BACKGROUND
sexual selection is responsible for much of the morphological diversity among animals.
this diversity is often expressed in the form of male ornamental display traits.
sexually selected characters are unusual among phenotypic traits in that they often maintain high levels of genetic variation despite being influenced by strong selection pressures  <cit> .
this rare combination of abundant intraspecific variation and rapid diversification means that these characters are particularly useful for examining the molecular basis of morphological evolution.
despite widespread study of sexually selected characters, few attempts have been made to identify variation at the molecular level associated with these ornamental traits.
because males and females share most of the same genes, differences in the timing and pattern of gene expression are likely to be the primary mechanism producing sexually dimorphic phenotypes.
sex is one of the major factors affecting variation in gene expression  <cit> .
studies on drosophila  <cit>  have estimated that between 1/ <dig> and 1/ <dig> of the genome show sex-specific expression, and that much of this variation is heritable  <cit> .
this variation is mirrored at the interspecific level where even more dramatic patterns of variation have been found.
ranz et al.
<cit>  showed that over 80% of the genes that exhibited expression differences between d. melanogaster and d. simulans have a sex-specific pattern of change.
half of these evolutionary changes involve the gain, loss or reversal of sex-biased expression and most of these differences involve genes with male-biased expression.
however, a subsequent study comparing these taxa did not find such substantial shifts in the pattern of sex-specific gene expression among species  <cit> .
the majority of drosophila microarray experiments examining sex-specific gene expression have focused on whole-body adult tissue.
attempts to separate the sex-specific expression patterns of gonads and somatic tissue  <cit>  have found substantially reduced sex-specific expression in somatic tissue relative to gonads.
few studies have examined differential gene expression with respect to a specific sexually dimorphic phenotype.
in one example, barmina et al.
<cit>  used microarray analysis to examine the pattern of gene expression in the developing first and second legs of males and females.
the first leg of d. melanogaster is sexually dimorphic with respect to bristle patterns while the second leg exhibits little dimorphism.
consistent with the phenotypic differences, they found over  <dig> genes with sex-specific expression in the first leg but no genes with sexual differences in expression in the second leg.
additional research focusing on the gene expression of specific sexually dimorphic tissue is critical to understanding the genetic factors responsible for the evolution of sex-specific phenotypes.
here, we report on the transcript profile of developing tissue for a highly exaggerated, sexually dimorphic character in the stalk-eyed fly, teleopsis dalmanni.
the elongation of the head into stalks, a condition known as hypercephaly, has evolved independently within diptera over  <dig> times  <cit> .
flies in the family diopsidae provide one of the most dramatic examples of this phenomenon.
in males of some species, the elongation is so extreme that the length of the eye-stalks exceeds the length of the body.
like drosophila, diopsids are part of a subsection of higher dipterans known as acalyptrate flies.
the acalyptrata only contain 20% of all described diptera, but include all of the hypercephalic species.
this clustered phylogenetic distribution suggests that acalyptrate flies may possess some morphological or developmental characteristic that makes them more likely to undergo head modification  <cit> .
there are nearly  <dig> described diopsid species in  <dig> genera  <cit> .
comparative analysis has revealed that extremely large, sexually dimorphic, male eyespan has evolved independently within the family several times  <cit> .
experiments examining the function of eye-stalks in the mating system of diopsids have provided considerable information about their adaptive significance.
they are critical as a signaling device in both male-male competition  <cit>  and female choice  <cit> .
in many sexually dimorphic species, males fight for and defend aggregation sites where matings occur.
both field and lab experiments have demonstrated that the size of a male's eyespan affects his ability to control these mating sites and that females tend to prefer sites controlled by males with larger eye-stalks.
among diopsids there is substantial variation both between sexes and among species in the amount of heritable genetic variation associated with eyespan  <cit> , and this heritable variation is more tightly linked to overall condition for male eyespan than for other morphological traits  <cit> .
in teleopsis dalmanni, artificial selection on male relative eyespan reveals that genes that influence male eyespan also influence female eyespan  <cit>  and exhibit x-linkage  <cit> .
male eyespan serves as an indicator of genetic quality due to an association between short eyespan and × chromosome segregation distortion  <cit> .
linkage mapping studies have identified quantitative trait loci  for eyespan on the x and on both autosomes in males  <cit> .
overall, quantitative genetic and phylogenetic analyses have demonstrated that the size and variance of eye-stalks is extremely labile, but the genetic mechanisms producing this diversity have not been investigated at the molecular level.
here we provide a catalogue of annotated transcripts present during development of this extraordinary morphological structure.
RESULTS
functional profile of est libraries
three non-normalized cdna libraries were made from the eye-antennal imaginal discs and optic lobes of t. dalmanni at three developmental stages: third instar wandering larva , early pupae  and mid pupae .
we generated  <dig>  high-quality expressed sequence tags  from these libraries with 66% of the ests coming from the larval library, 10% from the early pupal library and 24% from the mid-pupal library .
the ests are available through ncbi .
the ests were assembled into  <dig>  clusters containing a total of  <dig>  consensus sequences .
in general, multiple conseqs within a cluster resulted from the presence of non-overlapping sequence, allelic variation or alternative transcripts.
a total of  <dig>  of the clusters had two or more conseqs and  <dig> clusters had four or more conseqs.
<dig>  clusters contained at least one conseq that had a significant blast hit  to a protein in d. melanogaster and  <dig>  unique genes were represented in this list.
when blasted against the protein database for anopheles gambiae,  <dig>  clusters, comprising  <dig>  unique genes, had significant hits.
among the clusters that had a hit to d. melanogaster, but not a. gambiae, there were  <dig> unique genes, while  <dig> clusters had a hit to a. gambiae but not d. melanogaster.
an additional  <dig> clusters that did not have significant sequence similarity to d. melanogaster or a. gambiae proteins had significant hits to sequences in the non-redundant protein  and nucleotide  databases with approximately half of these hits being to transposable elements.
fourteen clusters had significant hits to microsatellites previously identified in t. dalmanni  <cit> .
† to d. melanogaster and a. gambiae protein databases
because eye-stalks are a complex structure involving modification of the brain, eye, optic nerves and head case, no single go category is likely to include all, or even most, of the genes involved in eye stalk development.
therefore, we selected from the gene ontology database a number of different biological process categories, such as cell growth and regulation of cell size and cell shape, that are likely to be important factors in eye-stalk development and calculated for each category the percentage of d. melanogaster genes within a given category for which homologues have been identified in the teleopsis est database .
for instance, about 56% of the genes known to affect cell growth in d. melanogaster have been identified in the t. dalmanni libraries.
overall, we have at least 50% of the genes in most go categories and  <dig> % of all the relevant genes across all categories.
this catalogue of genes provides a valuable framework for a comprehensive examination of gene expression in the developing eye disc.
in addition, these numbers likely underestimate the percentage of relevant genes discovered in our est database because the gene lists in drosophila include all genes that are expressed in any developmental stage or participating tissue.
only a subset of these genes is likely to be relevant during eye-antennal disc development.
developmental changes in gene expression
comparison between the est libraries from the larval, early pupal and mid-pupal developmental stages indicates some significant shifts in the type of genes expressed at each stage.
we identified several go categories that exhibited significant over-representation in one of the libraries relative to the est database as a whole .
in general, the larval stage is characterized by an over-representation of genes involved in anatomical structure formation, transcription, and cell proliferation while the pupal stage exhibits an increase in genes involved in neurogenesis and energy pathways followed by a substantial increase in cuticle production.
examination of the expression levels for individual genes also reveals substantial differences across libraries as numerous genes are expressed primarily in one developmental stage.
table  <dig> lists the genes with the largest stage-specific expression patterns as measured by the r-statistic  <cit> .
thirty additional genes  21ab , protein phosphatase 19c, ribonuclear protein at 97d , arginine methyltransferase  <dig>  cg <dig>  cg <dig>  cg <dig>  nopp <dig>  tenascin accessory, blown fuse, chd <dig>  chloride intracellular channel, eyes absent, frizzled  <dig>  glass, orf- <dig>  protein disulfide isomerase) were represented by eight or more cdnas in the database and were expressed exclusively in the larval library, but had lower r-statistic values due to the disproportionately high est sampling from this library.
in terms of gene families, all representatives of the enhancer of split complex  mα, e mβ, e m <dig>  e m <dig>  e m7) identified in the est libraries were expressed in the larval tissue.
in contrast, we identified  <dig> genes from the osiris complex  and  <dig> were expressed exclusively in the mid-pupal stage.
little is currently know about the function of the members of this gene family  <cit> , but their strong association with this pupal stage may provide insight about their role in development.
analysis of the go terms  and gene expression levels  indicate that the production of structural cuticle substantially increases during the middle of pupation.
in fact, we identified a total of  <dig> cuticular proteins  in the est database comprising over  <dig> cdnas and all but two of them were expressed exclusively in the mid-pupal library.
the distribution of transposable elements within the est database of t. dalmanni exhibited a particularly strong relationship with developmental stage.
we blasted  all the conseqs in the est libraries against the d. melanogaster transposable element  database and found  <dig> clusters with significant hits to a total of  <dig> different types of transposable elements .
these clusters are more likely to contain reads from the larval library than either of the pupal libraries.
of the  <dig> clusters with te hits,  <dig> are derived exclusively from cdnas from the larval library, while  <dig> clusters are exclusive to the early pupal library,  <dig> clusters from the mid pupal library and  <dig> clusters from more than one developmental stage.
if we look at the total number of cdnas within these clusters,  <dig> of the  <dig> te cdnas  come from the larval library whereas only 65% of the total number of cdnas was sequenced from this library.
furthermore, the mid-pupal library produced 24% of all cdnas but this stage contains only 4% of the te reads.
this distribution of te cdnas across libraries produces an r-statistic =  <dig>  and a χ <dig> =  <dig>  , indicating that the expression of tes is significantly biased toward the larval developmental stage.
developmental shifts in the pattern of te expression have been detected in drosophila  <cit>  and there are numerous host factors that can influence the spatial and temporal regulation of tes  <cit> .
most class i retrotransposable elements are dependent to some extent on the host transcriptional machinery or specific host transcription factors for their expression  <cit> .
the late larval developmental stage in t. dalmanni contains an overabundance of genes involved in transcription  that may be influencing the pattern of te expression either through the presence of specific transcription factors or a general increase in the core transcriptional machinery.
however, there is also a disproportionately high number of class ii dna transposons in the late larval library and these tes are less likely to be influenced by the dynamics of host transcription activity  <cit> .
an alternative explanation is that mechanisms involved in te suppression are more active during the pupal stages.
organisms possess a vast array of epigenetic mechanisms that suppress te activity  <cit> .
one such defense system involves the argonaute gene family.
proteins from these genes bind to small guide rnas to form te silencing complexes  <cit> .
argonaute- <dig>  which has been shown to form te silencing complexes in the somatic tissue of drosophila  <cit> , was identified in the est database  but not in sufficient quantity to determine if differences in relative distribution exist across developmental stages.
paralogous gene assessment
gene duplication is an important source of novel genetic variation that can facilitate morphological evolution  <cit> .
analysis of est databases provides a valuable tool for identifying gene duplications or gene expansions particular to the taxon of interest.
the central issue in this analysis is to differentiate paralogous gene pairs from allelic variation or alternative transcripts.
here, we have taken a conservative approach and tentatively labeled two or more clusters as paralogous genes established in the diopsid lineage if they meet two criteria:  they have greater than 10% amino acid divergence from each other and  they are both more closely related in a phylogenetic analysis to one specific drosophila gene than to any other drosophila gene.
using these criteria, we identified  <dig> pairs or sets of clusters that may have arisen from gene duplication events since the split between teleopsis and drosophila .
alternatively, the duplications may have occurred prior to the teleopsis-drosophila split with subsequent loss of one of the copies before the drosophila radiation.
the branch lengths for the trees in figure  <dig> provide some indication of the amount of divergence between the t. dalmanni clusters relative to the divergence across diptera.
in  <dig> of the genes, the divergence between the t. dalmanni clusters is greater than that between d. melanogaster and d. pseudoobscura suggesting that these are true paralogs and not different alleles of the same gene.
the lack of monophyly for a number of the cluster pairs may result from the short length of some of the est amino acid sequence.
sequencing of the entire protein coding region of these genes for t. dalmanni and a congeneric taxa will ultimately be necessary to confirm that these clusters are true paralogs and that both copies are functional across the entire length of their sequence.
examination of the gene ontology terms for these putative duplicates indicates that the set of  <dig> genes is significantly overrepresented for genes involved in spermatogenesis  and mrna binding
protein evolution
genes that play an important role in the rapid diversification of eye-stalks within diopsids may exhibit rapid rates of change at the protein level.
therefore, we used a measure of relative protein divergence specific to t. dalmanni in order to identify genes and go categories undergoing substantially faster evolution in this lineage.
this measure differs from blast identity percentage because it standardizes the amount of change in the lineage leading to t. dalmanni by the total amount of evolutionary change across other flies.
relative divergence is expressed as the percent of total tree length comprised by the branch leading to t. dalmanni.
translations of putative protein coding sequence data were obtained for  <dig>  contigs comprising  <dig>  unique genes with significant homology to a gene in d. melanogaster.
after alignment to drosophila and anopheles sequences, trimming of poorly aligned sequences and concatenation of multiple non-overlapping fragments, we were left with  <dig>  genes for analysis.
the gene set contained an average of  <dig> aligned amino acids  per gene and ranged from  <dig>  aa  to  <dig> aa .
based on a pairwise relative rate test,  <dig> genes were evolving significantly faster in t. dalmanni than in each of the three drosophila species included in the alignment.
figure  <dig> shows the branch lengths for the  <dig> most rapidly evolving genes within diopsids that had at least  <dig> aa in their alignment.
contrary to expectations, functional analysis of the relative gene divergence estimates did not indicate more rapid rates of evolutionary change for genes involved in biological processes expected to be important in eye-stalk development and evolution.
analysis based on the relative divergence rates for all  <dig>  genes indicated only a single category, transcription initiation from rna polymerase ii promoter , was evolving significantly faster than the t. dalmanni genes on average .
in contrast, several biological process categories–and some, such as neurological system process, compound eye photoreceptor fate commitment and regulation of cell growth, that are likely to be important in eye-stalk development–are evolving significantly slower than expected .
overall, when we combine all the genes that fall within the 'important eye-stalk' bp categories listed in figure  <dig>  these genes are evolving slightly slower, but not significantly different, than the rest of the genes .
the numerous slow-evolving categories that characterize the t. dalmanni lineage may result from strong stabilizing selection acting on genes in these categories in general.
if positive selection is operating on these biological processes at all it may be limited to only a few genes in each category.
for instance, the gene crooked legs  is involved in two of the slow-evolving categories–cell adhesion and tissue development–and, recently, has been shown to represent an important step in the pathway linking ecdysone signaling and cell proliferation  <cit> .
unlike other genes involved in these bp categories, crol is also undergoing extremely rapid protein evolution  and appears to have duplicated at least twice within the lineage leading to t. dalmanni .
alternatively, the gene set characterized by the list of 'important eye-stalk' bp categories may not reflect the processes that are actually important in eye-stalk evolution or rates of protein evolution may not provide a valuable indicator of their relevance to eye-stalk evolution.
it is also important to note that, because these estimates of gene divergence are derived from ests that represent only partial gene fragments, the estimated rates of evolution may be different when the entire protein coding sequence is evaluated.
furthermore, additional sampling of species within diptera is necessary to verify that apparent rate differences between t. dalmanni and drosophila are actually specific to diopsid lineages.
divergence in gene expression between selection lines
using the est sequences to construct probes for oligonucleotide microarrays, we conducted an experiment examining differences in gene expression between lines of flies selected for longer or shorter relative eyespan.
analysis of eight replicate arrays  revealed that  <dig> of  <dig>  genes exhibit differential expression based on a false discovery rate of 1%.
the d-statistics for the significant genes were either greater than  <dig>  or less than - <dig> .
of these genes,  <dig> had d-statistics exceeding  <dig> ,  <dig> exhibited more than a two-fold difference in expression between the lines, and one gene, cg <dig>  exhibited  <dig> times greater expression in flies from the most extreme high line than from the most extreme low line.
among the  <dig> significant genes,  <dig> exhibited no detectable homology to any gene in the drosophila database, and  <dig> belonged to the set of 'important eye-stalk' genes defined in figure  <dig>  fourteen of the differentially expressed genes  k <dig>  par- <dig>  ribonucleoside diphosphate reductase small subunit, small ribonucleoprotein particle protein b) are also evolving significantly faster in t. dalmanni than other flies and, of these,  <dig> genes  have duplicates in the est database.
cdc <dig>
† positive log ratios  indicate genes that are expressed at increased levels in the high line flies.
‡ genes that begin with 'orf-' or 'x-' do not have homologous sequences in d. melanogaster.
'orf-' genes have an open reading frame larger than  <dig> bp while 'x-' do not.
see methods for details on d-statistic.
functional analyses of the microarray results do not reveal a strong pattern of relationships among the set of  <dig> significant differentially expressed genes.
genemerge did not identify any go categories with significant overrepresentation when all differentially expressed genes were included in the analysis set.
when only genes that were significantly down-regulated  were analyzed, genes involved in rna splicing factor activity, transesterification mechanism  were significantly overrepresented .
no significant go categories were found among the set of up-regulated genes.
we also tested for differences in the mean d-statistic for each biological process category relative to all the genes.
this functional assay utilizes the expression values for all of the genes, not just those with significant expression difference between lines.
based on this analysis, genes that play a role in gamete generation , embryonic development , cell-cell adhesion  and neurological system process  have significantly higher mean d-statistics than an average gene set of similar size.
this result indicates that the high line flies had higher expression values for genes in these go categories than the low line flies.
it is likely that this pattern results from a shift in either the developmental timing or allometric relationship among body parts between lines.
for instance, genes involved in embryonic development are expressed relatively early in the metamorphic process compared with other genes in the est database, so if high line flies have delayed their differentiation to allow for extra imaginal disc growth relative to low line flies, an increase in the expression of embryonic development genes might occur.
consistent with this interpretation, flies from the high lines exhibit a steeper allometric relationship between eyespan and body length  <cit>  and take longer to develop  <cit>  than flies from the low lines.
similarly, the optic nerve may represent a larger proportion of the overall tissue in high line flies than in low line flies, which could result in a slight increase in the overall level of gene expression for neurological system process genes.
distinguishing between these possibilities will require additional experiments in which gene expression between replicate and control lines is compared at multiple time points during development.
quantitative rtpcr
relative expression of eight genes was estimated using quantitative reverse transcription pcr .
the correlation between the average relative expression detected by the microarray and by qrtpcr was  <dig>  .
variation was considerably greater for qrtpcr estimates because four, rather than eight, replicates were performed.
nevertheless, all four genes with greater high than low line expression showed increased high line expression by qrtpcr and the four genes with greater low than high line expression showed increased low line expression by qrtpcr.
CONCLUSIONS
stalk-eyed flies in the family diopsidae provide an excellent model system for studying sexual selection.
they possess a highly exaggerated ornamental character that plays an essential role in the mating system of numerous species and exhibits abundant intraspecific and interspecific variation.
elevated levels of heritable genetic variation is a common feature of sexually selected traits  <cit>  and has been demonstrated in the eye-stalks of diopsids  <cit> .
however, the molecular basis of this genetic variation is not well understood in any system.
developing a comprehensive and well-annotated catalogue of genes expressed in the tissue developing into ornamental morphologies is a critical step in this process.
overall, an understanding of the genetic architecture controlling eye-stalk development and evolution will provide critical insights concerning 1) the developmental mechanisms controlling allometry, 2) the genetic basis of sexual dimorphism and the convergent evolution of dimorphism and 3) the evolution of condition dependence of ornamental traits.
here, we report on the transcript profile of the eye-antennal imaginal disc of the sexually dimorphic stalk-eyed fly teleopsis dalmanni.
this est database will provide a valuable foundation for further investigations into the genetic and molecular basis of eye-stalk variation.
variation in gene expression that shapes morphological diversity is likely to be tissue and time dependent  <cit> .
by focusing our analysis of gene expression specifically within the eye-antennal discs and adjacent optic lobes at three developmental stages we are able to better isolate genes important in the development and evolution of eye-stalks.
a microarray experiment examined differences in gene expression between lines of flies that have been artificially selected for divergent eyespan.
while this experiment did not directly examine differences in gene expression between males and females, the selection lines do differ in their extent of eye-stalk sexual dimorphism.
numerous genes exhibited significantly divergent levels of expression between selection lines but no obvious causal genetic mechanism or pathway emerged from functional analysis of these genes.
the evolution of eye-stalk and sexual dimorphism in eye-stalks is undoubtedly a complicated morphological innovation involving numerous genes and pathways, so it is not surprising that a single gene expression comparison failed to pinpoint a primary genetic basis of eye-stalk variation.
additional microarray experiments are currently underway to quantify sex-biased expression in t. dalmanni and a congeneric monomorphic species, t. quinqueguttata.
despite a lack of clear signal when analyzed across pathways or higher level biological processes, analysis of gene expression across the est libraries and microarray experiment revealed several candidate genes that may play an important role in eye-stalk evolution.
some of the more noteworthy genes include crol, which has undergone at least two duplications and rapid protein evolution in diopsids, cg <dig>  which showed an 8-fold expression difference between selection lines and cdc <dig> and cg <dig> which both exhibit duplicate copies, rapid protein evolution and differential gene expression between selection line flies.
little is known about the function of cg <dig> and cg <dig> in drosophila, but both crol and cdc <dig> are important developmental genes influencing cell cycle progression  <cit> .
crol is of particular interest because it is regulated by ecdysone and mutations in the gene influence head eversion and appendage elongation  <cit> , phenotypic effects that are likely to be relevant to eye-stalk development.

 sexual selection is responsible for much of the morphological diversity among animals. this diversity is often expressed in the form of male ornamental display traits. sexually selected characters are unusual among phenotypic traits in that they often maintain high levels of genetic variation despite being influenced by strong selection pressures. this rare combination of abundant intraspecific variation and rapid diversification means that these characters are particularly useful for examining the molecular basis of morphological evolution. despite widespread study of sexually selected characters, few attempts have been made to identify variation at the molecular level associated with these ornamental traits. because males and females share most of the same genes, differences in the timing and pattern of gene expression are likely to be the primary mechanism producing sexually dimorphic phenotypes. sex is one of the major factors affecting variation in gene expression. studies on drosophila have estimated that between 1/ and 1/ of the genome show sex-specific expression, and that much of this variation is heritable. this variation is mirrored at the interspecific level where even more dramatic patterns of variation have been found. ranz et al. showed that over 80% of the genes that exhibited expression differences between d. melanogaster and d. simulans have a sex-specific pattern of change. half of these evolutionary changes involve the gain, loss or reversal of sex-biased expression and most of these differences involve genes with male-biased expression. however, a subsequent study comparing these taxa did not find such substantial shifts in the pattern of sex-specific gene expression among species. the majority of drosophila microarray experiments examining sex-specific gene expression have focused on whole-body adult tissue. attempts to separate the sex-specific expression patterns of gonads and somatic tissue have found substantially reduced sex-specific expression in somatic tissue relative to gonads. few studies have examined differential gene expression with respect to a specific sexually dimorphic phenotype. in one example, barmina et al. used microarray analysis to examine the pattern of gene expression in the developing first and second legs of males and females. the first leg of d. melanogaster is sexually dimorphic with respect to bristle patterns while the second leg exhibits little dimorphism. consistent with the phenotypic differences, they found over genes with sex-specific expression in the first leg but no genes with sexual differences in expression in the second leg. additional research focusing on the gene expression of specific sexually dimorphic tissue is critical to understanding the genetic factors responsible for the evolution of sex-specific phenotypes. here, we report on the transcript profile of developing tissue for a highly exaggerated, sexually dimorphic character in the stalk-eyed fly, teleopsis dalmanni. the elongation of the head into stalks, a condition known as hypercephaly, has evolved independently within diptera over times. flies in the family diopsidae provide one of the most dramatic examples of this phenomenon. in males of some species, the elongation is so extreme that the length of the eye-stalks exceeds the length of the body. like drosophila, diopsids are part of a subsection of higher dipterans known as acalyptrate flies. the acalyptrata only contain 20% of all described diptera, but include all of the hypercephalic species. this clustered phylogenetic distribution suggests that acalyptrate flies may possess some morphological or developmental characteristic that makes them more likely to undergo head modification. there are nearly described diopsid species in genera. comparative analysis has revealed that extremely large, sexually dimorphic, male eyespan has evolved independently within the family several times. experiments examining the function of eye-stalks in the mating system of diopsids have provided considerable information about their adaptive significance. they are critical as a signaling device in both male-male competition and female choice. in many sexually dimorphic species, males fight for and defend aggregation sites where matings occur. both field and lab experiments have demonstrated that the size of a male's eyespan affects his ability to control these mating sites and that females tend to prefer sites controlled by males with larger eye-stalks. among diopsids there is substantial variation both between sexes and among species in the amount of heritable genetic variation associated with eyespan, and this heritable variation is more tightly linked to overall condition for male eyespan than for other morphological traits. in teleopsis dalmanni, artificial selection on male relative eyespan reveals that genes that influence male eyespan also influence female eyespan and exhibit x-linkage. male eyespan serves as an indicator of genetic quality due to an association between short eyespan and × chromosome segregation distortion. linkage mapping studies have identified quantitative trait loci for eyespan on the x and on both autosomes in males. overall, quantitative genetic and phylogenetic analyses have demonstrated that the size and variance of eye-stalks is extremely labile, but the genetic mechanisms producing this diversity have not been investigated at the molecular level. here we provide a catalogue of annotated transcripts present during development of this extraordinary morphological structure.  functional profile of est libraries three non-normalized cdna libraries were made from the eye-antennal imaginal discs and optic lobes of t. dalmanni at three developmental stages: third instar wandering larva, early pupae and mid pupae. we generated high-quality expressed sequence tags from these libraries with 66% of the ests coming from the larval library, 10% from the early pupal library and 24% from the mid-pupal library. the ests are available through ncbi. the ests were assembled into clusters containing a total of consensus sequences. in general, multiple conseqs within a cluster resulted from the presence of non-overlapping sequence, allelic variation or alternative transcripts. a total of of the clusters had two or more conseqs and clusters had four or more conseqs. clusters contained at least one conseq that had a significant blast hit to a protein in d. melanogaster and unique genes were represented in this list. when blasted against the protein database for anopheles gambiae, clusters, comprising unique genes, had significant hits. among the clusters that had a hit to d. melanogaster, but not a. gambiae, there were unique genes, while clusters had a hit to a. gambiae but not d. melanogaster. an additional clusters that did not have significant sequence similarity to d. melanogaster or a. gambiae proteins had significant hits to sequences in the non-redundant protein and nucleotide databases with approximately half of these hits being to transposable elements. fourteen clusters had significant hits to microsatellites previously identified in t. dalmanni. † to d. melanogaster and a. gambiae protein databases because eye-stalks are a complex structure involving modification of the brain, eye, optic nerves and head case, no single go category is likely to include all, or even most, of the genes involved in eye stalk development. therefore, we selected from the gene ontology database a number of different biological process categories, such as cell growth and regulation of cell size and cell shape, that are likely to be important factors in eye-stalk development and calculated for each category the percentage of d. melanogaster genes within a given category for which homologues have been identified in the teleopsis est database. for instance, about 56% of the genes known to affect cell growth in d. melanogaster have been identified in the t. dalmanni libraries. overall, we have at least 50% of the genes in most go categories and % of all the relevant genes across all categories. this catalogue of genes provides a valuable framework for a comprehensive examination of gene expression in the developing eye disc. in addition, these numbers likely underestimate the percentage of relevant genes discovered in our est database because the gene lists in drosophila include all genes that are expressed in any developmental stage or participating tissue. only a subset of these genes is likely to be relevant during eye-antennal disc development. developmental changes in gene expression comparison between the est libraries from the larval, early pupal and mid-pupal developmental stages indicates some significant shifts in the type of genes expressed at each stage. we identified several go categories that exhibited significant over-representation in one of the libraries relative to the est database as a whole. in general, the larval stage is characterized by an over-representation of genes involved in anatomical structure formation, transcription, and cell proliferation while the pupal stage exhibits an increase in genes involved in neurogenesis and energy pathways followed by a substantial increase in cuticle production. examination of the expression levels for individual genes also reveals substantial differences across libraries as numerous genes are expressed primarily in one developmental stage. table lists the genes with the largest stage-specific expression patterns as measured by the r-statistic. thirty additional genes 21ab, protein phosphatase 19c, ribonuclear protein at 97d, arginine methyltransferase cg cg cg nopp tenascin accessory, blown fuse, chd chloride intracellular channel, eyes absent, frizzled glass, orf- protein disulfide isomerase) were represented by eight or more cdnas in the database and were expressed exclusively in the larval library, but had lower r-statistic values due to the disproportionately high est sampling from this library. in terms of gene families, all representatives of the enhancer of split complex mα, e mβ, e m e m e m7) identified in the est libraries were expressed in the larval tissue. in contrast, we identified genes from the osiris complex and were expressed exclusively in the mid-pupal stage. little is currently know about the function of the members of this gene family, but their strong association with this pupal stage may provide insight about their role in development. analysis of the go terms and gene expression levels indicate that the production of structural cuticle substantially increases during the middle of pupation. in fact, we identified a total of cuticular proteins in the est database comprising over cdnas and all but two of them were expressed exclusively in the mid-pupal library. the distribution of transposable elements within the est database of t. dalmanni exhibited a particularly strong relationship with developmental stage. we blasted all the conseqs in the est libraries against the d. melanogaster transposable element database and found clusters with significant hits to a total of different types of transposable elements. these clusters are more likely to contain reads from the larval library than either of the pupal libraries. of the clusters with te hits, are derived exclusively from cdnas from the larval library, while clusters are exclusive to the early pupal library, clusters from the mid pupal library and clusters from more than one developmental stage. if we look at the total number of cdnas within these clusters, of the te cdnas come from the larval library whereas only 65% of the total number of cdnas was sequenced from this library. furthermore, the mid-pupal library produced 24% of all cdnas but this stage contains only 4% of the te reads. this distribution of te cdnas across libraries produces an r-statistic = and a χ =, indicating that the expression of tes is significantly biased toward the larval developmental stage. developmental shifts in the pattern of te expression have been detected in drosophila and there are numerous host factors that can influence the spatial and temporal regulation of tes. most class i retrotransposable elements are dependent to some extent on the host transcriptional machinery or specific host transcription factors for their expression. the late larval developmental stage in t. dalmanni contains an overabundance of genes involved in transcription that may be influencing the pattern of te expression either through the presence of specific transcription factors or a general increase in the core transcriptional machinery. however, there is also a disproportionately high number of class ii dna transposons in the late larval library and these tes are less likely to be influenced by the dynamics of host transcription activity. an alternative explanation is that mechanisms involved in te suppression are more active during the pupal stages. organisms possess a vast array of epigenetic mechanisms that suppress te activity. one such defense system involves the argonaute gene family. proteins from these genes bind to small guide rnas to form te silencing complexes. argonaute- which has been shown to form te silencing complexes in the somatic tissue of drosophila, was identified in the est database but not in sufficient quantity to determine if differences in relative distribution exist across developmental stages. paralogous gene assessment gene duplication is an important source of novel genetic variation that can facilitate morphological evolution. analysis of est databases provides a valuable tool for identifying gene duplications or gene expansions particular to the taxon of interest. the central issue in this analysis is to differentiate paralogous gene pairs from allelic variation or alternative transcripts. here, we have taken a conservative approach and tentatively labeled two or more clusters as paralogous genes established in the diopsid lineage if they meet two criteria: they have greater than 10% amino acid divergence from each other and they are both more closely related in a phylogenetic analysis to one specific drosophila gene than to any other drosophila gene. using these criteria, we identified pairs or sets of clusters that may have arisen from gene duplication events since the split between teleopsis and drosophila. alternatively, the duplications may have occurred prior to the teleopsis-drosophila split with subsequent loss of one of the copies before the drosophila radiation. the branch lengths for the trees in figure provide some indication of the amount of divergence between the t. dalmanni clusters relative to the divergence across diptera. in of the genes, the divergence between the t. dalmanni clusters is greater than that between d. melanogaster and d. pseudoobscura suggesting that these are true paralogs and not different alleles of the same gene. the lack of monophyly for a number of the cluster pairs may result from the short length of some of the est amino acid sequence. sequencing of the entire protein coding region of these genes for t. dalmanni and a congeneric taxa will ultimately be necessary to confirm that these clusters are true paralogs and that both copies are functional across the entire length of their sequence. examination of the gene ontology terms for these putative duplicates indicates that the set of genes is significantly overrepresented for genes involved in spermatogenesis and mrna binding protein evolution genes that play an important role in the rapid diversification of eye-stalks within diopsids may exhibit rapid rates of change at the protein level. therefore, we used a measure of relative protein divergence specific to t. dalmanni in order to identify genes and go categories undergoing substantially faster evolution in this lineage. this measure differs from blast identity percentage because it standardizes the amount of change in the lineage leading to t. dalmanni by the total amount of evolutionary change across other flies. relative divergence is expressed as the percent of total tree length comprised by the branch leading to t. dalmanni. translations of putative protein coding sequence data were obtained for contigs comprising unique genes with significant homology to a gene in d. melanogaster. after alignment to drosophila and anopheles sequences, trimming of poorly aligned sequences and concatenation of multiple non-overlapping fragments, we were left with genes for analysis. the gene set contained an average of aligned amino acids per gene and ranged from aa to aa. based on a pairwise relative rate test, genes were evolving significantly faster in t. dalmanni than in each of the three drosophila species included in the alignment. figure shows the branch lengths for the most rapidly evolving genes within diopsids that had at least aa in their alignment. contrary to expectations, functional analysis of the relative gene divergence estimates did not indicate more rapid rates of evolutionary change for genes involved in biological processes expected to be important in eye-stalk development and evolution. analysis based on the relative divergence rates for all genes indicated only a single category, transcription initiation from rna polymerase ii promoter, was evolving significantly faster than the t. dalmanni genes on average. in contrast, several biological process categories–and some, such as neurological system process, compound eye photoreceptor fate commitment and regulation of cell growth, that are likely to be important in eye-stalk development–are evolving significantly slower than expected. overall, when we combine all the genes that fall within the'important eye-stalk' bp categories listed in figure these genes are evolving slightly slower, but not significantly different, than the rest of the genes. the numerous slow-evolving categories that characterize the t. dalmanni lineage may result from strong stabilizing selection acting on genes in these categories in general. if positive selection is operating on these biological processes at all it may be limited to only a few genes in each category. for instance, the gene crooked legs is involved in two of the slow-evolving categories–cell adhesion and tissue development–and, recently, has been shown to represent an important step in the pathway linking ecdysone signaling and cell proliferation. unlike other genes involved in these bp categories, crol is also undergoing extremely rapid protein evolution and appears to have duplicated at least twice within the lineage leading to t. dalmanni. alternatively, the gene set characterized by the list of'important eye-stalk' bp categories may not reflect the processes that are actually important in eye-stalk evolution or rates of protein evolution may not provide a valuable indicator of their relevance to eye-stalk evolution. it is also important to note that, because these estimates of gene divergence are derived from ests that represent only partial gene fragments, the estimated rates of evolution may be different when the entire protein coding sequence is evaluated. furthermore, additional sampling of species within diptera is necessary to verify that apparent rate differences between t. dalmanni and drosophila are actually specific to diopsid lineages. divergence in gene expression between selection lines using the est sequences to construct probes for oligonucleotide microarrays, we conducted an experiment examining differences in gene expression between lines of flies selected for longer or shorter relative eyespan. analysis of eight replicate arrays revealed that of genes exhibit differential expression based on a false discovery rate of 1%. the d-statistics for the significant genes were either greater than or less than -. of these genes, had d-statistics exceeding, exhibited more than a two-fold difference in expression between the lines, and one gene, cg exhibited times greater expression in flies from the most extreme high line than from the most extreme low line. among the significant genes, exhibited no detectable homology to any gene in the drosophila database, and belonged to the set of'important eye-stalk' genes defined in figure fourteen of the differentially expressed genes k par- ribonucleoside diphosphate reductase small subunit, small ribonucleoprotein particle protein b) are also evolving significantly faster in t. dalmanni than other flies and, of these, genes have duplicates in the est database. cdc † positive log ratios indicate genes that are expressed at increased levels in the high line flies. ‡ genes that begin with'orf-' or'x-' do not have homologous sequences in d. melanogaster.'orf-' genes have an open reading frame larger than bp while'x-' do not. see methods for details on d-statistic. functional analyses of the microarray results do not reveal a strong pattern of relationships among the set of significant differentially expressed genes. genemerge did not identify any go categories with significant overrepresentation when all differentially expressed genes were included in the analysis set. when only genes that were significantly down-regulated were analyzed, genes involved in rna splicing factor activity, transesterification mechanism were significantly overrepresented. no significant go categories were found among the set of up-regulated genes. we also tested for differences in the mean d-statistic for each biological process category relative to all the genes. this functional assay utilizes the expression values for all of the genes, not just those with significant expression difference between lines. based on this analysis, genes that play a role in gamete generation, embryonic development, cell-cell adhesion and neurological system process have significantly higher mean d-statistics than an average gene set of similar size. this result indicates that the high line flies had higher expression values for genes in these go categories than the low line flies. it is likely that this pattern results from a shift in either the developmental timing or allometric relationship among body parts between lines. for instance, genes involved in embryonic development are expressed relatively early in the metamorphic process compared with other genes in the est database, so if high line flies have delayed their differentiation to allow for extra imaginal disc growth relative to low line flies, an increase in the expression of embryonic development genes might occur. consistent with this interpretation, flies from the high lines exhibit a steeper allometric relationship between eyespan and body length and take longer to develop than flies from the low lines. similarly, the optic nerve may represent a larger proportion of the overall tissue in high line flies than in low line flies, which could result in a slight increase in the overall level of gene expression for neurological system process genes. distinguishing between these possibilities will require additional experiments in which gene expression between replicate and control lines is compared at multiple time points during development. quantitative rtpcr relative expression of eight genes was estimated using quantitative reverse transcription pcr. the correlation between the average relative expression detected by the microarray and by qrtpcr was. variation was considerably greater for qrtpcr estimates because four, rather than eight, replicates were performed. nevertheless, all four genes with greater high than low line expression showed increased high line expression by qrtpcr and the four genes with greater low than high line expression showed increased low line expression by qrtpcr.  stalk-eyed flies in the family diopsidae provide an excellent model system for studying sexual selection. they possess a highly exaggerated ornamental character that plays an essential role in the mating system of numerous species and exhibits abundant intraspecific and interspecific variation. elevated levels of heritable genetic variation is a common feature of sexually selected traits and has been demonstrated in the eye-stalks of diopsids. however, the molecular basis of this genetic variation is not well understood in any system. developing a comprehensive and well-annotated catalogue of genes expressed in the tissue developing into ornamental morphologies is a critical step in this process. overall, an understanding of the genetic architecture controlling eye-stalk development and evolution will provide critical insights concerning 1) the developmental mechanisms controlling allometry, 2) the genetic basis of sexual dimorphism and the convergent evolution of dimorphism and 3) the evolution of condition dependence of ornamental traits. here, we report on the transcript profile of the eye-antennal imaginal disc of the sexually dimorphic stalk-eyed fly teleopsis dalmanni. this est database will provide a valuable foundation for further investigations into the genetic and molecular basis of eye-stalk variation. variation in gene expression that shapes morphological diversity is likely to be tissue and time dependent. by focusing our analysis of gene expression specifically within the eye-antennal discs and adjacent optic lobes at three developmental stages we are able to better isolate genes important in the development and evolution of eye-stalks. a microarray experiment examined differences in gene expression between lines of flies that have been artificially selected for divergent eyespan. while this experiment did not directly examine differences in gene expression between males and females, the selection lines do differ in their extent of eye-stalk sexual dimorphism. numerous genes exhibited significantly divergent levels of expression between selection lines but no obvious causal genetic mechanism or pathway emerged from functional analysis of these genes. the evolution of eye-stalk and sexual dimorphism in eye-stalks is undoubtedly a complicated morphological innovation involving numerous genes and pathways, so it is not surprising that a single gene expression comparison failed to pinpoint a primary genetic basis of eye-stalk variation. additional microarray experiments are currently underway to quantify sex-biased expression in t. dalmanni and a congeneric monomorphic species, t. quinqueguttata. despite a lack of clear signal when analyzed across pathways or higher level biological processes, analysis of gene expression across the est libraries and microarray experiment revealed several candidate genes that may play an important role in eye-stalk evolution. some of the more noteworthy genes include crol, which has undergone at least two duplications and rapid protein evolution in diopsids, cg which showed an 8-fold expression difference between selection lines and cdc and cg which both exhibit duplicate copies, rapid protein evolution and differential gene expression between selection line flies. little is known about the function of cg and cg in drosophila, but both crol and cdc are important developmental genes influencing cell cycle progression. crol is of particular interest because it is regulated by ecdysone and mutations in the gene influence head eversion and appendage elongation, phenotypic effects that are likely to be relevant to eye-stalk development. 
many species of stalk-eyed flies possess highly-exaggerated, sexually dimorphic eye-stalks that play an important role in the mating system of these flies. the catalogue of genes identified in the est database provides a valuable framework for a comprehensive examination of the genetic basis of eye-stalk variation. based on alignments of the est fragments to homologous sequences in drosophila and anopheles, we identified putative gene duplication events in t. dalmanni and numerous genes undergoing significantly faster rates of evolution in t. dalmanni relative to the other dipteran species. eye-stalks are increasingly being used as a model system for studying sexual selection, but little is known about the genetic mechanisms producing variation in these ornamental traits. comparisons of the transcript profiles at different stages reveal a developmental shift in relative expression from genes involved in anatomical structure formation, transcription, and cell proliferation at the larval stage to genes involved in neurological processes and cuticle production during the pupal stages. therefore, we constructed an est database of genes expressed in the developing eye-antennal imaginal disc of the highly dimorphic species teleopsis dalmanni. we used this set of genes to construct microarray slides and compare patterns of gene expression between lines of flies with divergent eyespan. est assembly and annotation produced a total of clusters comprising unique genes with significant sequence similarity to a protein in either drosophila melanogaster or anopheles gambiae. several candidate genes, such as crooked legs, cdc cg and cg emerge from the analysis of gene duplication, protein evolution and microarray gene expression. microarray experiments identified over genes with significant differential expression between flies from lines selected for high and low relative eyespan but did not reveal any primary biological process or pathway that is driving the expression differences. additional comparisons of expression profiles between, for example, males and females, and species that differ in eye-stalk sexual dimorphism, are now enabled by these resources.
 many species of stalk-eyed flies possess highly-exaggerated, sexually dimorphic eye-stalks that play an important role in the mating system of these flies. eye-stalks are increasingly being used as a model system for studying sexual selection, but little is known about the genetic mechanisms producing variation in these ornamental traits. therefore, we constructed an est database of genes expressed in the developing eye-antennal imaginal disc of the highly dimorphic species teleopsis dalmanni. we used this set of genes to construct microarray slides and compare patterns of gene expression between lines of flies with divergent eyespan.  we generated high-quality ests from three non-normalized libraries made from the developing eye-stalk tissue at different developmental stages. est assembly and annotation produced a total of clusters comprising unique genes with significant sequence similarity to a protein in either drosophila melanogaster or anopheles gambiae. comparisons of the transcript profiles at different stages reveal a developmental shift in relative expression from genes involved in anatomical structure formation, transcription, and cell proliferation at the larval stage to genes involved in neurological processes and cuticle production during the pupal stages. based on alignments of the est fragments to homologous sequences in drosophila and anopheles, we identified putative gene duplication events in t. dalmanni and numerous genes undergoing significantly faster rates of evolution in t. dalmanni relative to the other dipteran species. microarray experiments identified over genes with significant differential expression between flies from lines selected for high and low relative eyespan but did not reveal any primary biological process or pathway that is driving the expression differences.  the catalogue of genes identified in the est database provides a valuable framework for a comprehensive examination of the genetic basis of eye-stalk variation. several candidate genes, such as crooked legs, cdc cg and cg emerge from the analysis of gene duplication, protein evolution and microarray gene expression. additional comparisons of expression profiles between, for example, males and females, and species that differ in eye-stalk sexual dimorphism, are now enabled by these resources. 