Laboratoire de Biologie Moléculaire de la Cellule, UMR CNRS5161, Ecole Normale Supérieure de Lyon, Lyon, France
Correspondence: E-mail: marc{at}sdsc.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: shark ray genome duplication 2R hypothesis phylogeny Chondrichthyes
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Lack of chondrichthyan genome data has led us to use the gene phylogeny approach to solve the question of when vertebrate-specific gene duplications did happen, by constructing phylogenetic trees of many protein-coding genes sequenced in Chondrichthyes. As mentioned above, if there were two major rounds of duplication, whether of genes or genomes, we would expect most gene families to show similar relative timing of speciation and duplication events. It should be noted that we are only interested in vertebrate-specific duplications here. Duplications that predate the chordate/arthropod/nematode split (approximately the origin of bilaterian animals), or more recent duplications such as frequently observed in actinopterygian fishes (Robinson-Rechavi et al. 2001), are outside the scope of this study.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gene families with duplications predating the arthropod/nematode/chordate divergence (fig. 2A) were split into subfamilies, which were then evaluated separately for vertebrate-specific duplications. In cases of a vertebrate gene without any known mammalian ortholog, additional Blast searches were done on the human genome (International Human Genome Sequencing Consortium 2001). In all Blast searches, an expect value of 0.01 and the default filter for repeated sequences were used, and potential new genes were assessed for relevance to our study by a phylogenetic analysis. Once gene trees were built (see below), 86 gene families were found to yield phylogenies that could not be interpreted for dating of events at the origin of vertebrates (see Results). Notably, insufficient phylogenetic resolution was diagnosed when the gene tree was strongly inconsistent with the expected species phylogeny (for example, lamprey grouping with chicken and mammals not monophyletic [NPY gene family]) with very low bootstrap support (i.e., under 50%).
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
It is possible that the observed distribution of gene duplications simply reflects the difference between the time intervals considered as "before the chondrichthyan/teleostome split" and "after the chondrichthyan/teleostome split." To test this, let us consider only the 27 gene families for which we have a vertebrate-specific duplication and a chordate outgroup (table 1: 16 + 9 + 2 = 27), since they allow a more precise dating of events. If we use paleontological datings (fig. 1), the interval between chordate diversification and the chondrichthyan/teleostome split is 98 Myr, whereas the interval between this and the sarcopterygian/actinopterygian split is 45 Myr. Then we expect 31% (45/[98 + 45]) of vertebrate-specific gene duplications to be after the chondrichthyan/teleostome (C/ T) split, under the assumption of a constant rate of gene duplication; the 95% confidence interval of this estimate is 14% to 49% ( f ± 1.96 var = f (1 - f )/N; f = 0.31; N = 27). If we use molecular clock estimates of divergence dates (fig. 1), we expect 26% of gene duplications after C/ T (confidence interval = 9.5% to 43%). The observed proportion of 7.4% (2/27) is significantly lower than expected by chance in either dating system (outside of the 95% confidence intervals). This conclusion holds true if we only use the 16 significantly supported phylogenies with a chordate outgroup (table 1): the observed proportion of duplications after the C/ T split is 0%, whereas the expected value's confidence interval is either 8.7% to 54% (paleontological dates), or 4.5% to 47% (molecular clock dates). Thus, gene duplications are significantly less frequent after than before the chondrichthyan/teleostome split, taking into account evolutionary time.
Although our data set is not meant for detailed testing of duplication hypotheses in other branches of the tree, it is interesting to compare duplications that appear specific to either of the two major branches of teleostomes: out of 48 gene families, there are three with sarcopterygian-specific duplications and eight with actinopterygian-specific duplications (see the second table in the Supplementary Material online at www.mbe.oupjournals.org), consistent with previous observations (Robinson-Rechavi et al. 2001). Interestingly, these more recent duplications concern 28% of the 32 gene families for which we have observed gene duplications ancestral to vertebrates but only 12.5% of the 16 gene families without vertebrate specific duplications.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is indeed noticeable that there has been no report of genome duplications ancestral to sarcopterygians (Pennisi 2001; Wolfe 2001; Durand 2003) or to any of the well-studied groups therein (e.g., tetrapodes, mammals, or sauropsids). Our own data are consistent with previous observations (Robinson-Rechavi et al. 2001; Taylor et al. 2001) that duplicate genes are significantly less abundant in sarcopterygians than in actinopterygians. Analysis of invertebrate chordate data also indicates that gene duplications are not abundant in these lineages (Dehal et al. 2002; Panopoulou et al. 2003).
Comparison of MHC-associated genes gave limited evidence for duplications before the chondrichthyan/teleostome split from two genes (Abi-Rached et al. 2002). Our results show that this pattern is general, with almost all vertebrate-specific gene duplications occurring before the chondrichthyan/teleostome split (table 1). This, added to all the previously published evidence, implies three waves of gene or genome duplications, two between the cephalochordate split and the chondrichthyan split and the other in actinopterygian fishes, separated by a period of "duplication calm" of about 45 Myr (which continued for 400 Myr in tetrapodes), which, although short, is significant. A major prediction of Ohno's (1970) original hypothesis, that of intense gene or genome duplication activity before the origin of vertebrates, is thus confirmed by the study.
Moreover, our results show that these gene duplications characterize all the jawed vertebrates and predict similar genetic complexity in sharks and rays as in tetrapodes. Consistent results are found for the evolution of hox clusters, which allow a direct connection between block duplications and morphological adaptations. Although hox genes are very poor phylogenetic markers, as illustrated by the difficulty in resolving the events that led to the different clusters of gnathostomes and lampreys (Force, Amores, and Postlethwait 2002; Irvine et al. 2002), partial sequences from the horn shark indicate that the duplications that led to four hox clusters in teleostomes occurred before the chondrichthyan/teleostome divergence (Kim et al. 2000). Moreover, horn shark and human hoxA clusters are remarkably conserved (Chiu et al. 2002). Thus, hox cluster analysis and our phylogenetic results are consistent in establishing no relation between gene duplications and the larger diversity of bony vertebrates than of cartilaginous vertebrates.
Although the basal branching of chondrichthyans among jawed vertebrates is considered extremely well supported by morphological and paleontological data (Janvier 1996), the analysis of complete mitochondrial sequences suggests a very different phylogeny, with chondrichthyans branching among bony ray-finned fishes (Actinopterygii) (Rasmussen and Arnason 1999). This surprising result has not been confirmed by any other source of data, and molecular phylogenies based on nuclear-encoded genes either are not informative (Martin 2001; this study) or strongly support the conventional branching position of chondrichthyans (Takezaki et al. 2003). In any case, our results show that vertebrate-specific gene duplications occurred before the divergence between chondrichthyans, actinopterygians, and sarcopterygians, whatever the order of these latter events.
Our results are at odds with a recent study that used a similar approach, dating gene duplications by their phylogenetic position relative to speciation events (Friedman and Hughes 2003). There are several differences between our methodology and that of Friedman and Hughes, but the main difference is the criterion for classifying gene duplications within speciation intervals. We consider genes to be duplicated within a given interval (i.e., between chordate diversification and the chondrichthyan/teleostome split) only if all relevant taxonomic groups (and thus speciations) are represented in the gene tree (i.e., a urochordate or a cephalochordate, a chondrichthyan, and a teleostome). Friedman and Hughes (2003) classify duplications as soon as they can be dated before or after one speciation. Moreover they used very distant dating points (i.e. the primate/rodent, amniote/amphibian, and deuterostome/protostome splits). It is unclear why they did not date duplications relative to the actinopterygian/sarcopterygian split, because this speciation would have been more relevant to the "2R" controversy, while taking advantage of genome data. As amphibians are the only lineage involved for which a genome sequence is not available, this may lead them to include in the "before primate/rodent" category gene duplications that occurred before the amphibian/amniote split but for which they do not have amphibian sequences in the tree. This in turn may introduce a bias in their argument that the abundance of "before primate/rodent" versus "before amniote/amphibian" duplications is evidence against a peak of gene duplications at the origin of vertebrates. We believe that in our study, the division of the sequences into major taxonomic units, and our separation of the results according to the outgroup sequences used (table 1), preserve our results from such biases. Thus, differences in the conclusions between that study (Friedman and Hughes 2003) and ours probably reflect different sampling strategies.
An interesting side observation from our data set is that observations of gene duplications at the origin of vertebrates, and more recently in either the actinopterygian or sarcopterygian lineage, appear correlated. This may be the result of sampling; for example, better detection of duplications in more studied genes. Alternatively, it may indicate that the function of certain genes makes them more prone to persisting as duplicate copies. Such a tendency has indeed been recently shown in yeasts, where certain genes are retained independently as duplicates in different species (Hughes and Friedman 2003).
This study and other recent studies draw an increasingly precise picture of gene or genome duplication waves in chordates (fig. 3), although questions remain. Among the six branches of the chordate tree for which sufficient data are available, three are characterized by abundant preservation of duplicate genes, all of them in vertebrates. It has also been suggested on the basis of chromosome counts that polyploidy played an important part in lamprey evolution (Potter and Rothwell 1970). Of course it is probable that small-scale duplications have been continuous on all branches of the tree (Lynch and Conery 2000; Gu, Wang, and Gu 2002). However, large-scale duplications seem to have been frequent in vertebrate evolution, and the branches where they are absent, such as the origin of bony vertebrates, appear as the exception rather than the rule.
|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
Hervé Phillippe, Associate Editor
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abi-Rached, L., A. Gilles, T. Shiina, P. Pontarotti, and H. Inoko. 2002. Evidence of en bloc duplication in vertebrate genomes. Nat. Genet. 22:22.
Adams, M. D., S. E. Celniker, and R. A. Holt, et al. (195 co-authors). 2000. The genome sequence of Drosophila melanogaster. Science 287:2185-2195.
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][ISI][Medline]
Basden, A. M., G. C. Young, M. I. Coates, and A. Ritchie. 2000. The most primitive osteichthyan braincase? Nature 403:185-188.[CrossRef][ISI][Medline]
Boeckmann, B., A. Bairoch, and R. Apweiler, et al. (12 co-authors). 2003. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res. 31:365-370.
Chiu, C.-H., C. Amemiya, K. Dewar, C.-B. Kim, F. H. Ruddle, and G. P. Wagner. 2002. Molecular evolution of the hoxA cluster in the three major gnathostome lineages. Proc. Natl. Acad. Sci. USA 99:5492-5497.
Dehal, P., Y. Satou, and R. K. Campbell, et al. (87 co-authors). 2002. The draft genome of Ciona intestinalis: insights into Chordate and Vertebrate origins. Science 298:2157-2167.
Durand, D. 2003. Vertebrate evolution: doubling and shuffling with a full deck. Trends Genet. 19:2-5.[CrossRef][ISI][Medline]
Duret, L., D. Mouchiroud, and M. Gouy. 1994. HOVERGEN: a database of homologous vertebrate genes. Nucleic Acids Res. 22:2360-2365.[Abstract]
Escriva, H., L. Manzon, J. Youzon, and V. Laudet. 2002. Analysis of lamprey and hagfish genes reveals a complex history of gene duplications during early vertebrate evolution. Mol. Biol. Evol. 19:1440-1450.
Force, A., A. Amores, and J. H. Postlethwait. 2002. Hox cluster organization in the jawless vertebrate Petromyzon marinus. J. Exp. Zool. 294:30-46.[CrossRef][ISI][Medline]
Friedman, R., and A. L. Hughes. 2003. The temporal distribution of gene duplication events in a set of highly conserved human gene families. Mol. Biol. Evol. 20:154-161.
Galtier, N., M. Gouy, and C. Gautier. 1996. SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput. Appl. Biosci. 12:543-548.[Abstract]
Gu, X., Y. Wang, and J. Gu. 2002. Age distribution of human gene families shows significant roles of both large- and small-scale duplications in vertebrate evolution. Nat. Genet. 31:205-209.[CrossRef][ISI][Medline]
Holland, P. W., J. Garcia-Fernandez, N. A. Williams, and A. Sidow. 1994. Gene duplications and the origins of vertebrate development. Development (suppl):125133.
Holt, R. A., G. M. Subramanian, and A. Halpern, et al. (123 co-authors). 2002. The genome sequence of the malaria mosquito Anopheles gambiae. Science 298:129-149.
Hughes, A. L. 1999. Phylogenies of developmentally important proteins do not support the hypothesis of two rounds of genome duplication early in vertebrate history. J. Mol. Evol. 48:565-576.[ISI][Medline]
Hughes, A. L., and R. Friedman. 2003. Parallel evolution by gene duplication in the genomes of two unicellular fungi. Genome Res. 13:794-799.
International Human Genome Sequencing Consortium. 2001. Initial sequencing and analysis of the human genome. Nature 409:860-921.[CrossRef][ISI][Medline]
Irvine, S. Q., J. L. Carr, W. J. Bailey, K. Kawasaki, N. Shimizu, C. T. Amemiya, and F. H. Ruddle. 2002. Genomic analysis of hox clusters in the sea lamprey Petromyzon marinus. J. Exp. Zool. 294:47-62.[CrossRef][ISI][Medline]
Janvier, P. 1996. Early vertebrates. Clarendon Press, Oxford.
Kim, C.-B., C. Amemiya, W. Bailey, K. Kawasaki, J. Mezey, W. Miller, S. Minoshima, N. Shimizu, G. Wagner, and F. Ruddle. 2000. Hox cluster genomics in the horn shark, Heterodontus francisci. Proc. Natl. Acad. Sci. USA 97:1655-1660.
Kumar, S., and S. B. Hedges. 1998. A molecular timescale for vertebrate evolution. Nature 392:917-920.[CrossRef][ISI][Medline]
Langkjaer, R. B., P. F. Cliften, M. Johnston, and J. Piskur. 2003. Yeast genome duplication was followed by asynchronous differentiation of duplicated genes. Nature 421:848-852.[CrossRef][ISI][Medline]
Lynch, M., and J. S. Conery. 2000. The evolutionary fate and consequences of duplicate genes. Science 290:1151-1155.
Martin, A. 2001. The phylogenetic placement of chondrichthyes: inferences from analysis of multiple genes and implications for comparative studies. Genetica 111:349-357.[CrossRef][ISI][Medline]
McLysaght, A., K. Hokamp, and K. H. Wolfe. 2002. Extensive genomic duplication during early chordate evolution. Nat. Genet. 31:200-204.[CrossRef][ISI][Medline]
Muller, T., and M. Vingron. 2000. Modeling amino acid replacement. J. Comput. Biol. 7:761-776.[CrossRef][ISI][Medline]
Nikoh, N., N. Iwabe, and K. Kuma, et al. (11 co-authors). 1997. An estimate of divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata by aldolase and triose phosphate isomerase clocks. J. Mol. Evol. 45:97-106.[ISI][Medline]
Ohno, S. 1970. Evolution by gene duplication. Springer-Verlag, Heidelberg.
Ono-Koyanagi, K., H. Suga, K. Katoh, and T. Miyata. 2000. Protein tyrosine phosphatases from amphioxus, hagfish, and ray: divergence of tissue-specific isoform genes in the early evolution of vertebrates. J. Mol. Evol. 50:302-311.[ISI][Medline]
Panopoulou, G., S. Hennig, D. Groth, A. Krause, A. J. Poustka, R. Herwig, M. Vingron, and H. Lehrach. 2003. New evidence for genome-wide duplications at the origin of vertebrates using an amphioxus gene set and completed animal genomes. Genome Res. 13:1056-1066.
Pennisi, E. 2001. Genome duplications: the stuff of evolution? Science 294:2458-2460.
Perrière, G., C. Combet, and S. Penel, et al. (11 co-authors). 2003. Integrated databanks access and sequence/structure analysis services at the PBIL. Nucleic Acids Res. 31:3393-3399.
Potter, I. C., and B. Rothwell. 1970. The mitotic chromosomes of the lamprey, Petromyzon marinus. L. Experientia 26:429-430.
Rasmussen, A. S., and U. Arnason. 1999. Molecular studies suggest that cartilaginous fishes have a terminal position in the piscine tree. Proc. Natl. Acad. Sci. USA 96:2177-2182.
Robinson-Rechavi, M., O. Marchand, H. Escriva, P.-L. Bardet, D. Zelus, S. Hughes, and V. Laudet. 2001. Euteleost fish genomes are characterized by expansion of gene families. Genome Res. 11:781-788.
Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]
Samson, I. J., M. M. Smith, and M. P. Smith. 1996. Scales of thelodont and shark-like fishes from the Ordovician of Colorado. Nature 379:628-630.[CrossRef][ISI]
Schmidt, H. A., K. Strimmer, M. Vingron, and A. von Haeseler. 2002. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18:502-504.
Shimodaira, H., and M. Hasegawa. 1999. Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol. Biol. Evol. 16:1114-1116.
Shu, D. G., L. Chen, J. Han, and X. L. Zhang. 2001. An early Cambrian tunicate from China. Nature 411:472-473.[CrossRef][ISI][Medline]
Shu, D. G., H. L. Luo, S. Conway Morris, X. L. Zhang, S. X. Hu, L. Chen, L. Han, M. Zhu, Y. Li, and L. Z. Chen. 1999. Lower Cambrian vertebrates from south China. Nature 402:42-46.[CrossRef][ISI]
Takezaki, N., F. Figueroa, Z. Zaleska-Rutczynska, and J. Klein. 2003. Molecular phylogeny of early vertebrates: monophyly of the agnathans revealed by sequences of 35 genes. Mol. Biol. Evol. 20:287-292.
Taylor, J. S., I. Braasch, T. Frickey, A. Meyer, and Y. Van de Peer. 2003. Genome duplication: a trait shared by 22,000 species of ray-finned fish. Genome Res. 13:382-390.
Taylor, J. S., Y. Van de Peer, I. Braasch, and A. Meyer. 2001. Comparative genomics provides evidence for an ancient genome duplication event in fish. Philos. Trans. R. Soc. Lond. B Biol. Sci. 356:1661-1679.[CrossRef][ISI][Medline]
The C. elegans Sequencing Consortium. 1998. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282:2012-2018.
Wittbrodt, J., A. Meyer, and M. Schartl. 1998. More genes in fish? Bioessays 20:511-515.[CrossRef][ISI]
Wolfe, K. H. 2001. Yesterday's polyploids and the mystery of diploidization. Nat. Rev. Genet. 2:333-341.[CrossRef][ISI][Medline]
Yang, Z. 1996. Among-site variation and its impact on phylogenetic analyses. Trends Ecol. Evol. 11:367-371.[CrossRef][ISI]
Zhu, M., Y. Xiaobo, and P. Janvier. 1999. A primitive fossil fish sheds light on the origin of bony fishes. Nature 397:607-610.[CrossRef][ISI]