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ARTICLE IN PRESS Opinion TRENDS in Genetics Vol.xx No.xx Monthxxxx Reconstruction of a 450-My-old ancestral vertebrate protokaryotype Matthias Kohn1,*, Josef Högel1,*, Walther Vogel1, Peter Minich1, Hildegard Kehrer-Sawatzki1, Jennifer A.M. Graves2 and Horst Hameister1 1 Department of Human Genetics, University of Ulm, D-89070 Ulm, Germany Comparative Genomics Research Group, Research School of Biological Sciences, Australian National University, Canberra ACT 2601, Australia 2 From recent work the putative eutherian karyotype from 100 Mya has been derived. Here, we have applied a new in silico technique, electronic chromosome painting (Epainting), on a large data set of genes whose positions are known in human, chicken, zebrafish and pufferfish. E-painting identifies conserved syntenies in the data set, and it enables a stepwise reconstruction of the ancestral vertebrate protokaryotype comprising 11 protochromosomes. During karyotype evolution in land vertebrates interchromosomal rearrangements by translocation are relatively frequent, whereas the karyotypes of birds and fish are much more conserved. Although the human karyotype is one of the most conserved in eutherians, it can no longer be considered highly conserved from a vertebrate-wide perspective. Introduction The genomes of different mammalian species are far more similar than has been expected from their karyotypic diversity. Overall these genomes share a similar number of w24 500–26 000 genes [1]. Mapping of orthologous genes and comparative painting techniques using DNA probes from single chromosomes has enabled the reconstruction of an ancestral eutherian founder karyotype from w100 million years ago (Mya) [2–6]. These reconstructions are based on ancestral chromosome characteristics shared between extant mammalian species. Most informative for these reconstructions are genomes from species with highly conserved karyotypes [4]. It was surprising to realize that the human karyotype is similar to this ancestral karyotype, implying that no major karyotype change occurred during the evolution of humans in the past 100 My. Similar comparisons have also been performed between the karyotypes of different fish species and human [7–11]. With the availability of the chicken genome [12] as a connecting link between mammals and fish, it is now possible to reconstruct the protokaryotype of a common vertebrate ancestor that lived 450 Mya. Because of the greater DNA divergence rate, chromosomal painting techniques are not applicable when comparing different Corresponding author: Hameister, H. ([email protected]). * These authors contributed equally to this article. vertebrate genomes. For karyotype reconstructions one has to switch to in silico analysis and search for shared ancestral syntenies of genes across multiple species. To do this, we established a gene orthology database containing w3300 genes at a density of one gene per Mb along all human (Homo sapiens, HSA) chromosomes. This database contains human genes and their orthologous genes together with their chromosomal assignment from chicken (Gallus gallus, GGA), green spotted pufferfish (Tetraodon nigrovirides, TNI) and zebrafish (Danio rerio, DRE). This database is an extension of the one we used previously to reconstruct the ancestor of the mammalian X chromosome [13]. Genes from chicken and fish were considered to be orthologous to the respective human gene if reciprocal BLAST best-hit searches identified them as such in the Ensembl database (Table S1 in supplementary online material). Another 801 genes were added from a third fish species, medaka (Oryzias latipes, OLA) [9]. Table 1 shows the numbers of orthologous genes that could be unequivocally identified in each genome. Because the chicken [12], pufferfish [10] and zebrafish genome analyses are still incomplete, the number of orthologous genes retrieved for each species differs. This database, which is described in more detail in Box 1, can be used for electronic chromosome painting (E-painting), which is highly informative. By E-painting conserved syntenies (for a definition, see Box 1) are identified and used to reconstruct the ancestral vertebrate protokaryotype. For the comparison only synteny groups with three or more genes in the sample were considered. First, the karyotype of the early tetrapod (TET), which is ancestral to both birds and mammals, was reconstructed. Second, a reconstruction was performed of the karyotype of an ancestral teleost fish (TEL), which lived before the extra whole-genome duplication that is characteristic for the teleost fish genomes [10,14]. Finally, a common early vertebrate (VER) protokaryotype from 450 Mya was established. Reconstruction of the ancestral tetrapod protokaryotype The reconstruction of the tetrapod karyotype was based on chicken and human genome data including the conserved chromosome characteristics that had already guided the reconstruction of the ancestral eutherian karyotype (e.g. Refs [2,6]). The following syntenic associations of www.sciencedirect.com 0168-9525/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2006.02.008 ARTICLE IN PRESS Opinion 2 TRENDS in Genetics Vol.xx No.xx Monthxxxx Table 1. The number of orthologous genes compared in different species Human Chicken Pufferfish Zebrafish Human 3283 Chicken 2751 Pufferfish 2149 1915 Zebrafish 2085 1825 1499 Medaka 801 667 543 550 neighboring chromosomal segments of the ancestral eutherian karyotype are also present in the chicken genome: 1/19p, 3/21, 4/8p, 7/16p, 12/22 (2 chromosomes), 14/15, 16q/19q (Table 2). Therefore, these associations and the well-established chromosomal fusion making up HSA chromosome 2 were integrated into the reconstruction of the tetrapod karyotype from 310 Mya. The genome data from pufferfish served as outgroup information. As an example, the reconstructions for the tetrapod chromosomes TET 6 and TET 18 are described. Comparative mapping studies in mammals have confirmed the association of segments from HSA 12p/q and 22q on two different chromosomes in the ancestral eutherian protokaryotype. Yang et al. [4], using chromosome paints, proposed that these associations represent the eutherian chromosomes EUT 7 and 21. The respective genes of the first and larger association of segments from HSA 12p/q and 22q map on the duplicated pair of pufferfish chromosomes TNI 13 and 19, as does a further segment from HSA 7 (Table 2). All these segments together are included in the large chicken chromosome GGA 1 and are derived from TET 6 (Figure 1). The second and smaller association, which represents the eutherian chromosome EUT 21 [4], is found on TNI 12. It makes up the whole GGA 15 and is derived from TET 18 (Table 2). In a similar manner all other tetrapod protochromosomes were reconstructed according to the parsimony principle and having in mind the whole-genome duplication in the teleost fish (Table 2). Reconstruction of the Box 1. The study of karyotype evolution in vertebrates Chromosomal banding techniques In silico whole-genome database comparisons The study of karyotype evolution in vertebrates was boosted when chromosomal banding techniques became available. The astounding similarity of the chromosome banding pattern between human and the other primates enabled Dutrillaux [22] to put forward a phylogenetic reconstruction of the primates based on karyotype similarity. But when species from different mammalian orders were compared, banding similarity was found to be restricted to a few chromosomal regions (e.g. human versus cat [23,24] and human versus mouse [25]). When whole genomes from evolutionarily distantly related species are compared one has to concentrate on the most conserved singlecopy sequences, which are the coding sequences of the genes. Pevzner and Tesler [18] have provided the GRIMM software, which enables the researcher to extract the minimal number of rearrangements that separate two genomes. The advantage of this tool is that intrachromosomal inversions are also counted. Both segments of conserved synteny and conserved gene order are taken into consideration. A highly dynamic picture of genome evolution is revealed, which could be biased because in the current applications the data from the greatly rearranged genomes from mouse and rat are integrated [16,17]. Gene mapping data The long and informative history of the human–mouse comparative gene-mapping initiative has dominated this area. In an early and highly influential article, Nadeau and Taylor [26] predicted on the basis of only w83 orthologous loci a total number of segments with conserved synteny and gene order of 178G39 between both species. The accuracy of this calculation remained unchallenged over the next 20 years. Similar comparative studies with human as the reference genome are reported for zebrafish [7,8], which is the other vertebrate species favored by developmental biologists. Again, a greatly rearranged genome is observed. A crude estimate arrived at 260 to 420 segments of conserved synteny [8]. Chromosomal painting techniques (Zoo–FISH analysis) This technique relies on DNA sequence similarity, which is revealed by fluorescence in situ hybridization (FISH) techniques. Preferably, DNA probes from single chromosomes are used, which under prolonged hybridization indicate the orthologous chromosome segments in other species [27]. This technique reveals segments with conserved synteny, but is not informative about gene order. With human DNA probes this technique works with all species of mammals. Outside of eutherians the technique has been successfully applied only with an X chromosome probe in marsupials [28]. It was immediately evident that most mammalian species show a much greater degree of karyotype conservation than expected from the human–mouse comparison [29]. Shared ancestral chromosome characteristics in the form of associations and fissions were observed as soon as more species were analyzed [30]. There are species with a relatively high rate of rearrangements (mouse, dog and gibbon) and other species with few rearrangements (aardvark, harbor seal and cat) [31]. These species bear a highly conserved karyotype and therefore are informative for karyotype reconstructions [4]. Several studies arrived at a eutherian protokaryotype with 21–22 chromosomes [2–6], which proves to be similar to the present-day human karyotype. www.sciencedirect.com In silico search for conserved syntenies In the studies that integrate fish genome data, orthologous gene pairs are registered in the databases together with their chromosomal assignment [9–11]. Conserved synteny is present when two or more orthologous loci map to a single chromosome in each of two or more different species, irrespective of conserved gene order [32]. Conserved synteny defines characteristics of ancestral chromosomes. This approach has been shown to be especially informative when integrating fish genome data, which show a high frequency of lineage-specific intrachromosomal inversions. In the present study we have developed the in silico analysis of orthologous gene pairs in the form of a universal E-painting tool (Table S1 in supplementary online material). For the four species to be compared in this study the genes and their chromosomal assignment were listed in an Excel spreadsheet. This enables us to order the genes according to different principles. An ordering preference can be given to the position of these genes on human chromosomes, or a preference can be given to their order on pufferfish, chicken or zebrafish chromosomes (Figure S1 in supplementary online material). This procedure reveals at once the segments of conserved synteny in all the other species. When ordered according to pufferfish chromosomes the respective segments of common synteny in zebrafish, chicken and human are revealed. Synteny can be assigned according to the species of choice, and the segments of common synteny in the other species are indicated. Therefore, this Excel database is highly informative and will be even more informative if further genes and more species are included. As has been successfully applied in the reconstructions of Naruse et al. [9] and Woods et al. [11], only synteny conservation was considered, that is, the positions of the respective genes on the same chromosome. Linkage conservation, or conserved gene order, was neglected. In this way the numerous and mostly lineage-specific intrachromosomal rearrangements by inversion are circumvented and the scenario of karyotype evolution is made much clearer. ARTICLE IN PRESS Opinion TRENDS in Genetics Vol.xx No.xx Monthxxxx Table 2. Reconstruction of the tetrapod protokaryotype (TET) Tetrapod protochromosome 1 Chicken chromosome 2, 9, 16 2 1, 24 3 3, 14 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 5, 10 4q, 22 1 17, Z 7 8, 28 6 18, 19, 27 4p 12 11 21, 23, 26, 32 13 20 15 Eutherian chromosome [4] 3, 4, 5, 7, 8, 13, 17 3, 6, 9, 12, 15, X 1, 2, 5, 12, 19, 20 6, 9 2 7, 8 4, 11, 17 10 1 14 16 Xq 1, 3, X 18 1 4 19 21 Human chromosome 3, 5, 6, 7, 8q, 10, 18 2p, 3, 21, 11, 13, 15, X 1, 2p, 6, 8, 7, 16p, 20 11, 14, 15 4, 8p 7, 12pq, 22 5, 9, 18 2q 1, 19p 10q 17 Xq 1, 3, X 16q, 19q 1 5 20 12q, 22q Pufferfish chromosome 6, 15, 8, 21 7, 16, 2, 3 2, 3, 10, 14, 17 5, 13, 10, 14 12, 18 13, 19 4, 12, 18 2, 3 1, 15 2, 17, 18 2, 3, 7, 16 1, 7 9, 11 5, 13 8, 21, 9, 11 1, 7 9, 11 12 large chromosomes TET 1–3, which are all found spread on several human chromosomes remains questionable. However, when the reconstruction is seen from the perspective of the chicken genome, it only needs one fusion of a microchromosome to a macrochromosome to form both TET 2 and 3 (Table 2). (For the reconstruction of TET 1, see Figure 3.) The reconstruction reduces the TET protokaryotype to nZ18 chromosomes. This protokaryotype includes large chromosomes, such as TET 9 (HSA 1 and 19p), and small chromosomes, for example, TET 18, which is made up of the small segments from HSA 12q and 22q (EUT 21). Reconstruction of the teleost protokaryotype Next we deduced the protokaryotype of an ancestral teleost fish that lived before the third and extra round of whole-genome duplication [10,14]. Again, only synteny Chicken (GGA) Pufferfish (TNI) Tetrapod (TET) 1 Eutheria (EUT) 13 19 6 7 8 Human (HSA) 12 22 7 TRENDS in Genetics Figure 1. Reconstruction of the tetrapod protochromosome TET 6. The association of segments from HSA 12p/q and 22q is known from the eutherian protokaryotype [2–6] and found in chicken on GGA 1. In pufferfish these associations are on the paralogous chromosome pair TNI 13 and 19, which bears also a conserved segment from HSA 7 as does chicken chromosome GGA 1. All three segments form the protochromosome TET 6. www.sciencedirect.com 3 segments with three or more genes in the sample were considered. Genome data from pufferfish [10] and medaka [9] provided most of the information. The principle used to reconstruct the teleost protokaryotype is illustrated in Figure 2 (Table 2, and Table S2 in supplementary online material). For simplicity, chromosomes are presented as boxes of the same size. Although genes from a single pufferfish chromosome are usually found spread on several zebrafish chromosomes, a pairwise homology is evident. For instance, TNI 5 and TNI 13 genes are each localized on DRE 7, 13, 18 and 25, and TNI 6 and TNI 15 genes are each localized on DRE 2, 7 and 24. This organization is easily explained by the independent evolution of the two chromosome duplicates that followed after the extra round of whole-genome duplication in pufferfish and zebrafish. The mapping data from medaka confirm these reconstructions, which lead to an ancestral teleost protokaryotype with nZ12 chromosomes. The ancestral teleost chromosomes were numbered according to the size relations known from the pufferfish genome; for example, protochromosome TEL 1 consists of the large TNI 2 and 3 chromosomes, which are included in chromosomes TET 2, 3, 8 and 11 (Table 2), implying that TEL 1 is a large chromosome. By contrast, protochromosome TEL 12 consists of segments of TNI 6 and 15 and corresponds to the small chromosome OLA 20. Similar reconstructions of the ancestral teleost protokaryotype with human included as outgroup species have been put forward [9–11]. There is extensive overlap between these different approaches. Jaillon et al. [10] also arrived at a karyotype of nZ12 chromosomes in the haploid state, but did not describe the smallest chromosome TEL 12, which is made up of TNI 6 and 15. Instead of this, these authors divided the largest chromosome, our TEL 1 made up of TNI 2 and 3, into two chromosomes. Woods et al. [11] arrived at a karyotype of nZ11 chromosomes. They did not describe TEL 5, which is made up of TNI 1 and 7 and corresponds to chromosome H of Jaillon et al. [10]. Naruse et al. [9] compared medaka with zebrafish and described our protochromosomes TEL 1, 2, 3, 7, 8 and 9. But the much smaller sample size of genes and markers used in this comparison is responsible for more discrepancies between the data of Naruse et al. [9] and the other studies [10,11]. Reconstruction of the vertebrate protokaryotype For the final reconstruction of the 450-My-old ancestral vertebrate protokaryotype (VER) no outgroup species is available to date. Two slightly different procedures, not independent of each other, were followed. To begin we went back to the original mapping data in human, chicken, pufferfish and zebrafish as given in Table S1 in supplementary online material. The map positions of w1500 genes were available in all four species and the reconstruction is based on this data set. The genes and their chromosomal assignment were listed in an Excel spreadsheet as described in detail in Box 1. By ordering these genes by E-painting according to their chromosomal assignment, it became evident that when the first preference is given to the chromosome position in pufferfish, and a second minor preference is given to ARTICLE IN PRESS Opinion 4 7 13 18 25 7 13 18 25 3 6 4 6 5 6 13 13 7 4 13 17 20 23 22 24 19 11 2 10 TRENDS in Genetics Vol.xx No.xx Monthxxxx 17 20 1 13 17 1 3 5 13 14 22 24 15 19 1 8 10 21 14 17 2 18 20 4 1 5 10 21 5 8 21 1 3 9 12 1 3 6 9 12 6 8 11 23 6 8 11 23 5 14 21 5 14 21 16 19 16 19 5 10 15 24 2 5 10 15 2 6 8 20 22 2 6 8 20 22 2 7 24 2 7 24 Zebrafish (DRE) 12 9 12 19 21 2 8 19 21 5 7 5 7 4 10 10 14 11 16 11 16 13 14 13 14 4 4 17 20 20 Medaka (OLA) 15 Green spotted pufferfish (TNI) 4 12 2 6 3 3 1 11 4 9 11 1 7 8 21 7 16 1 15 6 3 5 8 9 10 12 Teleost protokaryotype (TEL) 5 6 7 8 9 10 Vertebrate protokaryotype (VER) TRENDS in Genetics Figure 2. The reconstruction of the teleost and ancestral vertebrate protokaryotype from the combined genome data of Danio rerio (DRE), Oryzias latipes (OLA) and Tetraodon nigrovirides (TNI). From teleosts to the extant fish species a whole-genome duplication has taken place [10,14]. Therefore, each ancestral teleost chromosome is represented by a pair of paralogous pufferfish chromosomes. Genes from a pair of pufferfish chromosomes are found spread on defined pairs of medaka and zebrafish chromosome sets (the respective chromosomes are indicated by their numbering). chicken, the genome conservation between these four genomes is most apparent, that is, the largest segments with shared synteny are revealed (Figure S1 in supplementary online material). This observation is crucial for the reconstructions proposed here. One has to be aware that this procedure introduced a bias towards the fish genomes. This is justified, because from the genome data of the four species compared in this study the pufferfish genome is the most conserved. In some way the pufferfish genome served as an ‘index fossil genome’, as did the aardvark karyotype for the reconstruction of the ancestral eutherian protokaryotype [4]. Therefore, the paralogous pairs of duplicated pufferfish chromosomes, which correspond to single TEL protochromosomes (Figure 2), are key to reconstructing the vertebrate protokaryotype. The other slightly different but not independent procedure is shown here for the reconstruction of the protochromosome VER 7 (Figure 3). The respective genes reside on the duplicated pair of pufferfish chromosomes TNI 8 and 21 and zebrafish chromosomes DRE 16 and 19. In human these genes are spread on five different chromosomes, HSA 1, 3, 6, 7 and 8, but in chicken these genes are concentrated mostly on chromosome GGA 2 with one further syntenic segment on microchromosome 23. For this example the VER protochromosome reconwww.sciencedirect.com struction is evident at once, when the intermediate states of the teleost and tetrapod protochromosomes are combined into a common scheme (Figure 3). This example is informative, because it illustrates the advantage of the stepwise procedure during the reconstruction described here. Both the teleost and tetrapod protokaryotypes were assembled with the help of information from an outgroup species. These intermediate protokaryotypes from O 300 Mya each prove to be already so similar that the reconstruction of the common vertebrate genome is possible in spite of the lack of information from an outgroup species. One or the other reconstruction could be questionable. Two alternative reconstructions are possible for VER 6. In the first, the two TET chromosomes 12 and 16 are not fused in the VER protokaryotype (Table 3f and Figure S2 in supplementary online material) but remain as separate VER protochromosomes. Accordingly, fusion of these chromosomes occurred only in the teleost lineage. Consequently this VER protokaryotype would consist of 12 chromosomes. In the second scenario, TET 12 and 16 arise by fission of VER 6, as is favored in the version proposed here. There is independent evidence for the second scenario because the Xenopus tropicalis scaffold_105 contains genes from HSA Xq and 5 (Table S3l in ARTICLE IN PRESS Opinion TRENDS in Genetics Vol.xx No.xx Monthxxxx 5 Chicken Pufferfish 23 Tetrapod 8 21 Vertebrate Teleost 2 15 Zebrafish Human 8 7 Pufferfish 8 16 19 1 Zebrafish Chicken 16 2 Human 7 21 16 2 7 21 16 2 8 8 8 19 2 8 19 2 7 8 19 2 6 21 19 2 3 21 19 2 8 21 19 23 1 1 3 6 7 8 TRENDS in Genetics Figure 3. Reconstruction of the protochromosome VER 7. Two slightly different procedures were followed. In the first, the original mapping data (from Table S1 in supplementary online material) were evaluated by E-painting (long black arrows). In the second, the reconstruction is at once evident by pure inspection, when the teleost and tetrapod protochromosomes, TEL 8 and TET 1, are apposed (small gray arrows). These intermediate protochromosomes were each reconstructed with information from an outgroup species and already represent states from O300 Mya. The segments of conserved synteny that are informative for this reconstruction are color-coded as indicated in the table. supplementary online material). This can be taken as independent evidence that TET 12 and 16 are fused in the ancestral vertebrate genome. Overall, only three segments containing 12 genes (from a total of 1500 genes compared in this study) could not be directly assigned (Table S4 in supplementary online material). A vertebrate protokaryotype with 11 chromosome pairs is therefore proposed. Comparison of the vertebrate protokaryotype with the teleost and tetrapod karyotypes In most cases the teleost protochromosomes were shown to represent ancestral vertebrate protochromosomes (Figures 2 and 4). However, some fissions and fusions must have occurred. The largest chromosome, TEL 1, is composed of genes from TNI 2 and 3 and is the fusion product of the protochromosomes VER 4 and 11 (Figure 2). In zebrafish, chicken and human the genes of these two protochromosomes are separated on different sets of chromosomes (Table S3d,k in supplementary online material). In the proposal of Jaillon et al. [10] these genes were assigned to the two different protochromosomes C and D. Another complication brings in VER 2, which is separated into TEL 7 and 11, as is evident from their distribution on two different sets of chromosomes in www.sciencedirect.com zebrafish (Figure 2, and Table S3b in supplementary online material). This time in pufferfish the separation is not as evident, because genes from TNI 13 are found on both TEL 7 and 11. A more complicated situation is seen for the fission of VER 1 and 3, resulting in three teleost protochromosomes, TEL 2, 4 and 6 (Figure 2; and Table S3a,c in supplementary online material). Most parts of VER 1 are identical with TEL 2 and consist of genes from TNI 10 and 14. A smaller segment made up of TNI 17 genes and some TNI 18 genes is split away from VER 1 and fused with another small segment from VER 3, which also contains TNI 18 genes and some TNI 20 genes. The fusion product of both these small segments results in TEL 4. The remaining larger segment of VER 3 is made up of TNI 4 and 12 genes, which are found in the teleost karyotype as TEL 6. TEL 2, 4 and 6 correspond to the protochromosomes J, B and A in the proposal of Jaillon et al. [10]. Concluding remarks There are several initiatives underway to use the genome data from mammalian and vertebrate species to reconstruct by in silico analysis an ancestral founder genome [10,11,15–17]. An approach similar to that proposed here was taken by Jaillon et al. [10]. Data from 6684 genes with proven orthology in pufferfish and human were combined ARTICLE IN PRESS Opinion 6 TRENDS in Genetics Vol.xx No.xx Monthxxxx 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X 100 My Eutheria 1 2 4 3 5 6 180 My Human (Homo sapiens, HSA) Theria 7 9 8 10 11 12 Marsupialia 210 My 13 14 15 16 17 Mammalia 18 310 My Synapsidia Tetrapod protokaryotype (TET) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 26 27 28 32 Z Protheria Tetrapoda 3 Diapsidia 2 Sarcopterygians 1 4 Aves 450 My 6 7 8 9 10 Chicken (Gallus gallus, GGA) Actinopterygians 5 11 Vertebrate protokaryotype (VER) Zebrafish (Danio rerio, DRE) Medaka (Oryzias latipes, OLA) 3R Atherinomorpha Teleostei 280–160 My 1 2 3 4 5 6 7 8 9 10 11 Teleost protokaryotype (TEL) 12 Percomorpha 80–60 My 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Pufferfish (Tetraodon nigrovirides, TNI) TRENDS in Genetics www.sciencedirect.com ARTICLE IN PRESS Opinion 4 92 TRENDS in Genetics Vol.xx No.xx Monthxxxx VER 18 47 41 65 TET TEL 90 DRE 15 15 TNI GGA 40 HSA TRENDS in Genetics Figure 5. An analysis of the number of interchromosomal rearrangements using GRIMM. To use GRIMM [18], synteny blocks were formed for each pair of genomes considered in this study. The synteny blocks were numbered consecutively and entered into the online version of GRIMM. In the source genome (which is always the ancestral genome), numbers were in ascending order, separated by chromosome delimiters. In the destination genome, within each chromosome synteny blocks were also in ascending order. In the specific proposal of an optimal rearrangement scenario, we subtracted the number of reversals (inversions) from the total number of operations, thus only counting the number of fissions, fusions and translocations between chromosomes. The numbers are given next to the arrows. to reconstruct a protokaryotype with 12 chromosome pairs. Similar to the procedure used here, the genes were assembled in synteny segments, and only 110 segments with the most complete conserved synteny were used for the reconstruction. However, 20 of these 110 segments were left unassigned in this study. Because of the availability of the chicken genome the reconstruction presented here could be performed in a stepwise manner. In a first step the ancestral tetrapod and teleost protokaryotypes were reconstructed. They served as intermediates for the subsequent reconstruction of a more plausible ancestral vertebrate protokaryotype with 11 protochromosomes (Figures 2 and 4). We must emphasize that currently it is not possible to determine where this putative vertebrate protokaryotype is positioned exactly between teleosts and tetrapods. As is evident from Figure 5 it seems to be biased towards the fish genomes. On the basis of the conserved segments considered in our data set the number of interchromosomal rearrangements (fusions, fissions and translocations) was analyzed with the GRIMM software (http://www.cse. ucsd.edu/groups/bioinformatics/GRIMM/) [18]. The transition from the ancestral vertebrate to the tetrapod protokaryotype requires far more rearrangements than the transition to the teleost protokaryotype. Furthermore, the similarity between the chicken and teleost proteogenome is apparent, which proved to be highly informative for the reconstructions proposed during this study. Many more rearrangements are necessary for the transition from the tetrapod to the human karyotype. Thus the human karyotype can no longer be considered to be the most conserved, as was assumed when the eutherian founder karyotype was reconstructed [2–6]. Indeed, from the karyotypes of the three species shown in Figure 4 (pufferfish, chicken, human) the human karyotype is the 7 most rearranged one. This is apparent, for instance, for the sex chromosomes. The avian Z chromosome is made up of a single syntenic segment of one protochromosome (VER 3), whereas the mammalian X chromosome is a conglomeration of segments from three protochromosomes [13]. Nevertheless, four human chromosomes (9, 11, 14 and 18) each represent a single segment of a vertebrate protochromosome (Figure 4). These syntenies have been conserved for O450 My. The consequences and significance of the conservation of these syntenic relationships are currently unknown. The summary of the comparative mapping data in Figure 4 also shows that in fish and in birds the ancestral syntenies are best conserved and least interrupted by interchromosomal translocations. Following the extra round of whole-genome duplication in teleost fish a process of deletion, subfunctionalization and neofunctionalization of complements of the duplicated genes took place – a process termed divergent resolution [11] – which facilitated speciation. Today, the teleost fish have diverged into w20 000 different species, making them the most speciesrich order of vertebrates. However, they show a highly conserved karyotype with a uniform haploid chromosome number of nZ24–25. In birds, there have been many fissions into smaller segments, known as microchromosomes. But the bird karyotype turns out to be highly conserved, with proven homology down to turtle [19], the respective lines separating from each other 210 Mya. From their karyotypes the green spotted pufferfish and chicken are indeed ‘living fossils’ [6], and this has been enormously helpful for the karyotype reconstruction presented here (Figure 3). Typical karyotype evolution is shown in the land vertebrates, where diploid chromosome numbers vary from six in the Indian muntjac [20] to 102 in the red viscacha rat [21] and there have been many interchromosomal exchanges by translocation. Only land vertebrates can be effectively isolated, when living on different continents or separated by geographic barriers (e.g. the Indian and Chinese muntjacs, with six and 42 chromosomes, respectively, are separated by the Himalayan mountain range). It is suggested that karyotype evolution is more prominent during allopatric speciation events, when species diverge with local isolation. Supplementary data Supplementary data associated with this article can be found at doi:10.1016/j.tig.2006.02.008 References 1 Hedges, S.B. and Kumar, S. (2002) Vertebrate genomes compared. Science 297, 1283–1285 2 Chowdhary, B.P. et al. (1998) Emerging patterns of comparative genome organization in some mammalian species as revealed by ZooFISH. 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