these parental lines were derived from eastern jutland ( denmark ) -d48- , and melbourne ( australia ) -sh2- , respectively , selected for low ( d48 ) and high ( sh2 ) knockdown resistance to high temperature , and subsequently inbred . f1-females ( progeny of d48  sh2 ) were backcrossed to d48 males , and the backcross progeny were randomly mated for another two generations . after the last generation of random mating , individual pairs were set up , and their progeny were inbred by full - sib mating for 15 consecutive generations to form our  ril - d48  stocks . this procedure was also used to obtain ril - sh2 lines , with the only difference that f1-females ( progeny of d48  sh2 ) were backcrossed to sh2 males ( norry et al . ril from both reciprocal backcrosses ( two - way introgression ) rather than from a single backcross ( single - way introgression ) can increase the statistical power to detect qtl ( norry et al . 2008 ) . microsatellite loci were used as markers , resulting in a genetic map with markers throughout all major chromosomes ( supp fig . 1 [ online only ] ) . all stocks ( ril ) were maintained at 25  1  c in replicated vials containing a culture medium prepared with instant mashed potatoes plus water . in this study , we used 50 out of 54 currently available genotyped ril that were successfully reared ( i.e. free of bacterial growth contamination ) in all our experimental cultures ( 30 ril - d48 and 20 ril - sh2 ) . experimental individuals of 30 ril - d48 plus 20 ril - sh2 were simultaneously reared under standardized conditions to reduce the environmental variance at 25  c , with 30 12-h old larvae per standard culture vial and 5 replicate vials per line , under a photoperiod of 12:12 ( l : d ) h cycle . standard culture vials are 95  20-mm shell vials containing 6 ml of carolina culture medium ( biological supply , burlington , nc , usa ) , hereafter referred to as standard vials . twenty individuals ( four flies from each of five replicated vials ) were measured per sex and ril ( 20 flies  2 sexes  50 ril ) . measurement of more individuals did not affect the estimation of the mean value of each trait scored per ril when we examined several ril for a larger number ( 40 ) of flies measured in this study . the head was removed , placed on agar 1.5% into a small petri dish in the position to be observed from the front for measurements by one of us ( f.m.n . ) at 80 magnification . all measurements were performed with a binocular microscope fitted with an ocular micrometer ( 1  mm = 50 ocular units ) . head width ( hw ) is the distance between the left and the right side of the head capsule , and fw is the smallest distance between the eyes ( see fig . 1 ) . ew is the difference between hw and fw ( cowley and atchley 1990 , norry et al . 1 ) . additionally , we measured tl ( an index of body size ) in the parental lines d48 and sh2 , as the distance between the anterior margin of the thorax to the posterior tip of the scutellum at 50 magnification ( norry and loeschcke 2002 ) . 1.traits measured in d. melanogaster : hw , head with ; fw , face width ; el , eye length . the difference between hw and fw is ew . traits measured in d. melanogaster : hw , head with ; fw , face width ; el , eye length . differences between sets of lines were estimated for each trait using : dz = x ril - d48  x ril - sh2 , where x ril - d48 is the over - ril mean value in the ril - d48 set and x ril - sh2 is the over - ril mean value in the ril - sh2 set ( in mm ) . on average , ril - d48 flies are estimated to have 75% of d48 genes whereas ril - sh2 are estimated to have 75% of sh2 genes ( norry et al . 2008 ) , and dz should represent the difference in trait means between ril panels . these dz - values were consistent with dz - values between the parental lines , d48 and sh2 ( see supp tables 13 [ online only ] for mean values and anovas ) . body size ( as indexed by tl ) does not differ between the parental lines d48 and sh2 ( see supp tables 1 and 3 [ online only ] ) , and heat traits were thus not transformed for body size adjustments . transgressive segregation was apparent for some traits including fw and ew ( results not shown ) . significance for dz was tested by three - way anova with either ril panel or parental line and sex as fixed factors ( supp tables 2 and 3 [ online only ] ) , from which a single effect analysis was run to test for differences between ril panels for each sex . marker genotypes were the number of sh2-alleles ( 0 or 2 ) for both ril - d48 and ril - sh2 . for each trait , the mean values per ril were used in qtl analysis ( i.e. for each sex , the phenotypic value of each trait for each ril was the mean value of the 20 flies measured per ril ) . composite interval mapping was used to test the hypothesis that an interval flanked by two adjacent markers contains a qtl . this test was performed using model 6 in qtl - cartographer windows version 2.5 ( wang et al . 2010 ) , as well as the 1.3 version of this software , for ri2 design ( ril , sib mated ) , initially with five control markers and a window size of 10 cm . qtl positions that were found by using 10  cm as window size and five control markers were consistent across a wide range of parameter combinations . pairwise epistatic interactions were evaluated by using a linear model , with y = mx + my + mx my + e , where mx and my are the genotypes of markers x and y ( morgan and mackay 2006 ) . for each qtl region found in this study we subsequently explored the flybase gene ontology database ( flybase consortium 2003 ) for possible candidate genes with known or inferred functions on eye , wing , or body size phenotypes . besides , candidate loci were also genes for which information on their implication in the development of the head capsule is known ( bessa and casares 2001 , posnien et al . these parental lines were derived from eastern jutland ( denmark ) -d48- , and melbourne ( australia ) -sh2- , respectively , selected for low ( d48 ) and high ( sh2 ) knockdown resistance to high temperature , and subsequently inbred . f1-females ( progeny of d48  sh2 ) were backcrossed to d48 males , and the backcross progeny were randomly mated for another two generations . after the last generation of random mating , individual pairs were set up , and their progeny were inbred by full - sib mating for 15 consecutive generations to form our  ril - d48  stocks . this procedure was also used to obtain ril - sh2 lines , with the only difference that f1-females ( progeny of d48  sh2 ) were backcrossed to sh2 males ( norry et al . ril from both reciprocal backcrosses ( two - way introgression ) rather than from a single backcross ( single - way introgression ) can increase the statistical power to detect qtl ( norry et al . 2008 ) . microsatellite loci were used as markers , resulting in a genetic map with markers throughout all major chromosomes ( supp fig . 1 [ online only ] ) . all stocks ( ril ) were maintained at 25  1  c in replicated vials containing a culture medium prepared with instant mashed potatoes plus water . in this study , we used 50 out of 54 currently available genotyped ril that were successfully reared ( i.e. free of bacterial growth contamination ) in all our experimental cultures ( 30 ril - d48 and 20 ril - sh2 ) . experimental individuals of 30 ril - d48 plus 20 ril - sh2 were simultaneously reared under standardized conditions to reduce the environmental variance at 25  c , with 30 12-h old larvae per standard culture vial and 5 replicate vials per line , under a photoperiod of 12:12 ( l : d ) h cycle . standard culture vials are 95  20-mm shell vials containing 6 ml of carolina culture medium ( biological supply , burlington , nc , usa ) , hereafter referred to as standard vials . twenty individuals ( four flies from each of five replicated vials ) were measured per sex and ril ( 20 flies  2 sexes  50 ril ) . measurement of more individuals did not affect the estimation of the mean value of each trait scored per ril when we examined several ril for a larger number ( 40 ) of flies measured in this study . the head was removed , placed on agar 1.5% into a small petri dish in the position to be observed from the front for measurements by one of us ( f.m.n . ) at 80 magnification . all measurements were performed with a binocular microscope fitted with an ocular micrometer ( 1  mm = 50 ocular units ) . head width ( hw ) is the distance between the left and the right side of the head capsule , and fw is the smallest distance between the eyes ( see fig . 1 ) . ew is the difference between hw and fw ( cowley and atchley 1990 , norry et al . 1 ) . additionally , we measured tl ( an index of body size ) in the parental lines d48 and sh2 , as the distance between the anterior margin of the thorax to the posterior tip of the scutellum at 50 magnification ( norry and loeschcke 2002 ) . 1.traits measured in d. melanogaster : hw , head with ; fw , face width ; el , eye length . the difference between hw and fw is ew . traits measured in d. melanogaster : hw , head with ; fw , face width ; el , eye length . differences between sets of lines were estimated for each trait using : dz = x ril - d48  x ril - sh2 , where x ril - d48 is the over - ril mean value in the ril - d48 set and x ril - sh2 is the over - ril mean value in the ril - sh2 set ( in mm ) . on average , ril - d48 flies are estimated to have 75% of d48 genes whereas ril - sh2 are estimated to have 75% of sh2 genes ( norry et al . 2008 ) , and dz should represent the difference in trait means between ril panels . these dz - values were consistent with dz - values between the parental lines , d48 and sh2 ( see supp tables 13 [ online only ] for mean values and anovas ) . body size ( as indexed by tl ) does not differ between the parental lines d48 and sh2 ( see supp tables 1 and 3 [ online only ] ) , and heat traits were thus not transformed for body size adjustments . transgressive segregation was apparent for some traits including fw and ew ( results not shown ) . significance for dz was tested by three - way anova with either ril panel or parental line and sex as fixed factors ( supp tables 2 and 3 [ online only ] ) , from which a single effect analysis was run to test for differences between ril panels for each sex . marker genotypes were the number of sh2-alleles ( 0 or 2 ) for both ril - d48 and ril - sh2 . for each trait , the mean values per ril were used in qtl analysis ( i.e. for each sex , the phenotypic value of each trait for each ril was the mean value of the 20 flies measured per ril ) . composite interval mapping was used to test the hypothesis that an interval flanked by two adjacent markers contains a qtl . this test was performed using model 6 in qtl - cartographer windows version 2.5 ( wang et al . 2010 ) , as well as the 1.3 version of this software , for ri2 design ( ril , sib mated ) , initially with five control markers and a window size of 10 cm . qtl positions that were found by using 10  cm as window size and five control markers were consistent across a wide range of parameter combinations . pairwise epistatic interactions were evaluated by using a linear model , with y = mx + my + mx my + e , where mx and my are the genotypes of markers x and y ( morgan and mackay 2006 ) . for each qtl region found in this study we subsequently explored the flybase gene ontology database ( flybase consortium 2003 ) for possible candidate genes with known or inferred functions on eye , wing , or body size phenotypes . besides , candidate loci were also genes for which information on their implication in the development of the head capsule is known ( bessa and casares 2001 , posnien et al . two traits of the head capsule , fw and ew , substantially differed between ril panels as well as between parental lines ( mean values and anovas are given in supp tables 13 [ online only ] ) , as their respective dz - values were significant ( fig . 2 ) . ew showed an interesting variation in opposite direction to fw , as dz - values differed in sign between these traits ( fig . 2 ) . total hw did not differ between ril - d48 and ril - sh2 , as an increase in ew was associated to a decrease in fw ( fig . females were larger than males in all traits and there was no significant interaction between ril panel and sex ( see supp tables 1 and 2 [ online only ] ) . 2.the difference in the mean value ( dz ) between averaged ril - d48 lines ( n = 30 ) and averaged ril - sh2 lines ( n = 20 ) is shown for each trait ( in mm ) . dz - values are significant for cases where the name of the trait is indicated for each bar ( * p < 0.05 ) , as determined from two - tailed t - tests as well as from an analysis of simple effects from anova for each trait ( results not shown based on anova in supp table s2 [ online only ] ) . the difference in the mean value ( dz ) between averaged ril - d48 lines ( n = 30 ) and averaged ril - sh2 lines ( n = 20 ) is shown for each trait ( in mm ) . dz - values are significant for cases where the name of the trait is indicated for each bar ( * p < 0.05 ) , as determined from two - tailed t - tests as well as from an analysis of simple effects from anova for each trait ( results not shown based on anova in supp table s2 [ online only ] ) . abbreviations of the traits are as in table 1 . the genetic correlation between traits , as estimated from the among - ril covariance between traits , was computed by pooling both ril panels . significant correlations were negative between fw and ew in males , and positive between hw and ew ( table 1 ) . in contrast to ew , el was non - significantly correlated with other traits in this study ( table 1 ) . ew ( in mm ) is plotted against fw ( in mm ) in males from ril - d48 and ril - sh2 . data points represent the mean values of each individual ril for each trait in 30 lines ril - d48 and 20 lines ril - sh2 . table 1.across-ril correlations between traits are shown for females ( below diagonal ) and males ( above diagonal ) reared at 25  ctraithwfwewelhw0.110.800.20fw0.390.690.04ew0.700.340.16el0.430.280.17results are shown for data pooled across ril - d48 and ril - sh2 lines . abbreviations of traits are : head width ( hw ) , face width ( fw ) , eye width ( ew ) , eye length ( el ) . boldface values indicate significant correlations after correction for multiple comparisons by using a sequential bonferroni test ( p < 0.05 ) . ew ( in mm ) is plotted against fw ( in mm ) in males from ril - d48 and ril - sh2 . data points represent the mean values of each individual ril for each trait in 30 lines ril - d48 and 20 lines ril - sh2 . across - ril correlations between traits are shown for females ( below diagonal ) and males ( above diagonal ) reared at 25  c results are shown for data pooled across ril - d48 and ril - sh2 lines . abbreviations of traits are : head width ( hw ) , face width ( fw ) , eye width ( ew ) , eye length ( el ) . boldface values indicate significant correlations after correction for multiple comparisons by using a sequential bonferroni test ( p < 0.05 ) . the two negatively correlated traits , fw and ew , have diverged in opposite directions : on average , ril - d48 flies exhibited heads with more expanded eyes ( increased ew ) but shorter distance between the eyes ( reduced fw ) than ril - sh2 flies ( fig . composite interval mapping revealed a nonrandom distribution of qtl over the genome , as some of the qtl showed considerable overlapping across traits ( table 2 ; fig . qtl ranges are delimited by the following markers ( see supp fig . 1 [ online only ] ) : x2297267gt  dmu56661 ( q1 ) , dmtropini af017777 ( q2 ) , drogpdha  ac005889 ( q3 ) , ac009392droninac ( q4 ) , dmrhob  ac004658 ( q5 ) , ac005889ac004759 ( q6 ) , drosev  ac010705 ( q7 ) . one of the co - localized qtl ( q3 ) had additive effects that differed in sign between fw and ew in males ( table 2 ) , indicating antagonistic effects of the qtl on fw and ew in the same sex where the two traits were negatively correlated ( table 1 ) . in this antagonistic qtl , sh2-alleles ( from the melbourne line ) in contrast , el was controlled by one different qtl on chromosome x ( table 2 ; fig . 4likelihood ratio ( lr ) scores are plotted against map position ( in cm ) from composite interval mapping for each trait ( 1 lr  =  4.6052 lod ) . to avoid verlapping of lines of lr - scores , table 2qtl for four head capsule traits as identified by composite interval mapping in ril - d48 and ril - sh2 , in d. melanogaster reared at 25  ctraitsexril panelqtl rangea% varnameqver - qsome candidate genesewfemaleril - d483a4f20.0110q1n , ec , ilp7ewfemaleril - d4816f319f60.0121q2sw , upd1 , hs3st - b , amn , dome , merewmaleril - d4825f530a60.0216q3q4eya , wg , bchs , rca1 , csn8 , dfwmaleril - d4825f530a60.0215q3q4eya , wg , bchs , rca1 , cka , csn8 , dhwmaleril - d4823a28a30.0213q4q3eya , wg , slp1,mad , hydr2 , fred , rca1hwmaleril - d4862a63f10.028q5cct1 , bro , dlt , rho , ecd , sty , awhhwmaleril - sh230a338e90.0139q6dac , spi , pie , pelo , chico , nos , scar , crys , rab6 , nub , ifx , p38b , wb , elb , noc , su(h ) , ck , cul-3 , lace , sna , wor , cyce , gli , idgf3 , fas3 , drl , pax , nf - yb , bsh , dia , cadelfemaleril - sh210a112d0.0415q7limk1 , fw , dsh , hop , bif , amun , ade5 , set2cytological range is given for each qtl . a single name is given for qtl sharing identical range , and substantially overlapping qtl is indicated as  over - q  . additive effects ( a ) are given for traits in mm , which is the additive effect of substituting a sh allele by a d48 allele ( marker genotypes were the number of sh2-alleles , 0 or 2 , for both ril - d48 and ril - sh2 ) . abbreviations of traits are as in table 1 . likelihood ratio ( lr ) scores are plotted against map position ( in cm ) from composite interval mapping for each trait ( 1 lr  =  4.6052 lod ) . to avoid verlapping of lines of lr - scores , only cases that were significant for at least one qtl are shown . qtl for four head capsule traits as identified by composite interval mapping in ril - d48 and ril - sh2 , in d. melanogaster reared at 25  c cytological range is given for each qtl . a single name is given for qtl sharing identical range , and substantially overlapping qtl is indicated as  over - q  . additive effects ( a ) are given for traits in mm , which is the additive effect of substituting a sh allele by a d48 allele ( marker genotypes were the number of sh2-alleles , 0 or 2 , for both ril - d48 and ril - sh2 ) . epistasis was not apparent after correction for multiple comparisons for each trait , as tested by the interaction term in the linear model indicated in materials and methods ( p > 0.05 ) . in drosophila , the developmentally related traits fw and ew are negatively correlated and we found at least one qtl ( q3 ) for this antagonistic association in d. melanogaster . the traits fw and ew differed in opposite directions between ril panels ( and parental lines ) in this study , as expected if the negatively correlated traits change in opposite directions ( fig . the two parts of the head examined , fw and ew , are derived from a common precursor ( the eye - antennal discs ) , and were antagonistically affected by q3 , as indicated by the difference in sign of additive effects of the qtl ( tables 1 and 2 ) . this qtl ( q3 ) was significant in males only ( table 2 ) , the only sex exhibiting the significant and negative correlation between fw and ew in this study ( table 1 ) . this apparently pleiotropic qtl can be defined as a genomic region that influences the morphological scaling of the head . although pleiotropic qtl could contain multiple tightly linked trait - specific genes or a single gene with pleiotropic effects on the different traits , the antagonist qtl such as q3 for fw and ew can at least partially explain why these traits are negatively correlated in males ( tables 1 and 2 ) . these two traits can evolve in opposite directions in diverse drosophila species , presumably involving a genetic trade - off during development ( cowley and atchley 1990 , norry et al . fw and ew also have evolved in opposite directions in geographical populations of d. melanogaster ( e.g. compare the dz - sign between the traits in fig . it is suggested that negative correlations between fw and ew reflect subdivision of the imaginal disc cells into a population that forms the multifaceted eye and a population that forms tissue that is not destined to form eye ( norry et al . the developmentally - related traits ( fw and ew ) were influenced by some co - localized qtl in a pattern that is consistent with the sign of the genetic correlation between the traits . candidate genes in the regions of all qtl include many loci with either known or inferred functions in eye development and compound eye morphogenesis ( e.g. n , upd1 , dome , hs3st - b , mer , eya , wg , bchs , csn8 , mad ; ecd , sty , awh , pie , pelo , scar , ifx , limk1 , hop , amun , and several other of the genes listed in table 2 ) , but to find the genes that are responsible of each qtl requires future studies of fine - scale mapping ( e.g. deletion mapping in each qtl region ) . as noted earlier , the approach in this study was not to find the individual genes affecting each trait but rather to find qtls that influence the variation between strains that differ for the traits examined . other mapping populations might reveal other additional qtl , as all qtl studies depend on the differences in the putative alleles between the parental populations ( lynch and walsh 1998 ) . nevertheless , it is interesting that some qtl in this study were found to be trait specific whereas at least one qtl ( q3 ) was found to have antagonistic associations on fw and ew . eye and the adjacent face cuticle originate from the same pool of cells , involving an antagonistic subdivision of the imaginal disc cells into a population that forms the multifaceted eye and a population that forms head tissue that is not destined to form eye ( e.g. bessa and casares 2001 , posnien et al . , q3 partly explained the antagonistic association between fw and ew in males ( table 2 ; fig . q3 includes one of the candidate genes ( wg ) of two antagonistic signaling pathways which could be implicated in shaping negative genetic correlations between fw and ew , as wingless ( wg ) promotes head while decapentaplegic ( dpp ) promotes eye ( bessa and casares 2001 , neto et al . each qtl region can include 250 ( q1 ) to 570 ( q3 ) genes , and many of them are loci of still unknown functions . therefore , the present results should be interpreted not as indicating that wg and/or other genes listed in table 2 are responsible for negative correlations between fw and ew but rather as evidence that one qtl for these traits ( q3 ) is closely linked to these candidate genes . the genetic correlation between fw and ew is negative in diverse drosophila species ( cowley and atchley 1990 , norry et al . 2000 ) , indicating a pleiotropic association between these traits at the intra - specific level of variation . it is evident that the developmentally related traits , fw and ew , evolve typically in opposite directions ( norry et al . 2000 , arif et al . 2013 ) , implying some constraint on the pattern of inter - specific differentiation . the response to selection depends not only on the heritability of the traits but also on genetic correlations between traits ( lande 1979 ) , and the negative genetic correlation- or constraint- between fw and ew is therefore predicted to affect any adaptive change in these traits ( table 1 ) . nevertheless , inter - specific differentiation in fw and ew might also depend on a sub - set of qtl that are not pleiotropic on these traits ( arif et al . 2013 ) . although fw may be correlated with mating success , these traits could be influenced not only by development but also by functional effects ( e.g. norry and vilardi 1996 , arif et al . 2013 ) . the well - known case of drosophila 
 heteroneura and drosophila 
 silvestris , where fw extremely changed between species without an equivalent change in ew might suggest that constrained patterns in these traits can sometimes disappear by environmental stress ( rutherford and lindquist 1998 ) , and/or change by sexual selection ( templenton 1977 , price and wake 1995 ) . it would be interesting to also test associations between fw and ew in other diptherians as a constraint that could be not limited to drosophila . this research was supported by grants from the university of buenos aires , agencia nacional de promocin cientfica y tecnolgica , and consejo nacional de investigaciones cientficas y tcnicas to f.m.n .
in insects , some developmentally related traits are negatively correlated . here , we mapped quantitative trait loci ( qtl ) for traits of eye size and head capsule , in an intercontinental set of recombinant inbred lines ( rils ) of drosophila melanogaster . 
 composite interval mapping identified qtl on all major chromosomes . 
 two negatively correlated traits ( size of eyes and between - eyes distance ) were influenced by one qtl that appeared to be antagonistic between the traits ( qtl cytological range is 25f530a6 ) , consistent with a negative genetic correlation between these traits of the head capsule . 
 comparisons of qtl across traits indicated a nonrandom distribution over the genome , with a considerable overlap between some qtl across traits . 
 developmentally - related traits were influenced by qtl in a pattern that is consistent both with 1 ) the sign of the genetic correlation between the traits and 2 ) a constraint in the micro - evolutionary differentiation in the traits .