BACKGROUND
chemoperception is a central sense in nematodes, which lack vision and hearing.
detailed analysis of the cellular basis of chemosensory responses in caenorhabditis elegans has shown that a small number of sensory neurons mediate all known responses  <cit> .
despite this cellular simplicity, a huge number of genes encode candidate receptors for chemical stimuli.
the first indication of these genes came from an analysis of the partially completed genome sequence in  <dig>  this work identified several small genomic clusters of related genes encoding members of the rhodopsin superfamily of g-protein-coupled receptors , many of which were shown to be expressed in chemosensory neurons  <cit> .
subsequent analysis of the increasingly complete genome sequence identified additional large gene families with similar properties  <cit> .
a brief summary of the families has been published as a book chapter  <cit> .
here we report a full analysis of all the largest putative gpcr chemoreceptor gene families, which comprise about  <dig> genes and pseudogenes in the reference genome sequence, about  <dig> % of all genes in c. elegans.
although direct evidence for a specific chemosensory function is limited to a single gene  <cit> , expression patterns and other indirect evidence for many other members of the superfamily support a role predominantly in chemosensation  <cit> .
for simplicity, we refer to these genes as chemosensory receptor or chemoreceptor genes, although some of the genes have probably acquired other functions .
a small number of other genes may also encode chemoreceptors, notably members of the trp and guanylyl cyclase families  <cit> .
these other families are unrelated in structure to gpcrs and are not included in our analysis.
vertebrate chemoreceptor gene families have been the subject of intensive analysis.
they mediate olfaction, taste, and pheromone responses and show remarkably plastic evolution at the level of gene duplication and gene loss .
for example, nearly the entire complement of vomeronasal receptor genes  present in rodents has been deleted or pseudogenized on the primate lineage  <cit> .
this genetic change is probably connected with increased emphasis on visual cues for social behaviors in primates  <cit> .
drosophila melanogaster has a surprisingly small number of chemoreceptor genes, including just  <dig> odorant and  <dig> gustatory receptor genes  <cit> , which appear capable of mediating most of their known chemosensory responses .
comparisons across  <dig> newly available drosophila species genomes reveal that these insect chemoreceptors range in their evolutionary patterns from relatively conserved genes to relatively rapidly evolving gene lineages  <cit> .
more distant comparisons with mosquitoes, moths, beetles, and bees reveal hugely expanded and contracted lineages over time frames of 250– <dig> my  .
remarkably, none of the known taste and olfactory receptors from insects and vertebrates have conserved homologs in c. elegans, suggesting either that they evolved independently or that they have diverged too much for the relationship to be recognizable.
members of the c. elegans chemosensory receptor families form coherent sets of related sequences, falling into more than  <dig> families  <cit> , of which the  <dig> largest are analyzed in this paper.
a 20th family, srr, consists of only  <dig> genes in c. elegans, and may encode non-gpcr chemoreceptors .
within each chemoreceptor family, all of the genes share a common ancestor and the family has arisen through a long-term process of gene duplication, sequence divergence, and gene loss.
the largest of the families is called srh, which includes about  <dig> loci in c. elegans  <cit> .
these loci include genes that are probably functional, based on detailed gene annotation , and genes that appear to have nonfunctional alleles in the sequenced n <dig> genome .
about half of the apparently nonfunctional alleles in n <dig> have only a single apparent defect , and sequence analysis of several of these genes suggests that they have functional alleles in other wild isolates of c. elegans  <cit> .
the other  <dig> chemoreceptor families range in size from  <dig> loci  to  <dig> loci .
all of the families have a substantial frequency of apparently nonfunctional genes in the n <dig> genome and show strong gene clustering in the genome, suggesting recent gene duplication  <cit> .
as noted above for mammalian and insect chemoreceptors, a useful adjunct to studying a gene family in a specific organism is the capacity to compare the genes with those in related organisms with a variety of speciation dates.
in this respect nematodes provide a relatively weak data set, because relatively few nematodes are under active study in the laboratory and the closest known relatives to c. elegans diverged as much as  <dig> my ago  <cit> .
nevertheless, the nearly complete sequences of two relatives, c. briggsae and c. remanei, are available, a draft assembly of c. brenneri is available, and sequencing projects are underway for several additional nematodes, ranging from other close c. elegans relatives to distant parasitic species  <cit> .
for the sra, srab, and srz chemoreceptor gene families, published comparisons of c. elegans and c. briggsae indicate substantial differences in gene number and provide evidence of several expansions in gene number in c. elegans  <cit> .
here, we report an analysis of all  <dig> large chemoreceptor gene families in c. elegans, c. briggsae and c. remanei.
we show that all of the families are characterized by substantial rates of birth-death evolution, with large variation in the rates in different families.
we provide summaries of the evolution and conserved sequence features within each of the families.
RESULTS
curation of c. elegans chemoreceptor gene families
genes encoding potential gpcr proteins were identified using a variety of methods .
most of the gpcr gene candidates fell into families that are unrelated or distantly related to genes in other phyla.
these predicted genes were used as a starting point to manually correct existing gene predictions and to identify a small number of new genes .
among all of the predicted genes in a family, we identified probable full-length protein sequences and used these sequences as queries for tblastn searches to guide the correction of anomalous predictions.
the large number of paralogs within each family made this approach effective.
candidate nonfunctional genes were identified as genes that could not be modified to give good full-length alignments.
these included genes with in-frame stop codons, deletion of conserved parts of the family protein sequence, frameshifts in coding exons, probable splice junction defects, and missing start codons.
the  <dig> largest gene families were chosen for further analysis.
a complete list of encoded proteins for these families can be found in additional file  <dig>  sample full-length protein alignments for each family are shown in additional files  <dig> to  <dig>  alignments and analysis of nonfunctional genes were previously published for the srh and str families  <cit> .
c. briggsae and c. remanei annotation
using computational methods we completed an improved annotation of genes from each of the  <dig> chemoreceptor families in c. briggsae and c. remanei .
briefly, existing protein prediction sets for each species were combined with full-length c. elegans predictions and culled to identify all probable full-length proteins from the three species.
the full-length protein set for each gene family was used in a genewise prediction pipeline, which was then combined with existing predictions to identify a best prediction for each gene.
subsequent analysis was based on these best prediction sets.
defective genes and gene family sizes
it was difficult to define accurately what should count as a functional gene or a defective gene in these families; the issues are described in detail in methods.
for c. elegans, the reference genome sequence is complete and our manual annotation was intensive enough to permit good estimates of the number of defective genes for each family, as summarized in additional file  <dig>  the frequency of defective genes was weakly correlated with family size; this issue is discussed further in the next section.
although it was clear that the reference c. briggsae and c. remanei genomes also include many defective genes, we did not accurately determine their number.
instead, genes were included for further analysis if they had the potential to encode nearly full-length proteins, as described in methods.
these gene counts are listed in additional file  <dig>  with c. elegans gene counts based on the same method.
as some defective genes can encode near full-length proteins, the number of genes listed for c. elegans is larger than our estimate of the number of functional genes in additional file  <dig>  we presume that the number of functional genes in c. briggsae and c. remanei is similarly lower than the number of genes listed in additional file  <dig>  these near full-length protein predictions from the three species were used to construct protein trees; the protein sequences are given in additional files  <dig>   <dig>  and  <dig> .
protein trees
for each chemoreceptor family, proteins from all three species were combined and a maximum likelihood tree was built with approximate likelihood ratio test  branch supports  <cit> .
figures representing these trees for each of the  <dig> families are given in additional files  <dig> to  <dig>  a representative section of one of the trees is shown in figure  <dig>  all of the families were characterized by a substantial number of apparent gene duplications and probably a roughly balanced number of gene losses .
additional file  <dig> gives summary statistics that capture these properties for each family.
at one extreme, the c. elegans srbc, srw, and srz families have very few clear orthologs across the three species.
these trees instead are dominated by species-specific expansions, which presumably arose by a process of ongoing gene duplication and loss on each lineage.
at the other extreme, more than half of the genes in the c. elegans sre and srxa families have single orthologs in both c. briggsae and c. remanei, although even these relatively stable families have substantial numbers of apparent gene duplications and losses.
to test whether incomplete genome sequence or analysis problems might contribute to these patterns, we applied the same gene prediction and tree analysis to  <dig> metabotropic gpcrs and to  <dig> fmrfamide receptor-related genes from c. elegans.
nearly all of these genes had single orthologs in c. briggsae and c. remanei, as summarized in additional file  <dig>  similar analysis of other stable gene families gave similar results .
we conclude that very few genes are missing from the c. briggsae and c. remanei sequence assemblies and that our methods of gene annotation are of sufficiently high quality to give good estimates of family stability.
the fraction of defective genes in a family in c. elegans was well correlated with the family-specific rate of gene duplication and loss, as indirectly inferred from the fraction of orthologs in the protein tree .
this correlation fits with the idea that gene duplication is balanced by gene loss.
one route to gene loss is the fixation of a defective allele in the population followed by eventual deletion of the now neutrally evolving segment of dna.
although it is unclear what fraction of defective genes in the sequenced n <dig> genome are fixed pseudogenes  <cit> , it is likely that many are, since they have more than one obvious defect.
these are presumably gene family members on their way to complete gene loss.
assuming pseudogenes in different families are deleted from the genome at a similar rate, the frequency of such pseudogenes is expected to reflect the family-specific rate of gene loss.
gene duplication and genome position
patterns of genome location for all  <dig> families were analyzed in c. elegans, where the genome sequence is complete.
the degree of gene clustering and predominant chromosomal location for each family is summarized in additional file  <dig> and color-coded positions of genes in all families are shown in additional file  <dig>  most genes in each family were located on a single chromosome .
these patterns are consistent with an ancestral gene on one chromosome and largely local gene duplication events giving rise to the family over time.
even at the superfamily level, each of which presumably arose from an even older ancestral gene, this chromosome bias is apparent: most str and srg superfamily genes are located on chromosome v and most sra superfamily members are located on chromosome ii.
detailed inspection of protein trees and gene locations suggests that most exceptions to genes remaining on their ancestral chromosome arose by transposition of single genes followed by further duplication at the new chromosomal home.
these results support the conclusion that evolutionarily retained translocation events are extremely rare in caenorhabditis species  <cit>  and that the chromosome is a meaningful unit of long-term evolution in nematodes.
similar inferences are published for the srh, str, and srj families  <cit> .
lack of evidence for positive selection
genes in the srz chemoreceptor family are under positive selection in both c. elegans and c. briggsae  <cit> .
in that work, maximum likelihood dn/ds tests were conducted on paralog samples from most of the chemosensory receptor families described here.
we carried out additional systematic tests among all  <dig> chemoreceptor families.
these analyses confirmed positive selection in the srz family and produced no clear evidence of positive selection in any of the other families .
it is important to note that the dn/ds method used for these tests is sensitive only to persistent long-term positive selection.
methods for testing more recent selection events are not currently applicable with the limited population sequence data available in caenorhabditis.
promoter e-box distribution
one possible selective force for expansion in gene number is an increased need for diversified chemosensory receptors expressed in specific sensory neurons.
little is known about how promoter sequences control cell-type specific expression of chemoreceptor genes in c. elegans.
however, one promoter sequence, called the e-box, has been shown to be enriched in srh and sri chemoreceptor genes and to drive expression in the chemosensory neuron adl  <cit> .
to test the distribution of e-boxes among all chemoreceptor genes, we extracted  <dig>  promoter sequences that include  <dig> chemoreceptor family loci.
to reduce non-specific noise, analysis was restricted to the region from - <dig> to - <dig> from the predicted translation start, where e-boxes are concentrated .
each promoter was scanned for its best match to an e-box profile and the  <dig> best-scoring matches were analyzed for their distribution among gene families .
there was strong enrichment of e-boxes in chemoreceptor genes as a whole, and this was due entirely to high match frequencies in four families.
the other  <dig> chemoreceptor families had an e-box match frequency similar to the genome as a whole; we conclude that few if any genes in these families are likely to be regulated by e-box binding factors.
the sre, srh, sri, and srz families were strikingly enriched in e-boxes, with 25% to 50% of their promoters in the high-scoring set.
if e-box driven expression in adl neurons generalizes to the other chemoreceptor families, then adl likely expresses about  <dig> chemoreceptors, largely from the sre, srh, sri, and srz families.
these four families are very diverse, representing three different superfamilies.
these results suggest that the function of adl sensory neurons requires expression of a large and diverse set of chemoreceptors.
bitter taste sensory neurons in mammals and insects also express multiple receptors  <cit> , an unusual pattern that may be important for broadly tuned aversion to noxious compounds  <cit> .
we speculate that the aversion-mediating neuron adl has a similarly broad sensitivity in nematodes.
e-box hits are the  <dig> highest scoring motif matches in promoters from - <dig> to - <dig> from the translation start codon.
percent values based on zero or one hit are in parentheses to indicate high uncertainty.
the uncorrected p-value shown was determined by comparison with the number of hits in all promoters .
after bonferoni correction for multiple testing the sru and srw p-values are not significant.
the str and srj families are closely related and were analyzed together.
a few small chemoreceptor families are not shown individually, so numbers shown do not add up to the sr total.
sr, all members of putative chemoreceptor families; na, not applicable; ns, not significant.
brief family summaries
sra superfamily
sra family
this small published family  <cit>  includes a single large gene expansion in c. elegans, all of which are in a cluster on the right arm of chromosome i .
this expansion probably arose following the transposition of a single sra gene from chromosome ii, where most other members of the family reside.
apart from this part of the family, there is a relatively high frequency of orthology, suggesting that many sra gene functions are evolutionarily stable.
most sra proteins have two potential disulfide bonds that could join extracellular domain  <dig> with  <dig> and domain  <dig> with  <dig> .
five sra proteins lack the first cysteine pair, which suggests this pairing pattern.
there are no strongly characteristic sequence motifs conserved in the family.
the sra, srab, and srb families are closely related and a few genes are difficult to place in a specific family.
the family corresponds to existing pfam profile pf <dig>
srab family
this small published family  <cit>  includes a single large gene expansion in c. elegans, all but one of which are in a cluster on the left arm of chromosome v .
apart from this part of the family, there is a relatively high frequency of orthology, suggesting that many srab gene functions are evolutionarily stable.
srab proteins have two conserved potential disulfide bonds in the same positions as those in the sra family .
there is a protein segment in and near tm domain  <dig> that contains several highly conserved h, n, r, and d residues, which is characteristic of the srab family.
the family has been assigned to the new pfam profile pf <dig>  also see the sra family notes above.
srb family
this small family includes no large c. elegans gene expansions, but has one modest expansion in c. remanei and occasional duplications and losses throughout the tree .
the overall frequency of orthologs is relatively high, suggesting that many srb gene functions are evolutionarily stable.
there is one conserved potential disulfide bond that could join extracellular domains  <dig> and  <dig> in approximately the same position as one of the potential disulfide bonds in the sra and srab families .
at or near the inner end of tm domain  <dig> there is a nearly invariant fhxn sequence, which is shared with some srab and sre proteins but is otherwise characteristic of the srb family.
the family corresponds to existing pfam profile pf <dig>  also see the sra family notes above.
sre family
this medium-sized family includes two modest gene expansions in c. elegans, each located in a cluster on the right arm of chromosome ii .
in other parts of the tree there is a high frequency of orthology, suggesting that many sre gene functions are evolutionarily stable.
there are no conserved potential disulfide bonds in the family .
most sre proteins share the fhxn sequence with srb proteins and there is a highly conserved r/kfqxxen sequence in intracellular loop three that is characteristic of the family.
there is marked variation in the length of the second extracellular loop, with about half of sre proteins having an approximately  <dig> amino acid insertion relative to the others.
this variant is found in all three caenorhabditis species and correlates with other sequence characters in the protein tree.
the family corresponds to existing pfam profile pf <dig>
srg superfamily
srg family
this medium-sized family includes two modest gene expansions in c. elegans, one in a cluster on the left arm of chromosome v and another mostly in a cluster on the left arm of chromosome ii .
there are lower rates of gene duplication and gene loss in most of the rest of the tree, giving the family as a whole a moderately high frequency of orthologs.
there is a single potential disulfide bond in about half of srg proteins that could join extracellular domains  <dig> and  <dig> .
there is a nearly invariant w residue near the inside end of tm domain  <dig> that is characteristic of the family.
the family corresponds to existing pfam profile pf <dig>
srt family
this published family  <cit>  is medium sized and gene duplications and losses are fairly evenly distributed across the protein tree, with several small expansions in c. elegans, all located in clusters on the left arm of chromosome v .
there is a relatively low frequency of orthologs.
there is one potential disulfide bond in most srt proteins, which could join extracellular domains  <dig> and  <dig> .
there are two nearly invariant w residues, one in tm domain  <dig> and another in tm domain  <dig>  which are characteristic of the family.
the family has been assigned to the new pfam profile pf <dig>
sru family
instability in this medium-sized family is fairly evenly distributed across the protein tree, with several small expansions in c. elegans, located mostly in clusters on the left and right arms of chromosome v .
the family is notably larger in c. remanei, mostly as a result of two large expansions on the tree.
the c. remanei assembly is not adequate to fully assess genome clustering of these two gene expansions, but there is clearly some clustering on specific supercontigs.
there is a relatively low frequency of orthologs.
there is one potential disulfide bond in all but a few sru proteins that could join extracellular domains  <dig> and  <dig> .
there is a highly conserved protein segment in and near the inner end of tm domain  <dig> that is characteristic of the family.
the family corresponds to existing pfam profile pf <dig>
srv family
this medium-sized family includes a single small gene expansion in c. elegans that is in a cluster near the center of chromosome iv.
c. remanei has several small expansions scattered across the tree .
the rest of the tree is characterized by modest frequencies of gene duplication and loss, giving an overall frequency of orthology that is relatively low.
there are no conserved potential disulfide bonds in the family and no other obvious distinguishing sequence characteristics .
the family has been assigned to the new pfam profile pf <dig>
srx family
this large family includes several modest gene expansions in c. elegans, mostly in gene clusters on the left and right arms of chromosome v and on the left arm of chromosome ii .
outside of these regions of the tree there are many orthologs, giving the family overall an average frequency of orthology.
there is one conserved potential disulfide bond between two residues in extracellular domain  <dig> .
there is a nearly invariant nr motif in tm domain  <dig> that is found only in srx and srxa proteins.
the family has been assigned to the new pfam profile pf <dig>
srxa family
this small family includes a relatively small number of gene duplications and losses spread evenly across the tree , giving the family overall a relatively high frequency of orthologs and suggesting that many srxa functions are evolutionarily stable.
all srxa proteins have one potential disulfide bond between two residues in extracellular domain  <dig> , a characteristic that is shared only with the related srx family.
the family corresponds to existing pfam profile pf <dig>  also see the srx family notes above.
str superfamily
srd family
this medium-sized family includes two moderately large gene expansions in c. elegans .
one expansion corresponds to a gene cluster on the right arm of chromosome v, but the other includes genes on chromosomes iii and v and presumably involves one or more transposition events.
outside of these parts of the tree there are relatively few gene duplications and losses, giving the family as a whole a moderate frequency of orthologs.
most srd proteins have one potential disulfide bond between two residues in extracellular domain  <dig>  a pattern that is found only in this family .
it shares with the srh and sri families a highly conserved pyr sequence at or near the inner end of transmembrane  domain  <dig>  the family has been assigned to the new pfam profile pf <dig>
srh family
this published family  <cit>  is the largest chemoreceptor gene family in c. elegans.
the tree includes several large to moderately large gene expansions in c. elegans, mostly in gene clusters on the left and right arms of chromosome v .
there are no conserved potential disulfide bonds .
there is a conserved hg motif in tm domain  <dig> that characterizes most of the family.
several other short sequence motifs are shared only with the sri family.
the family has been assigned to the new pfam profile pf <dig>
sri family
this medium-sized family is closely related to the srh family, and includes several moderately large gene expansions in c. elegans, mostly in gene clusters in several places in the genome .
sri- <dig> and sri- <dig> are an outgroup to the rest of the family and in some analyses tree with the srh family.
there are no conserved potential disulfide bonds .
there is a conserved cf motif at or near the inner end of tm domain  <dig> that characterizes most members of the family.
the family has been assigned to the new pfam profile pf <dig>  also see srh family notes.
srj family
this medium-sized published family  <cit>  is closely related to the str family .
the family includes one large gene expansion in c. elegans mostly in one gene cluster on the left arm of chromosome v .
most of the rest of the tree includes lower frequencies of gene duplications and losses and the family overall has few orthologs.
srj- <dig> is highly divergent from all other srj genes but belongs in this family based on rooted trees.
there are no conserved potential disulfide bonds .
srj proteins can be distinguished from str proteins based on a nearly invariant rc motif near the center of tm domain  <dig>  the family has been assigned to the new pfam profile pf <dig>
str family
this published family  <cit>  is the second largest chemoreceptor gene family in c. elegans.
there are several moderately large gene expansions in c. elegans, mostly in gene clusters on the right arm of chromosome v, and several gene expansions in c. briggsae and c. remanei .
most of the rest of the tree is also characterized by a substantial frequency of gene duplications and losses, and overall the family has few orthologs.
this family includes odr- <dig>  the only chemoreceptor with a known specific function  <cit> .
odr- <dig> is on a relatively stable part of the protein tree and has orthologs in c. briggsae and c. remanei.
there are no conserved potential disulfide bonds but there are several nearly invariant residues in and near tm domain  <dig> that are shared only with the srj family .
the family has been assigned to the new pfam profile pf <dig>
other families
srbc family
this medium-sized family is dominated by three large gene expansions in c. elegans, mostly in gene clusters in several places on chromosome v .
there are also gene expansions in c. briggsae and c. remanei but they involve fewer genes, so that the srbc family is much larger in c. elegans.
there are very few orthologs in the family.
there are two invariant potential disulfide bonds, both among residues in extracellular domain  <dig>  a pattern that is characteristic of the family .
the spacing between these cysteine residues is nearly invariant but there is substantial variation in the specific sequences.
it seems likely that the disulfides form a framework for a ligand-binding domain unique to this family.
the family has been assigned to the new pfam profile pf <dig>
srsx family
this small family includes a few recent gene duplications in c. elegans, mostly in local pairs on the right arm of chromosome v .
most of the tree has very little gene duplication and loss, giving the family overall a relatively high frequency of orthologs and suggesting that many srsx functions are evolutionarily stable.
there is one invariant potential disulfide bond that could join extracellular domains  <dig> and  <dig> .
about 15% of srsx proteins have an approximately  <dig> amino acid insertion in intracellular domain  <dig> that is highly conserved, suggesting a distinct g-protein interaction for this group.
there is a nearly invariant gn sequence near the middle of tm domain  <dig> that is characteristic of the family.
the family has been assigned to the new pfam profile pf <dig>
srw family
this large family is the only one with a clear sequence relationship to other known gpcr proteins: it is weakly but clearly related to several groups of neuropeptide receptors, including the myosuppressin receptor family in insects and the fmrfamide receptor family in nematodes .
despite this relationship, it is unlikely that srw proteins function as neuropeptide receptors because they are subject to a high rate of gene duplication and loss, in stark contrast to members of the known peptide receptor families .
we speculate that srw proteins function as receptors for environmental peptides.
the family includes several moderate to large gene expansions in c. elegans and similar, albeit smaller, expansions in c. briggsae and c. remanei .
the expansions in c. elegans correspond to several gene clusters mostly on the left and right arms of chromosome v. there is one highly conserved potential disulfide bond that could link extracellular domains  <dig> and  <dig> .
there are two nearly invariant h residues that are characteristic of the family, one in tm domain  <dig> and the other in tm domain  <dig>  the family corresponds to existing pfam profile pf <dig>
srz family
this moderately large published family  <cit>  is characterized by a high rate of gene duplication and gene loss and is uniquely subject to strong positive selection on the extracellular face of the protein.
the tree is dominated by gene expansions specific to each species and indicates very few orthologs .
there are no conserved potential disulfide bonds and the proteins have diverse length and sequence in all four extracellular domains .
there are several conserved sequence motifs in or very near tm domains that are characteristic of the family.
the family has been assigned to the new pfam profile pf <dig>
discussion
gene number and diversity
the chemoreceptor genes in caenorhabditis are strikingly abundant and diverse.
in contrast, mammalian olfactory receptor genes are abundant but have relatively limited diversity, with nearly all of the genes belonging to a single gene family  <cit> .
for example, among putative functional olfactory receptors in human, dog, and mouse, the average pairwise amino acid identity is about  <dig>   refseq <dig>  data not shown).
this level of diversity is lower than within any single c. elegans family .
fish and amphibians have more diverse olfactory receptor genes, yet even in these groups all known genes fall into a single superfamily, with significant sequence relatedness between any given pair of olfactory receptor proteins  <cit> .
insects have much smaller chemoreceptor gene families  <cit> .
caenorhabditis species have seven superfamilies of putative chemosensory receptor genes, with no significant sequence similarity across superfamilies.
why do nematodes have such high diversity in their chemoreceptors?
we suggest that this is a result of sensory system emphasis.
unlike vertebrates and insects, nematodes lack vision and hearing.
we speculate that chemosensory diversity is thus more central to nematode sensory capabilities, leading to a high genetic investment and high receptor diversity.
a similar explanation for the smaller olfactory gene number in great apes relative to rodents has been proposed, based on an increased emphasis on a sophisticated visual system  <cit> .
the gray pawn hypothesis
totaled across all  <dig> families, the putative functional chemoreceptor genes account for about 7% of all c. elegans genes.
despite this large genetic investment in a single class of genes, our comparison among the three sequenced caenorhabditis species and gene knockdown studies  <cit>  suggest that few if any of the specific genes are essential for species viability, although subsets are implicated in specific physiological processes such as maintenance of fat content  <cit> .
instead, individual genes are lost at a high rate and this loss is roughly compensated by duplications among the remaining genes, similar to the olfactory receptor family in mammals  <cit> .
one possible explanation of these patterns is that new duplicate genes diverge and confer some specific selective advantage .
however, in both nematodes and mammals there is evidence for such positive selection in only small and specific subsets of chemoreceptor genes  <cit> .
although further analysis might reveal subtler signs of positive selection, it is unlikely to be a major evolutionary force in these families, in contrast to several other gene families in which similar dn/ds analysis reveals extensive evidence of positive selection .
what else could account for these patterns?
we propose that the function of most of these genes is not to be found in their individual contributions to fitness, but rather in their aggregate function in covering a large phenotype space.
in this model, each gene is a nearly insignificant and faceless pawn, but in aggregate these gray pawns form an effective army.
neutral processes may dominate many aspects of evolution in these families, but this alone cannot account for the retention of roughly similar numbers of genes in three highly divergent species.
the gray pawn hypothesis suggests that if the number of functional genes in a family drops too much, selective pressure increases to gain new genes by duplication and divergence, and gene number increases by selective retention of new duplicates.
if the number of genes increases too much, selective pressure to maintain individual genes decreases, and genes are lost by mutation and drift.
the number and diversity of functional genes results from this dynamic equilibrium combined with the species-specific importance of the process mediated by the gene family.
evolution of the immunoglobulin gene families in mammals may also be explained in part by the gray pawn hypothesis.
CONCLUSIONS
we have completed the annotation and description of all  <dig> large gpcr gene families in c. elegans and its relatives c. briggsae and c. remanei.
all of the families are characterized by a substantial number of nonfunctional genes in each species.
the rates of gene duplication and gene loss, inferred from protein trees in each family, are substantial in all the families.
despite these dynamics, evidence for positive selection is found in only one of the families.
we propose the gray pawn hypothesis to explain these patterns of molecular evolution: most individual genes are of little significance, but as a group they are required to cover a broad ligand space.
this is achieved by maintaining a large and diverse repertoire of chemoreceptors in which new genes arise by duplication and divergence and others are lost by mutation and genetic drift.

 chemoperception is a central sense in nematodes, which lack vision and hearing. detailed analysis of the cellular basis of chemosensory responses in caenorhabditis elegans has shown that a small number of sensory neurons mediate all known responses. despite this cellular simplicity, a huge number of genes encode candidate receptors for chemical stimuli. the first indication of these genes came from an analysis of the partially completed genome sequence in this work identified several small genomic clusters of related genes encoding members of the rhodopsin superfamily of g-protein-coupled receptors, many of which were shown to be expressed in chemosensory neurons. subsequent analysis of the increasingly complete genome sequence identified additional large gene families with similar properties. a brief summary of the families has been published as a book chapter. here we report a full analysis of all the largest putative gpcr chemoreceptor gene families, which comprise about genes and pseudogenes in the reference genome sequence, about % of all genes in c. elegans. although direct evidence for a specific chemosensory function is limited to a single gene, expression patterns and other indirect evidence for many other members of the superfamily support a role predominantly in chemosensation. for simplicity, we refer to these genes as chemosensory receptor or chemoreceptor genes, although some of the genes have probably acquired other functions. a small number of other genes may also encode chemoreceptors, notably members of the trp and guanylyl cyclase families. these other families are unrelated in structure to gpcrs and are not included in our analysis. vertebrate chemoreceptor gene families have been the subject of intensive analysis. they mediate olfaction, taste, and pheromone responses and show remarkably plastic evolution at the level of gene duplication and gene loss. for example, nearly the entire complement of vomeronasal receptor genes present in rodents has been deleted or pseudogenized on the primate lineage. this genetic change is probably connected with increased emphasis on visual cues for social behaviors in primates. drosophila melanogaster has a surprisingly small number of chemoreceptor genes, including just odorant and gustatory receptor genes, which appear capable of mediating most of their known chemosensory responses. comparisons across newly available drosophila species genomes reveal that these insect chemoreceptors range in their evolutionary patterns from relatively conserved genes to relatively rapidly evolving gene lineages. more distant comparisons with mosquitoes, moths, beetles, and bees reveal hugely expanded and contracted lineages over time frames of 250– my. remarkably, none of the known taste and olfactory receptors from insects and vertebrates have conserved homologs in c. elegans, suggesting either that they evolved independently or that they have diverged too much for the relationship to be recognizable. members of the c. elegans chemosensory receptor families form coherent sets of related sequences, falling into more than families, of which the largest are analyzed in this paper. a 20th family, srr, consists of only genes in c. elegans, and may encode non-gpcr chemoreceptors. within each chemoreceptor family, all of the genes share a common ancestor and the family has arisen through a long-term process of gene duplication, sequence divergence, and gene loss. the largest of the families is called srh, which includes about loci in c. elegans. these loci include genes that are probably functional, based on detailed gene annotation, and genes that appear to have nonfunctional alleles in the sequenced n genome. about half of the apparently nonfunctional alleles in n have only a single apparent defect, and sequence analysis of several of these genes suggests that they have functional alleles in other wild isolates of c. elegans. the other chemoreceptor families range in size from loci to loci. all of the families have a substantial frequency of apparently nonfunctional genes in the n genome and show strong gene clustering in the genome, suggesting recent gene duplication. as noted above for mammalian and insect chemoreceptors, a useful adjunct to studying a gene family in a specific organism is the capacity to compare the genes with those in related organisms with a variety of speciation dates. in this respect nematodes provide a relatively weak data set, because relatively few nematodes are under active study in the laboratory and the closest known relatives to c. elegans diverged as much as my ago. nevertheless, the nearly complete sequences of two relatives, c. briggsae and c. remanei, are available, a draft assembly of c. brenneri is available, and sequencing projects are underway for several additional nematodes, ranging from other close c. elegans relatives to distant parasitic species. for the sra, srab, and srz chemoreceptor gene families, published comparisons of c. elegans and c. briggsae indicate substantial differences in gene number and provide evidence of several expansions in gene number in c. elegans. here, we report an analysis of all large chemoreceptor gene families in c. elegans, c. briggsae and c. remanei. we show that all of the families are characterized by substantial rates of birth-death evolution, with large variation in the rates in different families. we provide summaries of the evolution and conserved sequence features within each of the families.  curation of c. elegans chemoreceptor gene families genes encoding potential gpcr proteins were identified using a variety of methods. most of the gpcr gene candidates fell into families that are unrelated or distantly related to genes in other phyla. these predicted genes were used as a starting point to manually correct existing gene predictions and to identify a small number of new genes. among all of the predicted genes in a family, we identified probable full-length protein sequences and used these sequences as queries for tblastn searches to guide the correction of anomalous predictions. the large number of paralogs within each family made this approach effective. candidate nonfunctional genes were identified as genes that could not be modified to give good full-length alignments. these included genes with in-frame stop codons, deletion of conserved parts of the family protein sequence, frameshifts in coding exons, probable splice junction defects, and missing start codons. the largest gene families were chosen for further analysis. a complete list of encoded proteins for these families can be found in additional file sample full-length protein alignments for each family are shown in additional files to alignments and analysis of nonfunctional genes were previously published for the srh and str families. c. briggsae and c. remanei annotation using computational methods we completed an improved annotation of genes from each of the chemoreceptor families in c. briggsae and c. remanei. briefly, existing protein prediction sets for each species were combined with full-length c. elegans predictions and culled to identify all probable full-length proteins from the three species. the full-length protein set for each gene family was used in a genewise prediction pipeline, which was then combined with existing predictions to identify a best prediction for each gene. subsequent analysis was based on these best prediction sets. defective genes and gene family sizes it was difficult to define accurately what should count as a functional gene or a defective gene in these families; the issues are described in detail in methods. for c. elegans, the reference genome sequence is complete and our manual annotation was intensive enough to permit good estimates of the number of defective genes for each family, as summarized in additional file the frequency of defective genes was weakly correlated with family size; this issue is discussed further in the next section. although it was clear that the reference c. briggsae and c. remanei genomes also include many defective genes, we did not accurately determine their number. instead, genes were included for further analysis if they had the potential to encode nearly full-length proteins, as described in methods. these gene counts are listed in additional file with c. elegans gene counts based on the same method. as some defective genes can encode near full-length proteins, the number of genes listed for c. elegans is larger than our estimate of the number of functional genes in additional file we presume that the number of functional genes in c. briggsae and c. remanei is similarly lower than the number of genes listed in additional file these near full-length protein predictions from the three species were used to construct protein trees; the protein sequences are given in additional files and. protein trees for each chemoreceptor family, proteins from all three species were combined and a maximum likelihood tree was built with approximate likelihood ratio test branch supports. figures representing these trees for each of the families are given in additional files to a representative section of one of the trees is shown in figure all of the families were characterized by a substantial number of apparent gene duplications and probably a roughly balanced number of gene losses. additional file gives summary statistics that capture these properties for each family. at one extreme, the c. elegans srbc, srw, and srz families have very few clear orthologs across the three species. these trees instead are dominated by species-specific expansions, which presumably arose by a process of ongoing gene duplication and loss on each lineage. at the other extreme, more than half of the genes in the c. elegans sre and srxa families have single orthologs in both c. briggsae and c. remanei, although even these relatively stable families have substantial numbers of apparent gene duplications and losses. to test whether incomplete genome sequence or analysis problems might contribute to these patterns, we applied the same gene prediction and tree analysis to metabotropic gpcrs and to fmrfamide receptor-related genes from c. elegans. nearly all of these genes had single orthologs in c. briggsae and c. remanei, as summarized in additional file similar analysis of other stable gene families gave similar results. we conclude that very few genes are missing from the c. briggsae and c. remanei sequence assemblies and that our methods of gene annotation are of sufficiently high quality to give good estimates of family stability. the fraction of defective genes in a family in c. elegans was well correlated with the family-specific rate of gene duplication and loss, as indirectly inferred from the fraction of orthologs in the protein tree. this correlation fits with the idea that gene duplication is balanced by gene loss. one route to gene loss is the fixation of a defective allele in the population followed by eventual deletion of the now neutrally evolving segment of dna. although it is unclear what fraction of defective genes in the sequenced n genome are fixed pseudogenes, it is likely that many are, since they have more than one obvious defect. these are presumably gene family members on their way to complete gene loss. assuming pseudogenes in different families are deleted from the genome at a similar rate, the frequency of such pseudogenes is expected to reflect the family-specific rate of gene loss. gene duplication and genome position patterns of genome location for all families were analyzed in c. elegans, where the genome sequence is complete. the degree of gene clustering and predominant chromosomal location for each family is summarized in additional file and color-coded positions of genes in all families are shown in additional file most genes in each family were located on a single chromosome. these patterns are consistent with an ancestral gene on one chromosome and largely local gene duplication events giving rise to the family over time. even at the superfamily level, each of which presumably arose from an even older ancestral gene, this chromosome bias is apparent: most str and srg superfamily genes are located on chromosome v and most sra superfamily members are located on chromosome ii. detailed inspection of protein trees and gene locations suggests that most exceptions to genes remaining on their ancestral chromosome arose by transposition of single genes followed by further duplication at the new chromosomal home. these results support the conclusion that evolutionarily retained translocation events are extremely rare in caenorhabditis species and that the chromosome is a meaningful unit of long-term evolution in nematodes. similar inferences are published for the srh, str, and srj families. lack of evidence for positive selection genes in the srz chemoreceptor family are under positive selection in both c. elegans and c. briggsae. in that work, maximum likelihood dn/ds tests were conducted on paralog samples from most of the chemosensory receptor families described here. we carried out additional systematic tests among all chemoreceptor families. these analyses confirmed positive selection in the srz family and produced no clear evidence of positive selection in any of the other families. it is important to note that the dn/ds method used for these tests is sensitive only to persistent long-term positive selection. methods for testing more recent selection events are not currently applicable with the limited population sequence data available in caenorhabditis. promoter e-box distribution one possible selective force for expansion in gene number is an increased need for diversified chemosensory receptors expressed in specific sensory neurons. little is known about how promoter sequences control cell-type specific expression of chemoreceptor genes in c. elegans. however, one promoter sequence, called the e-box, has been shown to be enriched in srh and sri chemoreceptor genes and to drive expression in the chemosensory neuron adl. to test the distribution of e-boxes among all chemoreceptor genes, we extracted promoter sequences that include chemoreceptor family loci. to reduce non-specific noise, analysis was restricted to the region from - to - from the predicted translation start, where e-boxes are concentrated. each promoter was scanned for its best match to an e-box profile and the best-scoring matches were analyzed for their distribution among gene families. there was strong enrichment of e-boxes in chemoreceptor genes as a whole, and this was due entirely to high match frequencies in four families. the other chemoreceptor families had an e-box match frequency similar to the genome as a whole; we conclude that few if any genes in these families are likely to be regulated by e-box binding factors. the sre, srh, sri, and srz families were strikingly enriched in e-boxes, with 25% to 50% of their promoters in the high-scoring set. if e-box driven expression in adl neurons generalizes to the other chemoreceptor families, then adl likely expresses about chemoreceptors, largely from the sre, srh, sri, and srz families. these four families are very diverse, representing three different superfamilies. these results suggest that the function of adl sensory neurons requires expression of a large and diverse set of chemoreceptors. bitter taste sensory neurons in mammals and insects also express multiple receptors, an unusual pattern that may be important for broadly tuned aversion to noxious compounds. we speculate that the aversion-mediating neuron adl has a similarly broad sensitivity in nematodes. e-box hits are the highest scoring motif matches in promoters from - to - from the translation start codon. percent values based on zero or one hit are in parentheses to indicate high uncertainty. the uncorrected p-value shown was determined by comparison with the number of hits in all promoters. after bonferoni correction for multiple testing the sru and srw p-values are not significant. the str and srj families are closely related and were analyzed together. a few small chemoreceptor families are not shown individually, so numbers shown do not add up to the sr total. sr, all members of putative chemoreceptor families; na, not applicable; ns, not significant. brief family summaries sra superfamily sra family this small published family includes a single large gene expansion in c. elegans, all of which are in a cluster on the right arm of chromosome i. this expansion probably arose following the transposition of a single sra gene from chromosome ii, where most other members of the family reside. apart from this part of the family, there is a relatively high frequency of orthology, suggesting that many sra gene functions are evolutionarily stable. most sra proteins have two potential disulfide bonds that could join extracellular domain with and domain with. five sra proteins lack the first cysteine pair, which suggests this pairing pattern. there are no strongly characteristic sequence motifs conserved in the family. the sra, srab, and srb families are closely related and a few genes are difficult to place in a specific family. the family corresponds to existing pfam profile pf srab family this small published family includes a single large gene expansion in c. elegans, all but one of which are in a cluster on the left arm of chromosome v. apart from this part of the family, there is a relatively high frequency of orthology, suggesting that many srab gene functions are evolutionarily stable. srab proteins have two conserved potential disulfide bonds in the same positions as those in the sra family. there is a protein segment in and near tm domain that contains several highly conserved h, n, r, and d residues, which is characteristic of the srab family. the family has been assigned to the new pfam profile pf also see the sra family notes above. srb family this small family includes no large c. elegans gene expansions, but has one modest expansion in c. remanei and occasional duplications and losses throughout the tree. the overall frequency of orthologs is relatively high, suggesting that many srb gene functions are evolutionarily stable. there is one conserved potential disulfide bond that could join extracellular domains and in approximately the same position as one of the potential disulfide bonds in the sra and srab families. at or near the inner end of tm domain there is a nearly invariant fhxn sequence, which is shared with some srab and sre proteins but is otherwise characteristic of the srb family. the family corresponds to existing pfam profile pf also see the sra family notes above. sre family this medium-sized family includes two modest gene expansions in c. elegans, each located in a cluster on the right arm of chromosome ii. in other parts of the tree there is a high frequency of orthology, suggesting that many sre gene functions are evolutionarily stable. there are no conserved potential disulfide bonds in the family. most sre proteins share the fhxn sequence with srb proteins and there is a highly conserved r/kfqxxen sequence in intracellular loop three that is characteristic of the family. there is marked variation in the length of the second extracellular loop, with about half of sre proteins having an approximately amino acid insertion relative to the others. this variant is found in all three caenorhabditis species and correlates with other sequence characters in the protein tree. the family corresponds to existing pfam profile pf srg superfamily srg family this medium-sized family includes two modest gene expansions in c. elegans, one in a cluster on the left arm of chromosome v and another mostly in a cluster on the left arm of chromosome ii. there are lower rates of gene duplication and gene loss in most of the rest of the tree, giving the family as a whole a moderately high frequency of orthologs. there is a single potential disulfide bond in about half of srg proteins that could join extracellular domains and. there is a nearly invariant w residue near the inside end of tm domain that is characteristic of the family. the family corresponds to existing pfam profile pf srt family this published family is medium sized and gene duplications and losses are fairly evenly distributed across the protein tree, with several small expansions in c. elegans, all located in clusters on the left arm of chromosome v. there is a relatively low frequency of orthologs. there is one potential disulfide bond in most srt proteins, which could join extracellular domains and. there are two nearly invariant w residues, one in tm domain and another in tm domain which are characteristic of the family. the family has been assigned to the new pfam profile pf sru family instability in this medium-sized family is fairly evenly distributed across the protein tree, with several small expansions in c. elegans, located mostly in clusters on the left and right arms of chromosome v. the family is notably larger in c. remanei, mostly as a result of two large expansions on the tree. the c. remanei assembly is not adequate to fully assess genome clustering of these two gene expansions, but there is clearly some clustering on specific supercontigs. there is a relatively low frequency of orthologs. there is one potential disulfide bond in all but a few sru proteins that could join extracellular domains and. there is a highly conserved protein segment in and near the inner end of tm domain that is characteristic of the family. the family corresponds to existing pfam profile pf srv family this medium-sized family includes a single small gene expansion in c. elegans that is in a cluster near the center of chromosome iv. c. remanei has several small expansions scattered across the tree. the rest of the tree is characterized by modest frequencies of gene duplication and loss, giving an overall frequency of orthology that is relatively low. there are no conserved potential disulfide bonds in the family and no other obvious distinguishing sequence characteristics. the family has been assigned to the new pfam profile pf srx family this large family includes several modest gene expansions in c. elegans, mostly in gene clusters on the left and right arms of chromosome v and on the left arm of chromosome ii. outside of these regions of the tree there are many orthologs, giving the family overall an average frequency of orthology. there is one conserved potential disulfide bond between two residues in extracellular domain. there is a nearly invariant nr motif in tm domain that is found only in srx and srxa proteins. the family has been assigned to the new pfam profile pf srxa family this small family includes a relatively small number of gene duplications and losses spread evenly across the tree, giving the family overall a relatively high frequency of orthologs and suggesting that many srxa functions are evolutionarily stable. all srxa proteins have one potential disulfide bond between two residues in extracellular domain, a characteristic that is shared only with the related srx family. the family corresponds to existing pfam profile pf also see the srx family notes above. str superfamily srd family this medium-sized family includes two moderately large gene expansions in c. elegans. one expansion corresponds to a gene cluster on the right arm of chromosome v, but the other includes genes on chromosomes iii and v and presumably involves one or more transposition events. outside of these parts of the tree there are relatively few gene duplications and losses, giving the family as a whole a moderate frequency of orthologs. most srd proteins have one potential disulfide bond between two residues in extracellular domain a pattern that is found only in this family. it shares with the srh and sri families a highly conserved pyr sequence at or near the inner end of transmembrane domain the family has been assigned to the new pfam profile pf srh family this published family is the largest chemoreceptor gene family in c. elegans. the tree includes several large to moderately large gene expansions in c. elegans, mostly in gene clusters on the left and right arms of chromosome v. there are no conserved potential disulfide bonds. there is a conserved hg motif in tm domain that characterizes most of the family. several other short sequence motifs are shared only with the sri family. the family has been assigned to the new pfam profile pf sri family this medium-sized family is closely related to the srh family, and includes several moderately large gene expansions in c. elegans, mostly in gene clusters in several places in the genome. sri- and sri- are an outgroup to the rest of the family and in some analyses tree with the srh family. there are no conserved potential disulfide bonds. there is a conserved cf motif at or near the inner end of tm domain that characterizes most members of the family. the family has been assigned to the new pfam profile pf also see srh family notes. srj family this medium-sized published family is closely related to the str family. the family includes one large gene expansion in c. elegans mostly in one gene cluster on the left arm of chromosome v. most of the rest of the tree includes lower frequencies of gene duplications and losses and the family overall has few orthologs. srj- is highly divergent from all other srj genes but belongs in this family based on rooted trees. there are no conserved potential disulfide bonds. srj proteins can be distinguished from str proteins based on a nearly invariant rc motif near the center of tm domain the family has been assigned to the new pfam profile pf str family this published family is the second largest chemoreceptor gene family in c. elegans. there are several moderately large gene expansions in c. elegans, mostly in gene clusters on the right arm of chromosome v, and several gene expansions in c. briggsae and c. remanei. most of the rest of the tree is also characterized by a substantial frequency of gene duplications and losses, and overall the family has few orthologs. this family includes odr- the only chemoreceptor with a known specific function. odr- is on a relatively stable part of the protein tree and has orthologs in c. briggsae and c. remanei. there are no conserved potential disulfide bonds but there are several nearly invariant residues in and near tm domain that are shared only with the srj family. the family has been assigned to the new pfam profile pf other families srbc family this medium-sized family is dominated by three large gene expansions in c. elegans, mostly in gene clusters in several places on chromosome v. there are also gene expansions in c. briggsae and c. remanei but they involve fewer genes, so that the srbc family is much larger in c. elegans. there are very few orthologs in the family. there are two invariant potential disulfide bonds, both among residues in extracellular domain a pattern that is characteristic of the family. the spacing between these cysteine residues is nearly invariant but there is substantial variation in the specific sequences. it seems likely that the disulfides form a framework for a ligand-binding domain unique to this family. the family has been assigned to the new pfam profile pf srsx family this small family includes a few recent gene duplications in c. elegans, mostly in local pairs on the right arm of chromosome v. most of the tree has very little gene duplication and loss, giving the family overall a relatively high frequency of orthologs and suggesting that many srsx functions are evolutionarily stable. there is one invariant potential disulfide bond that could join extracellular domains and. about 15% of srsx proteins have an approximately amino acid insertion in intracellular domain that is highly conserved, suggesting a distinct g-protein interaction for this group. there is a nearly invariant gn sequence near the middle of tm domain that is characteristic of the family. the family has been assigned to the new pfam profile pf srw family this large family is the only one with a clear sequence relationship to other known gpcr proteins: it is weakly but clearly related to several groups of neuropeptide receptors, including the myosuppressin receptor family in insects and the fmrfamide receptor family in nematodes. despite this relationship, it is unlikely that srw proteins function as neuropeptide receptors because they are subject to a high rate of gene duplication and loss, in stark contrast to members of the known peptide receptor families. we speculate that srw proteins function as receptors for environmental peptides. the family includes several moderate to large gene expansions in c. elegans and similar, albeit smaller, expansions in c. briggsae and c. remanei. the expansions in c. elegans correspond to several gene clusters mostly on the left and right arms of chromosome v. there is one highly conserved potential disulfide bond that could link extracellular domains and. there are two nearly invariant h residues that are characteristic of the family, one in tm domain and the other in tm domain the family corresponds to existing pfam profile pf srz family this moderately large published family is characterized by a high rate of gene duplication and gene loss and is uniquely subject to strong positive selection on the extracellular face of the protein. the tree is dominated by gene expansions specific to each species and indicates very few orthologs. there are no conserved potential disulfide bonds and the proteins have diverse length and sequence in all four extracellular domains. there are several conserved sequence motifs in or very near tm domains that are characteristic of the family. the family has been assigned to the new pfam profile pf discussion gene number and diversity the chemoreceptor genes in caenorhabditis are strikingly abundant and diverse. in contrast, mammalian olfactory receptor genes are abundant but have relatively limited diversity, with nearly all of the genes belonging to a single gene family. for example, among putative functional olfactory receptors in human, dog, and mouse, the average pairwise amino acid identity is about refseq data not shown). this level of diversity is lower than within any single c. elegans family. fish and amphibians have more diverse olfactory receptor genes, yet even in these groups all known genes fall into a single superfamily, with significant sequence relatedness between any given pair of olfactory receptor proteins. insects have much smaller chemoreceptor gene families. caenorhabditis species have seven superfamilies of putative chemosensory receptor genes, with no significant sequence similarity across superfamilies. why do nematodes have such high diversity in their chemoreceptors? we suggest that this is a result of sensory system emphasis. unlike vertebrates and insects, nematodes lack vision and hearing. we speculate that chemosensory diversity is thus more central to nematode sensory capabilities, leading to a high genetic investment and high receptor diversity. a similar explanation for the smaller olfactory gene number in great apes relative to rodents has been proposed, based on an increased emphasis on a sophisticated visual system. the gray pawn hypothesis totaled across all families, the putative functional chemoreceptor genes account for about 7% of all c. elegans genes. despite this large genetic investment in a single class of genes, our comparison among the three sequenced caenorhabditis species and gene knockdown studies suggest that few if any of the specific genes are essential for species viability, although subsets are implicated in specific physiological processes such as maintenance of fat content. instead, individual genes are lost at a high rate and this loss is roughly compensated by duplications among the remaining genes, similar to the olfactory receptor family in mammals. one possible explanation of these patterns is that new duplicate genes diverge and confer some specific selective advantage. however, in both nematodes and mammals there is evidence for such positive selection in only small and specific subsets of chemoreceptor genes. although further analysis might reveal subtler signs of positive selection, it is unlikely to be a major evolutionary force in these families, in contrast to several other gene families in which similar dn/ds analysis reveals extensive evidence of positive selection. what else could account for these patterns? we propose that the function of most of these genes is not to be found in their individual contributions to fitness, but rather in their aggregate function in covering a large phenotype space. in this model, each gene is a nearly insignificant and faceless pawn, but in aggregate these gray pawns form an effective army. neutral processes may dominate many aspects of evolution in these families, but this alone cannot account for the retention of roughly similar numbers of genes in three highly divergent species. the gray pawn hypothesis suggests that if the number of functional genes in a family drops too much, selective pressure increases to gain new genes by duplication and divergence, and gene number increases by selective retention of new duplicates. if the number of genes increases too much, selective pressure to maintain individual genes decreases, and genes are lost by mutation and drift. the number and diversity of functional genes results from this dynamic equilibrium combined with the species-specific importance of the process mediated by the gene family. evolution of the immunoglobulin gene families in mammals may also be explained in part by the gray pawn hypothesis.  we have completed the annotation and description of all large gpcr gene families in c. elegans and its relatives c. briggsae and c. remanei. all of the families are characterized by a substantial number of nonfunctional genes in each species. the rates of gene duplication and gene loss, inferred from protein trees in each family, are substantial in all the families. despite these dynamics, evidence for positive selection is found in only one of the families. we propose the gray pawn hypothesis to explain these patterns of molecular evolution: most individual genes are of little significance, but as a group they are required to cover a broad ligand space. this is achieved by maintaining a large and diverse repertoire of chemoreceptors in which new genes arise by duplication and divergence and others are lost by mutation and genetic drift. 
based on manual curation and sequence comparisons among putative g-protein-coupled chemoreceptor genes in the nematode caenorhabditis elegans, we identified approximately genes and pseudogenes in the largest gene families, most of which fall into larger superfamilies. chemoreceptor gene families in caenorhabditis species are large and evolutionarily dynamic as a result of gene duplication and gene loss. protein trees reveal family-specific and species-specific patterns of gene duplication and gene loss. to explain these patterns, we propose the gray pawn hypothesis: individual genes are of little significance, but the aggregate of a large number of diverse genes is required to cover a large phenotype space. in the related species c. briggsae and c. remanei, we identified most or all genes in each of the families. several families include large species-specific expansions, mostly in c. elegans and c. remanei. these dynamics shape the chemoreceptor gene complements in caenorhabditis species and define the receptor space available for chemosensory responses. for most families, c. elegans has the largest number of genes and c. briggsae the smallest number, suggesting changes in the importance of chemoperception among the species. animal genomes contain families of genes encoding chemoreceptors that mediate taste, olfaction, and pheromone responses.
 chemoreceptor proteins mediate the first step in the transduction of environmental chemical stimuli, defining the breadth of detection and conferring stimulus specificity. animal genomes contain families of genes encoding chemoreceptors that mediate taste, olfaction, and pheromone responses. the size and diversity of these families reflect the biology of chemoperception in specific species.  based on manual curation and sequence comparisons among putative g-protein-coupled chemoreceptor genes in the nematode caenorhabditis elegans, we identified approximately genes and pseudogenes in the largest gene families, most of which fall into larger superfamilies. in the related species c. briggsae and c. remanei, we identified most or all genes in each of the families. for most families, c. elegans has the largest number of genes and c. briggsae the smallest number, suggesting changes in the importance of chemoperception among the species. protein trees reveal family-specific and species-specific patterns of gene duplication and gene loss. the frequency of strict orthologs varies among the families, from just over 50% in two families to less than 5% in three families. several families include large species-specific expansions, mostly in c. elegans and c. remanei.  chemoreceptor gene families in caenorhabditis species are large and evolutionarily dynamic as a result of gene duplication and gene loss. these dynamics shape the chemoreceptor gene complements in caenorhabditis species and define the receptor space available for chemosensory responses. to explain these patterns, we propose the gray pawn hypothesis: individual genes are of little significance, but the aggregate of a large number of diverse genes is required to cover a large phenotype space. 