Background
Three types of asymmetry in bilateral characters have been recognized: directional
asymmetry, antisymmetry, and fluctuating asymmetry (FA). While both directional
asymmetry (the same side is consistently larger) and antisymmetry (one of the sides is
consistently larger) result from normal development, FA refers to subtle random deviations
from perfect symmetry in bilateral traits resulting from developmental perturbations, and is
often used as an indicator of stress and/or fitness e.g.. The assumption underlying this
practise is that FA reflects developmental instability (DI) an organisms inability to adjust
its development in an ideal symmetric pattern. Several studies have shown that high FA
levels are characteristics of individuals with low fitness e.g.. The link between FA and
various forms of stress has been repeatedly observed: habitat degradation , pollution ,
hybridisation, inbreeding, small population size, and marginal distribution have all been associated with increased levels of FA. Therefore, FA has been proposed to be
a useful bioindicator of individual quality and/or environmental stress. However,
despite these positive results, a number of studies have failed to find the expected
relationships between FA and stress or fitness, fuelling a debate about the general
applicability of FA as a bioindicator trait in conservation biology for reviews.
Numerous analytical and statistical issues, such as the proper control of measurement error in
metric traits, and the difficulty of reliably estimating DI using single traits, might provide at least partial explanation for the conflicting results. These
difficulties have also been proposed to account for the recent decrease in popularity of FA
studies.
While both theory and a number of observations align with the idea that the degree of FA at
the individual or population level is indicative of individual quality or degree of stress
experienced, relaxed selection against developmental perturbations is also expected to
increase FA in given population and/or trait. For instance, several studies have shown that the
levels of FA in functionally important bilateral traits is typically less than that in functionally
less important traits. Similarly, it is possible that the degree of canalizing selection
against developmental perturbations may differ among different populations. If so, this could
provide one explanation for heterogeneity in FA-stress associations in different studies: in
two populations experiencing the same incidence of stress induced developmental errors, the
one experiencing relaxed selection against FA will express a higher degree of FA on average
than a population that is under more stringent normalizing selection. However, to the best of
our knowledge, this hypothesis has not been tested to date.
The mechanosensory lateral line system, present in all fishes and aquatic amphibians, has
anatomical and functional properties which make it highly suitable and attractive for FA
studies. Firstly, the lateral line system consists of numerous sensory receptors (neuromasts)
located on the surface of the animal, either superficially (superficial or free neuromasts) or
under the skin in fluid-filled canals (canal neuromasts), which can be counted easily.
Meristic traits, such as neuromasts, have been shown to be superior over metric traits in
detecting correlations between FA and the environment, and can be counted with little error. Secondly, neuromasts are organised in anatomically distinct lines that are distributed
bilaterally along the head and trunk, and the existence of multiple traits (i.e. individual lines)
provides the possibility to determine the overall level of FA precisely, unlike most single-trait
estimations. Thirdly, the lateral line system is functionally very important, and likely to
influence individual fitness. This system senses weak water movements and mediates crucial
behaviours, including prey detection, predator avoidance, schooling,
orientation to water currents (rheotaxis), and localization of objects. Hence,
lateral line asymmetry is likely to reduce fitness, and as such, it is a potential target of natural
selection. Further, this effect could be expected to differ among populations living in
environments which differ in the demands on the lateral line system.
The goal of the present paper was to compare the degree of FA of marine (high piscine
predation risk) and pond (zero piscine predation risk) nine-spined stickleback (Pungitius
pungitius) populations differing both in the levels of genetic diversity and in the level of
expected selection (by piscine predation) for symmetry. Assuming that perfect symmetry in
the lateral line system is favoured by natural selection, we hypothesised that either (i) the
relaxed selection for symmetry in pond populations under negligible predation, and/or (ii) the
reduced genetic variability (= genetic stress) in pond populations, will result in reduced
developmental stability in ponds as compared to marine populations. In both cases, one
would expect to see higher FA levels in pond than in marine populations. However, because
genetic variability varies between populations within the same habitat [35], we also attempted
to disentangle the two alternative explanations for increased FA in pond environments.
Results
The GLMM on heterozygosity revealed a significant habitat effect (F1,6 = 10.17, P = 0.019),
but no population effect (Z = 1.45, P = 0.15). The average ( S.E.) heterozygosity in marine
populations (HE = 0.58 0.06) was approximately two times higher than in pond populations
(HE = 0.30 0.06; Figure 1). The GLM revealed a significant population effect (F7,176 = 14.47,
P < 0.001) and subsequent post hoc tests revealed no heterogeneity among marine populations
(all P > 0.22, Figure 1). The Mashinnoje pond population that has only recently become
isolated from the White Sea; White Sea Biological Station staff personal
communication] did not differ from the marine populations in terms of heterozygosity (all
P > 0.13, Figure 1). The remaining three ponds (Abbortjrn, Pyrelampi and Rytilampi) had
lower heterozygosity than the marine or Mashinnoje populations (all P < 0.01, Figure 1).
Pyrelampi had lower heterozygosity than any of the other populations (all P < 0.004, Figure
1). Therefore, while the marine populations had uniformly high heterozygosity, in the ponds
heterozygosity varied from being similar to the marine levels (Mashinnoje) to almost zero
(Pyrelampi; Figure 1.).
The GLMM revealed a significant habitat effect (F1,6 = 35.60, P < 0.001) on the composite
standardized relative FA-index, irrespective of sex (F1,156 = 0.27, P = 0.60), population
(Z = 1.05, P = 0.30), and heterozygosity (F1,5 = 2.19, P = 0.20). None of the interactions were significant (habitat*sex: F1,143 = 0.03, P = 0.86; habitat*heterozygosity: F1,4 = 0.10, P = 0.76).
The pond populations showed almost three times higher levels of asymmetry than the marine
populations (Least Squares means S.E.; marine = 6.55 1.3; pond = 17.45 1.3; Figure 1).
The multivariate GLM supported these results, revealing a strong habitat effect (Wilks
lambda 12,141 = 0.59, P < 0.001) on standardized relative asymmetry, irrespective of sex (Wilks
lambda 12,140 = 0.91, P = 0.36, habitat*sex: Wilks lambda 12,139 = 0.96, P = 0.91). The population effect was
also non-significant (Wilks lambda 72,773 = 0.54, P = 0.07), and the univariate tests showed
significant habitat effects for standardized relative asymmetry in all 12 traits (F1,150 > 5.38,
P < 0.02). The trends were similar to those revealed by the composite relative asymmetry
index: pond populations were generally more asymmetric than marine populations (data not
shown).
To explore further the (lack of) heterozygosity effect on FA in the GLMM (see above), we
performed simple correlation analyses using the population mean values of the two traits.
Using raw values, there was strong negative correlation between mean FA and mean
heterozygosity across the eight populations (rs = - 0.833, P = 0.01). However, if the average
effect of habitat type is controlled for by performing the correlation using heterozygosity
values standardized to a common mean across the habitat types, this correlation disappears
(rs = - 0.071, P = 0.35). These results are compatible with the results of the GLMM above, and
show that the association between FA and heterozygosity is mainly driven by the association
between habitat type and heterozygosity.
Discussion
The most salient finding of this study was that nine-spined sticklebacks from ponds exhibit
significantly and consistently higher levels of FA than their marine conspecifics.
Furthermore, while the pond sticklebacks in general had only about half of the genetic
variability of marine sticklebacks, the analyses did not support the idea that habitat
differences in levels of FA are explainable by differences in heterozygosity once the habitat
differences in heterozygosity are controlled for. Hence, the results support the conjecture that
high levels of FA in pond populations stem from decreased selection for perfect symmetry,
rather than from genetic stress.
Predation is a widely recognized mechanism of natural selection, and some studies have
shown that predated individuals express higher levels of FA than surviving individuals e.g.
[9,37-39]. Furthermore, decreased FA with age is also suggestive of poorer survival of more
asymmetric individuals e.g.[40]. Predation can decrease the population level FA in the prey
in at least three ways. First, predation can impose selection for individuals with low FA,
resulting in a high degree of developmental canalization and thereby in low FA. However,
this implies that there is an additive genetic basis for FA. Heritability of FA is a controversial
issue: initial meta-analyses yielded a relatively high average heritability estimate [41], but the
subsequent studies have since suggested that the heritability of FA is very low if not
negligible. Second, assuming that FA has no, or a weak, genetic basis, and is simply a
reflection of the growth environment experienced, predation might simply remove
asymmetric individuals from the population. Third, as negligible predation selects for larger
body size and higher growth rate predation has the potential to affect FA indirectly
through altering the growth intensity, where higher growth rate is coupled with higher DI
. Regardless of the mechanism, relaxation of predation pressure can be expected to
increase the average degree of FA in the population. While we are not aware of any studies that have compared FA levels among populations that differ in selection for perfect
symmetry, there are studies which show that less functionally important traits express higher
FA than important traits at the individual level. That said, it is also known that
strong directional selection can increase DI in selected traits. However, although the
mean number and organisation of lateral-line neuromast differ among marine and pond
populations in this species, the patterns of differentiation among populations are
heterogeneous and directional selection on lateral-line traits is indicated to occur mainly in
the marine environment. Likewise, the lateral plate number one of the traits analysed
in study is shown to be reduced in pond as compared marine populations presumably as
response relaxed piscine predation in pond environments. Hence, it seems unlikely that
the increased DI in pond populations lateral-line traits and lateral plate numbers would
results from directional selection.
Perhaps the most marked difference between pond and marine nine-spined stickleback
populations is the predation risk; marine sticklebacks are sympatric to a large number of
predatory fish species, while ponds lack predatory fish and the nine-spined stickleback is
often the only fish species present in ponds. Previous studies have demonstrated
marked behavioral and morphological differences in nine-spined sticklebacks in relation to
predation risk, including a recent study demonstrating habitat and population
specific differences in the lateral line system. The mechanosensory lateral line system of
fish and aquatic amphibians responds to weak water movements and is involved in avoidance
of predators and in schooling, which is an important antipredator behaviour. Hence the negligible predation in ponds might have resulted in relaxed selection for
perfect symmetry in the lateral line system and consequently, in the high levels of FA
observed in this habitat. While this is, to the best of our knowledge, the first study to suggest
this effect, we admit that the exact functions of the different lateral-line traits in this species
are as yet unknown. Hence, further functional studies about how the information from
the lateral-line is used in different contexts are needed. However, the fact that levels of FA in
lateral plate numbers a trait associated with variation in piscine predation showed
exactly the same patterns of FA as lateral-line traits supports the importance of predation in
dictating the observed patterns..
Inbreeding (mating among relatives) can increase homozygosity and result in inbreeding
depression, which can manifest itself in reduced survival and fertility Increased
FA levels have been linked to reduced heterozygosity both in the field , and in
controlled laboratory experiments with induced inbreeding. It has been shown that
pond nine-spined stickleback populations have lower genetic variability than marine
populations, and one explanation for the higher level of FA in pond sticklebacks could
be that reduced genetic variability in pond populations has resulted in increased DI, and
consequently increased FA. Based on the populations used in this study, the heterozygosity of
pond populations was on average half that of marine populations, with heterozygosity being
highly variable among pond populations, but similar among marine populations. However,
formal tests accounting for the on average lower heterozygosity in pond populations
failed to find association between heterozygosity and FA across the populations. This finding
is not completely surprising, as some other studies also found that heterozygosity had a weak,
or no effect on FA. However, given the fact heterozygosity and habitat type are
tightly associated in our study, their independent effects on FA cannot be fully disentangled.
Obviously, there are other factors that potentially affect FA that we could not directly address
here. For instance, there might be environmental stressors (e.g. water quality, temperature, oxygen levels, pH, etc.) that may differ among marine and pond populations, and cause
higher levels of FA in ponds. At the moment, too little is known about the variation in
relevant environmental parameters and their potential impact on FA in pond vs. marine
habitats to form informed arguments about their significance, but it is worth noting that there
is no a priori reason to suggest that pond fish would experience more stressful environmental
than the marine fish. In fact, pond fish live longer, grow faster and attain larger sizes than
marine fish both in laboratory and the wild. Nevertheless, more environmental data
coupled with experiments conducted under common garden settings would be needed to
study possible environmental determinants of high FA in pond fish. However, irrespectively
of the causes, the fact remains that the levels of FA in pond populations are markedly
elevated as compared to marine populations.
Conclusions
In conclusion, the results demonstrate that there is a three-fold difference in levels of FA between pond and marine nine-spined stickleback populations (pond > marine). While there is also a two-fold difference in heterozygosity (pond < marine), the loss of genetic variation did not explain the divergence in levels of FA once the habitat differences in heterozygosity were controlled for. We hypothesize that the negligible predation in pond populations (contrasted to the high predation in marine environments) is responsible for the increased FA in ponds.
October 2012
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