BACKGROUND
gypsy  is an endogenous retrovirus of d. melanogaster.
its structure is largely similar to the vertebrate retroviruses and it possesses infective properties  <cit> .
sequences homologous to gypsydm are widely distributed among drosophila species, found in both the subgenus sophophora as well as in the subgenus drosophila  <cit> .
in addition, they have been detected in some species of the genus scaptomyza  <cit> .
the phylogenetic relationships among gypsy sequences from drosophila species is not be always coincident with those of their hosts, which could be indicative of horizontal transfer during evolutionary history  <cit> .
horizontal transfer has been invoked to explain the evolutionary patterns of several families of transposable elements  <cit> .
in some cases, such as the p element, the horizontal transfer events are strongly documented  <cit> .
however, the underlying mechanisms to horizontal transfer remain elusive.
in the case of gypsydm, the horizontal transfer could be associated with the infectious properties of that element.
one way to evaluate the potential impact of the gypsy infectious ability on the evolutionary history of gypsy sequences from drosophila, is to analyze the infective ability of gypsy homologous sequences from species other than d. melanogaster.
the infectious ability of gypsydm is associated with the expression of a retroviral envelope-like protein encoded by the env gene  <cit> .
structural analysis of the env region of a given gypsy sequence is the first step in determining its potential infectious ability.
in previous reports, two complete gypsy elements from d. subosbcura and d. virilis were sequenced  <cit> .
although, the env region is preserved in its full-length, in both species single indels result in potentially truncated env proteins  <cit> .
however, as has been proposed in  <cit> , it is possible that both, defective and complete fully functional gypsy elements, coexist in the genome of these species.
this study describes the existence of full-length env genes in the genomes of d. subosbcura and d. virilis, as well as in the genomes of the several species closely related to d. melanogaster:d. simulans, d. erecta, d. orena, d. teissieri, and d. yakuba.
interestingly, these gyspy sequences can potentially invade the d. melanogaster genome, and escape the control normally exerted by the flamenco gene, whose product represses mobility <cit> .
it is generally accepted that species of the obscura group can be classified into five subgroups: the pseudobscura and affinis subgroups, consisting of neartic species, the subobscura and obscura subgroups, mainly consisting of paleartic species, and the microlabis subgroup encompassing afrotropical species  <cit> .
gypsy sequences homologous to gypsydm have been detected in several species of the neartic and paleartic subgroups, appearing as a monophyletic group that is highly diverged from the gypsydm prototypic element  <cit> .
with the exception of d. guanche, the 5' region of the env gene in all species examined could potentially produce a functional n-terminal region of the env protein.
to confirm the existence of potentially infective gypsy lineages other than gypsydm, gypsyds, and gypsydv, we report here an analysis of the complete env gene of ten species of the obscura group and one species from the genus scaptomyza.
most of the species analyzed contain intact copies of the env region and the env splicing sites, and in vitro can be translated into a protein of the predicted size.
RESULTS
sequences homologous to gypsy from d. melanogaster are widely distributed among drosophila species.
in previous studies, the complete gypsy elements from d. subobscura and d. virilis have been sequenced  <cit> .
these studies were carried out to understand the evolutionary behaviour of gypsy and indicate that the genetic organization of gypsy in those species is conserved.
moreover, in d. melanogaster do the gypsy elements have a complete functional env gene  <cit> .
in the case of d. virilis and d. subobscura, it has been shown that the genomes of these species contain at least one copy of gypsy putatively encoding a complete envelope protein  <cit> .
the main aim of this study is to examine whether potentially functional copies of the env region of gypsy exist in other species.
this was accomplished by searching genomes for the presence of full-length env coding sequences and assessing their functionality.
the analysis of the env sequences shows their infectious capability
to analyze the infectious capability of the gypsy element of each analyzed species  we amplified the env region with specific primers designed from the intergenic sequence .
in all species the size of the amplified sequence was  <dig>  kb, except for d. algonquin and d. tolteca, with sizes of  <dig>  and  <dig>  kb respectively.
the pcr products of all species, were cloned in pcr® <dig> -topo®  and sequenced with the specific primers of the vector, demonstrating that d. algonquin and d. tolteca had a deletion of  <dig>  kb and  <dig>  kb respectively.
in the same way, d. pseudoobscura and d. miranda had deletions of  <dig> pb and  <dig> pb respectively that produced a stop codon and an env truncated protein.
d. persimilis had a single nucleotide change, also producing a stop codon.
in contrast, d. madeirensis, d. obscura, d. ambigua, d. bifasciata, d. affinis and s. elmoi had different polymorphisms but none resulting in a truncated protein, so these env regions would produce potentially active proteins.
with these data, we can conclude that d. madeirensis, d. obscura, d. ambigua, d. bifasciata, d. affinis and s. elmoi could produce a complete env protein and therefore could maintain the infectious capability of gypsy in these species.
the env sequences obtained in different species have the capacity to produce complete envelope proteins
the rts system was used to scan for premature termination mutations and analyze the coding capacity of the gypsy env gene present in the genome of the different species studied.
we used genomic dna from each species to amplify the env sequence.
we cloned the obtained sequences into pcr® <dig> -topo  and then we used these clones to produce the pcr products for in vitro protein expression .
the major protein products obtained in this assay have molecular weights corresponding to the expected size for env protein of d. melanogaster, about  <dig> kda.
these protein products were obtained in the same species where the env nucleotide sequence was complete, without disruptions.
we obtained complete env protein from d. madeirensis, d. obscura, d. ambigua, d. bifasciata, d. affinis and s. elmoi, while we obtained truncated proteins from the species where the sequence was disrupted, d. pseudoobscura, d. persimilis and d. miranda .
in d. melanogaster, the splicing produces a start codon using at from 5' site in the gag region and a g from the 3' site in the env region  <cit> .
we have analyzed the sequences from the different species of this study and only d. madeirensis, d. obscura, d. ambigua, d. bifasciata, d. affinis and s. elmoi, have conserved the sequences that produce the start codon after the splicing.
these results show that these species contain at least one copy of gypsy element that can encode an envelope protein.
other data that support the existence of functional env proteins include the presence of n-glycosylation sites, conserved cysteine residues and endopeptidase cleavage sites in all the species with a complete env sequence and the atg codon , as described in  <cit> .
moreover, those species have the sequence pattern -r-x- -x- -x-g-x-d-x-d) for the detection in silico of endogenous retroviral envelope protein proposed in  <cit> .
only the signal peptide and the membrane-spanning domain sequence show differences on comparing to d. melanogaster gypsy sequence.
although sequences have the capability to produce complete envelope protein, their expression could be repressed in the species studied
to demonstrate the capability of the gypsy elements to produce an active envelope protein, we wanted to know if the env gene was expressed in vivo in the species with the complete env sequence.
in order to detect the production of the env mrna we performed a rt-pcr analysis using the gs <dig> and gs <dig> primers with the mrna extracted from ovarian chambers of the female flies.
since we also wanted to detect alternative splicing, necessary to produce env, we used the primers gypsy5'f and gs <dig> of the env gene using the same mrna as before .
amplification with the internal primers was successful in all species analysed as well as in the positive control .
however, alternative splicing was not detected .
these results suggest that the alternative splicing of gypsy in the obscura species group and s. elmoi would be repressed by a genomic gene as occurs in d. melanogaster with the flamenco gene  <cit> .
alternatively, the gypsy element may use an alternative start codon to express the env region or we simply could not detect the alternative splicing.
the gypsy phylogeny is not totally consistent with species phylogeny
since gypsy elements can potentially jump from one individual to another without the need of a vector, it has been proposed that horizontal transfers of gypsy elements is favoured by their ability to encoded full-length env proteins  <cit> .
in this way we performed a phylogeny analysis of the env gypsy sequences studied, looking for evidences of horizontal transfer events.
a neighbor-joining phylogenetic tree of envelope sequences is shown in figure  <dig>  the obscura sequences fall into four main clades that are generally consistent with species phylogeny.
clade a includes sequences from d. affinis of the affinis subgroup; clade b, d. ambigua, d. bifasciata, d. madeirensis, and d. obscura of the obscura subgroup; clade c, d. miranda, d. persimilis, and d. pseudoobscura of the pseudoobscura subgroup; and clade d, d. tolteca of the affinis subgroup.
the d. virilis sequence is responsible for the relatively low support for clade a; removing the d. virilis sequence from the analysis increases the bootstrap values to 98/ <dig> for a grouping of the d. hydei sequence with those from d. affinis, with the rest of the topology unchanged.
with the exception of the placement of sequences from s. elmoi, the topology of this tree is identical to that obtained by parsimony analysis, which yielded a shortest tree of  <dig> steps .
the distribution of sequence from d. affinis and d. tolteca into two distinct clades is an unexpected result that is consistently seen in all analyses.
constraining the analysis so that the sequences from d. tolteca and d. affinis are monophyletic adds an additional nine steps to the parsimony tree.
the potential effect of recombination on the phylogeny was assessed by using the gard algorithm, which uses maximum likelihood models to detect recombination events.
the bic model found no evidence of recombination, while the aic model identifies a potential breakpoint at position  <dig> in the  <dig> character alignment.
the potential effect of this event on the phylogeny was also examined using a sliding-window approach.
separate phylogenetic analyses were constructed from four 400-bp regions of the envelop alignments , and on a fifth dataset that excluded the region beyond the breakpoint.
the latter analysis resulted in an identical tree to that shown in figure  <dig>  as did two of the four sliding window analyses.
in the other two sliding-window trees, the d. virilis sequence was excluded from clade a, as it was in one of two alternate trees obtained from the aic analyses.
regardless of its precise affiliation, in an expanded phylogenetic analysis that includes  <dig> published gypsy sequences, d. virilis clearly belongs to a well-supported clade that includes all of the obscura sequences .
sequences from s. elmoi fall into clade b, in which they form two subclades.
although not monophyletic, the average amount of sequence divergence between the two subclades is just  <dig> %, and if the sequences from s. elmoi are constrained to be monophyletic the total length of the parsimony tree is unchanged.
if the gypsy sequences from s. elmoi are excluded from the analysis, the topology of the obsucra sequences is unchanged from that shown in figure  <dig>  to make the gypsy phylogeny conform completely to the species phylogeny  increases the parsimony tree length from  <dig> to  <dig> steps.
thus, there is clear incongruence between the gypsy phylogeny and that of the species from which the sequences were isolated.
the envelope sequences from s. elmoi differ on average by only  <dig> % from those found in the obscura species of clade b. as a hallmark of horizontal transfer is the existence of highly similar sequences in the genomes of divergent taxa  <cit> , transfer of gypsy from the obscura group to the genus scaptomyza seems likely.
because the sampling from s. elmoi is quite extensive, an explanation of the retention of an ancestral polymorphism for the phylogenetic incongruence seems unlikely.
this conclusion is further supported by comparison to the divergence of a non-mobile nuclear gene, adh.
over exon  <dig> of this gene, the average divergence between s. elmoi and the clade b adh sequences is  <dig> %.
a horizontal transfer involving the genus scaptomyza and the palearctic members of the obscura species group is of particular interest because of the well-documented horizontal transfer of the p transposable element between these taxa  <cit> .
in that case, the direction of transfer was almost certainly from scaptomyza to d. bifasciata.
in the case of gypsy, the phylogeny indicates a transfer in the opposite direction.
to provide some comparison to other studies of gypsy evolution, an extensive phylogenetic analysis was performed with an enlarged dataset of  <dig> sequences, combining those from this study with those of  <cit>  and  <cit> .
this allows us to assess the coherence of the two clades, a and b, which include postulated horizontal transfer events, and to increase the sample size considerably to examine the alternative hypothesis of ancient lineages.
this tree is presented in figure  <dig>  the positions of the d. hydei, d. virilis and s. elmoi sequences remain unchanged from that shown in figure  <dig>  providing additional evidence to support the hypothesis of horizontal transfer.
molecular evolutionary analysis of gypsy sequences is consistent with activity
to examine the evidence for selection on the gypsy sequences, the numbers of synonymous substitutions  and nonsynonymous substitutions  were calculated for each sequence.
the average dn/ds ratio was then calculated for each clade , providing some indication of whether purifying selection is or has been operating on these sequences.
for all major clades, the dn/ds ratio is similar to that obtained for non-mobile nuclear genes  <cit> .
similar results have been obtained in a number of studies of gypsy sequence evolution  <cit> .
together, these comparisons suggest that many gypsy elements are capable of activity, consistent with the expression data described above.
although the pattern is consistent with activity , this conclusion may be complicated by two factors:  codon bias, which is lower for the gypsy sequences, when compared to adh , and  the nature of selection on the gypsy sequences which may include functional constraints on the rna intermediate in addition to selection for a functional envelop protein.
calculations were based on the model of nei and gojobori  and include obscura sequences only.
clade designations are given in figure  <dig>
it is noteworthy that clade b includes the gypsy sequences that have intact envelope genes , whereas clades d and c consist of those with interrupted reading frames.
clade a comprises sequences from the neartic species d. affinis, which also encode full-length envelope proteins and are characterized by very low dn/ds ratios.
it is fascinating that in the two clades with open envelope reading frames and low dn/ds ratios, horizontal transfer has likely occurred.
even though the gypsy sequences examined from clade c are not likely functional, the low dn/ds ratio suggests that these elements have been active in the recent past.
the envelope coding regions of d. tolteca  are characterized by a substantial deletion at the beginning of the envelope gene and relatively higher dn/ds ratios, suggesting that they have been inactive for considerably longer than the clade c elements.
thus, the phylogenetic and sequence analyses together provide a snapshot of the evolutionary history of gypsy in the obscura species group that is consistent with their activity and potential infectivity.
horizontal transfer of gypsy has been suggested in other studies: among sequences from the obscura group and d. virilis/d.
hydei, which belong to a different subgenus  <cit> , and within the melanogaster species group  <cit> .
two additional studies, which are similar is scope to the one described here, concluded that horizontal transfer of gypsy was fairly widespread among species in the genus drosophila and sister genera  <cit> .
as these studies included a total of only three sequences from the obscura group, they were not able to identify precisely the species involved in the transfer events.
in the analysis presented here, the envelope sequence from d. hydei shows a strong affiliation to the sequences from d. affinis; a less well-supported clade includes the sequences from d. virilis as well.
as the envelope sequences from d. hydei, d. virilis, and d. affinis differ by an average of only  <dig> %, and the species sampling is now quite extensive , horizontal transfer remains the best explanation for the incongruent phylogeny.
the hypothesis of horizontal transfer finds additional support in a comparison of the divergence of the envelop region to the non-mobile gene, adh.
the average divergence of adh sequences from d. hydei/d.
virilis and d. affinis is  <dig> % .
using the values for synonymous nucleotide substitutions,  <cit>  provided an estimate of  <dig>  million years ago  for a transfer from the obscura group  to d. virilis.
when a similar analysis is done with the d. affinis sequences , an estimate of only  <dig>  mya is obtained for a transfer from d. affinis to d. hydei, and  <dig>  mya for a transfer from d. affinis to d. virilis.
in clade b, time estimates calculated from synonymous changes place the transfer from the paleartic obscura to s. elmoi at  <dig>  mya.
the fact that these transfers occurred relatively recently supports the idea that at least some genomes in the obscura group harbor active, infectious gypsy elements.
CONCLUSIONS
the genomes of six of the eleven species of analysed encode intact copies of the envelop coding region of gypsy and retain intact envelop splice sites.
their potential for expression was analyzed in vitro and each was found to encode a protein whose size is consistent with activity.
moreover, these elements possess two additional features of active gypsy genes, alternative splice sites and the use of alternative start codons.
evolutionary analysis of the envelop sequences shows that interspecies transfer of gypsy is associated with those obscura elements that can encode a functional envelop protein.
such horizontal transfer is supported by the incongruence of the gypsy and species phylogenies and by the low levels of gypsy divergence when compared to the divergence of host genes from the same species.

 gypsy is an endogenous retrovirus of d. melanogaster. its structure is largely similar to the vertebrate retroviruses and it possesses infective properties. sequences homologous to gypsydm are widely distributed among drosophila species, found in both the subgenus sophophora as well as in the subgenus drosophila. in addition, they have been detected in some species of the genus scaptomyza. the phylogenetic relationships among gypsy sequences from drosophila species is not be always coincident with those of their hosts, which could be indicative of horizontal transfer during evolutionary history. horizontal transfer has been invoked to explain the evolutionary patterns of several families of transposable elements. in some cases, such as the p element, the horizontal transfer events are strongly documented. however, the underlying mechanisms to horizontal transfer remain elusive. in the case of gypsydm, the horizontal transfer could be associated with the infectious properties of that element. one way to evaluate the potential impact of the gypsy infectious ability on the evolutionary history of gypsy sequences from drosophila, is to analyze the infective ability of gypsy homologous sequences from species other than d. melanogaster. the infectious ability of gypsydm is associated with the expression of a retroviral envelope-like protein encoded by the env gene. structural analysis of the env region of a given gypsy sequence is the first step in determining its potential infectious ability. in previous reports, two complete gypsy elements from d. subosbcura and d. virilis were sequenced. although, the env region is preserved in its full-length, in both species single indels result in potentially truncated env proteins. however, as has been proposed in, it is possible that both, defective and complete fully functional gypsy elements, coexist in the genome of these species. this study describes the existence of full-length env genes in the genomes of d. subosbcura and d. virilis, as well as in the genomes of the several species closely related to d. melanogaster:d. simulans, d. erecta, d. orena, d. teissieri, and d. yakuba. interestingly, these gyspy sequences can potentially invade the d. melanogaster genome, and escape the control normally exerted by the flamenco gene, whose product represses mobility. it is generally accepted that species of the obscura group can be classified into five subgroups: the pseudobscura and affinis subgroups, consisting of neartic species, the subobscura and obscura subgroups, mainly consisting of paleartic species, and the microlabis subgroup encompassing afrotropical species. gypsy sequences homologous to gypsydm have been detected in several species of the neartic and paleartic subgroups, appearing as a monophyletic group that is highly diverged from the gypsydm prototypic element. with the exception of d. guanche, the 5' region of the env gene in all species examined could potentially produce a functional n-terminal region of the env protein. to confirm the existence of potentially infective gypsy lineages other than gypsydm, gypsyds, and gypsydv, we report here an analysis of the complete env gene of ten species of the obscura group and one species from the genus scaptomyza. most of the species analyzed contain intact copies of the env region and the env splicing sites, and in vitro can be translated into a protein of the predicted size.  sequences homologous to gypsy from d. melanogaster are widely distributed among drosophila species. in previous studies, the complete gypsy elements from d. subobscura and d. virilis have been sequenced. these studies were carried out to understand the evolutionary behaviour of gypsy and indicate that the genetic organization of gypsy in those species is conserved. moreover, in d. melanogaster do the gypsy elements have a complete functional env gene. in the case of d. virilis and d. subobscura, it has been shown that the genomes of these species contain at least one copy of gypsy putatively encoding a complete envelope protein. the main aim of this study is to examine whether potentially functional copies of the env region of gypsy exist in other species. this was accomplished by searching genomes for the presence of full-length env coding sequences and assessing their functionality. the analysis of the env sequences shows their infectious capability to analyze the infectious capability of the gypsy element of each analyzed species we amplified the env region with specific primers designed from the intergenic sequence. in all species the size of the amplified sequence was kb, except for d. algonquin and d. tolteca, with sizes of and kb respectively. the pcr products of all species, were cloned in pcr® -topo® and sequenced with the specific primers of the vector, demonstrating that d. algonquin and d. tolteca had a deletion of kb and kb respectively. in the same way, d. pseudoobscura and d. miranda had deletions of pb and pb respectively that produced a stop codon and an env truncated protein. d. persimilis had a single nucleotide change, also producing a stop codon. in contrast, d. madeirensis, d. obscura, d. ambigua, d. bifasciata, d. affinis and s. elmoi had different polymorphisms but none resulting in a truncated protein, so these env regions would produce potentially active proteins. with these data, we can conclude that d. madeirensis, d. obscura, d. ambigua, d. bifasciata, d. affinis and s. elmoi could produce a complete env protein and therefore could maintain the infectious capability of gypsy in these species. the env sequences obtained in different species have the capacity to produce complete envelope proteins the rts system was used to scan for premature termination mutations and analyze the coding capacity of the gypsy env gene present in the genome of the different species studied. we used genomic dna from each species to amplify the env sequence. we cloned the obtained sequences into pcr® -topo and then we used these clones to produce the pcr products for in vitro protein expression. the major protein products obtained in this assay have molecular weights corresponding to the expected size for env protein of d. melanogaster, about kda. these protein products were obtained in the same species where the env nucleotide sequence was complete, without disruptions. we obtained complete env protein from d. madeirensis, d. obscura, d. ambigua, d. bifasciata, d. affinis and s. elmoi, while we obtained truncated proteins from the species where the sequence was disrupted, d. pseudoobscura, d. persimilis and d. miranda. in d. melanogaster, the splicing produces a start codon using at from 5' site in the gag region and a g from the 3' site in the env region. we have analyzed the sequences from the different species of this study and only d. madeirensis, d. obscura, d. ambigua, d. bifasciata, d. affinis and s. elmoi, have conserved the sequences that produce the start codon after the splicing. these results show that these species contain at least one copy of gypsy element that can encode an envelope protein. other data that support the existence of functional env proteins include the presence of n-glycosylation sites, conserved cysteine residues and endopeptidase cleavage sites in all the species with a complete env sequence and the atg codon, as described in. moreover, those species have the sequence pattern -r-x- -x- -x-g-x-d-x-d) for the detection in silico of endogenous retroviral envelope protein proposed in. only the signal peptide and the membrane-spanning domain sequence show differences on comparing to d. melanogaster gypsy sequence. although sequences have the capability to produce complete envelope protein, their expression could be repressed in the species studied to demonstrate the capability of the gypsy elements to produce an active envelope protein, we wanted to know if the env gene was expressed in vivo in the species with the complete env sequence. in order to detect the production of the env mrna we performed a rt-pcr analysis using the gs and gs primers with the mrna extracted from ovarian chambers of the female flies. since we also wanted to detect alternative splicing, necessary to produce env, we used the primers gypsy5'f and gs of the env gene using the same mrna as before. amplification with the internal primers was successful in all species analysed as well as in the positive control. however, alternative splicing was not detected. these results suggest that the alternative splicing of gypsy in the obscura species group and s. elmoi would be repressed by a genomic gene as occurs in d. melanogaster with the flamenco gene. alternatively, the gypsy element may use an alternative start codon to express the env region or we simply could not detect the alternative splicing. the gypsy phylogeny is not totally consistent with species phylogeny since gypsy elements can potentially jump from one individual to another without the need of a vector, it has been proposed that horizontal transfers of gypsy elements is favoured by their ability to encoded full-length env proteins. in this way we performed a phylogeny analysis of the env gypsy sequences studied, looking for evidences of horizontal transfer events. a neighbor-joining phylogenetic tree of envelope sequences is shown in figure the obscura sequences fall into four main clades that are generally consistent with species phylogeny. clade a includes sequences from d. affinis of the affinis subgroup; clade b, d. ambigua, d. bifasciata, d. madeirensis, and d. obscura of the obscura subgroup; clade c, d. miranda, d. persimilis, and d. pseudoobscura of the pseudoobscura subgroup; and clade d, d. tolteca of the affinis subgroup. the d. virilis sequence is responsible for the relatively low support for clade a; removing the d. virilis sequence from the analysis increases the bootstrap values to 98/ for a grouping of the d. hydei sequence with those from d. affinis, with the rest of the topology unchanged. with the exception of the placement of sequences from s. elmoi, the topology of this tree is identical to that obtained by parsimony analysis, which yielded a shortest tree of steps. the distribution of sequence from d. affinis and d. tolteca into two distinct clades is an unexpected result that is consistently seen in all analyses. constraining the analysis so that the sequences from d. tolteca and d. affinis are monophyletic adds an additional nine steps to the parsimony tree. the potential effect of recombination on the phylogeny was assessed by using the gard algorithm, which uses maximum likelihood models to detect recombination events. the bic model found no evidence of recombination, while the aic model identifies a potential breakpoint at position in the character alignment. the potential effect of this event on the phylogeny was also examined using a sliding-window approach. separate phylogenetic analyses were constructed from four 400-bp regions of the envelop alignments, and on a fifth dataset that excluded the region beyond the breakpoint. the latter analysis resulted in an identical tree to that shown in figure as did two of the four sliding window analyses. in the other two sliding-window trees, the d. virilis sequence was excluded from clade a, as it was in one of two alternate trees obtained from the aic analyses. regardless of its precise affiliation, in an expanded phylogenetic analysis that includes published gypsy sequences, d. virilis clearly belongs to a well-supported clade that includes all of the obscura sequences. sequences from s. elmoi fall into clade b, in which they form two subclades. although not monophyletic, the average amount of sequence divergence between the two subclades is just %, and if the sequences from s. elmoi are constrained to be monophyletic the total length of the parsimony tree is unchanged. if the gypsy sequences from s. elmoi are excluded from the analysis, the topology of the obsucra sequences is unchanged from that shown in figure to make the gypsy phylogeny conform completely to the species phylogeny increases the parsimony tree length from to steps. thus, there is clear incongruence between the gypsy phylogeny and that of the species from which the sequences were isolated. the envelope sequences from s. elmoi differ on average by only % from those found in the obscura species of clade b. as a hallmark of horizontal transfer is the existence of highly similar sequences in the genomes of divergent taxa, transfer of gypsy from the obscura group to the genus scaptomyza seems likely. because the sampling from s. elmoi is quite extensive, an explanation of the retention of an ancestral polymorphism for the phylogenetic incongruence seems unlikely. this conclusion is further supported by comparison to the divergence of a non-mobile nuclear gene, adh. over exon of this gene, the average divergence between s. elmoi and the clade b adh sequences is %. a horizontal transfer involving the genus scaptomyza and the palearctic members of the obscura species group is of particular interest because of the well-documented horizontal transfer of the p transposable element between these taxa. in that case, the direction of transfer was almost certainly from scaptomyza to d. bifasciata. in the case of gypsy, the phylogeny indicates a transfer in the opposite direction. to provide some comparison to other studies of gypsy evolution, an extensive phylogenetic analysis was performed with an enlarged dataset of sequences, combining those from this study with those of and. this allows us to assess the coherence of the two clades, a and b, which include postulated horizontal transfer events, and to increase the sample size considerably to examine the alternative hypothesis of ancient lineages. this tree is presented in figure the positions of the d. hydei, d. virilis and s. elmoi sequences remain unchanged from that shown in figure providing additional evidence to support the hypothesis of horizontal transfer. molecular evolutionary analysis of gypsy sequences is consistent with activity to examine the evidence for selection on the gypsy sequences, the numbers of synonymous substitutions and nonsynonymous substitutions were calculated for each sequence. the average dn/ds ratio was then calculated for each clade, providing some indication of whether purifying selection is or has been operating on these sequences. for all major clades, the dn/ds ratio is similar to that obtained for non-mobile nuclear genes. similar results have been obtained in a number of studies of gypsy sequence evolution. together, these comparisons suggest that many gypsy elements are capable of activity, consistent with the expression data described above. although the pattern is consistent with activity, this conclusion may be complicated by two factors: codon bias, which is lower for the gypsy sequences, when compared to adh, and the nature of selection on the gypsy sequences which may include functional constraints on the rna intermediate in addition to selection for a functional envelop protein. calculations were based on the model of nei and gojobori and include obscura sequences only. clade designations are given in figure it is noteworthy that clade b includes the gypsy sequences that have intact envelope genes, whereas clades d and c consist of those with interrupted reading frames. clade a comprises sequences from the neartic species d. affinis, which also encode full-length envelope proteins and are characterized by very low dn/ds ratios. it is fascinating that in the two clades with open envelope reading frames and low dn/ds ratios, horizontal transfer has likely occurred. even though the gypsy sequences examined from clade c are not likely functional, the low dn/ds ratio suggests that these elements have been active in the recent past. the envelope coding regions of d. tolteca are characterized by a substantial deletion at the beginning of the envelope gene and relatively higher dn/ds ratios, suggesting that they have been inactive for considerably longer than the clade c elements. thus, the phylogenetic and sequence analyses together provide a snapshot of the evolutionary history of gypsy in the obscura species group that is consistent with their activity and potential infectivity. horizontal transfer of gypsy has been suggested in other studies: among sequences from the obscura group and d. virilis/d. hydei, which belong to a different subgenus, and within the melanogaster species group. two additional studies, which are similar is scope to the one described here, concluded that horizontal transfer of gypsy was fairly widespread among species in the genus drosophila and sister genera. as these studies included a total of only three sequences from the obscura group, they were not able to identify precisely the species involved in the transfer events. in the analysis presented here, the envelope sequence from d. hydei shows a strong affiliation to the sequences from d. affinis; a less well-supported clade includes the sequences from d. virilis as well. as the envelope sequences from d. hydei, d. virilis, and d. affinis differ by an average of only %, and the species sampling is now quite extensive, horizontal transfer remains the best explanation for the incongruent phylogeny. the hypothesis of horizontal transfer finds additional support in a comparison of the divergence of the envelop region to the non-mobile gene, adh. the average divergence of adh sequences from d. hydei/d. virilis and d. affinis is %. using the values for synonymous nucleotide substitutions, provided an estimate of million years ago for a transfer from the obscura group to d. virilis. when a similar analysis is done with the d. affinis sequences, an estimate of only mya is obtained for a transfer from d. affinis to d. hydei, and mya for a transfer from d. affinis to d. virilis. in clade b, time estimates calculated from synonymous changes place the transfer from the paleartic obscura to s. elmoi at mya. the fact that these transfers occurred relatively recently supports the idea that at least some genomes in the obscura group harbor active, infectious gypsy elements.  the genomes of six of the eleven species of analysed encode intact copies of the envelop coding region of gypsy and retain intact envelop splice sites. their potential for expression was analyzed in vitro and each was found to encode a protein whose size is consistent with activity. moreover, these elements possess two additional features of active gypsy genes, alternative splice sites and the use of alternative start codons. evolutionary analysis of the envelop sequences shows that interspecies transfer of gypsy is associated with those obscura elements that can encode a functional envelop protein. such horizontal transfer is supported by the incongruence of the gypsy and species phylogenies and by the low levels of gypsy divergence when compared to the divergence of host genes from the same species. 
in this study we report molecular and phylogeny analysis of the complete env gene from ten species of the obscura group of the genus drosophila and one species from the genus scaptomyza. the results indicate that in most cases env sequences could produce a functional env protein and therefore maintain the infectious capability of gypsy in these species. the structure of gypsy includes an open reading frame resembling the retroviral gene env, which is responsible for the infectious properties of retroviruses. sequences homologous to the gypsy retroelement from drosophila melanogaster are widely distributed among drosophilids.
 sequences homologous to the gypsy retroelement from drosophila melanogaster are widely distributed among drosophilids. the structure of gypsy includes an open reading frame resembling the retroviral gene env, which is responsible for the infectious properties of retroviruses.  in this study we report molecular and phylogeny analysis of the complete env gene from ten species of the obscura group of the genus drosophila and one species from the genus scaptomyza.  the results indicate that in most cases env sequences could produce a functional env protein and therefore maintain the infectious capability of gypsy in these species. 