*Interdisciplinary Program in Genetics and
Department of Ecology and Evolutionary Biology, University of Arizona
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Abstract |
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Introduction |
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Despite their pervasiveness, many unanswered questions regarding TE evolution remain. Two central questions address the relative contributions of vertical and horizontal transmission to the taxonomic distribution of TE families and the role that positive selection plays in the long-term maintenance of TE functionality. Some TE families have widespread distributions, which often extend across genus, family, and even phylum boundaries (e.g., Stacey et al. 1986
; Daniels et al. 1990
; Montchamp-Moreau et al. 1993
; Robertson et al. 1998
). The distribution of a TE family in multiple taxa can result from vertical transmission of a TE present in their common ancestor or from the TE's spread among those taxa through horizontal transfer only. These are the two opposite extremes in a continuum of scenarios that differ with regard to the relative contributions of horizontal and vertical transmission. It has been argued that horizontal transfer occurs rarely, and that the transmission of TEs is predominantly vertical (Capy et al. 1997
, p. 3). However, horizontal transfer has been proposed to be an integral part of the life cycle of some eukaryotic class II TE families, such as the mariner-like and P families (Clark et al. 1995
; Robertson and Lampe 1995
). Recently, class I elements have been unquestionably shown to transfer horizontally as well (Jordan, Matyunina, and McDonald 1999
), an event with potentially wide-ranging implications (Flavell 1999
). Presently, it is unclear how frequent a process horizontal transfer among eukaryotes really is.
Horizontal transfer has traditionally been inferred when the high degree of similarity between TE sequences is impossible to reconcile with the long divergence time of their respective host species (e.g., Daniels et al. 1990
). Other observations can help corroborate that inference, such as the incongruence between TE and host phylogenies or the absence of the TE in question from taxa closely related to that into which the TE was supposedly transferred horizontally (e.g., Clark et al. 1995
). The identification of horizontal transfer events is not straightforward, as alternative explanations are often hard to dismiss conclusively, especially when TEs from closely related taxa are compared. Such explanations include TE ancestral polymorphism coupled with independent assortment of copies into the descendant species, inequality of substitution rates in TE sequences in different species, and the stochastic loss of TEs from a few taxa (Capy et al. 1997
, pp. 130135).
There is a situation, however, in which the inference of horizontal transfer events is less problematic. By comparing the divergence of TE nucleotide sequences with those observed for host genes evolving under similar or stronger selective constraints, a horizontal transfer event can be inferred whenever the divergence among TE sequences is significantly lower than that observed for the host genes. This procedure requires a careful assessment of the selective constraints on the TEs under study, as those constraints can vary by over an order of magnitude between and within TE families (e.g., Eickbush et al. 1995
; Robertson and Lampe 1995
; McAllister and Warren 1997
; Witherspoon et al. 1997
). Here, we used this method to estimate the frequency of horizontal transfer events in the canonical subfamily of the P elements.
The P family belongs to class II, as its elements transpose directly from DNA to DNA. A complete canonical P element was first isolated from Drosophila melanogaster. This 2,907-bp element has four open reading frames (ORFs), which encode a sequence-specific DNA-binding transposase that catalyzes transposition of the element (O'Hare and Rubin 1983
). The first three ORFs are also part of a truncated transcript that encodes a transposition repressor (Misra and Rio 1990
).
The taxonomic distribution of the P family of elements is patchy and relatively restricted. These elements are most prevalent in the subgenus Sophophora of the genus Drosophila (Daniels et al. 1990
). The subgenus Sophophora consists of four major species groups: the D. melanogaster and Drosophila obscura groups, which diverged in the Old World, and the Drosophila willistoni and Drosophila saltans groups, which are restricted to the New World (Throckmorton 1975
). The 200 P-element sequences obtained to date have been organized into 16 subfamilies (Hagemann, Miller, and Pinsker 1994
; Clark and Kidwell 1997
). Each subfamily corresponds to a monophyletic group of elements obtained from species of the same species group, with the exception of monophyletic elements from the closely related saltans and willistoni species groups, which are grouped into the same subfamily (Clark and Kidwell 1997
).
With one notable exception, the canonical subfamily of P elements appears to be restricted to the sophophoran New World species groups, saltans and willistoni, two sister taxa whose common ancestor dates back at least 15 Myr (Daniels et al. 1990
; Clark and Kidwell 1997
). The exceptional canonical elements found in D. melanogaster have been explained by a recent horizontal transfer event (Daniels et al. 1990
). The canonical subfamily is composed of closely related elements with a range of divergence between 0% and 10% at the sequence level (Clark et al. 1995
). The reason for this low divergence remains unclear. It might be the result of strong selective constraints, or it could be explained by multiple horizontal transfer events occurring both within and between the two species groups.
In the present study, we assess the roles played by selection and horizontal transfer in determining the similarity among canonical P elements from the saltans and willistoni species groups. The ratio of nonsynonymous to synonymous substitutions, as well as the amount of codon bias, in P elements allows us to determine the strength of selective constraints acting on the canonical elements. We compare the divergence of the P elements with that of host genes evolving under similar or stronger constraints than the P elements to determine the number of horizontal transfer events that are necessary to explain the current taxonomic distribution of canonical P elements. The relevance of selection and horizontal transfer in the history of the P canonical subfamily is discussed in the context of the evolution and long-term survival of the P family of TEs.
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Materials and Methods |
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Sources of Data Sets
Four data sets of DNA sequences were used for this study, a P-element data set and three others which correspond to host nuclear loci: alcohol dehydrogenase (Adh), period (per), and Cu/Zn superoxide dismutase (Sod). With the exception of the Adh sequences for Scaptomyza, these data were all gathered from the literature and represent all sequence information available in GenBank for at least one species in each of the two species groups.
The P-element data set consisted of 52 P-element partial sequences obtained from the literature (see table 1
). Four were sampled from species of the genus Scaptomyza: Spallida18 and Spallida02 (Simonelig and Anxolabéhère 1991
) and Selmoi4 and Selmoi12 (unpublished data). The remaining 48 elements corresponded to the canonical subfamily described by Clark et al. (1995)
, except that we excluded Daustrosaltans22 (identical to Daustrosal21), Dlusaltans37 (identical to Dlusal34), and Dsturtevanti24, Dsturtevanti25, and Dsturtevanti26 (identical to Dsturt13). The sequence DnebulosaN10 was obtained from Lansman et al. (1987)
. The sequences were 429 nt long and mapped to positions 13281757 in ORF2 of the canonical P element (O'Hare and Rubin 1983
).
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The per data set had 39 sequences (table 1
), of which 38 belonged to the willistoni species group (Gleason and Powell 1997
) and only one belonged to the saltans species group (Peixoto et al. 1993
). The willistoni group sequences consisted of a 1,231-nt fragment mapping to amino acids 518919 in exon 5 (Citri et al. 1987
). The D. saltans sequence consisted of a 198-nt fragment mapping to amino acids 582649, with a 6-bp gap corresponding to amino acids 618619.
The Sod data set consisted of two sequences, one from D. willistoni and one from D. saltans (Kwiatowski et al. 1994a, 1994b
). The D. willistoni sequence was 879 nt long; it consisted of a 417-nt intron and two exons of 66 and 396 nt in length. The D. saltans sequence had a shorter intron (352 nt), and the first 23 nt of the first exon were missing.
The Adh data set was the most complete, with sequences for all species listed in table 1
. It was therefore used to estimate the species phylogeny and its branch lengths. All three host genes were used to estimate DNA sequence divergence between species, species subgroups, and/or species groups. The per data were unbalanced (with 38 sequences available for the willistoni group and only 1 for the saltans group), and the Sod data set was much smaller than the other two, with only one sequence available for each species group. This is reflected in the standard deviation associated with the mean number of substitutions, which takes into account sequence length, number of sequences, and phylogeny (Nei and Jin 1989
).
Sequence Analyses
Alignment of P element, Adh, and Sod sequences was done by eye and was straightforward. Sixteen alignment gaps were required in the P-element data set to compensate for the presence of indels. The per sequences were aligned as in Gleason and Powell (1997)
.
Estimation of dN and dS
The numbers of synonymous substitutions per synonymous site, dS, and nonsynonymous substitutions per nonsynonymous site, dN, were estimated using the method of Nei and Gojobori (1986)
. Standard deviations for the average dS and dN between groups were calculated as in Nei and Jin (1989)
using the program dNdSwq (obtained from Jack da Silva). In order to preserve the reading frame of the functional canonical P element, the alignment gaps introduced in the P-element data set that represented insertions relative to the canonical P element were deleted prior to estimation of dS and dN. The three termination codons found (in sequences Dsalt51, DwilliS1, and Dsuci21) were recoded as missing data. The overall dN : dS ratio was also determined using a phylogenetics-oriented approach on a subset of the most likely topologies for the canonical P-element sequences (see below). The likelihood of each topology given the data was determined under two models, one in which dN/dS was free to vary, and a submodel in which dN/dS was set to unity. A significant difference in likelihood under the two models, identified with a likelihood ratio test, indicates that a dN : dS ratio different from 1 provides a significantly better fit to the data. This analysis was done using a codon-based model of substitution (Yang, Goldman, and Friday 1994
) implemented in PAML (Yang 1998
). Because of the inability of the program to handle deletions, two sets of analyses were performed. In one, the 14 P sequences with deletions were eliminated from the data set. In the other, codon positions containing indels were eliminated.
Estimation of Codon Bias and G+C in Synonymous Sites
Two indices of codon bias were determined for each sequence: the effective number of codons (Nc; Wright 1990
) and the codon bias index (CBI; Morton 1993
). Nc varies between 21 for maximum codon bias (when only one codon is used per amino acid) and 61 for minimum codon bias (synonymous codons for each amino acid used at similar frequencies). A CBI value of 0 corresponds to no bias, and a value of 1 corresponds to maximum bias.
The frequency of G and C nucleotides in third positions in synonymous codons was determined as the proportion of C- and G-ending codons, with the exclusion of termination, methionine, and tryptophan codons.
Inferring Horizontal Transfer
Horizontal transfer can be broadly defined as the introduction of genetic material into a species from which it had been previously absent. Potential mechanisms for horizontal transfer include vector-mediated transmission and introgression (Capy et al. 1997
, pp. 141143). In this study, horizontal transfer was investigated by comparing TE divergence with that of host genes across each node of the host phylogeny. A horizontal transfer event can be inferred when the estimated TE divergence is significantly lower than that of host genes under similar or higher levels of selective constraints than those operating on the TEs themselves.
DNA sequences from three host loci were used in this study: alcohol dehydrogenase (Adh), period (per), and Cu/Zn superoxide dismutase (Sod). The ADH enzyme is one of the most abundant proteins in Drosophila, in which it is used by both larvae and adults primarily to catabolize ethanol present in the fermenting fruits in which they develop and/or feed (Sullivan, Atkinson, and Starmer 1990
). per is a pleiotropic gene that plays a major role in essential functions such as male courtship behavior and circadian rhythm (Konopka and Benzer 1971
; Kyriacou and Hall 1989
). Finally, Sod encodes an antioxidant enzyme used in the pathway that converts oxygen radicals into hydrogen peroxide and water (Lindsley and Zimm 1992
and references therein). Given the functional significance of the three genes, selective constraints are expected to be high. Conservatively, horizontal transfer was inferred only when the divergence among P sequences was lower than that observed for all host genes.
Phylogenetic Analyses
P-Element Phylogeny
Phylogenetic analyses were done using parsimony and maximum likelihood (ML). Previous analyses had suggested the presence of many most-parsimonious trees (MPTs) for the canonical P-element sequences (Clark et al. 1995
). In order to obtain an exhaustive representation of those, we performed a parsimony search with 5,000 random-addition heuristic search replicates, keeping no more than 50 trees per replicate. The log-likelihood score of all MPTs was determined using the HKY85 model of substitution (Hasegawa, Kishino, and Yano 1985
) with rate heterogeneity between sites and parameters estimated from the data.
A full ML heuristic search is precluded by the large size of the P-element data set. Therefore, trees with the highest likelihood score were obtained by doing heuristic searches on all MPTs with the highest ML scores and on five additional trees among all MPTs, selected for the greatest differences. These five trees were obtained as follows: first, tree-to-tree distances (Waterman and Smith 1978
) were used to select the two most distant trees among all MPTs. Then, the distances of all other MPTs to the first two were calculated. A third tree was then selected from among the ones for which the sum of distances to the first two was highest. The process was repeated to obtain the fourth and fifth trees. The ML heuristic search was performed with the option NOMULPARS in effect, using tree bisection-reconnection (TBR) branch swapping and the HKY85 model of substitution with rate heterogeneity between sites; the parameter values of the model were estimated for each starting tree.
Adh Phylogeny
A parsimony analysis was performed with a heuristic search of 20 random-sequence-addition replicates and TBR branch swapping. An ML analysis was performed with 20 random-addition heuristic search replicates using TBR branch swapping; the HKY85 model of substitution was used with rate heterogeneity between sites and parameters estimated from the data. During the first replicate, a topological constraint was enforced which specified monophyly of both the saltans and willistoni species groups. During the following nine replicates, the parameters used for the model of evolution were the same as those estimated in the first replicate. These parameters were re-estimated on the first tree found in replicate 10, and replicates 1120 were performed using the re-estimated values for the model parameters. The same topological constraints enforced in replicate 1 were used in replicate 2, but not in any of the following 18 replicates.
Bootstrap analyses were performed for both P and Adh data sets using parsimony. Both analyses consisted of 100 bootstrap replicates, each with 10 random-addition replicates. In the P-element analysis, a maximum of 1,000 trees was saved during each replicate. All phylogenetic analyses were performed in PAUP*, version 4.0d64 (Swofford 1998
).
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Results and Discussion |
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Estimates for dN were similar for Adh and Sod, with dN = 0.042 ± 0.0053 and 0.042 ± 0.0115, respectively, and slightly higher for per, with dN = 0.059 ± 0.0206 (fig. 1 ). The difference in the standard errors for these estimates reflects the fact that the Sod comparison was based on only two sequences and that in the per comparison, only one very short sequence (198 nt) from the saltans species group was used. Values of dS were approximately one order of magnitude higher than those for dN and were more variable among genes: dS = 0.45 ± 0.051 for Adh, 0.69 ± 0.121 for Sod, and 1.03 ± 0.246 for per (fig. 1
). These values correspond to dN : dS ratios of 0.09 for Adh and 0.06 for both Sod and per. Differences in the magnitude of dS among genes have been found to be related to the intensity of natural selection on synonymous codon usage (Shields et al. 1988
; Moriyama and Gojobori 1992
). In order to test whether this was the case for the genes under study, we determined whether the magnitude of dS between the saltans and the willistoni species groups for the three host genes was correlated with the degree of codon bias (table 2
). Adh and Sod sequences were similar to each other in degree of codon bias (Nc = 44 and 45, respectively, and CBI = 0.54 for both) and were more biased than per sequences (Nc = 57 and CBI = 0.29). These indices are correlated (fig. 2
), so only the results obtained with CBI are presented. Figure 3
shows the relationship between the average CBI in each of the three host genes and the respective dS values between species groups. The relationship observed between codon bias and number of substitutions suggests that selection on codon composition is indeed a major factor in determining the rate of substitution at synonymous sites in these three genes.
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The Intensity of Selection in P Elements
The dN : dS ratio for P elements is high when compared with that of host genes (fig. 1
). This may be due to either weak selection at nonsynonymous sites or very strong selection at synonymous sites. The low to moderate level of codon bias in the sequences of canonical P elements, indicated by an Nc value of 52 and a CBI value of 0.47 (table 2
), suggests the former. Furthermore, the nature of codon bias in canonical P elements indicates that the bias present is due to mutation bias rather than selection-induced bias, for the reasons that follow. Mutation bias in Drosophila leads to a preferential accumulation of A and T nucleotides (63%) over C and G (Shields et al. 1988
), leading to an increase in A- and T-ending codons. Conversely, selection-induced codon bias in the Sophophora subgenus produces an excess of G- and especially C-ending codons (Shields et al. 1988
; Starmer and Sullivan 1989
; Akashi 1994
). The proportion of G's and C's at synonymous positions in canonical P elements is approximately 40% (table 2
). This is very similar to the frequency expected due to genome mutation bias. It is, however, possible that the discrepancy in synonymous-base composition between P elements and host genes could be due to a pattern of mutation or selection characteristic of P elements (Lerat, Biémont, and Capy 2000
). The presence of termination codons in three of the 48 canonical elements and the presence of deletions that unambiguously disrupt the reading frame in eight of those elements further support a scenario of weak selection acting on P elements.
Comparison of dN and dS values between canonical P elements and host genes (fig. 1
) estimated between species groups reveals that the average dN for P elements is similar to that observed for host genes. Also, the average value of dS observed for P elements is 5 to 10 times as small as the dS observed for host genes. Given our above conclusion of reduced selective constraints acting on P, the smaller value of dS for P elements than for host genes is unexpected. As we argued above, the small number of synonymous substitutions in P is not due to selective pressure on codon usage. Selection-induced codon bias is clearly higher in both Adh and Sod than in the P element. Another possible explanation for the small number of substitutions in P elements is that dS between species groups is larger in host genes because of a radical shift in codon preference between species groups in these genes, but not in P elements. Shifts in codon bias between the subgenera Drosophila and Sophophora, and well as between D. melanogaster and D. willistoni (subgenus Sophophora), have been documented (Starmer and Sullivan 1989
; Anderson, Carew, and Powell 1993
). If associated with strong selection on codon usage in host genes, different codon preferences in the two species groups might be radical enough to cause a large increase in dS. Under this hypothesis, host genes in which codon usage is under stronger selective pressure should have experienced the strongest shift, and a positive correlation between degree of codon bias and number of substitutions (as measured by dS) would be expected. Figure 3
shows that this is not the case. dS is negatively correlated with CBI, therefore providing evidence to reject this second explanation.
Selection Versus Recent Horizontal Transfer
An alternative explanation for the low level of divergence among canonical P sequences in the two species groups is that these elements shared a common ancestor more recently than did their host species. In other words, P elements have been transferred horizontally between species groups more recently than these species groups have shared a common ancestor. In this section, we formulate the predictions that allow us to distinguish between selection and horizontal transfer as possible explanations for the high similarity among all canonical P elements.
Selection (of an ad hoc nature) and recent horizontal transfer have very different consequences both for the level of congruence expected between the P-element and host phylogenies and for the proportionality of P-element divergence relative to host gene divergence, when sister groups of different ages are compared. Namely, if the low P-element divergence resulted from selection and the elements were transmitted vertically, TE and host phylogenies are expected to be congruent. In addition, P-element divergence and host gene divergence should be correlated. If, on the other hand, recent horizontal transfer caused the low P-element divergence, then the P-element phylogeny is not necessarily expected to be congruent with that of the host species. Also, P-element and host gene divergence should not be correlated. When the taxa compared have diverged recently, P-element divergence should be high relative to the divergence of host genes evolving under strong selective constraints. As we compare taxa that are progressively older, P-element divergence should become smaller in relation to the divergence of host genes. These predictions are investigated in the following two sections.
P-Element Versus Host Gene Phylogenies
Host Phylogeny
The phylogenetic relationships within the saltans and willistoni species groups have been studied before (Bicudo 1973
; Throckmorton 1975
; Gleason, Griffith, and Powell 1998
; O'Grady 1998
; O'Grady, Clark, and Kidwell 1998
). We analyzed the phylogeny of the Adh sequences to assess whether the relationships among them reflect those previously determined for the species and to obtain branch length information with which to compare similar information obtained for the P-element sequences. Our parsimony analysis yielded seven MPTs, which were found in all 20 heuristic search replicates. All of these trees had higher likelihood scores than those resulting from the original ML search and were therefore used as the initial trees in another ML search. Three trees resulted from this search, which correspond to the three possible resolutions of the polytomy that includes D. austrosaltans, D. lusaltans, and D. saltans (in all three cases, the internal branch has length 0); the three trees were otherwise identical (fig. 4a
). These results agree with previous hypotheses concerning the relationships in the saltans group (Bicudo 1973
; Throckmorton 1975
; O'Grady, Clark, and Kidwell 1998
), with one exception. The position of the elliptica and neocordata subgroups is unusual in that they are believed to be the most anciently derived subgroups in the saltans group (Throckmorton 1975
). The relationships within the willistoni group are also in agreement with previous analyses of the group (Gleason, Griffith, and Powell 1998
), with two exceptions. The willistoni species group is paraphyletic with respect to the saltans group due to the position of D. nebulosa, and the willistoni subgroup is paraphyletic with respect to D. fumipennis. These discrepancies do not affect our results concerning P-element evolution.
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One tree selected arbitrarily among the 24 with the highest log-likelihood score is presented in figure 4b. In the canonical clade, most DNA mutations occur within 13 clades identified in the bootstrap analysis. In the deeper part of the tree, branch lengths are small and the structure of the tree is largely unresolved.
These phylogenetic analyses yield two main conclusions. First, P-element and species phylogenies are not congruent (fig. 4 ). Second, the fact that in the P-element phylogeny so many of the deeper branches (above species level) have length 0 suggests that the transmission of P elements among species occurred rapidly, preventing the accumulation of substitutions in the early stages of the diversification of this P-element subfamily. Both conclusions are fully compatible with the scenario of recent horizontal transfer of P elements into the two Sophophoran species groups but are improbable under a scenario of vertical transmission according to which P elements were present in the common ancestor of all species and evolved under strong selective constraints.
P-Element Versus Host Gene Divergence
P-element and host gene divergences between pairs of taxa were estimated to determine if P elements are at least as divergent as host genes and if those divergences are correlated.
Drosophila willistoni Species Group
Figure 6a
shows the results of a comparison of the willistoni subgroup P elements with all others from the willistoni group. dN for P (0.05) lies between the values for host genes (dN = 0.08 for per and 0.04 for Adh). dS, however, is much smaller for P (0.13) than for per (0.62) or Adh (0.54). Similar comparisons were performed across progressively younger nodes of the species tree (see fig. 4a
): D. tropicalis versus its sister clade (fig. 6b
), D. willistoni versus its sister clade (fig. 6c
), D. equinoxialis versus its sister clade (fig. 6d
), and, finally, between D. paulistorum and D. pavlovskiana (fig. 6e
). As expected, host gene divergence decreases with the age of the node. P-element divergence, on the other hand, not only remains approximately constant in the first three comparisons, but also is always smaller than that of host genes (fig. 6bd
). This relationship changes when D. paulistorum and D. pavlovskiana are compared. The divergence between the two pairs of closely related elements (Dpauli13-Dpavlo16 and Dpauli10-Dpavlo21) is now intermediate between that of the two host genes (fig. 6e
). These results are contrary to the expectations under vertical transmission and constant selective pressure operating on these sequences.
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The same rationale was used to interpret the comparison between D. sucinea and D. capricorni. The most closely related elements in the two species were compared (Dcapri15 and Dsuci1). The divergence of the P elements is intermediate between that of each of the two host genes (fig. 6f ). Therefore, we conclude that P elements could have been transmitted vertically to these two species from their common ancestor. The lineages leading to D. nebulosa and D. fumipennis were present before the introduction of canonical elements into the willistoni group, which, according to our results, took place at the time of the divergence of D. pavlovskiana from D. paulistorum and that of D. capricorni from D. sucinea (fig. 4 ). In summary, the distribution of P elements within the willistoni species group can be explained by seven horizontal transfer events, one into D. nebulosa, one into D. fumipennis, one into the ancestor of D. sucinea and D. capricorni, and four within the willistoni subgroup.
These results rule out the possibility that the monophyly of the P elements from the willistoni subgroup was due to the presence of canonical elements in the ancestor of the subgroup. The observation that some of the crosses between species within the willistoni subgroup produce fertile offspring (reviewed by Bock 1984
) indicates that gene flow between these species could have been present until fairly recently. This suggests introgression as a possible horizontal transfer mechanism for the spread of P elements among the species in the willistoni subgroup, and as the reason why these elements form a monophyletic group.
Drosophila saltans Species Group
Similar analyses were performed on P elements in the saltans group. First, we compared the elements from D. sturtevanti with those from D. saltans and D. subsaltans (fig. 7a
). Then, the two elements from D. subsaltans and Dsalt28 from D. saltans were compared (fig. 7b
). As P elements were less divergent than the host gene in both comparisons, we conclude that P elements have been transmitted horizontally between the three species subgroups in question. We further compared the four species in the saltans subgroup, D. saltans, D. prosaltans, D. austrosaltans, and D. lusaltans (fig. 7c and d
). In both pairwise comparisons performed, P element divergence was higher than that observed for Adh, suggesting that canonical elements might have been present in the saltans subgroup prior to its divergence. In conclusion, at least three instances of horizontal transfer are detectable in the saltans group: one into the sturtevanti subgroup, one into the parasaltans subgroup, and one into the ancestor of the saltans subgroup. Finally, the close similarity of D. austrosaltans elements to two others from D. fumipennis is clearly the result of horizontal transfer between those species (fig. 8
).
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Age of the Most Recent Common Ancestor of Canonical P Elements
We determined the age of the most recent common ancestor of all canonical P elements (MRCA-P) to assess if the age of the subfamily was consistent with the conclusion of the previous section. The age of the MRCA-P was determined in three ways: (1) by calibrating dS between P elements with the rate of synonymous substitution estimated for Drosophila genes with low codon bias, (2) by calibrating dS between P elements with the rate of synonymous substitution in the R1 and R2 TE families, and (3) by examining the relationship between codon bias and dS. The results of these three methods are as follows:
These three estimates, even though not completely independent (they all depend in part on the number of substitutions in the Adh gene), are very similar to one another. All point to an age for the MRCA-P of approximately 23 Myr, an estimate in very good agreement with the conclusion of the previous section. The willistoni subgroup originated between 8 and 12 MYA (Clark and Kidwell 1997
). This date is consistent with the spread of canonical elements through this subgroup by horizontal transfer within the last 13 Myr, as most extant species in the subgroup were likely present by the time the MRCA-P invaded these taxa. The saltans subgroup is very young (O'Grady, Clark, and Kidwell 1998
), and so it is conceivable that its diversification could have occurred subsequent to the invasion by the MRCA-P.
Divergence Between P-Element Subfamilies
Autonomous elements, defined as those capable of catalyzing transposition, are known in two P-element subfamilies: the canonical subfamily and that containing the Scaptomyza elements (table 1 ; O'Hare and Rubin 1983
; Simonelig and Anxolabéhère 1991
). We estimated dN and dS between these two subfamilies to determine the strength of the selective constraints in intersubfamily divergence. The values obtained were dN = 0.195 and dS = 1.25 (fig. 9
), for a dN/dS ratio of 0.16. This ratio is probably overestimated, given that synonymous sites are saturated. This ratio is much smaller than that observed within both the canonical subfamily (0.75 > dN/dS > 0.42) and the subfamily of Scaptomyza elements (dN/dS = 0.26). dN and dS were also estimated between the canonical subfamily and other more divergent subfamilies of P elements present in the saltans and willistoni subgroups. Even though the divergence between these and the canonical subfamily was too large to allow accurate estimation of dS for all pairwise comparisons of elements between subfamilies, the comparisons that were possible suggest that dS is again about one order of magnitude higher than dN (results not shown). These results indicate that the divergence between Pelement subfamilies is characterized by stronger constraints in nonsynonymous sites than those observed in the divergence between closely related elements.
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Conclusions |
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While horizontal transfer clearly plays a major part in the evolution of the canonical subfamily, the role of selection is not as easy to ascertain. Our results show that when canonical P elements are compared among species groups, the dN/dS ratio is approximately 0.45. However, when this ratio is estimated over the entire canonical P-element phylogeny, it increases to 0.75 (and is not significantly different from 0). This suggests that the evolution of P elements in younger branches of the P-element phylogeny, namely among and within species, is characterized by weaker selective constraints in nonsynonymous sites than those in older branches. This was confirmed by estimating the average dN/dS ratio within the 10 clades formed by elements from a single species. dN/dS in these intraspecific comparisons varied between 0.39 and 2.9, revealing a general lack of constraints in nonsynonymous sites. These results contrast sharply with those observed when subfamilies of P elements are compared; the dN/dS ratio among subfamilies is one order of magnitude smaller, revealing strong selective constraints in the evolution of P-element sequences when the elements compared are only distantly related.
Several questions are raised. First, is there a relationship between the high rate of horizontal transfer observed for the canonical subfamily and the "patchy" distribution of P elements in the saltans and willistoni species groups? Second, what is the origin of the canonical P elements, given that their presence in the New World Sophophora groups is so recent? Third, why do selective constraints, as reflected in the dN/dS ratio, vary through different levels of P-element divergence? And finally, how do the scenarios of frequent horizontal transfer and low selective constraints fit into the life cycle of the P family? These questions will be addressed in the following sections.
Horizontal Transfer and the Distribution of P Elements
The distribution of P elements in the subgenus Sophophora is not homogeneous, but "patchy"; while some species lack P elements altogether, others seem to host multiple P subfamilies (Clark and Kidwell 1997
).
Within the willistoni species group, D. insularis, a species restricted to a few islands in the Lesser Antilles (Spassky et al. 1971
), has no canonical P elements (Daniels and Strausbaugh 1986
; Clark et al. 1995
). Daniels et al. (1990)
proposed that this could be due to the spread of canonical P elements by horizontal transfer after the geographical isolation of D. insularis was established. Alternatively, Clark et al. (1995)
proposed that these elements could have been lost following the geographic isolation of this species. Our results provide support for the first hypothesis. Drosophila insularis is the oldest species in the willistoni subgroup (Gleason, Griffith, and Powell 1998
), which is 812 Myr old (Clark and Kidwell 1997
). Hence, it seems more plausible that this species was already formed when the canonical P element spread took place 23 MYA and that its geographical isolation shielded the species from invasion.
In the elliptica and cordata subgroups, represented here by D. emarginata and D. neocordata, all species surveyed are devoid of any discernible P elements (Daniels et al. 1990
; Clark et al. 1995
). These two subgroups, thought to be the most anciently derived within the saltans group, are believed to have diversified in North America (Throckmorton 1975
). Meanwhile, and prior to the formation of the Isthmus of Panama, an ancestor of the sturtevanti, parasaltans, and saltans subgroups crossed to South America, where these subgroups later diversified (Throckmorton 1975
). Daniels et al. (1990)
suggested that P elements could have spread throughout the saltans and willistoni groups in South America, at a time when the elliptica and cordata subgroups were still geographically isolated in North America. Alternatively, Clark et al. (1995)
proposed that P elements could have been lost from the elliptica and cordata subgroups early in the history of the saltans group, when those two subgroups were isolated from the lineages leading to the more recently derived saltans subgroups. However, we now know that the spread of canonical P elements within the group took place within the last 3 Myr, at a time when North and South America were no longer disconnected. The current distributions of the elliptica and cordata subgroups through Central and South America overlap those of the other saltans and willistoni subgroups (Patterson and Mainland 1944
; Magalhães 1962
; Spassky et al. 1971
). Therefore, the absence of canonical P elements in the elliptica and cordata subgroups does not seem to be due to their geographical isolation. An alternative hypothesis is that there might be host factors in the elliptica and cordata lineages that confer resistance to P-element invasion. For example, recent simulation studies show that the inability of a species to cope with the type of mutations induced by transposition could prevent P-element invasion (Quesneville and Anxolabéhère 1997
). This hypothesis, which can explain the complete absence of P-homologous sequences in these species, could potentially be tested with transformation studies.
Our analyses suggest that the patchy taxonomic distribution of canonical P elements is due not to the loss of vertically transmitted elements, but to their spread by horizontal transfer. A few other instances of horizontal transfer involving P elements have already been documented (Clark, Maddison, and Kidwell 1994
; Hagemann, Haring, and Pinsker 1996
; Clark and Kidwell 1997
; Loreto et al. 1998
), which suggests that this phenomenon is present in other taxa carrying P elements. Also, preliminary analyses of additional P subfamilies present in the saltans and willistoni groups indicate that horizontal transfer is common in other P subfamilies (unpublished data). Detailed analyses, similar to the ones performed here for the canonical elements, of all P subfamilies could be done to ascertain if the history of the canonical subfamily is representative of that of the whole P family.
Where Did Canonical P Elements Come From?
This question has been previously asked, in the context of the recent introduction of canonical P elements into D. melanogaster (Engels 1989
). The same question resurfaces now that we know that the invasion of the willistoni and saltans groups by canonical P elements, even though earlier than that of D. melanogaster, was still fairly recent. A detailed phylogenetic analysis of P-element sequences in the subgenus Sophophora places the clade of canonical elements as the sister group to all other subfamilies of Drosophila and Scaptomyza P elements (Clark and Kidwell 1997
). In addition to the canonical subfamily, that study identified three other P subfamilies in species of the saltans and willistoni groups. The four subfamilies form distantly related clades, as indicated by mutational saturation at third codon positions when elements in different subfamilies are compared (unpublished data). However, within P subfamilies, elements sampled from different species, and even species groups, are often very closely related (unpublished data). The discreteness of these subfamilies coexisting in the same genome provides a strong indication that the presence of multiple P subfamilies in the saltans and willistoni species groups is the result of multiple waves of horizontal transfer events. This further emphasizes the relevance of this mode of transmission in the evolution of the P TE family. The source of these waves of horizontal transfer is unknown. It is possible that lineages leading to each subfamily were present but dormant in a New World sophophoran species and at some point spread through the two species groups. Alternatively, a reservoir may exist in another Drosophila species or perhaps even in a different taxonomic group. This could potentially be investigated by PCR analysis using pools of DNA from multiple species. These species could be sampled among the species that have easy access to rotting fruits in which the eggs of these Drosophila species are laid, such as soil organisms, nematodes, or other fruit-feeding dipterans.
Selection in the P Family of TEs
Five mechanisms through which selection can be effective on TE sequences have recently been summarized (Witherspoon 1999
): selection on TEs is expected if (1) transposition increases host fitness, (2) transcripts from functional TEs increase host fitness, (3) functional elements transpose at a higher frequency within a genome, (4) functional elements transpose at a higher frequency within a population, and/or (5) only functional elements can spread by horizontal transfer. According to the first four mechanisms, one would expect selection to be effective on TE intraspecific evolution. However, if selection operates only at the time of horizontal transfer, as described by the fifth mechanism, selective constraints should be detected only when elements from different species are compared, as horizontal transfer between taxa creates a sieve through which functional elements pass more easily than nonfunctional elements.
Our results show that the dN/dS ratio in intraspecific P-element comparisons does not differ significantly from 1 but that this ratio decreases for interspecific P comparisons (0.4 < dN/dS < 1), providing strong support for the fifth selection mechanism. The fact that it is even smaller (dN/dS < 0.2) in intersubfamily P comparisons is also easily explained: lineages leading to distantly related elements have survived for longer periods of evolutionary time, with the concurrent accrual of mutations at synonymous sites. These elements would likely have survived through further rounds of horizontal transfer, which would have imposed selective constraints in nonsynonymous positions. Consequently, the dN/dS ratio among elements of different subfamilies is expected to be lower than that observed for comparisons among the closely related elements that compose a subfamily.
Long-Term Maintenance, Horizontal Transfer, and the Life Cycle of P Elements
The life cycle of P elements is similar to that described for members of other class II TE families, such as the mariner-like elements (Engels 1989
; Hartl et al. 1997
). This cycle starts with the invasion of a naive host, a rapid increase in copy number, the spread through the host population, and the eventual stabilization of copy number once repression of transposition arises. The frequency of functional elements then decreases, as functional copies are lost through mutation, excision, drift, and selection. Extinction is the eventual fate of TE systems such as P, in which the transposition of full-length, functional elements can give rise to both functional and nonfunctional elements; nonfunctional elements can also transpose, and transposition is a negative function of copy number (Kaplan, Darden, and Langley 1985
). Horizontal transfer of a functional element into a naive host, with its concomitant high rates of unrepressed transposition, provides the source for a large number of functional copies and a chance to escape extinction (Lohe et al. 1995
; Robertson and Lampe 1995
). Overall, our results, together with those of Witherspoon (1999)
, provide strong empirical evidence that horizontal transfer is the main, and maybe the unique, source of selective constraint on the P-element transposase sequence as a whole, at least in the hosts surveyed to date. Whether this selection mechanism is sufficient to have maintained the P-element family for over 40 Myr is unknown. The existence of a reservoir species is possible in which selection acts during intraspecific evolution of P elements. Simulation studies are needed to address this question from a theoretical standpoint. At the same time, surveys of the genomes of species that share the habitat of sophophoran drosophilids are needed to assay for the presence of canonical P elements.
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Footnotes |
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1 Abbreviations: ML, maximum likelihood; MPT, most-parsimonious tree; MRCA-P, most recent common ancestor of canonical P elements; ORF, open reading frame; TBR, tree bisection-reconnection; TE, transposable element.
2 Keywords: P element
horizontal transfer
selection on transposable elements
Drosophila
Sophophora
3 Address for correspondence and reprints: Joana Silva, Genetics Program, BioSciences West #310, University of Arizona, Tucson, Arizona 85721. E-mail: joana{at}u.arizona.edu
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