BACKGROUND
a defining feature of the early development of non-eutherian vertebrates is a cleidoic egg endowed with variable amounts of yolk.
until very recently, the major products of the vitellogenin  genes that encode yolk proteins have been considered to be simple precursors of the energy reserve of vertebrate eggs, but the latest studies have demonstrated several non-nutritional roles for vtg  <cit> .
similarly, the recent observation that remnants of vtg genes exist in eutheria, including humans, but have sequentially been lost through co-evolution with casein genes  <cit>  elegantly demonstrated that the three known vtg genes in birds represent a conserved gene complement.
in a previous study, finn & kristoffersen  <cit>  proposed a model for the evolution and neofunctionalization of vtg genes in acanthomorph teleosts.
we identified vtgc as an ancestral gene, and argued that the dual vtgaa/vtgab system, first noted by la fleur et al.
<cit>  was derived from a single form, the a-type vtg.
in this model, the separation of the vtgc- and vtga-type genes occurred following the second round  of whole genome duplication .
subsequently vtga duplicated and formed paragolous vtgaa and vtgab genes in acanthomorph teleosts.
this phylogenetic model has been corroborated by other investigators  <cit> .
most recently however, babin  <cit>  has provided a syntenic map of vertebrate vtg genes, which shows that the three forms of vtg are encoded in a vtg gene cluster  in non-eutherian vertebrates.
a major goal of the present study was to integrate the statistical, biochemical and physical models of vtg gene evolution in vertebrates.
through a series of studies, we and other laboratories have shown that the pelagic nature of a marine teleost egg is an evolved feature  <cit>  that primarily results from the maturational influx of water due to differential degradation of vtgaa-type yolk proteins , and the temporal insertion of novel aquaporins  in the microvillous portion of the plasmalemma  <cit> .
the neofunctionalization of the vtgaa form in acanthomorph teleosts, has sensitized the heavy chain domain  to catheptic proteolysis that generates a large organic osmolyte pool of free amino acids  in the ovulated egg  <cit> .
in contrast the lvh domains derived from vtgab and vtgc genes may be partially cleaved, but remain mostly intact following the maturational proteolytic event, and thus contribute minimally to oocyte hydration  <cit> .
in teleosts that spawn benthic eggs , a character that we have argued to be the ancestral condition due to an ancient freshwater heritage , yps may be cleaved or partially processed to generate a small pool of faa during oocyte hydration  <cit> .
in order to reconcile the differences in the phylogenetic, biochemical and syntenic models we have examined the evolution and expression of the vtg gene complement in modern  and old  teleosts that spawn pelagic and benthic eggs.
we were interested in determining how the expression of different vtg transcripts relates to the character of the egg in a single family of closely related teleosts, and whether lineage-specific gene duplication resulted in neofunctionalization of the aa-type vtgs.
to determine the orthologies and ancestry of the novel labrid vtg genes cloned in the present study, we anchored the results of phylogenetic inference with the syntenic arrangement of genomic vtgs in model vertebrates.
this approach allowed us to identify the proto-chromosomal origin of the vgc that is conserved between the actinopterygii  and the sarcopterygii .
it further allowed us to conclude that neofunctionalization of the vtgaa genes in acanthomorph teleosts occurred long after the duplication event.
RESULTS
multiple transcripts
a total of eleven vtg transcripts  were cloned from vitellogenic livers of the labrid species investigated .
ncbi blast searches of the deduced proteins verified that all sequences are members of the vtg family.
four distinct vtg cdna sequences were cloned from rock cook  and goldsinny wrasse , two of which  were entirely novel for vertebrates.
three transcripts  were obtained from cuckoo wrasse .
repeated attempts using gene-specific primers to extract a second vtgab-type transcript from livers of cuckoo wrasse did not yield any novel transcripts.
the deduced amino acid sequences of the full-length rock cook and cuckoo wrasse vtgs revealed that the vtgaa and vtgab products are complete pentapartite type proteins  while vtgc belongs to the phosvitinless class of vtg   <cit> .
since it is now postulated that the vertebrate vtg gene complement represents a conserved cluster in sarcopterygii  and actinopterygii   <cit> , we aligned the longest partial segment of the labrid sequences to the genomic variants in chicken  and medaka , respectively .
relative homology scores of the aligned amino acids and codons revealed three forms with highest identity to medaka olvtgaa <dig>  olvtgab and olvtgc, respectively .
the relationship between the teleost and chicken genes was less obvious, with essentially equal identity scores for the ggvtgii and ggvtgiii products.
interestingly, ggvtgi had slightly lower scores to the teleost c-type products compared to the aa- or ab-type products over the aligned lvh sub-region .
this suggests that despite their orthology  the minor ggvtgi and vtgc genes have functionally diverged in association with the loss of the phosvitin  and c-terminal domains in teleosts.
within the labrid species, sequences had highest identities to their homologs in closely related goldsinny wrasse.
since only a single ab-type transcript was detected in cuckoo wrasse, and it showed 100% identity to the novel cevtgab <dig> and crvtgab <dig> sequences over the ~ <dig> aligned aa and ~ <dig> aligned nt , we named the cuckoo wrasse ab-type sequence lmvtgab <dig>
to further classify the labrid vtgs, we conducted large-scale phylogenetic analyses of the deduced amino acid and codon alignments of available  and novel genomic  variants.
for the genomic variants, we identified three genes in 3-spined stickleback , torafugu  and spotted green pufferfish , respectively, and four genes in medaka, of which two have been independently sequenced .
in zebrafish eight genes are found in the genome, of which several have been fully  <cit>  or partially sequenced  <cit> , and each of which is expressed and deposited in the growing oocyte  <cit> .
validation of the tree topology was achieved through multiple methods of phylogenetic inference.
each method consistently clustered the labrid sequences as three forms: vtgaa, vtgab and vtgc, respectively .
for bayesian, maximum parsimony and neighbour-joining analyses the majority of branches were supported by 100% posterior probabilities, and 100% bootstrap values, respectively, with only minor branch rearrangements within gene groups.
these data verify that three forms of vtg exist with acanthomorph teleosts, two within protacanthomorph teleosts  <cit> , and three within ostariophysan teleosts.
the data further revealed that a fourth novel gene in medaka is an aa-type product, and we thus classified it as olvtgaa <dig>  in a separate study, we have identified three novel gene variants in a basal clupeocephalan teleost, the atlantic herring .
these atlantic herring transcripts clustered as a basal node to all ostariophysan vtgs in full agreement with the clupeocephalan phylogenetic rank.
the present analyses thus fully corroborate our earlier study of vertebrate vtgs  <cit>  wherein the major and minor transcripts cluster according to taxonomic group.
we further verified the inferred duplication of the vtgaa and vtgab clusters using the method of zmasek and eddy  <cit> .
hence the tree topology is inconsistent with the syntenic arrangement of vertebrate vtg genes, and suggests that different functions have evolved within the major clades.
specifically for acanthomorph teleosts, the lvh-aa domains have evolved a sensitivity to acidic degradation following activation of v-class proton pumps during oocyte maturation  <cit> .
the data for labrid teleosts that spawn benthic and pelagic eggs support this view .
in order to understand the disparity between the phylogenetic and syntenic arrangement of vertebrate vtg genes, we increased the phylogenetic data set to include all known sarcopterygian vtgs , including amphibian, bird, reptile and platypus variants.
in addition we examined the syntenic positions of vtg genes within non-metatherian vertebrates.
the larger data set did not affect the topology of the teleost branches, but did place ggvtgi and the partial platypus transcript  at the base of the tetrapoda and closer to, but on a separate branch to teleost vtgc variants .
similarly, two further partial platypus transcripts  clustered as a basal node to ggvtgiii and ggvtgii, but after amphibian variants.
these findings agree with the recent studies of brawand et al.
<cit>  and babin  <cit>  who demonstrated that three genes exist in monotremata and are putative orthologs of the three bird genes.
since these latter platypus genes are not yet localized to any chromosome, but are annotated in contigs  <dig>  and  <dig> , respectively, it was not possible to discern their true orthology, or syntenic arrangement in relation to chicken.
however, by combining the results of the present phylogenetic data with the syntenic positions of the chicken and teleost vtgs, it can be stated that teleost vtgc genes are the putative orthologs of ggvtgi, and that teleost vtgaa and vtgab genes are the putative orthologs of ggvtgii and ggvtgiii, respectively.
previously we showed that the evolution of vtg sub-domains is neither clock-like, nor under strong functional constraint  <cit> .
we further highlighted the disparate retention of the sub-domain structures of the mature proteins.
teleost vtgc proteins have all lost the highly phosphorylated polyserine pv region and the c-terminal domains that are homologous to human vwfd.
in chicken, however, all three vtgs, including ggvtgi, are complete type proteins containing all encoded domains.
the same appears to be true for other tetrapod vtgs, exemplified by amphibia and the platypus, although brawand et al.
<cit>  have shown that premature stop codons or indels have led to loss of function in ggvtgii/iii orthologs in the platypus.
the loss of sub-domains in teleosts is not restricted to the vtgc class.
amongst all ostariophysan teleosts, which represent the second largest superorder of fishes  <cit> , the major gene expressed encodes a truncated form of vtg that lacks the vwfd region  <cit> .
however, a complete-type gene is present in zebrafish as vtg <dig>  and a novel juxtaposed gene  encodes a protein that lacks the pv and ct domains, i.e.
a tripartite protein .
to address the vtg orthologies in ostariophysi, we thus included all known zebrafish coding variants in the phylogenetic data set and examined their loci.
unlike other acanthoperygian and sarcopterygian animals for which genomic data are available, the chromosomal loci of zebrafish vtgs are complex.
seven genes  are located in a tight cluster on dre <dig> , while vtg <dig> is located on dre <dig> .
however, none of the upstream or downstream genes that flank the ggvtgii/iii or vtgaa/ab clusters in sarcopterygians or teleosts, respectively, are currently annotated on dre <dig>  the zfvtg1- <dig> cluster thus represents an island of vtg genes that has either lost the flanking genes, or translocated from another chromosome such as dre <dig>  alternatively, the chromosomal loci may reflect the remnants of wgd, where differential loss via diploidization  <cit> , or germline fusion and fission events have only left traces of the ancestral condition  <cit> .
indeed kasahara et al.
<cit>  have argued that tni <dig> in spotted green pufferfish, which has retained the vgc, is derived from fusion of proto-chromosomes f, g and m, while ola <dig> in medaka, which also has retained the vgc, is derived from proto-chromosome m. interestingly medaka ola <dig> which has lost the vgc, but retained the flanking genes, and ola <dig> which also is derived from proto-chromosome m, as are tni <dig> and tni <dig> in spotted green pufferfish are the likely genomic remnants of the lost vgc paralogons.
in zebrafish the derivatives of proto-chromosome m have translocated to dre <dig>   <dig>   <dig> and  <dig>  and fission variants have been retained in dre <dig> and  <dig>  <cit> .
thus orthologs of the genes that flank the vgc in chicken should be localized in these chromosomes.
this happens to be the case .
however, since none of the orthologs that flank the chicken vgc are annotated on dre <dig>  deciphering the orientation of the zfvtg1- <dig> remains challenging.
the synovial sarcoma, x breakpoint  <dig> interacting protein  that is juxtaposed to ggvtgii and olvtgaa <dig>  as in all other vertebrates  <cit> , is currently annotated in scaffold zv7_na <dig> in zebrafish.
a blast search for this protein revealed multiple hits throughout the genome.
however, one hit  was found on dre <dig> at position  <dig>  mb.
we have therefore oriented the zfvgc1- <dig> as shown in fig.
<dig>  which also corresponds to the increasing bp positions observed in other vertebrates.
to ascertain the local cis-duplication events, we integrated the results of the phylogentic analyses.
the topology of the zfvtg1- <dig> shown in fig.
<dig> precisely replicates the chromosomal loci of each gene in the genome shown in fig.
<dig>  this is also true for the meadaka vgc, where olvtgaa <dig> represents an internal duplication of the independently sequenced olvtgaa <dig> .
in fact each of the major genes that are expressed in vertebrates are located at the outer edge of the ggvtgii/iii and vtgaa/ab vgc.
this arrangement appears to have implications for the differential expression of the cluster, where cis-regulatory elements associated with estrogen induction  <cit>  and recruitment of the transcription initiation complexes are concerned.
in zebrafish, vtg <dig> is the major gene, which can be expressed at levels  <dig> – 1000× higher than other variants  <cit> .
similarly, in other teleosts, expression ratios of the aa- or ab-type vtgs vary according to species  <cit> .
this is also true for the major ggvtgii gene in chicken  <cit> .
previously we have shown that in goldsinny wrasse, extreme levels of aa-type yps are incorporated in the growing oocyte  <cit> .
these yps are substantially degraded to faa during pre-ovulatory maturation, leaving only a lipovitellin light chain  fragment for embryonic development.
due to the neofunctionalization of vtgaa  <cit> , the degree of yp proteolysis may depend upon the relative expression levels of each vtg, and it has been predicted that a dynamic expression ratio of vtgaa and vtgab forms should exist in relation to ambient temperature and salinity, egg size and the presence of oil globules  <cit> .
to investigate this possibility, we performed northern blots of hepatically expressed transcripts in the three labrid species .
the selected model of labrid teleosts are best known for their cleaning behaviour, a character that is currently being exploited as an environmentally-friendly means of biological control of ectopic parasites in mariculture.
members of this family, the third largest of vertebrates  <cit>  with more than  <dig> species in  <dig> genera  <cit>  are also known for their remarkable sex lives  <cit> , where sex change, transvestitism and male-dominated parental care is prevalent.
although only one species spawns pelagic eggs in the temperate coastal waters of our norwegian study area, it is noteworthy that all labrid teleosts are marine species, with the vast majority spawning pelagic eggs in a subtropical environment  <cit> .
an understanding of the differential expression in the non-disrupted vtg-gene complement in this family of fishes is an important step towards development of sex-specific molecular markers for this group.
probes were designed from the highly conserved n-terminal area of each cdna sequence and their gene specificity verified by subsequent cloning and sequencing.
with the exception of crvtgab <dig>  a single band of relevant size was detected after hybridization in each species.
although several levels of regulation lie between an expressed hepatic transcript and deposited yp product in the growing oocyte, it is noteworthy that vtg band intensities were highly correlated to the type of yp deposited in the oocytes and the pelagic or benthic nature of the spawned egg.
for the goldsinny wrasse, which spawns pelagic eggs and generates the largest pool of faa during oocyte hydration, an exceptional level of vtgaa is expressed in the liver compared to the other vtg transcripts.
these data strongly support our earlier findings where virtually all of the oocyte yps are maturationally proteolysed to faa that subsequently drive the osmotic flow of water into the highly hydrated egg  <cit> .
in the benthophil rock cook, more even band intensities of the four vtg transcripts are found, although higher levels of vtgaa are also expressed in this species.
the rock cook is a close relative of the corkwing wrasse , which generates a small pool of faa from partial yp proteolysis during oocyte hydration  <cit> .
we have recently conducted a proteomic analysis of the oocyte and egg yps in the rock cook and cuckoo wrasse and found that moderate oocyte hydration is associated with proteolysis of mainly vtgaa derived yp products .
in cuckoo wrasse, a species that spawns benthic eggs and shows very limited maturational proteolysis, higher levels of vtgab-type yps are deposited in the oocyte.
these latter observations are further supported by the fact that although greater amounts of rna were inadvertently loaded in the vtgaa lane, the vtgab <dig> band had the highest intensity.
the significance of higher expression levels of ab-type vtgs relates to the fact that these genes have not neofunctionalized  <cit>  and their yp products remain mostly intact in the hydrated egg as the major protein reserve for the developing embryo.
taken together, these data are in line with the notion that differential expression of non-neofunctionalized and neofunctionalized vtgs in acanthomorph teleosts is related to the benthic or pelagic character of the spawned egg.
CONCLUSIONS
we find that labrid teleosts differentially express up to four vtg genes that are orthologous to an ancient vtg gene cluster that existed prior to the separation of actinopterygii from sarcopterygii.
with the exception of zebrafish, the vertebrate vtg gene cluster remains linked on single chromosomes that arose in close association with the second round of whole genome duplication  > <dig> million years ago.
our model for lineage-specific duplication of the major vtg genes in teleosts shows that they comprise a variable subcluster.
the copy number of this variable subcluster, which comprises the ggvtgiii/vtgab and ggvtgii/vtgaa orthologs, is likely to be the combined result of the third round of wgd in teleosts with subsequent gene loss due to chromosomal rearrangements followed by lineage-specific gene duplications.
the topology of the phylogenetic tree for the  <dig> zebrafish vtg genes precisely replicates their chromosomal loci in the genome and suggests that lineage-specific duplications can occur within the teleost subcluster.
the expression data for the labrid transcripts demonstrated that the more ancestral vtgc genes that are orthologous to chicken ggvtgi are the least expressed and we argue that these minor genes have functionally diverged in the teleost lineage due to loss of the pv and c-terminal domains.
in the closely related family of labrid teleosts, the expression ratios of the major vtgab and neofunctionalized vtgaa transcripts reflect the benthic or pelagic character of the spawned egg.

 a defining feature of the early development of non-eutherian vertebrates is a cleidoic egg endowed with variable amounts of yolk. until very recently, the major products of the vitellogenin genes that encode yolk proteins have been considered to be simple precursors of the energy reserve of vertebrate eggs, but the latest studies have demonstrated several non-nutritional roles for vtg. similarly, the recent observation that remnants of vtg genes exist in eutheria, including humans, but have sequentially been lost through co-evolution with casein genes elegantly demonstrated that the three known vtg genes in birds represent a conserved gene complement. in a previous study, finn & kristoffersen proposed a model for the evolution and neofunctionalization of vtg genes in acanthomorph teleosts. we identified vtgc as an ancestral gene, and argued that the dual vtgaa/vtgab system, first noted by la fleur et al. was derived from a single form, the a-type vtg. in this model, the separation of the vtgc- and vtga-type genes occurred following the second round of whole genome duplication. subsequently vtga duplicated and formed paragolous vtgaa and vtgab genes in acanthomorph teleosts. this phylogenetic model has been corroborated by other investigators. most recently however, babin has provided a syntenic map of vertebrate vtg genes, which shows that the three forms of vtg are encoded in a vtg gene cluster in non-eutherian vertebrates. a major goal of the present study was to integrate the statistical, biochemical and physical models of vtg gene evolution in vertebrates. through a series of studies, we and other laboratories have shown that the pelagic nature of a marine teleost egg is an evolved feature that primarily results from the maturational influx of water due to differential degradation of vtgaa-type yolk proteins, and the temporal insertion of novel aquaporins in the microvillous portion of the plasmalemma. the neofunctionalization of the vtgaa form in acanthomorph teleosts, has sensitized the heavy chain domain to catheptic proteolysis that generates a large organic osmolyte pool of free amino acids in the ovulated egg. in contrast the lvh domains derived from vtgab and vtgc genes may be partially cleaved, but remain mostly intact following the maturational proteolytic event, and thus contribute minimally to oocyte hydration. in teleosts that spawn benthic eggs, a character that we have argued to be the ancestral condition due to an ancient freshwater heritage, yps may be cleaved or partially processed to generate a small pool of faa during oocyte hydration. in order to reconcile the differences in the phylogenetic, biochemical and syntenic models we have examined the evolution and expression of the vtg gene complement in modern and old teleosts that spawn pelagic and benthic eggs. we were interested in determining how the expression of different vtg transcripts relates to the character of the egg in a single family of closely related teleosts, and whether lineage-specific gene duplication resulted in neofunctionalization of the aa-type vtgs. to determine the orthologies and ancestry of the novel labrid vtg genes cloned in the present study, we anchored the results of phylogenetic inference with the syntenic arrangement of genomic vtgs in model vertebrates. this approach allowed us to identify the proto-chromosomal origin of the vgc that is conserved between the actinopterygii and the sarcopterygii. it further allowed us to conclude that neofunctionalization of the vtgaa genes in acanthomorph teleosts occurred long after the duplication event.  multiple transcripts a total of eleven vtg transcripts were cloned from vitellogenic livers of the labrid species investigated. ncbi blast searches of the deduced proteins verified that all sequences are members of the vtg family. four distinct vtg cdna sequences were cloned from rock cook and goldsinny wrasse, two of which were entirely novel for vertebrates. three transcripts were obtained from cuckoo wrasse. repeated attempts using gene-specific primers to extract a second vtgab-type transcript from livers of cuckoo wrasse did not yield any novel transcripts. the deduced amino acid sequences of the full-length rock cook and cuckoo wrasse vtgs revealed that the vtgaa and vtgab products are complete pentapartite type proteins while vtgc belongs to the phosvitinless class of vtg. since it is now postulated that the vertebrate vtg gene complement represents a conserved cluster in sarcopterygii and actinopterygii, we aligned the longest partial segment of the labrid sequences to the genomic variants in chicken and medaka, respectively. relative homology scores of the aligned amino acids and codons revealed three forms with highest identity to medaka olvtgaa olvtgab and olvtgc, respectively. the relationship between the teleost and chicken genes was less obvious, with essentially equal identity scores for the ggvtgii and ggvtgiii products. interestingly, ggvtgi had slightly lower scores to the teleost c-type products compared to the aa- or ab-type products over the aligned lvh sub-region. this suggests that despite their orthology the minor ggvtgi and vtgc genes have functionally diverged in association with the loss of the phosvitin and c-terminal domains in teleosts. within the labrid species, sequences had highest identities to their homologs in closely related goldsinny wrasse. since only a single ab-type transcript was detected in cuckoo wrasse, and it showed 100% identity to the novel cevtgab and crvtgab sequences over the ~ aligned aa and ~ aligned nt, we named the cuckoo wrasse ab-type sequence lmvtgab to further classify the labrid vtgs, we conducted large-scale phylogenetic analyses of the deduced amino acid and codon alignments of available and novel genomic variants. for the genomic variants, we identified three genes in 3-spined stickleback, torafugu and spotted green pufferfish, respectively, and four genes in medaka, of which two have been independently sequenced. in zebrafish eight genes are found in the genome, of which several have been fully or partially sequenced, and each of which is expressed and deposited in the growing oocyte. validation of the tree topology was achieved through multiple methods of phylogenetic inference. each method consistently clustered the labrid sequences as three forms: vtgaa, vtgab and vtgc, respectively. for bayesian, maximum parsimony and neighbour-joining analyses the majority of branches were supported by 100% posterior probabilities, and 100% bootstrap values, respectively, with only minor branch rearrangements within gene groups. these data verify that three forms of vtg exist with acanthomorph teleosts, two within protacanthomorph teleosts, and three within ostariophysan teleosts. the data further revealed that a fourth novel gene in medaka is an aa-type product, and we thus classified it as olvtgaa in a separate study, we have identified three novel gene variants in a basal clupeocephalan teleost, the atlantic herring. these atlantic herring transcripts clustered as a basal node to all ostariophysan vtgs in full agreement with the clupeocephalan phylogenetic rank. the present analyses thus fully corroborate our earlier study of vertebrate vtgs wherein the major and minor transcripts cluster according to taxonomic group. we further verified the inferred duplication of the vtgaa and vtgab clusters using the method of zmasek and eddy. hence the tree topology is inconsistent with the syntenic arrangement of vertebrate vtg genes, and suggests that different functions have evolved within the major clades. specifically for acanthomorph teleosts, the lvh-aa domains have evolved a sensitivity to acidic degradation following activation of v-class proton pumps during oocyte maturation. the data for labrid teleosts that spawn benthic and pelagic eggs support this view. in order to understand the disparity between the phylogenetic and syntenic arrangement of vertebrate vtg genes, we increased the phylogenetic data set to include all known sarcopterygian vtgs, including amphibian, bird, reptile and platypus variants. in addition we examined the syntenic positions of vtg genes within non-metatherian vertebrates. the larger data set did not affect the topology of the teleost branches, but did place ggvtgi and the partial platypus transcript at the base of the tetrapoda and closer to, but on a separate branch to teleost vtgc variants. similarly, two further partial platypus transcripts clustered as a basal node to ggvtgiii and ggvtgii, but after amphibian variants. these findings agree with the recent studies of brawand et al. and babin who demonstrated that three genes exist in monotremata and are putative orthologs of the three bird genes. since these latter platypus genes are not yet localized to any chromosome, but are annotated in contigs and, respectively, it was not possible to discern their true orthology, or syntenic arrangement in relation to chicken. however, by combining the results of the present phylogenetic data with the syntenic positions of the chicken and teleost vtgs, it can be stated that teleost vtgc genes are the putative orthologs of ggvtgi, and that teleost vtgaa and vtgab genes are the putative orthologs of ggvtgii and ggvtgiii, respectively. previously we showed that the evolution of vtg sub-domains is neither clock-like, nor under strong functional constraint. we further highlighted the disparate retention of the sub-domain structures of the mature proteins. teleost vtgc proteins have all lost the highly phosphorylated polyserine pv region and the c-terminal domains that are homologous to human vwfd. in chicken, however, all three vtgs, including ggvtgi, are complete type proteins containing all encoded domains. the same appears to be true for other tetrapod vtgs, exemplified by amphibia and the platypus, although brawand et al. have shown that premature stop codons or indels have led to loss of function in ggvtgii/iii orthologs in the platypus. the loss of sub-domains in teleosts is not restricted to the vtgc class. amongst all ostariophysan teleosts, which represent the second largest superorder of fishes, the major gene expressed encodes a truncated form of vtg that lacks the vwfd region. however, a complete-type gene is present in zebrafish as vtg and a novel juxtaposed gene encodes a protein that lacks the pv and ct domains, i.e. a tripartite protein. to address the vtg orthologies in ostariophysi, we thus included all known zebrafish coding variants in the phylogenetic data set and examined their loci. unlike other acanthoperygian and sarcopterygian animals for which genomic data are available, the chromosomal loci of zebrafish vtgs are complex. seven genes are located in a tight cluster on dre, while vtg is located on dre. however, none of the upstream or downstream genes that flank the ggvtgii/iii or vtgaa/ab clusters in sarcopterygians or teleosts, respectively, are currently annotated on dre the zfvtg1- cluster thus represents an island of vtg genes that has either lost the flanking genes, or translocated from another chromosome such as dre alternatively, the chromosomal loci may reflect the remnants of wgd, where differential loss via diploidization, or germline fusion and fission events have only left traces of the ancestral condition. indeed kasahara et al. have argued that tni in spotted green pufferfish, which has retained the vgc, is derived from fusion of proto-chromosomes f, g and m, while ola in medaka, which also has retained the vgc, is derived from proto-chromosome m. interestingly medaka ola which has lost the vgc, but retained the flanking genes, and ola which also is derived from proto-chromosome m, as are tni and tni in spotted green pufferfish are the likely genomic remnants of the lost vgc paralogons. in zebrafish the derivatives of proto-chromosome m have translocated to dre and and fission variants have been retained in dre and. thus orthologs of the genes that flank the vgc in chicken should be localized in these chromosomes. this happens to be the case. however, since none of the orthologs that flank the chicken vgc are annotated on dre deciphering the orientation of the zfvtg1- remains challenging. the synovial sarcoma, x breakpoint interacting protein that is juxtaposed to ggvtgii and olvtgaa as in all other vertebrates, is currently annotated in scaffold zv7_na in zebrafish. a blast search for this protein revealed multiple hits throughout the genome. however, one hit was found on dre at position mb. we have therefore oriented the zfvgc1- as shown in fig. which also corresponds to the increasing bp positions observed in other vertebrates. to ascertain the local cis-duplication events, we integrated the results of the phylogentic analyses. the topology of the zfvtg1- shown in fig. precisely replicates the chromosomal loci of each gene in the genome shown in fig. this is also true for the meadaka vgc, where olvtgaa represents an internal duplication of the independently sequenced olvtgaa. in fact each of the major genes that are expressed in vertebrates are located at the outer edge of the ggvtgii/iii and vtgaa/ab vgc. this arrangement appears to have implications for the differential expression of the cluster, where cis-regulatory elements associated with estrogen induction and recruitment of the transcription initiation complexes are concerned. in zebrafish, vtg is the major gene, which can be expressed at levels – 1000× higher than other variants. similarly, in other teleosts, expression ratios of the aa- or ab-type vtgs vary according to species. this is also true for the major ggvtgii gene in chicken. previously we have shown that in goldsinny wrasse, extreme levels of aa-type yps are incorporated in the growing oocyte. these yps are substantially degraded to faa during pre-ovulatory maturation, leaving only a lipovitellin light chain fragment for embryonic development. due to the neofunctionalization of vtgaa, the degree of yp proteolysis may depend upon the relative expression levels of each vtg, and it has been predicted that a dynamic expression ratio of vtgaa and vtgab forms should exist in relation to ambient temperature and salinity, egg size and the presence of oil globules. to investigate this possibility, we performed northern blots of hepatically expressed transcripts in the three labrid species. the selected model of labrid teleosts are best known for their cleaning behaviour, a character that is currently being exploited as an environmentally-friendly means of biological control of ectopic parasites in mariculture. members of this family, the third largest of vertebrates with more than species in genera are also known for their remarkable sex lives, where sex change, transvestitism and male-dominated parental care is prevalent. although only one species spawns pelagic eggs in the temperate coastal waters of our norwegian study area, it is noteworthy that all labrid teleosts are marine species, with the vast majority spawning pelagic eggs in a subtropical environment. an understanding of the differential expression in the non-disrupted vtg-gene complement in this family of fishes is an important step towards development of sex-specific molecular markers for this group. probes were designed from the highly conserved n-terminal area of each cdna sequence and their gene specificity verified by subsequent cloning and sequencing. with the exception of crvtgab a single band of relevant size was detected after hybridization in each species. although several levels of regulation lie between an expressed hepatic transcript and deposited yp product in the growing oocyte, it is noteworthy that vtg band intensities were highly correlated to the type of yp deposited in the oocytes and the pelagic or benthic nature of the spawned egg. for the goldsinny wrasse, which spawns pelagic eggs and generates the largest pool of faa during oocyte hydration, an exceptional level of vtgaa is expressed in the liver compared to the other vtg transcripts. these data strongly support our earlier findings where virtually all of the oocyte yps are maturationally proteolysed to faa that subsequently drive the osmotic flow of water into the highly hydrated egg. in the benthophil rock cook, more even band intensities of the four vtg transcripts are found, although higher levels of vtgaa are also expressed in this species. the rock cook is a close relative of the corkwing wrasse, which generates a small pool of faa from partial yp proteolysis during oocyte hydration. we have recently conducted a proteomic analysis of the oocyte and egg yps in the rock cook and cuckoo wrasse and found that moderate oocyte hydration is associated with proteolysis of mainly vtgaa derived yp products. in cuckoo wrasse, a species that spawns benthic eggs and shows very limited maturational proteolysis, higher levels of vtgab-type yps are deposited in the oocyte. these latter observations are further supported by the fact that although greater amounts of rna were inadvertently loaded in the vtgaa lane, the vtgab band had the highest intensity. the significance of higher expression levels of ab-type vtgs relates to the fact that these genes have not neofunctionalized and their yp products remain mostly intact in the hydrated egg as the major protein reserve for the developing embryo. taken together, these data are in line with the notion that differential expression of non-neofunctionalized and neofunctionalized vtgs in acanthomorph teleosts is related to the benthic or pelagic character of the spawned egg.  we find that labrid teleosts differentially express up to four vtg genes that are orthologous to an ancient vtg gene cluster that existed prior to the separation of actinopterygii from sarcopterygii. with the exception of zebrafish, the vertebrate vtg gene cluster remains linked on single chromosomes that arose in close association with the second round of whole genome duplication > million years ago. our model for lineage-specific duplication of the major vtg genes in teleosts shows that they comprise a variable subcluster. the copy number of this variable subcluster, which comprises the ggvtgiii/vtgab and ggvtgii/vtgaa orthologs, is likely to be the combined result of the third round of wgd in teleosts with subsequent gene loss due to chromosomal rearrangements followed by lineage-specific gene duplications. the topology of the phylogenetic tree for the zebrafish vtg genes precisely replicates their chromosomal loci in the genome and suggests that lineage-specific duplications can occur within the teleost subcluster. the expression data for the labrid transcripts demonstrated that the more ancestral vtgc genes that are orthologous to chicken ggvtgi are the least expressed and we argue that these minor genes have functionally diverged in the teleost lineage due to loss of the pv and c-terminal domains. in the closely related family of labrid teleosts, the expression ratios of the major vtgab and neofunctionalized vtgaa transcripts reflect the benthic or pelagic character of the spawned egg. 
sequencing of a novel fourth vtg transcript in labrid species, and the presence of duplicated paralogs in certain model organisms supports the notion that lineage-specific gene duplications frequently occur in teleosts. the differential expression of the labrid vtg genes are further consistent with the notion that neofunctionalized aa-type vtgs are important determinants of the pelagic or benthic character of the eggs in acanthomorph teleosts. the differential expression and latent neofunctionalization of vtg genes in acanthomorph teleosts is an adaptive feature associated with oocyte hydration and spawning in the marine environment. we argue that the vertebrate vtg gene cluster originated in proto-chromosome m, but that vtg genes have subsequently duplicated and rearranged following whole genome duplications. the data show that the vtg gene cluster is more conserved between acanthomorph teleosts and tetrapods, than in ostariophysan teleosts such as the zebrafish. the vertebrate vtg gene cluster existed prior to the separation of sarcopterygii from actinopterygii > million years ago, a period associated with the second round of whole genome duplication. by integrating phylogenetics of novel vtg transcripts from old and modern teleosts with syntenic analyses of all available genomic variants of non-metatherian vertebrates we identify the gene orthologies between the sarcopterygii and actinopterygii.
 the multiplicity or loss of the vitellogenin gene family in vertebrates has been argued to have broad implications for the mode of reproduction, cleavage pattern and character of the egg. earlier proposals for the existence of three forms of vertebrate vtgs present conflicting models for their origin and subsequent duplication.  by integrating phylogenetics of novel vtg transcripts from old and modern teleosts with syntenic analyses of all available genomic variants of non-metatherian vertebrates we identify the gene orthologies between the sarcopterygii and actinopterygii. we argue that the vertebrate vtg gene cluster originated in proto-chromosome m, but that vtg genes have subsequently duplicated and rearranged following whole genome duplications. sequencing of a novel fourth vtg transcript in labrid species, and the presence of duplicated paralogs in certain model organisms supports the notion that lineage-specific gene duplications frequently occur in teleosts. the data show that the vtg gene cluster is more conserved between acanthomorph teleosts and tetrapods, than in ostariophysan teleosts such as the zebrafish. the differential expression of the labrid vtg genes are further consistent with the notion that neofunctionalized aa-type vtgs are important determinants of the pelagic or benthic character of the eggs in acanthomorph teleosts.  the vertebrate vtg gene cluster existed prior to the separation of sarcopterygii from actinopterygii > million years ago, a period associated with the second round of whole genome duplication. the presence of higher copy numbers in a more highly expressed subcluster is particularly prevalent in teleosts. the differential expression and latent neofunctionalization of vtg genes in acanthomorph teleosts is an adaptive feature associated with oocyte hydration and spawning in the marine environment. 