Complex Evolution of gypsy in Drosophilid Species
http://www.100md.com
分子生物学进展 2004年第10期
* Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; and Departamento de Biologia, Instituto de Ciências Exatas e Naturais, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
E-mail: elgion@base.ufsm.br.
Abstract
In an endeavor to contribute to the comprehension of the evolution of transposable elements (TEs) in the genome of host species, we investigated the phylogenetic relationships of sequences homologous to the retrotransposon gypsy of Drosophila melanogaster in 19 species of Drosophila, in Scaptodrosophila latifasciaeformis, and in Zaprionus indianus. This phylogenetic study was based on approximately 500 base pairs of the env gene. Our analyses showed considerable discrepancy between the phylogeny of gypsy elements and the relationship of their host species, and they allow us to infer a complex evolutionary pattern that could include ancestral polymorphism, vertical transmission, and several cases of horizontal transmission.
Key Words: gypsy ? Drosophila ? retrotransposon ? phylogeny ? horizontal transfer
Introduction
Retrotransposable elements have been found in all Eukaryota investigated so far. The superfamily of retrotransposons Ty3/gypsy is widely distributed among living organisms (Miller et al. 1999; Marín and Lloréns 2000), and its relationship with retroviruses has been inferred in several studies (Xiong and Eickbush 1990; Kim et al. 1994; Pélisson et al. 1997; Lerat and Capy 1999).
The gypsy retroelement, also known as mdg4, was first described in Drosophila melanogaster. It is 7.5 kb long and has 482 base pair (bp) long terminal repeats (LTRs; Georgiev et al. 1981; Bayev et al. 1984). Kim et al. (1994) reported the first evidence that culminated in the characterization of gypsy as the first retrovirus in invertebrates. Gypsy retroelements are now classified as members of the Metaviridae family, retrovirus genus Errantivirus (Boeke et al. 1998). As occurs in the genomes of other known retroviruses, that of gypsy has three open-reading frames (ORFs) called gag, pol, and env, encoding proteins responsible for its replication and infectivity (Kim et al. 1994). Gypsy was also isolated and sequenced from the genomes of D. virilis and D. subobscura (Mizrokhi and Mazo 1991; Alberola and De Frutos 1996). When the phylogenetic relationships were established for these three gypsy sequences, incongruities were observed relative to the relationship among their host species (Alberola and De Frutos 1996).
Gypsy is stable in almost all D. melanogaster strains studied so far, and its copies are usually located in the centromeric and/or pericentromeric regions. However, in hypermutable strains such as MS and MG, gypsy presents a high number of copies and significant activity due to the existence of permissive alleles in homozygosis of the gene flamenco, whose product is known for its ability to repress the activity exhibited by this retrotransposon (Bucheton 1995; Prud'homme et al. 1995; Pélisson et al. 1997).
The ubiquity of the sequences homologous to gypsy in the genus Drosophila has been attributed to its presence in ancestral genomes, before the separation of the main radiation branches known nowadays, with further expansion by vertical transmission (Alberola and De Frutos 1996). Some evidence obtained with Southern blot and phylogenetic analyses of gypsy sequences performed within groups of species, however, pointed to a more complex evolutionary picture, including the possibility of horizontal transfer events (Stacey et al. 1986; De Frutos, Peterson, and Kidwell 1992; Alberola and De Frutos 1993a, 1993b, 1996; Terzian et al. 2000; Vázquez-Manrique et al. 2000).
Recently, Mejlumian et al. (2002) reported the existence of DNA sequences putatively encoding full-length, functional env proteins in the genome of various species closely related to D. melanogaster and in more distant species such as D. virilis and D. subobscura. These data support the hypothesis that these sequences are potentially infectious gypsy copies able to spread between sexually isolated species.
Aiming to gain a more comprehensive insight into the evolutionary history of gypsy in the genus Drosophila and related species, we performed a phylogenetic analysis of gypsy homologous sequences based in a 500-bp region of ORF 3 of this element in 19 species of genus Drosophila, in Zaprionus indianus, and in Scaptodrosophila latifasciaeformis. Our results indicate that gypsy elements from these species show a complex evolutionary pattern, with phylogenetic relationships inconsistent with those of their hosts. Horizontal transmission is shown to be an important feature of the evolutionary history of gypsy, one that is required to explain the wide taxonomic distribution of this element among Drosophila species.
Materials and Methods
Drosophila Stocks Used and Rearing Conditions
Isofemale lines of all studied species were established and reared in cornmeal medium (Marques et al. 1966) at constant temperature and humidity (17°C ± 1°C; 60% r.h.). A list of all species and populations employed is shown in table 1.
Table 1 List of Drosophila Species and Strains Used in This Study
DNA Extraction and PCR Amplification
Approximately 100 adult flies per sample were macerated in a 1.5-ml microcentrifuge tube with liquid nitrogen and the genomic DNA was extracted according to Jowett (1986).
The pair of degenerate primers used in this work was designed based on the alignment of the sequences of gypsy available in GenBank (NCBI) for D. melanogaster (accession number M12927), D. virilis (accession number M38438), and D. subobscura (accession number X72390). The primers obtained were GYP3S2 (sense) 5'-AAAGGCGAYTTGGTTGACACTCC 3' and GYP3AS2 (antisense) 5'-CARGTGGCTRGGTTGRGTGTG3', and they correspond, respectively, to nucleotide positions 6026–6048 and 6491–6511 of the D. melanogaster gypsy element, producing a fragment of 485 nucleotides that maps to ORF 3. For the amplification, 50 ng of DNA were submitted to a reaction containing 1U Taq polymerase (Invitrogen, San Diego, Calif.), 50 mM of each nucleotide, 20 pmol of each primer, and 1.5 mM of MgCl2 in a volume of 50 μl. The amplification conditions used in all reactions were: 96°C (2 min), followed by 35 cycles of 15 s at 96°C, 30 s at 55°C, and 90 s at 72°C, and finally 72°C (5 min).
Cloning and Sequencing
The PCR products with the expected length were purified with GFXTM PCR DNA and Gel Band Purification kit (Amersham Biosciences, Buckinghamshire, England) and cloned in the pCR-TOPO plasmid using the TOPO TA cloning kit for sequencing (Invitrogen). Plasmids of the colonies obtained were extracted by the alkaline lysis mini-prep method and purified with GFXTM. Two or three clones were selected and sequenced on one strand only, using standard protocols. Automated sequencing was performed at the Molecular Genetics Instrumentation Facility, University of Georgia (Athens, G.A.).
For D. willistoni, we extended the study of gypsy-homologous sequences to two strains to confirm the identity of this sequence, using the Morro Santana (Southern Brazil) and Pará (Northern Brazil) strains, the most geographically distant ones available in our laboratory.
Phylogenetic Analyses
The nucleotide sequences obtained, and their respective amino acid sequences, were aligned using the program ClustalV 1.3b (Higgins, Bleasby, and Fuchs 1992) and hand adjustments of the alignments were performed when necessary. Accession numbers in GenBank are AF548143–AF548203. The alignment is available in the Supplementary Material online (Fasta file). The deduced amino acid sequences were obtained using GeneDoc version 2.6.001 (Nicholas and Nicholas 1997). All the phylogenetic analyses were carried out using the MEGA version 2.1 (Kumar et al. 2001). The methods utilized for tree inference were maximum parsimony and Neighbor-Joining. Bootstrap tests with 500 samplings were performed for all trees obtained. For maximum parsimony, we used the Close-Neighbor-Interchange (CNI) method, level 3 and "random addition" option (10 samplings). Distance matrices for the Neighbor-Joining analyses were based on the number of substitutions calculated by the Kimura two-parameter model (Kimura 1980) for the nucleotide sequences and on P distance for the amino acid sequences. The number of nonsynonymous substitutions per nonsynonymous site (dN) and the number of synonymous substitutions per synonymous site (dS) of the translated sequences were calculated using Nei and Gojobori's method (1986). A Z test was used to determine whether dN and dS differed significantly from each other. A value of dN significantly lower than dS suggests that purifying selection is acting on the sequence. The remaining parameters of analysis were those established by default by the program used. Construction of phylogenetic trees using the PAUP program version 3.1.1 (Swofford 1993) gave similar results.
Results
The sequences used to establish the phylogenetic inferences were 499 positions (including gaps) from 64 samples analyzed.
The average percent difference among all taxa was 32.7%, with values ranging between 0.5% (minimum) and 63.2% (maximum). The sequences of D. willistoni were the most different from the remaining samples and the percent difference found between this species and the others varied from 51.2% (D. griseolineata) to 63.2% (D. mediopunctata, clones 49 and 86). Thus, to assess whether the high substitution level found for this species would influence the analysis, we compared the clusters obtained with and without D. willistoni sequences. This comparison showed that the main clusters remained constant and so D. willistoni sequences were kept in the analysis. However, to confirm the degree of nucleotide divergence obtained for the clones analyzed from D. willistoni, we sequenced and analyzed the same gypsy region in individuals of geographically distant populations from this species. These two Brazilian D. willistoni populations were collected in places located about 4,000 km apart, but, in spite of this, they are still extremely homogeneous with regard to nucleotide sequence of the gypsy element (from 0%–1% of divergence).
The tree obtained via the Neighbor-Joining method (fig. 1) consists of four clades that can be subdivided into eight groups (designated by letters A to H) according to their degree of divergence. We considered as distinct groups those with degrees of divergence higher than 20%. The average divergence found within and between these groups is shown in table 2.
FIG. 1.— Phylogenetic analysis of gypsy element nucleotide sequences. Neighbor-Joining tree with Kimura two-parameter distances. Numbers above branches are percentage bootstrap values based on 500 replications. The species names are followed by clone number. Different symbols represent distinct subgenera of genus Drosophila: circle, Drosophila; triangle, Sophophora; square, Dorsilopha. Different patterns inside the symbols represent distinct species group.
Table 2 Percent Difference of Nucleotides (Below and Left in the Diagonal) and Amino Acids (Above and Right in the Diagonal) Found Within and Between Groups of gypsy Sequences
In the genome of the related species D. mediopunctata and D. mediopicta, the sequences homologous to gypsy presented a degree of divergence that placed them into distinct groups in the phylogenetic tree. In D. mediopunctata, the divergence between clone 45 and the other clones of this species was 34.8%. In D. mediopicta, the nucleotide average distance between clone 34 of group F and the remaining clones (1 and 35 of group D) was 30.8%.
The strict consensus tree obtained from 208 other equally parsimonious trees (CI = 0,54 e RI = 0,9), all containing 594 steps, resulted in the same eight groups mentioned above (see fig. 2 in the Supplementary Material online). It is important to stress the fact that the nodes that form the eight clusters are well supported by bootstrap values.
FIG. 2.— Evolution scenario proposed in this work involving multiple cases of horizontal transfer to explain the incongruities between gypsy element phylogeny and species phylogeny. Open circles and rectangles correspond to ancestral family. Solid circles and rectangles correspond to different current subfamilies according to the legend. The arrows indicate the horizontal transfer events and the number above of these arrows means time estimation, in Myr, of the occurrence of these events.
For the analyses of the amino acids sequences, some taxa were excluded. The sequences of D. willistoni were excluded because they presented seven "stop" codons and a high degree of divergence in relation to all the other species. In addition, each species was represented only by clones that differed by more than 1% of their nucleotide sequences. Thus, we obtained 40 sequences that were aligned in all codon positions, except for a few "gaps" to compensate for existing insertions/deletions. The region of the env gene analyzed in this study corresponds to a portion potentially able to encode 151 amino acids.
The phylogenies obtained (by both Neighbor-Joining and maximum parsimony methods) from the analysis of the amino acids sequences resulted in very similar topologies, with seven of groups initially observed in the first analysis (fig. 3 in the Supplementary Material online). The average percent divergence obtained for these data was 30.6%. Table 2 shows the divergence found within and among groups also for amino acids sequences.
dN and dS were estimated for coding sequences (tables 1 and 2 in the Supplementary Material online). The average values obtained for these parameters were 61.5% and 17.7%, respectively, and the ratio dN/dS was 0.29. This ratio indicates that purifying selection is under way in the analyzed region. A selection test was performed for each pair of sequences and evidenced neutrality in the following comparisons: between gypsy of D. hydei and D. virilis; between clones of S. latifasciaeformis, Z. indianus, D. annulimana, and D. griseolineata; between the species D. ornatifrons and D. griseolineata; and, finally, among all the sequences to the genomes of species clustered in group G (fig. 1). All the other comparisons ruled out the null hypothesis (neutrality), suggesting purifying selection (table 3 in the Supplementary Material online).
Discussion
The wide distribution of some class I transposable elements, such as copia (Stacey et al. 1986), 412 (Cizeron et al. 1998), 1731 (Montchamp-Moreau et al. 1993), and bilbo (Blesa, Gandía, and Martínez-Sebastián 2001), has been well documented. This scenario is usually explained by existence of the transposable element (TE) in the ancestral species. For bilbo and 1731, the authors concluded that vertical transmission was the main mechanism responsible for the expansion of those mobile sequences. Elements such as 412 and copia showed more complex divergence patterns, where the phylogenetic relationships of the species is, sometimes, incompatible with the element phylogeny. In these cases, horizontal transmission events were referred to as important to explain these discordant findings. This last evolutionary process appears to also explain our results with the gypsy element, in accordance with other studies (Stacey et al. 1986; De Frutos et al. 1992; Terzian et al. 2000; Mejlumian et al. 2002).
The data available about gypsy in Drosophila have shown these sequences to be extensively present in this genus. However, this retrotransposon appears to be inactive in most species. Does gypsy's wide distribution result from its presence in a Drosophila ancestor? This question may be addressed by comparing the relationship among the transposon sequences present in several different host genomes with the phylogeny of the hosts themselves.
Phylogeny of the Species Versus Phylogeny of gypsy
For a comparative approach we used the phylogenetic relationships among Drosophila species and Scaptodrosophila latifasciaeformis inferred and corroborated in studies by Kwiatowski and Ayala (1999), Tatarenkov et al. (1999), Tarrio, Rodriguez-Trelles, and Ayala (2001), Tatarenkov, Zurovcova, and Ayala (2001), and Remsen and O'Grady (2002).
Scaptodrosophila and Zaprionus are traditionally classified by Wheeler (1981) as a subgenus and genus, respectively. In this paper we refer to them as genus and subgenus, respectively, according to the review by Grimaldi (1990) that raised the first to genus and combined molecular data that appoint Zaprionus as an internal clade inside of the Drosophila genus (reviewed in papers cited above).
Comparison of the phylogenetic relationships of the host species with those of their gypsy elements as depicted in figure 1 revealed several incongruencies. We observed, in fact, the clustering of gypsy sequences sampled from different subgenera, e.g., in clusters A, C, and G. More detailed comparisons about the position of the species groups in the different sections of the tree point to more incongruities. Cluster A joins gypsy elements from D. subobscura and D. busckii, two species of different subgenera. Cluster B contains sequences of clones of two species from the tripunctata group; however, they are only distantly related to other gypsy sampled from species of this species group. In cluster C, sequences of Z. indianus appear in close proximity with gypsy sequences of sampled from the melanogaster group. Cluster F brings together all studied species of the guarani group, with species of groups tripunctata, repleta, and cardini. Although all these species are members of the subgenus Drosophila, the relationships among their gypsy element established are not compatible with those observed in the phylogeny of the species.
The incongruities are strengthened when the rates of divergence for the gypsy sequences were compared to those of certain nuclear genes available in the literature (Pelandakis and Solignac [1993] for rDNA-28S and Kwiatowski et al. [1994] and Kwiatowski and Ayala [1999] for the Sod gene). The sequences of these genes were obtained from PubMed (accession numbers 7545938 and 8283482, respectively; see table 4 in the Supplementary Material online), and the distances were calculated as described above. By comparing the values obtained for Sod and rDNA of the same pairs of taxa we found that the rDNA sequences were approximately three times more conserved than those of Sod. Substitution rates of retroviral sequences as gypsy are in general higher than those of nuclear genes, due to the high rates of misincorporation of nucleotides by reverse transcriptase when compared with DNA polymerase (Drake 1993). Taking these findings into account, we expected a priori higher values of divergence for gypsy than for other nuclear sequences, with the magnitude of this difference depending on the rate of transposition of gypsy in the host genome and on characteristics of the host genome (Eickbush et al. 1995).
The majority of the comparisons between the sequences of gypsy and nuclear genes, shown in tables 3 and 4, demonstrate a higher evolutionary distance for gypsy, as expected for transposable elements. However, some of these comparisons are discrepant, mainly those results shown in bold in the tables. Sequences like Sod and rDNA are expected to undergo purifying selection because they are essential for the organisms. Once the purifying selection is not predicted to act over transposable elements, it is not expected to obtain similar values of divergence among gypsy sequences and nuclear genes, and values of divergence among gypsy sequences very similar (and sometimes lower) to that among nuclear genes are unexpected (Silva and Kidwell 2000). Furthermore, the evolutionary distances seen among gypsy sequences are not compatible with the evolutionary history of the species, as inferred from evolutionary distances of the nuclear sequences. Some of the most intriguing cases were obtained from comparisons between D. busckii and D. subobscura (0.27 for Sod and 0.05 for gypsy), D. simulans and Z. indianus (0.33 for Sod and 0.08 for rDNA compared with 0.07 for gypsy), and D. subobscura and D. hydei (0.36 for Sod and 0.08 for gypsy). Even species that are members of the same species radiation, such as D. virilis and D. hydei, presented much smaller substitution rates for gypsy than those found for Sod (0.06 and 0.19, respectively). If we try to explain these findings through an evolutionary pattern driven by vertical transmission of gypsy elements, we need to assume a very high coefficient of selection, several times higher than those occurring in one of the most conserved regions of the ribosomal DNA (28S). Moreover, we will not be able to explain the close relationship between gypsy sequences of distantly related species, whereas gypsy sequences of closely related species, as those of the tripunctata group, are so divergent and scattered throughout the phylogeny reconstructed in the present report.
Table 3 Comparative Analysis Between Genetic Distances (Kimura's Two-Parameter Method) of Sod Sequences and gypsy Sequences
Table 4 Comparative Analysis Between Genetic Distances (Kimura's Two-Parameter Method) of rDNA Sequences and gypsy Sequences
Several authors that faced such patterns of divergence have maintained that horizontal transmission events are the most likely explanation for the relationships observed in their studies (Maruyama and Hartl 1991; Robertson and MacLeod 1993; Lohe et al. 1995; Clark and Kidwell 1997; Gonzales and Lessios 1999; Jordan, Matyunina, and McDonald 1999; Silva and Kidwell 2000; Loreto et al. 2001). Such appears to be the case for the history of the gypsy element reported here.
Subfamilies of gypsy
According to criteria used by other authors (Lohe et al. 1995; Clark and Kidwell 1997; Capy et al. 1998; Jordan and McDonald 1998; Blesa, Gandía, and Martínez-Sebastián 2001), the distances obtained between clusters such as A and G (fig. 1) reveal the existence of multiple subfamilies of gypsy. Pinsker et al. (2001) suggests that care should be taken in the establishment of families and subfamilies of transposable elements when only one region of the element is investigated. Alberola and De Frutos (1996), however, detected very similar percentages of divergence in the different regions of the three ORFs of complete gypsy retrotransposons of D. melanogaster, D. subobscura, and D. virilis. This finding gives support to our estimates of divergence based only in one region of the gypsy.
Therefore, we suggest that the clusters from A to G represent different subfamilies of gypsy, as their percent divergence varies from 23% to 40.8% (tables 2 and 3). The distances found between cluster H and the remaining clusters varied from 52.3% to 62.1 %, suggesting that this clade represents a distinct family of gypsy. However, more detailed analyses of other regions of gypsy are necessary to evaluate the homology of this sequence with those of other families of transposable elements related to gypsy already described in literature.
The high sequence divergence between gypsy sequences amplified from others species relative to those found in the genome of D. willistoni is more interesting still when its conservation among distant populations of this species is considered. This observation contradicts the high evolutionary rate of this gypsy strain in this genome in particular. More plausible seems to be the hypothesis of this distant origin and ancestral polymorphism followed by losses of this type of sequence in all other species, including D. paulistorum. However, we cannot discard the presence this gypsy-like sequence found in D. willistoni in other species not detected in our analysis by sampling error. Horizontal transfer involving a donor species not included in our analyses could also explain the pattern presented by D. willistoni. Nevertheless, sequences homologous to gypsy in this species, although presenting a high degree of divergence, form clusters with species phylogenetically related.
Complex Evolution
To prevent a superestimation of the horizontal transfer events we established that only one case of horizontal transfer (HT) is computed when two or more closely related species are involved in the phenomenon. In this case, to characterize the event we chose the most discrepant value in the comparison between the divergence values for gypsy and nuclear genes among the involved species. The application of this criterion was necessary because HT cases could be attributed to the low evolutionary divergence among the receipt species gypsy sequences and the sequences of the others species, related to the donor. Thus, we established as the donor species, in the group of phylogenetically related species that share gypsy sequences, the most similar one to the distant species (receipt). Therefore, we postulated nine horizontal transmission cases (fig. 2). The most prominent involved the transfer of gypsy-homologous sequences among: (1) D. nebulosa and D. neocardini, (2) D. nebulosa and D. paulistorum; (3) D. subobscura and D. busckii; (4) D. subobscura and D. hydei; (5) D. simulans and Zaprionus indianus; (6) D. simulans and S. latifasciaeformis; (7) D. pallidipennis and D. bandeirantorum; (8) D. virilis and D. hydei; (9) D. mediopicta and D. zotti. All comparisons between data of divergence of gypsy and of nuclear genes in these nine cases seem be inconsistent with vertical transmission. In several of these comparisons dN is not significantly lower than dS, excluding the possibility that very strong selection produced this high conservation after the event of horizontal transmission.
The occurrence of vertical transmission can be postulated if two criterions are followed: (1) the gypsy sequences are grouped in the phylogeny following the phylogenetic relationships of the host species, or (2) evolutionary distances among gypsy sequences are proportional to the one obtained with the nuclear genes. Examples of possible events of gypsy vertical transmission are evidenced by the evolutionary distance values between the species of the guarani group as well as some species of the tripunctata group.
The general picture depicted here suggests that there were two subfamilies of gypsy sequences in the ancestor of the genus Drosophila. One of the two can be involved in all independent horizontal transfers described here. Moreover, our results point to a recent invasion of the genomes of a large number of species by gypsy sequences.
Estimates of the Occurrence of Time of the Horizontal Transmission Events
Using a substitution rate of 1.6% per Myr estimated for nuclear genes with low preferential use of codons (Sharp and Li 1989), we estimated the time of divergence of gypsy sequences involved in the nine cases of horizontal transmission proposed in the present report. This approach had already been implemented by other authors (Alberola and De Frutos 1993b; Silva and Kidwell 2000; Loreto et al. 2001). The estimated time of divergence of gypsy in our samples are shown in figure 2, with the arrows indicating the possible cases of horizontal transmission. These values provide a good notion of the time when the invasion of the genomes by gypsy occurred, the more recent event being around 1.2 MYA and the oldest around 3.4 MYA.
Analyses of dN/dS
The proposal of recent invasion of the Drosophila genome by gypsy appears to be corroborated by the values of dN and dS. The average value of dN/dS obtained for all comparisons among the 40 sequences utilized in our analyses was 0.29 (varying around 0.03 and 2.5). These values are similar to those calculated for nuclear genes (Akashi 1994). Besides this, the selection tests performed showed that in the majority of comparisons, purifying selection is operating (or operated in a recent past) on the gypsy sequences (at the significance level of 0.05). Terzian et al. (2000) investigated gypsy in species of the subgroup melanogaster and also identified purifying selection, relating their findings to recent events of transposition. Comparing gypsy of D. melanogaster, D. virilis, and D. subobscura, Alberola and De Frutos (1996) also pointed to low dN/dS values, which implies that selection is operating on these sequences. In addition, Martínez-Sebastián et al. (2002) analyzed the evolutionary constraints of the gypsy in species of the obscura group, and their data support that gypsy sequences are subjected to purifying selection. Our data of dN and dS are comparable with those obtained for Adh (Silva and Kidwell 2000), allowing us to infer that the gypsy sequences are evolving or have evolved under strong selection in a recent past.
Different Subfamilies Coexisting in the Same Genome
Another important subject to be discussed is the existence of very divergent sequences of gypsy in the genome of one same species as observed in D. mediopicta and D. mediopunctata. For gypsy, the coexistence of more than one subfamily in a host genome has been previously reported (Alberola and De Frutos 1996; Hochstenbach et al. 1996; Terzian et al. 2000; Vázquez-Manrique et al. 2000; Martínez-Sebastián et al. 2002). The phenomenon of multiple subfamilies of TEs in the same species was observed in other elements, such as in mariner (Robertson and MacLeod 1993), P (Clark and Kidwell 1997), Minos (Arcà and Savakis 2000), or R1 (Gentile, Burke, and Eickbush 2001).
The coexistence of different subfamilies of a TE in a host genome has been given by some authors as evidence of independent invasions of a genome by a mobile element, probably via horizontal transfer (Zelentsova et al. 1999; Arcà and Savakis 2000; Silva and Kidwell 2000). Ancestral polymorphism, however, cannot be disregarded as a possible explanation. This could be the case for the divergent sequences of gypsy found in the genome of D. mediopunctata. The genetic distance calculated between gypsy of clone 45 and the sequences of D. maculifrons could be considered as compatible with the hypothesis of vertical transmission of the gypsy sequences along the evolutionary divergence of these species.
Conclusion
Several authors have suggested that horizontal transmission is an essential step in the life-cycle of some TE families (Lohe et al. 1995; Jordan and McDonald 1998; Silva and Kidwell 2000; Pinsker et al. 2001). Convincing cases of horizontal transmission were limited, until a few years ago, to elements of class II, such as P, mariner, and hobo (Daniels, Chovnick, and Boussy 1990; Simmons 1992; Clark, Maddison, and Kidwell 1994; Lohe et al. 1995). Even though evidence of this phenomenon in retrotransposons has accumulated in the past few years (Alberola and De Frutos 1996; Flavell et al. 1997; Cizeron et al. 1998; Jordan and McDonald 1998; Gonzales and Lession 1999; Jordan, Matyunina, and McDonald 1999; Terzian et al. 2000; Vázquez-Manrique et al. 2000, Martínez-Sebastián et al. 2002), horizontal transfer still seem to be more common among class I TEs (reviewed in Silva, Loreto, and Clark 2004).
The occurrence of horizontal transmission usually requires the existence of a vector, such as viruses (Friesen and Nissen 1990), parasitic mites (Houck et al. 1991), or endosymbiotic bacteria (Kidwell 1993), to shuttle the DNA between hosts. However, the evolutionary scenario of the gypsy retrotransposon is remarkable in that the several events of horizontal transfer proposed here do not require a vector. If gypsy is able to encode the envelope protein that allows it to behave like a retrovirus (Kim et al. 1994; Mejlumian et al. 2002), then the pattern observed can be attributed to the occurrence of infective waves. This would explain why horizontal transfer is more prevalent among gypsy than among other class I elements (Silva, Loreto, and Clark 2004). This capacity was already demonstrated experimentally by Mejlumian et al. (2002) for a few species other than D. melanogaster and deserves to be expanded to more species. The perspective of these results will help us to find answers regarding the role of retroviral sequences and their horizontal transmission in the evolution of eukaryotic genomes.
This work represents an attempt to describe some aspects of an extremely complex evolutionary pattern. We do not propose to stress or finish the analysis or to discard alternative models that might explain the observed evolutionary aspects. The different phenomena (vertical transmission, horizontal transmission, different evolutionary rate, ancestral polymorphism) discussed here are not mutually exclusive and probably occur simultaneously. Therefore, analysis using different regions of the element, as well as a larger number of species, is necessary to complete a more realistic picture of the evolution of the gypsy retroelement.
Supplementary Materials
All figures and tables cited as Supplementary Material are available on the journal's Web site.
Acknowledgements
This work was supported by grants from Conselho Nacional de Pesquisa, PROPESQ-UFRGS, Financiadora de Estudos e Projetos, and Funda??o de Amparo a Pesquisa do Estado do Rio Grande do Sul. We thank Daniela Cristina De Toni and Luciano Basso da Silva for fly stocks. We are grateful to Joana C. Silva and Pierre Capy for critical comments on the manuscript and two anonymous reviewers for their valuable suggestions.
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E-mail: elgion@base.ufsm.br.
Abstract
In an endeavor to contribute to the comprehension of the evolution of transposable elements (TEs) in the genome of host species, we investigated the phylogenetic relationships of sequences homologous to the retrotransposon gypsy of Drosophila melanogaster in 19 species of Drosophila, in Scaptodrosophila latifasciaeformis, and in Zaprionus indianus. This phylogenetic study was based on approximately 500 base pairs of the env gene. Our analyses showed considerable discrepancy between the phylogeny of gypsy elements and the relationship of their host species, and they allow us to infer a complex evolutionary pattern that could include ancestral polymorphism, vertical transmission, and several cases of horizontal transmission.
Key Words: gypsy ? Drosophila ? retrotransposon ? phylogeny ? horizontal transfer
Introduction
Retrotransposable elements have been found in all Eukaryota investigated so far. The superfamily of retrotransposons Ty3/gypsy is widely distributed among living organisms (Miller et al. 1999; Marín and Lloréns 2000), and its relationship with retroviruses has been inferred in several studies (Xiong and Eickbush 1990; Kim et al. 1994; Pélisson et al. 1997; Lerat and Capy 1999).
The gypsy retroelement, also known as mdg4, was first described in Drosophila melanogaster. It is 7.5 kb long and has 482 base pair (bp) long terminal repeats (LTRs; Georgiev et al. 1981; Bayev et al. 1984). Kim et al. (1994) reported the first evidence that culminated in the characterization of gypsy as the first retrovirus in invertebrates. Gypsy retroelements are now classified as members of the Metaviridae family, retrovirus genus Errantivirus (Boeke et al. 1998). As occurs in the genomes of other known retroviruses, that of gypsy has three open-reading frames (ORFs) called gag, pol, and env, encoding proteins responsible for its replication and infectivity (Kim et al. 1994). Gypsy was also isolated and sequenced from the genomes of D. virilis and D. subobscura (Mizrokhi and Mazo 1991; Alberola and De Frutos 1996). When the phylogenetic relationships were established for these three gypsy sequences, incongruities were observed relative to the relationship among their host species (Alberola and De Frutos 1996).
Gypsy is stable in almost all D. melanogaster strains studied so far, and its copies are usually located in the centromeric and/or pericentromeric regions. However, in hypermutable strains such as MS and MG, gypsy presents a high number of copies and significant activity due to the existence of permissive alleles in homozygosis of the gene flamenco, whose product is known for its ability to repress the activity exhibited by this retrotransposon (Bucheton 1995; Prud'homme et al. 1995; Pélisson et al. 1997).
The ubiquity of the sequences homologous to gypsy in the genus Drosophila has been attributed to its presence in ancestral genomes, before the separation of the main radiation branches known nowadays, with further expansion by vertical transmission (Alberola and De Frutos 1996). Some evidence obtained with Southern blot and phylogenetic analyses of gypsy sequences performed within groups of species, however, pointed to a more complex evolutionary picture, including the possibility of horizontal transfer events (Stacey et al. 1986; De Frutos, Peterson, and Kidwell 1992; Alberola and De Frutos 1993a, 1993b, 1996; Terzian et al. 2000; Vázquez-Manrique et al. 2000).
Recently, Mejlumian et al. (2002) reported the existence of DNA sequences putatively encoding full-length, functional env proteins in the genome of various species closely related to D. melanogaster and in more distant species such as D. virilis and D. subobscura. These data support the hypothesis that these sequences are potentially infectious gypsy copies able to spread between sexually isolated species.
Aiming to gain a more comprehensive insight into the evolutionary history of gypsy in the genus Drosophila and related species, we performed a phylogenetic analysis of gypsy homologous sequences based in a 500-bp region of ORF 3 of this element in 19 species of genus Drosophila, in Zaprionus indianus, and in Scaptodrosophila latifasciaeformis. Our results indicate that gypsy elements from these species show a complex evolutionary pattern, with phylogenetic relationships inconsistent with those of their hosts. Horizontal transmission is shown to be an important feature of the evolutionary history of gypsy, one that is required to explain the wide taxonomic distribution of this element among Drosophila species.
Materials and Methods
Drosophila Stocks Used and Rearing Conditions
Isofemale lines of all studied species were established and reared in cornmeal medium (Marques et al. 1966) at constant temperature and humidity (17°C ± 1°C; 60% r.h.). A list of all species and populations employed is shown in table 1.
Table 1 List of Drosophila Species and Strains Used in This Study
DNA Extraction and PCR Amplification
Approximately 100 adult flies per sample were macerated in a 1.5-ml microcentrifuge tube with liquid nitrogen and the genomic DNA was extracted according to Jowett (1986).
The pair of degenerate primers used in this work was designed based on the alignment of the sequences of gypsy available in GenBank (NCBI) for D. melanogaster (accession number M12927), D. virilis (accession number M38438), and D. subobscura (accession number X72390). The primers obtained were GYP3S2 (sense) 5'-AAAGGCGAYTTGGTTGACACTCC 3' and GYP3AS2 (antisense) 5'-CARGTGGCTRGGTTGRGTGTG3', and they correspond, respectively, to nucleotide positions 6026–6048 and 6491–6511 of the D. melanogaster gypsy element, producing a fragment of 485 nucleotides that maps to ORF 3. For the amplification, 50 ng of DNA were submitted to a reaction containing 1U Taq polymerase (Invitrogen, San Diego, Calif.), 50 mM of each nucleotide, 20 pmol of each primer, and 1.5 mM of MgCl2 in a volume of 50 μl. The amplification conditions used in all reactions were: 96°C (2 min), followed by 35 cycles of 15 s at 96°C, 30 s at 55°C, and 90 s at 72°C, and finally 72°C (5 min).
Cloning and Sequencing
The PCR products with the expected length were purified with GFXTM PCR DNA and Gel Band Purification kit (Amersham Biosciences, Buckinghamshire, England) and cloned in the pCR-TOPO plasmid using the TOPO TA cloning kit for sequencing (Invitrogen). Plasmids of the colonies obtained were extracted by the alkaline lysis mini-prep method and purified with GFXTM. Two or three clones were selected and sequenced on one strand only, using standard protocols. Automated sequencing was performed at the Molecular Genetics Instrumentation Facility, University of Georgia (Athens, G.A.).
For D. willistoni, we extended the study of gypsy-homologous sequences to two strains to confirm the identity of this sequence, using the Morro Santana (Southern Brazil) and Pará (Northern Brazil) strains, the most geographically distant ones available in our laboratory.
Phylogenetic Analyses
The nucleotide sequences obtained, and their respective amino acid sequences, were aligned using the program ClustalV 1.3b (Higgins, Bleasby, and Fuchs 1992) and hand adjustments of the alignments were performed when necessary. Accession numbers in GenBank are AF548143–AF548203. The alignment is available in the Supplementary Material online (Fasta file). The deduced amino acid sequences were obtained using GeneDoc version 2.6.001 (Nicholas and Nicholas 1997). All the phylogenetic analyses were carried out using the MEGA version 2.1 (Kumar et al. 2001). The methods utilized for tree inference were maximum parsimony and Neighbor-Joining. Bootstrap tests with 500 samplings were performed for all trees obtained. For maximum parsimony, we used the Close-Neighbor-Interchange (CNI) method, level 3 and "random addition" option (10 samplings). Distance matrices for the Neighbor-Joining analyses were based on the number of substitutions calculated by the Kimura two-parameter model (Kimura 1980) for the nucleotide sequences and on P distance for the amino acid sequences. The number of nonsynonymous substitutions per nonsynonymous site (dN) and the number of synonymous substitutions per synonymous site (dS) of the translated sequences were calculated using Nei and Gojobori's method (1986). A Z test was used to determine whether dN and dS differed significantly from each other. A value of dN significantly lower than dS suggests that purifying selection is acting on the sequence. The remaining parameters of analysis were those established by default by the program used. Construction of phylogenetic trees using the PAUP program version 3.1.1 (Swofford 1993) gave similar results.
Results
The sequences used to establish the phylogenetic inferences were 499 positions (including gaps) from 64 samples analyzed.
The average percent difference among all taxa was 32.7%, with values ranging between 0.5% (minimum) and 63.2% (maximum). The sequences of D. willistoni were the most different from the remaining samples and the percent difference found between this species and the others varied from 51.2% (D. griseolineata) to 63.2% (D. mediopunctata, clones 49 and 86). Thus, to assess whether the high substitution level found for this species would influence the analysis, we compared the clusters obtained with and without D. willistoni sequences. This comparison showed that the main clusters remained constant and so D. willistoni sequences were kept in the analysis. However, to confirm the degree of nucleotide divergence obtained for the clones analyzed from D. willistoni, we sequenced and analyzed the same gypsy region in individuals of geographically distant populations from this species. These two Brazilian D. willistoni populations were collected in places located about 4,000 km apart, but, in spite of this, they are still extremely homogeneous with regard to nucleotide sequence of the gypsy element (from 0%–1% of divergence).
The tree obtained via the Neighbor-Joining method (fig. 1) consists of four clades that can be subdivided into eight groups (designated by letters A to H) according to their degree of divergence. We considered as distinct groups those with degrees of divergence higher than 20%. The average divergence found within and between these groups is shown in table 2.
FIG. 1.— Phylogenetic analysis of gypsy element nucleotide sequences. Neighbor-Joining tree with Kimura two-parameter distances. Numbers above branches are percentage bootstrap values based on 500 replications. The species names are followed by clone number. Different symbols represent distinct subgenera of genus Drosophila: circle, Drosophila; triangle, Sophophora; square, Dorsilopha. Different patterns inside the symbols represent distinct species group.
Table 2 Percent Difference of Nucleotides (Below and Left in the Diagonal) and Amino Acids (Above and Right in the Diagonal) Found Within and Between Groups of gypsy Sequences
In the genome of the related species D. mediopunctata and D. mediopicta, the sequences homologous to gypsy presented a degree of divergence that placed them into distinct groups in the phylogenetic tree. In D. mediopunctata, the divergence between clone 45 and the other clones of this species was 34.8%. In D. mediopicta, the nucleotide average distance between clone 34 of group F and the remaining clones (1 and 35 of group D) was 30.8%.
The strict consensus tree obtained from 208 other equally parsimonious trees (CI = 0,54 e RI = 0,9), all containing 594 steps, resulted in the same eight groups mentioned above (see fig. 2 in the Supplementary Material online). It is important to stress the fact that the nodes that form the eight clusters are well supported by bootstrap values.
FIG. 2.— Evolution scenario proposed in this work involving multiple cases of horizontal transfer to explain the incongruities between gypsy element phylogeny and species phylogeny. Open circles and rectangles correspond to ancestral family. Solid circles and rectangles correspond to different current subfamilies according to the legend. The arrows indicate the horizontal transfer events and the number above of these arrows means time estimation, in Myr, of the occurrence of these events.
For the analyses of the amino acids sequences, some taxa were excluded. The sequences of D. willistoni were excluded because they presented seven "stop" codons and a high degree of divergence in relation to all the other species. In addition, each species was represented only by clones that differed by more than 1% of their nucleotide sequences. Thus, we obtained 40 sequences that were aligned in all codon positions, except for a few "gaps" to compensate for existing insertions/deletions. The region of the env gene analyzed in this study corresponds to a portion potentially able to encode 151 amino acids.
The phylogenies obtained (by both Neighbor-Joining and maximum parsimony methods) from the analysis of the amino acids sequences resulted in very similar topologies, with seven of groups initially observed in the first analysis (fig. 3 in the Supplementary Material online). The average percent divergence obtained for these data was 30.6%. Table 2 shows the divergence found within and among groups also for amino acids sequences.
dN and dS were estimated for coding sequences (tables 1 and 2 in the Supplementary Material online). The average values obtained for these parameters were 61.5% and 17.7%, respectively, and the ratio dN/dS was 0.29. This ratio indicates that purifying selection is under way in the analyzed region. A selection test was performed for each pair of sequences and evidenced neutrality in the following comparisons: between gypsy of D. hydei and D. virilis; between clones of S. latifasciaeformis, Z. indianus, D. annulimana, and D. griseolineata; between the species D. ornatifrons and D. griseolineata; and, finally, among all the sequences to the genomes of species clustered in group G (fig. 1). All the other comparisons ruled out the null hypothesis (neutrality), suggesting purifying selection (table 3 in the Supplementary Material online).
Discussion
The wide distribution of some class I transposable elements, such as copia (Stacey et al. 1986), 412 (Cizeron et al. 1998), 1731 (Montchamp-Moreau et al. 1993), and bilbo (Blesa, Gandía, and Martínez-Sebastián 2001), has been well documented. This scenario is usually explained by existence of the transposable element (TE) in the ancestral species. For bilbo and 1731, the authors concluded that vertical transmission was the main mechanism responsible for the expansion of those mobile sequences. Elements such as 412 and copia showed more complex divergence patterns, where the phylogenetic relationships of the species is, sometimes, incompatible with the element phylogeny. In these cases, horizontal transmission events were referred to as important to explain these discordant findings. This last evolutionary process appears to also explain our results with the gypsy element, in accordance with other studies (Stacey et al. 1986; De Frutos et al. 1992; Terzian et al. 2000; Mejlumian et al. 2002).
The data available about gypsy in Drosophila have shown these sequences to be extensively present in this genus. However, this retrotransposon appears to be inactive in most species. Does gypsy's wide distribution result from its presence in a Drosophila ancestor? This question may be addressed by comparing the relationship among the transposon sequences present in several different host genomes with the phylogeny of the hosts themselves.
Phylogeny of the Species Versus Phylogeny of gypsy
For a comparative approach we used the phylogenetic relationships among Drosophila species and Scaptodrosophila latifasciaeformis inferred and corroborated in studies by Kwiatowski and Ayala (1999), Tatarenkov et al. (1999), Tarrio, Rodriguez-Trelles, and Ayala (2001), Tatarenkov, Zurovcova, and Ayala (2001), and Remsen and O'Grady (2002).
Scaptodrosophila and Zaprionus are traditionally classified by Wheeler (1981) as a subgenus and genus, respectively. In this paper we refer to them as genus and subgenus, respectively, according to the review by Grimaldi (1990) that raised the first to genus and combined molecular data that appoint Zaprionus as an internal clade inside of the Drosophila genus (reviewed in papers cited above).
Comparison of the phylogenetic relationships of the host species with those of their gypsy elements as depicted in figure 1 revealed several incongruencies. We observed, in fact, the clustering of gypsy sequences sampled from different subgenera, e.g., in clusters A, C, and G. More detailed comparisons about the position of the species groups in the different sections of the tree point to more incongruities. Cluster A joins gypsy elements from D. subobscura and D. busckii, two species of different subgenera. Cluster B contains sequences of clones of two species from the tripunctata group; however, they are only distantly related to other gypsy sampled from species of this species group. In cluster C, sequences of Z. indianus appear in close proximity with gypsy sequences of sampled from the melanogaster group. Cluster F brings together all studied species of the guarani group, with species of groups tripunctata, repleta, and cardini. Although all these species are members of the subgenus Drosophila, the relationships among their gypsy element established are not compatible with those observed in the phylogeny of the species.
The incongruities are strengthened when the rates of divergence for the gypsy sequences were compared to those of certain nuclear genes available in the literature (Pelandakis and Solignac [1993] for rDNA-28S and Kwiatowski et al. [1994] and Kwiatowski and Ayala [1999] for the Sod gene). The sequences of these genes were obtained from PubMed (accession numbers 7545938 and 8283482, respectively; see table 4 in the Supplementary Material online), and the distances were calculated as described above. By comparing the values obtained for Sod and rDNA of the same pairs of taxa we found that the rDNA sequences were approximately three times more conserved than those of Sod. Substitution rates of retroviral sequences as gypsy are in general higher than those of nuclear genes, due to the high rates of misincorporation of nucleotides by reverse transcriptase when compared with DNA polymerase (Drake 1993). Taking these findings into account, we expected a priori higher values of divergence for gypsy than for other nuclear sequences, with the magnitude of this difference depending on the rate of transposition of gypsy in the host genome and on characteristics of the host genome (Eickbush et al. 1995).
The majority of the comparisons between the sequences of gypsy and nuclear genes, shown in tables 3 and 4, demonstrate a higher evolutionary distance for gypsy, as expected for transposable elements. However, some of these comparisons are discrepant, mainly those results shown in bold in the tables. Sequences like Sod and rDNA are expected to undergo purifying selection because they are essential for the organisms. Once the purifying selection is not predicted to act over transposable elements, it is not expected to obtain similar values of divergence among gypsy sequences and nuclear genes, and values of divergence among gypsy sequences very similar (and sometimes lower) to that among nuclear genes are unexpected (Silva and Kidwell 2000). Furthermore, the evolutionary distances seen among gypsy sequences are not compatible with the evolutionary history of the species, as inferred from evolutionary distances of the nuclear sequences. Some of the most intriguing cases were obtained from comparisons between D. busckii and D. subobscura (0.27 for Sod and 0.05 for gypsy), D. simulans and Z. indianus (0.33 for Sod and 0.08 for rDNA compared with 0.07 for gypsy), and D. subobscura and D. hydei (0.36 for Sod and 0.08 for gypsy). Even species that are members of the same species radiation, such as D. virilis and D. hydei, presented much smaller substitution rates for gypsy than those found for Sod (0.06 and 0.19, respectively). If we try to explain these findings through an evolutionary pattern driven by vertical transmission of gypsy elements, we need to assume a very high coefficient of selection, several times higher than those occurring in one of the most conserved regions of the ribosomal DNA (28S). Moreover, we will not be able to explain the close relationship between gypsy sequences of distantly related species, whereas gypsy sequences of closely related species, as those of the tripunctata group, are so divergent and scattered throughout the phylogeny reconstructed in the present report.
Table 3 Comparative Analysis Between Genetic Distances (Kimura's Two-Parameter Method) of Sod Sequences and gypsy Sequences
Table 4 Comparative Analysis Between Genetic Distances (Kimura's Two-Parameter Method) of rDNA Sequences and gypsy Sequences
Several authors that faced such patterns of divergence have maintained that horizontal transmission events are the most likely explanation for the relationships observed in their studies (Maruyama and Hartl 1991; Robertson and MacLeod 1993; Lohe et al. 1995; Clark and Kidwell 1997; Gonzales and Lessios 1999; Jordan, Matyunina, and McDonald 1999; Silva and Kidwell 2000; Loreto et al. 2001). Such appears to be the case for the history of the gypsy element reported here.
Subfamilies of gypsy
According to criteria used by other authors (Lohe et al. 1995; Clark and Kidwell 1997; Capy et al. 1998; Jordan and McDonald 1998; Blesa, Gandía, and Martínez-Sebastián 2001), the distances obtained between clusters such as A and G (fig. 1) reveal the existence of multiple subfamilies of gypsy. Pinsker et al. (2001) suggests that care should be taken in the establishment of families and subfamilies of transposable elements when only one region of the element is investigated. Alberola and De Frutos (1996), however, detected very similar percentages of divergence in the different regions of the three ORFs of complete gypsy retrotransposons of D. melanogaster, D. subobscura, and D. virilis. This finding gives support to our estimates of divergence based only in one region of the gypsy.
Therefore, we suggest that the clusters from A to G represent different subfamilies of gypsy, as their percent divergence varies from 23% to 40.8% (tables 2 and 3). The distances found between cluster H and the remaining clusters varied from 52.3% to 62.1 %, suggesting that this clade represents a distinct family of gypsy. However, more detailed analyses of other regions of gypsy are necessary to evaluate the homology of this sequence with those of other families of transposable elements related to gypsy already described in literature.
The high sequence divergence between gypsy sequences amplified from others species relative to those found in the genome of D. willistoni is more interesting still when its conservation among distant populations of this species is considered. This observation contradicts the high evolutionary rate of this gypsy strain in this genome in particular. More plausible seems to be the hypothesis of this distant origin and ancestral polymorphism followed by losses of this type of sequence in all other species, including D. paulistorum. However, we cannot discard the presence this gypsy-like sequence found in D. willistoni in other species not detected in our analysis by sampling error. Horizontal transfer involving a donor species not included in our analyses could also explain the pattern presented by D. willistoni. Nevertheless, sequences homologous to gypsy in this species, although presenting a high degree of divergence, form clusters with species phylogenetically related.
Complex Evolution
To prevent a superestimation of the horizontal transfer events we established that only one case of horizontal transfer (HT) is computed when two or more closely related species are involved in the phenomenon. In this case, to characterize the event we chose the most discrepant value in the comparison between the divergence values for gypsy and nuclear genes among the involved species. The application of this criterion was necessary because HT cases could be attributed to the low evolutionary divergence among the receipt species gypsy sequences and the sequences of the others species, related to the donor. Thus, we established as the donor species, in the group of phylogenetically related species that share gypsy sequences, the most similar one to the distant species (receipt). Therefore, we postulated nine horizontal transmission cases (fig. 2). The most prominent involved the transfer of gypsy-homologous sequences among: (1) D. nebulosa and D. neocardini, (2) D. nebulosa and D. paulistorum; (3) D. subobscura and D. busckii; (4) D. subobscura and D. hydei; (5) D. simulans and Zaprionus indianus; (6) D. simulans and S. latifasciaeformis; (7) D. pallidipennis and D. bandeirantorum; (8) D. virilis and D. hydei; (9) D. mediopicta and D. zotti. All comparisons between data of divergence of gypsy and of nuclear genes in these nine cases seem be inconsistent with vertical transmission. In several of these comparisons dN is not significantly lower than dS, excluding the possibility that very strong selection produced this high conservation after the event of horizontal transmission.
The occurrence of vertical transmission can be postulated if two criterions are followed: (1) the gypsy sequences are grouped in the phylogeny following the phylogenetic relationships of the host species, or (2) evolutionary distances among gypsy sequences are proportional to the one obtained with the nuclear genes. Examples of possible events of gypsy vertical transmission are evidenced by the evolutionary distance values between the species of the guarani group as well as some species of the tripunctata group.
The general picture depicted here suggests that there were two subfamilies of gypsy sequences in the ancestor of the genus Drosophila. One of the two can be involved in all independent horizontal transfers described here. Moreover, our results point to a recent invasion of the genomes of a large number of species by gypsy sequences.
Estimates of the Occurrence of Time of the Horizontal Transmission Events
Using a substitution rate of 1.6% per Myr estimated for nuclear genes with low preferential use of codons (Sharp and Li 1989), we estimated the time of divergence of gypsy sequences involved in the nine cases of horizontal transmission proposed in the present report. This approach had already been implemented by other authors (Alberola and De Frutos 1993b; Silva and Kidwell 2000; Loreto et al. 2001). The estimated time of divergence of gypsy in our samples are shown in figure 2, with the arrows indicating the possible cases of horizontal transmission. These values provide a good notion of the time when the invasion of the genomes by gypsy occurred, the more recent event being around 1.2 MYA and the oldest around 3.4 MYA.
Analyses of dN/dS
The proposal of recent invasion of the Drosophila genome by gypsy appears to be corroborated by the values of dN and dS. The average value of dN/dS obtained for all comparisons among the 40 sequences utilized in our analyses was 0.29 (varying around 0.03 and 2.5). These values are similar to those calculated for nuclear genes (Akashi 1994). Besides this, the selection tests performed showed that in the majority of comparisons, purifying selection is operating (or operated in a recent past) on the gypsy sequences (at the significance level of 0.05). Terzian et al. (2000) investigated gypsy in species of the subgroup melanogaster and also identified purifying selection, relating their findings to recent events of transposition. Comparing gypsy of D. melanogaster, D. virilis, and D. subobscura, Alberola and De Frutos (1996) also pointed to low dN/dS values, which implies that selection is operating on these sequences. In addition, Martínez-Sebastián et al. (2002) analyzed the evolutionary constraints of the gypsy in species of the obscura group, and their data support that gypsy sequences are subjected to purifying selection. Our data of dN and dS are comparable with those obtained for Adh (Silva and Kidwell 2000), allowing us to infer that the gypsy sequences are evolving or have evolved under strong selection in a recent past.
Different Subfamilies Coexisting in the Same Genome
Another important subject to be discussed is the existence of very divergent sequences of gypsy in the genome of one same species as observed in D. mediopicta and D. mediopunctata. For gypsy, the coexistence of more than one subfamily in a host genome has been previously reported (Alberola and De Frutos 1996; Hochstenbach et al. 1996; Terzian et al. 2000; Vázquez-Manrique et al. 2000; Martínez-Sebastián et al. 2002). The phenomenon of multiple subfamilies of TEs in the same species was observed in other elements, such as in mariner (Robertson and MacLeod 1993), P (Clark and Kidwell 1997), Minos (Arcà and Savakis 2000), or R1 (Gentile, Burke, and Eickbush 2001).
The coexistence of different subfamilies of a TE in a host genome has been given by some authors as evidence of independent invasions of a genome by a mobile element, probably via horizontal transfer (Zelentsova et al. 1999; Arcà and Savakis 2000; Silva and Kidwell 2000). Ancestral polymorphism, however, cannot be disregarded as a possible explanation. This could be the case for the divergent sequences of gypsy found in the genome of D. mediopunctata. The genetic distance calculated between gypsy of clone 45 and the sequences of D. maculifrons could be considered as compatible with the hypothesis of vertical transmission of the gypsy sequences along the evolutionary divergence of these species.
Conclusion
Several authors have suggested that horizontal transmission is an essential step in the life-cycle of some TE families (Lohe et al. 1995; Jordan and McDonald 1998; Silva and Kidwell 2000; Pinsker et al. 2001). Convincing cases of horizontal transmission were limited, until a few years ago, to elements of class II, such as P, mariner, and hobo (Daniels, Chovnick, and Boussy 1990; Simmons 1992; Clark, Maddison, and Kidwell 1994; Lohe et al. 1995). Even though evidence of this phenomenon in retrotransposons has accumulated in the past few years (Alberola and De Frutos 1996; Flavell et al. 1997; Cizeron et al. 1998; Jordan and McDonald 1998; Gonzales and Lession 1999; Jordan, Matyunina, and McDonald 1999; Terzian et al. 2000; Vázquez-Manrique et al. 2000, Martínez-Sebastián et al. 2002), horizontal transfer still seem to be more common among class I TEs (reviewed in Silva, Loreto, and Clark 2004).
The occurrence of horizontal transmission usually requires the existence of a vector, such as viruses (Friesen and Nissen 1990), parasitic mites (Houck et al. 1991), or endosymbiotic bacteria (Kidwell 1993), to shuttle the DNA between hosts. However, the evolutionary scenario of the gypsy retrotransposon is remarkable in that the several events of horizontal transfer proposed here do not require a vector. If gypsy is able to encode the envelope protein that allows it to behave like a retrovirus (Kim et al. 1994; Mejlumian et al. 2002), then the pattern observed can be attributed to the occurrence of infective waves. This would explain why horizontal transfer is more prevalent among gypsy than among other class I elements (Silva, Loreto, and Clark 2004). This capacity was already demonstrated experimentally by Mejlumian et al. (2002) for a few species other than D. melanogaster and deserves to be expanded to more species. The perspective of these results will help us to find answers regarding the role of retroviral sequences and their horizontal transmission in the evolution of eukaryotic genomes.
This work represents an attempt to describe some aspects of an extremely complex evolutionary pattern. We do not propose to stress or finish the analysis or to discard alternative models that might explain the observed evolutionary aspects. The different phenomena (vertical transmission, horizontal transmission, different evolutionary rate, ancestral polymorphism) discussed here are not mutually exclusive and probably occur simultaneously. Therefore, analysis using different regions of the element, as well as a larger number of species, is necessary to complete a more realistic picture of the evolution of the gypsy retroelement.
Supplementary Materials
All figures and tables cited as Supplementary Material are available on the journal's Web site.
Acknowledgements
This work was supported by grants from Conselho Nacional de Pesquisa, PROPESQ-UFRGS, Financiadora de Estudos e Projetos, and Funda??o de Amparo a Pesquisa do Estado do Rio Grande do Sul. We thank Daniela Cristina De Toni and Luciano Basso da Silva for fly stocks. We are grateful to Joana C. Silva and Pierre Capy for critical comments on the manuscript and two anonymous reviewers for their valuable suggestions.
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