BACKGROUND
the origin of novel traits and their subsequent diversification have been central themes in evolutionary biology ever since the discipline's inception over  <dig> years ago  <cit> .
specifically, the genetic, developmental, and ecological mechanisms, and the interactions between them, that allow novel phenotypes and functions to arise from pre-existing variation, continue to represent major frontiers in our understanding of phenotypic diversity.
with the advent of modern -omics approaches, researchers have increasingly departed from a candidate gene or pathway approach and begun to explore organismal development and evolution from a genome, transcriptome, or proteome perspective, focusing in large part on existing genetic model systems such as drosophila or caenorhabditis.
however, many key questions in evolutionary biology, including the mechanisms underlying organismal innovation, the role of plasticity in diversification, and the interplay between ecology and developmental evolution, are often difficult to address solely within the confines of classic model systems.
recent efforts have therefore begun to generate genomic and developmental genetic resources for organisms with promise as future model systems in evolutionary developmental biology and ecological genetics .
here we present and apply the first genomic resources to advance the study of a class of traits that is both novel and highly diverse in a group of organisms that is ecologically complex and experimentally tractable: beetle horns and horned beetles.
beetle horns possess many characteristics that make them interesting models for integrating genetic, developmental, and environmental perspectives on the development and evolution of complex, novel traits .
first, beetle horns are major structures, often dominating the phenotype of their bearers.
second, beetle horns function as weapons of sexual selection, thus playing a major role in the behavioral ecology of individuals and populations.
third, beetle horns are inordinately variable, both within and between species, including differences in number, size, shape, and location.
moreover, diversity in horn expression is paralleled by amazing species richness.
for instance, the genus onthophagus currently contains over  <dig>  extant species, making it the most speciose genus in the animal kingdom  <cit> .
forth, beetle horns are influenced in their expression by both genetic and environmental factors, ranging from absence of environmental sensitivity to complete determination by nutritional condition.
in some cases, both extremes of environmental sensitivity can be found in different horn types expressed by the same individual  <cit> .
finally, beetle horns lack any obvious homology to structures in other insects or non-insect arthropods.
beetle horns are not modified antennae or mouthparts, but instead horns were "invented" by beetles in addition to their traditional appendages  <cit> , and now provide their bearers with an important new function: a weapon used in male-male competition.
beetle horns and horned beetles therefore offer rich opportunities to explore the mechanisms of organismal innovation and diversification.
beetle horns are rigid outgrowths of the exoskeleton that originate as epidermal outbuddings of the head or prothoracic epithelium.
horns lack joints, muscles, and nervous tissue.
several recent studies have begun to shed light on how beetle horns develop and differentiate during ontogeny  <cit>  and showed that two developmentally dissociated processes contribute to the final degree of horn expression seen in adults: a prepupal growth phase late in larval development followed by a pupal remodeling phase just prior to the final adult molt .
as such, the development of horns shows many qualitative similarities to the development of traditional appendages, but also exhibits important differences.
for instance, prothoracic horn primordia are frequently resorbed during the pupal stage in a sex- and species-specific manner, a phenomenon not usually associated with regular appendages  <cit> .
furthermore, earlier studies have also begun to question whether horns that develop in different body regions, such as the head vs. prothorax, constitute serial homologs, or instead may have evolved and diversified independently of each other  <cit> .
here we present the first steps toward a systematic analysis of the genetic and genomic basis of horn development and diversification in the genus onthophagus.
we first present the results of a comprehensive est analysis of two normalized cdna libraries obtained from two disparate developmental stages of onthophagus taurus: larva and pupa.
second, using microarrays developed from our est libraries we contrast the transcription profiles of the primordia of developing prothoracic horns, head horns, and legs right after the transition from larva to pupa.
we then use these contrasts to identify candidate genes involved in the development and diversification of beetle horns.
furthermore, we examine two basic questions regarding the origin and diversification of horns.
are horns highly simplified versions of more traditional appendages such as legs?
if so, transcription profiles of developing horn primordia should largely match those of developing legs.
if not, transcription profiles of developing horn primordia should only partly match those of developing legs and also include horn specific transcription signatures.
are different horn types produced in different body regions homonomous, i.e.
serial homologs of the same ancestral structure?
if so, different horn types should exhibit highly similar transcription profiles.
however, if different horn types originated and diversified independently of each other, transcription profiles may be predicted to exhibit important horn-type specific differences.
we discuss the significance of our findings in the context of the biology of horned beetles in particular, and the origins and diversification of novel traits in general.
RESULTS
production and analysis of est sequences
we constructed two normalized, size selected, and directionally cloned cdna libraries from  heads and thoraces dissected from larvae and prepupae  and  whole pupae .
individual 5'- sequencing reads were generated from  <dig>  randomly selected cdna clones .
the set of est sequences were trimmed of vector sequence, adaptor sequence, and poly tails, and filtered to remove sequences that were either low quality, chimeric, or shorter than  <dig> nucleotides .
this yielded  <dig>  high-quality sequences  that are available at genbank .
we then used estpiper  <cit>  to assemble these sequences into contigs .
a total of  <dig>  of these sequences were assembled into  <dig> contigs with an average of  <dig>  clones per contig and a maximum of  <dig> clones per contig.
the remaining  <dig>  sequences did not assemble into contigs and are referred to as singletons.
thus, the  <dig>  sequences collapse into  <dig>  distinct sequences  that we refer to as "non-redundant" sequences.
it is likely that some of the non-redundant sequences derive from the same transcript but do not overlap, possibly due to 5'-truncated cdna clones.
in order to estimate the magnitude of this redundancy, we aligned the onthophagus non-redundant sequences to drosophila proteins, filtered the alignments for highly similar matches , and then manually examined the alignments for separate onthophagus sequences that align to distinct regions of the same drosophila protein.
among  <dig> non-redundant onthophagus sequences we found  <dig> pairs of sequences that aligned to the same drosophila protein.
of these,  <dig> pairs aligned with highly similar matches to different regions of the same protein, indicating they derive from non-overlapping regions of the same transcript;  <dig> pairs had co-linear alignments with 95-98% sequence identities, suggesting that they either derive from the same gene with polymorphisms and/or sequencing errors, or derive from highly similar duplicate genes; and  <dig> pairs appear to be splice variants.
thus, this sample of  <dig> onthophagus non-redundant sequences represents approximately  <dig> distinct genes .
while this is not a random sample and thus can't be extrapolated to full set of non-redundant sequences, it does however indicate that false-negative assemblies are not a pervasive problem among the non-redundant sequences.
functional annotation of assembled sequences
given that insects express a broad diversity of genes during metamorphosis  <cit> , we expected that the larval and pupal ests would be a rich source of gene discovery.
in order to provide a first pass annotation for the putative function of the onthophagus gene sequences, we annotated the non-redundant sequences using the uniprotkb/trembl protein sequence database .
this successfully annotated  <dig> % of the non-redundant sequences.
as expected, these annotations covered a wide diversity of biological and molecular functions including the major expected categories such as cellular processes, metabolic processes, biological regulation, multicellular organismal processes, and developmental processes .
this, coupled with the low redundancy within the onthophagus libraries, indicates the set of ests as a rich source for gene discovery.
given that the cdna libraries derive from animals undergoing metamorphosis, which involves dramatic remodeling of the insect body accompanied by complex patterns of gene expression, it was expected that the est libraries would include genes involved in a wide range of developmental processes.
indeed the assembled sequences included over  <dig> genes with close sequence similarity to genes with important functions in the development of other arthropods .
briefly, these included the following major groups: 1) genes involved in axis-specification, patterning and morphogenesis, including many transcription factors ; 2) proteins involved in several signaling pathways, including mapk pathways  the wnt receptor signaling pathway , the notch signaling pathway , the hedgehog signaling pathway , the tgf-beta related pathway , and the toll signaling pathway ; 3) genes involved in endocrine regulation of development including ecdysone signaling  and juvenile hormone signaling .
these clones represent a rich set of annotated genes for future studies investigating the function of the respective pathways in onthophagus development and evolution.
<dig> = contigs consisting of larval ests only;  <dig> = contigs consisting of pupal ests only; and  <dig> = contigs consisting of larval and pupal ests.
comparative analysis of the onthophagus transcriptome
while the beetle order is incredibly species rich and diverse, this study represents only the second systematic study of beetle genes  <cit> .
comparative analyses of gene content in tribolium castaneum revealed that the proportion of universal and insect specific genes is similar to that in other insects  <cit> .
however, the proportion of genes without similarity to other organisms is higher in red flour beetles than in other insects  <cit> .
in order to explore the conservation of onthophagus genes across metazoans, we aligned the  <dig>  non-redundant onthophagus sequences to the protein sequences derived from the annotated genomes of tribolium castaneum , drosophila melanogaster , caenorhabditis elegans , human , as well as non-redundant protein dataset  from genbank .
we also aligned our translated sequences with combined "invertebrate protein datasets" from ncbi .
in order to group the onthophagus sequences according to patterns of conservation and divergence across these datasets we filtered them for those with blastx sequence matches with proteins in the various datasets , and then clustered them according to the bit scores .
a total of  <dig>  non-redundant onthophagus sequences  had sequence matches to proteins in all the datasets searched .
a further  <dig> of the non-redundant sequences  had matches to proteins in the tribolium dataset, as well as to proteins in one or more of the other datasets .
within group  <dig> there are two prominent sub-groups.
first, there were  <dig> non-redundant  with matches to fly, "invertebrate", and nr proteins, but no matches to worms and humans, and are thus putatively restricted to insects.
we manually inspected the matched sequences and confirmed that there are no protein matches from non-insect species.
while gene ontology annotations of these sequences show no striking enrichment for specific biological processes, they do include proteins with functions specific to insects, such as cuticle proteins .
second, there were  <dig>  non-redundant sequences with matches to proteins in either only tribolium, or tribolium and either/both of "invertebrate" and nr proteins.
in order to test if these sequences are indeed restricted to beetles we filtered them to meet the following criteria:  the onthophagus sequences did not have a sequence match  with proteins from non-beetle species in the nr dataset;  the tribolium protein to which the onthophagus sequence had the best alignment did not have a sequence match  with proteins from non-beetle species in the nr dataset.
in total,  <dig> of the non-redundant sequences  met these criteria and thus are restricted to beetle species among the currently available protein sequence information.
this suggests that these genes may have arisen de novo, or may be fast evolving, in beetles.
a total of  <dig> translated non-redundant sequences  did not match proteins from tribolium, but did have matches in one or more of flies, worms, humans, invertebrates or nr .
among these,  <dig> sequences had matches in all other protein datasets, and  <dig> additional sequences had matches to drosophila, "invertebrate", and nr.
combined, these data raise the possibility that at least some of these  <dig> sequences may either have been lost from the tribolium lineage, or alternatively, may be conserved but simply not yet annotated in tribolium.
indeed, we found  <dig> cases where the onthophagus sequences matched against regions of the tribolium genome  with no gene annotations.
these may represent genes that are unannotated in the tribolium genome.
thus, the onthophagus expressed sequences reported here would be useful in refining the annotation of the tribolium genome.
our analysis also identified  <dig> of the non-redundant sequences  to have "no-hit"  to any of the proteomes.
this is consistent with the finding that approximately 23% of genes annotated in the tribolium genome lack sequence matches in a wide range of other species  <cit> .
however, our estimate of onthophagus specific sequences is likely to be inflated by  sequences that are largely, or entirely, within the utrs of protein coding transcripts, or  sequences that may be non-coding transcripts.
resolving the question of whether these sequences do in fact represent genes that are unique to onthophagus must await large-scale sequencing of the transcriptomes and/or genomes of onthophagus and related species.
however, the observation that 44% of theses sequences include orfs of greater than  <dig> nucleotides , suggests that at least some of these represent protein-coding genes that have not yet been identified in the species sequenced to date.
gene expression profiles in pupal appendage primordia
while our est analysis identified many genes homologous to interesting drosophila developmental genes, and such an approach to identify candidate genes has been successful in beetles  <cit> , this approach is limited to identifying obvious candidates.
given that onthophagus horns appear to be novel structures invented in beetles, it is highly likely that unexpected, or indeed previously uncharacterized genes may be important in their development.
we therefore developed a custom microarray spotted with the  <dig>  cdna clones from which the ests were derived , undertook gene expression profiling of developing horns  as an unbiased means of identifying such candidates.
since there is evidence that head horns and prothoracic horn are quite distinct structures , we analyzed gene expression in each of these organs separately.
since there is evidence that some, but not all, appendage patterning genes play a role in horn development  <cit> , we included legs in our analysis in order to distinguish similarities and differences between horns and a canonical appendage.
finally, since beetle horns and legs both develop by out-budding of the epithelium, we use non-appendage bearing epithelium  as a common reference sample.
the design of the expression profiling experiments included three comparisons, each done with five independent biological replicates .
the complete microarray data are available at geo , accession number gpl7555).
the percentage of array elements that detected signal  was uniformly high across the four tissues interrogated: an average of  <dig> % in head horns,  <dig> % in prothoracic horns,  <dig> % in legs, and  <dig> % in abdominal epithelium.
signal intensities were also reproducible across both technical replicates hybridized on the same microarray , and independent biological replicates hybridized on different microarrays .
these data indicate the microarrays were sensitive and the experiments were reproducible.
a total of  <dig>  of the  <dig>  cdna array elements detected statistically significant differential expression  in one or more of the three comparisons - head horns, prothoracic horns and legs all compared to abdomen.
in order to examine the overall similarities and differences in the patterns of these differentially expressed genes we used two-dimensional hierarchical clustering .
this revealed that expression patterns of head horns, prothoracic horns, and legs are remarkably similar .
similar results were obtained when the data were collapsed into non-redundant sequences .
83% of the array elements  detected enriched or depleted expression in all three tissues compared to abdominal epithelium.
despite the high degree of similarity, the expression patterns in head horns and prothoracic horns are still significantly closer to each other than they are to those in legs .
11% of the array elements  detected enriched or depleted expression in both head and prothoracic horns and not legs.
thus, in terms of overall patterns of gene expression, head and prothoracic pupal horn primordia are similar to but distinct from pupal leg primordia.
identifying candidate genes based on expression in horn primordia
we expected that at least some genes involved in horn development would show differential expression in pupal horn primordia.
in order to focus on these genes, we collapsed the data from array elements into non-redundant sequences , and then filtered the non-redundant sequences for those that were both statistically differentially expressed and showed at least two-fold changes in either head horns, prothoracic horn or both types of horns.
a total of  <dig> non-redundant sequences met these criteria ;  <dig> in head horns only,  <dig> in prothoracic horns only, and  <dig> in both head and prothoracic horns .
these  <dig> non-redundant sequences included  <dig> with no blastx matches  to protein sequences in uniprotkb, tribolium castaneum , drosophila melanogaster , caenorhabditis elegans , human , or non-redundant protein dataset  from genbank.
the remaining  <dig> were putatively annotated based on matches to proteins from other species .
the candidate genes for the development of beetle horns can be grouped into three conceptual categories .
first, we identified genes whose expression in the context of horn development could be expected given existing insights into the developmental biology of horns, and knowledge about the function of these genes in other organisms.
for instance, the hox gene sex combs reduced  is enriched in the prothoracic horn  and legs  relative to abdominal epithelium .
in drosophila and tribolium scr patterns the identity of the labial and first thoracic segment  <cit> .
preliminary results showed that onthophagus scr executes similar functions during labial and thoracic development in addition to playing a major role in the regulation of prothoracic horn development .
secondly, we identified genes that although functionally well characterized in drosophila or elsewhere, would not readily be expected to be expressed in the context of horn development.
genes in this category include the putative ortholog of drosophila doublesex , enriched more than 2-fold in the head and prothoracic horns relative to abdominal epithelium .
in drosophila the expression of sex-specific dsx isoforms regulate somatic sex-determination sexually dimorphic differentiation  <cit> .
while onthophagus horns are sexually dimorphic, our observation that the putative dsx ortholog is expressed preferentially in the male horn tissue when compared to male abdominal tissue was unexpected.
expression and functional studies are now under way to identify the role of dsx in the development and diversification of horns.
similarly, we found that the putative onthophagus orthologs of yellow-c, -e, and -f were enriched more than 2-fold in head and/or prothoracic horns relative to abdominal epithelium .
the functions of yellow family genes are remarkably diverse and include the regulation of pigmentation  <cit> , the production of a major component of royal jelly in the honeybee  <cit>  as well as expression of normal male courtship behavior in drosophila  <cit> .
combined, these observations suggest that yellow genes may be involved in the regulation of a wide array of sex- or caste-specific functions, at least among insects, though it remains to be determined, what, if any, function the gene family may be executing in onthophagus beetles.
lastly, we identified  <dig> genes that were significantly differentially expressed in either head horns or prothoracic horns, or both, that lack obvious homology to proteins in any of the datasets used in this study.
of those  <dig>  at least  <dig>  contained predicted orfs with longer than  <dig> nucleotides .
discussion
horned beetles, most notably in the genus onthophagus, are increasingly being recognized as an emerging model system in evo-devo and eco-devo studies  <cit> .
below we discuss the major findings of our study and their applicability to ongoing and future research efforts in horned beetles and beyond.
onthophagus taurus expressed sequences as a resource
the expressed sequences and the corresponding cdnas presented here provide a valuable entry point for studies of gene function in onthophagus taurus.
the sequences derived from normalized larval and pupal cdna libraries had a low level of redundancy.
the  <dig>  high quality est sequences from both libraries assembled into  <dig>  non-redundant sequences .
the low level of redundancy resulted in a sample of sequences derived from a wide range of biological functions.
the onthophagus transcriptome
this study provides a first pass survey of genes found in onthophagus.
prior to this study, tribolium castaneum was the only species of beetle for which comprehensive sequence information was available  <cit> .
comparative analyses indicate that the gene repertoire of tribolium is consistent with the general trends seen across sequenced insects and vertebrates  <cit> .
our estimates of the proportions of onthophagus sequences that are common to other species are consistent with those in tribolium  <cit> .
for instance, we found that 39% of onthophagus sequences had sequence matches to proteins in all the datasets searched, which is consistent with the tribolium genome in which ~35% of genes have orthologs in all species examined  <cit> .
of particular interest are the 23% of onthophagus sequences that lack orthology  to proteins from six proteomes including the non-redundant dataset which is very close to the corresponding estimate of 23% of annotated tribolium genes  <cit> .
about 40% of these group  <dig> onthophagus sequences exhibited appreciable putative open reading frames and thus need to be considered potentially protein-coding.
this group of genes likely contains genes unique to, or fast evolving in, onthophagus beetles, and studies are under way to further characterize and analyze the significance of these genes for the evolution, diversification, and radiation of horned beetles.
from ests to candidate genes for the evolutionary biology of beetle horns and horned beetles
beetle horns and horned beetles are attractive system to address several current frontiers in evolutionary biology.
the est resources and array results presented here provide the first genomic resources to identify candidate genes, pathways, and networks underlying morphological, behavioral, and developmental aspects of the biology of horned beetles, as well as providing insights into their respective evolutionary histories.
below we briefly highlight two broad categories of current research efforts and how they are being advanced by the results presented here.
the origins of horns
beetle horns have attracted attention because they lack obvious homology to other appendages or outgrowths in the insects.
horns therefore constitute an evolutionary novelty.
understanding how novel traits arise from pre-existing variation remains one of the most challenging and poorly understood questions in evolutionary biology.
one hypothesis that has been proposed toward explaining the origin of horns is based on the observation that horns share many morphological and developmental features with traditional appendages .
furthermore, in several other respects horns are much simpler than legs or mouthparts .
horns may therefore have evolved via the large-scale co-option of genes ancestrally used to instruct appendage development.
our microarray results suggest that horns and legs are indeed highly similar in gene expression profiles and support the hypothesis that many genes involved in leg formation may also play a role in horn development.
earlier research has begun to implicate a small subset of appendage patterning genes in horn development .
the results presented here add a substantial list of gene candidates  that may have mediated the origin of horns via co-option from traditional appendage development.
at the same time, horn-specific transcription profiles also included genes not represented in developing legs, suggesting that horns should not be viewed solely as being simplified appendages.
while this fraction of genes was small in comparison, it nevertheless highlights a possible class of genes involved in developmental processes of horn formation that are not represented, or at least not to the same degree, during the development of traditional appendages.
if correct, this would suggest that the origin of horns may have been mediated by the co-option of appendage patterning genes alongside integration of genes and pathways unrelated to appendage formation.
clearly, additional contrasts including the sampling of other developmental time points, as well as gene function studies, are needed to establish the general validity of these conclusions.
the diversification of beetle horns and horned beetles
beetle horns and horned beetles are attractive study organisms because they permit investigation of the mechanisms underlying phenotypic diversification on many interesting levels.
first, species differ in the body region involved in horn expression: horns may extend from the head, prothorax, or both, and while their function as weapons in male combat appears to be conserved across species, recent studies suggest that different horn types may have originated and diversified at least in part independently of one another  <cit> .
our results support this scenario by identifying a list of genes whose expression differs significantly across horn types such as yellow-e , tailup , or scr .
while the function, if any, of these candidate genes in the context of horn development remains to be explored our results presented here provide an important starting point toward untangling shared, independent, and convergent aspects in the evolution of different horn types across horned beetles.
substantial diversity in horn expression also exists within species in the form of sexual and male dimorphisms.
sexual dimorphisms are brought about via sex-specific regulation of horn expression whereas male dimorphisms are predominantly the product of nutritional differences experienced during larval life .
endocrine factors such as juvenile hormone  are likely to play important roles in the regulation of both types of diversity  <cit> .
furthermore, the same nutritional or hormonal manipulations affect sexual and male dimorphisms differently in different species and populations, suggesting that evolutionary changes in the interplay between endocrine factors, nutrition, and sexual differentiation have contributed to the diversification of horned beetles  <cit> .
our est resources and microarray results provide an important starting point to begin exploring putative candidate genes that may be associated with sex-specific  or nutrition-dependent  expression of horns.
moreover, the resources presented here should support the development of experiments towards characterizing sex- and morph-specific transcriptomes in o. taurus and closely related species in the genus .
CONCLUSIONS
in conclusion, the est resources and microarray results present here provide a first step toward a systematic analysis of the molecular basis of horn development and diversification in beetles with the potential to inform several major frontiers in evolutionary developmental biology.

 the origin of novel traits and their subsequent diversification have been central themes in evolutionary biology ever since the discipline's inception over years ago. specifically, the genetic, developmental, and ecological mechanisms, and the interactions between them, that allow novel phenotypes and functions to arise from pre-existing variation, continue to represent major frontiers in our understanding of phenotypic diversity. with the advent of modern -omics approaches, researchers have increasingly departed from a candidate gene or pathway approach and begun to explore organismal development and evolution from a genome, transcriptome, or proteome perspective, focusing in large part on existing genetic model systems such as drosophila or caenorhabditis. however, many key questions in evolutionary biology, including the mechanisms underlying organismal innovation, the role of plasticity in diversification, and the interplay between ecology and developmental evolution, are often difficult to address solely within the confines of classic model systems. recent efforts have therefore begun to generate genomic and developmental genetic resources for organisms with promise as future model systems in evolutionary developmental biology and ecological genetics. here we present and apply the first genomic resources to advance the study of a class of traits that is both novel and highly diverse in a group of organisms that is ecologically complex and experimentally tractable: beetle horns and horned beetles. beetle horns possess many characteristics that make them interesting models for integrating genetic, developmental, and environmental perspectives on the development and evolution of complex, novel traits. first, beetle horns are major structures, often dominating the phenotype of their bearers. second, beetle horns function as weapons of sexual selection, thus playing a major role in the behavioral ecology of individuals and populations. third, beetle horns are inordinately variable, both within and between species, including differences in number, size, shape, and location. moreover, diversity in horn expression is paralleled by amazing species richness. for instance, the genus onthophagus currently contains over extant species, making it the most speciose genus in the animal kingdom. forth, beetle horns are influenced in their expression by both genetic and environmental factors, ranging from absence of environmental sensitivity to complete determination by nutritional condition. in some cases, both extremes of environmental sensitivity can be found in different horn types expressed by the same individual. finally, beetle horns lack any obvious homology to structures in other insects or non-insect arthropods. beetle horns are not modified antennae or mouthparts, but instead horns were "invented" by beetles in addition to their traditional appendages, and now provide their bearers with an important new function: a weapon used in male-male competition. beetle horns and horned beetles therefore offer rich opportunities to explore the mechanisms of organismal innovation and diversification. beetle horns are rigid outgrowths of the exoskeleton that originate as epidermal outbuddings of the head or prothoracic epithelium. horns lack joints, muscles, and nervous tissue. several recent studies have begun to shed light on how beetle horns develop and differentiate during ontogeny and showed that two developmentally dissociated processes contribute to the final degree of horn expression seen in adults: a prepupal growth phase late in larval development followed by a pupal remodeling phase just prior to the final adult molt. as such, the development of horns shows many qualitative similarities to the development of traditional appendages, but also exhibits important differences. for instance, prothoracic horn primordia are frequently resorbed during the pupal stage in a sex- and species-specific manner, a phenomenon not usually associated with regular appendages. furthermore, earlier studies have also begun to question whether horns that develop in different body regions, such as the head vs. prothorax, constitute serial homologs, or instead may have evolved and diversified independently of each other. here we present the first steps toward a systematic analysis of the genetic and genomic basis of horn development and diversification in the genus onthophagus. we first present the results of a comprehensive est analysis of two normalized cdna libraries obtained from two disparate developmental stages of onthophagus taurus: larva and pupa. second, using microarrays developed from our est libraries we contrast the transcription profiles of the primordia of developing prothoracic horns, head horns, and legs right after the transition from larva to pupa. we then use these contrasts to identify candidate genes involved in the development and diversification of beetle horns. furthermore, we examine two basic questions regarding the origin and diversification of horns. are horns highly simplified versions of more traditional appendages such as legs? if so, transcription profiles of developing horn primordia should largely match those of developing legs. if not, transcription profiles of developing horn primordia should only partly match those of developing legs and also include horn specific transcription signatures. are different horn types produced in different body regions homonomous, i.e. serial homologs of the same ancestral structure? if so, different horn types should exhibit highly similar transcription profiles. however, if different horn types originated and diversified independently of each other, transcription profiles may be predicted to exhibit important horn-type specific differences. we discuss the significance of our findings in the context of the biology of horned beetles in particular, and the origins and diversification of novel traits in general.  production and analysis of est sequences we constructed two normalized, size selected, and directionally cloned cdna libraries from heads and thoraces dissected from larvae and prepupae and whole pupae. individual 5'- sequencing reads were generated from randomly selected cdna clones. the set of est sequences were trimmed of vector sequence, adaptor sequence, and poly tails, and filtered to remove sequences that were either low quality, chimeric, or shorter than nucleotides. this yielded high-quality sequences that are available at genbank. we then used estpiper to assemble these sequences into contigs. a total of of these sequences were assembled into contigs with an average of clones per contig and a maximum of clones per contig. the remaining sequences did not assemble into contigs and are referred to as singletons. thus, the sequences collapse into distinct sequences that we refer to as "non-redundant" sequences. it is likely that some of the non-redundant sequences derive from the same transcript but do not overlap, possibly due to 5'-truncated cdna clones. in order to estimate the magnitude of this redundancy, we aligned the onthophagus non-redundant sequences to drosophila proteins, filtered the alignments for highly similar matches, and then manually examined the alignments for separate onthophagus sequences that align to distinct regions of the same drosophila protein. among non-redundant onthophagus sequences we found pairs of sequences that aligned to the same drosophila protein. of these, pairs aligned with highly similar matches to different regions of the same protein, indicating they derive from non-overlapping regions of the same transcript; pairs had co-linear alignments with 95-98% sequence identities, suggesting that they either derive from the same gene with polymorphisms and/or sequencing errors, or derive from highly similar duplicate genes; and pairs appear to be splice variants. thus, this sample of onthophagus non-redundant sequences represents approximately distinct genes. while this is not a random sample and thus can't be extrapolated to full set of non-redundant sequences, it does however indicate that false-negative assemblies are not a pervasive problem among the non-redundant sequences. functional annotation of assembled sequences given that insects express a broad diversity of genes during metamorphosis, we expected that the larval and pupal ests would be a rich source of gene discovery. in order to provide a first pass annotation for the putative function of the onthophagus gene sequences, we annotated the non-redundant sequences using the uniprotkb/trembl protein sequence database. this successfully annotated % of the non-redundant sequences. as expected, these annotations covered a wide diversity of biological and molecular functions including the major expected categories such as cellular processes, metabolic processes, biological regulation, multicellular organismal processes, and developmental processes. this, coupled with the low redundancy within the onthophagus libraries, indicates the set of ests as a rich source for gene discovery. given that the cdna libraries derive from animals undergoing metamorphosis, which involves dramatic remodeling of the insect body accompanied by complex patterns of gene expression, it was expected that the est libraries would include genes involved in a wide range of developmental processes. indeed the assembled sequences included over genes with close sequence similarity to genes with important functions in the development of other arthropods. briefly, these included the following major groups: 1) genes involved in axis-specification, patterning and morphogenesis, including many transcription factors; 2) proteins involved in several signaling pathways, including mapk pathways the wnt receptor signaling pathway, the notch signaling pathway, the hedgehog signaling pathway, the tgf-beta related pathway, and the toll signaling pathway; 3) genes involved in endocrine regulation of development including ecdysone signaling and juvenile hormone signaling. these clones represent a rich set of annotated genes for future studies investigating the function of the respective pathways in onthophagus development and evolution. = contigs consisting of larval ests only; = contigs consisting of pupal ests only; and = contigs consisting of larval and pupal ests. comparative analysis of the onthophagus transcriptome while the beetle order is incredibly species rich and diverse, this study represents only the second systematic study of beetle genes. comparative analyses of gene content in tribolium castaneum revealed that the proportion of universal and insect specific genes is similar to that in other insects. however, the proportion of genes without similarity to other organisms is higher in red flour beetles than in other insects. in order to explore the conservation of onthophagus genes across metazoans, we aligned the non-redundant onthophagus sequences to the protein sequences derived from the annotated genomes of tribolium castaneum, drosophila melanogaster, caenorhabditis elegans, human, as well as non-redundant protein dataset from genbank. we also aligned our translated sequences with combined "invertebrate protein datasets" from ncbi. in order to group the onthophagus sequences according to patterns of conservation and divergence across these datasets we filtered them for those with blastx sequence matches with proteins in the various datasets, and then clustered them according to the bit scores. a total of non-redundant onthophagus sequences had sequence matches to proteins in all the datasets searched. a further of the non-redundant sequences had matches to proteins in the tribolium dataset, as well as to proteins in one or more of the other datasets. within group there are two prominent sub-groups. first, there were non-redundant with matches to fly, "invertebrate", and nr proteins, but no matches to worms and humans, and are thus putatively restricted to insects. we manually inspected the matched sequences and confirmed that there are no protein matches from non-insect species. while gene ontology annotations of these sequences show no striking enrichment for specific biological processes, they do include proteins with functions specific to insects, such as cuticle proteins. second, there were non-redundant sequences with matches to proteins in either only tribolium, or tribolium and either/both of "invertebrate" and nr proteins. in order to test if these sequences are indeed restricted to beetles we filtered them to meet the following criteria: the onthophagus sequences did not have a sequence match with proteins from non-beetle species in the nr dataset; the tribolium protein to which the onthophagus sequence had the best alignment did not have a sequence match with proteins from non-beetle species in the nr dataset. in total, of the non-redundant sequences met these criteria and thus are restricted to beetle species among the currently available protein sequence information. this suggests that these genes may have arisen de novo, or may be fast evolving, in beetles. a total of translated non-redundant sequences did not match proteins from tribolium, but did have matches in one or more of flies, worms, humans, invertebrates or nr. among these, sequences had matches in all other protein datasets, and additional sequences had matches to drosophila, "invertebrate", and nr. combined, these data raise the possibility that at least some of these sequences may either have been lost from the tribolium lineage, or alternatively, may be conserved but simply not yet annotated in tribolium. indeed, we found cases where the onthophagus sequences matched against regions of the tribolium genome with no gene annotations. these may represent genes that are unannotated in the tribolium genome. thus, the onthophagus expressed sequences reported here would be useful in refining the annotation of the tribolium genome. our analysis also identified of the non-redundant sequences to have "no-hit" to any of the proteomes. this is consistent with the finding that approximately 23% of genes annotated in the tribolium genome lack sequence matches in a wide range of other species. however, our estimate of onthophagus specific sequences is likely to be inflated by sequences that are largely, or entirely, within the utrs of protein coding transcripts, or sequences that may be non-coding transcripts. resolving the question of whether these sequences do in fact represent genes that are unique to onthophagus must await large-scale sequencing of the transcriptomes and/or genomes of onthophagus and related species. however, the observation that 44% of theses sequences include orfs of greater than nucleotides, suggests that at least some of these represent protein-coding genes that have not yet been identified in the species sequenced to date. gene expression profiles in pupal appendage primordia while our est analysis identified many genes homologous to interesting drosophila developmental genes, and such an approach to identify candidate genes has been successful in beetles, this approach is limited to identifying obvious candidates. given that onthophagus horns appear to be novel structures invented in beetles, it is highly likely that unexpected, or indeed previously uncharacterized genes may be important in their development. we therefore developed a custom microarray spotted with the cdna clones from which the ests were derived, undertook gene expression profiling of developing horns as an unbiased means of identifying such candidates. since there is evidence that head horns and prothoracic horn are quite distinct structures, we analyzed gene expression in each of these organs separately. since there is evidence that some, but not all, appendage patterning genes play a role in horn development, we included legs in our analysis in order to distinguish similarities and differences between horns and a canonical appendage. finally, since beetle horns and legs both develop by out-budding of the epithelium, we use non-appendage bearing epithelium as a common reference sample. the design of the expression profiling experiments included three comparisons, each done with five independent biological replicates. the complete microarray data are available at geo, accession number gpl7555). the percentage of array elements that detected signal was uniformly high across the four tissues interrogated: an average of % in head horns, % in prothoracic horns, % in legs, and % in abdominal epithelium. signal intensities were also reproducible across both technical replicates hybridized on the same microarray, and independent biological replicates hybridized on different microarrays. these data indicate the microarrays were sensitive and the experiments were reproducible. a total of of the cdna array elements detected statistically significant differential expression in one or more of the three comparisons - head horns, prothoracic horns and legs all compared to abdomen. in order to examine the overall similarities and differences in the patterns of these differentially expressed genes we used two-dimensional hierarchical clustering. this revealed that expression patterns of head horns, prothoracic horns, and legs are remarkably similar. similar results were obtained when the data were collapsed into non-redundant sequences. 83% of the array elements detected enriched or depleted expression in all three tissues compared to abdominal epithelium. despite the high degree of similarity, the expression patterns in head horns and prothoracic horns are still significantly closer to each other than they are to those in legs. 11% of the array elements detected enriched or depleted expression in both head and prothoracic horns and not legs. thus, in terms of overall patterns of gene expression, head and prothoracic pupal horn primordia are similar to but distinct from pupal leg primordia. identifying candidate genes based on expression in horn primordia we expected that at least some genes involved in horn development would show differential expression in pupal horn primordia. in order to focus on these genes, we collapsed the data from array elements into non-redundant sequences, and then filtered the non-redundant sequences for those that were both statistically differentially expressed and showed at least two-fold changes in either head horns, prothoracic horn or both types of horns. a total of non-redundant sequences met these criteria; in head horns only, in prothoracic horns only, and in both head and prothoracic horns. these non-redundant sequences included with no blastx matches to protein sequences in uniprotkb, tribolium castaneum, drosophila melanogaster, caenorhabditis elegans, human, or non-redundant protein dataset from genbank. the remaining were putatively annotated based on matches to proteins from other species. the candidate genes for the development of beetle horns can be grouped into three conceptual categories. first, we identified genes whose expression in the context of horn development could be expected given existing insights into the developmental biology of horns, and knowledge about the function of these genes in other organisms. for instance, the hox gene sex combs reduced is enriched in the prothoracic horn and legs relative to abdominal epithelium. in drosophila and tribolium scr patterns the identity of the labial and first thoracic segment. preliminary results showed that onthophagus scr executes similar functions during labial and thoracic development in addition to playing a major role in the regulation of prothoracic horn development. secondly, we identified genes that although functionally well characterized in drosophila or elsewhere, would not readily be expected to be expressed in the context of horn development. genes in this category include the putative ortholog of drosophila doublesex, enriched more than 2-fold in the head and prothoracic horns relative to abdominal epithelium. in drosophila the expression of sex-specific dsx isoforms regulate somatic sex-determination sexually dimorphic differentiation. while onthophagus horns are sexually dimorphic, our observation that the putative dsx ortholog is expressed preferentially in the male horn tissue when compared to male abdominal tissue was unexpected. expression and functional studies are now under way to identify the role of dsx in the development and diversification of horns. similarly, we found that the putative onthophagus orthologs of yellow-c, -e, and -f were enriched more than 2-fold in head and/or prothoracic horns relative to abdominal epithelium. the functions of yellow family genes are remarkably diverse and include the regulation of pigmentation, the production of a major component of royal jelly in the honeybee as well as expression of normal male courtship behavior in drosophila. combined, these observations suggest that yellow genes may be involved in the regulation of a wide array of sex- or caste-specific functions, at least among insects, though it remains to be determined, what, if any, function the gene family may be executing in onthophagus beetles. lastly, we identified genes that were significantly differentially expressed in either head horns or prothoracic horns, or both, that lack obvious homology to proteins in any of the datasets used in this study. of those at least contained predicted orfs with longer than nucleotides. discussion horned beetles, most notably in the genus onthophagus, are increasingly being recognized as an emerging model system in evo-devo and eco-devo studies. below we discuss the major findings of our study and their applicability to ongoing and future research efforts in horned beetles and beyond. onthophagus taurus expressed sequences as a resource the expressed sequences and the corresponding cdnas presented here provide a valuable entry point for studies of gene function in onthophagus taurus. the sequences derived from normalized larval and pupal cdna libraries had a low level of redundancy. the high quality est sequences from both libraries assembled into non-redundant sequences. the low level of redundancy resulted in a sample of sequences derived from a wide range of biological functions. the onthophagus transcriptome this study provides a first pass survey of genes found in onthophagus. prior to this study, tribolium castaneum was the only species of beetle for which comprehensive sequence information was available. comparative analyses indicate that the gene repertoire of tribolium is consistent with the general trends seen across sequenced insects and vertebrates. our estimates of the proportions of onthophagus sequences that are common to other species are consistent with those in tribolium. for instance, we found that 39% of onthophagus sequences had sequence matches to proteins in all the datasets searched, which is consistent with the tribolium genome in which ~35% of genes have orthologs in all species examined. of particular interest are the 23% of onthophagus sequences that lack orthology to proteins from six proteomes including the non-redundant dataset which is very close to the corresponding estimate of 23% of annotated tribolium genes. about 40% of these group onthophagus sequences exhibited appreciable putative open reading frames and thus need to be considered potentially protein-coding. this group of genes likely contains genes unique to, or fast evolving in, onthophagus beetles, and studies are under way to further characterize and analyze the significance of these genes for the evolution, diversification, and radiation of horned beetles. from ests to candidate genes for the evolutionary biology of beetle horns and horned beetles beetle horns and horned beetles are attractive system to address several current frontiers in evolutionary biology. the est resources and array results presented here provide the first genomic resources to identify candidate genes, pathways, and networks underlying morphological, behavioral, and developmental aspects of the biology of horned beetles, as well as providing insights into their respective evolutionary histories. below we briefly highlight two broad categories of current research efforts and how they are being advanced by the results presented here. the origins of horns beetle horns have attracted attention because they lack obvious homology to other appendages or outgrowths in the insects. horns therefore constitute an evolutionary novelty. understanding how novel traits arise from pre-existing variation remains one of the most challenging and poorly understood questions in evolutionary biology. one hypothesis that has been proposed toward explaining the origin of horns is based on the observation that horns share many morphological and developmental features with traditional appendages. furthermore, in several other respects horns are much simpler than legs or mouthparts. horns may therefore have evolved via the large-scale co-option of genes ancestrally used to instruct appendage development. our microarray results suggest that horns and legs are indeed highly similar in gene expression profiles and support the hypothesis that many genes involved in leg formation may also play a role in horn development. earlier research has begun to implicate a small subset of appendage patterning genes in horn development. the results presented here add a substantial list of gene candidates that may have mediated the origin of horns via co-option from traditional appendage development. at the same time, horn-specific transcription profiles also included genes not represented in developing legs, suggesting that horns should not be viewed solely as being simplified appendages. while this fraction of genes was small in comparison, it nevertheless highlights a possible class of genes involved in developmental processes of horn formation that are not represented, or at least not to the same degree, during the development of traditional appendages. if correct, this would suggest that the origin of horns may have been mediated by the co-option of appendage patterning genes alongside integration of genes and pathways unrelated to appendage formation. clearly, additional contrasts including the sampling of other developmental time points, as well as gene function studies, are needed to establish the general validity of these conclusions. the diversification of beetle horns and horned beetles beetle horns and horned beetles are attractive study organisms because they permit investigation of the mechanisms underlying phenotypic diversification on many interesting levels. first, species differ in the body region involved in horn expression: horns may extend from the head, prothorax, or both, and while their function as weapons in male combat appears to be conserved across species, recent studies suggest that different horn types may have originated and diversified at least in part independently of one another. our results support this scenario by identifying a list of genes whose expression differs significantly across horn types such as yellow-e, tailup, or scr. while the function, if any, of these candidate genes in the context of horn development remains to be explored our results presented here provide an important starting point toward untangling shared, independent, and convergent aspects in the evolution of different horn types across horned beetles. substantial diversity in horn expression also exists within species in the form of sexual and male dimorphisms. sexual dimorphisms are brought about via sex-specific regulation of horn expression whereas male dimorphisms are predominantly the product of nutritional differences experienced during larval life. endocrine factors such as juvenile hormone are likely to play important roles in the regulation of both types of diversity. furthermore, the same nutritional or hormonal manipulations affect sexual and male dimorphisms differently in different species and populations, suggesting that evolutionary changes in the interplay between endocrine factors, nutrition, and sexual differentiation have contributed to the diversification of horned beetles. our est resources and microarray results provide an important starting point to begin exploring putative candidate genes that may be associated with sex-specific or nutrition-dependent expression of horns. moreover, the resources presented here should support the development of experiments towards characterizing sex- and morph-specific transcriptomes in o. taurus and closely related species in the genus.  in conclusion, the est resources and microarray results present here provide a first step toward a systematic analysis of the molecular basis of horn development and diversification in beetles with the potential to inform several major frontiers in evolutionary developmental biology. 
we have laid the foundation for a systematic analysis of the genetic basis of horned beetle development and diversification with the potential to contribute significantly to several major frontiers in evolutionary developmental biology. our experiments identify a first comprehensive list of candidate genes for the evolution and diversification of beetle horns. we find that developing horns and legs show many similarities as well as important differences in their transcription profiles, suggesting that the origin of horns was mediated partly, but not entirely, by the recruitment of genes involved in the formation of more traditional appendages such as legs. furthermore, we find that horns developing from the head and prothorax differ in their transcription profiles to a degree that suggests that head and prothoracic horns are not serial homologs, but instead may have evolved independently from each other. microarrays developed from the combined libraries were then used to contrast the transcriptome of developing primordia of head horns, prothoracic horns, and legs. the origin of novel traits and their subsequent diversification represent central themes in evo-devo and evolutionary ecology. we developed two high quality, normalized cdna libraries for larval and pupal onthophagus taurus and sequenced ests that assembled into contigs and singletons. here we explore the genetic and genomic basis of a class of traits that is both novel and highly diverse, in a group of organisms that is ecologically complex and experimentally tractable: horned beetles. we present the annotation and a comparative analysis of the conservation of the sequences.
 the origin of novel traits and their subsequent diversification represent central themes in evo-devo and evolutionary ecology. here we explore the genetic and genomic basis of a class of traits that is both novel and highly diverse, in a group of organisms that is ecologically complex and experimentally tractable: horned beetles.  we developed two high quality, normalized cdna libraries for larval and pupal onthophagus taurus and sequenced ests that assembled into contigs and singletons. we present the annotation and a comparative analysis of the conservation of the sequences. microarrays developed from the combined libraries were then used to contrast the transcriptome of developing primordia of head horns, prothoracic horns, and legs. our experiments identify a first comprehensive list of candidate genes for the evolution and diversification of beetle horns. we find that developing horns and legs show many similarities as well as important differences in their transcription profiles, suggesting that the origin of horns was mediated partly, but not entirely, by the recruitment of genes involved in the formation of more traditional appendages such as legs. furthermore, we find that horns developing from the head and prothorax differ in their transcription profiles to a degree that suggests that head and prothoracic horns are not serial homologs, but instead may have evolved independently from each other.  we have laid the foundation for a systematic analysis of the genetic basis of horned beetle development and diversification with the potential to contribute significantly to several major frontiers in evolutionary developmental biology. 