BACKGROUND
alternative splicing  of transcripts is common in diverse eukaryotic lineages.
by this mechanism, a variety of transcripts and proteins are produced from a single gene, contributing to increased transcriptome and proteome diversity.
as has been reported in a wide range of eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi  <cit> .
however, it is unclear and hard to assess whether this process has arisen independently in the different lineages  or whether it was already present in their last common ancestor.
the spliceosome, the machinery responsible for the splicing of introns in eukaryotic genes, is ancestral to all extant eukaryotic groups with the last common ancestor possessing a complex machinery, similar to that found in most modern organisms  <cit> .
in addition, we recently argued that eukaryotic ancestors had weak 5' splice site boundary consensus sequences  <cit> , a characteristic that is linked to the presence of as in modern organisms  <cit> .
these ancestral traits thus allow for the possibility that as arose early in eukaryotic evolution.
how can we begin to address this issue?
if as arose independently in different lineages, we might expect that different classes of genes would show varying levels of as in separate lineages, reflecting differential evolutionary histories.
in particular, genes with regulatory functions, such as transcription factors  <cit>  or signal transducers  <cit> , exhibit high levels of alternative splicing in mammals, consistent with a central role for as in generating the complexity of mammalian ontology  <cit> , while basic enzymatic functions show less splice variation  <cit> .
by the same reasoning, if as arose along with the rise of organismal complexity in different multicellular lineages, genes central to this complexity would likely have high as frequencies, while conserved ancient eukaryotic gene functions might have lower as frequencies.
notably, the finding of significant as in the intron-rich pathogenic unicellular fungus cryptococcus neoformans  <cit>  demonstrates that widespread as is not restricted to multicellular or highly-differentiated organisms.
indeed, from an evolutionary viewpoint, it is not likely that as would evolve "in order for" multicellularity to develop; rather, it is possible that as already existed  and was then later co-opted for multicellular development.
intron/exon structure may be an important determinant for evolution of as.
genes with more introns have more opportunities for as.
this would be consistent with vertebrates' higher intron numbers and as frequencies  <cit> .
however, recent results have shown that vertebrate intron number is not particularly high by historical metazoan standards  <cit> , and that early eukaryotic ancestors likely harbored relatively high intron numbers  <cit> .
we studied patterns of as in  <dig> well-annotated genomes from plants, fungi and animals.
we compared frequencies of as of genes of different classes according to their gene structure, evolutionary origins, phylogenetic distribution and functionality.
our major findings include:  ancient genes  are equally likely to have known as as 'newer' genes;  ancient functions are carried by genes that show relatively high levels of as;  genes found across all lineages  are no more likely to show as than are genes that have been lost in one or more lineages; and  there is a strong relationship between intron number and the existence of known as across genes.
we interpret our results to support the notion that a potentially widespread as may have been present at least as early as the unicellular ancestor of animals, fungi and plants.
RESULTS
intron/exon structure and as frequency
we found a clear positive relationship between average intron number per gene and occurrence of as across  <dig> animal, fungus, and plant species .
this relationship is also seen across genes within each of the eight species with significant frequencies of as: higher intron number is associated with higher as levels within each genome .
in particular, there is a steady increase in incidence of as among genes with up to 6– <dig> introns.
given estimates of high intron densities in the plant animal ancestor , this finding is consistent with frequent ancestral as.
age of alternatively spliced genes
the kog database  <cit>  includes groups of orthologous genes for seven animal, fungus, and plant species.
for each of the four species with significant as levels in the kog database  we divided gene families into four groups: 1) common  to plants, animals, and fungi ; 2) common to fungi and animals ; 3) specific to animals ; 4) specific to a single lineage  .
gene dispensability and alternative splicing
among kog's shared between a. thaliana and animals and/or fungi, we determined as in 'indispensable' genes  and 'dispensable' genes ; both classes of genes showed high as levels .
furthermore, no correlation was found between a kog's pgl  and as in any species .
thus, genes encoding basic and highly conserved cellular functions are no less likely to be alternatively spliced than are other genes .
cellular location of alternatively spliced genes
the level of as by cellular location for  <dig> eukaryotes is shown in figure  <dig> and in detail in additional file  <dig>  genes for proteins in most cellular locations showed as.
in particular, we were interested in the level of as of genes encoding extracellular proteins, since many of these genes are likely to be important in the intercellular structures and communication vital to multicellularity  than in the multicellular species ).
such extracellular-associated genes did not show elevated as rates.
instead, genes encoding proteins located in the nucleus and plasma membrane generally have higher proportions of as.
functional profile of alternatively spliced genes
as levels across species for molecular function  and biological process  go categories are shown in figures  <dig> and  <dig>  respectively, and in detail in additional files  <dig> and  <dig>  respectively.
again, gene categories generally associated with multicellularity  did not show elevated as rates.
among molecular functions, protein kinase activity, rna binding and calcium ion binding generally had high as frequencies while monooxygenase activity, receptor activity, transporter activity and heme binding had much lower as across all species.
biological processes showed greater variation across species
functional profiling of alternatively spliced genes thus shows that most genes encoding most cellular functions exhibit as.
some functions seem to be especially amenable to as, perhaps due to these gene functions being particularly improved by the production of multiple products.
as is particularly prevalent in genes associated with regulation and signaling, consistent with previous observations  <cit> .
interestingly, the overrepresentation of as in genes with regulatory functions previously observed in mammals is observed across lineages.
discussion
patterns of genome-wide as usage are similar in different eukaryotic lineages
we studied patterns of genome-wide as in  <dig> eukaryotic genomes.
our major findings include:
1) a correspondence between intron number and frequency of known as, both within and between species;
2) evidence that ancient genes show relatively high levels of known as.
3) no evidence for elevated as in recently evolved genes – in fact, the most recently evolved genes are less likely to have known as;
4) no clear evidence for elevated as in classes of genes thought to be important in the rise of multicellularity;
5) a variety of similarities in the patterns of as across diverse species, which could reflect patterns inherited from a putative as-rich plant-animal ancestor.
alternatively spliced genes in animals, plants and fungi have several common features.
in all studied species, as is widely found across genes with different functions, although those associated with regulation  show consistently higher as levels.
also, the species studied showed similar patterns of as usage in genes of different evolutionary age.
finally, intron number per gene was related with as frequency in a similar manner in all species.
the simplest hypothesis to explain these similarities is that as is homologous in these groups, inherited from their common ancestor, and that as patterns in the common ancestor might have been similar.
however, caution is necessary in interpreting this result.
given the high rates of gains and losses of as events, the alternative hypothesis of convergent evolution of as patterns in the different lineages, although less parsimonious, cannot be excluded.
ancient genes and functions show relatively high levels of as
also consistent with a relatively early origin of as is our finding that recently evolved gene families  showed the lowest frequency of as in all species .
many of these genes are likely associated with newly evolved and complex lineage-specific traits in plants and animals.
on the other hand, ancient gene families , which were already present in the ancestor, are highly alternatively spliced in modern organisms  and a wide range of fundamental ancient functions are currently performed in eukaryotic cells by alternatively spliced genes , indicating no constraints for ancient genes to be alternatively spliced.
finally, gene functions that show consistently higher  or lower  levels of as across eukaryotes could have also had similar relative levels in the common ancestor.
alternatively splicing and proteomic networks
we found no clear relationship between incidence of as and gene dispensability .
indispensable genes tend to have large numbers of interaction partners, occupying central positions in proteomic networks, while dispensable genes usually occupy external positions in the interacting networks  <cit> .
these results thus suggest that as may be integrated across all levels of eukaryotic proteomic networks.
intron-rich gene structures is the main requirement for as
we show that intron/exon number correlates strongly with the frequency of as within and between genomes .
in accordance, most intron-reduced genomes, such as those of most microsporidia and ascomycetes, show no as  <cit>  and other relatively reduced genomes, such as amoebas  <cit>  or apicomplexans  <cit>  do not exhibit high frequencies of as.
interestingly, lse genes, found to have significantly lower levels of as, show lower average intron numbers than the other groups.
our and others' previous work has shown that the plant-animal ancestor was at least moderately intron-rich , and that lower modern densities in some lineages reflect widespread intron loss  <cit> .
taken with present results, there are two important potential implications.
first, retention of ancestral intron densities was likely an important condition for modern as.
thus, if in fact as played an important role in the emergence of organismal complexity  <cit> , differential retention of ancestral introns would have profound consequences for morphological evolution across lineages.
second, intron-rich ancestors are likely to have had significant as.
all thoroughly studied intron-rich genomes show relatively high frequencies of as, suggesting both that a complex gene structure favors as and that as could have an important role in most non-reduced genomes, with high numbers of introns per gene.
as we mention above, this is especially interesting in light of accumulating evidence that the last common ancestor of plants and opisthokonts was at least moderately intron rich  <cit>   and that it had weak consensus 5' splice site boundaries  <cit> .
among modern eukaryotes, both high intron number and weak 5'ss are characteristic of diverse species with widespread alternative splicing  <cit> .
therefore, these studies together strongly suggest the presence of as in plant-animal ancestor
intron numbers, spliceosomal errors, functionality and origin of as
it is important to note that our present results do not address the functionality of alternative splice variants  either in early eukaryotes or in modern organisms, a topic currently under debate  <cit> .
alternative transcripts produced from the same gene might:  encode different functions,  reflect nonfunctional  variants or  represent rare spliceosomal errors, which will all appear in est databases and thus in est-based as annotations.
it seems likely that all three cases contribute to modern transcriptome variability.
if in fact our argument is correct , it would be interesting to know how levels of as functionality have changed through time.
increased requirements on proteome and regulatory flexibility could have driven an increase in functional as.
in tandem, refinements in the spliceosomal machinery could have increased splicing fidelity through eukaryotic evolution, disproportionately decreasing nonfunctional as variants.
interestingly, the evolutionary origin of functional as is likely related to mis-splicing .
as might have evolved from mis-splicing as the early eukaryotic cells evolved to use and benefit from multiple splicing outputs.
therefore, the widespread production of multiple splice forms could be a main requirement for the origin of functional as.
thus, our results along with the likely existence of weak splice sites in early eukaryotes  <cit>  do not prove that early eukaryotes had functional as, but they strongly suggest that the last plant-animal ancestor had at least such additional splice variants available for potential participation and recruitment in biological processes.
as in unicellular organisms and the origin of multicellularity
a striking potential implication of our results is that as already existed in the plant-animal ancestor, a rather ancient and "primitive" unicellular eukaryote.
as seen in some modern unicellular organisms , as could have played an extensive role in the biology and evolution of these ancestral unicellular eukaryotes.
in this case, as would have predated multicellularity and could perhaps have been recruited to allow the rise of multicellular complexity.
this would resemble other biological processes, like apoptosis, whose origin precedes the rise of multicellular organisms, although the co-option of this ability was crucial for the advent of multicellularity  <cit> .
reliability of as databases to answer evolutionary questions
though we restricted our analysis to well-annotated genomes from long- and deeply-studied species with wide cdna/est coverage  <cit> , it is likely that many alternative transcripts are not represented in current annotations, introducing the possibility of sampling biases.
for instance, some gene types have been more thoroughly studied, and therefore may show higher proportions of annotated as.
however, such differences are unlikely to explain our central conclusions, since they are largely based on shared similarities, not differences , and clear associations across large numbers of genes.
indeed, est coverage in c. elegans does not correlate with fractions of predicted alternatively spliced genes across different go categories or gene ages, suggesting that the observed patterns are not due to est sampling .
another potential problem associated with est based annotation is the source of these data, especially in the case of humans.
many human est libraries derive from cancerous or abnormal cell lineages, thought to contain aberrant, disease related alternative splice variants  <cit> .
if these variants are more frequent among some groups of genes, this could introduce a bias in our results.
however, our results for functional ontologies are based on data from a variety of species and the patterns presented here are consistent among all the species despite this potential source of noise in human databases.
we used genome annotation databases for this analysis because they are constructed using very similar approaches and so they might be more suitable for comparing these species.
supporting the quality of the studies databases, we found in the thorough analysis of the c. elegans wormbase dataset that the vast majority of alternatively spliced isoforms included are well supported by experimental evidence, and only very few cases represent annotation mistakes.
finally, it should be noted that the current results concern only presence/absence of as, rather than number of alternative transcripts.
since alternatively spliced genes may produce from  <dig> to hundreds of isoforms, the effects on the transcriptome output will be quite different across genes, and further studies should address this important issue.
instead, we have concentrated on known as presence/absence in a gene, which is likely to be less sensitive to differences in est sampling.
similarly, different positions of the as events in each gene may produce very different outputs with different effects on the organism's fitness.
our analysis does not take differences in function between splice variants into account.
however, these considerations are unlikely to affect our conclusions about as in early eukaryotes.
to verify our hypotheses on the emergence of as, further studies of conservation of as mechanisms , splicing boundaries, and expression patterns will be necessary.
characterization of levels and patterns of as in diverse additional eukaryotes, particularly unicellular intron-rich species, will also be important.
species of apicomplexans  <cit>  and diatoms  <cit>  have already been shown to have as.
est and genome sequencing projects will provide data to assess whether as was an ancestral feature of eukaryotic organisms, playing another important role in the complex rna processing of early eukaryotes  <cit> .
CONCLUSIONS
we find similar patterns of genome-wide as usage in different eukaryotic lineages.
we show that ancient genes and functions  have high levels of as in modern organisms indicating no bias against as of these genes.
these genes were also likely intron-rich in the common ancestor  <cit> , which we find to be the main requirement for as.
since the spliceosomal machinery is widely conserved throughout eukaryotes  <cit> , our results favor the hypothesis that some form of as appeared relatively early in eukaryotic evolution, at least in the unicellular common ancestor of plants, animals and fungi .
this implies as appeared before the rise of multicellular organisms, and could therefore have an important role in the biology of ancient unicellular organisms.

 alternative splicing of transcripts is common in diverse eukaryotic lineages. by this mechanism, a variety of transcripts and proteins are produced from a single gene, contributing to increased transcriptome and proteome diversity. as has been reported in a wide range of eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. however, it is unclear and hard to assess whether this process has arisen independently in the different lineages or whether it was already present in their last common ancestor. the spliceosome, the machinery responsible for the splicing of introns in eukaryotic genes, is ancestral to all extant eukaryotic groups with the last common ancestor possessing a complex machinery, similar to that found in most modern organisms. in addition, we recently argued that eukaryotic ancestors had weak 5' splice site boundary consensus sequences, a characteristic that is linked to the presence of as in modern organisms. these ancestral traits thus allow for the possibility that as arose early in eukaryotic evolution. how can we begin to address this issue? if as arose independently in different lineages, we might expect that different classes of genes would show varying levels of as in separate lineages, reflecting differential evolutionary histories. in particular, genes with regulatory functions, such as transcription factors or signal transducers, exhibit high levels of alternative splicing in mammals, consistent with a central role for as in generating the complexity of mammalian ontology, while basic enzymatic functions show less splice variation. by the same reasoning, if as arose along with the rise of organismal complexity in different multicellular lineages, genes central to this complexity would likely have high as frequencies, while conserved ancient eukaryotic gene functions might have lower as frequencies. notably, the finding of significant as in the intron-rich pathogenic unicellular fungus cryptococcus neoformans demonstrates that widespread as is not restricted to multicellular or highly-differentiated organisms. indeed, from an evolutionary viewpoint, it is not likely that as would evolve "in order for" multicellularity to develop; rather, it is possible that as already existed and was then later co-opted for multicellular development. intron/exon structure may be an important determinant for evolution of as. genes with more introns have more opportunities for as. this would be consistent with vertebrates' higher intron numbers and as frequencies. however, recent results have shown that vertebrate intron number is not particularly high by historical metazoan standards, and that early eukaryotic ancestors likely harbored relatively high intron numbers. we studied patterns of as in well-annotated genomes from plants, fungi and animals. we compared frequencies of as of genes of different classes according to their gene structure, evolutionary origins, phylogenetic distribution and functionality. our major findings include: ancient genes are equally likely to have known as as'newer' genes; ancient functions are carried by genes that show relatively high levels of as; genes found across all lineages are no more likely to show as than are genes that have been lost in one or more lineages; and there is a strong relationship between intron number and the existence of known as across genes. we interpret our results to support the notion that a potentially widespread as may have been present at least as early as the unicellular ancestor of animals, fungi and plants.  intron/exon structure and as frequency we found a clear positive relationship between average intron number per gene and occurrence of as across animal, fungus, and plant species. this relationship is also seen across genes within each of the eight species with significant frequencies of as: higher intron number is associated with higher as levels within each genome. in particular, there is a steady increase in incidence of as among genes with up to 6– introns. given estimates of high intron densities in the plant animal ancestor, this finding is consistent with frequent ancestral as. age of alternatively spliced genes the kog database includes groups of orthologous genes for seven animal, fungus, and plant species. for each of the four species with significant as levels in the kog database we divided gene families into four groups: 1) common to plants, animals, and fungi; 2) common to fungi and animals; 3) specific to animals; 4) specific to a single lineage. gene dispensability and alternative splicing among kog's shared between a. thaliana and animals and/or fungi, we determined as in'indispensable' genes and'dispensable' genes; both classes of genes showed high as levels. furthermore, no correlation was found between a kog's pgl and as in any species. thus, genes encoding basic and highly conserved cellular functions are no less likely to be alternatively spliced than are other genes. cellular location of alternatively spliced genes the level of as by cellular location for eukaryotes is shown in figure and in detail in additional file genes for proteins in most cellular locations showed as. in particular, we were interested in the level of as of genes encoding extracellular proteins, since many of these genes are likely to be important in the intercellular structures and communication vital to multicellularity than in the multicellular species ). such extracellular-associated genes did not show elevated as rates. instead, genes encoding proteins located in the nucleus and plasma membrane generally have higher proportions of as. functional profile of alternatively spliced genes as levels across species for molecular function and biological process go categories are shown in figures and respectively, and in detail in additional files and respectively. again, gene categories generally associated with multicellularity did not show elevated as rates. among molecular functions, protein kinase activity, rna binding and calcium ion binding generally had high as frequencies while monooxygenase activity, receptor activity, transporter activity and heme binding had much lower as across all species. biological processes showed greater variation across species functional profiling of alternatively spliced genes thus shows that most genes encoding most cellular functions exhibit as. some functions seem to be especially amenable to as, perhaps due to these gene functions being particularly improved by the production of multiple products. as is particularly prevalent in genes associated with regulation and signaling, consistent with previous observations. interestingly, the overrepresentation of as in genes with regulatory functions previously observed in mammals is observed across lineages. discussion patterns of genome-wide as usage are similar in different eukaryotic lineages we studied patterns of genome-wide as in eukaryotic genomes. our major findings include: 1) a correspondence between intron number and frequency of known as, both within and between species; 2) evidence that ancient genes show relatively high levels of known as. 3) no evidence for elevated as in recently evolved genes – in fact, the most recently evolved genes are less likely to have known as; 4) no clear evidence for elevated as in classes of genes thought to be important in the rise of multicellularity; 5) a variety of similarities in the patterns of as across diverse species, which could reflect patterns inherited from a putative as-rich plant-animal ancestor. alternatively spliced genes in animals, plants and fungi have several common features. in all studied species, as is widely found across genes with different functions, although those associated with regulation show consistently higher as levels. also, the species studied showed similar patterns of as usage in genes of different evolutionary age. finally, intron number per gene was related with as frequency in a similar manner in all species. the simplest hypothesis to explain these similarities is that as is homologous in these groups, inherited from their common ancestor, and that as patterns in the common ancestor might have been similar. however, caution is necessary in interpreting this result. given the high rates of gains and losses of as events, the alternative hypothesis of convergent evolution of as patterns in the different lineages, although less parsimonious, cannot be excluded. ancient genes and functions show relatively high levels of as also consistent with a relatively early origin of as is our finding that recently evolved gene families showed the lowest frequency of as in all species. many of these genes are likely associated with newly evolved and complex lineage-specific traits in plants and animals. on the other hand, ancient gene families, which were already present in the ancestor, are highly alternatively spliced in modern organisms and a wide range of fundamental ancient functions are currently performed in eukaryotic cells by alternatively spliced genes, indicating no constraints for ancient genes to be alternatively spliced. finally, gene functions that show consistently higher or lower levels of as across eukaryotes could have also had similar relative levels in the common ancestor. alternatively splicing and proteomic networks we found no clear relationship between incidence of as and gene dispensability. indispensable genes tend to have large numbers of interaction partners, occupying central positions in proteomic networks, while dispensable genes usually occupy external positions in the interacting networks. these results thus suggest that as may be integrated across all levels of eukaryotic proteomic networks. intron-rich gene structures is the main requirement for as we show that intron/exon number correlates strongly with the frequency of as within and between genomes. in accordance, most intron-reduced genomes, such as those of most microsporidia and ascomycetes, show no as and other relatively reduced genomes, such as amoebas or apicomplexans do not exhibit high frequencies of as. interestingly, lse genes, found to have significantly lower levels of as, show lower average intron numbers than the other groups. our and others' previous work has shown that the plant-animal ancestor was at least moderately intron-rich, and that lower modern densities in some lineages reflect widespread intron loss. taken with present results, there are two important potential implications. first, retention of ancestral intron densities was likely an important condition for modern as. thus, if in fact as played an important role in the emergence of organismal complexity, differential retention of ancestral introns would have profound consequences for morphological evolution across lineages. second, intron-rich ancestors are likely to have had significant as. all thoroughly studied intron-rich genomes show relatively high frequencies of as, suggesting both that a complex gene structure favors as and that as could have an important role in most non-reduced genomes, with high numbers of introns per gene. as we mention above, this is especially interesting in light of accumulating evidence that the last common ancestor of plants and opisthokonts was at least moderately intron rich and that it had weak consensus 5' splice site boundaries. among modern eukaryotes, both high intron number and weak 5'ss are characteristic of diverse species with widespread alternative splicing. therefore, these studies together strongly suggest the presence of as in plant-animal ancestor intron numbers, spliceosomal errors, functionality and origin of as it is important to note that our present results do not address the functionality of alternative splice variants either in early eukaryotes or in modern organisms, a topic currently under debate. alternative transcripts produced from the same gene might: encode different functions, reflect nonfunctional variants or represent rare spliceosomal errors, which will all appear in est databases and thus in est-based as annotations. it seems likely that all three cases contribute to modern transcriptome variability. if in fact our argument is correct, it would be interesting to know how levels of as functionality have changed through time. increased requirements on proteome and regulatory flexibility could have driven an increase in functional as. in tandem, refinements in the spliceosomal machinery could have increased splicing fidelity through eukaryotic evolution, disproportionately decreasing nonfunctional as variants. interestingly, the evolutionary origin of functional as is likely related to mis-splicing. as might have evolved from mis-splicing as the early eukaryotic cells evolved to use and benefit from multiple splicing outputs. therefore, the widespread production of multiple splice forms could be a main requirement for the origin of functional as. thus, our results along with the likely existence of weak splice sites in early eukaryotes do not prove that early eukaryotes had functional as, but they strongly suggest that the last plant-animal ancestor had at least such additional splice variants available for potential participation and recruitment in biological processes. as in unicellular organisms and the origin of multicellularity a striking potential implication of our results is that as already existed in the plant-animal ancestor, a rather ancient and "primitive" unicellular eukaryote. as seen in some modern unicellular organisms, as could have played an extensive role in the biology and evolution of these ancestral unicellular eukaryotes. in this case, as would have predated multicellularity and could perhaps have been recruited to allow the rise of multicellular complexity. this would resemble other biological processes, like apoptosis, whose origin precedes the rise of multicellular organisms, although the co-option of this ability was crucial for the advent of multicellularity. reliability of as databases to answer evolutionary questions though we restricted our analysis to well-annotated genomes from long- and deeply-studied species with wide cdna/est coverage, it is likely that many alternative transcripts are not represented in current annotations, introducing the possibility of sampling biases. for instance, some gene types have been more thoroughly studied, and therefore may show higher proportions of annotated as. however, such differences are unlikely to explain our central conclusions, since they are largely based on shared similarities, not differences, and clear associations across large numbers of genes. indeed, est coverage in c. elegans does not correlate with fractions of predicted alternatively spliced genes across different go categories or gene ages, suggesting that the observed patterns are not due to est sampling. another potential problem associated with est based annotation is the source of these data, especially in the case of humans. many human est libraries derive from cancerous or abnormal cell lineages, thought to contain aberrant, disease related alternative splice variants. if these variants are more frequent among some groups of genes, this could introduce a bias in our results. however, our results for functional ontologies are based on data from a variety of species and the patterns presented here are consistent among all the species despite this potential source of noise in human databases. we used genome annotation databases for this analysis because they are constructed using very similar approaches and so they might be more suitable for comparing these species. supporting the quality of the studies databases, we found in the thorough analysis of the c. elegans wormbase dataset that the vast majority of alternatively spliced isoforms included are well supported by experimental evidence, and only very few cases represent annotation mistakes. finally, it should be noted that the current results concern only presence/absence of as, rather than number of alternative transcripts. since alternatively spliced genes may produce from to hundreds of isoforms, the effects on the transcriptome output will be quite different across genes, and further studies should address this important issue. instead, we have concentrated on known as presence/absence in a gene, which is likely to be less sensitive to differences in est sampling. similarly, different positions of the as events in each gene may produce very different outputs with different effects on the organism's fitness. our analysis does not take differences in function between splice variants into account. however, these considerations are unlikely to affect our conclusions about as in early eukaryotes. to verify our hypotheses on the emergence of as, further studies of conservation of as mechanisms, splicing boundaries, and expression patterns will be necessary. characterization of levels and patterns of as in diverse additional eukaryotes, particularly unicellular intron-rich species, will also be important. species of apicomplexans and diatoms have already been shown to have as. est and genome sequencing projects will provide data to assess whether as was an ancestral feature of eukaryotic organisms, playing another important role in the complex rna processing of early eukaryotes.  we find similar patterns of genome-wide as usage in different eukaryotic lineages. we show that ancient genes and functions have high levels of as in modern organisms indicating no bias against as of these genes. these genes were also likely intron-rich in the common ancestor, which we find to be the main requirement for as. since the spliceosomal machinery is widely conserved throughout eukaryotes, our results favor the hypothesis that some form of as appeared relatively early in eukaryotic evolution, at least in the unicellular common ancestor of plants, animals and fungi. this implies as appeared before the rise of multicellular organisms, and could therefore have an important role in the biology of ancient unicellular organisms. 
we find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. to better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in eukaryotic species, comparing rates of alternative splicing across genes of different functional classes, cellular locations, intron/exon structures and evolutionary origins. ancient genes show high levels of alternative splicing. however, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor, and may therefore predate multicellularity, is still unknown. genes with products expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing.
 alternative splicing has been reported in various eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. however, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor, and may therefore predate multicellularity, is still unknown. to better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in eukaryotic species, comparing rates of alternative splicing across genes of different functional classes, cellular locations, intron/exon structures and evolutionary origins.  for each species, we find that genes from most functional categories are alternatively spliced. ancient genes show high levels of alternative splicing. genes with products expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. we find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. in general, we find several similarities in patterns of alternative splicing across these diverse eukaryotes.  along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. a role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process. 