BACKGROUND
populations of organisms confined to islands often evolve extensive morphological and behavioral changes often over a short time  <cit> .
the best known of these changes are gigantism in small vertebrates and dwarfism in larger species, the so-called island rule  <cit> .
in addition there are changes in body shape.
insular mammals often have reduced limb lengths and enlarged cheek teeth  <cit> .
many insular birds evolve flightlessness  <cit> , dull plumage coloration , and larger bills  <cit> .
insular rodents exhibit an "island syndrome"  <cit>  involving the co-occurrence of most  of these traits: reduced aggressiveness, gigantism, reduced litter size, greater life expectancy, and delayed sexual maturity.
mcnab  <cit>  further noted that organisms affected by the syndrome have a reduced basal metabolic rate, and a propensity to enter torpor, which entails reduced energy expenditure  <cit> .
adler and levins  <cit>  linked the emergence of the island syndrome to the reduced interspecific competition and predation pressure that, they claimed, are typical of islands, and to the size and degree of isolation of the island itself.
when predation pressure is low insular population size increases rapidly .
this makes it adaptive to invest more in somatic growth and less in reproduction, laying smaller litters/clutches of larger offspring  <cit> .
in crowded conditions larger adults and larger offspring usually have higher fitness and better survival  <cit> .
since adler and levins's seminal work, it has been suggested that a similar "syndrome" occurs in insular passerine birds .
according to this theory these birds tend to evolve larger body size, grow larger bills, are less sexually dimorphic and less aggressive towards conspecifics, reach sexual maturity later in life, lay smaller clutches of larger offspring, live at higher density, and have higher habitat fidelity than their mainland relatives.
it is apparent that when living at high and stable population density, many insular vertebrates show a number of correlated trait shifts including, at least, decreased sexual dimorphism, decreased energy allocation to reproduction, and decreased aggressiveness toward conspecifics.
here we refer to these trait shifts as the "island syndrome" expanding upon the original definition given for rodents  <cit> .
similar observations have been made for reptiles.
for instance, lizards on islands show a host of trait changes including melanism, modified limb lengths and head shapes, size change , smaller clutch size, frequent shifts to herbivorous diets, and reduced aggressiveness  <cit> .
buckley and jetz  <cit>  reported that insular lizards often live at very high densities  hence density compensation probably takes place in many insular lizard populations.
under the island syndrome fecundity, aggressiveness and sexual dimorphism decrease because the population density is high and stable, and the environment is predictable.
here we hypothesize that when the environment is highly unpredictable and there are strong fluctuations in population size, investment of more resources into reproduction, aggressive behavior, and sexual competition are favored  <cit> .
sexual dimorphism is expected to increase because of the greater emphasis on sexual selection  <cit> .
the same set of trait shifts should theoretically occur when predators are present and keep the population density low.
we name this hypothesis the "reversed island syndrome" .
shifts in food intake rate and pigmentation  are not directly predicted by the ris hypothesis and are only true if ris is triggered by changes in melanocortin receptors activity, and these are not polymorphic.
in the last column we report the trait shifts mediated by increase in pomc expression and their relationship with ris.
in mammals, clutch size generally decreases with increasing body size  <cit> , thus gigantism is likely to be associated with lower reproductive effort/fewer offspring.
in reptiles, on the other hand, large size is associated with larger clutch and litter sizes  <cit> .
moreover, large insular reptiles may have simply lived for longer  <cit> .
consequently, we predict ris applies may apply to dwarf insular mammals and to large insular reptiles.
we provide explicit tests of the ris-predicted phenotypic and behavioral trait shifts , by using the licosa island wall lizard podarcis sicula klemmeri as a model.
this is a large-sized, melanistic variant of the italian wall lizard p. sicula from mainland italy  <cit> .
p. s. klemmeri population size fluctuates yearly by up to 40% .
individuals of both sexes from the insular population are bright blue and no normal colored lizard  has ever been observed on licosa .
we predicted that p. s. klemmeri would be more aggressive, more sexually dimorphic, and develop faster than mainland p. sicula.
we investigated the genetic basis of p. s. klemmeri body coloration.
the blue color is a form of melanism in reptiles  <cit> .
in vertebrates, melanistic coloration is often associated with increased aggressiveness and sexual activity  <cit> .
in a recent review ducrest et al.
<cit>  suggest the existence of a behavioral syndrome resulting from the pleiotropic effects of the proopiomelanocortin  gene, the products of which regulate genes for melanocortin receptors, mcrs.
pomc produces the melanocortins α-, β- and γ-msh and acth, which bind to five melanocortin receptors .
in vertebrates, pomc and the five mcr genes are highly conserved and their tissue distribution and functions are similar across species  <cit> .
mcrs are involved in the regulation of pigmentation, aggressiveness, food intake, energy expenditure, sexual activity, and immunological responses to stress factors.
since these coordinated traits partly overlap with the trait-shifts we predicted to occur under ris , we predicted that increased mcr expression levels could be the proximal, physiological causes of the ris.
we tested this hypothesis by comparing levels of expression of three different melanocortin receptors: mc1r, 3r, and 4r; which directly influence skin pigmentation , food intake rate, body size, and energy expenditure , and sexual activity  .
these predictions hold only if there are no mutations in mcr genes  <cit> .
therefore, we sequenced and compared the mc1r locus in the two populations in order to rule out the possibility that the striking difference in pigmentation in our lizards populations is due to mutation in mc1r in the island population.
we predict that activity for all of these mcrs is higher in the insular lizards.
increased aggressiveness and sexual activity are expected to occur if an over-expression of the melanocortin system is the molecular basis of the ris, whereas changes in food intake rate and darker pigmentation are expected to occur irrespective of ris, as a consequence of the pleiotropy in the melanocortin system .
finally, we investigated head shape ontogeny in licosa and mainland p. sicula to test for differences in developmental and growth rates.
ontogeny is particularly relevant here because life history theory predicts a tradeoff between growth and reproduction  <cit> .
changes in ontogeny may be reflected in adult shape differences because individuals that mature earlier may appear paedomorphic  <cit> .
paedomorphosis is linked to increased reproductive investment in ephemeral habitats in amphibians  <cit> , and was proposed to drive gigantism in canary island lizards gallotia stehlini and g. simonyi  <cit>  and dwarfism in sauropod dinosaurs  <cit>  and dwarf elephants  <cit> .
on the other hand, positive selection for sexually-selected traits may produce peramorphosis, since sexually selected traits usually appear later in ontogeny  <cit> .
under ris, we predicted that the insular lizard  develops faster in order to mature early,  is more sexually dimorphic as a consequence of strong sexual selection, and  has slower growth rate as a consequence of re-allocation of resources from growth into reproduction  <cit> .
RESULTS
behavioral traits shifts
insular lizards were much more aggressive, spent less time basking and had higher food intake rate .
in the field , we observed island lizards either threatening or attacking other individuals, on average,  <dig>  times per hour, compared to  <dig>  times on the mainland.
a randomization test indicates this difference in attack rate  is highly significant .
in the lab , insular males were nearly four times as likely to attack other males as mainland males .
attacks towards the same sex were significantly more common for insular than for mainland females .
sexual interactions in the lab were quite rare .
we counted just three interactions in mainland lizards and five in insular lizards.
the difference is not significant .
plainly, though, the power of the test is very low given the small numbers.
lab experiments revealed that insular lizards had higher food intake rate than mainland lizards.
mainland lizards ate wax worms, on average, at 7'45" minute intervals, insular lizards at 3' 2" intervals.
on average, insular lizards attacked their first prey after 4' 6'' on average , whereas mainland lizards did not begin feeding until, on average, 12' 26'' .
all insular lizards consumed all the wax worms they were given within  <dig> to  <dig> minutes while none of the mainland lizards consumed all the wax worms, and the 4th wax worm was consumed after  <dig> to  <dig> minutes .
regressing time  to prey consumption versus the number of prey consumed gives a slope of  <dig>  for insular lizards  and  <dig>  for mainland lizards .
the two slopes are statistically different .
in the field, the difference between the two population in the time spent basking is not significant .
yet, in the lab mainland individuals of both sexes spent about twice as much time close to a heat source  than insular ones .
insular lizards spent about equal amounts of time under different levels of radiation while mainland lizards spent 68% of the time directly under the light bulb.
the difference between the two populations is highly significant .
mc1r gene polymorphism and selection test
unlike all other vertebrates studied, we discovered that the mc1r gene in p. sicula consists of two exones.
the first codes for the entire protein , and the second includes the stop codon and 3' utr .
three nucleotide polymorphic sites occur in the two populations.
one of them  results in a charge-changing amino acid variant in the first extracellular loop of mc1r.
the extracellular regions of the mc1r-encoded receptor are important for ligand binding and regulation of mc1r reactivity.
this amino acid variant creates two different alleles, but we did not find statistical association between the t-110-i polymorphism and pigmentation .
thus, the absence of association between mc1r polymorphism and skin color rules out the possibility that mutations in mc1r caused the differences in pigmentation we observed between our study populations.
fifty-five percent of the sequences  were associated with isoleucine at amino acid site  <dig> in the licosa island blue lizard, whereas 45% of sequences were associated with threonine at this position.
the corresponding figures in the mainland population were 25% and 75%, respectively .
patterns of nucleotide variation within lizard populations were consistent with a neutral model of molecular evolution: the distribution of allele frequencies at mc1r as reflected in tajima's d is generally consistent with neutral expectations for mainland and island populations.
for the island population we found tajima d =  <dig> , number of segregating sites s =  <dig>  nucleotide diversity π =  <dig> ; for mainland tajima d =  <dig> , number of segregating sites s =  <dig>  nucleotide diversity π =  <dig> .
differences in mcrs expression
mc1r expression varied between the populations with the insular population showing a roughly four-fold greater gene expression compared to the mainland population  , suggesting that podarcis mc1r gene expression could be involved in skin pigmentation.
western blot analyses showed significant differences in the expression of mc3r and mc4r in the lizards' brain and testes, using actin as an internal standard .
mc3r protein expression in the brain of insular lizards was about three times higher than in mainland lizards' brains .
no appreciable difference in mc3r density was noted in male testes between the two populations .
we found that mc4r expression in testes of insular male lizards was almost twice that observed in mainland lizards .
brain expression of mc4r was not significantly different between the two populations .
growth and development
age, size, and growth
insular lizards had larger maximum svl .
all three lizards >  <dig> mm svl belonged to the insular population, and they were also the only lizards in our sample bearing  <dig> lines of arrested growth  in their long bones.
island lizards were a little older, on average .
in addition, the sex ratio was different, being nearly 1: <dig> on the mainland, but about 1: <dig>  on the island .
we are unaware of a sampling bias that might have increased the likelihood of capturing females on the island.
growth rates did not differ between the two populations .
when sexes were analyzed separately, however, females grew significantly slower on the island.
in fact, insular males grew faster than insular females.
this accounts for the significant body size dimorphism on the island .
ci = confidence intervals.
changes in ontogeny
a standardized major axis regression of procrustes distances calculated from a common reference  indicated that the slope  of somatic development did not differ between populations .
the multivariate angle between regression vectors  of the two populations was  <dig> , the angle within licosa island population was  <dig> , and within punta licosa was  <dig> .
this means that although the between-populations angle was rather large, the ontogenetic trajectories of the two populations were the same .
procrustes distances between age groups confirm this notion .
the distances between juvenile individuals of the two populations, and between adults of the two populations are similar, implying the ontogenetic trajectories are parallel .
importantly, the distance between the two groups of juveniles is significantly >  <dig>  indicating that consistent difference in development between the two populations occurs early in morphogenesis  <cit> .
the largest distance  was between licosa adults and punta licosa juveniles.
this distance was much larger than between licosa juveniles and punta licosa adults .
finally, the lengths of ontogenetic vectors were  <dig>  and  <dig>  on licosa and punta licosa, respectively.
the metric of shape distance is the procrustes distance .
juveniles are assumed to be all the individuals of age 0- <dig> lags.
individuals with n >  <dig> lag are considered adults.
p values are reported in the upper-right triangle, procrutes distances  are in the lower triangle.
j = juveniles; a = adults.
taken together, these results indicate that although the rate and trajectory of somatic development is similar between populations, insular juveniles head shapes appear more adult-like, and insular adult head shapes develop "beyond" mainland adults, in keeping with the notion of peramorphosis.
the shift occurs early in morphogenesis .
sexual dimorphism
in two-factor anova, residuals of ols regression of log svl against log head width revealed significant differences between populations  and sexes .
the interaction term is significant .
insular male heads are much larger than insular female heads.
insular male lizard heads are also larger than mainland lizard heads in both sexes .
sexual size dimorphism  is not particularly pronounced in podarcis lizards  <cit> .
we found significant ssd only in three year old insular lizards.
males' mean svl at three years was  <dig>  mm on licosa, whereas females' mean svl was  <dig>  mm.
the svl difference was statistically significant .
d = procrustes distances.
number of individuals is reported in parentheses.
for ssd we report the mean svl of females  and the mean svl of males .
*t =  <dig> , p <  <dig> .
sexual head shape dimorphism was large.
the procrustes  distance between insular adult male and female individuals was  <dig>  .
on the mainland, this distance was smaller and only marginally significant .
discussion
despite the fact that the two populations analyzed here diverged a short time ago, and that there is sizeable genetic introgression between them  <cit> , we found striking evolutionary change in the licosa lizards.
compared to their mainland relatives, these blue lizards were much more aggressive, voracious and dimorphic, as predicted by the ris hypothesis .
at first glance the increased activity in both mc3r and 4r is expected to decrease food intake.
yet, mc3r is believed to function as an inhibitory autoreceptor on pomc neurons: experimental injections of mc3r-specific agonists stimulate feeding in mice  <cit> .
moreover, food deprivation increases mc4r expression in the liver of barfin flounder verasper moseri  <cit> .
in our insular lizards, the complex pathways that regulate food intake via modification of mc3r and 4r activity deserve further investigation.
however this is beyond the scope of the present study.
the island syndrome predicts that sexual selection should be less intense in insular vertebrates living at high and stable population density, because mates are a non-limited resource in this condition  <cit> .
conversely, with either low or strongly fluctuating population size, as with the ris hypothesis, higher investment in  reproduction should favor positive selection of sexually-selected traits, due to an unpredictable mortality schedule  <cit> .
in keeping with this, insular males had larger heads, and sexual size and shape dimorphism were increased on the island .
moreover, in insular females body size growth rate was low , which is expected when energy allocation toward reproduction is increased.
as predicted, somatic development proceeded at a faster rate early in insular lizards' ontogeny, as indicated by head morphogenesis .
the notion that the licosa lizard is a large-sized variant of mainland podarcis sicula  <cit>  was confirmed by our data.
however, insular lizards were, on average, older, and the higher mean svl on the island could be a result of different age structure  rather than the result of direct selection on size.
the increased density of mcr receptors in insular lizard tissues provides a proximate explanation for the phenotypic trait shifts we observed in these organisms .
it is remarkable that we found neutral selection on the mc1r gene both in insular and mainland individuals.
this suggests that selection for color per se probably does not occur.
although we found some evidence that the darker coloration of insular lizards decrease the time spent basking the blue color may simply be a by-product of concerted increase in mcr activity levels.
consequently, we argue the blue coloration may have been recruited adaptively for better thermoregulatory activity, as a 'spandrel'  <cit> .
we argue that increased aggressiveness and food intake rates are the likely targets of natural selection in our insular lizards.
aggressive individuals are very often the most successful  in natural populations, although they incur a greater risk of injury and greater exposure to predators.
however, the risk of becoming prey is very low on licosa.
behaving aggressively and increasing food intake rate should be very advantageous in a restricted habitat with limited and probably inconstant access to both resources and mates, as is the case with licosa.
in lizards, color morphs are quite common  <cit>  and often underlie differences in life history traits and reproductive strategies even in the same population  <cit> .
in insular lizards, black and blue morphs are frequent , and evolutionary mechanisms similar to what we describe for p. s. klemmeri may have been working in many of them.
bauwens and castilla  <cit>  noted that all the largest podarcis lilfordi males on cabrera island have black or deep blue skin, and indicated that this change in coloration is ontogenetic, occurring at svl of  <dig> mm.
furthermore, they noted that the largest adults of all of the  <dig> islets surrounding cabrera are black.
males of bonaire island whiptail cnemidophorus murinus occur in two color morphs, brown and blue.
baird et al.
<cit>  found that blue males are far more aggressive than browns.
their sexual activity is much increased, and their testes are larger.
of all the courtship encounters they observed  <dig> % were initiated by blue males, vs.  <dig> % initiated by brown males and  <dig> % initiated by females.
individuals of a translocated population of st. lucia whiptail cnemidophorus vanzoi on praslin island  <cit>  evolved increased growth rates, and high intensity of intraspecific competition .
praslin whiptails also have increased sexual size dimorphism and sexual dichromatism.
moreover, very surprisingly, praslin whiptails are territorial although cnemidophorus belongs to a family  of typically non-territorial lizards  <cit> .
some adult males also maintain juvenile colors  <cit> .
in practice, these studies suggest that the occurrence of the 'reversed island syndrome' in insular lizards may well extend to other melanistic forms.
however, we emphasize, that the link between ris and melanism is not necessary .
the mechanism behind the emergence of ris is the reallocation of energy from growth to reproduction.
this reallocation is expected to take place when resource supply and/or mortality risk are unpredictable, thereby favoring aggressiveness and voracity to secure resources and mates in the short term  <cit> .
for example, the okada broad-headed skink plestiodon okadae occurs in a number of islands of the izu archipelago, japan.
hasegawa  <cit>  noted that some p. okadae populations have: "a suite of traits including small body size, early maturity, production of small and many eggs, low frequency of failure in follicle development, annual reproduction, high relative clutch mass and low post-reproductive mass of females.
whereas others have a suite of the opposite character states".
fast life histories occur on the island with the largest predator fauna , whereas slow life histories characterize islands with higher density and the lowest mortality of hatchlings .
still, fast-living ohshima skinks are the smallest and the most dimorphic, with males having disproportionately large heads with a distinctive color pattern  <cit> .
hasegawa manipulated lizard density in miyakejima  <cit> , removing adults in order to simulate increased mortality.
as expected, after the experiment, both males and females started reproducing at a younger age, and females laid larger clutches, whereas egg size and body size at first reproduction went almost unaltered.
this experiment demonstrates how decreasing survival rates ignited ris-like changes in life history traits.
plestiodon okadae from different islands may well represent both the island syndrome and ris.
a similar pattern of variation has been shown for canary islands gallotia  <cit> .
island syndrome and ris describe a number of phenotypic traits that often, but by no means always, change together in insular species.
we think one should not expect that all these traits co-occur.
for instance, the most obvious trait shift linked to the island syndrome  is change in body size.
taking into account possible changes in ontogeny , the relationship between body size and the island syndrome may in fact appear weak.
ontogenetic shifts may produce early-maturing, fast-growing small individuals via paedomorphosis.
this is suggested to occur in the extinct dwarf elephant elephas falconeri  <cit>  and in dwarf dinosaurs .
slow growth was found to occur in new zealand's gigantic moas  <cit> , extinct giant lemurs of madagascar  <cit> , females of the licosa island lizard , and extinct dwarf balearic goats .
bunce et al.
<cit> , however, showed that dinornis moas were exceptionally dimorphic, and suggested that these birds invested a great deal of energy into egg production.
this goes against the island syndrome predictions, despite dinornis being huge even by moa standards.
lancaster et al.
<cit>  correctly noted that resource allocation trade-offs are best understood at multiple levels, and pointed out that "the more resources are available, the less trade-offs will be observed on any level.
this occurs over an individual's lifespan for plastic resource allocation traits  and over evolutionary time for those mediated by antagonistic pleiotropy or other genetic mechanisms".
this means that looking at one aspect alone  when comparing an insular species to its mainland relative may be misleading.
spencer et al.
<cit>  emphasized that although growth is expected to be slow when population density is high, rapid growth beyond a critical size reduces predation risk and could be favored .
roth  <cit>  argued that density compensation might explain dwarfism in insular pigmy elephants.
bonaire whiptails live at huge population density, yet the blue morph males allocate a great deal of resources to reproduction, which is consistent with ris hypothesis  <cit> .
bonaire whiptails are herbivorous, and so food is not a limiting factor on bonaire  <cit> .
we remark once more that it is not density  per se, but the set of ecological conditions favoring either small or large allocation of available resources toward reproduction that produces the island syndrome or the ris, respectively.
ris is most likely to occur on small islets where environmental variation  from one year to the next can be high.
the possible instances of ris described above all relate to species living alone or almost alone on islets and cliffs such as licosa, brusnik, praslin, isla de la reya, the islets off the main island in the cabrera archipelago, sombrero, the faraglioni cliffs off the island of capri and vetara islet .
we showed that the ris is linked to increased activity of melanocortin receptors in our insular study population.
melanocortins pleiotropically regulate a number of processes including pigmentation, sexual activity, aggressiveness, bodily growth, and food intake.
we find it highly likely that the same pattern of change in mcr activity occurs in other melanistic insular organisms.
we believe that shifts in growth rate, body size, age and size at maturity, and clutch size will depend on how these traits trade off with each other  <cit> .
furthermore, an insular species may well express the ris without being melanistic.
despite all of the evidence we present in favor of ris, there are drawbacks to two-taxon comparative studies  <cit> .
in these studies, it is possible to interpret as adaptation what really is the phenotypic difference between taxa due to other factors or even to genetic drift.
in this study, we did not demonstrate that ris applies to other insular populations/species.
however, blue podarcis sicula are common on other islands, and it would be very surprising if the basis for this coloration changed from one island to another.
roulin and salamin  <cit>  recently demonstrated that insular barn owls tyto alba of the world have lighter pheomelanic coloration and are less sexually dimorphic than continental barn owls.
they interpreted this observation as a component of the island syndrome, mediated by changes in melanocortin expression.
this suggests that, exactly as we predict in our study, pomc involvement in the island syndrome and its opposite, the ris, is common in insular vertebrates.
CONCLUSIONS
we predicted that insular vertebrates facing unpredictable resource supply and mortality schedules should evolve aggressive behavior, high food intake rate, and increased energy allocation to reproduction as a mean to increase fitness.
we refer to these predictions as the reversed island syndrome  hypothesis, in contrast to the island syndrome  <cit> .
on the proximate level, we demonstrated that the increased activity of melanocortin receptors underlies ris in our study organism, the insular wall lizard podarcis sicula klemmeri.
although much study is needed to fully understand patterns of trait evolution and covariation in insular vertebrates, we hope to have shed some light on this complex issue, by showing how disparate factors must be taken into account in order to gain a meaningful depiction of the evolution of life on islands  <cit> .

 populations of organisms confined to islands often evolve extensive morphological and behavioral changes often over a short time. the best known of these changes are gigantism in small vertebrates and dwarfism in larger species, the so-called island rule. in addition there are changes in body shape. insular mammals often have reduced limb lengths and enlarged cheek teeth. many insular birds evolve flightlessness, dull plumage coloration, and larger bills. insular rodents exhibit an "island syndrome" involving the co-occurrence of most of these traits: reduced aggressiveness, gigantism, reduced litter size, greater life expectancy, and delayed sexual maturity. mcnab further noted that organisms affected by the syndrome have a reduced basal metabolic rate, and a propensity to enter torpor, which entails reduced energy expenditure. adler and levins linked the emergence of the island syndrome to the reduced interspecific competition and predation pressure that, they claimed, are typical of islands, and to the size and degree of isolation of the island itself. when predation pressure is low insular population size increases rapidly. this makes it adaptive to invest more in somatic growth and less in reproduction, laying smaller litters/clutches of larger offspring. in crowded conditions larger adults and larger offspring usually have higher fitness and better survival. since adler and levins's seminal work, it has been suggested that a similar "syndrome" occurs in insular passerine birds. according to this theory these birds tend to evolve larger body size, grow larger bills, are less sexually dimorphic and less aggressive towards conspecifics, reach sexual maturity later in life, lay smaller clutches of larger offspring, live at higher density, and have higher habitat fidelity than their mainland relatives. it is apparent that when living at high and stable population density, many insular vertebrates show a number of correlated trait shifts including, at least, decreased sexual dimorphism, decreased energy allocation to reproduction, and decreased aggressiveness toward conspecifics. here we refer to these trait shifts as the "island syndrome" expanding upon the original definition given for rodents. similar observations have been made for reptiles. for instance, lizards on islands show a host of trait changes including melanism, modified limb lengths and head shapes, size change, smaller clutch size, frequent shifts to herbivorous diets, and reduced aggressiveness. buckley and jetz reported that insular lizards often live at very high densities hence density compensation probably takes place in many insular lizard populations. under the island syndrome fecundity, aggressiveness and sexual dimorphism decrease because the population density is high and stable, and the environment is predictable. here we hypothesize that when the environment is highly unpredictable and there are strong fluctuations in population size, investment of more resources into reproduction, aggressive behavior, and sexual competition are favored. sexual dimorphism is expected to increase because of the greater emphasis on sexual selection. the same set of trait shifts should theoretically occur when predators are present and keep the population density low. we name this hypothesis the "reversed island syndrome". shifts in food intake rate and pigmentation are not directly predicted by the ris hypothesis and are only true if ris is triggered by changes in melanocortin receptors activity, and these are not polymorphic. in the last column we report the trait shifts mediated by increase in pomc expression and their relationship with ris. in mammals, clutch size generally decreases with increasing body size, thus gigantism is likely to be associated with lower reproductive effort/fewer offspring. in reptiles, on the other hand, large size is associated with larger clutch and litter sizes. moreover, large insular reptiles may have simply lived for longer. consequently, we predict ris applies may apply to dwarf insular mammals and to large insular reptiles. we provide explicit tests of the ris-predicted phenotypic and behavioral trait shifts, by using the licosa island wall lizard podarcis sicula klemmeri as a model. this is a large-sized, melanistic variant of the italian wall lizard p. sicula from mainland italy. p. s. klemmeri population size fluctuates yearly by up to 40%. individuals of both sexes from the insular population are bright blue and no normal colored lizard has ever been observed on licosa. we predicted that p. s. klemmeri would be more aggressive, more sexually dimorphic, and develop faster than mainland p. sicula. we investigated the genetic basis of p. s. klemmeri body coloration. the blue color is a form of melanism in reptiles. in vertebrates, melanistic coloration is often associated with increased aggressiveness and sexual activity. in a recent review ducrest et al. suggest the existence of a behavioral syndrome resulting from the pleiotropic effects of the proopiomelanocortin gene, the products of which regulate genes for melanocortin receptors, mcrs. pomc produces the melanocortins α-, β- and γ-msh and acth, which bind to five melanocortin receptors. in vertebrates, pomc and the five mcr genes are highly conserved and their tissue distribution and functions are similar across species. mcrs are involved in the regulation of pigmentation, aggressiveness, food intake, energy expenditure, sexual activity, and immunological responses to stress factors. since these coordinated traits partly overlap with the trait-shifts we predicted to occur under ris, we predicted that increased mcr expression levels could be the proximal, physiological causes of the ris. we tested this hypothesis by comparing levels of expression of three different melanocortin receptors: mc1r, 3r, and 4r; which directly influence skin pigmentation, food intake rate, body size, and energy expenditure, and sexual activity. these predictions hold only if there are no mutations in mcr genes. therefore, we sequenced and compared the mc1r locus in the two populations in order to rule out the possibility that the striking difference in pigmentation in our lizards populations is due to mutation in mc1r in the island population. we predict that activity for all of these mcrs is higher in the insular lizards. increased aggressiveness and sexual activity are expected to occur if an over-expression of the melanocortin system is the molecular basis of the ris, whereas changes in food intake rate and darker pigmentation are expected to occur irrespective of ris, as a consequence of the pleiotropy in the melanocortin system. finally, we investigated head shape ontogeny in licosa and mainland p. sicula to test for differences in developmental and growth rates. ontogeny is particularly relevant here because life history theory predicts a tradeoff between growth and reproduction. changes in ontogeny may be reflected in adult shape differences because individuals that mature earlier may appear paedomorphic. paedomorphosis is linked to increased reproductive investment in ephemeral habitats in amphibians, and was proposed to drive gigantism in canary island lizards gallotia stehlini and g. simonyi and dwarfism in sauropod dinosaurs and dwarf elephants. on the other hand, positive selection for sexually-selected traits may produce peramorphosis, since sexually selected traits usually appear later in ontogeny. under ris, we predicted that the insular lizard develops faster in order to mature early, is more sexually dimorphic as a consequence of strong sexual selection, and has slower growth rate as a consequence of re-allocation of resources from growth into reproduction.  behavioral traits shifts insular lizards were much more aggressive, spent less time basking and had higher food intake rate. in the field, we observed island lizards either threatening or attacking other individuals, on average, times per hour, compared to times on the mainland. a randomization test indicates this difference in attack rate is highly significant. in the lab, insular males were nearly four times as likely to attack other males as mainland males. attacks towards the same sex were significantly more common for insular than for mainland females. sexual interactions in the lab were quite rare. we counted just three interactions in mainland lizards and five in insular lizards. the difference is not significant. plainly, though, the power of the test is very low given the small numbers. lab experiments revealed that insular lizards had higher food intake rate than mainland lizards. mainland lizards ate wax worms, on average, at 7'45" minute intervals, insular lizards at 3' 2" intervals. on average, insular lizards attacked their first prey after 4' 6'' on average, whereas mainland lizards did not begin feeding until, on average, 12' 26''. all insular lizards consumed all the wax worms they were given within to minutes while none of the mainland lizards consumed all the wax worms, and the 4th wax worm was consumed after to minutes. regressing time to prey consumption versus the number of prey consumed gives a slope of for insular lizards and for mainland lizards. the two slopes are statistically different. in the field, the difference between the two population in the time spent basking is not significant. yet, in the lab mainland individuals of both sexes spent about twice as much time close to a heat source than insular ones. insular lizards spent about equal amounts of time under different levels of radiation while mainland lizards spent 68% of the time directly under the light bulb. the difference between the two populations is highly significant. mc1r gene polymorphism and selection test unlike all other vertebrates studied, we discovered that the mc1r gene in p. sicula consists of two exones. the first codes for the entire protein, and the second includes the stop codon and 3' utr. three nucleotide polymorphic sites occur in the two populations. one of them results in a charge-changing amino acid variant in the first extracellular loop of mc1r. the extracellular regions of the mc1r-encoded receptor are important for ligand binding and regulation of mc1r reactivity. this amino acid variant creates two different alleles, but we did not find statistical association between the t-110-i polymorphism and pigmentation. thus, the absence of association between mc1r polymorphism and skin color rules out the possibility that mutations in mc1r caused the differences in pigmentation we observed between our study populations. fifty-five percent of the sequences were associated with isoleucine at amino acid site in the licosa island blue lizard, whereas 45% of sequences were associated with threonine at this position. the corresponding figures in the mainland population were 25% and 75%, respectively. patterns of nucleotide variation within lizard populations were consistent with a neutral model of molecular evolution: the distribution of allele frequencies at mc1r as reflected in tajima's d is generally consistent with neutral expectations for mainland and island populations. for the island population we found tajima d =, number of segregating sites s = nucleotide diversity π =; for mainland tajima d =, number of segregating sites s = nucleotide diversity π =. differences in mcrs expression mc1r expression varied between the populations with the insular population showing a roughly four-fold greater gene expression compared to the mainland population, suggesting that podarcis mc1r gene expression could be involved in skin pigmentation. western blot analyses showed significant differences in the expression of mc3r and mc4r in the lizards' brain and testes, using actin as an internal standard. mc3r protein expression in the brain of insular lizards was about three times higher than in mainland lizards' brains. no appreciable difference in mc3r density was noted in male testes between the two populations. we found that mc4r expression in testes of insular male lizards was almost twice that observed in mainland lizards. brain expression of mc4r was not significantly different between the two populations. growth and development age, size, and growth insular lizards had larger maximum svl. all three lizards > mm svl belonged to the insular population, and they were also the only lizards in our sample bearing lines of arrested growth in their long bones. island lizards were a little older, on average. in addition, the sex ratio was different, being nearly 1: on the mainland, but about 1: on the island. we are unaware of a sampling bias that might have increased the likelihood of capturing females on the island. growth rates did not differ between the two populations. when sexes were analyzed separately, however, females grew significantly slower on the island. in fact, insular males grew faster than insular females. this accounts for the significant body size dimorphism on the island. ci = confidence intervals. changes in ontogeny a standardized major axis regression of procrustes distances calculated from a common reference indicated that the slope of somatic development did not differ between populations. the multivariate angle between regression vectors of the two populations was, the angle within licosa island population was, and within punta licosa was. this means that although the between-populations angle was rather large, the ontogenetic trajectories of the two populations were the same. procrustes distances between age groups confirm this notion. the distances between juvenile individuals of the two populations, and between adults of the two populations are similar, implying the ontogenetic trajectories are parallel. importantly, the distance between the two groups of juveniles is significantly > indicating that consistent difference in development between the two populations occurs early in morphogenesis. the largest distance was between licosa adults and punta licosa juveniles. this distance was much larger than between licosa juveniles and punta licosa adults. finally, the lengths of ontogenetic vectors were and on licosa and punta licosa, respectively. the metric of shape distance is the procrustes distance. juveniles are assumed to be all the individuals of age 0- lags. individuals with n > lag are considered adults. p values are reported in the upper-right triangle, procrutes distances are in the lower triangle. j = juveniles; a = adults. taken together, these results indicate that although the rate and trajectory of somatic development is similar between populations, insular juveniles head shapes appear more adult-like, and insular adult head shapes develop "beyond" mainland adults, in keeping with the notion of peramorphosis. the shift occurs early in morphogenesis. sexual dimorphism in two-factor anova, residuals of ols regression of log svl against log head width revealed significant differences between populations and sexes. the interaction term is significant. insular male heads are much larger than insular female heads. insular male lizard heads are also larger than mainland lizard heads in both sexes. sexual size dimorphism is not particularly pronounced in podarcis lizards. we found significant ssd only in three year old insular lizards. males' mean svl at three years was mm on licosa, whereas females' mean svl was mm. the svl difference was statistically significant. d = procrustes distances. number of individuals is reported in parentheses. for ssd we report the mean svl of females and the mean svl of males. *t =, p <. sexual head shape dimorphism was large. the procrustes distance between insular adult male and female individuals was. on the mainland, this distance was smaller and only marginally significant. discussion despite the fact that the two populations analyzed here diverged a short time ago, and that there is sizeable genetic introgression between them, we found striking evolutionary change in the licosa lizards. compared to their mainland relatives, these blue lizards were much more aggressive, voracious and dimorphic, as predicted by the ris hypothesis. at first glance the increased activity in both mc3r and 4r is expected to decrease food intake. yet, mc3r is believed to function as an inhibitory autoreceptor on pomc neurons: experimental injections of mc3r-specific agonists stimulate feeding in mice. moreover, food deprivation increases mc4r expression in the liver of barfin flounder verasper moseri. in our insular lizards, the complex pathways that regulate food intake via modification of mc3r and 4r activity deserve further investigation. however this is beyond the scope of the present study. the island syndrome predicts that sexual selection should be less intense in insular vertebrates living at high and stable population density, because mates are a non-limited resource in this condition. conversely, with either low or strongly fluctuating population size, as with the ris hypothesis, higher investment in reproduction should favor positive selection of sexually-selected traits, due to an unpredictable mortality schedule. in keeping with this, insular males had larger heads, and sexual size and shape dimorphism were increased on the island. moreover, in insular females body size growth rate was low, which is expected when energy allocation toward reproduction is increased. as predicted, somatic development proceeded at a faster rate early in insular lizards' ontogeny, as indicated by head morphogenesis. the notion that the licosa lizard is a large-sized variant of mainland podarcis sicula was confirmed by our data. however, insular lizards were, on average, older, and the higher mean svl on the island could be a result of different age structure rather than the result of direct selection on size. the increased density of mcr receptors in insular lizard tissues provides a proximate explanation for the phenotypic trait shifts we observed in these organisms. it is remarkable that we found neutral selection on the mc1r gene both in insular and mainland individuals. this suggests that selection for color per se probably does not occur. although we found some evidence that the darker coloration of insular lizards decrease the time spent basking the blue color may simply be a by-product of concerted increase in mcr activity levels. consequently, we argue the blue coloration may have been recruited adaptively for better thermoregulatory activity, as a'spandrel'. we argue that increased aggressiveness and food intake rates are the likely targets of natural selection in our insular lizards. aggressive individuals are very often the most successful in natural populations, although they incur a greater risk of injury and greater exposure to predators. however, the risk of becoming prey is very low on licosa. behaving aggressively and increasing food intake rate should be very advantageous in a restricted habitat with limited and probably inconstant access to both resources and mates, as is the case with licosa. in lizards, color morphs are quite common and often underlie differences in life history traits and reproductive strategies even in the same population. in insular lizards, black and blue morphs are frequent, and evolutionary mechanisms similar to what we describe for p. s. klemmeri may have been working in many of them. bauwens and castilla noted that all the largest podarcis lilfordi males on cabrera island have black or deep blue skin, and indicated that this change in coloration is ontogenetic, occurring at svl of mm. furthermore, they noted that the largest adults of all of the islets surrounding cabrera are black. males of bonaire island whiptail cnemidophorus murinus occur in two color morphs, brown and blue. baird et al. found that blue males are far more aggressive than browns. their sexual activity is much increased, and their testes are larger. of all the courtship encounters they observed % were initiated by blue males, vs. % initiated by brown males and % initiated by females. individuals of a translocated population of st. lucia whiptail cnemidophorus vanzoi on praslin island evolved increased growth rates, and high intensity of intraspecific competition. praslin whiptails also have increased sexual size dimorphism and sexual dichromatism. moreover, very surprisingly, praslin whiptails are territorial although cnemidophorus belongs to a family of typically non-territorial lizards. some adult males also maintain juvenile colors. in practice, these studies suggest that the occurrence of the'reversed island syndrome' in insular lizards may well extend to other melanistic forms. however, we emphasize, that the link between ris and melanism is not necessary. the mechanism behind the emergence of ris is the reallocation of energy from growth to reproduction. this reallocation is expected to take place when resource supply and/or mortality risk are unpredictable, thereby favoring aggressiveness and voracity to secure resources and mates in the short term. for example, the okada broad-headed skink plestiodon okadae occurs in a number of islands of the izu archipelago, japan. hasegawa noted that some p. okadae populations have: "a suite of traits including small body size, early maturity, production of small and many eggs, low frequency of failure in follicle development, annual reproduction, high relative clutch mass and low post-reproductive mass of females. whereas others have a suite of the opposite character states". fast life histories occur on the island with the largest predator fauna, whereas slow life histories characterize islands with higher density and the lowest mortality of hatchlings. still, fast-living ohshima skinks are the smallest and the most dimorphic, with males having disproportionately large heads with a distinctive color pattern. hasegawa manipulated lizard density in miyakejima, removing adults in order to simulate increased mortality. as expected, after the experiment, both males and females started reproducing at a younger age, and females laid larger clutches, whereas egg size and body size at first reproduction went almost unaltered. this experiment demonstrates how decreasing survival rates ignited ris-like changes in life history traits. plestiodon okadae from different islands may well represent both the island syndrome and ris. a similar pattern of variation has been shown for canary islands gallotia. island syndrome and ris describe a number of phenotypic traits that often, but by no means always, change together in insular species. we think one should not expect that all these traits co-occur. for instance, the most obvious trait shift linked to the island syndrome is change in body size. taking into account possible changes in ontogeny, the relationship between body size and the island syndrome may in fact appear weak. ontogenetic shifts may produce early-maturing, fast-growing small individuals via paedomorphosis. this is suggested to occur in the extinct dwarf elephant elephas falconeri and in dwarf dinosaurs. slow growth was found to occur in new zealand's gigantic moas, extinct giant lemurs of madagascar, females of the licosa island lizard, and extinct dwarf balearic goats. bunce et al., however, showed that dinornis moas were exceptionally dimorphic, and suggested that these birds invested a great deal of energy into egg production. this goes against the island syndrome predictions, despite dinornis being huge even by moa standards. lancaster et al. correctly noted that resource allocation trade-offs are best understood at multiple levels, and pointed out that "the more resources are available, the less trade-offs will be observed on any level. this occurs over an individual's lifespan for plastic resource allocation traits and over evolutionary time for those mediated by antagonistic pleiotropy or other genetic mechanisms". this means that looking at one aspect alone when comparing an insular species to its mainland relative may be misleading. spencer et al. emphasized that although growth is expected to be slow when population density is high, rapid growth beyond a critical size reduces predation risk and could be favored. roth argued that density compensation might explain dwarfism in insular pigmy elephants. bonaire whiptails live at huge population density, yet the blue morph males allocate a great deal of resources to reproduction, which is consistent with ris hypothesis. bonaire whiptails are herbivorous, and so food is not a limiting factor on bonaire. we remark once more that it is not density per se, but the set of ecological conditions favoring either small or large allocation of available resources toward reproduction that produces the island syndrome or the ris, respectively. ris is most likely to occur on small islets where environmental variation from one year to the next can be high. the possible instances of ris described above all relate to species living alone or almost alone on islets and cliffs such as licosa, brusnik, praslin, isla de la reya, the islets off the main island in the cabrera archipelago, sombrero, the faraglioni cliffs off the island of capri and vetara islet. we showed that the ris is linked to increased activity of melanocortin receptors in our insular study population. melanocortins pleiotropically regulate a number of processes including pigmentation, sexual activity, aggressiveness, bodily growth, and food intake. we find it highly likely that the same pattern of change in mcr activity occurs in other melanistic insular organisms. we believe that shifts in growth rate, body size, age and size at maturity, and clutch size will depend on how these traits trade off with each other. furthermore, an insular species may well express the ris without being melanistic. despite all of the evidence we present in favor of ris, there are drawbacks to two-taxon comparative studies. in these studies, it is possible to interpret as adaptation what really is the phenotypic difference between taxa due to other factors or even to genetic drift. in this study, we did not demonstrate that ris applies to other insular populations/species. however, blue podarcis sicula are common on other islands, and it would be very surprising if the basis for this coloration changed from one island to another. roulin and salamin recently demonstrated that insular barn owls tyto alba of the world have lighter pheomelanic coloration and are less sexually dimorphic than continental barn owls. they interpreted this observation as a component of the island syndrome, mediated by changes in melanocortin expression. this suggests that, exactly as we predict in our study, pomc involvement in the island syndrome and its opposite, the ris, is common in insular vertebrates.  we predicted that insular vertebrates facing unpredictable resource supply and mortality schedules should evolve aggressive behavior, high food intake rate, and increased energy allocation to reproduction as a mean to increase fitness. we refer to these predictions as the reversed island syndrome hypothesis, in contrast to the island syndrome. on the proximate level, we demonstrated that the increased activity of melanocortin receptors underlies ris in our study organism, the insular wall lizard podarcis sicula klemmeri. although much study is needed to fully understand patterns of trait evolution and covariation in insular vertebrates, we hope to have shed some light on this complex issue, by showing how disparate factors must be taken into account in order to gain a meaningful depiction of the evolution of life on islands. 
we predicted that when population density is low and/or fluctuating insular vertebrates may evolve correlated trait shifts running opposite to the island syndrome, which we collectively refer to as the'reversed island syndrome' hypothesis. the growth rate of insular females was slower than growth rates of mainland individuals of both sexes, and of insular males. insular lizards have a higher food intake rate than mainland individuals, which is consistent with the increased activity of melanocortin receptors. melanocortins are postranslational products of the proopiomelanocortin gene, which controls pleiotropically pigmentation, aggressiveness, sexual activity, and food intake in vertebrates. on the proximate level, we hypothesized that ris is caused by increased activity levels in melanocortin receptors. as predicted, melanocortin receptors were much more active in individuals of the insular population. this set of observations is referred to as the'island syndrome'. in keeping with our predictions, insular lizards were more aggressive and sexually dimorphic than their mainland relatives. insular lizards of both sexes spent less time basking than their mainland relatives. many small vertebrates on islands grow larger, mature later, lay smaller clutches/litters, and are less sexually dimorphic and aggressive than their mainland relatives. this may be adaptive in an unpredictable environment such as licosa island. we contend that when population density is either low or fluctuating annually as a result of environmental unpredictability, it may be advantageous to individuals to behave more aggressively, to raise their rate of food intake, and allocate more energy into reproduction. consequently, size and shape dimorphism are higher on the island. we tested the ris hypothesis performing a number of behavioral, genetic, and ontogenetic tests on a blue colored insular variant of the italian wall lizard podarcis sicula, living on a small island off the southern italian coast. we suspect this is a by-product of the positive selection for increased activity of melanocortins receptors. insular males had wide, peramorphic heads. the syndrome is linked to high population density on islands. the population density of this blue-colored variant was generally low and highly fluctuating from one year to the next.
 many small vertebrates on islands grow larger, mature later, lay smaller clutches/litters, and are less sexually dimorphic and aggressive than their mainland relatives. this set of observations is referred to as the'island syndrome'. the syndrome is linked to high population density on islands. we predicted that when population density is low and/or fluctuating insular vertebrates may evolve correlated trait shifts running opposite to the island syndrome, which we collectively refer to as the'reversed island syndrome' hypothesis. on the proximate level, we hypothesized that ris is caused by increased activity levels in melanocortin receptors. melanocortins are postranslational products of the proopiomelanocortin gene, which controls pleiotropically pigmentation, aggressiveness, sexual activity, and food intake in vertebrates.  we tested the ris hypothesis performing a number of behavioral, genetic, and ontogenetic tests on a blue colored insular variant of the italian wall lizard podarcis sicula, living on a small island off the southern italian coast. the population density of this blue-colored variant was generally low and highly fluctuating from one year to the next. in keeping with our predictions, insular lizards were more aggressive and sexually dimorphic than their mainland relatives. insular males had wide, peramorphic heads. the growth rate of insular females was slower than growth rates of mainland individuals of both sexes, and of insular males. consequently, size and shape dimorphism are higher on the island. as predicted, melanocortin receptors were much more active in individuals of the insular population. insular lizards have a higher food intake rate than mainland individuals, which is consistent with the increased activity of melanocortin receptors. this may be adaptive in an unpredictable environment such as licosa island. insular lizards of both sexes spent less time basking than their mainland relatives. we suspect this is a by-product of the positive selection for increased activity of melanocortins receptors.  we contend that when population density is either low or fluctuating annually as a result of environmental unpredictability, it may be advantageous to individuals to behave more aggressively, to raise their rate of food intake, and allocate more energy into reproduction. 