdr, leading to a replacement of the amino
acid leucine with phenylalanine in this protein, has been
isolated from a number of insect species that have
developed resistance (Soderlund & Knipple 2003).
The highly predictable nature of insecticide-resistance
evolution not only carries across species, but is also capable
of being replicated in the laboratory. McKenzie &
Batterham (1998) reviewed evidence that resistance to
organophosphate in the sheep blowfly, Lucilia cuprina,
involves allelic substitution at the Rop-1 locus. Rop-1 encodes
a carboxylesterase enzyme, which is involved in the
detoxification of organophosphates. The allelic substitution
at Rop-1 leads to an amino acid change that alters the
substrate specificity of the enzyme and effective detoxification (Newcomb et al. 1997). This change has been observed
not only in the field, but has also been generated in the
laboratory. Flies from a susceptible laboratory population of
L. cuprina were exposed to chemicals that cause mutations
(mutagenesis), and this was followed by selection for
diazinon resistance, which led to the same resistance
Genetic markers and environmental change 67
Review and Synthesis
(a)
Cyp6g1
Cyp6g1
(b)
Cyp6g1
no selection
Cyp6g1
selective sweep
Figure 1 A beneficial mutation and the resulting selective sweep, as exemplified by the Drosophila melanogaster insecticide resistance locus
Cyp6g1. (a) A beneficial mutation occurs in a particular genetic background, in this example the insertion of the Accord transposable element
(triangle) upstream of Cyp6g1 leading to insecticide resistance via Cyp6g1 overexpression. (b) A selective sweep occurs when the Accord
carrying allele of Cyp6g1 is selected. In individuals without the beneficial mutation neutral polymorphisms (circles) are evenly, but randomly
distributed across the chromosomal region because of recombination and mutation. In lines carrying the Accord insertion mutation, selection
of the beneficial mutation (in this example the Accord insertion) results in a reduction of neutral polymorphisms at sites closely linked to
Cyp6g1. Both the presence of the Accord insertion and the reduction in polymorphisms are signatures of selection.
outcomes as seen in resistant field populations (McKenzie
et al. 1992). Similar experiments in L. cuprina involving
mutagenesis and selection have also generated resistance to
dieldrin, with the resistance mechanism involving a mutation in Rdl that matched the change in Rdl in field
populations discussed above (Smyth et al. 1992).
There is little information on the genetic basis of
evolutionary responses to other types of environmental
toxins, although an exception is the evolution of mercury
resistance in mosquito fish. The time taken for fish to die
under mercury exposure has been related to genotypes at a
locus encoding the enzyme glucose phosphate isomerase.
The distribution of allozyme genotypes at this locus has
been related to mercury levels in a contaminated environment (Heagler et al. 1993). A series of studies which
included long-term mesocosm experiments on polymorphic
populations have also implicated the same locus in mercury
responses (Tatara et al. 2002).
While these types of studies can identify genes under
selection, there is an additional approach available to detect
selection which focuses on molecular variation around the
genes rather than directly on trait associations. This
approach can be illustrated by the evolution of resistance
to insecticides in D. melanogaster involving the gene Cyp6g1.
This gene encodes one of the many types of enzymes
known as cytochrome P450s that detoxify toxins in animals.
Cyp6g1 is overexpressed in D. melanogaster lines from natural
populations that exhibit resistance due to the insertion of a
transposable element known as Accord into the promoter
region that regulates expression of the gene (Daborn et al.
2002). Analysis of the DNA region around Cyp6g1 shows
that there is a sharp reduction in the level of molecular
variation in this region. This reduced variation is a molecular
signature of the recent spread of the allele containing Accord
in D. melanogaster populations (Catania et al. 2004) as outlined
in Fig. 1. When the allele with the transposable element first
arose, it would have existed within a particular region of
DNA and associated with other alleles at adjacent loci. As
the new allele was favoured by selection due to the
overexpression of the detoxification mechanism, alleles at
adjacent loci would also have spread along with it,
decreasing genetic variation in this genomic region in the
population (a !selective sweep"). This loss of variation is only
gradually restored because of recombination and mutation.
In the case of Cyp6g1, there is also evidence of a selective
sweep around this gene in a related species, Drosophila
simulans. However, in this case the sweep is associated with a
different transposable element known as Doc, but also
inserted in the promoter region of Cyp6g1 (Schlenke &
Begun 2004). In both species these changes are likely to
reflect a recent history of exposure to pesticides from
agricultural activities. Patterns of molecular variation can
therefore be used to recognize areas of the genome under
recent selection around genes that are being selected
because of particular environmental changes.
What about evolutionary responses to climatic variables?
Several longitudinal studies indicate that genetic markers can
be linked to climate change. One case involving allozyme
markers (Table 1) is selection on the alcohol dehydrogenase
(Adh) gene in D. melanogaster. There are two common alleles
at the Adh locus, AdhF and AdhS. These alleles show a strong
cline along the east coast of Australia, with the S allele at a
! 2006 Blackwell Publishing Ltd/CNRS
68 A. A. Hoffmann and P. J. Daborn
relatively higher frequency in the tropics (Oakeshott et al.
1982). This Adh cline is also present on other continents
around the world suggesting that the AdhS allele is favoured
in tropical conditions rather than temperate conditions,
consistent with the results of laboratory experiments
(Oakeshott et al. 1982). Clinal variation in Adh in eastern
Australia was first established based on collections from
1979, and an identical pattern was observed in flies collected
a few years later (Anderson et al. 1987). However, when the
cline was resampled in 2000 and 2002, a different pattern
emerged (Umina et al. 2005). While the slope of the
association between latitude and Adh had not changed over
this time, there had been a marked shift in the intercept of
the relationship. This corresponded to a shift of several
100 km in latitude. In contrast, there was no change in the
latitudinal association of the Gpdh gene, which showed a
much weaker clinal pattern.
Several chromosomal regions in D. melanogaster and
many other Diptera may exist in two forms: a non-inverted
(standard) form, and an inverted form where the arrangement of genes in the chromosomal region is reversed.
Chromosomal inversions lead to a reduction in viable
recombinants in the inverted region of DNA causing
alleles of genes within the inversion to be inherited
together (Schaeffer et al. 2003; Hoffmann et al. 2004;
Kennington et al. 2006). Drosophila melanogaster has several
of these inversions that are cosmopolitan and populations
can be polymorphic for the standard or inverted region.
Changes in latitudinal patterns over time have now been
established for these inversion polymorphisms (Table 1).
One of these cosmopolitan inversions is known as In(3R)P
and is located on the right arm of chromosome 3. The
In(3R)P inverted chromosome arrangement increases
sharply in frequency with latitude in Australia and on
other continents (Knibb 1983), from near fixation for the
standard arrangement at high latitudes to near fixation of
the inverted arrangement towards the equator. On the east
coast of Australia, the cline in this inversion has changed
sharply over the last 20 years (Umina et al. 2005). While
the slope of the cline has not changed, the intercept has
moved such that populations now have inversion frequencies resembling those of populations 700 km nearer the
equator 20 years ago.
Levitan & Etges (2005) and Etges et al. (2006) have
related changes in the frequency of inversion polymorphisms in Drosophila robusta to temperature shifts. These
inversions have been studied in North American populations since the 1940s and have exhibited latitudinal and
altitudinal clines over several decades. The clines have
changed over the last few decades. Since the 1970s, the
frequency of several inversion arrangements have increased,
matching increases in minimum temperature at several sites
(Levitan & Etges 2005). Along an altitudinal transect in the
! 2006 Blackwell Publishing Ltd/CNRS
Review and Synthesis
Smoky Mountains, the frequency of high-altitude inversion
arrangements has increased from the 1940s to the 1980s,
matching locally cooler temperatures, but these trends have
reversed recently as temperatures have warmed again.
Changes in inversion frequencies in D. robusta are therefore
rapid and closely track temperature shifts.
Finally, the frequency of inversions in the O chromosome
of Drosophila subobscura in Spain have changed over the last
15 years (Rodriguez-Trelles & Rodriguez 1998). Two of the
chromosomal arrangements that normally increase in
frequency in summer have become more common in
populations. These changes are associated with temperature,
which has increased at a rate of 0.081 "C per year since the
mid-1970s. Chromosomal diversity has decreased at the
same time (Rodriguez-Trelles & Rodriguez 1998). In
southwestern Europe, latitudinal patterns in four arrangements have changed over 30 years; arrangements common
in warmer southern areas are now relatively more common
in all populations even though latitudinal patterns have been
maintained (Sole et al. 2002).
The chromosomal arrangements of D. subobscura have
also been studied in the Americas which were recently
invaded by this species. Following invasion, latitudinal clines
in inversions were rapidly established in populations and
converged on clines in the Old World where the species
originated (Prevosti et al. 1990). However, this convergence
was not supported by later sampling (Balanya et al. 2003)
which suggested that, while latitudinal patterns in the New
and Old Worlds were similar, convergence stopped for
many of the polymorphisms. Thus the inversions may have
developed a different dynamic following invasion, perhaps
dependent upon the genetic makeup of the invading
population.
Genes within inverted chromosomal regions that are
responsible for these patterns have not yet been isolated. As
mentioned above, inversion polymorphisms lead to regions
of association between alleles along the inverted region,
particularly near points where the inverted region starts and
finishes, but also extending away from these points
(Schaeffer et al. 2003; Kennington et al. 2006). Therefore,
they help to lock together a number of alleles, which singly
or together might influence fitness under different climatic
conditions. Interactions among alleles within inversions
might influence climatic adaptation. Under the classical
explanation of inversion polymorphisms advanced by
Dobzhansky, inversions in a population help to lock up
alleles that are co-adapted. However, alleles within an
inversion that are coadapted in one population may not
necessarily be coadapted in a different population. If this
explanation is correct (and there is presently only limited
evidence for it), then changes in latitudinal patterns of
inversions might be difficult to interpret because the allele
contents of the inversion varies with latitude.
Review and Synthesis
What about climatic variation in other species? In the
Sierra willow beetle, Chrysomela aeneicollis, genetic variation at
the locus coding for the enzyme phosphoglucose isomerase
has been related by Rank & Dahlhoff (2002) to climatic
variation. This species experiences large daily fluctuations in
temperature from )5 to >32 "C. One of the alleles (Pgi-1)
increased by 11% from 1988 to 1996, while there were no
changes in allele frequency at two other allozyme loci. This
increase was related to cooler conditions at the study site
prevailing prior to 1996. Physiological studies indicated that
female beetles homozygous for the Pgi-1 allele survived a
laboratory cold shock better than beetles with other
genotypes, suggesting that the change in allele frequency
was associated with shifting climatic conditions. In addition,
transplant studies (McMillian et al. 2005) were used to
demonstrate that the Pgi-1 allele was at a disadvantage in
terms of survival and development time when beetles were
moved to a warm location. These effects may be partly
mediated through an effect of Pgi genotypes on expression
of HSP70, one of the heat-shock proteins that protects cells
against heat-shock damage.
There are numerous other polymorphisms that could
form the basis of longitudinal studies in future attempts to
link environmental shifts to genetic changes. For instance, in
deermice there is a well-known altitudinal pattern for
polymorphism in the a-chain of haemoglobin (Chappell &
Snyder 1984) and this might change as conditions become
warmer. There are also other polymorphisms that can be
easily scored and could be worth considering, such as snailbanding polymorphisms that are affected by heat stress
(Richardson 1984), and melanism patterns in ladybirds that
have been related to climatic selection (Honek et al. 2005)
and pollution (Brakefield & Liebert 2000). Any polymorphism that shows a clear latitudinal and/or altitudinal pattern
could be re-examined to test for associations with climate
change. Surveys of clinal markers (e.g. Sezgin et al. 2004;
Kennington et al. 2006) can be used to identify genes or
genomic regions for monitoring in longitudinal studies. In
fact, any screen of markers across an environmental gradient
could be used to develop candidates. Comparisons of
expression patterns of genes across gradients using microarray technology (that allows changes in expression of genes
throughout the genome to be characterized) provide an
additional approach for isolating candidates (Whitehead &
Crawford 2006).
A hazard in all this work is that any associations between
markers and environmental changes could be spurious.
Identification of candidate loci needs to encompass multiple
lines of evidence. For instance, if an association between
variation in a gene and an environmental change is
established in one organism, the impact of mutants in the
gene could be examined in that organism or a related model
species to help establish a causal association. For this reason,
Genetic markers and environmental change 69
the examples discussed above involve accumulated knowledge based on diverse data sources that include mutant
studies, expression patterns, fitness experiments and monitoring genetic changes in experimental systems.
Finally, changes in the frequency of clonal lineages in
parthenogenetic organisms may be useful for environmental
monitoring. Selection on these lineages can be extremely
strong as demonstrated by rapid shifts in the clonal
composition of field populations of parthenogenetic populations over just a few generations (Weeks & Hoffmann
1998; Vorburger 2006). This reflects the fact that, in the
absence of recombination, the entire genome of a clonal
lineage is under selection. Once associations between clones
and particular environmental conditions are established,
changes in the clonal composition of populations could be
used to monitor for these conditions. For instance, in
Daphnia, clonal composition has been linked to seasonal
variation in climate (Carvalho & Crisp 1987) and levels of
acid pollution (Lopes et al. 2004).
MARKER LISTS AND MARKER SIGNATURES
The potential of markers as monitors is illustrated by their
growing application in the area of insecticide resistance.
Traditionally, insecticide-resistance bioassays have been
used to detect the frequency of resistant individuals.
Molecular markers offer the advantage of being able to
detect heterozygous individuals, otherwise undetectable for
recessive traits, and also allow higher numbers of field
individuals to be assayed. Several markers have now been
developed to monitor the frequency of resistance in natural
populations. For example, in the pink bollworm, Pectinophora
gossypiella, polymerase chain reaction (PCR)-based assays
have been developed to detect mutant alleles which are
involved in resistance to Bt toxins (Morin et al. 2004). These
toxins are produced by B. thuringiensis and bind to receptor
proteins including cadherin proteins. However, in resistant
mutants, alleles in the BtR cadherin gene fail to produce a
cadherin protein that would normally bind to one of the Bt
toxins, producing resistance. PCR-based technologies
capable of allele discrimination have also been adapted to
detect specific point mutations in the kdr gene that lead to
pyrethroid resistance in aphids (Anstead et al. 2004), horn fly
(Li et al. 2003) and mosquitoes (Tripet et al. 2006), as well as
the detection of the single mutation in Rdl that is associated
with resistance to the chemicals fipronil and dieldrin in the
cat flea (Daborn et al. 2004) and the German cockroach
(Hansen et al. 2005).
One advantage of using genetic markers in this context is
that they provide a method of early detection of resistance,
so that management programmes can be implemented prior
to a high level of resistance developing. The assays
described above are extremely specific, monitoring specific
! 2006 Blackwell Publishing Ltd/CNRS
70 A. A. Hoffmann and P. J. Daborn
resistance alleles of particular genes. For resistance markers
to work in both the context of management and monitoring
environmental change, all possible mechanisms of resistance
need to be monitored. Assays would need to have the
flexibility to detect new resistance-conferring mutations in
the genes currently being monitored, and to detect new
types of resistance mechanisms as these evolve. The
increasing understanding of potential resistance mechanisms
makes it more likely that reliable markers for resistance
detection can be developed.
The molecular marker approach can be applied to other
toxin responses and traits as information accumulates on
genes that may influence variation in particular traits
(!candidate" genes). For some types of stresses candidate
genes are already emerging, even when organisms are not
well-known genetically. In earthworms, the mechanisms
underlying resistance to cadmium pollution have been
largely identified, as reviewed in Sturzenbaum et al. (2004).
Metallothionein proteins are used to bind and compartmentalize the cadmium, and these have been isolated and
the genes controlling them have been identified. Metallothionein expression has also been linked to cadmium tolerance
in springtails (Timmermans et al. 2005), although in this case
additional mechanisms of cadmium resistance are likely to
be involved. Where genes in organisms of interest to a
researcher are not known information from model organisms can be used to derive candidates. For instance, heavy
metal resistance in D. melanogaster has been associated with
metallothioneins, based on molecular studies of lines and
natural variants with different levels of heavy metal
resistance (Maroni et al. 1987). Drosophila is already used to
detect candidates for insecticide resistance based on the
knowledge that the identified genes are good candidates for
resistance to the same stresses in non-model organisms
(Wilson 2005).
For complex traits there is rapid progress in the
identification of genes underlying natural variation. Currently, there is a large focus on understanding the genetic
basis of variation in a range of traits by using model
organisms such as mice, Drosophila and Arabidopsis. These
efforts include a focus on traits likely to be of interest for
environmental monitoring. In D. melanogaster a great deal of
progress has been made in understanding the genetic basis
of responses to stresses such as temperature extremes
(Hoffmann et al. 2003; Norry et al. 2004; Morgan & Mackay
2006) and starvation (Harbison et al. 2005). Selection
experiments indicate that different sets of genes tend to
be involved in adaptive responses to stresses like desiccation, heat and cold resistance (Bubliy & Loeschcke 2005).
Traits like heat resistance can be broken down further into
genetically independent set of traits (Hoffmann et al. 1997;
Bubliy & Loeschcke 2005). Mapping and tests on candidate
genes have yielded lists of candidate genes likely to underlie
! 2006 Blackwell Publishing Ltd/CNRS
Review and Synthesis
these independent traits. For instance, phenotypic analysis
of laboratory lines has indicated that high-temperature
resistance in larvae is associated with levels of one of the
heat-shock proteins, HSP70 (Sorensen et al. 2003). Natural
variation in HSP70 levels have also been linked to variation
in heat resistance within populations and across thermal
gradients (Sorensen et al. 2003). However, while HSP70
levels and induction are involved in resistance, the exact
nature of genetic variation underlying changes in these levels
is not understood. Crosses between strains have been used
to map areas of the genome where loci influencing heat
resistance [quantitative trait loci (QTL)] are located. This has
indicated that variation in heat resistance to a knockdown
stress maps to QTL around Hsp70 (Norry et al. 2004), but
variation in the Hsp70 gene itself has not been convincingly
linked to heat resistance (Weeks et al. 2002). Eventually, a
combination of mapping, candidate gene identification and
correlated responses in selection lines, as well as genetic
dissection of latitudinal patterns, can help isolate the
contribution of specific genes to genetic variation in thermal
resistance.
Identifying suitable candidates will be aided by molecular
analysis that determines if genes have been exposed to
recent selective sweeps (Fig. 1). Because recombination and
genetic drift remove evidence for selective sweeps relatively
rapidly (Przeworski 2002), selective sweeps can provide
evidence of recent positive selection on genes, as in the case
of Cyp6g1 (Catania et al. 2004) and Pgm (Verrelli & Eanes
2000). Hsp70 genes also show evidence of selective sweeps
in populations (Bettencourt & Feder 2002). More examples
are emerging as variation in and surrounding candidate
genes are examined further (Pool et al. 2006).
As well as being useful for verifying candidates under
selection, monitoring for areas of the genome experiencing
selective sweeps can itself detect genetic change, without a
priori knowledge of candidate genes. The presence of a low
level of variation can indicate that selection has taken place
on a particular allele of a gene recently. Monitoring the
intensity of the sweep over time can be a means of
assessing the intensity of selective forces acting in the
population. Beisswanger et al. (2006) scanned for selective
sweeps to identify candidates of selection in a European
population of D. melanogaster. They initially found regions
with decreased variation and then undertook finer level
analyses to isolate small regions of the genome with
particularly low variation. One of these regions, on the
X chromosome, completely lacked variation. One of the
seven genes in this region is likely to be responsible for this
sweep, although the mechanism remains to be identified. In
another selective sweep study on the X chromosome of
D. melanogaster, a single gene with unknown function was
identified to have been under selection (DuMont &
Aquadro 2005).
Review and Synthesis
Both mutations directly influencing gene function and
mutations affecting gene expression can potentially affect
traits involved in environmental responses. Most of these
are single base mutations and therefore difficult to detect.
However, one group of mutations that is easier to detect
involves transposable element insertions, discussed above in
the context of the Cyp6g1 gene (Fig. 1). Transposable
elements play an important role in driving and shaping
genome evolution (Kazazian 2004). They can cause mutation by inserting into the coding regions of genes and
disrupting or altering gene function, or by inserting into the
regulatory regions of genes and disrupting or altering gene
expression. They often contain DNA sequences that can
alter gene expression (Marino-Ramirez et al. 2005). Some
transposable element insertions increase to a high frequency
in populations because the mutant phenotype provides a
selective advantage. Detecting these events and the genes
under positive selection can be as simple as detecting
transposable element insertions present at high frequencies
in populations. For example, in many populations of
D. melanogaster a Doc element insertion into the CHKov1
gene has been observed. This insertion results in a truncated
form of the protein and is under recent strong selection,
sweeping to high frequencies in D. melanogaster populations
(Aminetzach et al. 2005). A Bari-1 transposable element
inserted in the Cyp12a4 gene also increases expression
of this gene. This insertion allele is fixed in natural
D. melanogaster populations (Marsano et al. 2005). Monitoring
the frequency of transposable element insertions throughout
the entire genome may be a feasible approach in the future
to identify parts of the genome that are involved in adapting
to environmental changes (Franchini et al. 2004), although
detailed follow-up work will be required to establish causal
links.
GENETIC MARKERS: POTENTIAL OR LOST CAUSE?
Genetic markers have two advantages as environmental
monitors. First, gene frequency shifts are likely to occur well
before population extinction. As the majority of traits are
thought to be heritable, there is the potential for evolution
in response to almost all types of environmental changes.
Allele frequencies at loci that underlie directional trait shifts
can be monitored as long as enough is known about the
underlying genetic basis of trait variation. Second, gene
frequency shifts are likely to be highly specific depending on
the nature of the environmental variable driving selection.
This means that the genetic changes can indicate a particular
type of environmental change.
Despite these advantages, there has been limited use of
genetic markers to monitor environmental changes, even in
ecotoxicology. One reason is that adaptive shifts may not
always occur when an environment changes. Klerks & Weis
Genetic markers and environmental change 71
(1987) reviewed the early literature and found numerous
cases where animals appeared to show physiological
differences between polluted and unpolluted sites, but often
it was difficult to distinguish between acclimation and
genetic change. In cases where genetic factors could be
isolated, it appears that many species did not successfully
adapt to pollutants like heavy metals (Klerks 2002). Animals
may also fail to adapt to climate change. The most common
response of bird populations to climate change involves a
shift in breeding date; however, while there is heritable
variation for breeding date in bird populations, there is no
evidence that this trait has yet evolved, and instead shifts
appear to be environmentally based (Pulido & Berthold
2004). If genetic approaches are to be successful, they need
to focus on organisms likely to adapt and traits with a high
evolvability.
Another reason is that adaptation can involve subtle
changes that are difficult to detect and characterize.
Adaptation to pollutants can involve life-history changes
rather than physiological tolerance (Postma et al. 1995).
These can be more difficult to detect than tolerance,
requiring comparisons of an organism’s development and
reproductive allocation. Adaptation to climatic change can
also involve life-history shifts, as is the case of clinal
variation in Drosophila (Mitrovski & Hoffmann 2001) and
latitudinal adaptation in mosquitoes (Bradshaw et al. 2004).
The genetic basis underlying the adaptive shift can be
difficult to identify even when there are candidate genes. In
the mummichog, Fundulus heteroclitus, an estuarine fish found
along the east coast of the USA, several populations have
evolved resistance to pollutants including PCBs and the
resistance mechanism acts toxicologically through the arylhydrocarbon receptor (reviewed in Nacci et al. 2002).
However, attempts to isolate genes underlying resistance
have proved difficult, despite a considerable effort focusing
on a variety of candidates and some positive associations
being detected (Roark et al. 2005).
The task of isolating genes underlying an adaptive
response can be onerous. Genomic regions identified via
mapping of traits may depend on the exact nature of
environmental conditions being tested, as well as sex, and
the nature of the trait itself (Mackay 2004). When genomic
regions are identified, it can be difficult to isolate specific
genes because the phenotype depends on interactions
among the genes. However, very rapid progress is now
being made in identifying genes and pathways underlying
traits. An increased understanding of the relationship
between the physiological makeup of organisms, and the
underlying genetics, will enhance our ability to select
candidate genes, leading to numerous targets that can
indicate the effects of selection.
Yet another reason why genetic markers have had limited
use to date is that the same selection targets might be
! 2006 Blackwell Publishing Ltd/CNRS
72 A. A. Hoffmann and P. J. Daborn
reached by multiple pathways, making it difficult to predict
the specific genes involved in selection responses across
populations and species. In the case of insecticide resistance,
there is clearly a high degree of predictability across
populations and even across unrelated species. Whether
selection responses in complex traits involve predictable sets
of genes remains to be seen. However, there is evidence
from bacterial studies that selection to thermal extremes
involves predictable genetic changes (Counago et al. 2006).
In animal populations, independent selection responses
often map to the same genomic regions. For instance, for
heat resistance in D. melanogaster, two mapping exercises
have been undertaken so far and produced consistent
results. One of these (Norry et al. 2004) involved crosses
between heat selected and control lines, while the other
(Morgan & Mackay 2006) was based on crosses between
two inbred lines that differed in heat resistance. Despite this
difference in the genetic background of the stocks, there
was a high degree of overlap in the areas of the genome
identified as controlling heat resistance (Morgan & Mackay
2006). Both studies also identified regions where genes
known to influence variation in heat resistance were located.
A high degree of overlap is also evident in the mapping of
size genes from the ends of different D. melanogaster clines:
size genes have been localized to the right arm of
chromosome 3 in mapping experiments involving lines
from Australia and South America (Calboli et al. 2003); in
males, this region accounts for 60% of the difference in size
between cline ends (Rako et al. 2006). It remains to be seen
if lists of candidate genes for complex traits show substantial
overlap between model and related species.
What about monitoring using quantitative traits? This
approach is feasible if traits can be assessed repeatedly. In
the case of photoperiodic responses studied by Bradshaw &
Holzapfel (2001), the responses were highly repeatable
across assays so that results obtained in 1 year can be related
to those from a different year. Body size and other
morphological assessments are also likely to be highly
repeatable. However, physiological traits are often variable
across assays, making it more difficult to detect phenotypic
changes across years. In the case of insecticide resistance,
estimates of resistance levels can also vary across assays, and
a common solution is to compare resistance levels to those
from a sensitive laboratory culture. Nevertheless, there is a
danger that resistance will change in this culture due to
inbreeding or laboratory adaptation. This problem will also
arise when testing for changes in physiological responses or
life-history traits.
CONCLUDING REMARKS
There is potential for identifying alleles associated with
specific environmental changes. The examples discussed
! 2006 Blackwell Publishing Ltd/CNRS
Review and Synthesis
above illustrate that genetic changes can occur over a short
time frame, that there are molecular signatures of such
changes having occurred in the past and that highly
specific information can often be obtained by monitoring
such changes. A number of marker systems are available
for linking environmental changes to genetic markers and
each of these systems has advantages and disadvantages in
relation to the number of markers available, ease of
scoring and difficulties in linking markers to phenotypes
(Table 1).
Genetic markers need to be compared with other
methods for detecting environmental change and used in
conjunction with them. For the detection of pollutants,
genetic markers need to be compared with a range of
biomarkers including biochemical and physiological parameters, changes in population growth rate and shifts in species
distributions. Genetic markers are likely to provide a useful
adjunct to these other approaches once the range of genes
that respond to specific pollutants have been identified, and
once there is information on the likelihood of adaptive
responses in groups of species.
An experimental programme to further develop links
between environmental change and genetic markers might
include some of the following components.
(1) Identification of candidate markers from model organisms. Lists of markers for any likely environmental
response should become available from these organisms.
This includes markers for specific chemical stresses
including pesticides, heavy metals, hydrocarbon pollutants and specific toxins-like PCBs and PAHs (polycyclic
aromatic hydrocarbons). They also include markers for
traits underlying temperature extremes, desiccation and
starvation resistance, at least some life-history shifts and
responses to other stressors like radiation.
(2) Comparative genomic data to indicate which markers
have been under recent selection. Achieving this aim
will depend on comparisons of genomic data from
populations or closely related species that have been
exposed to different conditions, or genomic data from
longitudinal samples.
(3) Development of screening techniques for comparing
genetic variation at numerous candidate loci. This
technology is under development for screening human
genetic variation. Microarrays can be used for comparing levels of gene expression where connections
between expression and particular phenotypes have
been made.
(4) Information about the response of clonal organisms to
environmental variables, along with the development of
genetic markers for distinguishing the clones.
(5) Longitudinal samples for testing hypotheses about
genetic responses to environmental changes. These
Review and Synthesis
samples already exist for some organisms. For instance,
Daphnia egg banks exist in the sediment of lakes and
DNA can be extracted from these eggs for genetic
analysis and links to environmental variables (Reid et al.
2002). For other model organisms, efforts can now be
made to collect and preserve samples from environmental gradients.
ACKNOWLEDGEMENTS
The authors were supported by the Australian Research
Council whilst preparing this review through their Special
Research Centre, Federation Fellowship and Linkage Postdoctoral Fellowship schemes. We thank Bill Bradshaw and
Volker Loeschcke for discussions about issues raised in this
article.
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Editor, Craig Moritz
Manuscript received 23 June 2006
First decision made 14 August 2006
Manuscript accepted 21 August 2006