*Department of Biology
NERC Centre for Population Biology, Imperial College, Silwood Park, Ascot, Berkshire, U.K
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Abstract |
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Introduction |
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If fixation is followed by degeneration, then the only way for VDE to persist as a HEG over long evolutionary time spans would be to occasionally move to a new location, on average at least once before degeneration. In principle, movement might be to a new place in the same genome (transposition) or to a different species (horizontal transmission). For mitochondrial HEGs associated with self-splicing introns, horizontal transmission is much more common than transposition (Cho et al. 1998
; Goddard and Burt 1999
). In no case is the mechanism of horizontal transmission known, and unlike transposable elements, there is no extrachromosomal phase in the normal propagation of a HEG which might facilitate horizontal transmission. For yeasts, perhaps the simplest scenario is that there is occasionally some leakage of mtDNA during the preliminary phase of interspecific matings that are subsequently aborted. Such direct transfer might be more difficult for a nuclear HEG, as it would seem to require full nuclear fusion, and hybrids of even close relatives are usually sterile (Naumov 1987
; Marinoni et al. 1999
). It is for these reasons that the study of a nuclear HEG is of interest. Furthermore, note that, whatever the mechanism of crossing the species barrier, a key prerequisite for the successful transfer and spread of a HEG is that the recognition sequence exists in the recipient species. If the ability to undergo horizontal transmission is an important factor in the persistence of a HEG, then one expects there to have been selection against HEGs with poorly conserved recognition sequences. VDE, which has persisted, should therefore have an unusually well-conserved recognition sequence.
To test whether VDE shows horizontal transmission, we surveyed 22 species of saccharomycete yeasts from four closely related genera (Saccharomyces, Torulaspora, Zygosaccharomyces, and Kluyveromyces) for the presence or absence of VDE in the VMA1 gene or elsewhere in the genome and combined our results with published data from two more distantly related yeasts, Candida tropicalis (Gu et al. 1993
) and C. albicans (Stanford DNA Sequencing and Technology Centre; http://www-sequence.Stanford.edu/group/candida). We then analyzed the distribution of VDE in the 24 species and compared the phylogenetic relationships among all VDEs recovered with those of the host species. To examine whether VDE might be specifically adapted for horizontal transmission, we tested whether the VDE recognition site is an unusually well-conserved region of the genome.
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Materials and Methods |
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Phylogenetic Analyses
DNA sequences were translated using the ABI Sequence Navigator program and amino acid sequences aligned with Clustal W (Thompson, Higgins, and Gibson 1994
). To reconstruct the host phylogeny, nuclear 18S, 26S, and ITS1-5.8S-ITS2 data were obtained from GenBank (Cai, Roberts, and Collins 1996
; James et al. 1997
; Kurtzman and Robnet 1998
; Goddard and Burt 1999
). Sequence alignments can be obtained from the authors. Phylogenetic analyses were performed with PAUP* (Version 4.0d64) (Swofford 1999
), MacClade (Version 3.07) (Maddison and Maddison 1992
), MultiLocus (Version 1.0b; http://www.bio.ic.ac.uk/evolve/software/multilocus), and Mathematica (Version 3.0.1) (Wolfram 1996
).
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Results and Discussion |
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Horizontal Transmission
The distribution of VDE in the Saccharomycetaceae appears to be random, without significant clustering along specific clades, indicating multiple independent gains or losses (or both) (fig. 1 ). Significant clustering of states on the tree would have suggested that gains and losses are relatively infrequent events. The most parsimonious character reconstruction for the observed distribution of VDE on the host tree infers eight steps and is no shorter than the length obtained when presence/absence is randomized on the tree (P = 0.66, from 100 randomizations). We also failed to find significant clustering if gains or losses are modeled as irreversible (P = 0.94 and 0.59, from 100 randomizations, respectively). To estimate the rates of VDE gain and loss, we used a maximum likelihood approach, combining information from the observed distribution of VDE and the maximum parsimony host tree (fig. 1
; Pagel 1994
). Branch lengths were estimated by maximum likelihood while enforcing a molecular clock (Swofford 1999
) and were calibrated following Berbee and Taylor (1993)
. The fit of a two-parameter model, in which rates of gain and loss are allowed to differ, is not significantly better than the one-parameter model with equal rates (
lnL = 0.33, df = 1, NS [not significant]) but is significantly better than unidirectional models in which only gains or only losses are allowed (
lnL = 19.46 and 16.62, respectively, df = 1, P < 0.001). Therefore, to estimate the rates of gain and loss we fitted a one-parameter model with equal rates. The estimate maximizing the likelihood of the data is
(infinity), with a lower bound of 0.17 events per million years (from the 2-unit support limit, i.e., the value at which the lnL is 2 units less than the maximum, which is equivalent to the 95% confidence limit; Edwards 1992
; note that this does not take into account error in the phylogeny; fig. 1
). This is equivalent to a maximum average waiting time of 6 Myr for each transition. As the total time represented in the entire phylogeny is estimated to be 766 Myr, this implies at least 128 gains and losses in the ancestry of our 24 species. Note this last estimate is independent of the temporal calibration of the host tree. It indicates that VDE has been an extremely dynamic element, continuously moving in and out of species throughout its evolutionary history.
A history of horizontal transmission should mean that the phylogenies of host and VDE are significantly different. Before testing this prediction, we first determined whether the self-splicing and endonuclease domains of VDE themselves have different phylogenetic histories (Dalgaard et al. 1997a
). Separate phylogenies for the two domains have no well-supported branches (i.e., with bootstrap values >70%) that are incompatible between trees (not shown), and the data sets do not differ by the partition homogeneity test (P = 0.11; Farris et al. 1995
). However, the host and VDE phylogenies are strikingly different (fig. 2
). First, each tree has well-supported branches that are incompatible with the other tree. Second, each of the two data sets fits its own tree significantly better than the alternative tree (winning sites test; 83 of 95 sites, P < 0.0001, and 63 of 71 sites, P < 0.0001, for host and VDE data sets, respectively; Swofford 1999
). Finally, the two data sets are significantly different by the partition-homogeneity test (P << 0.001; fig. 2
; Farris et al. 1995
; Swofford 1999
), despite having similar amounts of homoplasy (consistency index [CI] = Lmin/Lshortest: 0.73 = 475/649 and 0.78 = 472/605, for the host and VDE trees, respectively, with character changes distributed over 153 and 126 informative sites). These incompatibilities provide compelling evidence for horizontal transmission of VDE across yeast species. An absolute lower bound on the number of horizontal transmission events can be obtained by calculating how many branches of one tree have to be cut off and reconnected in a new position to transform it into the other tree (Hein 1993
). Starting with the better resolved VDE tree, it takes four such rearrangements to get the host tree or three rearrangements to get a tree that is not significantly worse than the best host tree (table 1
). However, such an approach is likely to substantially underestimate the actual number of rearrangements (Goddard and Burt 1999
).
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Adaptation for Horizontal Transfer
Three lines of evidence indicate that the recognition sequence of VDE is particularly well conserved. First, the host gene, VMA1, is unusually well conserved among yeasts. Complete or virtually complete genomes are now available for two yeasts, S. cerevisiae and C. albicans, and we used these to compute an average amino acid divergence of genes: 20 genes of known function were chosen randomly from the S. cerevisiae genome directory (Goffeau et al. 1997
) and used as queries to search the C. albicans genome data base (Stanford DNA sequencing and technology centre; http://www-sequence.Stanford.edu/group/candida). VMA1 is 12% divergent, significantly better conserved than random genes in the two species (one-tailed t-test on arcsine-transformed values, t = 2.76, P < 0.01; fig. 3a
). Second, an alignment of VMA1 genes from six ascomycete fungi shows that the 31-bp recognition sequence of VDE is significantly better conserved than random 31-bp regions within the gene (P = 0.03; fig. 3b
). This last result could be due in part to the homogenization of the recognition sequence, through coconversion of flanking regions during homing (Dujon 1989
; Cho et al. 1998
), though the high divergence of VDE among species suggests that this cannot be a strong effect. Finally, within VDEs 31-bp recognition sequence, there are nine sites which have been identified by site-directed mutagenesis as being critical for effective homing (Gimble and Wang 1996
). Our VMA1 sequences include the recognition site for all 24 species, and these nine sites are perfectly conserved in all 24 species and are significantly better conserved than the other 22 sites (G = 9.83, P < 0.005, fig. 3c
). These results indicate that VDE has evolved to use as its homing site a region that is very highly conserved and thus likely to be present in a large variety of species.
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Conclusions |
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If horizontal transmission is critical in determining the distribution of HEGs, then within taxa there ought to be selection among HEGs for those that are better able to transfer successfully, which in turn would produce adaptation for horizontal transmission. For transfer to be successful (i.e., the transferred gene spreads through the recipient population), the recipient population must contain the HEG recognition site. Therefore, HEGs with highly conserved recognition sequences should transfer successfully more frequently and become more widespread. This line of reasoning suggests that extant HEGs should target unusually well-conserved regions of the genome. The fact that VDE is in a gene instead of free standing in noncoding DNA supports this idea, but our analyses go further, indicating that VDE is in an unusually well-conserved region of an unusually well-conserved gene. This positioning presumably reflects selection among HEGs targeting different regions of the genome. Such selection probably also explains why HEGs are typically found in functionally important parts of genes (Garret et al. 1991
; Dalgaard et al. 1997b
; Edgell, Belfort, and Shub 2000
). Moreover, within the recognition site, VDE targets particularly well-conserved base pairs, and presumably this adaptation arose and is maintained by selection among variant VDEs at the same site, favoring those with increased binding affinity for highly-conserved nonsynonymous sites. These features of VDE biology we take to be adaptations for successful horizontal transmission and evidence that horizontal transmission is a key parameter in the distribution of these selfish genes. There are no adaptations for horizontal transmission, of which we are aware, other than the operon structure of prokaryotic genomes (Lawrence and Roth 1996
).
The mechanism of horizontal transfer is unclear as infectious viruses and plasmids appear to be absent from yeasts. Possibilities include interspecific hybridization (Marinoni et al. 1999
), vectoring by predacious yeasts (Lachance and Pang 1997
), and uptake of naked DNA from the environment (Nevoigt, Fassbender, and Stahl 2000)
. Once inside the cell, fragments of DNA are readily integrated into the yeast genome by homologous recombination. Note that this last step could also select for HEGs in well-conserved regions of the genome. Also unclear is the mechanism of intein loss, as imprecise deletions would disrupt the host gene and be highly deleterious.
More generally, the results also show that horizontal transmission in yeasts is not restricted to mitochondrial DNA but that nuclear DNA is also transferred intact from one species to another. HEGs have no extrachromosomal phase in their life cycle (unlike, say, transposable elements), and therefore normal host-benefiting genes probably also get transferred occasionally. The extent to which these show evidence of past horizontal transmission should then be determined by selection, rather than opportunity. Some degree of horizontal transmission of nuclear genes has been shown for filamentous fungi (Rosewich and Kistler 2000)
. For yeasts we are not yet aware of any compelling examples, but interestingly some ecologically contingent genes occur in clusters, for example, those involved in biotin synthesis (Phalip et al. 1999
), galactose metabolism (Greger and Proudfoot 1998
), and arsenic tolerance (Bobrowicz et al. 1997
). By analogy with prokaryotes, these would be excellent candidates for horizontal transmission.
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Acknowledgements |
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Footnotes |
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Abbreviations: VDE, VMA1-derived endonuclease; HEG, homing endonuclease gene; VMA1, vacuolar membrane H+-ATPase.
Keywords: horizontal (lateral) transfer (transmission)
homing endonuclease
adaptation
yeast
selfish gene
VDE
distribution
Address for correspondence and reprints: Vassiliki Koufopanou, Department of Biology, Imperial College, Silwood Park, Ascot, Berkshire SL5 7PY, UK. v.koufopanou{at}ic.ac.uk
.
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