BACKGROUND
comparative studies of spliceosomal intron densities have suggested relatively high rates of intron gain during eukaryote evolution  <cit> .
the establishment of introns within fast-evolving genes appears to be an infrequent, but common process involving, for example, intronization of exonic sequences  <cit> .
however, recent gains of introns inside of conserved coding sequences , often equated with the usage of novel intron positions, appear to be a rare and poorly understood phenomenon  <cit> .
at least six mechanisms  have been proposed to explain novel intron positions within conserved open reading frames :  insertion of a self-splicing type ii intron via reverse splicing  <cit> ;  insertion of a spliceosomal intron via reverse splicing into a new position  <cit> ;  partial tandem duplication of an exon including a cryptic ag/gy splice motif  <cit> ;  insertion of a transposable element  <cit> ;  gene conversion from an intron-containing site into a previously intron-less paralogous site  <cit> ; and  intron sliding  <cit> .
only the last three pathways are supported by undisputed, albeit anecdotal, evidence  <cit> .
recently, a study on daphnia populations  <cit>  suggested another intron gain mechanism: the repair of dna double-strand breaks using small segmental insertions.
the analysis of near intron pairs  <cit>  allows a systematic investigation of intron gain mechanisms.
a near intron pair  consists of two intron positions that exist in orthologous genes at nearby locations.
exon sizes smaller than about  <dig> nt are relatively rare  <cit>  and in general functionally detrimental  <cit> .
thus, such nearby introns typically exclude each other within a single gene.
accordingly, one of these introns must be evolutionarily younger and should define a monophyletic group.
by using nips to determine the time at which the younger introns were gained, it can be avoided to erroneously identify those introns as novel that have been lost independently in multiple lineages.
here we use the relatively recently diverged genomes of  <dig> drosophila species  <cit>  to identify recent intron gain events in a comparative analysis of gene structures and evaluate possible mechanisms of their origin.
in contrast to previous attempts to identify intron loss and gain in drosophila  <cit> , we chose the nip approach instead of dollo parsimony to restrict our analysis to introns for which the evidence of intron gain is more unambiguous.
we could identify  <dig> nips within drosophila.
their distribution supports the known species phylogeny.
both introns of a nip were evaluated for sequence similarity to introns, neighboring exons and transposable elements and screened for repetitiveness and potentially meaningful secondary structures, using the ancient, plesiomorphic intron of the nip as control.
in addition, we looked for cryptic splice signals in the adjacent exonic and intronic sequences.
there is evidence for intron sliding in  <dig> of the  <dig> drosophila nips, while  <dig> other cases probably arose by tandem duplications within the orf.
in contrast, we found no evidence for any intron insertion mechanism based on alien sequences in our data set.
RESULTS
compilation and characterization of the nip data set
we started with  <dig> sets of orthologous protein-coding genes of drosophila species .
after an automatic nip extraction alignment procedure, we obtained  <dig> nip regions containing two or more near introns .
these were manually inspected for splice site, alignment and conservation validity, resulting in  <dig> regions comprising  <dig> nips.
based on the frequency of short exons and nip distances within drosophila , we decided to decrease the maximal allowed intron distance to  <dig> nt, which resulted in  <dig> alignment regions comprising  <dig> nips .
since there was an alternative site consistent with the old  intron position that could not be clearly excluded by splice site sequence analysis, we evaluated seven nip candidates experimentally.
as no est data supported these nips, we performed rt-pcr or genomic pcr experiments in some crucial species .
our analysis confirmed  <dig> of the investigated nips .
the remaining  <dig> cases were reducible to one intron position.
three of these cases were based on sequence errors that could be corrected by genomic pcr or rt-pcr .
one nip candidate represented a frame shift mutation that led to an incorrectly annotated splice site .
in the last case , intron sliding has happened only at the 3' splice site.
age of nips
not all of the remaining  <dig> nips contain introns that necessarily have been gained during the evolution of the genus drosophila.
using the intron distributions among drosophila and the arthropod outgroup species glossina morsitans, aedes aegypti, anopheles gambiae, culex pipiens, bombyx mori, tribolium castaneum, apis mellifera, nasonia vitripennis, acyrthosiphon pisum, pediculus humanus, daphnia pulex and ixodes scapularis, we discovered that at least  <dig> intron positions of the  <dig> nips have arisen during drosophila radiation.
<dig> nips contain exactly one novel intron position, whereas  <dig> other nips consist of two different novel intron positions, which are supported by other nearby introns in the outgroup species.
the phase distribution of these  <dig> novel introns is similar to the average intron phase distribution in drosophila  <cit> :  <dig> of these introns are in phase  <dig> , and  <dig> in phase  <dig> and phase  <dig>  respectively .
for the  <dig> remaining nips, the relative age of intron positions could not be determined either due to a lack of sufficient local sequence similarity to orthologous gene structures outside of drosophila or due to a lack of nearby introns in outgroup sequences.
nips are suitable phylogenetic markers for drosophila species
nips were primarily introduced as reliable phylogenetic markers for insect evolution  <cit> .
the well-known phylogeny of drosophila  <cit>  opens the possibility to verify the suitability of nips for phylogenetic analyses of recent radiations .
phylogenetically informative nips within drosophila were identified using the aforementioned arthropod outgroup species.
together,  <dig> synapomorphic  and  <dig> autapomorphic  nip characters appeared during the evolution of the genus drosophila.
the subgenus drosophila, the species groups obscura and melanogaster, the sister relationship between the melanogaster and the obscura species groups and as well as the melanogaster species subgroup are supported by at least one synapomorphically distributed nip .
for  <dig> remaining internal nodes  and  <dig> remaining external nodes  of the tree no supporting nip evidence was found.
out of  <dig> nips, only one character distribution  contradicts the established tree of the  <dig> drosophila species.
this nip is clearly associated with a sliding event .
the local nature of sliding makes independent changes of intron position with identical results much more likely than any of the other mechanisms, which require independent targeting of the same genomic position.
we conclude that nips could be reliable phylogenetic markers also for recent radiations.
however, short branch lengths, as observed between drosophila species, could critically limit the number of available characters.
intron sliding accounts for a low but detectable level of homoplasy in this type of character.
intron migration into novel positions
next, we evaluated possible evolutionary mechanisms that had produced introns at novel positions.
for all the introns of the  <dig> nips, we could not find any relevant sequence conservation to other introns, to transposons, or to exons .
to control whether some of the intron position changes resulted from intron sliding, we evaluated all introns of the nips concerning properties that would be expected if a coordinated migration of both splice sites occurred in relation to the former intron and the cds.
these properties are:  there is no intermediate, intron-less state in the tree;  nip distance is a multiple of  <dig> allowing a stepwise intron shift;  cryptic splice sites occur at nip distances and are supported by amino acid conservation ;  one-sided shifts or gyrgyr/nagnag splice sites in some species ; and  significant sequence similarity  between introns of both positions .
the last criterion is sufficient on its own to support intron sliding and was found in  <dig> cases .
here, we observed that the intron sequence had stayed in place, but that the splice sites seem to have been migrated.
"nt" means nip distance in nucleotides.
note that each of the  <dig> nips resulting supposedly from intron sliding shows at least three of the expected properties and/or a significant sequence similarity between introns of both positions .
introns gained by tandem duplication show a proto-splice site.
in addition, a plesiomorphic intron is not identifiable because both introns probably have been gained by the same duplication.
in another  <dig> nips, at least three of the other four criteria are fulfilled.
while the first two conditions are also consistent with mechanisms of intron gain , cryptic splice sites at the corresponding distance and one-sided shifts of intron borders in some species are specific requirements for intron sliding .
thus, the fulfillment of at least three criteria argues for sliding also in these cases.
it should be noted that only one case of sliding  has occurred within about  <dig> million years of evolution   <cit>  and was, therefore, expected to show sequence conservation between both introns of the nip.
all other putative sliding events have occurred between drosophila lineages that diverged at least  <dig> million years ago .
no footprint of internal sequence conservation across the two intron positions has been retained in any of these cases.
in summary,  <dig> out of  <dig> nips most likely originated by intron sliding.
this is surprising since earlier studies concluded that intron sliding is a very rare event  <cit> .
on the other hand, frequent intron sliding can be expected as a consequence of the high abundance of tandem splice sites  in eukaryotic genes  <cit> .
detection of strong proto-splice sites in a subset of novel introns
donor and acceptor site consensus sequences of spliceosomal splicing also include exonic parts.
at the donor  splice site, the last two exonic nucleotides  could bind to the u <dig> snrna  <cit>  and the corresponding nucleotide positions are essential in some human splice sites  <cit> .
in contrast, the acceptor  splice site appears to be functionally independent from exonic parts of the consensus  <cit> .
surprisingly, there are nucleotide preferences nevertheless, typically 5'-rt-3'  <cit> .
sverdlov et al.
<cit>  reported that younger introns exhibit a stronger signal in the exonic part of both splice consensus parts than older ones, while the intronic part of the splice signal is stronger for older introns than for younger ones.
both observations are to be expected if novel introns sometimes originate through tandem duplications within coding exons .
following such an event, a duplicated proto-splice site  could turn the sequence between both sites into an intron   <cit> .
in drosophila, the role of the branch site and of additional enhancer and silencer sequences is very limited  <cit>  and typically does not interfere with an intron definition by donor and acceptor site alone.
thus, an intron could immediately emerge by tandem duplication of a proto-splice site and will have the size of the duplication.
concomitantly, such a duplication will allow differential splice patterns  as a temporary or permanent alternative to the establishment of a constitutive intron if the duplication size is a multiple of three.
alternatively, unspliced mrna variants may be degraded by nonsense-mediated decay   <cit> .
thus, nmd may often serve as a backup for weak splicing of such novel introns as recently suggested by farlow et al.
<cit> .
the mature mrna will remain qualitatively unchanged during such a process.
later on, the splice signals could be improved by selection, whereas the other parts of the novel intronic sequence are free to change by insertions, deletions and substitutions.
thus, we searched for coincidences of introns and proto-splice sites supported by amino acid conservation.
we found  <dig> introns in  <dig> nips that are surrounded by an ag/gy proto-splice site.
for  <dig> of these introns , the proto-splice sites are conserved also in drosophila species having no intron there, and should thus already have been present at the time of intron origin.
the potential functionality of these sites both as donor and acceptor sites is supported by their splice site scores  that reside within the variability of functional splice sites of d. melanogaster, but well above the splice site scores of typical exonic sequences surrounding drosophila introns .
for two of these  <dig> td introns, regional repetitiveness enhances the probability of duplications in the cds .
in all  <dig> individual sequences of td introns we found a premature termination codon, suggesting that nmd might have played a role during their evolution.
for details of the reference data set and score generation see the methods section.
percentile values in brackets are with respect to the rp reference set.
consensus nts: b = not a, d = not c, h = not g, k = g or t, m = a or c, r = a or g, s = c or g, u = not t, w = a or t, y = c or t.
identified proto-splice sites support tandem duplication rather than reverse splicing
the concept of the proto-splice site was proposed more than  <dig> years ago  <cit> .
despite the simplicity of the tandem duplication mechanism , intron gain was seldom explained in this way .
instead, proto-splice sites were introduced as preferential insertion sites for reverse-spliced introns  <cit> .
this pathway requires four successive, rate-limiting steps, namely  germ line transcription of the target gene;  reverse splicing of an - occasionally retained - intron lariat into a novel site of the target mrna;  reverse transcription of this now intron-containing mrna; and  homologous recombination of this cdna with the target gene.
to our knowledge, this reverse-splicing pathway has never been shown to have produced a spliceosomal intron.
in contrast, tandem duplications frequently occur in natural populations.
emerson et al.
<cit>  compared  <dig> natural isofemale lines of d. melanogaster and detected  <dig> duplications, mostly in tandem, with a median size of  <dig> nt in at least one line.
irrespective of the strong evidence for purifying selection against this type of mutations,  <dig> of these duplications included some exonic sequences, but not a whole gene.
given this significant amount of function-challenging mutations, the gain of an intron may compensate for a duplication simply if a proto-splice site is included.
in order to further support the hypothesis that the  <dig> td introns arose by tandem duplication rather than by reverse splicing, we evaluated their proto-splice sites.
if splice sites had emerged directly by duplication of proto-splice sites,  proto-splice and splice sites should be similar to each other and  both should be functional splice sites.
if intronic splice sites stemmed from a reverse-spliced intron, proto-splice sites would not need to be similar to splice sites but should have provided a preferential binding site for the spliceosome that has inserted the intron.
the sequence specificity of spliceosome-binding during forward splicing is mediated by the binding of the u <dig> snrnp to the donor site  <cit> .
however, the initial interactions of a spliceosome capable of reverse splicing are unknown.
thus, alternatively, the u <dig> snrnp complex maybe binds first because this complex interacts during the second splicing reaction with both exon ends to ligate them  <cit> .
for u <dig> snrnp, known sequence preferences are weak and not consistent with proto-splice sites  <cit> .
other sequence-specific binding components of the spliceosome such as the u <dig>  u <dig> and u <dig> snrnps exclusively interact with the intron.
reverse splicing, therefore, might have produced proto-splice sites that remember donor sites  or no proto-splice sites at all.
specifically, the acceptor site consensus 5' of ag would not necessarily be included in such a proto-splice site.
to distinguish between the two alternative pathways, we evaluated the splice sites, the proto-splice sites and the relative location of the new introns in more detail:
we determined scores for reference splice sites and proto-splice sites excluding the central ag/gy motif.
these scores are based on the nts + <dig> to + <dig>  and the nts - <dig> to - <dig> .
we found that such partial proto-splice acceptor sites of td introns show intermediate scores between reference splice sites  on the one hand, and potential proto-splice sites surrounding reference introns  and intron-less aggy proto-splice site motifs of exons   on the other hand.
moreover, the partial proto-splice acceptor site scores of the td introns are statistically significantly different  from the corresponding scores of other nip introns  , reference introns   and exonic aggy motifs  , respectively.
in contrast, the scores of partial proto-splice donor sites show no differences .
this argues against reverse splicing that would predict an exclusive similarity to the donor site.
during splicing, the acceptor-binding parts of the spliceosome are occupied by intronic sequences, thus, these parts cannot be involved in target site selection in case of reverse splicing.
if the supposedly duplicated sequences of both intron/exon borders of one intron are compared under exclusion of the central ag/gy motif, these regions of td introns are significantly more similar to each other compared to other nip introns  or reference introns  .
this finding is based on the same data as the first one but they were used here in a slightly different way.
this result argues in favour of tandem duplication.
the location of the td introns within the genes does not support an origin by reverse splicing.
a reverse-transcription mediated pathway would imply a 3' biased distribution of novel introns as the reverse transcriptase should start primarily at the 3' end of the mrna  <cit> .
apomorphic nip introns, however, show a biased location in favour of the 5' and, to a lesser extent, 3' ends of the genes .
whereas the average locations of apomorphic introns of the td and on sets across the  <dig> bins were not significantly different , there is a moderate significance  to reject the null hypothesis that the  <dig> novel introns  are equally distributed between the 5' -region and the 3' -region.
this is more consistent with an origin by tandem duplication, which is indifferent to location, than by reverse splicing.
the above suggested cases of exon duplications are not supported by sequence conservation of  exonic and unconstrained intronic sequences.
this could be due to the relatively large evolutionary age of all cases of exon duplications reported here.
the youngest intron that may have originated by duplication has evolved in the d. melanogaster lineage after divergence from the d. erecta lineage and thus appears to be between  <dig> and  <dig> million years old .
accordingly, the footprint of duplication might have been lost.
it has to be noted that intron gain by exon duplication typically cannot be detected using the nip approach because in most cases an ancient intron will not be present.
thus, tandem duplication may be a more common mechanism of intron gain than suggested by our specifically selected data  <cit> .
however, two different intron positions may arise in one duplicated region if two proto-splice sites are included.
in this case, the intron gains are not independent.
this may have occurred in fbgn <dig>  and in fbgn <dig>  before the divergence of the subgenera drosophila and sophophora.
alternatively, there might have been an ancient intron that was lost before the duplication .
in the remaining two cases, the other intron seems to be novel, too, but has emerged in another lineage by an unknown mechanism .
CONCLUSIONS
during our study of novel intron positions in evolutionarily conserved genes of drosophila we confirmed that near intron pairs  are reliable phylogenetic markers.
furthermore, our results support that intron sliding  is one of the causes of recently emerged intron positions within conserved protein-coding drosophila genes.
we also found evidence for the rise of novel introns by tandem duplication of exonic dna.
the origin for  <dig> out of  <dig> identified nips remains unknown .
contrary to expectations, the gain of novel introns by other mechanisms could not be proved, for example, by insertion of a spliceosomal intron via reverse splicing into a new position, by insertion of a transposable element or by gene conversion with an intron-containing paralog.
recent origins of spliceosomal introns in eukaryotic genes, therefore, often might be local mutations  or insertions of alien sequences   <cit> , but not insertions of reverse-transcribed sequences.
this is supported by a recent analysis of intron gain through intronization in caenorhabditis  <cit>  and should be evaluated also for other eukaryotes.

 comparative studies of spliceosomal intron densities have suggested relatively high rates of intron gain during eukaryote evolution. the establishment of introns within fast-evolving genes appears to be an infrequent, but common process involving, for example, intronization of exonic sequences. however, recent gains of introns inside of conserved coding sequences, often equated with the usage of novel intron positions, appear to be a rare and poorly understood phenomenon. at least six mechanisms have been proposed to explain novel intron positions within conserved open reading frames: insertion of a self-splicing type ii intron via reverse splicing; insertion of a spliceosomal intron via reverse splicing into a new position; partial tandem duplication of an exon including a cryptic ag/gy splice motif; insertion of a transposable element; gene conversion from an intron-containing site into a previously intron-less paralogous site; and intron sliding. only the last three pathways are supported by undisputed, albeit anecdotal, evidence. recently, a study on daphnia populations suggested another intron gain mechanism: the repair of dna double-strand breaks using small segmental insertions. the analysis of near intron pairs allows a systematic investigation of intron gain mechanisms. a near intron pair consists of two intron positions that exist in orthologous genes at nearby locations. exon sizes smaller than about nt are relatively rare and in general functionally detrimental. thus, such nearby introns typically exclude each other within a single gene. accordingly, one of these introns must be evolutionarily younger and should define a monophyletic group. by using nips to determine the time at which the younger introns were gained, it can be avoided to erroneously identify those introns as novel that have been lost independently in multiple lineages. here we use the relatively recently diverged genomes of drosophila species to identify recent intron gain events in a comparative analysis of gene structures and evaluate possible mechanisms of their origin. in contrast to previous attempts to identify intron loss and gain in drosophila, we chose the nip approach instead of dollo parsimony to restrict our analysis to introns for which the evidence of intron gain is more unambiguous. we could identify nips within drosophila. their distribution supports the known species phylogeny. both introns of a nip were evaluated for sequence similarity to introns, neighboring exons and transposable elements and screened for repetitiveness and potentially meaningful secondary structures, using the ancient, plesiomorphic intron of the nip as control. in addition, we looked for cryptic splice signals in the adjacent exonic and intronic sequences. there is evidence for intron sliding in of the drosophila nips, while other cases probably arose by tandem duplications within the orf. in contrast, we found no evidence for any intron insertion mechanism based on alien sequences in our data set.  compilation and characterization of the nip data set we started with sets of orthologous protein-coding genes of drosophila species. after an automatic nip extraction alignment procedure, we obtained nip regions containing two or more near introns. these were manually inspected for splice site, alignment and conservation validity, resulting in regions comprising nips. based on the frequency of short exons and nip distances within drosophila, we decided to decrease the maximal allowed intron distance to nt, which resulted in alignment regions comprising nips. since there was an alternative site consistent with the old intron position that could not be clearly excluded by splice site sequence analysis, we evaluated seven nip candidates experimentally. as no est data supported these nips, we performed rt-pcr or genomic pcr experiments in some crucial species. our analysis confirmed of the investigated nips. the remaining cases were reducible to one intron position. three of these cases were based on sequence errors that could be corrected by genomic pcr or rt-pcr. one nip candidate represented a frame shift mutation that led to an incorrectly annotated splice site. in the last case, intron sliding has happened only at the 3' splice site. age of nips not all of the remaining nips contain introns that necessarily have been gained during the evolution of the genus drosophila. using the intron distributions among drosophila and the arthropod outgroup species glossina morsitans, aedes aegypti, anopheles gambiae, culex pipiens, bombyx mori, tribolium castaneum, apis mellifera, nasonia vitripennis, acyrthosiphon pisum, pediculus humanus, daphnia pulex and ixodes scapularis, we discovered that at least intron positions of the nips have arisen during drosophila radiation. nips contain exactly one novel intron position, whereas other nips consist of two different novel intron positions, which are supported by other nearby introns in the outgroup species. the phase distribution of these novel introns is similar to the average intron phase distribution in drosophila: of these introns are in phase, and in phase and phase respectively. for the remaining nips, the relative age of intron positions could not be determined either due to a lack of sufficient local sequence similarity to orthologous gene structures outside of drosophila or due to a lack of nearby introns in outgroup sequences. nips are suitable phylogenetic markers for drosophila species nips were primarily introduced as reliable phylogenetic markers for insect evolution. the well-known phylogeny of drosophila opens the possibility to verify the suitability of nips for phylogenetic analyses of recent radiations. phylogenetically informative nips within drosophila were identified using the aforementioned arthropod outgroup species. together, synapomorphic and autapomorphic nip characters appeared during the evolution of the genus drosophila. the subgenus drosophila, the species groups obscura and melanogaster, the sister relationship between the melanogaster and the obscura species groups and as well as the melanogaster species subgroup are supported by at least one synapomorphically distributed nip. for remaining internal nodes and remaining external nodes of the tree no supporting nip evidence was found. out of nips, only one character distribution contradicts the established tree of the drosophila species. this nip is clearly associated with a sliding event. the local nature of sliding makes independent changes of intron position with identical results much more likely than any of the other mechanisms, which require independent targeting of the same genomic position. we conclude that nips could be reliable phylogenetic markers also for recent radiations. however, short branch lengths, as observed between drosophila species, could critically limit the number of available characters. intron sliding accounts for a low but detectable level of homoplasy in this type of character. intron migration into novel positions next, we evaluated possible evolutionary mechanisms that had produced introns at novel positions. for all the introns of the nips, we could not find any relevant sequence conservation to other introns, to transposons, or to exons. to control whether some of the intron position changes resulted from intron sliding, we evaluated all introns of the nips concerning properties that would be expected if a coordinated migration of both splice sites occurred in relation to the former intron and the cds. these properties are: there is no intermediate, intron-less state in the tree; nip distance is a multiple of allowing a stepwise intron shift; cryptic splice sites occur at nip distances and are supported by amino acid conservation; one-sided shifts or gyrgyr/nagnag splice sites in some species; and significant sequence similarity between introns of both positions. the last criterion is sufficient on its own to support intron sliding and was found in cases. here, we observed that the intron sequence had stayed in place, but that the splice sites seem to have been migrated. "nt" means nip distance in nucleotides. note that each of the nips resulting supposedly from intron sliding shows at least three of the expected properties and/or a significant sequence similarity between introns of both positions. introns gained by tandem duplication show a proto-splice site. in addition, a plesiomorphic intron is not identifiable because both introns probably have been gained by the same duplication. in another nips, at least three of the other four criteria are fulfilled. while the first two conditions are also consistent with mechanisms of intron gain, cryptic splice sites at the corresponding distance and one-sided shifts of intron borders in some species are specific requirements for intron sliding. thus, the fulfillment of at least three criteria argues for sliding also in these cases. it should be noted that only one case of sliding has occurred within about million years of evolution and was, therefore, expected to show sequence conservation between both introns of the nip. all other putative sliding events have occurred between drosophila lineages that diverged at least million years ago. no footprint of internal sequence conservation across the two intron positions has been retained in any of these cases. in summary, out of nips most likely originated by intron sliding. this is surprising since earlier studies concluded that intron sliding is a very rare event. on the other hand, frequent intron sliding can be expected as a consequence of the high abundance of tandem splice sites in eukaryotic genes. detection of strong proto-splice sites in a subset of novel introns donor and acceptor site consensus sequences of spliceosomal splicing also include exonic parts. at the donor splice site, the last two exonic nucleotides could bind to the u snrna and the corresponding nucleotide positions are essential in some human splice sites. in contrast, the acceptor splice site appears to be functionally independent from exonic parts of the consensus. surprisingly, there are nucleotide preferences nevertheless, typically 5'-rt-3'. sverdlov et al. reported that younger introns exhibit a stronger signal in the exonic part of both splice consensus parts than older ones, while the intronic part of the splice signal is stronger for older introns than for younger ones. both observations are to be expected if novel introns sometimes originate through tandem duplications within coding exons. following such an event, a duplicated proto-splice site could turn the sequence between both sites into an intron. in drosophila, the role of the branch site and of additional enhancer and silencer sequences is very limited and typically does not interfere with an intron definition by donor and acceptor site alone. thus, an intron could immediately emerge by tandem duplication of a proto-splice site and will have the size of the duplication. concomitantly, such a duplication will allow differential splice patterns as a temporary or permanent alternative to the establishment of a constitutive intron if the duplication size is a multiple of three. alternatively, unspliced mrna variants may be degraded by nonsense-mediated decay. thus, nmd may often serve as a backup for weak splicing of such novel introns as recently suggested by farlow et al.. the mature mrna will remain qualitatively unchanged during such a process. later on, the splice signals could be improved by selection, whereas the other parts of the novel intronic sequence are free to change by insertions, deletions and substitutions. thus, we searched for coincidences of introns and proto-splice sites supported by amino acid conservation. we found introns in nips that are surrounded by an ag/gy proto-splice site. for of these introns, the proto-splice sites are conserved also in drosophila species having no intron there, and should thus already have been present at the time of intron origin. the potential functionality of these sites both as donor and acceptor sites is supported by their splice site scores that reside within the variability of functional splice sites of d. melanogaster, but well above the splice site scores of typical exonic sequences surrounding drosophila introns. for two of these td introns, regional repetitiveness enhances the probability of duplications in the cds. in all individual sequences of td introns we found a premature termination codon, suggesting that nmd might have played a role during their evolution. for details of the reference data set and score generation see the methods section. percentile values in brackets are with respect to the rp reference set. consensus nts: b = not a, d = not c, h = not g, k = g or t, m = a or c, r = a or g, s = c or g, u = not t, w = a or t, y = c or t. identified proto-splice sites support tandem duplication rather than reverse splicing the concept of the proto-splice site was proposed more than years ago. despite the simplicity of the tandem duplication mechanism, intron gain was seldom explained in this way. instead, proto-splice sites were introduced as preferential insertion sites for reverse-spliced introns. this pathway requires four successive, rate-limiting steps, namely germ line transcription of the target gene; reverse splicing of an - occasionally retained - intron lariat into a novel site of the target mrna; reverse transcription of this now intron-containing mrna; and homologous recombination of this cdna with the target gene. to our knowledge, this reverse-splicing pathway has never been shown to have produced a spliceosomal intron. in contrast, tandem duplications frequently occur in natural populations. emerson et al. compared natural isofemale lines of d. melanogaster and detected duplications, mostly in tandem, with a median size of nt in at least one line. irrespective of the strong evidence for purifying selection against this type of mutations, of these duplications included some exonic sequences, but not a whole gene. given this significant amount of function-challenging mutations, the gain of an intron may compensate for a duplication simply if a proto-splice site is included. in order to further support the hypothesis that the td introns arose by tandem duplication rather than by reverse splicing, we evaluated their proto-splice sites. if splice sites had emerged directly by duplication of proto-splice sites, proto-splice and splice sites should be similar to each other and both should be functional splice sites. if intronic splice sites stemmed from a reverse-spliced intron, proto-splice sites would not need to be similar to splice sites but should have provided a preferential binding site for the spliceosome that has inserted the intron. the sequence specificity of spliceosome-binding during forward splicing is mediated by the binding of the u snrnp to the donor site. however, the initial interactions of a spliceosome capable of reverse splicing are unknown. thus, alternatively, the u snrnp complex maybe binds first because this complex interacts during the second splicing reaction with both exon ends to ligate them. for u snrnp, known sequence preferences are weak and not consistent with proto-splice sites. other sequence-specific binding components of the spliceosome such as the u u and u snrnps exclusively interact with the intron. reverse splicing, therefore, might have produced proto-splice sites that remember donor sites or no proto-splice sites at all. specifically, the acceptor site consensus 5' of ag would not necessarily be included in such a proto-splice site. to distinguish between the two alternative pathways, we evaluated the splice sites, the proto-splice sites and the relative location of the new introns in more detail: we determined scores for reference splice sites and proto-splice sites excluding the central ag/gy motif. these scores are based on the nts + to + and the nts - to -. we found that such partial proto-splice acceptor sites of td introns show intermediate scores between reference splice sites on the one hand, and potential proto-splice sites surrounding reference introns and intron-less aggy proto-splice site motifs of exons on the other hand. moreover, the partial proto-splice acceptor site scores of the td introns are statistically significantly different from the corresponding scores of other nip introns, reference introns and exonic aggy motifs, respectively. in contrast, the scores of partial proto-splice donor sites show no differences. this argues against reverse splicing that would predict an exclusive similarity to the donor site. during splicing, the acceptor-binding parts of the spliceosome are occupied by intronic sequences, thus, these parts cannot be involved in target site selection in case of reverse splicing. if the supposedly duplicated sequences of both intron/exon borders of one intron are compared under exclusion of the central ag/gy motif, these regions of td introns are significantly more similar to each other compared to other nip introns or reference introns. this finding is based on the same data as the first one but they were used here in a slightly different way. this result argues in favour of tandem duplication. the location of the td introns within the genes does not support an origin by reverse splicing. a reverse-transcription mediated pathway would imply a 3' biased distribution of novel introns as the reverse transcriptase should start primarily at the 3' end of the mrna. apomorphic nip introns, however, show a biased location in favour of the 5' and, to a lesser extent, 3' ends of the genes. whereas the average locations of apomorphic introns of the td and on sets across the bins were not significantly different, there is a moderate significance to reject the null hypothesis that the novel introns are equally distributed between the 5' -region and the 3' -region. this is more consistent with an origin by tandem duplication, which is indifferent to location, than by reverse splicing. the above suggested cases of exon duplications are not supported by sequence conservation of exonic and unconstrained intronic sequences. this could be due to the relatively large evolutionary age of all cases of exon duplications reported here. the youngest intron that may have originated by duplication has evolved in the d. melanogaster lineage after divergence from the d. erecta lineage and thus appears to be between and million years old. accordingly, the footprint of duplication might have been lost. it has to be noted that intron gain by exon duplication typically cannot be detected using the nip approach because in most cases an ancient intron will not be present. thus, tandem duplication may be a more common mechanism of intron gain than suggested by our specifically selected data. however, two different intron positions may arise in one duplicated region if two proto-splice sites are included. in this case, the intron gains are not independent. this may have occurred in fbgn and in fbgn before the divergence of the subgenera drosophila and sophophora. alternatively, there might have been an ancient intron that was lost before the duplication. in the remaining two cases, the other intron seems to be novel, too, but has emerged in another lineage by an unknown mechanism.  during our study of novel intron positions in evolutionarily conserved genes of drosophila we confirmed that near intron pairs are reliable phylogenetic markers. furthermore, our results support that intron sliding is one of the causes of recently emerged intron positions within conserved protein-coding drosophila genes. we also found evidence for the rise of novel introns by tandem duplication of exonic dna. the origin for out of identified nips remains unknown. contrary to expectations, the gain of novel introns by other mechanisms could not be proved, for example, by insertion of a spliceosomal intron via reverse splicing into a new position, by insertion of a transposable element or by gene conversion with an intron-containing paralog. recent origins of spliceosomal introns in eukaryotic genes, therefore, often might be local mutations or insertions of alien sequences, but not insertions of reverse-transcribed sequences. this is supported by a recent analysis of intron gain through intronization in caenorhabditis and should be evaluated also for other eukaryotes. 
intron migration and exon duplication together may account for a significant amount of novel intron positions in conserved coding sequences. other novel introns seem to have been gained by regional tandem duplications of coding sequences containing a proto-splice site. we identified unambiguous cases of novel intron positions in drosophila genes that exhibit near intron pairs. here, a nip consists of an ancient and a novel intron position that are separated by less than nt. a survey for nips among drosophila genomes identifies intron sliding as one of the more frequent causes of novel intron positions. recent intron gains sometimes appear to have arisen by duplication of exonic sequences and subsequent intronization of one of the copies. positions of spliceosomal introns are often conserved between remotely related genes. however, introns verified as novel are needed to evaluate contemporary processes of intron gain.
 positions of spliceosomal introns are often conserved between remotely related genes. introns that reside in non-conserved positions are either novel or remnants of frequent losses of introns in some evolutionary lineages. a recent gain of such introns is difficult to prove. however, introns verified as novel are needed to evaluate contemporary processes of intron gain.  we identified unambiguous cases of novel intron positions in drosophila genes that exhibit near intron pairs. here, a nip consists of an ancient and a novel intron position that are separated by less than nt. within a single gene, such closely-spaced introns are very unlikely to have coexisted. in most cases, therefore, the ancient intron position must have disappeared in favour of the novel one. a survey for nips among drosophila genomes identifies intron sliding as one of the more frequent causes of novel intron positions. other novel introns seem to have been gained by regional tandem duplications of coding sequences containing a proto-splice site.  recent intron gains sometimes appear to have arisen by duplication of exonic sequences and subsequent intronization of one of the copies. intron migration and exon duplication together may account for a significant amount of novel intron positions in conserved coding sequences. 