BACKGROUND
the ais in jawed vertebrates is a sophisticated system mediated by numerous genes in specialized cells  <cit> .
because the ais is conserved widely among jawed vertebrates but not invertebrates  <cit> , it is widely held that the ais appeared suddenly, as an immunologic "big bang," in the common ancestor of jawed vertebrates  <cit> .
the recently sequenced genome of stringylocentrotus purpuratus  <cit> , one of the closest relatives of chordates, has revealed that although they lack an ais, sea urchins have almost all of the vertebrate gene repertoire, including many genes involved in the ais  <cit> .
this situation indicates that many of the genes involved in the ais arose long before the emergence of jawed vertebrates  and thereafter evolved concurrently to gain new functions in the ais at the immunologic big bang.
gene duplications are important evolutionary paths for the acquisition of new functions, because one copy of a duplicated gene can accumulate mutations and acquire novel functions while the other retains the original function.
such duplications can involve individual genes, genomic segments, or whole genomes.
because they generate a large number of gene duplicates concurrently, whole-genome duplications  are considered particularly important evolutionary events  <cit> .
it has long been hypothesized that a wgd occurred twice in early vertebrates  <cit> .
this hypothesis has recently been supported by clear patterns of four-way paralogous regions occurring throughout the human genome  <cit> .
phylogenetic analysis indicates that the occurrence of 2r-wgd preceded emergence of the ais  <cit> ; therefore, 2r-wgd may have been important in the acquisition of ais-related functions by numerous genes at immunologic big bang.
however, 2r-wgd has been directly linked to limited ais-related genes that are located in only a few paralogons in the human genome  <cit> , indicating that the genes arose from 2r-wgd.
these currently available examples are too limited to reveal the precise relationship between 2r-wgd and the acquisition of ais-related functions by the numerous genes involved in this system.
we focus here on many signaling genes in the ais and investigate the extent to which 2r-wgd contributed to their gains of function in the ais.
in addition, to provide insight into the ancestral status of the ais-related genes before 2r-wgd, we compared these genes with their orthologs in an invertebrate  that lacks an ais.
RESULTS
molecular functions of paralogs in paralogons formed in early vertebrates
by using an anchoring strategy combined with a phylogenetic approach  <cit> , we identified in the human genome  <dig> paralogons including  <dig> bv paralogous genes .
an important first step in demonstrating that 2r-wgd indeed was important in the evolution of genes involved in the ais would be to show the types of genes that were retained after duplication by 2r-wgd.
to this end, we used gene ontology  annotations to investigate the molecular roles of the bv paralogous genes.
go terms such as "regulation of biological process"  and "signal transduction"  were over-represented among the bv paralogous genes , as indicated in previous wgd studies  <cit> .
we therefore focused on genes in the ais with molecular roles related to the over-represented go terms and examined the relationship between the evolution of these genes and 2r-wgd.
* p values are calculated by using hypergeometric distribution.
a bv paralogous gene is a paralogous gene that was formed at the base of the vertebrate lineage.
detection of ais subfamilies
in the ais, signal transduction machineries play indispensable roles as mediators between the diverse extracellular stimuli received by membrane-bound receptors and various induced biological processes.
we focused on an antigen-recognition receptor pathway, chemokine receptor pathway, and cytokine receptor pathway as representatives of the various signal transduction machineries in the ais.
we prepared a list of the  <dig> families of human genes  that comprise these three signaling pathways  and that include many genes essential to the ais  <cit> .
if the genes in an ais family have emerged at or before the divergence of protostomes and deuterostomes, then the ais family will encompass one or more subfamilies, each of which will include all the descendants of a single ancestral gene in the common ancestor of protostomes and deuterostomes .
we comprehensively identified such subfamilies by applying a clustering algorithm to the complete gene sets of one invertebrate  and three vertebrates  and investigated whether any of the ais families contained one or more resulting subfamilies.
consequently, 93%  of the ais families encompassed at least one subfamily , indicating that their ancestral genes existed in the ancestral bilaterian.
however, in the case of ais families with multiple subfamilies, one or more subfamilies may not contain any ais-related genes, because each ais family can include genes that are not involved in the ais.
by using published information on the ais-related functions of each gene in the subfamilies of all ais families, we identified  <dig> subfamilies that each included at least one member gene involved in the ais ; the  <dig> subfamilies were defined as "ais subfamilies" .
because each subfamily included all the descendants of a single ancestral gene in the common ancestor of protostomes  and deuterostomes , an ais subfamily included at least one human ais gene, its paralog , and its drosophila ortholog.
timing of gene duplications in ais subfamilies
we found that 84%  of the ais subfamilies included multiple human genes , indicating that the numbers of subfamily members increased through gene duplication after the divergence of protostomes and deuterostomes.
to clarify the timing of the duplications, we conducted phylogenetic analyses of the ais subfamilies.
examination of the resulting phylogenetic trees showed that 88% of the total duplications  in the ais subfamilies occurred in the early vertebrate lineage .
genomic mapping analysis
to determine whether gene duplications in the ais subfamilies occurred as a result of 2r-wgd, we conducted a genomic mapping analysis, in which we considered that if two genes of a given paralogous pair were located in the separate two regions of a paralogon, the genes were retained as duplicates from 2r-wgd.
for example, three members of the jak  subfamily – jak <dig>  jak <dig>  and jak <dig> – are located on human chromosomes  <dig>   <dig>  and  <dig>  respectively.
the analysis revealed that the pairs of these members were part of paralogons , indicating that these genes were retained as duplicates from 2r-wgd.
to visualize the relationships of the members' locations and paralogons, we used red lines to connect the genomic map positions of the members, whereas we used gray lines to connect the genes in each bv paralogous pair of paralogons in the human genome.
the red lines lay within the groups of gray lines , indicating that all of the paralog pairs in the jak subfamily were part of paralogons.
a total of 67%  of the paralog pairs in all ais subfamilies were part of paralogons , and this rate is significantly higher than that expected due to chance .
use of large-scale microarray analysis to detect tissues and organs in which the genes in ais subfamilies are expressed at high levels
in the widely accepted "gene duplication and divergence" model  <cit> , after gene duplication, a new function will be acquired by one of the duplicates as a result of accumulated mutations while the original function is retained by the other.
because ais-related functions were acquired after 2r-wgd, ancestral non-ais-related functions might be retained by the human genes in ais subfamilies.
to investigate the functional diversity among the human genes in the ais subfamilies, we used large-scale microarray expression profiles of various human organs and tissues  <cit> .
for a broad overview of tissue- and organ-specific expression, we used a classification scheme  that is a simplified version of cell catalogue  <cit>  to classify all  <dig> tissues and organs used in the microarray data into  <dig> system-level function categories; we then assigned these categories to the human genes in the ais subfamilies: if a gene was specifically expressed in at least one of the tissues or organs in a system-level function category, then the system-level function category was assigned to the gene .
we then assigned a system-level function category to an ais subfamily if the ais subfamily included at least one member gene having the system-level function category.
consequently we found that the categories "nervous system" , "innate immunity", "muscle tissue", ais, and "blood" were significantly enriched  among ais subfamilies as compared with the overall distribution of these categories among all subfamilies  detected among the complete gene sets of three vertebrates  and one invertebrate .
this result indicates that the human genes in these ais subfamilies have not only ais-related functions but also other functions, particularly those related to the ns, innate immunity, muscle tissue, and blood, all of which can be considered as candidates of ancestral functions of ais subfamilies.
outgroup comparison for inferring the ancestral functions of genes in ais subfamilies
we assigned the categories ns, innate immunity, muscle tissue, and blood to the drosophila genes in the ais subfamilies according to their flybase annotations  <cit>  .
to infer the ancestral categories of the ais subfamilies, we used the outgroup comparison method  <cit> , which assumes that the categories shared between the ingroup and outgroup are relatively primitive with respect to those found only in the ingroup.
that is, by comparing assigned categories of human  and drosophila  genes in an ais subfamily, we assigned the shared categories across these two species as ancestral functions of the subfamily.
consequently we found that at least one category was assigned to each of 71%  of the ais subfamilies; particularly, the ns category was assigned to most  of the ais subfamilies .
furthermore, we found that the ns category was significantly over-represented among the ais subfamilies as compared with the overall distribution of this category among all subfamilies ; this over-representation of the ns category was more significant than that of any other category .
these results suggest that an ancestral function of the ais subfamilies was associated with the ns.
ais, adaptive immune system.
*p values are calculated by using hypergeometric distribution.
gene usage of ais subfamilies in the ns and ais
large-scale microarray data  <cit>  enabled us to examine the statistical significance of the enrichment of the system-level function categories among the ais subfamilies and revealed that the most significantly enriched category among the ais subfamilies was ns.
to precisely assess gene usage in the ns and ais among the ais subfamilies, we listed the ns- and ais-related functions of the human genes in the ais subfamilies according to data in the published literature .
most of the human genes in the ais families  were used in either the ns or ais, suggesting that the functions of these genes are specialized for the respective system .
in contrast,  <dig> genes  were used in both the ns and ais , although whether the genes have different or similar functions in each system is unclear.
discussion
because most traces of 2r-wgd have been deleted by subsequent gene loss and genomic rearrangement  <cit> , direct evidence linking 2r-wgd and expansion of ais subfamilies is difficult to obtain by analyzing the membership of ais subfamilies.
a 4: <dig> ratio in the number of vertebrate to invertebrate genes in a family  is considered to reflect an effect of 2r-wgd.
however, because of massive gene loss after 2r-wgd, few ais subfamilies include four human genes .
likewise, the phylogenetic timing of duplications in ais subfamilies is inconclusive, because duplications can occur on every branch.
although the number of duplications was increased somewhat in the early vertebrate lineage , this increase may simply indicate that this evolutionary period was associated with accelerated small-scale gene duplication or a reduced rate of gene loss, rather than wgd.
however, the genomic mapping analysis, which examined the relationship between the genomic map positions of members in ais subfamilies and those of bv paralogous genes in paralogons, yielded conclusive evidence of the role of 2r-wgd in the expansion of the ais subfamilies.
this analysis revealed that most of the paralogous pairs in ais subfamilies were part of paralogons in the human genome , indicating that most ais genes were retained as duplicates from 2r-wgd.
our outgroup comparison analysis suggested the category ns as the ancestral function of most ais genes , as in a previous study  <cit> .
although suggested by several previous studies  <cit> , the apparent association between the ais and ns may have arisen simply because the number of genes involved in the ns is larger than that of genes involved in other systems.
we therefore examined the relative enrichment of assigned categories among ais subfamilies as compared with the overall distribution of these categories among all subfamilies.
this analysis revealed that the ns category was significantly over-represented as an ancestral function among ais subfamilies, supporting statistically the hypothesis  <cit>  that ais-related signaling genes were ancestrally involved in the development and/or function of the ns in urbilaterians.
our study did not address the specific evolutionary roles of each round of 2r-wgd.
because of the unavailability of the genome sequence of jawless vertebrates, which presumably diverged from jawed vertebrates before the second round of 2r-wgd  <cit> , it is difficult to differentiate the effect of the first round of 2r-wgd in vertebrate genomes from the second.
it has recently been shown that jawless vertebrates evolved their own ais, which is different from the ais of jawed vertebrates  <cit> .
we anticipate that the ongoing genome project  of a jawless vertebrate  will clarify the individual roles of each round of 2r-wgd in the evolution of the different aiss of these organisms.
to date, studies using small-scale gene sets have hinted at relationships between the ais and 2r-wgd  <cit>  and between the ais and ns  <cit> .
although such studies are informative, they cannot yield conclusions that are broadly applicable to the role of 2r-wgd.
however, our use of large-scale data sets  enabled us to address these relationships in a genome-wide fashion.
in future research, integration of other large-scale data sets  into similar analyses will facilitate efforts to describe the evolution of the ais precisely and in detail and will help to further unravel the biologic significance of 2r-wgd in that process.
CONCLUSIONS
the occurrence of 2r-wgd preceding emergence of the ais enhances our understanding of the biologic significance of wgd.
comprehensive identification of the paralogons in the human genome enabled us to conduct a genomic mapping analysis to examine the relationship between 2r-wgd and the evolution of many of the genes involved in the ais.
this analysis revealed that numerous pairs of signaling genes in the ais  and their paralogs were part of paralogons, indicating that the genes were retained as duplicates from 2r-wgd.
the large-scale biologic data enabled us to examine the statistical significance of enrichment of system-level function categories among the ais subfamilies as compared with the overall distribution of these categories among all subfamilies.
this examination revealed that the category ns was significantly over-represented among ais subfamilies, supporting statistically the hypothesis that the ais-related signaling genes were involved in the nervous system of ancestral bilaterians.
our analysis uncovered the evolutionary role of 2r-wgd in duplicating numerous signaling genes used in the ancestral ns and thereafter in leading to the coordinated evolution of the resulting duplicates to gain new functions in the ais.
we believe that our findings provide a basis for further detailed exploration of the diverse roles of 2r-wgd in the evolutionary success of the vertebrate lineage.

 the ais in jawed vertebrates is a sophisticated system mediated by numerous genes in specialized cells. because the ais is conserved widely among jawed vertebrates but not invertebrates, it is widely held that the ais appeared suddenly, as an immunologic "big bang," in the common ancestor of jawed vertebrates. the recently sequenced genome of stringylocentrotus purpuratus, one of the closest relatives of chordates, has revealed that although they lack an ais, sea urchins have almost all of the vertebrate gene repertoire, including many genes involved in the ais. this situation indicates that many of the genes involved in the ais arose long before the emergence of jawed vertebrates and thereafter evolved concurrently to gain new functions in the ais at the immunologic big bang. gene duplications are important evolutionary paths for the acquisition of new functions, because one copy of a duplicated gene can accumulate mutations and acquire novel functions while the other retains the original function. such duplications can involve individual genes, genomic segments, or whole genomes. because they generate a large number of gene duplicates concurrently, whole-genome duplications are considered particularly important evolutionary events. it has long been hypothesized that a wgd occurred twice in early vertebrates. this hypothesis has recently been supported by clear patterns of four-way paralogous regions occurring throughout the human genome. phylogenetic analysis indicates that the occurrence of 2r-wgd preceded emergence of the ais; therefore, 2r-wgd may have been important in the acquisition of ais-related functions by numerous genes at immunologic big bang. however, 2r-wgd has been directly linked to limited ais-related genes that are located in only a few paralogons in the human genome, indicating that the genes arose from 2r-wgd. these currently available examples are too limited to reveal the precise relationship between 2r-wgd and the acquisition of ais-related functions by the numerous genes involved in this system. we focus here on many signaling genes in the ais and investigate the extent to which 2r-wgd contributed to their gains of function in the ais. in addition, to provide insight into the ancestral status of the ais-related genes before 2r-wgd, we compared these genes with their orthologs in an invertebrate that lacks an ais.  molecular functions of paralogs in paralogons formed in early vertebrates by using an anchoring strategy combined with a phylogenetic approach, we identified in the human genome paralogons including bv paralogous genes. an important first step in demonstrating that 2r-wgd indeed was important in the evolution of genes involved in the ais would be to show the types of genes that were retained after duplication by 2r-wgd. to this end, we used gene ontology annotations to investigate the molecular roles of the bv paralogous genes. go terms such as "regulation of biological process" and "signal transduction" were over-represented among the bv paralogous genes, as indicated in previous wgd studies. we therefore focused on genes in the ais with molecular roles related to the over-represented go terms and examined the relationship between the evolution of these genes and 2r-wgd. * p values are calculated by using hypergeometric distribution. a bv paralogous gene is a paralogous gene that was formed at the base of the vertebrate lineage. detection of ais subfamilies in the ais, signal transduction machineries play indispensable roles as mediators between the diverse extracellular stimuli received by membrane-bound receptors and various induced biological processes. we focused on an antigen-recognition receptor pathway, chemokine receptor pathway, and cytokine receptor pathway as representatives of the various signal transduction machineries in the ais. we prepared a list of the families of human genes that comprise these three signaling pathways and that include many genes essential to the ais. if the genes in an ais family have emerged at or before the divergence of protostomes and deuterostomes, then the ais family will encompass one or more subfamilies, each of which will include all the descendants of a single ancestral gene in the common ancestor of protostomes and deuterostomes. we comprehensively identified such subfamilies by applying a clustering algorithm to the complete gene sets of one invertebrate and three vertebrates and investigated whether any of the ais families contained one or more resulting subfamilies. consequently, 93% of the ais families encompassed at least one subfamily, indicating that their ancestral genes existed in the ancestral bilaterian. however, in the case of ais families with multiple subfamilies, one or more subfamilies may not contain any ais-related genes, because each ais family can include genes that are not involved in the ais. by using published information on the ais-related functions of each gene in the subfamilies of all ais families, we identified subfamilies that each included at least one member gene involved in the ais; the subfamilies were defined as "ais subfamilies". because each subfamily included all the descendants of a single ancestral gene in the common ancestor of protostomes and deuterostomes, an ais subfamily included at least one human ais gene, its paralog, and its drosophila ortholog. timing of gene duplications in ais subfamilies we found that 84% of the ais subfamilies included multiple human genes, indicating that the numbers of subfamily members increased through gene duplication after the divergence of protostomes and deuterostomes. to clarify the timing of the duplications, we conducted phylogenetic analyses of the ais subfamilies. examination of the resulting phylogenetic trees showed that 88% of the total duplications in the ais subfamilies occurred in the early vertebrate lineage. genomic mapping analysis to determine whether gene duplications in the ais subfamilies occurred as a result of 2r-wgd, we conducted a genomic mapping analysis, in which we considered that if two genes of a given paralogous pair were located in the separate two regions of a paralogon, the genes were retained as duplicates from 2r-wgd. for example, three members of the jak subfamily – jak jak and jak – are located on human chromosomes and respectively. the analysis revealed that the pairs of these members were part of paralogons, indicating that these genes were retained as duplicates from 2r-wgd. to visualize the relationships of the members' locations and paralogons, we used red lines to connect the genomic map positions of the members, whereas we used gray lines to connect the genes in each bv paralogous pair of paralogons in the human genome. the red lines lay within the groups of gray lines, indicating that all of the paralog pairs in the jak subfamily were part of paralogons. a total of 67% of the paralog pairs in all ais subfamilies were part of paralogons, and this rate is significantly higher than that expected due to chance. use of large-scale microarray analysis to detect tissues and organs in which the genes in ais subfamilies are expressed at high levels in the widely accepted "gene duplication and divergence" model, after gene duplication, a new function will be acquired by one of the duplicates as a result of accumulated mutations while the original function is retained by the other. because ais-related functions were acquired after 2r-wgd, ancestral non-ais-related functions might be retained by the human genes in ais subfamilies. to investigate the functional diversity among the human genes in the ais subfamilies, we used large-scale microarray expression profiles of various human organs and tissues. for a broad overview of tissue- and organ-specific expression, we used a classification scheme that is a simplified version of cell catalogue to classify all tissues and organs used in the microarray data into system-level function categories; we then assigned these categories to the human genes in the ais subfamilies: if a gene was specifically expressed in at least one of the tissues or organs in a system-level function category, then the system-level function category was assigned to the gene. we then assigned a system-level function category to an ais subfamily if the ais subfamily included at least one member gene having the system-level function category. consequently we found that the categories "nervous system", "innate immunity", "muscle tissue", ais, and "blood" were significantly enriched among ais subfamilies as compared with the overall distribution of these categories among all subfamilies detected among the complete gene sets of three vertebrates and one invertebrate. this result indicates that the human genes in these ais subfamilies have not only ais-related functions but also other functions, particularly those related to the ns, innate immunity, muscle tissue, and blood, all of which can be considered as candidates of ancestral functions of ais subfamilies. outgroup comparison for inferring the ancestral functions of genes in ais subfamilies we assigned the categories ns, innate immunity, muscle tissue, and blood to the drosophila genes in the ais subfamilies according to their flybase annotations. to infer the ancestral categories of the ais subfamilies, we used the outgroup comparison method, which assumes that the categories shared between the ingroup and outgroup are relatively primitive with respect to those found only in the ingroup. that is, by comparing assigned categories of human and drosophila genes in an ais subfamily, we assigned the shared categories across these two species as ancestral functions of the subfamily. consequently we found that at least one category was assigned to each of 71% of the ais subfamilies; particularly, the ns category was assigned to most of the ais subfamilies. furthermore, we found that the ns category was significantly over-represented among the ais subfamilies as compared with the overall distribution of this category among all subfamilies; this over-representation of the ns category was more significant than that of any other category. these results suggest that an ancestral function of the ais subfamilies was associated with the ns. ais, adaptive immune system. *p values are calculated by using hypergeometric distribution. gene usage of ais subfamilies in the ns and ais large-scale microarray data enabled us to examine the statistical significance of the enrichment of the system-level function categories among the ais subfamilies and revealed that the most significantly enriched category among the ais subfamilies was ns. to precisely assess gene usage in the ns and ais among the ais subfamilies, we listed the ns- and ais-related functions of the human genes in the ais subfamilies according to data in the published literature. most of the human genes in the ais families were used in either the ns or ais, suggesting that the functions of these genes are specialized for the respective system. in contrast, genes were used in both the ns and ais, although whether the genes have different or similar functions in each system is unclear. discussion because most traces of 2r-wgd have been deleted by subsequent gene loss and genomic rearrangement, direct evidence linking 2r-wgd and expansion of ais subfamilies is difficult to obtain by analyzing the membership of ais subfamilies. a 4: ratio in the number of vertebrate to invertebrate genes in a family is considered to reflect an effect of 2r-wgd. however, because of massive gene loss after 2r-wgd, few ais subfamilies include four human genes. likewise, the phylogenetic timing of duplications in ais subfamilies is inconclusive, because duplications can occur on every branch. although the number of duplications was increased somewhat in the early vertebrate lineage, this increase may simply indicate that this evolutionary period was associated with accelerated small-scale gene duplication or a reduced rate of gene loss, rather than wgd. however, the genomic mapping analysis, which examined the relationship between the genomic map positions of members in ais subfamilies and those of bv paralogous genes in paralogons, yielded conclusive evidence of the role of 2r-wgd in the expansion of the ais subfamilies. this analysis revealed that most of the paralogous pairs in ais subfamilies were part of paralogons in the human genome, indicating that most ais genes were retained as duplicates from 2r-wgd. our outgroup comparison analysis suggested the category ns as the ancestral function of most ais genes, as in a previous study. although suggested by several previous studies, the apparent association between the ais and ns may have arisen simply because the number of genes involved in the ns is larger than that of genes involved in other systems. we therefore examined the relative enrichment of assigned categories among ais subfamilies as compared with the overall distribution of these categories among all subfamilies. this analysis revealed that the ns category was significantly over-represented as an ancestral function among ais subfamilies, supporting statistically the hypothesis that ais-related signaling genes were ancestrally involved in the development and/or function of the ns in urbilaterians. our study did not address the specific evolutionary roles of each round of 2r-wgd. because of the unavailability of the genome sequence of jawless vertebrates, which presumably diverged from jawed vertebrates before the second round of 2r-wgd, it is difficult to differentiate the effect of the first round of 2r-wgd in vertebrate genomes from the second. it has recently been shown that jawless vertebrates evolved their own ais, which is different from the ais of jawed vertebrates. we anticipate that the ongoing genome project of a jawless vertebrate will clarify the individual roles of each round of 2r-wgd in the evolution of the different aiss of these organisms. to date, studies using small-scale gene sets have hinted at relationships between the ais and 2r-wgd and between the ais and ns. although such studies are informative, they cannot yield conclusions that are broadly applicable to the role of 2r-wgd. however, our use of large-scale data sets enabled us to address these relationships in a genome-wide fashion. in future research, integration of other large-scale data sets into similar analyses will facilitate efforts to describe the evolution of the ais precisely and in detail and will help to further unravel the biologic significance of 2r-wgd in that process.  the occurrence of 2r-wgd preceding emergence of the ais enhances our understanding of the biologic significance of wgd. comprehensive identification of the paralogons in the human genome enabled us to conduct a genomic mapping analysis to examine the relationship between 2r-wgd and the evolution of many of the genes involved in the ais. this analysis revealed that numerous pairs of signaling genes in the ais and their paralogs were part of paralogons, indicating that the genes were retained as duplicates from 2r-wgd. the large-scale biologic data enabled us to examine the statistical significance of enrichment of system-level function categories among the ais subfamilies as compared with the overall distribution of these categories among all subfamilies. this examination revealed that the category ns was significantly over-represented among ais subfamilies, supporting statistically the hypothesis that the ais-related signaling genes were involved in the nervous system of ancestral bilaterians. our analysis uncovered the evolutionary role of 2r-wgd in duplicating numerous signaling genes used in the ancestral ns and thereafter in leading to the coordinated evolution of the resulting duplicates to gain new functions in the ais. we believe that our findings provide a basis for further detailed exploration of the diverse roles of 2r-wgd in the evolutionary success of the vertebrate lineage. 
phylogenetic analysis indicates that emergence of the ais followed the occurrence of two rounds of whole-genome duplication in early vertebrates, but little direct evidence linking these two events is available. the adaptive immune system of jawed vertebrates is a sophisticated system mediated by numerous genes in specialized cells. to analyze the evolution of the many genes related to signal transduction in the ais, we identified groups of genes that included at least one human ais gene, its paralogs, and its drosophila ortholog. outgroup comparison analysis revealed that subfamilies in which human and fly genes shared a nervous system-related function were significantly enriched among ais subfamilies, as compared with the overall incidence of shared nervous system-related functions among all subfamilies in bilaterians. the current results suggest that 2r-wgd played a major role in the duplication of many signaling genes, ancestrally used in nervous system development and function, that were later co-opted for new functions during evolution of the ais. genomic mapping revealed that numerous pairs of ais genes and their paralogs were part of paralogons – series of paralogous regions that derive from a common ancestor – throughout the human genome, indicating that the genes were retained as duplicates after 2r-wgd. this finding statistically supports the hypothesis that ais-related signaling genes were ancestrally involved in the nervous system of urbilaterians. we examined the relationship between 2r-wgd and the gain of ais-related functions by numerous genes.
 the adaptive immune system of jawed vertebrates is a sophisticated system mediated by numerous genes in specialized cells. phylogenetic analysis indicates that emergence of the ais followed the occurrence of two rounds of whole-genome duplication in early vertebrates, but little direct evidence linking these two events is available.  we examined the relationship between 2r-wgd and the gain of ais-related functions by numerous genes. to analyze the evolution of the many genes related to signal transduction in the ais, we identified groups of genes that included at least one human ais gene, its paralogs, and its drosophila ortholog. genomic mapping revealed that numerous pairs of ais genes and their paralogs were part of paralogons – series of paralogous regions that derive from a common ancestor – throughout the human genome, indicating that the genes were retained as duplicates after 2r-wgd. outgroup comparison analysis revealed that subfamilies in which human and fly genes shared a nervous system-related function were significantly enriched among ais subfamilies, as compared with the overall incidence of shared nervous system-related functions among all subfamilies in bilaterians. this finding statistically supports the hypothesis that ais-related signaling genes were ancestrally involved in the nervous system of urbilaterians.  the current results suggest that 2r-wgd played a major role in the duplication of many signaling genes, ancestrally used in nervous system development and function, that were later co-opted for new functions during evolution of the ais. 