BACKGROUND
transferring functional annotations between species constitutes an integral part of modern biomedical research that tries to prioritize candidate genes for various diseases  <cit>  as well as to better understand phenotypic evolution  <cit> .
although genes for which similar functions are described in one species  usually represent good candidate genes in other species of interest , still such strategy leads to an inherent bias in a way that only conserved genes with identifiable orthologs are pursued by functional follow up studies.
this practice bears the danger of ignoring a large number of genes that either do not have identifiable one-to-one orthologs  or completely lack any traces of homology in other species .
as a consequence the latter class of taxonomically restricted genes that is as commonly referred to as orphan genes  <cit> , new genes  <cit>  or pioneer genes  <cit>  has long been neglected in genetic research.
thus, it is poorly understood what role orphan genes play in the biology of their host species.
however, numerous studies in vertebrates, worms and flies have shown substantial phenotypic and genomic divergence , which strongly suggests that adaptation to new biological niches is reflected by differences in gene repertoires  <cit> .
along these lines, our lab has recently shown that a developmental decision even if conserved between nematodes, can still be under the control of unrelated genes that have absolutely no homology between species  <cit> .
depending on the definition and the phylogenetic resolution, up to one third of genes in current genome sequencing projects are usually identified as orphan genes .
the number of orphan genes identified in a study largely depends on the definition of orphan gene as well as on the phylogenetic resolution of available genomic data.
thus, genomes of taxonomic groups that are vastly underrepresented in genomic data predictably reveal higher number of orphan genes.
currently, we lack a comprehensive understanding of how orphan genes evolve.
strong divergence, de novo gene formation and horizontal gene transfer are frequently discussed explanations  <cit> .
however, since most genomes are at least partially annotated by gene predictions, it has been shown that orphan genes can arise from poorly assembled genomes  <cit> .
an alternative hypothesis is that many orphan genes represent non-coding rnas and might evolve without the constraints that are typical for protein-coding sequences.
in order to investigate whether orphan genes are indeed protein-coding genes, prediction artifacts or non-coding rnas we analysed genomic, transcriptomic and proteomic data for the nematode pristionchus pacificus.
p. pacificus has been established as a satellite model organism to caenorhabditis elegans for comparative studies involving developmental biology, immunity  <cit>  and population genomics  <cit> .
the genome of p. pacificus has been sequenced using sanger technology  <cit>  and has been improved using  <dig> data  <cit> .
despite its relatively large size , in terms of contiguity it is either superior or at least comparable  to many other recently published nematode genomes  <cit> .
more importantly, it has proven of great use for several studies to identify and clone causative genes for various different phenotypes  <cit> .
various gene finding programs predicted  <dig> - <dig>  genes that have been evaluated several times using transcriptomic and comparative genomic data indicating that current gene annotations are largely complete  <cit> .
in this study, we try to clarify the nature of orphan genes in p. pacificus.
our basic strategy is to first check whether they are expressed and we consider expression as positive evidence that a gene prediction is not an artifact.
in addition, strong support towards the protein coding nature of a predicted gene can be gathered if it either has a match in available proteomic data or shows evidence of negative selection that manifests in depletion of nonsynomous vs. synonymous substitutions.
gene predictions that are expressed but do not fulfill any of the two criteria are candidates for non-coding rnas.
based on the two criteria, our results show that between 40– <dig> % of orphan genes are actually truly protein-coding, indicating that orphan genes likely represent a substantial factor in the biology of p. pacificus.
application of our methodology on genomic data from other taxonomically under-sampled groups will further support the validity of orphan genes and their biological significance.
RESULTS
the majority of orphan genes are transcribed
we used previously defined sets of  <dig> orphan genes and  <dig>  conserved genes  <cit>  and  <dig> previously published rna-seq experiments in p. pacificus to assess up to what extent both gene sets are transcribed and to examine the dependence between the amount of expressed genes and the number of included rna-seq samples .
thereby, we defined two different thresholds on the magnitude of expression, which was measured as fragments per kilobase transcript per million fragments  sequenced.
while a value of fpkm > = <dig> defines robust expression, a value of fpkm > = <dig> can still be interpreted as reliable evidence of expression as many functionally characterized genes  <cit>  display fpkm values well below  <dig> fig.
<dig> transcription and differential expression of orphan genes.
a rna sequencing data from  <dig> experiments are used to determine the number of expressed orphan and conserved genes as a function of the number of rna-seq samples  and different expression thresholds .
the boxplot shows the variation in the number of expressed genes from ten random permutations of the order of rna-seq samples b) saturation analysis for the number of differentially expression pattern of both orphan and conserved genes from six transcriptome profiling studies  <cit>
even though conserved genes show in general higher levels of expressed  and robustly expressed genes , still in our analysis 4129– <dig>  of orphan genes are transcribed in at least one of the samples .
orphan genes are embedded in gene-regulatory networks
in addition to using transcription as a necessary criterion for a real gene, we also quantified the amount of conserved and orphan genes that are differentially expressed under variable conditions like different developmental stages or exposure to pathogens.
the rational behind this analysis is based on the assumption that a gene that can be differentially expressed under various conditions has likely been integrated into a gene-regulatory network.
therefore such a gene is a more suitable candidate for a real gene rather than a gene , which is constitutively expressed at very low levels.
this would also suggest that older genes would more likely be integrated into the gene network and they will have stronger differential expression pattern compared to new genes.
consistent with this assumption, we have previously shown that orphan genes are significantly depleted among genes that are developmentally regulated and conserved genes are significantly enriched  <cit> .
however, analysis of previously conducted transcriptome profiling studies   <cit>  shows that not only  <dig>  conserved genes but even  <dig>  orphan genes are differentially expressed in p. pacificus.
this indicates that a subset of orphan genes is differentially expressed under various conditions and implies that such an orphan gene must have persisted a sufficient amount of time in a genome for it to be integrated into a regulatory circuit.
a subset of orphan genes shows direct evidence for translation
next we tested to what extent conserved and orphan genes showed direct evidence of translation by testing for matches in available proteomic data  <cit> .
using  <dig> % sequence identity over the full peptide length as criteria to search through the peptide data  we found peptide evidence for  <dig>  orphan genes.
this number is quite low when compared with the  <dig>  conserved genes that have peptide evidence, but it is consistent with previous study that reported  <cit>  the under-representation of orphan genes in transcriptome and peptide data.
however, we also observe that even three quarters of conserved genes do not have peptide evidence, thus indicating that the lack of peptide evidence is not a sufficient criterion to distinguish truly protein-coding genes from potential artifacts or non-coding rnas.
for this reason we employ other genomic resources to measure indirect evidence for protein-coding genes, i.e.
selection against non-synonymous substitutions.
comparative genomics of orphan genes
as most orphan genes do not display matches in peptide data, we use negative selection in protein-coding genes as an indirect evidence for translation.
however, to estimate evolutionary constraint  at least two sequences are needed which is problematic for orphan genes since they do not have homologs in other species.
this impediment can be circumvented by comparison with a species that is within the taxonomic group of the focal organism.
in our case, we can make use of the recently sequenced sister species p. exspectatus.
the genome of p. exspectatus shows roughly  <dig> % sequence divergence and was previously used to polarize intra-species comparisons within p. pacificus  <cit> .
to this end, all predicted genes from both species p. pacificus and p. exspectatus were segregated into  <dig>  different orthologous clusters based on protein sequence homology using orthomcl  <cit> .
figure 2a shows the distribution of all the clusters into six different categories based on the number of the p. pacificus and p. exspectatus genes present in the clusters.
evidently, the majority of clusters have only two members one each from p. pacificus and p. exspectatus genomes.
after filtering out p. exspectatus specific clusters, hybrid clusters and short peptide containing clusters or poorly aligning clusters , we separated  <dig>  clusters containing one or more p. pacificus conserved genes and  <dig> clusters containing one or more p. pacificus orphan genes for further investigation.
these two sets represent  <dig> orphan and  <dig>  conserved genes from p. pacificus.
conserved and orphan gene clusters were further subdivided into two different datasets; the first dataset called ‘orthologous clusters’ had the clusters that contain at least one p. pacificus and one p. exspectatus gene, the second dataset called ‘paralogous clusters’ consisted of all the clusters containing more than one p. pacificus genes minus all the p. exspectatus genes from the clusters.
we decided to distinguish the two types of clusters in order to assess to what extent we gain evidence for negative selection with the help of a second species.
the combination of both the datasets represent all p. pacificus genes except the singletons genes that are present only in a single copy in p. pacificus and do not have a corresponding homolog in the p. exspectatus.
in order to accommodate the p. pacificus singletons and study selection at a closer time scale we created a third dataset called ‘clade a1-a <dig> orthologs’  where we compared divergence of orthologous gene pairs  across two geographically isolated p.pacificus lineages  <cit> .fig.
<dig> orphan genes are under strong negative selection.
a distribution of gene clusters based on the number of orthologs between p. pacificus and p. exspectatus.
b the graph shows the cumulative proportion of orphan and conserved gene clusters with evidence for negative selection relative to different ω thresholds in the p. pacificus – p. exspectatus orthologous clusters dataset.
using an abitrary cutoff of ω <  <dig> ,  <dig> % of all orphan clusters  show evidence of negative selection.
c comparison of the proportion of negatively selected orphan gene clusters and conserved gene clusters  based on the analysis of paralogous gene clusters.
given, that only  <dig> % of conserved clusters and  <dig> % of all orphan clusters contain p. pacificus paralogs, this analysis shows that even in the absence of other genomic data, negative selection can be investigated in a subset of orphan genes.
d cumulative proportion of orphan and conserved gene clusters under given ω value in clade a <dig> – clade a <dig> ortholog dataset
orphan and conserved genes are under strong negative selection
selection pressure was measured as the ratio of synonymous and non-synonymous rate of amino acid substitution  also called omega  for each cluster of the three above-mentioned datasets using the paml suit  <cit> .
an ω =  <dig> would indicate neutral evolution and ω <  <dig> can be interpreted as evidence for negative selection.
the measurement of ω on orthologous clusters showed that conserved gene clusters are under stronger negative selection than the orphan gene clusters .
nonetheless, the majority of orphan gene clusters are also under relatively strong negative selection.
the result of similar analysis on paralogous conserved and orphan clusters were also comparable, keeping in mind that only  <dig> % of conserved clusters and  <dig> % of orphan clusters are present in this dataset .
the selection pressure results for clade a1-a <dig> dataset also follows the trend of showing stronger negative selection on conserved genes when compared with orphan genes; nevertheless, the orphan genes also appear to be under robust negative selection .
ortholog, paralog and intra-species comparison are highly complementary
similar to the analysis of the expression data, we define a range between a conservative and a more liberal threshold to estimate the amount of genes under negative selection.
first, we arbitrarily chose ω <  <dig>  as a liberal cutoff to call a cluster as evolving under negative selection.
in addition, we defined a more conservative cutoff by calculating the statistical significance of a negative-selection model compared with a neutral model.
for each cluster the codeml package of paml was run twice, once allowing a single ω value for the entire cluster tree but free to change  and the second time ω fixed at  <dig> for the entire cluster tree .
likelihood ratio test was conducted with one degree of freedom and at false discovery rate  adjusted p-value <  <dig>  for each cluster to determine the significance of the alternate model.
a combination of ω <  <dig> and fdr adjusted p-value <  <dig>  was used as second more conservative criterion for calling a cluster as evolving under negative selection.
please note that the lack of significance does not imply the lack of evolutionary constraint and in some cases might just be indicative of low statistical power due to the comparison of small proteins or little divergence between the sequences.
in order to investigate the overlap between the different datasets, we decided to compare orphan candidates found to be under negative selection in all the three datasets.
taking the cutoff of ω value lower than  <dig>  in any one of the three datasets  identified  <dig>  of orphan genes as being under negative selection.
figure 3a shows that the largest contribution towards identified negative selection  comes from the intra-species comparison.
however, since the evolutionary distances in this comparison are rather small, random fluctuations in the number of non-synonymous and synonymous sites can easily generate ω values below  <dig> .
given that the signal for positive selection is unlikely to affect complete genes, ω >  <dig> are most likely due to such random fluctuations; based on this we estimate that around one third of the identified orphan genes with ω <  <dig>  can be due to random noise.
therefore, we would like to emphasize that the cutoff of ω <  <dig>  has to be regarded as upper limit of the estimated number of negatively selected orphan genes.fig.
<dig> ortholog, paralog and population genomic data are highly complementary.
a venn diagram for three different data sets using a definition of ω <  <dig> .
as most genes with evidence of negative selection are unique to one specific data set, this indicates that all three data sets are highly complementary.
b venn diagram for three different data sets using a definition of ω <  <dig> and p <  <dig> .
again, most genes with evidence of negative selection are unique to one specific data set.
c overlap between different thresholds to define negative selection and expressed genes.
d identification of a candidate gene set of orphan genes that lack any expression data, are under significant negative selection and also have ω value less than  <dig>  in at least two of the three datasets
using the stringent likelihood ratio test we identified  <dig>  orphan genes with significant evidence for negative selection in at least one of the three data sets .
more precisely, out of  <dig> orthologous orphan clusters  <dig> clusters  show significantly better goodness of fit for negative selection  compared with the neutral evolution model .
the corresponding figure for the conserved clusters is  <dig> out of  <dig>  .
among paralogous clusters,  <dig> out of  <dig>  orphan clusters and  <dig> out of  <dig>  conserved clusters showed significant goodness of fit for negative selection.
in clade a1-a <dig> dataset, only  <dig> out of  <dig> orphan gene clusters and  <dig> out of  <dig>  conserved gene clusters had significant goodness of fit supporting negative selection, indicating that the divergence between two p. pacificus lineages in general is not enough to identify robust evidence for negative selection at a single gene level.
in order to further evaluate the use of ω <  <dig>  as less conservative cutoff, we compared the identified gene sets from both methods with available expression data .
this demonstrates that a cutoff of ω <  <dig>  on one side captures almost all examples of significant negative selection and it minimizes the fraction of genes that are neither expressed nor show significant likelihood results.
we have observed that changing this cutoff value to  <dig>  or  <dig>  drastically impairs this balance.
in summary, our analysis suggests that a large proportion  of orphan genes are under strong negative selection.
evolutionary constraint predicts expression
our previous analysis has indicated that a considerable number of orphan genes show evidence for negative selection.
we combined our expression data with the selection analysis to screen for orphan genes that show negative selection but lack expression evidence .
more precisely, the list of  <dig> orphan genes showing significant negative selection were intersected with two other lists: first the list of  <dig> orphan genes without expression data and second the list of genes that had ω value less than  <dig>  in at least  <dig> datasets .
from the  <dig> genes found to be present in all three lists eleven genes that had more than  <dig> exons in their predicted open reading frame were selected as candidates for validation by pcr.
pcr products from three candidate genes were found in the expected nucleotide range when pcr was done against cdna but not against genomic dna .
sequencing of pcr products resulted in expressed sequence tags that exactly matched the gene prediction .
these results demonstrate, that even in the absence of expression evidence in  <dig> rna-seq samples, pcr was able to detect expression of orphan genes in standard mixed-stage worm cultures.
as expression profiles can be highly stage and tissue-specific, we speculate that further optimization of protocols and more specific rna samples could validate predicted gene structures in many more cases.fig.
<dig> validation of orphan genes.
a pcr validation experiments for eleven candidate orphan genes.
genomic dna  and cdna  was amplified using the same primer pairs.
in three cases, we obtained bands in the expected size range.
b sequencing of amplification products resulted in ests that exactly confirmed the gene structure
differences between various gene classes
based on the obtained expression data and estimates for negative selection, we divided all p. pacificus genes into four classes.
first class is made of  <dig> potential prediction artifacts or pseudogenes , second class contains  <dig> candidates for non-coding rnas , conserved genes and orphans that showed either significant negative selection or peptide evidence constitute the third and fourth classes.
four gene features  were compared across all four gene classes .
while conserved and negatively selected orphans tend to be longer and contain more exons than potential prediction artifacts/pseudogenes and non-coding rna candidates , at the level of gc content and contig size percentile no obvious differences are detectable .
given that fragmented assemblies were identified as potential source of prediction artifacts, it is interesting to see that in all four gene classes around  <dig> % of genes reside in the top  <dig> % of largest contigs.
there are no significant trends towards accumulation of potential artifacts on smaller contigs.
thus, partial gene model as a result of smaller contigs are an unlikely source for the majority of prediction artifact or pseudogene candidates.
however, further analysis is needed to characterize these gene sets in greater detail.fig.
<dig> differences between various gene classes.
a comparison of transcript length for potential prediction artifacts/pseudogenes, non-coding rna candidates, negatively selected orphan genes, and conserved genes.
the y-axis denotes the fraction of genes at a given transcript length.
b comparison of the number of exons for different gene classes.
c gc content distribution for all four gene classes.
d distribution of contig size percentiles among all four classes.
the top  <dig> % of largest contigs harbors roughly  <dig> % of genes for all four gene classes
discussion
the drastic reduction in sequencing cost has facilitated whole genome sequencing of several non-model species.
however, current genome sequencing projects constantly identify large fraction of genes without homology in related species.
the amount of identified orphan genes depends on the definition as well as on the phylogenetic resolution of available genomic data.
as a consequence, non-model or non-classical model organisms such as p. pacificus, which has been estimated to share a common ancestor with c. elegans 280– <dig> mya  <cit> , exhibit a much higher number of orphan genes in comparison to vertebrate genomes such as humans and mouse.
orphan genes can arise from technical  but also biological reasons .
in addition, it has been shown that orphan genes can arise from genome assembly and gene prediction problems  <cit> .
as gene prediction is still an integral part of current gene annotation pipelines  <cit> , we need to first clarify whether orphan genes are truly protein-coding genes before we can study their biology and evolutionary history.
in this study, we use expression data to first test whether orphan genes are real biological entities or non-functional pseudogenes or gene prediction artifacts.
first, we showed that up to  <dig> % of orphan genes are indeed transcribed.
second, using available proteomic and genomic data, we found that 40– <dig> % of orphan genes are actually truly protein-coding as they either show direct support in proteomic data or evolve under constraints that are expected for protein-coding genes.
although problems in gene annotation  still persist that might eventually be overcome by improvements in genome assembly and annotation, our study indicates that this will not eliminate the presence of orphan genes.
as the most prominent conclusion of our study, that orphan genes are protein-coding genes under negative selection is based on phylogenetic analysis, we would like to point out that most technical artifacts such as mispredictions, pseudogenes or incorrect alignment would drive the signal towards neutral selection.
this suggests that further correcting these types of errors will increase the amount of detected evidence for negative selection.
furthermore, the finding of evolutionary constraint acting on orphan genes is consistent with other studies from insects, fish and primates .
along with the accumulating functional evidence suggesting that taxonomically restricted genes can influence various phenotypic traits  <cit> , recognizing orphan genes as true biological entities will further emphasize their importance and thus lead to a more holistic perspective in modern biomedical research.
given that most orphan genes are protein-coding, the next questions we need to ask are: how do they evolve, how old are they, and what distinguishes conserved and orphan genes.
three possible scenarios for the emergence of orphan genes are frequently discussed in literature  <cit> : strong divergence, horizontal gene transfer, and de novo gene formation.
in p. pacificus, horizontal gene transfer has already been extensively studied  <cit>  revealing strong evidence only for three different gene families.
thus, horizontal gene transfer is an unlikely explanation for the bulk of orphan genes in p. pacificus.
it is difficult to decide on the remaining two scenarios because a clear proof of one scenario was only obtained by detailed case studies of individual genes  <cit>  and not by genome-wide analysis.
based on our analysis, we can only state that even if a period of strong divergence has lead to the loss of detectable homology, the evolutionary pressure that initially caused this divergence is not acting anymore at the moment since most orphan genes show evidence of strong negative selection.
as orphan genes have only been generally defined as taxonomically restricted genes  <cit> , each individual study has to somewhat arbitrarily decide on a practical definition of orphan genes and the obtained results may depend on this definition.
here, we make use of previously identified orphan genes  <cit> , which were defined using  <dig> non-diplogastrid genomes as outgroup species.
as the family of diplogastridae is comprised of several dozens of genera and is at least  <dig> million years old  <cit> , the age of the identified orphan genes is probably quite variable and not comparable to other studies that defined orphans at a species-specific level  <cit> .
to further investigate the age of p. pacificus orphan genes, future studies will have to incorporate genomic data at a much higher phylogenetic resolution.
our study also supports the hypothesis that the age of genes might be linked to the probability of a gene being embedded in gene-regulatory networks.
a recent study in human and mouse  <cit>  suggested that lineage-specific genes are initially loosely connected to the network periphery but with time become highly connected by acquiring essential and pleiotropic functions.
it can be stipulated that the failure of an orphan gene to become an integral part of gene networks increases the possibility of its loss over long evolutionary periods.
based on our analysis we suggest that a substantial fraction of orphan genes exhibiting differential expression under various conditions are well integrated into gene networks and might be therefore much older than other recently evolved orphan genes that may have a transient functional role but eventually will be lost from the genome.
such a mechanism augurs well with the fact that even though new orphan genes are added to the genome still the genome size does not show proportionate increase.
contrasting conserved genes with orphans, we find that orphan genes are shorter, have fewer exons, are less likely to be expressed, and are under less evolutionary constraint.
again, these findings are consistent with other studies from nematodes, insects, fish and plants .
although some of these features maybe related to the age of orphan genes, still further analysis is needed to investigate these trends and possible explanations in greater detail.
taken together, studies that prove the validity of orphan genes in individual genomes set up the starting point for further research of the biology and evolutionary history of orphan genes.
CONCLUSIONS
our study investigates the nature of orphan genes, which lack homology in other evolutionary lineages.
in the case of the nematode p. pacificus, we show that most of the predicted orphan genes are transcribed and evolve under constraints that are typical for protein-coding genes.
comparing paralogous and orthologous clusters with population genomic data suggests that the different data sets are highly complementary and demonstrates that even though evolutionary constraint acting on orphan genes can be studied within a single genome, the presence of a closely related genome has the greatest power for phylogenetic analysis.
our approach can be transferred to other genome data of non-model organisms in order to ascertain the biological importance of orphan genes.

 transferring functional annotations between species constitutes an integral part of modern biomedical research that tries to prioritize candidate genes for various diseases as well as to better understand phenotypic evolution. although genes for which similar functions are described in one species usually represent good candidate genes in other species of interest, still such strategy leads to an inherent bias in a way that only conserved genes with identifiable orthologs are pursued by functional follow up studies. this practice bears the danger of ignoring a large number of genes that either do not have identifiable one-to-one orthologs or completely lack any traces of homology in other species. as a consequence the latter class of taxonomically restricted genes that is as commonly referred to as orphan genes, new genes or pioneer genes has long been neglected in genetic research. thus, it is poorly understood what role orphan genes play in the biology of their host species. however, numerous studies in vertebrates, worms and flies have shown substantial phenotypic and genomic divergence, which strongly suggests that adaptation to new biological niches is reflected by differences in gene repertoires. along these lines, our lab has recently shown that a developmental decision even if conserved between nematodes, can still be under the control of unrelated genes that have absolutely no homology between species. depending on the definition and the phylogenetic resolution, up to one third of genes in current genome sequencing projects are usually identified as orphan genes. the number of orphan genes identified in a study largely depends on the definition of orphan gene as well as on the phylogenetic resolution of available genomic data. thus, genomes of taxonomic groups that are vastly underrepresented in genomic data predictably reveal higher number of orphan genes. currently, we lack a comprehensive understanding of how orphan genes evolve. strong divergence, de novo gene formation and horizontal gene transfer are frequently discussed explanations. however, since most genomes are at least partially annotated by gene predictions, it has been shown that orphan genes can arise from poorly assembled genomes. an alternative hypothesis is that many orphan genes represent non-coding rnas and might evolve without the constraints that are typical for protein-coding sequences. in order to investigate whether orphan genes are indeed protein-coding genes, prediction artifacts or non-coding rnas we analysed genomic, transcriptomic and proteomic data for the nematode pristionchus pacificus. p. pacificus has been established as a satellite model organism to caenorhabditis elegans for comparative studies involving developmental biology, immunity and population genomics. the genome of p. pacificus has been sequenced using sanger technology and has been improved using data. despite its relatively large size, in terms of contiguity it is either superior or at least comparable to many other recently published nematode genomes. more importantly, it has proven of great use for several studies to identify and clone causative genes for various different phenotypes. various gene finding programs predicted - genes that have been evaluated several times using transcriptomic and comparative genomic data indicating that current gene annotations are largely complete. in this study, we try to clarify the nature of orphan genes in p. pacificus. our basic strategy is to first check whether they are expressed and we consider expression as positive evidence that a gene prediction is not an artifact. in addition, strong support towards the protein coding nature of a predicted gene can be gathered if it either has a match in available proteomic data or shows evidence of negative selection that manifests in depletion of nonsynomous vs. synonymous substitutions. gene predictions that are expressed but do not fulfill any of the two criteria are candidates for non-coding rnas. based on the two criteria, our results show that between 40– % of orphan genes are actually truly protein-coding, indicating that orphan genes likely represent a substantial factor in the biology of p. pacificus. application of our methodology on genomic data from other taxonomically under-sampled groups will further support the validity of orphan genes and their biological significance.  the majority of orphan genes are transcribed we used previously defined sets of orphan genes and conserved genes and previously published rna-seq experiments in p. pacificus to assess up to what extent both gene sets are transcribed and to examine the dependence between the amount of expressed genes and the number of included rna-seq samples. thereby, we defined two different thresholds on the magnitude of expression, which was measured as fragments per kilobase transcript per million fragments sequenced. while a value of fpkm > = defines robust expression, a value of fpkm > = can still be interpreted as reliable evidence of expression as many functionally characterized genes display fpkm values well below fig. transcription and differential expression of orphan genes. a rna sequencing data from experiments are used to determine the number of expressed orphan and conserved genes as a function of the number of rna-seq samples and different expression thresholds. the boxplot shows the variation in the number of expressed genes from ten random permutations of the order of rna-seq samples b) saturation analysis for the number of differentially expression pattern of both orphan and conserved genes from six transcriptome profiling studies even though conserved genes show in general higher levels of expressed and robustly expressed genes, still in our analysis 4129– of orphan genes are transcribed in at least one of the samples. orphan genes are embedded in gene-regulatory networks in addition to using transcription as a necessary criterion for a real gene, we also quantified the amount of conserved and orphan genes that are differentially expressed under variable conditions like different developmental stages or exposure to pathogens. the rational behind this analysis is based on the assumption that a gene that can be differentially expressed under various conditions has likely been integrated into a gene-regulatory network. therefore such a gene is a more suitable candidate for a real gene rather than a gene, which is constitutively expressed at very low levels. this would also suggest that older genes would more likely be integrated into the gene network and they will have stronger differential expression pattern compared to new genes. consistent with this assumption, we have previously shown that orphan genes are significantly depleted among genes that are developmentally regulated and conserved genes are significantly enriched. however, analysis of previously conducted transcriptome profiling studies shows that not only conserved genes but even orphan genes are differentially expressed in p. pacificus. this indicates that a subset of orphan genes is differentially expressed under various conditions and implies that such an orphan gene must have persisted a sufficient amount of time in a genome for it to be integrated into a regulatory circuit. a subset of orphan genes shows direct evidence for translation next we tested to what extent conserved and orphan genes showed direct evidence of translation by testing for matches in available proteomic data. using % sequence identity over the full peptide length as criteria to search through the peptide data we found peptide evidence for orphan genes. this number is quite low when compared with the conserved genes that have peptide evidence, but it is consistent with previous study that reported the under-representation of orphan genes in transcriptome and peptide data. however, we also observe that even three quarters of conserved genes do not have peptide evidence, thus indicating that the lack of peptide evidence is not a sufficient criterion to distinguish truly protein-coding genes from potential artifacts or non-coding rnas. for this reason we employ other genomic resources to measure indirect evidence for protein-coding genes, i.e. selection against non-synonymous substitutions. comparative genomics of orphan genes as most orphan genes do not display matches in peptide data, we use negative selection in protein-coding genes as an indirect evidence for translation. however, to estimate evolutionary constraint at least two sequences are needed which is problematic for orphan genes since they do not have homologs in other species. this impediment can be circumvented by comparison with a species that is within the taxonomic group of the focal organism. in our case, we can make use of the recently sequenced sister species p. exspectatus. the genome of p. exspectatus shows roughly % sequence divergence and was previously used to polarize intra-species comparisons within p. pacificus. to this end, all predicted genes from both species p. pacificus and p. exspectatus were segregated into different orthologous clusters based on protein sequence homology using orthomcl. figure 2a shows the distribution of all the clusters into six different categories based on the number of the p. pacificus and p. exspectatus genes present in the clusters. evidently, the majority of clusters have only two members one each from p. pacificus and p. exspectatus genomes. after filtering out p. exspectatus specific clusters, hybrid clusters and short peptide containing clusters or poorly aligning clusters, we separated clusters containing one or more p. pacificus conserved genes and clusters containing one or more p. pacificus orphan genes for further investigation. these two sets represent orphan and conserved genes from p. pacificus. conserved and orphan gene clusters were further subdivided into two different datasets; the first dataset called ‘orthologous clusters’ had the clusters that contain at least one p. pacificus and one p. exspectatus gene, the second dataset called ‘paralogous clusters’ consisted of all the clusters containing more than one p. pacificus genes minus all the p. exspectatus genes from the clusters. we decided to distinguish the two types of clusters in order to assess to what extent we gain evidence for negative selection with the help of a second species. the combination of both the datasets represent all p. pacificus genes except the singletons genes that are present only in a single copy in p. pacificus and do not have a corresponding homolog in the p. exspectatus. in order to accommodate the p. pacificus singletons and study selection at a closer time scale we created a third dataset called ‘clade a1-a orthologs’ where we compared divergence of orthologous gene pairs across two geographically isolated p.pacificus lineages.fig. orphan genes are under strong negative selection. a distribution of gene clusters based on the number of orthologs between p. pacificus and p. exspectatus. b the graph shows the cumulative proportion of orphan and conserved gene clusters with evidence for negative selection relative to different ω thresholds in the p. pacificus – p. exspectatus orthologous clusters dataset. using an abitrary cutoff of ω < , % of all orphan clusters show evidence of negative selection. c comparison of the proportion of negatively selected orphan gene clusters and conserved gene clusters based on the analysis of paralogous gene clusters. given, that only % of conserved clusters and % of all orphan clusters contain p. pacificus paralogs, this analysis shows that even in the absence of other genomic data, negative selection can be investigated in a subset of orphan genes. d cumulative proportion of orphan and conserved gene clusters under given ω value in clade a – clade a ortholog dataset orphan and conserved genes are under strong negative selection selection pressure was measured as the ratio of synonymous and non-synonymous rate of amino acid substitution also called omega for each cluster of the three above-mentioned datasets using the paml suit. an ω =  would indicate neutral evolution and ω <  can be interpreted as evidence for negative selection. the measurement of ω on orthologous clusters showed that conserved gene clusters are under stronger negative selection than the orphan gene clusters. nonetheless, the majority of orphan gene clusters are also under relatively strong negative selection. the result of similar analysis on paralogous conserved and orphan clusters were also comparable, keeping in mind that only % of conserved clusters and % of orphan clusters are present in this dataset. the selection pressure results for clade a1-a dataset also follows the trend of showing stronger negative selection on conserved genes when compared with orphan genes; nevertheless, the orphan genes also appear to be under robust negative selection. ortholog, paralog and intra-species comparison are highly complementary similar to the analysis of the expression data, we define a range between a conservative and a more liberal threshold to estimate the amount of genes under negative selection. first, we arbitrarily chose ω <  as a liberal cutoff to call a cluster as evolving under negative selection. in addition, we defined a more conservative cutoff by calculating the statistical significance of a negative-selection model compared with a neutral model. for each cluster the codeml package of paml was run twice, once allowing a single ω value for the entire cluster tree but free to change and the second time ω fixed at for the entire cluster tree. likelihood ratio test was conducted with one degree of freedom and at false discovery rate adjusted p-value <  for each cluster to determine the significance of the alternate model. a combination of ω <  and fdr adjusted p-value <  was used as second more conservative criterion for calling a cluster as evolving under negative selection. please note that the lack of significance does not imply the lack of evolutionary constraint and in some cases might just be indicative of low statistical power due to the comparison of small proteins or little divergence between the sequences. in order to investigate the overlap between the different datasets, we decided to compare orphan candidates found to be under negative selection in all the three datasets. taking the cutoff of ω value lower than in any one of the three datasets identified of orphan genes as being under negative selection. figure 3a shows that the largest contribution towards identified negative selection comes from the intra-species comparison. however, since the evolutionary distances in this comparison are rather small, random fluctuations in the number of non-synonymous and synonymous sites can easily generate ω values below. given that the signal for positive selection is unlikely to affect complete genes, ω >  are most likely due to such random fluctuations; based on this we estimate that around one third of the identified orphan genes with ω <  can be due to random noise. therefore, we would like to emphasize that the cutoff of ω <  has to be regarded as upper limit of the estimated number of negatively selected orphan genes.fig. ortholog, paralog and population genomic data are highly complementary. a venn diagram for three different data sets using a definition of ω < . as most genes with evidence of negative selection are unique to one specific data set, this indicates that all three data sets are highly complementary. b venn diagram for three different data sets using a definition of ω <  and p < . again, most genes with evidence of negative selection are unique to one specific data set. c overlap between different thresholds to define negative selection and expressed genes. d identification of a candidate gene set of orphan genes that lack any expression data, are under significant negative selection and also have ω value less than in at least two of the three datasets using the stringent likelihood ratio test we identified orphan genes with significant evidence for negative selection in at least one of the three data sets. more precisely, out of orthologous orphan clusters clusters show significantly better goodness of fit for negative selection compared with the neutral evolution model. the corresponding figure for the conserved clusters is out of. among paralogous clusters, out of orphan clusters and out of conserved clusters showed significant goodness of fit for negative selection. in clade a1-a dataset, only out of orphan gene clusters and out of conserved gene clusters had significant goodness of fit supporting negative selection, indicating that the divergence between two p. pacificus lineages in general is not enough to identify robust evidence for negative selection at a single gene level. in order to further evaluate the use of ω <  as less conservative cutoff, we compared the identified gene sets from both methods with available expression data. this demonstrates that a cutoff of ω <  on one side captures almost all examples of significant negative selection and it minimizes the fraction of genes that are neither expressed nor show significant likelihood results. we have observed that changing this cutoff value to or drastically impairs this balance. in summary, our analysis suggests that a large proportion of orphan genes are under strong negative selection. evolutionary constraint predicts expression our previous analysis has indicated that a considerable number of orphan genes show evidence for negative selection. we combined our expression data with the selection analysis to screen for orphan genes that show negative selection but lack expression evidence. more precisely, the list of orphan genes showing significant negative selection were intersected with two other lists: first the list of orphan genes without expression data and second the list of genes that had ω value less than in at least datasets. from the genes found to be present in all three lists eleven genes that had more than exons in their predicted open reading frame were selected as candidates for validation by pcr. pcr products from three candidate genes were found in the expected nucleotide range when pcr was done against cdna but not against genomic dna. sequencing of pcr products resulted in expressed sequence tags that exactly matched the gene prediction. these results demonstrate, that even in the absence of expression evidence in rna-seq samples, pcr was able to detect expression of orphan genes in standard mixed-stage worm cultures. as expression profiles can be highly stage and tissue-specific, we speculate that further optimization of protocols and more specific rna samples could validate predicted gene structures in many more cases.fig. validation of orphan genes. a pcr validation experiments for eleven candidate orphan genes. genomic dna and cdna was amplified using the same primer pairs. in three cases, we obtained bands in the expected size range. b sequencing of amplification products resulted in ests that exactly confirmed the gene structure differences between various gene classes based on the obtained expression data and estimates for negative selection, we divided all p. pacificus genes into four classes. first class is made of potential prediction artifacts or pseudogenes, second class contains candidates for non-coding rnas, conserved genes and orphans that showed either significant negative selection or peptide evidence constitute the third and fourth classes. four gene features were compared across all four gene classes. while conserved and negatively selected orphans tend to be longer and contain more exons than potential prediction artifacts/pseudogenes and non-coding rna candidates, at the level of gc content and contig size percentile no obvious differences are detectable. given that fragmented assemblies were identified as potential source of prediction artifacts, it is interesting to see that in all four gene classes around % of genes reside in the top % of largest contigs. there are no significant trends towards accumulation of potential artifacts on smaller contigs. thus, partial gene model as a result of smaller contigs are an unlikely source for the majority of prediction artifact or pseudogene candidates. however, further analysis is needed to characterize these gene sets in greater detail.fig. differences between various gene classes. a comparison of transcript length for potential prediction artifacts/pseudogenes, non-coding rna candidates, negatively selected orphan genes, and conserved genes. the y-axis denotes the fraction of genes at a given transcript length. b comparison of the number of exons for different gene classes. c gc content distribution for all four gene classes. d distribution of contig size percentiles among all four classes. the top % of largest contigs harbors roughly % of genes for all four gene classes discussion the drastic reduction in sequencing cost has facilitated whole genome sequencing of several non-model species. however, current genome sequencing projects constantly identify large fraction of genes without homology in related species. the amount of identified orphan genes depends on the definition as well as on the phylogenetic resolution of available genomic data. as a consequence, non-model or non-classical model organisms such as p. pacificus, which has been estimated to share a common ancestor with c. elegans 280– mya, exhibit a much higher number of orphan genes in comparison to vertebrate genomes such as humans and mouse. orphan genes can arise from technical but also biological reasons. in addition, it has been shown that orphan genes can arise from genome assembly and gene prediction problems. as gene prediction is still an integral part of current gene annotation pipelines, we need to first clarify whether orphan genes are truly protein-coding genes before we can study their biology and evolutionary history. in this study, we use expression data to first test whether orphan genes are real biological entities or non-functional pseudogenes or gene prediction artifacts. first, we showed that up to % of orphan genes are indeed transcribed. second, using available proteomic and genomic data, we found that 40– % of orphan genes are actually truly protein-coding as they either show direct support in proteomic data or evolve under constraints that are expected for protein-coding genes. although problems in gene annotation still persist that might eventually be overcome by improvements in genome assembly and annotation, our study indicates that this will not eliminate the presence of orphan genes. as the most prominent conclusion of our study, that orphan genes are protein-coding genes under negative selection is based on phylogenetic analysis, we would like to point out that most technical artifacts such as mispredictions, pseudogenes or incorrect alignment would drive the signal towards neutral selection. this suggests that further correcting these types of errors will increase the amount of detected evidence for negative selection. furthermore, the finding of evolutionary constraint acting on orphan genes is consistent with other studies from insects, fish and primates. along with the accumulating functional evidence suggesting that taxonomically restricted genes can influence various phenotypic traits, recognizing orphan genes as true biological entities will further emphasize their importance and thus lead to a more holistic perspective in modern biomedical research. given that most orphan genes are protein-coding, the next questions we need to ask are: how do they evolve, how old are they, and what distinguishes conserved and orphan genes. three possible scenarios for the emergence of orphan genes are frequently discussed in literature: strong divergence, horizontal gene transfer, and de novo gene formation. in p. pacificus, horizontal gene transfer has already been extensively studied revealing strong evidence only for three different gene families. thus, horizontal gene transfer is an unlikely explanation for the bulk of orphan genes in p. pacificus. it is difficult to decide on the remaining two scenarios because a clear proof of one scenario was only obtained by detailed case studies of individual genes and not by genome-wide analysis. based on our analysis, we can only state that even if a period of strong divergence has lead to the loss of detectable homology, the evolutionary pressure that initially caused this divergence is not acting anymore at the moment since most orphan genes show evidence of strong negative selection. as orphan genes have only been generally defined as taxonomically restricted genes, each individual study has to somewhat arbitrarily decide on a practical definition of orphan genes and the obtained results may depend on this definition. here, we make use of previously identified orphan genes, which were defined using non-diplogastrid genomes as outgroup species. as the family of diplogastridae is comprised of several dozens of genera and is at least million years old, the age of the identified orphan genes is probably quite variable and not comparable to other studies that defined orphans at a species-specific level. to further investigate the age of p. pacificus orphan genes, future studies will have to incorporate genomic data at a much higher phylogenetic resolution. our study also supports the hypothesis that the age of genes might be linked to the probability of a gene being embedded in gene-regulatory networks. a recent study in human and mouse suggested that lineage-specific genes are initially loosely connected to the network periphery but with time become highly connected by acquiring essential and pleiotropic functions. it can be stipulated that the failure of an orphan gene to become an integral part of gene networks increases the possibility of its loss over long evolutionary periods. based on our analysis we suggest that a substantial fraction of orphan genes exhibiting differential expression under various conditions are well integrated into gene networks and might be therefore much older than other recently evolved orphan genes that may have a transient functional role but eventually will be lost from the genome. such a mechanism augurs well with the fact that even though new orphan genes are added to the genome still the genome size does not show proportionate increase. contrasting conserved genes with orphans, we find that orphan genes are shorter, have fewer exons, are less likely to be expressed, and are under less evolutionary constraint. again, these findings are consistent with other studies from nematodes, insects, fish and plants. although some of these features maybe related to the age of orphan genes, still further analysis is needed to investigate these trends and possible explanations in greater detail. taken together, studies that prove the validity of orphan genes in individual genomes set up the starting point for further research of the biology and evolutionary history of orphan genes.  our study investigates the nature of orphan genes, which lack homology in other evolutionary lineages. in the case of the nematode p. pacificus, we show that most of the predicted orphan genes are transcribed and evolve under constraints that are typical for protein-coding genes. comparing paralogous and orthologous clusters with population genomic data suggests that the different data sets are highly complementary and demonstrates that even though evolutionary constraint acting on orphan genes can be studied within a single genome, the presence of a closely related genome has the greatest power for phylogenetic analysis. our approach can be transferred to other genome data of non-model organisms in order to ascertain the biological importance of orphan genes. 
using genomic, transcriptomic and proteomic data for the nematode pristionchus pacificus, we show that between 4129– of predicted orphan genes are expressed and 3818– of orphan genes are under negative selection. our study shows that the great majority of previously identified orphan genes in p. pacificus are indeed protein-coding genes. current genome sequencing projects reveal substantial numbers of taxonomically restricted, so called orphan genes that lack homology with genes from other evolutionary lineages. however, even in the absence of other genomic data, the availability of paralogous sequences was enough to show negative selection in 8– % of orphan genes. even though this work represents a case study on a single species, our approach can be transferred to genomic data of other non-model organisms in order to ascertain the protein-coding nature of orphan genes. second, every sequence that is supported by proteome data or shows a depletion of non-synonymous substitutions is a protein-coding gene. comparing different data sets to infer selection on orphan gene clusters, we find that the presence of a closely related genome provides the most powerful resource to robustly identify evidence of negative selection. orphan genesnegative selectionorthologparalognematodesgene expressiondn/dsissue-copyright-statement© the author 2016 in three cases that exhibited strong evolutionary constraint but lacked expression evidence in rna-seq samples, we could experimentally validate the predicted gene structures. however, it is not clear to what extent orphan genes are real, genomic artifacts, or represent non-coding rnas. here, we use a simple set of assumptions to test the nature of orphan genes.
 current genome sequencing projects reveal substantial numbers of taxonomically restricted, so called orphan genes that lack homology with genes from other evolutionary lineages. however, it is not clear to what extent orphan genes are real, genomic artifacts, or represent non-coding rnas.  here, we use a simple set of assumptions to test the nature of orphan genes. first, a sequence that is transcribed is considered a real biological entity. second, every sequence that is supported by proteome data or shows a depletion of non-synonymous substitutions is a protein-coding gene. using genomic, transcriptomic and proteomic data for the nematode pristionchus pacificus, we show that between 4129– of predicted orphan genes are expressed and 3818– of orphan genes are under negative selection. in three cases that exhibited strong evolutionary constraint but lacked expression evidence in rna-seq samples, we could experimentally validate the predicted gene structures. comparing different data sets to infer selection on orphan gene clusters, we find that the presence of a closely related genome provides the most powerful resource to robustly identify evidence of negative selection. however, even in the absence of other genomic data, the availability of paralogous sequences was enough to show negative selection in 8– % of orphan genes.  our study shows that the great majority of previously identified orphan genes in p. pacificus are indeed protein-coding genes. even though this work represents a case study on a single species, our approach can be transferred to genomic data of other non-model organisms in order to ascertain the protein-coding nature of orphan genes. keywords orphan genesnegative selectionorthologparalognematodesgene expressiondn/dsissue-copyright-statement© the author 2016 