*Physiological Chemistry I, Biocenter, University of Würzburg, Würzburg, Germany;
Department of Biology, University of Konstanz, Konstanz, Germany
The fish nonlong-terminal-repeat (non-LTR) retrotransposon Rex3 has recently been isolated from the platyfish Xiphophorus maculatus (Volff et al. 1999
). Complete versions of Rex3 encode a reverse transcriptase (RT) and an apurinic/apyrimidinic endonuclease (fig. 1
). Rex3 belongs to the RTE family of non-LTR retrotransposons (Malik and Eickbush 1998
; Volff et al. 1999
). From all autonomous fish retrotransposons reported to date, Rex3 has the widest distribution observed in teleosts and is present in fish species having diverged 150200 MYA. We report here a large PCR- and Southern blotbased survey of Rex3 evolution including 21 representative teleost species (fig. 1
) and 115 Rex3 partial reverse transcriptase sequences (fig. 2
). The species chosen include a panel of economically important fishes (salmon Salmo salar, trout Oncorhynchus mykiss, carp Cyprinus carpio, sturgeon Acipenser sturio, mandarin fish Siniperca chuatsi) and several small aquarium teleosts used as models for developmental biology, cancer research and evolutionary studies (zebrafish Danio rerio, medakafish Oryzias latipes, platyfish Xiphophorus maculatus, and other Poeciliidae), as well as the genome project fish, the Japanese pufferfish Fugu rubripes.
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There are about 1,000 Rex3 copies in the haploid genome of Xiphophorus species (Volff et al. 1999
). All other Poeciliidae species included in this study and the related Fundulus displayed a high level of Rex3 reiteration as well (data not shown). Rex3 is present in high copy numbers in the genomes of O. latipes, Oreochromis niloticus (Nile tilapia), Batrachocottus baikalensis, A. anguilla (Volff et al. 1999
), Esox lucius (pike), Cichlasoma labridens, and Siniperca chuatsi (not shown). The Rex3 copy numbers per haploid genome were estimated by quantitative slot blot as described (Volff et al. 1999
) and found to be approximately 50 for the carp C. carpio and 500 for the related zebrafish D. rerio using carp- and zebrafish-specific probes, respectively (data not shown).
A total of 115 unique sequences covering a common 420-nt part of the RT-encoding domain were obtained for phylogenetic analysis (fig. 2
), including three Xiphophorus sequences isolated from a genomic cosmid library (Volff et al. 1999
; AF125981AF125983), one database sequence from an intron of the membrane guanylyl cyclase gene of the medakafish O. latipes (AB021490), and one database sequence from the immunoglobulin heavy-chain gene cluster of the pufferfish F. rubripes (AF108422), as well as several sequences from the F. rubripes genome project (Elgar et al. 1996
; http://fugu.hgmp.mrc.ac.uk). According to morphological and molecular fish phylogenies (Nelson 1994
; Forey et al. 1996
; Orti and Meyer 1996
), Rex3 A. anguilla sequences were chosen as the outgroup in phylogenetic analysis because they were the most divergent elements.
In most cases, one sequence was more related to the other sequences from the same fish species than to sequences from other species. This showed the occurrence of numerous independent bursts of retrotransposition from distinct master copies during teleost genome evolution. Different waves of retrotransposition were detected even between different members of the same family, as observed, for example, between O. niloticus (an Old world cichlid) and C. labridens (a New world cichlid). In comparisons between phylogenetic groups of sequences having different last common ancestors, the rate of substitutions between Rex3 RT genes was clearly higher at synonymous sites (Ks) than at nonsynonymous sites (Ka) (table 1
). This indicated purifying selection maintaining Rex3 RT activity and again suggested frequent retrotransposition of Rex3 during fish evolution. The Ks/Ka ratio was closer to unity in comparisons between closely related sequences. This indicated that Rex3 elements were first influenced by pseudogene-like evolution after retrotransposition, as observed for other retroelements (McAllister and Werren 1997
).
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The phylogeny of Rex3 (fig. 2
) diverges significantly from the classical fish phylogeny (P < 0.0001, Kishino-Hasegawa test; Kishino and Hasegawa 1989
). In particular, D. rerio, C. carpio, and E. lucius elements should have diverged before those of O. niloticus, B. baikalensis, and C. labridens, and the Poeciliidae/Fundulus sequences should be more related to the O. latipes elements than to the F. rubripes C sequences. Taking different segments of the 420-nt sequence for phylogenetic analysis (the first, last, and middle 200 nt) did not introduce any significant change compared with the phylogeny of the whole sequence and did not result in a phylogeny more compatible with classical phylogenies.
Such phylogenetic discrepancies can have different causes (Capy, Anxolabéhère, and Langin 1994
; Cummings 1994
). The topology of Rex3 phylogeny might be explained by the presence of several different ancient Rex3 lineages that diverged before their actual host genomes did. Loss or nondetection by PCR of certain Rex3 lineages could lead to comparison between paralogous sequences and introduce major differences between host and transposon phylogenies. Accordingly, two lineages were detected in the PCR-independent Fugu sequences (fig. 2
). Nevertheless, we observed that the rates of synonymous substitutions between Rex3 elements from different species were generally not higher than those for other nuclear genes presenting similar levels of codon bias, as observed for the Drosophila non-LTR retrotransposon R1 (Lathe et al. 1995
), and were frequently even lower (data not shown). This suggests that if several Rex3 lineages are present in teleost genomes, they are probably not very ancient. Alternatively, horizontal transfer of Rex3 retrotransposons might be compatible with the generally low rates of synonymous substitutions found for Rex3. Nevertheless, although horizontal transfer of a non-LTR retrotransposon has recently been suggested in teleosts (Volff, Körting, and Schartl 2000
), the large number of interspecific transmissions necessary to explain the Rex3 phylogeny appears inconsistent with the presumed extreme rarity of such events (Malik, Burke, and Eickbush 1999
). Apparent anomalies observed in transposon phylogenies could also result from differences in evolutionary rates. Sequences with higher rates of evolution can typically be "pulled down" to the root in a tree and will give the impression that they diverged from a lineage earlier than they evolutionarily did. Finally, it also appears possible that several mechanisms have acted together during the evolution of Rex3 sequences in fish genomes.
Acknowledgements
We are grateful to G. Schneider, H. Schwind, and P. Weber for fish maintenance, to S. Chen (University of Würzburg, Germany, and Huanghai Fisheries Research Institute, Qingdao, China), Y. Hong, and C. Winkler (University of Würzburg) and S. Kirilchik and M. Grachev (Institute of Limnology, Irkutsk, Russia) for fish organs and DNA, to K. T. Cuong and A. K. Meyer for DNA sequencing, to Y. Hong for helping to prepare fish for DNA isolation, and to the Fugu genome project for making genome survey sequences public. This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 465 "Entwicklung und Manipulation pluripotenter Zellen"), the European Commission (FAIR Project PL 97-3796, "Basis of sex determination and gonadal sex differentiation for sex control in aquaculture"), and the Fonds der Chemischen Industrie.
Footnotes
Thomas Eickbush, Reviewing Editor
2 Keywords: non-LTR retrotransposon
reverse transcriptase
phylogeny
Xiphophorus
teleost evolution
1 Address for correspondence and reprints: Jean-Nicolas Volff, Physiological Chemistry I, Biocenter, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany. E-mail: volff{at}biozentrum.uni-wuerzburg.de
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