a custom nimblegen 12x135k microarray designed from d. serrata expressed sequence tags ( ests ) ( frentiu et al . 2009 ) was used to measure expression of 11,631 ests ( supplementary file s3 , supplementary material online ) . a panel of 43 wild - derived inbred lines of d. serrata from a single population ( st lucia , brisbane , australia ) were used for the whole - body samples ( n = 2 hybridizations per sex for each line for a total of 168 ) , and a laboratory stock from the same location was used to obtain samples of several body parts : head ( n = 4 per sex ) , thorax ( female n = 3 ; male n = 4 ) , gonadectomized abdomen ( n = 4 per sex ) , ovaries ( n = 3 ) , testes ( n = 4 ) , and accessory glands ( n = 4 ) ; all replicate hybridizations are biological replicates from independent rna extractions of different groups of flies . flies were reared in 50 ml holding vials containing standard yeast medium and maintained at 25 c with a 12-h day / night cycle . offspring were collected as virgins with the use of light co2 anesthesia and held for 3 days in same - sex groups of five flies . two replicate pools of 30 flies per line per sex and four replicate pools of 100 flies for the whole - body and tissue samples , respectively , were snap - frozen in liquid nitrogen , without the use of co2 anesthesia . rna extractions were performed by using the trizol procedure followed by rna isolation using rneasy minikits . cdna synthesis , labeling , hybridization , and microarray scanning were performed by the centre for genomics and bioinformatics , bloomington , in . 2004 ; carvalho and irizarry 2010 ; draghici 2012 ) and the experimental metrics report provided by nimblegen . for the whole - body samples , this reduced the data set from n = 168 to n = 142 hybridizations , but no hybridizations were excluded for the tissue - specific samples ( n = 34 hybridizations ) . expression measurements were normalized via a log2 transformation and summarized by taking the median of a probe set ( draghici 2012 ) where probe - level observations were the mean of the two replicates of that probe on the array . the expression data have been submitted to the ncbi gene expression omnibus ( gse45801 ) . genes were tested for sex - biased expression using welch s t - tests and applying a false discovery rate ( fdr ) of 0.05 ( benjamini and hochberg 1995 ) . 2008 ) , there is strong chromosome level conservation of orthologous genes between d. serrata and d. melanogaster ( stocker et al . stand - alone blast ( version 2.2.27 + ) was used to perform tblastx ( default settings ) between our est sequences and d. melanogaster chromosome , coding , gene , transcript , and pseudogene sequences obtained from flybase . genes associated with chromosomes 2 ( 4,323 genes , 37% ) , 3 ( 4,938 genes , 43% ) , and x ( 1,706 genes , 15% ) were used in the analysis ; these chromosomes accounted for 10,967 ( 95% ) of the genes in our microarray . a further 547 ( 4.7% ) of the genes in our array had poor - quality blast hits ( e - value > 0.1 ) and were therefore omitted from further analyses . due to intrinsic variability in microarray data , , we identified genes that are likely expressed exclusively in a single sex by using a sex - specific metric of tissue specificity : 
 ( 1 ) 
 where ei is mean expression of tissue i and emax is the maximum of the tissue - specific mean expression across all tissues ( yanai et al . 2005 ) . this metric ranges from 0 , for genes expressed at the same level in all tissues , to 1 , for genes that are highly expressed in one tissue only , and has previously been used to classify genes as sex - limited if expression was highly biased toward sex - limited tissues such as the ovaries and testes ( meiklejohn and presgraves 2012 ; meisel et al . we calculated  using measures of gene expression for nine different tissue types ( male- and female - only dissections of head , thorax , and abdomen plus ovaries , testes , and accessory gland ) . using a stringent  threshold of 0.9 , genes were classified as sex - specific if expression was highly biased for any of the sex - limited tissues or , for example , head of females only . likewise , genes were classified as co - expressed in both sexes if they fell below the  threshold . to test whether sex - biased genes were distributed nonrandomly across chromosomes , we used permutation tests where the chromosomal location of each gene was permuted 1,000 times ( meisel et al . 2012 ) . the number of sex - biased genes per chromosome was deemed to not differ from random if more than 5% of the permuted estimates were greater / less than the observed value . the same permutation approach was used to assess demasculinization and feminization of the x chromosome by testing whether the chromosomal distribution of testis- , accessory gland- , abdomen- , and ovary - specific genes was nonrandom . to test for differences in global expression between the x chromosome and autosomal genes , mann  whitney tests were performed on males and females separately ( meisel et al . 2012 ) . mann whitney tests were further performed on sex - specific and co - expressed subsets of the male and female data to determine whether observed x  autosome expression differences were specific to one or both of these gene classes . a custom nimblegen 12x135k microarray designed from d. serrata expressed sequence tags ( ests ) ( frentiu et al . 2009 ) was used to measure expression of 11,631 ests ( supplementary file s3 , supplementary material online ) . a panel of 43 wild - derived inbred lines of d. serrata from a single population ( st lucia , brisbane , australia ) were used for the whole - body samples ( n = 2 hybridizations per sex for each line for a total of 168 ) , and a laboratory stock from the same location was used to obtain samples of several body parts : head ( n = 4 per sex ) , thorax ( female n = 3 ; male n = 4 ) , gonadectomized abdomen ( n = 4 per sex ) , ovaries ( n = 3 ) , testes ( n = 4 ) , and accessory glands ( n = 4 ) ; all replicate hybridizations are biological replicates from independent rna extractions of different groups of flies . flies were reared in 50 ml holding vials containing standard yeast medium and maintained at 25 c with a 12-h day / night cycle . offspring were collected as virgins with the use of light co2 anesthesia and held for 3 days in same - sex groups of five flies . two replicate pools of 30 flies per line per sex and four replicate pools of 100 flies for the whole - body and tissue samples , respectively , were snap - frozen in liquid nitrogen , without the use of co2 anesthesia . rna extractions were performed by using the trizol procedure followed by rna isolation using rneasy minikits . cdna synthesis , labeling , hybridization , and microarray scanning were performed by the centre for genomics and bioinformatics , bloomington , in . 2004 ; carvalho and irizarry 2010 ; draghici 2012 ) and the experimental metrics report provided by nimblegen . for the whole - body samples , this reduced the data set from n = 168 to n = 142 hybridizations , but no hybridizations were excluded for the tissue - specific samples ( n = 34 hybridizations ) . expression measurements were normalized via a log2 transformation and summarized by taking the median of a probe set ( draghici 2012 ) where probe - level observations were the mean of the two replicates of that probe on the array . the expression data have been submitted to the ncbi gene expression omnibus ( gse45801 ) . genes were tested for sex - biased expression using welch s t - tests and applying a false discovery rate ( fdr ) of 0.05 ( benjamini and hochberg 1995 ) . the chromosomal location of ests was established based on homology with d. melanogaster . as observed among other drosophila species ( bhutkar et al . 2008 ) , there is strong chromosome level conservation of orthologous genes between d. serrata and d. melanogaster ( stocker et al . stand - alone blast ( version 2.2.27 + ) was used to perform tblastx ( default settings ) between our est sequences and d. melanogaster chromosome , coding , gene , transcript , and pseudogene sequences obtained from flybase . genes associated with chromosomes 2 ( 4,323 genes , 37% ) , 3 ( 4,938 genes , 43% ) , and x ( 1,706 genes , 15% ) were used in the analysis ; these chromosomes accounted for 10,967 ( 95% ) of the genes in our microarray . a further 547 ( 4.7% ) of the genes in our array had poor - quality blast hits ( e - value > 0.1 ) and were therefore omitted from further analyses . due to intrinsic variability in microarray data , it is difficult to define genes as not expressed . to overcome this issue , we identified genes that are likely expressed exclusively in a single sex by using a sex - specific metric of tissue specificity : 
 ( 1 ) 
 where ei is mean expression of tissue i and emax is the maximum of the tissue - specific mean expression across all tissues ( yanai et al . 2005 ) . this metric ranges from 0 , for genes expressed at the same level in all tissues , to 1 , for genes that are highly expressed in one tissue only , and has previously been used to classify genes as sex - limited if expression was highly biased toward sex - limited tissues such as the ovaries and testes ( meiklejohn and presgraves 2012 ; meisel et al . we calculated  using measures of gene expression for nine different tissue types ( male- and female - only dissections of head , thorax , and abdomen plus ovaries , testes , and accessory gland ) . using a stringent  threshold of 0.9 , genes were classified as sex - specific if expression was highly biased for any of the sex - limited tissues or , for example , head of females only . likewise , genes were classified as co - expressed in both sexes if they fell below the  threshold . to test whether sex - biased genes were distributed nonrandomly across chromosomes , we used permutation tests where the chromosomal location of each gene was permuted 1,000 times ( meisel et al . 2012 ) . the number of sex - biased genes per chromosome was deemed to not differ from random if more than 5% of the permuted estimates were greater / less than the observed value . the same permutation approach was used to assess demasculinization and feminization of the x chromosome by testing whether the chromosomal distribution of testis- , accessory gland- , abdomen- , and ovary - specific genes was nonrandom . to test for differences in global expression between the x chromosome and autosomal genes , mann  whitney tests were performed on males and females separately ( meisel et al . 2012 ) . mann whitney tests were further performed on sex - specific and co - expressed subsets of the male and female data to determine whether observed x  autosome expression differences were specific to one or both of these gene classes . supplementary files s1s3 and figures s1 and s2 are available at genome biology and evolution online ( http://www.gbe.oxfordjournals.org/ ) .
the chromosomal distribution of genes with sex - biased expression is often nonrandom , and in species with xy sex chromosome systems , it is common to observe a deficit of x - linked male - biased genes and an excess of x - linked female - biased genes . 
 one explanation for this pattern is that sex - specific selection has shaped the gene content of the x. alternatively , the deficit of male - biased and excess of female - biased genes could be an artifact of differences between the sexes in the global expression level of their x chromosome(s ) , perhaps brought about by a lack of dosage compensation in males and hyperexpression in females . in the montium fruit 
 fly , drosophila serrata , both these explanations can account for a deficit of male - biased and excess of female - biased x - linked genes . using genome - wide expression data from multiple male and female tissues ( n = 176 hybridizations ) 
 , we found that testis- and accessory gland - specific genes are underrepresented whereas female ovary - specific genes are overrepresented on the x chromosome , suggesting that x - linkage is disfavored for male function genes but favored for female function genes . 
 however , genes with such sex - specific functions did not fully account for the deficit of male - biased and excess of female - biased x - linked genes . 
 we did , however , observe sex differences in the global expression level of the x chromosome and autosomes . 
 surprisingly , and in contrast to other species where a lack of dosage compensation in males is responsible , we found that hyperexpression of x - linked genes in both sexes leads to this imbalance in d. serrata . 
 our results highlight how common genomic distributions of sex - biased genes , even among closely related species , may arise via quite different evolutionary processes .