BACKGROUND
toll-like receptors  perform a vital role as sentinels of the innate immune system in their host organism through the recognition of pathogen associated molecular patterns .
pamp recognition is achieved through binding to the extracellular leucine rich repeat  containing domain, specific to each receptor type  <cit> .
in response to ligand binding, signalling is activated by the cytoplasmic toll/interleukin i resistance  domain resulting in an inflammatory response and release of inflammatory cytokines.
this is an evolutionarily highly conserved domain, present in a wide range of taxa from plants to animals indicating the first toll-like gene existed in the unicellular ancestor of plants and animals over  <dig> billion years ago  <cit> .
the first toll gene to be discovered was in drosophila melanogaster, which encodes a receptor involved in developmental patterning, but more importantly also in resistance to fungal infections  <cit> .
in animals, these genes can be sub-divided into two simple categories based on taxonomy; the toll genes of protostome taxa and the tlrs of deuterostomes  <cit> .
it is likely that toll originally had a purely developmental role and that this during the course of evolution became combined with disease resistance functions in coelomate organisms, to protect the body cavity from infectious pathogens  <cit> .
although tlrs have been identified in many animal lineages the majority of research has been carried out on eutherian mammals, especially humans and mice  <cit> .
in recent years, however, advances in teleost genomics have led to the rapid discovery of tlr genes in fugu and zebrafish  <cit> .
moreover, recent analyses of chicken ests and genome sequences have revealed tlrs in chicken  <cit> .
gene gain and gene loss are important evolutionary processes, especially with respect to gene family dynamics  <cit> .
gene duplication is believed to be the principal cause by which new genes are created.
gene gain can occur either on a large scale from a whole genome duplication or on a small scale when chromosomal sections or individual genes are copied.
single gene duplications occur by tandem duplications as a result of either unequal crossing over or by retrotransposition  <cit> .
retrogenes are clearly recognised by their lack of introns and are often found to be non-functional paralogs, as regulatory sequences are usually missing  <cit> .
gene gain is frequently followed by differential gene loss either by mutation in one gene isoform leading to the creation of a pseudogene or by complete deletion  <cit> .
it is believed that gene deletion closely follows duplication in the majority of cases  <cit> .
the process of gene gain and gene loss is believed to occur at a constant rate with the exception of an episode of whole genome duplication in vertebrates ~ <dig> mya  <cit> .
on occasions where both genes remain active one may be freed from purifying selection and be able to evolve a new function, or it may retain its original function but alter its tissue expression profile  <cit> .
to define tlr orthologs and uncover the pattern of gene duplication and/or gene loss in vertebrate lineages, we performed a phylogenetic analysis of all known tlr sequences.
our results confirm all of the chicken tlr genes found in previous studies  <cit>  and do not detect any more.
our findings also confirm and extend the sequence and structure of these genes and corroborate the presence of duplicated genes as being separate genes and not splice variants of the same gene.
aims
the major aims of this investigation were to determine what tlrs are present in chicken and determine where unmapped tlr genes are located.
these data then allowed us to define orthologs in other species and examine tlr gene family evolution using phylogenetic methods.
these analyses allowed us to characterise the variation within and between vertebrate lineages, as well as the dynamics of tlr gene gain/gene loss.
in addition, tlr gene nomenclature issues were also addressed.
RESULTS
definition of orthologs and tlr gene nomenclature
sequences newly identified as tlrs and those that have previously been named are listed in table  <dig>  suggested alterations to nomenclature are devised in accordance with the standards of human genome nomenclature.
of particular note is the sequence genbank: ay <dig>  which has sometimes previously been described as mouse tlr <dig>  <cit>  although it shares its sequence with genbank: ak <dig>  ak <dig> and ay <dig>  all of which are described as tlr <dig>  phylogenetic and structural analyses combined with gene mapping data in mouse and rat have led to this investigation regarding ay <dig> as mouse tlr <dig>  our phylogenetic analyses showed the lethenteron japonicum  tlrs to be duplicated forms of tlr <dig> and various fish tlrs to be tlr <dig>  a tlr <dig> in xenopus laevis was also defined .
current name indicates those used by this paper following detailed phylogenetic and structural analyses.
tlr gene discovery in chicken and other vertebrates
we used a total of  <dig> non-redundant tlr sequences  in this study, representing  <dig> tlr genes from  <dig> different species as described in table  <dig>
tlrs are listed in numerical order along with pseudonyms found by this investigation and example genbank accession numbers.
taxonomic coverage is inferred by the presence of sequences found in this investigation and corresponding phylogenetic data.
the pamp and pathogen data are reported as found in the cited literature.
* incorrectly described as tlr <dig> in the literature
this investigation confirms the presence of the  <dig> tlrs in the chicken found in previous studies  <cit>  and found no further tlrs.
this is the same number as found in human and two fewer than in mouse.
chicken tlrs  <dig>   <dig>   <dig> and  <dig> are directly orthologus to those found in other vertebrates.
the duplicated genes, tlrs 2a and 2b found in the chicken are both orthologs of the single tlr <dig> of mammals.
chicken tlr <dig> is an ortholog of tlr <dig> in fish and amphibians.
it appears that tlrs 1la, 1lb and  <dig> are unique to birds .
the chicken tlr repertoire contains tandem duplications of the genes for tlrs 1l and  <dig>  <cit> .
a partial sequence of chicken tlr1lb has been described previously by yilmaz et al.
<cit> .
using rt-pcr we have sequenced and cloned the full length mrna .
this sequence includes two further exons which, although transcribed, are not translated.
using synonymous substitution rates  we estimated the gene duplication events that gave rise to tlr1la, 1lb and 2a, 2b to have taken place  <dig> and  <dig> mya respectively.
the chicken is also found to be missing a number of tlrs, which are present in most mammals.
the tlr <dig>   <dig> and  <dig> subfamily is present in fish and mammals but is only represented by tlr <dig> in the chicken  <cit> .
tlr <dig> is present as a pseudogene  <cit>  and tlr <dig> has been deleted.
genetic mapping of chicken tlr1la and b genes
the chromosomal locations of most chicken tlrs are known  <cit> , with the exception of tlr1la and tlr1lb which, based on a single sequence contig  map close together on an unmapped chromosome  <cit> .
sequencing of a genomic fragment from tlr1lb locus from parents of the east lansing reference mapping cross, identified two snps in tlr1lb.
genetic linkage analysis of tlr1lb showed linkage to chromosome  <dig> .
structural analysis of proteins and selective constraints on functional domains
comparisons of structural predictions for the chicken tlrs show them to have a varied structure  <cit> .
the newly discovered tlr <dig> has the unusual feature of many lrrs clustered towards the c-terminus of the molecule and few at the n-terminus .
tlr <dig> has the uncharacteristic feature of a splice variant with two predicted trans-membrane  domains only previously found in mammalian tlr <dig> .
the predicted structure of the tlr1lb fragment described by yilmaz et al.
<cit>  is shown to have a further predicted tm domain towards the n-terminus whereas the majority of tlrs have a single tm domain between the extracellular and intracellular domains .
the predicted structure of this tlr is otherwise very similar to tlr1la only missing the two lrrs at the n-terminus .
by contrast the duplications of tlr <dig> are marked by the absence of an lrr from tlr2a, which is present in all other known tlr2s .
phylogeny of vertebrate tlrs
molecular phylogenies based on sequence alignments are only as accurate as the alignment data from which they are produced; consequently it is important that the alignment data quality is determined  <cit> .
in order to assess the alignment's tree like structure, likelihood mapping was carried out and showed the sequence alignment data to have strong phylogenetic signal .
both phylogenetic methods used produced near identical topologies, which were strongly supported with bootstrapping .
close up images of individual clades in fig.
<dig> are shown in additional files  <dig>   <dig> and  <dig>  close up images of individual clades in fig.
<dig> are shown in additional files  <dig>   <dig> and  <dig>
the majority of tlrs form specific clades, with each species containing one copy of each receptor .
the main exception to this trend is the assemblage of receptors named tlr <dig>  which have independently evolved in fish, xenopus, chicken, opossum and eutherian mammals .
these genes are not orthologs but have often been given the same name, which creates some confusion .
the same is true for tlr5s in fish, where a soluble tlr <dig> has independently evolved in different fish species  <cit>  .
the presence of most tlr gene families across so great a range of vertebrate taxa indicates that the majority of tlr families must have been established prior to the evolutionary radiation of vertebrates in the cambrian period 540- <dig> mya  <cit> .
phylogenetic analyses show that chicken tlr <dig> is an ortholog of fugu and xenopus tlr <dig>  chicken tlr <dig> shares a clade with tlrs  <dig>   <dig>   <dig>   <dig> and  <dig> , but the precise nature of this relationship remains unresolved due to the small number sequences available.
tlrs  <dig>   <dig>   <dig>   <dig> and  <dig> form a clade that mainly follow the expected species tree; the exceptions to this being the loss of chicken tlrs  <dig> and  <dig> and the gain of soluble versions of tlr <dig> in teleost fish.
gene gain has been comparatively common in the clade consisting of tlrs  <dig>   <dig> and the  <dig>   <dig>   <dig> assemblage.
the tlrs in this clade are found to respond to bacterial pathogens, and in mammals and chicken have been shown to function heterodimerically  <cit> .
no specific ligand has been identified for the clade containing tlr  <dig>   <dig> and  <dig> .
the neighbour joining tree  attaches the tlr 11– <dig> clade to this group.
the positioning of this clade is not as well supported by neighbour joining as it is using maximum parsimony, which places the tlr11– <dig> clade basal to the tlr <dig>   <dig>   <dig>   <dig> and  <dig> group .
both the tlr11– <dig> clade and tlr13-21- <dig> clade share a history of multiple gene loss events.
tlrs  <dig>   <dig> and  <dig> have been found to have been lost from all land animals .
discussion
structural and phylogenetic analyses suggest the chicken tlr repertoire to consist of ten genes.
phylogenetic analyses show these to consist of six orthologs of mammals and fish, one fish ortholog and three unique to chicken.
chromosome mapping of chicken tlr genes shows them to be in syntenic positions compared to other animals.
half of the chicken tlr genes are located on chromosome  <dig> .
this large proportion of tlr genes on a single chromosome is due in part to the gene duplication tlrs 1l and  <dig> in the chicken .
the homologues of these tlr genes in the human also map to a single chromosome  <cit>  although this tendency is not found in other vertebrates.
comparison of the predicted structure of toll-like receptors
from protein alignments as well as smart predictions it is evident that the tir domain is highly conserved across many different receptor families whereas the extracellular lrr region shows great variation in the positioning of lrrs between receptors although a strong level of structural conservation across taxa for a given receptor .
chicken tlr <dig> has a predicted tm domain at the c-terminus as have the majority of tlrs, but also has an extra tm domain at the n-terminus.
similarly an extra tm domain at the n-terminus tip is predicted for chicken tlr1lb as well as tlr <dig>  a receptor present only in eutherian mammals.
a tm domain is predicted at the n-terminal in fugu tlr <dig> but in this case the usual tm domain is not found at the c-terminal .
as the predicted n-terminal tm domain of tlr 1lb is also found in a number of related tlrs it is possible that this is a conserved feature.
however, the absence of such a predicted structure from all examples of tlr <dig> other than chicken may mean that this feature is a result of the smart program falsely interpreting a hydrophobic region.
a pseudogene caused by an insertion event for tlr <dig> in chicken has been identified but tlr <dig> in chicken has been lost leaving only flanking regions where the tlr <dig> locus is predicted  <cit> .
phylogenetic analyses of the toll-like receptor gene family
there have been previous phylogenetic analyses of tlr data  <cit> .
since the inclusion of more sequences improves the data quality of the alignment and consequently the phylogenies produced  <cit> , this investigation has been able to improve on phylogenetic analyses carried out by previous studies owing to the increased number of sequences available.
this is corroborated by the likelihood mapping analysis  and the high bootstrap values by both neighbour joining  and parsimony  methods for the major tlr family groups.
also very similar trees are produced from neighbour joining and maximum parsimony analyses .
gain of toll-like receptor genes
the vast majority of duplications of whole tlr genes occurring since the cambrian period have occurred in the clade containing tlrs  <dig>   <dig>   <dig> and  <dig>  the first of these took place  <dig> mya leading to the establishment of tlr  <dig> in the lineage that gave rise to the mammals.
this was followed by tlrs  <dig> and  <dig> in the eutherian mammals  <dig> mya  <cit> ; data which are supported by figures  <dig>   <dig> and roach et al.
<cit> .
a further duplication has occurred  <dig> mya in the lineage that gave rise to modern birds, leading to avian tlr1la and tlr1lb followed by the latest duplication  <dig> mya producing tlr2a and tlr2b in the chicken.
the duplication of tlr <dig> in the chicken lineage is not a unique event, other independent duplication events of tlr <dig> have occurred in x. tropicalis , the american alligator  <cit>  and the ancestor of marsupial and eutherian mammals  <cit>  the timing of these duplications, however, remains unknown.
all of the lrrs of tlr1la are in positions shared by closely related tlrs .
the much shorter tlr1lb has an almost identical 3' sequence and structure, but the two 5' exons and the start of the third and final exon are disrupted by stop codons and frame shifts.
a start codon is found a short way into the third exon producing a product lacking the two n-terminal lrrs and including an extra predicted tm domain.
the predicted structure of this receptor is the same as that reported in yilmaz et al.
<cit>  although the complete coding sequence of this gene is now complete  .
loss of toll-like receptor genes
compared to the paucity of gene gain in the evolution of the tlr family there are many instances of gene loss.
tlrs  <dig>   <dig> and  <dig> are not found in any land dwelling vertebrates and tlr <dig> has been lost in the majority of studied fish species, yet is preserved in land animals.
tlrs  <dig>   <dig> and  <dig> found in a small number of animals are lost in birds and many mammals, however a pseudogene for human tlr <dig> has been reported  <cit> .
although tlr <dig> of the zebrafish and tlr <dig> of x. tropicalis clearly represent ancestral sequences of tlrs  <dig> and  <dig> , there is no trace of a receptor of this kind to be found either in other fish, mammals or the chicken.
this indicates that members of this group must have been lost on more that one occasion.
functional genes for tlrs  <dig> and  <dig> are absent from the chicken; tlr <dig> is present as a pseudogene  <cit>  and tlr <dig> has been deleted from the genome.
the inclusion of certain taxonomic groups would assist in our understanding of these gene losses.
the inclusion of fish species  and more basal tetrapods  would help to give a more precise idea of the point at which tlrs  <dig>   <dig> and  <dig> were lost.
CONCLUSIONS
we found chicken to have a total of ten tlrs, the same number as in humans but with only five human orthologs.
phylogenetic analyses show tlr families to be of ancient origin, predating the divergence of major vertebrate lineages.
the chicken genome highlights recent instances of both gene loss and gene gain within tlrs which allows an insight into the patterns and processes of gene family evolution.

 toll-like receptors perform a vital role as sentinels of the innate immune system in their host organism through the recognition of pathogen associated molecular patterns. pamp recognition is achieved through binding to the extracellular leucine rich repeat containing domain, specific to each receptor type. in response to ligand binding, signalling is activated by the cytoplasmic toll/interleukin i resistance domain resulting in an inflammatory response and release of inflammatory cytokines. this is an evolutionarily highly conserved domain, present in a wide range of taxa from plants to animals indicating the first toll-like gene existed in the unicellular ancestor of plants and animals over billion years ago. the first toll gene to be discovered was in drosophila melanogaster, which encodes a receptor involved in developmental patterning, but more importantly also in resistance to fungal infections. in animals, these genes can be sub-divided into two simple categories based on taxonomy; the toll genes of protostome taxa and the tlrs of deuterostomes. it is likely that toll originally had a purely developmental role and that this during the course of evolution became combined with disease resistance functions in coelomate organisms, to protect the body cavity from infectious pathogens. although tlrs have been identified in many animal lineages the majority of research has been carried out on eutherian mammals, especially humans and mice. in recent years, however, advances in teleost genomics have led to the rapid discovery of tlr genes in fugu and zebrafish. moreover, recent analyses of chicken ests and genome sequences have revealed tlrs in chicken. gene gain and gene loss are important evolutionary processes, especially with respect to gene family dynamics. gene duplication is believed to be the principal cause by which new genes are created. gene gain can occur either on a large scale from a whole genome duplication or on a small scale when chromosomal sections or individual genes are copied. single gene duplications occur by tandem duplications as a result of either unequal crossing over or by retrotransposition. retrogenes are clearly recognised by their lack of introns and are often found to be non-functional paralogs, as regulatory sequences are usually missing. gene gain is frequently followed by differential gene loss either by mutation in one gene isoform leading to the creation of a pseudogene or by complete deletion. it is believed that gene deletion closely follows duplication in the majority of cases. the process of gene gain and gene loss is believed to occur at a constant rate with the exception of an episode of whole genome duplication in vertebrates ~ mya. on occasions where both genes remain active one may be freed from purifying selection and be able to evolve a new function, or it may retain its original function but alter its tissue expression profile. to define tlr orthologs and uncover the pattern of gene duplication and/or gene loss in vertebrate lineages, we performed a phylogenetic analysis of all known tlr sequences. our results confirm all of the chicken tlr genes found in previous studies and do not detect any more. our findings also confirm and extend the sequence and structure of these genes and corroborate the presence of duplicated genes as being separate genes and not splice variants of the same gene. aims the major aims of this investigation were to determine what tlrs are present in chicken and determine where unmapped tlr genes are located. these data then allowed us to define orthologs in other species and examine tlr gene family evolution using phylogenetic methods. these analyses allowed us to characterise the variation within and between vertebrate lineages, as well as the dynamics of tlr gene gain/gene loss. in addition, tlr gene nomenclature issues were also addressed.  definition of orthologs and tlr gene nomenclature sequences newly identified as tlrs and those that have previously been named are listed in table suggested alterations to nomenclature are devised in accordance with the standards of human genome nomenclature. of particular note is the sequence genbank: ay which has sometimes previously been described as mouse tlr although it shares its sequence with genbank: ak ak and ay all of which are described as tlr phylogenetic and structural analyses combined with gene mapping data in mouse and rat have led to this investigation regarding ay as mouse tlr our phylogenetic analyses showed the lethenteron japonicum tlrs to be duplicated forms of tlr and various fish tlrs to be tlr a tlr in xenopus laevis was also defined. current name indicates those used by this paper following detailed phylogenetic and structural analyses. tlr gene discovery in chicken and other vertebrates we used a total of non-redundant tlr sequences in this study, representing tlr genes from different species as described in table tlrs are listed in numerical order along with pseudonyms found by this investigation and example genbank accession numbers. taxonomic coverage is inferred by the presence of sequences found in this investigation and corresponding phylogenetic data. the pamp and pathogen data are reported as found in the cited literature. * incorrectly described as tlr in the literature this investigation confirms the presence of the tlrs in the chicken found in previous studies and found no further tlrs. this is the same number as found in human and two fewer than in mouse. chicken tlrs and are directly orthologus to those found in other vertebrates. the duplicated genes, tlrs 2a and 2b found in the chicken are both orthologs of the single tlr of mammals. chicken tlr is an ortholog of tlr in fish and amphibians. it appears that tlrs 1la, 1lb and are unique to birds. the chicken tlr repertoire contains tandem duplications of the genes for tlrs 1l and. a partial sequence of chicken tlr1lb has been described previously by yilmaz et al.. using rt-pcr we have sequenced and cloned the full length mrna. this sequence includes two further exons which, although transcribed, are not translated. using synonymous substitution rates we estimated the gene duplication events that gave rise to tlr1la, 1lb and 2a, 2b to have taken place and mya respectively. the chicken is also found to be missing a number of tlrs, which are present in most mammals. the tlr and subfamily is present in fish and mammals but is only represented by tlr in the chicken. tlr is present as a pseudogene and tlr has been deleted. genetic mapping of chicken tlr1la and b genes the chromosomal locations of most chicken tlrs are known, with the exception of tlr1la and tlr1lb which, based on a single sequence contig map close together on an unmapped chromosome. sequencing of a genomic fragment from tlr1lb locus from parents of the east lansing reference mapping cross, identified two snps in tlr1lb. genetic linkage analysis of tlr1lb showed linkage to chromosome. structural analysis of proteins and selective constraints on functional domains comparisons of structural predictions for the chicken tlrs show them to have a varied structure. the newly discovered tlr has the unusual feature of many lrrs clustered towards the c-terminus of the molecule and few at the n-terminus. tlr has the uncharacteristic feature of a splice variant with two predicted trans-membrane domains only previously found in mammalian tlr. the predicted structure of the tlr1lb fragment described by yilmaz et al. is shown to have a further predicted tm domain towards the n-terminus whereas the majority of tlrs have a single tm domain between the extracellular and intracellular domains. the predicted structure of this tlr is otherwise very similar to tlr1la only missing the two lrrs at the n-terminus. by contrast the duplications of tlr are marked by the absence of an lrr from tlr2a, which is present in all other known tlr2s. phylogeny of vertebrate tlrs molecular phylogenies based on sequence alignments are only as accurate as the alignment data from which they are produced; consequently it is important that the alignment data quality is determined. in order to assess the alignment's tree like structure, likelihood mapping was carried out and showed the sequence alignment data to have strong phylogenetic signal. both phylogenetic methods used produced near identical topologies, which were strongly supported with bootstrapping. close up images of individual clades in fig. are shown in additional files and close up images of individual clades in fig. are shown in additional files and the majority of tlrs form specific clades, with each species containing one copy of each receptor. the main exception to this trend is the assemblage of receptors named tlr which have independently evolved in fish, xenopus, chicken, opossum and eutherian mammals. these genes are not orthologs but have often been given the same name, which creates some confusion. the same is true for tlr5s in fish, where a soluble tlr has independently evolved in different fish species. the presence of most tlr gene families across so great a range of vertebrate taxa indicates that the majority of tlr families must have been established prior to the evolutionary radiation of vertebrates in the cambrian period 540- mya. phylogenetic analyses show that chicken tlr is an ortholog of fugu and xenopus tlr chicken tlr shares a clade with tlrs and, but the precise nature of this relationship remains unresolved due to the small number sequences available. tlrs and form a clade that mainly follow the expected species tree; the exceptions to this being the loss of chicken tlrs and and the gain of soluble versions of tlr in teleost fish. gene gain has been comparatively common in the clade consisting of tlrs and the assemblage. the tlrs in this clade are found to respond to bacterial pathogens, and in mammals and chicken have been shown to function heterodimerically. no specific ligand has been identified for the clade containing tlr and. the neighbour joining tree attaches the tlr 11– clade to this group. the positioning of this clade is not as well supported by neighbour joining as it is using maximum parsimony, which places the tlr11– clade basal to the tlr and group. both the tlr11– clade and tlr13-21- clade share a history of multiple gene loss events. tlrs and have been found to have been lost from all land animals. discussion structural and phylogenetic analyses suggest the chicken tlr repertoire to consist of ten genes. phylogenetic analyses show these to consist of six orthologs of mammals and fish, one fish ortholog and three unique to chicken. chromosome mapping of chicken tlr genes shows them to be in syntenic positions compared to other animals. half of the chicken tlr genes are located on chromosome. this large proportion of tlr genes on a single chromosome is due in part to the gene duplication tlrs 1l and in the chicken. the homologues of these tlr genes in the human also map to a single chromosome although this tendency is not found in other vertebrates. comparison of the predicted structure of toll-like receptors from protein alignments as well as smart predictions it is evident that the tir domain is highly conserved across many different receptor families whereas the extracellular lrr region shows great variation in the positioning of lrrs between receptors although a strong level of structural conservation across taxa for a given receptor. chicken tlr has a predicted tm domain at the c-terminus as have the majority of tlrs, but also has an extra tm domain at the n-terminus. similarly an extra tm domain at the n-terminus tip is predicted for chicken tlr1lb as well as tlr a receptor present only in eutherian mammals. a tm domain is predicted at the n-terminal in fugu tlr but in this case the usual tm domain is not found at the c-terminal. as the predicted n-terminal tm domain of tlr 1lb is also found in a number of related tlrs it is possible that this is a conserved feature. however, the absence of such a predicted structure from all examples of tlr other than chicken may mean that this feature is a result of the smart program falsely interpreting a hydrophobic region. a pseudogene caused by an insertion event for tlr in chicken has been identified but tlr in chicken has been lost leaving only flanking regions where the tlr locus is predicted. phylogenetic analyses of the toll-like receptor gene family there have been previous phylogenetic analyses of tlr data. since the inclusion of more sequences improves the data quality of the alignment and consequently the phylogenies produced, this investigation has been able to improve on phylogenetic analyses carried out by previous studies owing to the increased number of sequences available. this is corroborated by the likelihood mapping analysis and the high bootstrap values by both neighbour joining and parsimony methods for the major tlr family groups. also very similar trees are produced from neighbour joining and maximum parsimony analyses. gain of toll-like receptor genes the vast majority of duplications of whole tlr genes occurring since the cambrian period have occurred in the clade containing tlrs and the first of these took place mya leading to the establishment of tlr in the lineage that gave rise to the mammals. this was followed by tlrs and in the eutherian mammals mya; data which are supported by figures and roach et al.. a further duplication has occurred mya in the lineage that gave rise to modern birds, leading to avian tlr1la and tlr1lb followed by the latest duplication mya producing tlr2a and tlr2b in the chicken. the duplication of tlr in the chicken lineage is not a unique event, other independent duplication events of tlr have occurred in x. tropicalis, the american alligator and the ancestor of marsupial and eutherian mammals the timing of these duplications, however, remains unknown. all of the lrrs of tlr1la are in positions shared by closely related tlrs. the much shorter tlr1lb has an almost identical 3' sequence and structure, but the two 5' exons and the start of the third and final exon are disrupted by stop codons and frame shifts. a start codon is found a short way into the third exon producing a product lacking the two n-terminal lrrs and including an extra predicted tm domain. the predicted structure of this receptor is the same as that reported in yilmaz et al. although the complete coding sequence of this gene is now complete. loss of toll-like receptor genes compared to the paucity of gene gain in the evolution of the tlr family there are many instances of gene loss. tlrs and are not found in any land dwelling vertebrates and tlr has been lost in the majority of studied fish species, yet is preserved in land animals. tlrs and found in a small number of animals are lost in birds and many mammals, however a pseudogene for human tlr has been reported. although tlr of the zebrafish and tlr of x. tropicalis clearly represent ancestral sequences of tlrs and, there is no trace of a receptor of this kind to be found either in other fish, mammals or the chicken. this indicates that members of this group must have been lost on more that one occasion. functional genes for tlrs and are absent from the chicken; tlr is present as a pseudogene and tlr has been deleted from the genome. the inclusion of certain taxonomic groups would assist in our understanding of these gene losses. the inclusion of fish species and more basal tetrapods would help to give a more precise idea of the point at which tlrs and were lost.  we found chicken to have a total of ten tlrs, the same number as in humans but with only five human orthologs. phylogenetic analyses show tlr families to be of ancient origin, predating the divergence of major vertebrate lineages. the chicken genome highlights recent instances of both gene loss and gene gain within tlrs which allows an insight into the patterns and processes of gene family evolution. 
phylogenetic analysis shows the absence of tlrs and in chicken to be the result of gene loss. the notable exception to the tendency of gene loss in tlr evolution is found in chicken tlrs and each of which underwent gene duplication about and mya, respectively. comparative phylogenetic analysis of vertebrate tlr genes provides insight into their patterns and processes of gene evolution, with examples of both gene gain and gene loss. this study takes advantage of the recently sequenced chicken genome to determine the complete chicken tlr repertoire and place it in context of vertebrate genomic evolution. the chicken tlr repertoire consists of ten genes. phylogenetic analyses show that six of these genes have orthologs in mammals and fish, while one is only shared by fish and three appear to be unique to birds. toll-like receptors perform a vital role in disease resistance through their recognition of pathogen associated molecular patterns. recent advances in genomics allow comparison of tlr genes within and between many species. in addition, these comparisons clarify the nomenclature of tlr genes in vertebrates.
 toll-like receptors perform a vital role in disease resistance through their recognition of pathogen associated molecular patterns. recent advances in genomics allow comparison of tlr genes within and between many species. this study takes advantage of the recently sequenced chicken genome to determine the complete chicken tlr repertoire and place it in context of vertebrate genomic evolution.  the chicken tlr repertoire consists of ten genes. phylogenetic analyses show that six of these genes have orthologs in mammals and fish, while one is only shared by fish and three appear to be unique to birds. furthermore the phylogeny shows that tlr1-like genes arose independently in fish, birds and mammals from an ancestral gene also shared by tlr and tlr all other tlrs were already present prior to the divergence of major vertebrate lineages mya and have since been lost in certain lineages. phylogenetic analysis shows the absence of tlrs and in chicken to be the result of gene loss. the notable exception to the tendency of gene loss in tlr evolution is found in chicken tlrs and each of which underwent gene duplication about and mya, respectively.  comparative phylogenetic analysis of vertebrate tlr genes provides insight into their patterns and processes of gene evolution, with examples of both gene gain and gene loss. in addition, these comparisons clarify the nomenclature of tlr genes in vertebrates. 