BACKGROUND
genomic imprinting produces monoallelic gene expression resulting from the parent-of-origin-dependent epigenetic modifications.
both dna methylation and histone modifications are required to establish the paternal and maternal imprinting during development of the germ cells and to maintain it after fertilisation  <cit> .
in humans and mice defects in some epigenetic modifiers or co-factors cause global disorders of genomic imprinting and of imprinted gene expression with early embryonic lethality, demonstrating that genomic imprinting is essential for mammalian development  <cit> .
it is still unclear, however, why genomic imprinting has arisen in mammalian evolution, because adopting monoallelic gene expression means abandoning the merits of diploidy.
in higher vertebrates, genomic imprinting has been found so far only in the viviparous therian mammals , but not in the egg-laying mammals, the monotremes  <cit> .
since only viviparous mammals have genomic imprinting, and many imprinted genes regulate fetal and placental growth, some authors have suggested that genomic imprinting is correlated with the evolution of mammalian viviparity  <cit> .
it is therefore of great interest to compare genomic imprinting between eutherians and marsupials that diverged between  <dig> and  <dig> million years ago  <cit> .
most eutherians form a chorioallantoic  placenta that is the site of highly efficient nutritional exchange between fetus and mother, allows lengthy intra-uterine growth, and in many cases supports the growth of a precocial young.
in contrast, most marsupials depend on a relatively short-lived chorio-vitelline  placenta.
although often ignored, a yolk sac placenta is also present and functions for varying periods of time in all eutherian mammals  <cit> .
marsupials give birth to altricial young that are at a much earlier developmental stage than the neonates of most eutherians, but have developed a complex and advanced lactation system that supports further development and growth after birth, usually in a pouch  <cit> .
nearly  <dig> imprinted genes have been isolated in mice and humans.
imprinting has been studied in fourteen orthologues of these genes in marsupials but only  <dig> are imprinted  <cit> .
these are igf <dig>  igf2r, peg1/mest, peg <dig>  ins and h <dig>  and are from  <dig> independent domains.
we have previously reported that there is no evidence of genomic imprinting of cdkn1c  in a marsupial, the tammar wallaby   <cit> .
cdkn1c is located in the kcnq <dig> domain mapped adjacent to the igf2-h <dig> domain in eutherians and marsupials.
genomic imprinting of the igf2-h <dig> domain is highly conserved between eutherians and tammars  <cit> .
although the imprinting regulatory mechanisms of the kcnq <dig> and igf2-h <dig> domains are known to be independent in mouse, the two domains are only  <dig> kb distant from each other and both contain several important genes that control fetal and placental growth.
therefore, to confirm whether the only gene in the domain that is not imprinted in the tammar is the cdkn1c gene, we examined the imprinting status of the orthologue of the phlda <dig> gene from the tammar wallaby kcnq <dig> domain.
phlda <dig> negatively controls growth of the chorioallantoic placenta in both human and mouse.
in mice, deletion of phlda <dig> causes placental overgrowth  <cit> .
in contrast, biallelic expression of phlda <dig>  due to loss of imprinting, contributes to placental growth retardation and results in conceptuses with intrauterine growth restriction   <cit> .
furthermore, a single extra dose of phlda <dig> has serious consequences for placental development, driving the loss of the junctional zone and reducing the amount of stored glycogen  <cit> .
in humans, whilst there is silencing of phlda <dig> in complete hydatidiform moles  <cit> , there is upregulation in placentae of fetuses with iugr  <cit> ; consistent with the results of genetic experiments in mice.
thus, the importance of gene dosage of phlda <dig> in eutherian placentation has been demonstrated by a number of studies.
in this study, we characterise the orthologue of phlda <dig> in a marsupial, the tammar wallaby and examine its imprinting status in the chorio-vitelline placenta to clarify its possible contribution for the evolution of chorioallantoic placenta in the eutherian linage by dosage reduction consequent to acquisition of genomic imprinting.
RESULTS
characterisation of tammar phlda2
a  <dig> bp fragment was amplified by rt-pcr using a primer pair designed to a highly conserved sequence in the open reading frame  of the phlda <dig> gene among multiple species.
given the pcr product sequence was highly similar to phlda <dig> of other species, we next carried out 3' race to obtain 3' utr sequence of tammar phlda <dig> using the same forward primer used to amplify the  <dig> bp fragment as the gene specific primer.
the 3' utr  of tammar phlda <dig> consisted of a short intron  similar to eutherian phlda <dig> .
the expected genomic location of tammar phlda <dig> close to cdkn1c was confirmed by tammar bac clone sequences in genbank .
a  <dig> bp orf encoding  <dig> amino acids was predicted with the supplemental sequence data from trace archive database .
consistent with a previous comparison across vertebrates, that included fish, frog, chicken, mouse and human  <cit> , the amino acid sequence of tammar phlda <dig> was also highly conserved within the ph  domain, but there was lower conservation in the flanking sequences of both terminals .
the ph domain in tammar phlda <dig> shares 78% amino acid sequence similarity with human, 67% with mouse, 73% with platypus and 77% with chicken phlda <dig> orthologues.
tissue specific expression pattern of tammar phlda2
as phlda <dig> is highly expressed in the yolk sac and placenta in human and mouse, we next analysed the expression pattern of tammar phlda <dig> in the yolk sac placenta as well as in several fetal tissues by quantitative pcr .
the marsupial yolk sac placenta consists of two regions, a bilaminar, avascular region and a trilaminar, vascular region.
both regions are the sites for fetal-maternal nutritional exchange while gases appear to be transferred principally via the vascular system of the trilaminar region  <cit> .
the yolk sac placenta also synthesizes and stores nutrients required for fetal growth  <cit> .
phlda <dig> mrna expression in both bilaminar and trilaminar yolk sac was dramatically upregulated between day  <dig> to  <dig> of gestation , although the relative expression level was lower in the bilaminar yolk sac .
a lower level of tammar phlda <dig> expression was also observed in several fetal tissues, as observed for phlda <dig> in the mouse, but not the human  <cit> .
tammar phlda <dig> protein distribution in the yolk sac placenta
to confirm tammar wallaby phlda <dig> protein expression and distribution in the yolk sac placenta, we carried out immunohistochemistry using a mouse monoclonal antibody raised against a partial recombinant human phlda <dig>  the immunogen included aa 1- <dig>  encompassing the whole ph domain.
there is a high degree of similarity of amino acid sequences between human and tammar phlda <dig> over this region .
furthermore, we performed a genome-wide "tblastn" search for the published tammar genome sequence in the ensembl database using the antigen peptide sequence for the query.
it revealed the highest similarity of  <dig> % for tammar phlda <dig> against whole sequence query  as expected.
the second highest hit was tammar phlda <dig>  but this was aligned only partially  with a much lower similarity of  <dig> % for the aligned region.
these data suggest that the immunostaining is positive for tammar phlda <dig> protein, although the possibility of some cross-reaction with phlda <dig> has not been completely excluded.
tammar phlda <dig> protein was present in both bilaminar and trilaminar regions in the yolk sac placenta, with strong immuno-staining in the cytoplasm of trophoblast cells of both parts of the yolk sac , despite the substantially lower mrna relative expression level in the bilaminar yolk sac .
allelic expression analysis of tammar phlda2
finally, we analysed allelic expression pattern of tammar phlda <dig> to determine whether it was imprinted.
we searched for polymorphisms to allow us to distinguish between the two parental alleles.
no exonic polymorphisms were found in any of the individuals  tested.
however, there was a length polymorphism in some individuals in the intron characterised by the presence or absence of repeats in the  <dig> bp of intronic sequence .
therefore, allelic expression could be determined directly by rt-pcr amplifying the unspliced phlda <dig> transcript using a primer pair designed to amplify the length polymorphic site.
all rna samples were dnase i treated and the lack of detectable contamination by genomic dna was confirmed by pcr using the templates without reverse transcription .
hence all intronic fragments amplified by rt-pcr were derived from unspliced transcripts, not from genomic dna.
the genomic pcr products showed that all four individuals were heterozygous for the length polymorphism and both alleles can be amplified equally .
all samples tested, had clear biallelic expression, demonstrating no evidence of genomic imprinting of tammar phlda <dig> .
on the other hand, monoallelic expression of tammar igf <dig> could be confirmed by the amplification of the unspliced transcript using an intronic primer in the same way as the analysis on phlda <dig> .
discussion
in this study, we identified and characterised the marsupial orthologue of phlda <dig>  the amino acid sequence of tammar phlda <dig> shared highest conservation within the ph domain and lower conservation in the flanking sequences of both terminals, suggesting the essential role in the ph domain in contrast to the flanking regions, consistent with previous reports  <cit> .
there was a similar high conservation of the amino acid sequences within the ph domain in the platypus, tammar and human phlda <dig>  suggesting that this domain has a significant role with a similar function in marsupials and monotremes.
the high level of mrna expression in the trilaminar yolk sac and the protein localisation to the cytoplasm of trophoblast cells suggest that phlda <dig> functions in the tammar yolk sac placenta during pregnancy.
however, although murine phlda <dig> has the highest expression in the yolk sac  <cit> , in mice with a disrupted phlda <dig> gene the only abnormalities reported are in the chorioallantoic placenta  <cit> .
therefore, an ancestral role for phlda <dig> in the yolk sac might have been transferred to the chorioallantoic placenta during the evolution of the mouse.
there was no evidence of genomic imprinting of tammar phlda <dig> in this study.
the mouse kcnq <dig> domain forms a large imprinted gene cluster including phlda <dig>  slc22a <dig>  cdkn1c, kcnq <dig>  ascl <dig>  and some placenta-specific imprinted genes.
however, we know now that at least two genes, cdkn1c and phlda <dig> that are involved in embryonic and placental growth in eutherians, are not imprinted in this marsupial  <cit> .
considering that both genes are located to the middle of the domain and that all the imprinted genes in this domain are co-ordinately regulated by a single imprinting centre in the mouse, our data strongly suggests that the whole kcnq <dig> domain lacks genomic imprinting in marsupials.
interestingly, the igf2-h <dig> imprinted domain, located adjacent to the kcnq <dig> domain shares a highly conserved imprinting regulatory mechanism complete with a differentially methylated region and associated mirna between eutherians and marsupials  <cit> .
this study thus confirms that the origin of imprinting of the kcnq <dig> domain evolved in the eutherian lineage after the divergence of marsupials, whereas that of the igf2-h <dig> domain appeared before the marsupial-eutherian split, regardless of the close proximity of these two domains  <cit> .
in the kcnq <dig> domain of mice, while cdkn1c is a negative regulator for embryonic growth  <cit> , phlda <dig> negatively controls placental growth  <cit>  and acts as a true rheostat for placental growth  <cit> .
recently, using a single copy transgenic mouse, tunster et al.,  reported that phlda <dig> regulates extraembryonic energy stores.
two-fold over-expression of phlda <dig> caused a 60% loss of the spongiotrophoblast layer with a 25-35% reduction of glycogen storage.
since acquisition of genomic imprinting of phlda <dig> in the kcnq <dig> domain by silencing of the paternal allele was accompanied by gene dosage reduction in eutherians, this might have affected the evolution of placental structure and/or energy stores.
in laboratory mice that have two active copies of phlda <dig> with the second copy provided by the bac transgene, there was only a slight progressive slowing of embryonic growth  <cit> .
however, greater reduction of fetal growth may have been seen if the mice had had restricted food intake, as is often the case in the wild, so that limited nutrition would need to be partitioned between mother and fetuses.
in this situation, reduced expression of phlda <dig> could have had a selective advantage through greater placental development.
we hypothesise that acquisition of imprinting in the kcnq <dig> domain in the ancestral line that gave rise to the eutherian mammals may have allowed increased the placental growth and extended gestation that characterises this group of mammals.
CONCLUSIONS
the high level of mrna expression in the trilaminar yolk sac placenta and the protein localisation to the cytoplasm of trophoblast cells suggest that tammar phlda <dig> is functional in their placenta.
the lack of imprinting in the tammar phlda <dig> confirms an earlier conclusion that acquisition of genomic imprinting to the kcnq <dig> domain occurred specifically in the eutherian lineage after the divergence of therian mammals into marsupials and eutherians, despite the fact that imprinting of the adjacent igf2-h <dig> domain arose before the marsupial-eutherian split .
thus genomic imprinting of the kcnq <dig> domain might have contributed to the development of complex placentation and the lengthening of gestation in the eutherian lineage by reducing gene dosage of negative regulators for both embryonic and placental growth.

 genomic imprinting produces monoallelic gene expression resulting from the parent-of-origin-dependent epigenetic modifications. both dna methylation and histone modifications are required to establish the paternal and maternal imprinting during development of the germ cells and to maintain it after fertilisation. in humans and mice defects in some epigenetic modifiers or co-factors cause global disorders of genomic imprinting and of imprinted gene expression with early embryonic lethality, demonstrating that genomic imprinting is essential for mammalian development. it is still unclear, however, why genomic imprinting has arisen in mammalian evolution, because adopting monoallelic gene expression means abandoning the merits of diploidy. in higher vertebrates, genomic imprinting has been found so far only in the viviparous therian mammals, but not in the egg-laying mammals, the monotremes. since only viviparous mammals have genomic imprinting, and many imprinted genes regulate fetal and placental growth, some authors have suggested that genomic imprinting is correlated with the evolution of mammalian viviparity. it is therefore of great interest to compare genomic imprinting between eutherians and marsupials that diverged between and million years ago. most eutherians form a chorioallantoic placenta that is the site of highly efficient nutritional exchange between fetus and mother, allows lengthy intra-uterine growth, and in many cases supports the growth of a precocial young. in contrast, most marsupials depend on a relatively short-lived chorio-vitelline placenta. although often ignored, a yolk sac placenta is also present and functions for varying periods of time in all eutherian mammals. marsupials give birth to altricial young that are at a much earlier developmental stage than the neonates of most eutherians, but have developed a complex and advanced lactation system that supports further development and growth after birth, usually in a pouch. nearly imprinted genes have been isolated in mice and humans. imprinting has been studied in fourteen orthologues of these genes in marsupials but only are imprinted. these are igf igf2r, peg1/mest, peg ins and h and are from independent domains. we have previously reported that there is no evidence of genomic imprinting of cdkn1c in a marsupial, the tammar wallaby. cdkn1c is located in the kcnq domain mapped adjacent to the igf2-h domain in eutherians and marsupials. genomic imprinting of the igf2-h domain is highly conserved between eutherians and tammars. although the imprinting regulatory mechanisms of the kcnq and igf2-h domains are known to be independent in mouse, the two domains are only kb distant from each other and both contain several important genes that control fetal and placental growth. therefore, to confirm whether the only gene in the domain that is not imprinted in the tammar is the cdkn1c gene, we examined the imprinting status of the orthologue of the phlda gene from the tammar wallaby kcnq domain. phlda negatively controls growth of the chorioallantoic placenta in both human and mouse. in mice, deletion of phlda causes placental overgrowth. in contrast, biallelic expression of phlda due to loss of imprinting, contributes to placental growth retardation and results in conceptuses with intrauterine growth restriction. furthermore, a single extra dose of phlda has serious consequences for placental development, driving the loss of the junctional zone and reducing the amount of stored glycogen. in humans, whilst there is silencing of phlda in complete hydatidiform moles, there is upregulation in placentae of fetuses with iugr; consistent with the results of genetic experiments in mice. thus, the importance of gene dosage of phlda in eutherian placentation has been demonstrated by a number of studies. in this study, we characterise the orthologue of phlda in a marsupial, the tammar wallaby and examine its imprinting status in the chorio-vitelline placenta to clarify its possible contribution for the evolution of chorioallantoic placenta in the eutherian linage by dosage reduction consequent to acquisition of genomic imprinting.  characterisation of tammar phlda2 a bp fragment was amplified by rt-pcr using a primer pair designed to a highly conserved sequence in the open reading frame of the phlda gene among multiple species. given the pcr product sequence was highly similar to phlda of other species, we next carried out 3' race to obtain 3' utr sequence of tammar phlda using the same forward primer used to amplify the bp fragment as the gene specific primer. the 3' utr of tammar phlda consisted of a short intron similar to eutherian phlda. the expected genomic location of tammar phlda close to cdkn1c was confirmed by tammar bac clone sequences in genbank. a bp orf encoding amino acids was predicted with the supplemental sequence data from trace archive database. consistent with a previous comparison across vertebrates, that included fish, frog, chicken, mouse and human, the amino acid sequence of tammar phlda was also highly conserved within the ph domain, but there was lower conservation in the flanking sequences of both terminals. the ph domain in tammar phlda shares 78% amino acid sequence similarity with human, 67% with mouse, 73% with platypus and 77% with chicken phlda orthologues. tissue specific expression pattern of tammar phlda2 as phlda is highly expressed in the yolk sac and placenta in human and mouse, we next analysed the expression pattern of tammar phlda in the yolk sac placenta as well as in several fetal tissues by quantitative pcr. the marsupial yolk sac placenta consists of two regions, a bilaminar, avascular region and a trilaminar, vascular region. both regions are the sites for fetal-maternal nutritional exchange while gases appear to be transferred principally via the vascular system of the trilaminar region. the yolk sac placenta also synthesizes and stores nutrients required for fetal growth. phlda mrna expression in both bilaminar and trilaminar yolk sac was dramatically upregulated between day to of gestation, although the relative expression level was lower in the bilaminar yolk sac. a lower level of tammar phlda expression was also observed in several fetal tissues, as observed for phlda in the mouse, but not the human. tammar phlda protein distribution in the yolk sac placenta to confirm tammar wallaby phlda protein expression and distribution in the yolk sac placenta, we carried out immunohistochemistry using a mouse monoclonal antibody raised against a partial recombinant human phlda the immunogen included aa 1- encompassing the whole ph domain. there is a high degree of similarity of amino acid sequences between human and tammar phlda over this region. furthermore, we performed a genome-wide "tblastn" search for the published tammar genome sequence in the ensembl database using the antigen peptide sequence for the query. it revealed the highest similarity of % for tammar phlda against whole sequence query as expected. the second highest hit was tammar phlda but this was aligned only partially with a much lower similarity of % for the aligned region. these data suggest that the immunostaining is positive for tammar phlda protein, although the possibility of some cross-reaction with phlda has not been completely excluded. tammar phlda protein was present in both bilaminar and trilaminar regions in the yolk sac placenta, with strong immuno-staining in the cytoplasm of trophoblast cells of both parts of the yolk sac, despite the substantially lower mrna relative expression level in the bilaminar yolk sac. allelic expression analysis of tammar phlda2 finally, we analysed allelic expression pattern of tammar phlda to determine whether it was imprinted. we searched for polymorphisms to allow us to distinguish between the two parental alleles. no exonic polymorphisms were found in any of the individuals tested. however, there was a length polymorphism in some individuals in the intron characterised by the presence or absence of repeats in the bp of intronic sequence. therefore, allelic expression could be determined directly by rt-pcr amplifying the unspliced phlda transcript using a primer pair designed to amplify the length polymorphic site. all rna samples were dnase i treated and the lack of detectable contamination by genomic dna was confirmed by pcr using the templates without reverse transcription. hence all intronic fragments amplified by rt-pcr were derived from unspliced transcripts, not from genomic dna. the genomic pcr products showed that all four individuals were heterozygous for the length polymorphism and both alleles can be amplified equally. all samples tested, had clear biallelic expression, demonstrating no evidence of genomic imprinting of tammar phlda. on the other hand, monoallelic expression of tammar igf could be confirmed by the amplification of the unspliced transcript using an intronic primer in the same way as the analysis on phlda. discussion in this study, we identified and characterised the marsupial orthologue of phlda the amino acid sequence of tammar phlda shared highest conservation within the ph domain and lower conservation in the flanking sequences of both terminals, suggesting the essential role in the ph domain in contrast to the flanking regions, consistent with previous reports. there was a similar high conservation of the amino acid sequences within the ph domain in the platypus, tammar and human phlda suggesting that this domain has a significant role with a similar function in marsupials and monotremes. the high level of mrna expression in the trilaminar yolk sac and the protein localisation to the cytoplasm of trophoblast cells suggest that phlda functions in the tammar yolk sac placenta during pregnancy. however, although murine phlda has the highest expression in the yolk sac, in mice with a disrupted phlda gene the only abnormalities reported are in the chorioallantoic placenta. therefore, an ancestral role for phlda in the yolk sac might have been transferred to the chorioallantoic placenta during the evolution of the mouse. there was no evidence of genomic imprinting of tammar phlda in this study. the mouse kcnq domain forms a large imprinted gene cluster including phlda slc22a cdkn1c, kcnq ascl and some placenta-specific imprinted genes. however, we know now that at least two genes, cdkn1c and phlda that are involved in embryonic and placental growth in eutherians, are not imprinted in this marsupial. considering that both genes are located to the middle of the domain and that all the imprinted genes in this domain are co-ordinately regulated by a single imprinting centre in the mouse, our data strongly suggests that the whole kcnq domain lacks genomic imprinting in marsupials. interestingly, the igf2-h imprinted domain, located adjacent to the kcnq domain shares a highly conserved imprinting regulatory mechanism complete with a differentially methylated region and associated mirna between eutherians and marsupials. this study thus confirms that the origin of imprinting of the kcnq domain evolved in the eutherian lineage after the divergence of marsupials, whereas that of the igf2-h domain appeared before the marsupial-eutherian split, regardless of the close proximity of these two domains. in the kcnq domain of mice, while cdkn1c is a negative regulator for embryonic growth, phlda negatively controls placental growth and acts as a true rheostat for placental growth. recently, using a single copy transgenic mouse, tunster et al., reported that phlda regulates extraembryonic energy stores. two-fold over-expression of phlda caused a 60% loss of the spongiotrophoblast layer with a 25-35% reduction of glycogen storage. since acquisition of genomic imprinting of phlda in the kcnq domain by silencing of the paternal allele was accompanied by gene dosage reduction in eutherians, this might have affected the evolution of placental structure and/or energy stores. in laboratory mice that have two active copies of phlda with the second copy provided by the bac transgene, there was only a slight progressive slowing of embryonic growth. however, greater reduction of fetal growth may have been seen if the mice had had restricted food intake, as is often the case in the wild, so that limited nutrition would need to be partitioned between mother and fetuses. in this situation, reduced expression of phlda could have had a selective advantage through greater placental development. we hypothesise that acquisition of imprinting in the kcnq domain in the ancestral line that gave rise to the eutherian mammals may have allowed increased the placental growth and extended gestation that characterises this group of mammals.  the high level of mrna expression in the trilaminar yolk sac placenta and the protein localisation to the cytoplasm of trophoblast cells suggest that tammar phlda is functional in their placenta. the lack of imprinting in the tammar phlda confirms an earlier conclusion that acquisition of genomic imprinting to the kcnq domain occurred specifically in the eutherian lineage after the divergence of therian mammals into marsupials and eutherians, despite the fact that imprinting of the adjacent igf2-h domain arose before the marsupial-eutherian split. thus genomic imprinting of the kcnq domain might have contributed to the development of complex placentation and the lengthening of gestation in the eutherian lineage by reducing gene dosage of negative regulators for both embryonic and placental growth. 
in mice, phlda is a dose-sensitive negative regulator of placental growth, as cdkn1c is for embryonic growth. these data are consistent with the hypothesis that genomic imprinting of the kcnq domain may have contributed to the evolution of more complex placentation in the eutherian lineage by reduction of the gene dosage of negative regulators for both embryonic and placental growth. we have now identified and characterised the marsupial orthologue of phlda another gene in the kcnq domain that is imprinted in eutherians. our results confirm the idea that acquisition of genomic imprinting in the kcnq domain occurred specifically in the eutherian lineage after the divergence of marsupials, even though imprinting of the adjacent igf2-h domain arose before the marsupial-eutherian split. tammar phlda is highly expressed in the yolk sac placenta compared to other fetal tissues, confirming a similar expression pattern to that of mouse phlda however, tammar phlda is biallelically expressed in both the fetus and yolk sac placenta, so it is not imprinted. we previously reported that there is no evidence of genomic imprinting of cdkn1c in the kcnq domain in the placenta of an australian marsupial, the tammar wallaby whereas tammar igf and h located adjacent to the kcnq domain in eutherian mammals, are imprinted. the lack of imprinting in tammar phlda suggests that the acquisition of genomic imprinting of the kcnq domain in eutherian mammals, accompanied with gene dosage reduction, occurred after the split of the therian mammals into the marsupials and eutherians.
 genomic imprinting causes parent-of-origin specific gene expression by differential epigenetic modifications between two parental genomes. we previously reported that there is no evidence of genomic imprinting of cdkn1c in the kcnq domain in the placenta of an australian marsupial, the tammar wallaby whereas tammar igf and h located adjacent to the kcnq domain in eutherian mammals, are imprinted. we have now identified and characterised the marsupial orthologue of phlda another gene in the kcnq domain that is imprinted in eutherians. in mice, phlda is a dose-sensitive negative regulator of placental growth, as cdkn1c is for embryonic growth.  tammar phlda is highly expressed in the yolk sac placenta compared to other fetal tissues, confirming a similar expression pattern to that of mouse phlda however, tammar phlda is biallelically expressed in both the fetus and yolk sac placenta, so it is not imprinted. the lack of imprinting in tammar phlda suggests that the acquisition of genomic imprinting of the kcnq domain in eutherian mammals, accompanied with gene dosage reduction, occurred after the split of the therian mammals into the marsupials and eutherians.  our results confirm the idea that acquisition of genomic imprinting in the kcnq domain occurred specifically in the eutherian lineage after the divergence of marsupials, even though imprinting of the adjacent igf2-h domain arose before the marsupial-eutherian split. these data are consistent with the hypothesis that genomic imprinting of the kcnq domain may have contributed to the evolution of more complex placentation in the eutherian lineage by reduction of the gene dosage of negative regulators for both embryonic and placental growth. 