© 2006 Society of Systematic Biologists
Phylogenomic Analysis of the L1 Retrotransposons in Deuterostomia
Edited by Andrew Shedlock: Associate Editor
san Kordi1in2ek1,2
1 Department of Biochemistry and Molecular Biology, Jo
ef Stefan Institute Ljubljana, Slovenia E-mail: dusan.kordis{at}ijs.si (D.K.)
2 Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana Slovenia
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Abstract |
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L1 retrotransposons constitute the largest single component of mammalian genomes. In contrast to the single remaining lineage of L1 retrotransposons in mammalian genomes, some teleost fishes contain a highly diverse L1 retrotransposon repertoire. Major evolutionary changes in L1 retrotransposon repertoires have therefore taken place in the land vertebrates (Tetrapoda). The lack of sequence data for L1 retrotransposons in the basal living Tetrapoda lineages prompted an investigation of their distribution and evolution in the genomes of the key tetrapod lineages, amphibians and reptiles, and in lungfishes. In this study, we combined genome database searches with PCR analysis to demonstrate that L1 retrotransposons are present in the genomes of lungfishes, amphibians, and lepidosaurs. Phylogenomic analysis shows that the genomes of Deuterostomia possess three highly divergent groups of L1 retrotransposons, with distinct distribution patterns. The analysis of L1 diversity shows the presence of a very large number of diverse L1 families, each with very low copy numbers, at the time of the origin of tetrapods. During the evolution of synapsids, all but one L1 lineage have been lost. This study establishes that the loss of L1 diversity and explosion in copy numbers occurred in the synapsid ancestors of mammals, and was most probably caused by severe population bottlenecks.
Keywords: Deuterostomia; L1 retrotransposon; non-LTR retrotransposon; phylogenomics; Tetrapoda
Received May 5, 2005; Revised May 15, 2006; Accepted June 14, 2006
Retrotransposons are class I transposable elements (TEs) that transpose through reverse transcription of an RNA intermediate. They are present in all eukaryotic genomes, where they constitute the most abundant class of mobile DNA. In many cases, they comprise over 50% of the nuclear DNA, a state that may have arisen in just a few million years. Retrotransposons play a central role in the structure, evolution, and function of eukaryotic genomes (Brosius, 2003; Kidwell and Lisch, 2000).
The L1 clade is one of the most diverse and widespread clades of non-LTR retrotransposons in eukaryotes (Malik et al., 1999). L1 retrotransposons have been studied extensively in humans and their exceptional role in genome remodeling has been demonstrated (Deininger et al., 2003; Ostertag and Kazazian, 2001). L1s alone constitute > 20% of the human genome and, through the mobilization of Alu repeats and 3' flanking DNA, have created > 40% of the human genome (International Human Genome Sequencing Consortium, 2001). Although the vast majority of L1s are inactive, as a result of 5' truncations, internal rearrangements, and mutations, a small proportion of active L1 retrotransposons, the hot L1s, are involved in in vivo human retrotranspositions (Brouha et al., 2003). Human L1 elements provide the replication machinery for the highly repetitive Alu elements (Dewannieux et al., 2003), and also contribute to the formation of processed pseudogenes (Esnault et al., 2000). L1 retrotransposons can transduce adjacent genomic sequences at their 3' end, facilitating exon shuffling (Moran et al., 1999; Goodier et al., 2000). L1 retrotransposons may alter transcriptional profiles and generate novel mRNA and protein isoforms in mammals (Han and Boeke, 2005). The presence and activity of L1 retrotransposons constitute substantial sources of genomic instability, and so are associated with major changes in genomic structure, that result in large genomic deletions (Gilbert et al., 2002; Symer et al., 2002). By providing templates for recombination, they are responsible for large-scale genome rearrangements (Burwinkel and Kilimann, 1998; Buzdin et al., 2003). L1 insertions have also been implicated in a number of human genetic diseases (Kazazian and Moran, 1998).
In contrast to the extensive understanding of retrotransposition mechanisms and their action at the genome level, we know very little about the evolution of the L1 clade in Deuterostomia and vertebrates, especially the basal living land vertebrates (Tetrapoda). The first insight into the diversity of L1 retrotransposons in the genomes of three fish species (Danio, Takifugu, and Tetraodon) was gained recently (Volff et al., 2003). An attempt has been made to solve the problem of the transition from the diverse L1 retrotransposon repertoire in zebrafish to the single remaining L1 lineage in mammals (Furano et al., 2004). The conclusions of both papers suffer from the problem of inadequate taxon sampling, because no genome sequence data nor the larger sample of data from tetrapods and lungfishes were included.
Current knowledge about L1 retrotransposons in deuterostomes is limited to mammals (Smit et al., 1995; Smit, 1999; International Human Genome Sequencing Consortium, 2001; Mouse Genome Sequencing Consortium, 2002) and teleost fishes (Duvernell and Turner, 1998; Volff et al., 2003; Furano et al., 2004; Duvernell et al., 2004). The numerous ongoing genome projects in Deuterostomia provide an unprecedented opportunity to obtain the complete L1 repertoires from the genomes of diverse deuterostomes. The aim of this study was to obtain a global picture of the distribution, diversity, evolution, and origin of L1 retrotransposons in Deuterostomia and Vertebrata. Phylogenomic analysis of the complete L1 repertoires from the genomes of diverse deuterostomes has provided a deep insight into the diversity and evolution of L1s in deuterostomes. This has provided an answer to one of the unanswered fundamental questions of mammalian genomics as to why the TE content of mammals is so unique among animals and what kind of evolutionary changes have occurred in the genomes of land vertebrates that have led to the simultaneous loss of L1 diversity and enormous explosion of their copy numbers in mammalian genomes.
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Data Mining
All database searches were performed online and were completed in February 2006. The databases analyzed were the nonredundant (NR), EST, GSS, HTGS (NISC comparative sequencing of vertebrates), WGS, as well as TraceDb databases at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) (Table S1) (All supplementary tables [S1 to S5] are available online at a http://systematicbiology.org/.) In addition to the Ensembl (http://www.ensembl.org) and National Institute of Genetics (NIG) Sequencing Center Medaka genome BLAST (http://dolphin.lab.nig.ac.jp/medaka/index.php), we searched the Joint Genome Institute (JGI) (http://www.jgi.doe.gov) databases. Taxon-specific genome databases were searched at the ENSEMBL and JGI websites (Silurana (Xenopus) tropicalis), whereas diverse taxon-specific databases were searched at the NCBI for all major deuterostome lineages. In order to detect all the available L1 retrotransposon sequences, database searches were performed iteratively, using first the diverse L1 retrotransposons (medaka Swimmer 1 and human L1 element), and then the novel L1 elements. At the NCBI website, comparisons were performed using different BLAST programs (Altschul et al., 1997). At the JGI and Ensembl websites, the TBLASTN program was used with E-values set at 10– 5 and otherwise with default settings. Diverse full-length L1 retrotransposons or their reverse transcriptase (RT) domains have been used as queries. For translation of the DNA sequences Translate program (http://www.expasy.org/tools/dna.html) was used. Closely related groups of full-length L1 retrotransposons displaying > 90% amino acid identity between their reverse transcriptases have been designated as families. The amino acid sequences of deuterostome L1 retrotransposons used for phylogenetic analysis are available online as an appendix at a http://systematicbiology.org/.
PCR Amplification, Cloning, and Sequencing of L1 Retrotransposons
The species examined, along with their taxonomic classification, are shown in Table S2; the reptilian genomic DNA samples were those examined previously (Kordi and Gubenek, 1998). Genomic DNA from amphibians and a lungfish was extracted using the standard proteinase K digestion—phenol/chloroform cleaning—ethanol precipitation method, or were provided by Dr. R. Zardoya.
The oligonucleotide primers were derived from regions encoding the conserved amino acid motifs in the reverse transcriptase: KAFD (L1KAFDs, 5'-GATGCA GAGAAGGCATTTGAC-3') and VKM (L1VKMas, 5'-CAT(CT)TT(AG)GG(CT)AAGATATTCATTTTCAC-3'), that are conserved in medaka Swimmer 1 and human L1 retrotransposon. PCR amplification of the 650-bp fragment was performed on 1 µg of genomic DNA from each species in 100 µl of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, 0.2 µ M of each oligonucleotide, and 2.5 units of AmpliTaq polymerase (Perkin Elmer). After an initial denaturation step of 10 min at 96°C, the PCR reactions were subjected to 30 cycles of amplification consisting of 1 min denaturation at 96°C, 1 min annealing at 56°C, and 1 min extension at 72°C, with a 10-min final extension at 72°C. Ten microliters of each reaction solution containing the amplified DNA fragments were electrophoresed on a 1% agarose gel, stained with ethidium bromide and visualized with UV light. The resulting PCR products were ligated directly into a pGEM vector, using pGEM-T-easy cloning kit (Promega), for sequence determination. The inserts were sequenced on both strands with an ABI fluorescent sequencing kit on an ABI 310 sequencer (Applied Biosystems). The sequence data from this study have been submitted to GenBank database under accession numbers AY528456 [GenBank] to AY528505.
Phylogenetic Analysis of L1 Retrotransposons
We have included in our analysis all the nonredundant deuterostome full-length L1 sequences as well as a number of nucleotide sequences of PCR amplified L1 retrotransposons. The amino acid and nucleotide sequences of the RT domains of deuterostome L1 retrotransposons were aligned using Clustal W (Thompson et al., 1994). We tested all the available correction distance models, but found that all the complex correction methods performed significantly worse than simple correction methods. Only the simplest models of evolution led to tree topologies that are in agreement with the established mammalian phylogeny (Murphy et al., 2001), similary as reported previously for retroviruses (Posada and Crandall, 2001). We therefore used uncorrected p distances for deduced amino acid sequences to measure the extent of sequence divergence. Due to their smaller variance these distances give better results than more complicated distances when the number of sequences is large, when sequences are very divergent, and when the number of positions used is large (Nei and Kumar, 2000; Takahashi and Nei, 2000). For distance estimations of nucleotide sequences of PCR amplified L1 RTs, we excluded indels and used all alignment positions and transversions only. Phylogenetic trees were reconstructed from these distances using the neighbor-joining (NJ) method (Saitou and Nei, 1987). The reliability of the resulting topologies was tested by the bootstrap method. CR1 retrotransposon from X. tropicalis (accession number AC144967
[GenBank]
) was used as outgroup. In order to confirm that the novel elements belong to the L1 clade, we included diverse representatives of the Tx1 subclade (Kojima and Fujiwara, 2004). All analyses were performed with the computer programs MEGA 3.1 (Kumar et al., 2004) and Treecon (van de Peer and De Wachter, 1997).
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Numerous Novel L1 Retrotransposon Families in the Genomes of Deuterostomia
A comprehensive survey of L1 retrotransposons was carried out, using the extensive genome sequence data available for all major deuterostome lineages, in order to obtain a global picture about L1 origin, distribution, diversity, and evolution in Deuterostomia. We collected all available L1 retrotransposon families from the 35 completed deuterostome genomes and from numerous deuterostome genomes with reasonable amounts (at least several Mb) of genome sequence data (Table S1). We obtained the largest collection of the available full-length L1 elements from the basal deuterostomes and vertebrates, constituting the complete L1 repertoires of all deuterostome species with completed genomes (Fig. 1 and Table S3).
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The major finding of this analysis is that L1 diversity is not limited to teleost fishes (Furano et al., 2004), but is typical of all deuterostomes, the only exceptions being mammals. Our data set of complete L1 repertoires from diverse deuterostome genomes contains more than 300 diverse L1 families. The most surprising finding was the extreme diversity of L1s in the most basal living tetrapods, the amphibians, the X. tropicalis genome containing 126 low copy number L1 families (Fig. 1 and Table S3). This is currently the largest L1 diversity to be observed in deuterostomes, as well as in eukaryotes. The finding of diverse L1s in the genome of tuatara (Sphenodon punctatus) is also very important. Among the basal deuterostomes, such as echinoderms and urochordates, only very small L1 repertoires can be found. We have found a surprisingly rich L1 repertoire in the cephalochordates, the lancelet (B. floridae) genome containing 40 diverse L1 families (Fig. 1 and Table S3). In a strong contrast to cephalochordates, we found a surprising complete absence of L1 retrotransposons from the genome of sea lamprey (Petromyzon marinus), the representative of the most basal living vertebrates, the cyclostomes. A large variation in the size of L1 repertoires can be seen in teleost fishes, from zero (T. nigroviridis, Gasterosteus aculeatus), a single L1 family (T. rubripes), 17 L1 families in medaka (Oryzias latipes), to 59 L1 families in a zebrafish (D. rerio) genome (Fig. 1 and Table S3).
Although such a large and diverse collection of L1 families will be quite useful, it is far from showing the complete extent of L1 diversity in Deuterostomia. Genome data are still missing for very important deuterostome taxonomic groups, such as hemichordates, cyclostomes, cartilaginous fishes, latimeria, lungfishes, and for diverse amphibian and reptilian orders. Despite this lack, our data set of deuterostome L1 repertoires has enabled an in depth phylogenomic analysis of L1 retrotransposons, a classification of highly divergent deuterostome L1 groups and a simple explanation of the evolution of diverse L1 groups (origins of diverse L1 groups, diversification and loss of L1 groups). These will now be considered in detail.
Three Highly Divergent Groups of L1 Retrotransposon are Present in the Genomes of Deuterostomia
Phylogenomic analysis of the L1 retrotransposon repertoires in deuterostomes (Fig. 2) emphasizes, firstly, the large diversity of L1 retrotransposons in teleost fishes and amphibians, reflected in the lengths of their branches. All mammalian L1 retrotransposons have very short branch lengths, indicating that these orthologs are very similar. Secondly, the analysis shows that the genomes of Deuterostomia possess three groups of L1 retrotransposons, having distinct distribution patterns (Figs. 2 and 3, Table S4). Group A is the oldest, because it contains representatives from echinoderms to teleost fishes, but none from tetrapods. Group B is vertebrate specific, because it is present at least in cartilaginous and teleost fishes, latimeria, and amphibians, but not in mammals. Group C is also vertebrate specific, and is present at least in teleost fishes, lungfishes, amphibians, lepidosaurs, and mammals (Fig. 2, Table S4). Thirdly, the replicative and explosive nature of the L1 transposition process is revealed by the presence of species-specific clusters in all three L1 groups (e.g., Figs. 2 and 3). Fourthly, the phylogenomic analysis clearly shows also the tempo and mode of the origin and disappearance of distinct L1 groups, because the distribution patterns of three L1 groups are different. These data provide a useful framework for analyzing the molecular evolution of L1 retrotransposons in the well defined taxonomic group Deuterostomia, over a very long period of time (more than 600 Myr).
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Diversity of L1 Retrotransposon Repertoires
A number of selective mechanisms that maintain TEs at low frequencies and abundance have been proposed. TEs can disrupt gene function or normal cellular activity, there may be deleterious effects of ectopic exchange between elements located at different genomic positions, or deleterious consequences of TEs may arise through their effects on genome size, rates of cell division, and overall metabolism (Wright and Schoen, 2000). L1 repertoires in diverse deuterostome genomes exhibit high diversity among families and low copy numbers within families. This is the first comprehensive analysis of the diversity of L1 retrotransposons in deuterostomes and uses the largest number of L1 representatives, from echinoderms to mammals.
Echinoderms, urochordates, and cephalochordates
The new data on the presence and diversity of L1 retrotransposons in living lower deuterostomes demonstrates their widespread distribution in echinoderms, urochordates and cephalochordates (Table S4, Fig. 3). Data mining of the echinoderm (S. purpuratus), urochordate (C. intestinalis and C. savignyi), and cephalochordate (B. floridae) genomes shows quite different levels of L1 diversity. The lowest level of diversity has remained in the compact urochordate genomes, with only five to six low copy number families being present. Much higher L1 diversity is present in the echinoderm genomes, whereas in a cephalochordate genome we found an unexpectedly high level of L1 diversity (Fig. 3). Species-specific clusters were found in the genomes of echinoderms, urochordates, cephalochordates, and teleost fishes, indicating their origins by transposition bursts (Fig. 3). In contrast to the vertebrates, all living lower deuterostomes contain only a single L1 group, the ancestral L1 group A.
Cyclostomes
No L1 retrotransposons can be found in the first available genome data of the sea lamprey (P. marinus) (
3.5 million sequence reads and genome size of 2.1 Gb), indicating their loss, at least in this species.
Teleost fishes
A highly diverse L1 clade repertoire has recently been found in the zebrafish genome (Furano et al., 2004). We have now found that 59 distinct L1 families coexist in the zebrafish genome (Fig. 4), twice as many as reported previously. Zebrafish L1 retrotransposons are structurally intact and highly conserved, indicating recent retrotransposition; they also differ in their copy number (Furano et al., 2004). The numerous clusters in all three L1 groups in zebrafish genome indicates that they originated by transposition bursts (Fig. 4).
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The availability of medaka genome data enables an insight into the L1 diversity of teleost fishes, whose genome sizes differ from that of the compact pufferfish or from much larger zebrafish genome. The genome size of medaka is 800 Mb, half that of zebrafish (1.7 Gb). Phylogenetic studies place the medaka in close relationship to the pufferfish (T. rubripes and T. nigroviridis), with an estimated divergence time of 60 to 80 Myr, less than the evolutionary distance between man and mouse. Medaka and the zebrafish are relatively distant cousins that have evolved separately for at least 110 Myr (Wittbrodt et al., 2002). The previously analyzed Swimmer 1 element (Duvernell and Turner, 1998), the low copy L1 element from medaka, is only one of the 17 diverse L1 families that exist in the medaka genome. We have found, however, that the diversity of the L1 repertoire of medaka is very similar to that of the zebrafish, despite the large difference in their genome sizes (Fig. 5). Thus, diverse L1 repertoires are not limited to the zebrafish (Furano et al., 2004), but are typical of the majority of teleost fishes, except the species, which have compact genomes. This study also shows that among vertebrates only teleost fishes possess all three highly divergent L1 groups (Figs. 4 and 5).
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The availability of genome sequence data for several fish species with very diverse genome sizes provides an insight into the evolutionary dynamics of L1 retrotransposons in the compact and normal size genomes of teleost fishes. In contrast to the zebrafish and medaka, some teleost species have lost L1 retrotransposons. A single remaining full-length L1 retrotransposon, not reported previously, was found in the T. rubripes genome (Figs. 2 and 5). The second pufferfish species, T. nigroviridis, has, however, lost all its L1 retrotransposons. Surprisingly, all three L1 groups in the genome of stickleback (G. aculeatus), with the relatively compact genome size of 675 Mb, have also been lost. The presence of the three diverse L1 groups in the genomes of teleost fishes represents the ancestral state for Tetrapoda.
Amphibians
The first data on the presence and diversity of L1 retrotransposons in Amphibia are reported. PCR analysis of L1 distribution shows for the first time their widespread distribution in all three extant orders of Amphibia, in caecilians (Gymnophiona), frogs (Anura) and salamanders (Caudata) (Table S2, Fig. 6). Data mining of the X. tropicalis genome shows an unexpectedly high level of diversity, the highest among all available vertebrate genomes (Fig. 7). It now becomes clear that L1 diversity is considerably greater in amphibians than in teleost fishes. In the X. tropicalis genome, we found a highly diverse L1 repertoire, containing 126 diverse L1 families (Fig. 7), that contains structurally intact L1 elements, indicating recent retrotransposition. The explosive transposition bursts in X. tropicalis genome have produced numerous clusters in both L1 groups (Fig. 7). The X. tropicalis genome is very informative, because it demonstrates the enormous L1 diversity in Amphibia. Thus, the highly diverse L1 repertoire observed in the most basal living tetrapods, constitutes the ancestral state for Amniota (reptiles, birds, and mammals).
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Sauropsids (reptiles and birds)
The key taxonomic group that can give information about the loss of L1 retrotransposon diversity are the sauropsids, but only a very limited amount of genome sequence data exists, preventing a closer insight into their L1 diversity. Because the amphibians possess highly diverse L1 repertoires, whereas all the mammals have only a single L1 lineage, the loss of the diverse L1 repertoire should have occurred in the mammalian ancestors (synapsids), after they split from the sauropsids. PCR analysis of L1s in Reptilia shows their presence only in diverse Lepidosauria representatives, in a lacertid lizard, and in four snake families: Boidae, Viperidae, Elapidae, and Colubridae (Table S2, Fig. 6). The analysis of the partial genome data of tuatara (S. punctatus) shows for the first time the presence of a few diverse L1s (Fig. 8, Table S3), indicating that L1 repertoire of the most basal living sauropsid lineage (Lepidosauria) is still diverse. Resolving the question of L1 diversity in sauropsids and the precise timing of the loss of diverse L1 repertoires will require finding diverse L1 families in the genomes of Lepidosauria, which are the key taxonomic group for understanding L1 retrotransposon evolution in mammals.
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Mammals
Numerous novel full length L1 retrotransposons have been found in 43 species that belong to marsupials and all four eutherian superorders, by data mining of the novel genome sequence data for numerous mammals (Table S3, Fig. 9). This widespread distribution indicates that L1 retrotransposons have been present in mammalian genomes for more than 170 million years. We searched for the presence of diverse L1 retrotransposon lineages in mammals, but found no evidence for their presence. All mammals contain only a single L1 retrotransposon lineage, the only exception being the extant monotremes, because both platypus and echidna have lost the L1 retrotransposons (Table S3, Fig. 2).
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Tempo and Mode of Evolution of Diverse L1 Retrotransposon Groups
The evolution of L1 retrotransposons can be described very simply using the phylogenomic approach (Eisen and Hanawalt, 1999), for which knowledge of the correct taxonomy is crucial. Vertebrate-specific L1 groups have evolved from the ancestral to 16pt L1 group A that was present in the last common ancestor (LCA) of deuterostomes.
L1 group A
This is the most ancestral L1 group in Deuterostomia, because only group A can be found in the genomes of living lower deuterostomes (echinoderms, urochordates, and to 16pt cephalochordates). Group A has survived, at least until to teleost fishes (Fig. 3, Table S4 and Table S5). On the basis of the strict vertical transmission (SVT) hypothesis, we expect that this L1 group is present also in the genomes of cyclostomes and cartilaginous fishes, because it is present both in basal deuterostomes and in teleost fishes, but not in amphibians. Thus, the L1 group A has been lost in the lineage leading to the land vertebrates. No remains of L1 group A are seen in the genomes of land vertebrates.
L1 group B
This group is present exclusively in vertebrates (Figs. 2, 4, and 7, Tables S4 and S5). The oldest taxonomic lineage in which we confirmed their presence are cartilaginous fishes. L1 group B has highly diverse repertoires in the genomes of teleost fishes and amphibians (Figs. 4 and 7). We found the first representatives of group B in genomes of cartilaginous fishes (Leucoraja erinacea C0051542 and DT726706
[GenBank]
) and in Latimeria menadoensis (AC150284
[GenBank]
), and, on the basis of the SVT hypothesis, we expect its presence in the lungfish genomes. This group is not present in the genomes of living lower deuterostomes, but most probably originated in the LCA of vertebrates. Group B is not present in mammalian genomes, and has thus been lost, either in the LCA of Amniota or in the synapsid ancestor of mammals.
L1 Group C
This is the key L1 group for understanding the evolutionary fate of L1s in mammals. Group C is quite widespread, being present in the genomes of teleost fishes, lungfishes, amphibians, lepidosaurs and mammals (Figs. 2, 4, 6 and 7, Tables S4 and S5). From the currently available genome data we can see that teleost fishes are the oldest vertebrate class that possess L1 group C. On the basis of the SVT hypothesis, we might expect that this L1 group was already present in the LCA of the vertebrates (originated either in agnathans or in gnathostomes). In teleost fishes, group C is still small in extent because only two families can be found in the medaka genome and nine in the zebrafish genome (Table S3, Figs. 4 and 5). Surprisingly, this group exploded in the most basal living tetrapods (amphibians), in which we observed a huge diversity, the largest among the vertebrate genomes available. Despite the presence of more than 70 diverse families of L1 group C in the X. tropicalis genome (Fig. 7), they are unusually small in size, because only one to five members/family can be found. We demonstrated that the most basal living sauropsid lineage, the Lepidosauria (tuataras, lizards and snakes), still contains a diverse L1 repertoire, and we expect it to be similar to that observed in X. tropicalis (diverse and low copy number L1 elements), although the L1 diversity might be significantly reduced.
Origin of vertebrate-specific L1 groups in the LCA of vertebrates
Cyclostomes, the most basal living vertebrates are crucial for the understanding the origin of vertebrate-specific L1 groups in the LCA of vertebrates. However, the analysis of quite a large number of sea lamprey genome sequence data clearly shows the absence of any of the L1 groups, indicating that L1s have been completely lost, at least in sea lamprey. Unfortunately, the first genome data available for any cyclostome cannot provide any insight into the presence and diversity of L1s in agnathan genomes. The phylogenomic analysis of L1 retrotransposons suggests that the agnathans may contain all three distinct L1 groups (Tables S4 and S5). On the basis of the SVT hypothesis, group A, at least, is expected to be present in the genomes of cyclostomes. If, however, the groups B and C have not originated in the LCA of agnathans, then they originated later in the LCA of the jawed vertebrates (Gnathostomata). On the basis of the SVT hypothesis, the L1 group A is also expected to be present in the genomes of cartilaginous fishes, and we have confirmed the presence of the group B also in their genomes (Table S4). Thus, the clues about the origin and loss of L1 retrotransposon diversity in vertebrates are to be found in the key taxonomic groups, such as cyclostomes, cartilaginous fishes, and lepidosaurs.
Evolutionary Dynamics of L1 Retrotransposons in Vertebrates
The question as to what has happened during the long-term evolution, over 450 Myr, of the L1 retrotransposons in large and well-defined taxonomic groups, such as vertebrates and tetrapods, was until this study hidden in their genomes.
Strict vertical transfer
In this study we have observed the presence of L1 retrotransposons in most of the vertebrate lineages that were tested by PCR analysis or by searching genome and EST databases. We have found them in the genomes of cartilaginous fishes, latimeria, lungfishes (Dipnoi), and the majority of tetrapod lineages, such as Amphibia, Lepidosauria, and most mammalian orders (except monotremes) (Table S2; Figs. 2 and 6). The majority of the novel vertebrate L1 sequence data is from the diverse Tetrapoda lineages (Table S2; Figs. 2 and 6). We have found L1 retrotransposons in the lungfishes (Dipnoi), which are the most basal living sarcopterygians (Table S2, Fig. 6). Among the Amphibia, we have found them in the genomes of all three extant orders, Gymnophiona, Anura, and Caudata. Such a widespread distribution indicates that L1 retrotransposons are a widespread genome component of amphibians. Among the sauropsids, we found L1 retrotransposons only in the most basal living sauropsid lineage, Lepidosauria, clearly showing that L1 retrotransposons are not a widespread genome component of sauropsids. In the class Mammalia they are present in all available marsupial and all four eutherian superorders (Fig. 9), although not in Monotremata, thus confirming their widespread distribution in the class Mammalia. Such a widespread presence of L1 retrotransposons in the genomes of lungfishes, amphibians, lepidosaurs, and mammals confirms their ubiquity in tetrapod genomes and their evolution by strict vertical transfer.
Independent stochastic loss
During this study we found a number of independent, stochastic losses of L1 retrotransposons in diverse vertebrate lineages. One of the most surprising losses of L1 retrotransposons was in cyclostomes, in the genome of sea lamprey (P. marinus). In the teleost fishes, the compact fish genomes show the complete (T. nigroviridis and G. aculeatus) or nearly complete (T. rubripes) loss of the L1 repertoire. Although the L1 retrotransposons constitute the major part of mammalian genomes, they were lost, uniquely, from the genomes of platypus (O. anatinus) and echidna (Tachyglossus aculeatus), the representatives of the most basal living mammals, the monotremes. In both monotreme genomes only very high copy number L2 retrotransposons (Lovin et al., 2001) are present. The only other case for the extinction of L1s in mammals was reported for the genus Oryzomys (Rodentia) (Grahn et al., 2005). PCR analysis of L1 distribution in amphibians and sauropsids also shows several possible cases of stochastic loss (Table S2). The distribution pattern of L1 retrotransposons in sauropsids is interesting, their being present in the most basal living sauropsid lineage (Lepidosauria), but absent from the genomes of the evolutionarily younger extant turtles and all archosaurs (birds and crocodiles) (Table S2 to Table S3 Table S4). It is obvious that in vertebrates the L1 retrotransposons have undergone several cases of independent stochastic loss.
In the above examples the rate of loss of L1 retrotransposons by random genetic drift exceeded the rate of gain by transposition, until no L1 retrotransposons remained in their genomes. Only in the case of rare stochastic events will active L1 retrotransposons be lost from a population in which their rate of transposition substantially exceeds that of their mutational decay. The rate of TE decay caused by nucleotide substitutions and indels is of the order of 10–6 per generation. Since the insertion rate of L1s in human genome is about 10–3 to 10–4 insertions per active L1 element per gamete (Kazazian 2004), the transposition rates of active human L1 elements have been substantially greater than the rates of mutational decay. However, it appears that this was not the case, in a number of the vertebrates listed above.
Variable presence and absence of L1s in diverse vertebrates
If the L1 retrotransposons were polymorphic in some of the ancestral species than a speciation or population bottleneck events could lead to the fixation of a variant source L1 gene incapable of retroposition and this appear as a loss of L1 in this species; such type of event has been suggested in rodents (Grahn et al., 2005). Very recently a number of studies demonstrated that mammalian L1s possess hot and cold driver genes (Brouha et al., 2003; Seleme Mdel et al., 2006) with different retroposition rates. It appears that the lineage sorting of ancestral polymorphic driver L1 sequences is most likely responsible for the variable presence and absence of L1s in diverse vertebrate species. The size of the L1 family will depend on the balance between the generation of new active elements and their loss or inactivation. Stochastic effects will have a major role in L1 evolutionary dynamics when the numbers of active elements are small.
Transposition bursts and diversity of L1s in deuterostome genomes
During their coexistence with a host genome, L1s are subjected to short bursts of activity, followed by long-term inactivation by host-directed silencing mechanisms (Deininger et al., 2003; Han et al., 2005). Induction of L1 retroposition by environmental, genomic, or demographic stresses (Capy, 2000) might cause a sudden mobilization of L1s as a response to a stressful conditions. Mammalian L1 elements exist as active elements with lower levels of amplification (Han et al., 2005). Such evolutionary strategy of L1s, analogous to the low virulence of parasites evolving by vertical transmission, might enabled L1s to bypass mutational inactivation, negative selection and host defense mechanisms that could have limited their expansion (Han et al., 2005). When a burst of L1 retrotransposition occurs, the resulting negative selection can result in two general types of controlling forces: the control of its own amplification or the selection may eliminate all but the relatively inefficient L1 elements that cause minimal damage to the host. Thus the selective processes may allow survival of only the "smart" L1 elements that control the level of damage they cause a genome (Deininger et al., 2003). Selective pressure against active L1 elements will result in self regulation. As a consequence, an effective L1 retroposition strategy, termed "stealth driver," can be envisioned (Han et al., 2005). In this scenario, succsesful L1 lineages will remain largely inactive over extended periods of evolutionary time due to quiescent source. The change in the efficiency of the natural selection in the periods of rapid expansion of overactive L1 elements may be related to population bottlenecks or by fluctuation in retroposition rates. The stealth driver model may explain why the L1s have been subjected to periods of retropositional quiescence interspersed with episodic bursts of amplification (Han et al., 2005).
Huge diversification of L1s in deuterostome genomes is very likely a by-product of the arms race (coevolution) of L1s with the host-directed silencing mechanisms. Such a rapid sequence diversification of deuterostome L1s might be the consequence of a strong selective pressure caused by the risk of ectopic recombination (Petrov et al., 2003). This might be one of the reasons for the evolution of such a large number of L1 families in diverse deuterostomes (especially in cephalochordates, teleost fishes and amphibians) as observed in this study (Fig. 1). A number of possible explanations for the reduced L1 retrotransposon diversity in mammals in terms of a reduced ectopic recombination rate (Eickbush and Furano, 2002; Furano et al., 2004), competition for the host factors required for L1 replication, that resulted in a single L1 lineage (Furano et al., 2004; Eickbush and Furano, 2002) or higher permissivity of mammals for TEs than any other organism (Furano et al., 2004) has been proposed. A variable levels of gene conversion or sequence homogenization might also explain the differences in the sequence diversity of L1 elements in mammals and other genomes. With higher levels of gene conversion more homogenous families of L1 elements could evolve in mammals.
A Severe Population Bottleneck was Responsible for the Loss of L1 Diversity and the Explosion of a Single Remaining L1 Lineage in Mammals
From this study it is evident that the most basal living tetrapods, the amphibians, possess the most diverse L1 retrotransposon repertoire among vertebrates. In the amphibians, group C is much more diverse than in teleost fishes (Figs. 4, 5, and 7). The major difference between teleost fishes and amphibians is, however, not in L1 diversity, but in their copy numbers. In zebrafish, a typical representative of teleost fishes, the L1 families contain moderate numbers, from a few to a few tens, of full-length L1 copies (Furano et al., 2004). In contrast to the teleost fishes, all L1 families in amphibians (represented by X. tropicalis) contain very low copy numbers, from a single copy to a few full-length L1 copies only. This means that, in genomes of basal extant tetrapods, L1s are present as single or rare (low frequency) alleles, which are extremely vulnerable to demographic effects, mutational inactivation, and stochastic loss. Population bottlenecks can indeed very quickly eliminate many low frequency alleles (Nei et al., 1975), which provides the simplest explanation for the loss of L1 diversity in mammals and the enormous increase of their copy numbers. We found that severe population bottlenecks in the early history of synapsids caused the dramatic changes in L1 repertoires of mammals and consequently reshaped the ancestral mammalian genomes by the simultaneous loss of L1 diversity and explosion of the single surviving L1 lineage; details and full results will be presented elsewhere.
What can L1 Retrotransposons tell us about Vertebrate Phylogeny?
Mammals
We provide the first evidence that L1 retrotransposons can be used as a new phylogenetic marker for therian mammals (Fig. 9). An NJ tree of L1s from 43 species provided quite good resolution of mammalian relationships. However, for better resolution of the L1-based mammalian phylogeny, much denser sampling will be needed. All therian species analyzed have partial or complete genome data available, therefore we used for the phylogenetic analysis only the RTs from the full-length elements. Despite their enormous copy numbers in therian mammals, the L1 retrotransposons still behave as single-copy genes. The reason for that is the fact that the L1s are represented in each mammalian genome by a single family possessing very high copy numbers. The majority of these families represent the "dead on arrival" (DOA) pseudogenes, which, however, possess very limited phylogenetic information. The prerequisite for good resolution of therian phylogeny is to obtain the full-length L1 retrotransposons in the genome databases or by careful PCR amplification. We studied the largest mammalian L1 data set so far available; however, the major problem is the highly biased representation of mammalian orders and superorders. The order Primates is the best one covered in the genome databases, all other orders not being equally well covered by the genome sequence data.
We tested all available amino acid correction methods in the MEGA 3.1 (Kumar et al., 2004) and in Treecon (van de Peer and De Wachter, 1997) and came to surprising conclusion. None of the complex or more advanced correction methods can infer the NJ tree that corresponds to the established relationships of therian mammals. We used the Bayesian mammalian tree as a true one (Murphy et al., 2001). We found that the best tree topologies, very similar to the true mammalian tree, can only be inferred by using the simple uncorrected distances. The use of complex correction methods produced very strange topologies, showing numerous erroneous sister group relationships, like that of primates and marsupials. Transposable elements, and especially retroelements, are very different from the normal nuclear genes and gene families, due to very high levels of sequence divergence. However, in the case of mammals, L1s are highly conserved, therefore divergence cannot be the prime reason for the very poor performance of the complex correction methods.
A very similar finding has been reported for retroviruses (Posada and Crandall, 2001), where the best trees (most similar to the true trees) were inferred only by using the simplest models of sequence evolution. The authors tried to explain their unusual findings by the difficulties in multiple alignments, due to the very high level of sequence divergence in retroviruses. In contrast, no such problem was recognized in the multiple alignment of L1s from therian mammals; even the distances were in the range from 0.2 to 0.45. It appears that retroelements have their special mode of sequence evolution that is not well covered yet with the existing correction models. Indeed the favored use of Bayesian or maximum likelihood phylogenies with very complex models of sequence evolution will produce erroneous phylogenies of retroelements or erroneous species phylogenies (as in the case of L1s) with incorrect biological explanations. In the era of comparative genomics, now is the right time to find the robust model of sequence evolution that will apply to all or most retroelements. We believe that on a relatively short evolutionary timescale (< 200 Myr) the L1s have the strong potential to resolve some of the problematic evolutionary relationships among therian mammals.
Tetrapods
Because the majority of vertebrates possess diverse L1 groups with numerous species-specific families, such L1s have very limited potential to resolve some problematic evolutionary relationships of basal living sarcopterygians, amphibians and sauropods. The first problem is the lack of genome data for above mentioned taxa. The next is the absence of knowledge about complete L1 repertoires for most of these taxa (except X. tropicalis), therefore PCR is the current method of choice. However, as we found (Fig. 6), such an approach has very limited phylogenetic potential, due to biased amplification of L1 pseudogenes (which is indeed typical for all non-LTR retrotransposons). The sequences of L1 pseudogenes obtained from a number of basal living tetrapods and from a lungfish contain numerous stops and indels, causing difficulties in multiple alignments and inference of their phylogenies. The amplification of L1s in these taxa was useful just as the first evidence for their presence in the analysed taxa. However, in the phylogenetic analysis, the use of L1 pseudogenes raises numerous problems, therefore they are not recommended for answering any longstanding questions of tetrapod relationships.
Turtles and archosaurs
Even the L1 distribution pattern (presence/absence) in some archosaur and turtle genomes can still be very informative. The classical reptilian phylogeny (based mostly on paleontological data) states that turtles (anapsids) have the most basal position in any reptilian tree (with extinct or with extant species). However, a number of studies has rejected such a view, showing that turtles are the sister group to extant archosaurs (Iwabe et al., 2005). Even the L1 data confirm such a reptilian phylogeny, because the absence of L1s (by PCR and from the partial genome data) from the turtle genomes follows the genome pattern of archosaurs. If turtles are indeed the most basal reptilian lineage, their genomes must contain a rich and diverse L1 repertoire, as we found for the tuatara (Lepidosauria). These findings, and our additional unpublished data, suggest that the genome data are much more powerful and informative, even for systematists, than the mere presence/absence of a hole in amniote skulls. These holes are indeed only derived adaptations for attachment of muscles or mobility of the head, and the real biological value of skull holes is highly overestimated. The genome and osteocranial data of turtles are thus in conflict; however, the genome data are the more informative and indeed support the archosaurian affinity of turtles and confirm the most recently reported phylogenetic position of turtles (Iwabe et al., 2005).
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TopAbstractMethodsResults and DiscussionConclusionsAcknowledgmentsReferences |
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By phylogenomic analysis we provided the first global insight into the origin, distribution, diversity, and evolution of L1 retrotransposons in Deuterostomia and Vertebrata. We demonstrated that L1 retrotransposons in Deuterostomia belong to three highly divergent L1 groups that coexist in the genomes of vertebrates. We assembled the largest currently available data set of complete L1 retrotransposon repertoires from echinoderms, urochordates, cephalochordates, teleost fishes, amphibians, and numerous mammals and provided the first evidence for the presence of diverse L1 retrotransposons in the genomes of Lepidosauria. During the evolution of synapsids, all but one L1 lineage have been lost. This study establishes that the loss of L1 diversity and the explosion in copy numbers occurred in the synapsid ancestors of mammals, and was most probably caused by severe population bottlenecks. Finally, we have provided the evidence that the tempo and mode of L1 retrotransposon evolution in tetrapods is completely different from that shown by comparing only teleost fishes and mammals.
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We thank Prof. R. H. Pain for critical reading of the manuscript and Benjamin Gorin
ek for help in figure preparation. We thank anonymous reviewers for critical comments on an earlier version of this manuscript. The Ministry of Science and Technology of Slovenia supported this work by program P0-0501-0106. We greatly appreciate the help of the following colleagues in supplying genomic DNA or tissue samples of different vertebrate species: Dr. R. Zardoya, Museo Nacional de Ciencias Naturales, Madrid, and Prof. J.M. Joss, Macquarie University, Sydney. The Xenopus tropicalis and Branchiostoma floridae genome sequence data were produced by the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/). The medaka (Oryzias latipes) genome sequence data were produced by the National Institute of Genetics (NIG) Sequencing Center (http://dolphin.lab.nig.ac.jp/). |
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