Chromosome evolution in eukaryotes: a multi-kingdom perspective Avril Coghlan1,2,* , Evan E. Eichler3,* , Stephen G. Oliver4,* , Andrew H. Paterson5,* and Lincoln Stein6,* 1 Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland 2 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK, CB10 1SA 3 Box 357730, Department of Genome Sciences, University Washington School of Medicine, Seattle, WA 98145, USA 4 Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester, UK, M13 9PT 5 Plant Genome Mapping Laboratory, University of Georgia, 111 Riverbend Road Rm 228, Athens, GA 30602, USA 6 Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA In eukaryotes, chromosomal rearrangements, such as inversions, translocations and duplications, are common and range from part of a gene to hundreds of genes. Lineage-specific patterns are also seen: translocations are rare in dipteran flies, and angiosperm genomes seem prone to polyploidization. In most eukaryotes, there is a strong association between rearrangement breakpoints and repeat sequences. Current data suggest that some repeats promoted rearrangements via non-allelic homologous recombination, for others the association might not be causal but reflects the instability of particular genomic regions. Rearrangement polymorphisms in eukaryotes are correlated with phenotypic differences, so are thought to confer varying fitness in different habitats. Some seem to be under positive selection because they either trap favorable allele combinations together or alter the expression of nearby genes. There is little evidence that chromosomal rearrangements cause speciation, but they probably intensify reproductive isolation between species that have formed by another route. Darwin's theory of natural selection [1,2], required that there must be naturally occurring variation among individuals of a species, and that this variation was stably inherited from parent to child. However, the nature of this genetic variation was a mystery until the discovery [3] that the chromosomes of Drosophila melanogaster and D. simulans differ by a large inversion in chromosome III. Then Dobzhansky reported that flies in wild populations of D. pseudoobscura have numerous gross rearrangements in their chromosomes that are polymorphic between strains. He realized that these rearrangements provided crucial evidence for Darwin's theory because chromosomal changes can `supply the raw materials for evolution', thereby enabling populations to evolve rapidly as environmental conditions change [4]. Common types of `gross' chromosomal rearrangement (usually several megabases long) can be detected at the microscope level and include deletions, inversions, duplications and translocations (Box 1). Rearrangements often occur in complex combinations of these basic types. Advances in molecular biology enabled the detection of fine-scale changes in chromosomal structure, shifting researchers' interest from gross rearrangements to smaller changes, such as single base pair substitutions and indels. Recently, with the sequencing of many eukaryotic genomes, the pendulum has swung back, bringing a renewed appreciation of how frequently chromosomal rearrangements occur, and how rearrangement rates vary between species. The rearrangements can reshuffle genes relative to regulatory elements, delete several genes or part of a single gene or even duplicate the entire genome (i.e. polyploidization ­ associated with changes in organism size and fitness [5]). Patterns of chromosomal rearrangement Fungi The whole-genome sequences (WGS) of w20 species of yeasts and filamentous fungi either are, or soon will be, available.Theseorganisms cover a wideevolutionary range: the hemiascomycetes are more evolutionarily diverged than the chordate phylum of the animal kingdom [6]. Within the fungi, the yeasts (e.g. Saccharomyces clade) represent excellent subjects for chromosome evolutionary studies. Saccharomyces, Kluyveromyces and Candida are all members of the hemiascomycete fungi that adopt, in at least part of their life cycle, a unicellular or yeast-like form. Kluyveromyces has a smaller genome than the other two genera and has 50% fewer chromosomes in its haploid complement than Saccharomyces [7]. This difference reflects the whole-genome duplication that occurred in an ancestor of Saccharomyces some point after it split with the lineage leading to Kluyveromyces. Candida has a genome size w30% larger than that of Saccharomyces but, similar to Kluyveromyces, has only eight chromosomes. (Although a large-scale genome duplication event might have occurred in the evolution of Candida, this was a separate and distinct event to that in the Saccharomyces lineage [7­10]). It is unclear whether the Saccharomyces duplication was the result of autopolyploidization or allopolyploidization Corresponding author: Stein, L. (lstein@cshl.edu). * All authors contributed equally to this article. Available online 19 October 2005 Review TRENDS in Genetics Vol.21 No.12 December 2005 www.sciencedirect.com 0168-9525/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2005.09.009 (Box 1; [8,11]). However, several forces have remodeled the genomes of Saccharomyces yeasts since this ancient duplication event and, in other yeast clades, extensive genome expansions have occurredby segmental rather than whole-genome duplication [10]. Smaller scale inversions, translocations and duplications, involving segments containingonlyafew genes, arealsocommoninfungalgenomes [9]. For further information on the organisms discussed in this article, see Table 1. Invertebrates The genomes of five invertebrate species have been sequenced: two nematodes (Caenorhabditis elegans and C. briggsae) and three arthropods (D. melanogaster, Anopheles gambiae and Bombyx mori). Most is known about gross chromosomal rearrangement in Drosophila because rearrangements are easily detected in the chromosomes of their giant salivary glands. The most common type of gross chromosomal rearrangement are paracentric inversions, which do not span the centromere. Paracentric inversions are common polymorphisms within the fly species: different populations of D. melanogaster alone harbor O500 inversion polymorphisms [12]. Most other Drosophila species also have many known paracentric inversion polymorphisms [13]. Paracentric inversions are also common as fixed differences between fly species [14]. Comparison of the WGS of D. melanogaster and A. gambiae has revealed that, although their chromosomal arms have retained similar gene content since they diverged, numerous gross and fine-scale paracentric inversions have occurred within each arm [15]. Thus, the same pattern of frequent paracentric inversions is seen within Drosophila species and between Drosophila and other dipterans, such as Anopheles. Little was known about the evolution of nematode chromosomes until recently, when the genome of Caenorhabditis briggsae was sequenced and compared with that of C. elegans [16]. As in fly genomes, fine-scale inversions are common in these nematodes' genomes (although their inversions cannot be classified as paracentric because nematode chromosomes lack localized centromeres). Conversely, translocations of whole chromosome arms or small segments of arms are mysteriously rare in fly genomes, but have occurred frequently in the Caenorhabditis genomes ­ there has been almost one translocation for every five inversions [16]. Polyploidy is relatively rare in animals (compared with plants); however hundreds of polyploid invertebrates are known, including arthropods, nematodes, molluscs and flatworms [5]. Indeed, two diploid species that have been chosen for sequencing ­ the root knot nematode Box 1. Gross rearrangements ­ a primer Gross rearrangements are large-scale changes in chromosome structure between species or between individuals in a species. Large rearrangements can be seen in light microscope examinations of chromosomal spreads. Smaller rearrangements can be identified by genetic mapping or genome sequencing. Deletions: are the loss of a piece of chromosome, and can range in size from microdeletions of a few base pairs to the deletion of a chromosome arm or even an entire chromosome. Duplications: are the creation of an extra copy of a piece of chromosome. These are further subclassified into tandem duplications, in which the copy is adjacent to the original segment and has the same orientation, and inverted duplications in which the copy is reversed in orientation relative to the original. Inversions: occur when a region of the chromosome is flipped `head to tail' but its position is unchanged. Translocations: occur when a region of the chromosome moves from one location to another. The two most common type of translocation are reciprocal translocations, when two non-homologous chromosomes exchange chunks of DNA, and transpositions, when a chromosomal region simply moves from one location to another without a reciprocal change. Chromosomal fusions and fissions: occur when two chromosomes fuse together, forming a single chromosome, or, conversely, when a single chromosome splits, forming two new chromosomes. Polyploidization: occurs when the entire genome is duplicated, doubling or trebling the number of chromosomes. It can be followed by an evolutionary period in which the genome is gradually reduced in size, sometimes back to its pre-polyploidization size and chromosome count, a process known as diploidization. However, the traces of ancient polyploidization are preserved in the form of multiple copies of genes. Polyploidization can occur by either autopolyploidization, in which the genome is duplicated within a single species, or allopolyploidization, in which the genome is duplicated when two closely related species cross-hybridize [7­11]. Table 1. Genomes sizes and karyotypes of model organismsa Species Genome size Number of chromosomes Number of genes Drosophila melanogaster [124] 180 Mb (including heterochromatin) 3 A, XY w13 600 Anopheles gambiae [125] 278 Mb 2 A, XY w13 600 Bombyx mori [126] 429 Mb 28 A, ZW w18 500 Caenorhabditis elegans [127] 100 Mb 5 A, X w19 100 Caenorhabditis briggsae [16] 104 Mb 5 A, X w19 500 Homo sapiens [59,128] 3100 Mb 22 A, XY w20 000­25 000 Mus musculus [56] 2500 Mb 19 A, XY w22 000 Rattus norvegicus [60] 2750 Mb 20 A, XY w21 000 Gallus gallus [61] 1100 Mb 38 A, ZW w20 000­23 000 Takifugu rubripes [129] 365 Mb 22 Ab w31 100 Arabidopsis thaliana [37] 125 Mb 5 A w25 500 Oryza sativa [130,131] 420­470 Mb 12 A w32 000­55 600 Saccharomyces cerevisiae [73] 12.1 Mb 16 A 5538 (genes of R100 codons) [132] 5773 [133] a The karyotype is given in terms of the haploid number of autosomes (A) and sex chromosomes (X,Y,Z or W) b The sex chromosome has not yet been identified for Takifugu rubripes. Review TRENDS in Genetics Vol.21 No.12 December 2005674 www.sciencedirect.com Meloidogyne hapla and the water flea Daphnia pulex have both diploid and polyploid populations in the wild ([17,18]; D. Bird and J. Colbourne, personal communication). Vertebrates With the exception of teleost fish and some amphibian species [19,20] whole-genome duplications are not a common occurrence in the recent evolution of most vertebrate lineages. Most vertebrate genome sequence analyses, however, support at least one ancient wholegenome duplication occurring w500 million years ago (Mya). Instead, chromosomal rearrangements (including fissions, fusions and translocations) and the differential expansion of repetitive sequences have been the primary forces of chromosomal change among the vertebrates [21]. Several studies have revealed numerous short intrachromosomal rearrangements in mammals [22]. For example, comparisons of the human, mouse and rat genome sequences uncovered thousands of tiny `microrearrangements', which range from part of a gene or intergenic region to several genes long. Although a small proportion of these might be artefacts stemming from errors in the draft genome assemblies of mouse and rat [22], the number of intrachromosomal rearrangements observed is consistent with studies showing that inversions are common in invertebrate animals [14,16]. Even if we disregard putative microrearrangements, mammalian genomes contain far more short syntenic blocks than we would expect to see if rearrangement breakpoints occurred at random sites throughout the genome [23]. This has led to the controversial suggestion that chromosomal breakages tend to reoccur at `fragile sites' or `hotspots' in mammalian chromosomes [22,24]. This hypothesis is also supported by evidence that several other types of rearrangement tend to recur at particular sites (e.g. movements of centromeres [25,26] and segmental duplications [27­29]). Plants (angiosperms) Polyploidization and subsequent diploidization seem to have a greater role in the evolution of angiosperms than in other eukaryotes [30]. It has long been suspected that many angiosperms have undergone polyploidization events during their evolutionary history [31], and recent findings suggest that virtually all angiosperms are ancient polyploids. Early evidence for ancient segmental duplications in Arabidopsis [32,33], Oryza [34,35] and Sorghum [36] has been confirmed by analysis of the nearly completed Arabidopsis [30,37­41] and Oryza [42,43] genome sequences. In Arabidopsis, there seem to have been three different episodes of duplication [30,44], but only 30% of the genes duplicated by the most recent event (!83 Mya) still retain a duplicate copy. Similarly, in Oryza, only w21% of genes have retained duplicate copies since the polyploidization event that occurred w70 Mya [43,45]. Polyploidization can obscure other types of rearrangement in plant genomes because the loss of different copies of a duplicated gene in different lineages will lead to incongruities in the comparative maps of plant taxa [45]. However, computer programs can reconstruct the ancestral gene order that probably existed before polyploidization, thereby enabling the identification of regions of synteny and colinearity between distantly related plants [30]. After reconstruction of the ancestral state, we see striking parallels in the gene order among diverse plants, which are peppered by many exceptions. These changes in gene order have arisen because of transpositions of single genes (generally to positions nearby on the chromosome), movements of transposable elements (which in some cases have carried unrelated sequences with them) and diploidization of ancient segmental duplications [46]. Comparisons of the sequences of BACs from genomes that have not yet been fully sequenced have provided further insights into recent plant chromosomal evolution [46­53]. For example, analysis of sequenced BACs from a pair of Zea mays inbred strains that diverged !1 Mya showed the loss of colinearity in 27/72 (30%) of protein-coding genes, representing a dramatic rate of genomic change [54]. Dramatic expansion in the genome sizes of plants has occurred over short evolutionary times as a result of amplification of rapidly evolving repeat families, without destroying the underlying synteny [48]. Plant repetitive DNA seems to be different from that of many other taxa, in that individual repeat families only have a life span of a few million years [55]. This is in sharp contrast with mammalian repeat families: for example, 70% of L1 elements have been retained since humans diverged from mice 75 Mya [56]. Rearrangement rates Although difficult to estimate reliably (Box 2), the rates of genomic rearrangement appear to vary between different species and can vary significantly throughout the evolutionary history of a species. Invertebrates The rate of chromosomal rearrangement in invertebrates is almost twice that in vertebrates: Drosophila has w0.05­ 0.07 breakpoints per Mb per million years (Myr) [14], compared with only w0.03 breakpoints per Mb per Myr in rodents (including both gross and fine-scale rearrangements [22]). This difference is probably largely because of life history traits. Drosophila species have a larger effective population size than rodents (w106 versus w105 ), and more generations per year than rodents (wten generations per year versus wtwo generations per year [57]). There are also large differences in rate among other invertebrates. Caenorhabditis has a surprisingly high rate of w0.5­0.7 breakpoints per Mb per Myr [16], which could be due to an even larger effective population size and shorter generation time in Caenorhabditis than in Drosophila. However, chromosomal rearrangements can be less deleterious in nematode genomes because nematodes have holocentric chromosomes [58]. Vertebrates The availability of WGS from several vertebrate species [56,59­61] has provided unprecedented resolution in Review TRENDS in Genetics Vol.21 No.12 December 2005 675 www.sciencedirect.com studies of vertebrate chromosomal evolution. For example, a four-way comparison of the human, mouse, rat and chicken genomes revealed that rearrangements in the chicken lineage have been relatively infrequent and mainly intrachromosomal in nature (Figure 1). A similarly slow rate of chromosomal evolution was observed in fish, by comparing gene order in Tetraodon and Fugu to that in human [61,62]. In contrast to chicken and fish, the rate of interchromosomal rearrangements in mammals has accelerated [61]. Rodent lineages, in particular, have undergone far more rapid evolutionary change than primates since the two lineages diverged (3.2­3.5 rearrangements per Myr in rodent versus 1.6 rearrangements per Myr in humans). Consequently, the gene order is more highly conserved between the human and chicken genomes than between humans and rodents. It is possible to reconstruct the gene order along the chromosomes of the ancestral placental mammal with considerable confidence [22,61]. For example, from sequence and comparative mapping data, we know that human chromosomes 3 plus 21; 4 plus 8; 12 plus 22a; and 12 plus 22b were originally part of larger chromosomes that later underwent fissions in several mammalian lineages. Comparative gene maps indicate that the rate of rearrangement in vertebrates has varied by more than an order of magnitude during the past 500 Myr [61,63]. Using chicken as an outgroup, Burt et al. [63] distinguished three different phases in vertebrates' chromosome evolution. During the initial phase (100­300 Mya) chromosome rearrangement was slow (0.2 fixed rearrangements per Myr). This was followed by an episode of elevated chromosomal rearrangement (65­100 Mya), during which the rate increased to O1.1 rearrangements per Myr. Since the mammalian radiation, rates have varied radically in a lineage-specific fashion. In some lineages (e.g. the human branch), chromosomal rearrangement has slowed to a crawl (w0.1 events per Myr), whereas in others (e.g. the New World Monkeys and hylobatid primate branches), rates of chromosomal rearrangement have suddenly surged (1.5­2.3 events per Myr). Plants The extensive variation in angiosperm genome sizes makes it challenging to devise rate estimates that are directly comparable to those of taxa with smaller size variation. For example, rice and wheat differ by at least 20 major rearrangements; they last shared common ancestry w50 Mya and have genome sizes of 420 Mb and w15 000 Mb, respectively. The 40-fold difference in genome size means that the comparison of rice with wheat can be used to make two rate estimates that are 40fold apart (0.0008 breakpoints per Mb per Myr, or 0.00002 breakpoints per Mb per Myr). This is an extreme case, but by no means the only one. For example, rate estimates for the more rapidly rearranging plant genomes, Arabidopsis TRENDS in Genetics Chicken Human Lemur Mouse Shrew Horse Pig Dolphin Millions of years ago 0.2 1.1 0.1­2.3 Rearrangements per million years : 300 200 100 65­85 Figure 1. Using chicken as an outgroup, Burt et al. [63] distinguished three phases in vertebrate chromosomal evolution. During the initial phase (100­300 Mya) chromosome rearrangement was slow. This was followed by an episode of elevated rearrangement (65­100 Mya). Since the mammalian radiation, rates have varied radically in a lineage-specific fashion. Box 2. Estimating the rate of chromosomal rearrangement To estimate the rate of chromosomal rearrangement, we can divide the number of rearrangements that have occurred since two species diverged by their divergence date. However, genome size and the type of chromosomal rearrangement vary between lineages, so Ranz et al. [14] estimated the rate of rearrangement as the number of chromosomal breakages caused by rearrangements (rather than the number of rearrangements itself) per Mb per Myr. Each reciprocal translocation causes two breakpoints, each inversion two breakpoints and each transposition three breakpoints [121]. Using this approach, Ranz et al. [14] estimated that fly genomes evolve two orders of magnitude faster than those of mammals, and at least fivefold faster than the most dynamic plant genomes, in the Arabidopsis­Brassica clade. The calculation of rearrangement rates is fraught with problems. The scarcity of fossils means that dates must often be estimated using the unreliable molecular clock assumption, leading to errors in divergence date estimates (discussed in detail by Graur and Martin [122]). The estimate of the number of rearrangements can also have large margins of error, sometimes because of low data quality, and sometimes because of ascertainment issues. For example, rearrangement rates estimated using genetic maps will miss rearrangements that are smaller than the spacing between markers. Conversely, partially sequenced genomes might miss large rearrangements that are larger than the average contig. When two finished genomes are compared, assembly errors can incorrectly inflate the apparent number of rearrangements. Even when comparing two finished, high-quality genomes, it can be difficult to accurately estimate the number of rearrangements owing to inaccuracy in the methods used to detect syntenic regions. A standard approach is to make nucleotide-level alignments, and to merge contiguous alignments into larger syntenic regions, but the number of syntenic regions found depends on the alignment and `merging' algorithms used. Furthermore, estimating the number of rearrangements from a set of syntenic regions is not trivial, because it can be impossible to distinguish a transposition from overlapping inversions [123] or a translocation from a fusion followed by a fission event. Improved methods for finding syntenic regions, and inferring rearrangements from them, can reduce these sources of error. Review TRENDS in Genetics Vol.21 No.12 December 2005676 www.sciencedirect.com and Brassica, vary by approximately fivefold (0.002 ­ 0.009 breakpoints per Mb per Myr). Mechanisms of chromosomal rearrangement Fungi Even in the relatively small yeast genomes, there are several repeated sequences: rRNA genes, tRNA genes, telomeres, telomere-associated genes and transposons (Ty elements). These repeated sequences can act as regions of homology that can promote gene duplication or genome rearrangement events. For example, recombination between the long terminal repeats (LTRs or delta sequences) of two Ty elements on the same chromosome arm will result in the excision of a chromosomal segment, which might be subsequently reinserted into another chromosome through delta!delta recombination. Mating events could then result in the duplication of the segment (a so-called chromosome homology region or CHR), with the gene order frequently being circularly permuted (see Refs [64,65]). Unequal meiotic crossing-over between Ty elements located at different sites within a chromosome arm will produce a duplication-deletion event, with the chromosome bearing the tandem duplication usually surviving [66­68]. Repeated sequences can also promote major karyotypic changes through ectopic homologous recombination [66,69]. Rearrangements in Saccharomyces cerevisiae are frequently associated with Ty elements or their LTRs [70­72]; a survey of interspecific chromosomal translocations in species of the Saccharomyces `sensu stricto' clade [70] revealed that the breakpoints were always associated with repeated sequences, such as Tys, their LTRs or tRNA genes in the paradigmatic S. cerevisiae genome sequence [73]. Studies on filamentous fungi also point to a key role for transposable elements in promoting chromosomal rearrangements [74,75]. Invertebrates Several lines of evidence suggest that repetitive elements also trigger chromosomal rearrangements in invertebrates. Ca´ceres et al. [76] found compelling evidence that repetitive elements are responsible for Drosophila species' rich inversion polymorphisms, when they sequenced the breakpoints of the 2j polymorphic inversion from D. buzzattii. They found that chromosomes carrying the 2j inversion contained transposon insertions at both breakpoints, and hypothesized that the inversion arose by unequal meiotic crossing-over between the two copies of the transposon. They subsequently sequenced the 2j region in 39 different flies, and found that the inversion breakpoints are genetically unstable hotspots that are rapidly accumulating transposons and other microrearrangements [77]. Inversion polymorphisms in D. melanogaster and in Anopheles species probably originate by a similar mechanism [78,79]. There is evidence that repetitive elements also generate chromosomal rearrangements in nematodes: the breakpoints of translocations that have occurred since the divergence of C. elegans and C. briggsae are associated with repetitive elements in the C. elegans genome (although it is not known which species they occurred in), and most of these rearrangements occur in the repeat-rich chromosome ends [16,80]. Vertebrates Similar to fungi and invertebrates, mammalian breakpoint regions also tend to be enriched for various classes of repeats [27,81,82]. For example, breakpoints in conserved synteny between chicken and mammalian chromosomes map to repeat-dense centromeric locations [61]. Similarly, an analysis of conserved synteny between human and mouse for human chromosome 19 revealed that ten out of fifteen breakpoint regions examined mapped to clusters of tandem gene duplications [81]. There is also a striking association between synteny breakpoints and large segmental duplications. Global analyses of human and mouse breakpoints have found that 25­53% of all breakpoints map to regions of segmental duplication within either species' genome [29,82]. This association is equally prevalent among interchromosomal and intrachromosomal rearrangements. A similar trend is seen among great-ape and human chromosomes, where sites of chromosome fusion (human chromosome 2), reciprocal translocation [gorilla chromosome t(4:19)] and pericentric inversion (human chromosomes 12, 15 and 18) are enriched for segmental duplications [83­87]. It is tempting to explain the association of large-scale rearrangements and segmental duplications by speculating that such regions promote such events by non-allelic homologous recombination. However, comparisons among the mouse, human and rat genomes do not support such a model [29]. When only rodent or primate lineage-specific breakpoints are considered, the association between rearrangement breakpoints and segmental duplications disappears. In other words, chromosomal rearrangements and segmental duplications are associated with each other between different lineages but not within the same lineage. This suggests that segmental duplications do not themselves cause chromosomal rearrangements, but that both are manifestations of the instability of particular chromosomal regions. Plants Inversions of entire chromosome arms are found even in closely related angiosperm taxa, such as Lycopersicon (tomato family), Solanum (potato) [88­90] and recently diverged diploids of the Gossypium (cotton) genus [91,92]. A variety of mechanisms have been proposed to explain gene gain, loss, inversions and tandem duplications in plants, including transposition and illegitimate recombination [46,47,50,51]. Local regions of repetitive DNA, such as centromeres, have also been implicated in many largescale rearrangements. Indeed, the difficulty of detecting any traces of ancient duplication in the centromeric regions of both Arabidopsis and Oryza might be due to the instability of these regions. Functional consequences of rearrangement Are chromosomal rearrangements merely a nuisance for the genome, or do they have functional significance in the short term (e.g. by enabling a species to adapt to changing Review TRENDS in Genetics Vol.21 No.12 December 2005 677 www.sciencedirect.com environmental conditions) or in the long term (e.g. by facilitating speciation)? Fungi In fungi, chromosomal rearrangements might be expected to promote speciation by imposing post-zygotic isolation between strains (each translocation event results in a 50% drop in spore viability in hybrids of strains with wild-type and rearranged genomes). However, there is no correlation between the incidence of chromosomal translocations and the sequence-based phylogeny of Saccharomyces `sensu stricto' species. Instead, it appears that species arise through another mechanism, although chromosomal translocations can intensify the reproductive isolation between species that has already formed by another route [70]. The problem with such retrospective surveys is that it is not possible to distinguish the impact on fertility of genetic differences caused by sequence divergence, from that of chromosome rearrangements. However, in the genetically malleable S. cerevisiae, it has proved possible to separate the contribution of these two factors, using an interventionist strategy. Delneri et al. [11] inserted loxP sites into specific regions of S. cerevisiae chromosomes and mediated chromosome breakage by inducing the Cre nuclease [93]. Next, they screened for survivors in which the same chromosomal translocations found in S. mikatae [70] had been produced in S. cerevisiae. They found, using inter-specific crosses, that the rearrangement of the S. cerevisiae genome so that it was collinear with that of S. mikatae resulted in the elevation of spore viability from ! 3% to 20­30% in some zygote clones. Thus chromosomal rearrangements have a significant, although not dominant, role in the reproductive isolation between yeast species. Moreover, analysis of the engineered S. cerevisiae strains [94], and evidence from natural isolates [95], shows that such chromosomal rearrangements can have adaptive significance, thus accelerating the speciation process. Invertebrates Coluzzi et al. [96] observed three non-interbreeding populations of A. gambiae that live in the same region of Mali (and differ by chromosomal inversions) and speculated that chromosomal rearrangements might be contributing to speciation in the A. gambiae species complex (Figure 2). This is difficult to prove: even a highly significant coincidence in time between chromosomal rearrangements and speciation does not prove a causal relationship. However, some evidence suggests that inversions have contributed to speciation between the close relatives D. pseudoobscura and D. persimilis, because inversions are found within the genomic regions associated with hybrid sterility [97]. There are also some hints that chromosomal inversions might be contributing to speciation in the apple maggot fly (Rhagoletis pomonella), because genic differences between two reproductively isolated races of fly seem to be disproportionately located within inversions [98]. These observations suggest that chromosomal rearrangements reduce recombination between the genomes of incipient species, thereby enabling genetic differences to accumulate within the rearranged regions [99]. Some inversion polymorphisms in Drosophila and Anopheles species are correlated with seasonal changes and altitude, and so are thought to confer varying fitness in different habitats [100,101]. Other Drosophila inversions are associated with variations in body size and shape [102], whereas A. gambiae has polymorphic inversions associated with differences in Plasmodium infection rates [103]. In the Australian grasshopper Keyacris scurra, two inversion polymorphisms located on different chromosomes are associated with differences in body size and viability (Figure 3; [13]). At the molecular level, Schaeffer et al. [104] have shown that some Drosophila inversions are under positive selection because they keep favorable allele combinations together. However, at least one Drosophila inversion seems to be adaptive because it alters the expression level of a nearby gene [105]. Vertebrates Among vertebrates, the relationship between chromosomal rearrangement and speciation has been the subject of considerable speculation [106]. The observation that closely related vertebrate species frequently differ in chromosome number or morphology (as shown by a change in the centromere position) might be viewed as weak, indirect evidence of a cause-and-effect relationship. Direct evidence, particularly experimental data, is sparse for vertebrates. Most of the advances in this area have TRENDS in Genetics Savanna : 2R b or 2R cu or 2R bcu or 2R + Bamako : 2R jcu or 2R jbcu Mopti : 2R bc or 2R u or 2R + (standard) 2Rj 2Rl 2Rbk 2Rd 2Rb 2Rc 2Ru (a) Polymorphic inversions in A. gambiae 2R (b) Inversion genotypes of non-interbreeding populations in Mali Figure 2. Do chromosomal rearrangements contribute to speciation in Anopheles gambiae? (a) The main polymorphic paracentric inversions in A. gambiae chromosome arm 2R [96]. All inversions are based on the standard 2R genotype, except for inversion 2Rk, which is based on a pre-existing 2Rb inversion. For example, a chromosome with arrangement 2Rjcu has inversions j, c and u. (b) Coluzzi et al. [96] observed three non-interbreeding populations of A. gambiae (named Bamako, Savanna and Mopti) that live in the same region of Mali (shown in red). The three populations differ by chromosomal inversions that might be contributing to speciation in A. gambiae. Review TRENDS in Genetics Vol.21 No.12 December 2005678 www.sciencedirect.com been theoretical, with a particular emphasis on modeling the reduced evolutionary fitness of chromosomal hybrids thought to form as a result of such events. In one model by Pardo-Manuel de Villena and Sapienza [107], a bias in the segregation of rearranged chromosomes during maternal meiosis helped to explain the rapidity with which such events become fixed. Navarro and Barton [108] argued for another model (parapatric speciation), in which the suppression of recombination that results from chromosomal rearrangement serves as a genetic barrier that leads to the accumulation of post-zygotic isolation genes genes that have alleles that are compatible within species but incompatible across species ­ as suggested for Rhagoletis. The finding of an increased number of positively selected genes on rearranged as opposed to collinear chromosomes between chimpanzee and human [109] provides some controversial [110,111] support for this new model of chromosomal speciation. Studies of natural large-scale structural variation (50 kb­1000 kb) within the human genome [112,113] suggest that rearrangements (deletions and duplications) are common in the human population. It is possible that the deleterious effects of chromosomal rearrangements and their role in speciation have been over-estimated and that such rearrangements might confer a selective advantage on evolving populations [114,115]. Plants The exact relationship between chromosomal rearrangement and speciation remains unclear in plants; but, in at least one example, recombination between two divergent diploid species appears to have provoked a speciation event in sunflowers [116]. Although there is little direct evidence for the impact of gene or chromosomal rearrangements on phenotype in angiosperms, the occurrence (even in large genomes) of `gene-rich' regions that lack repetitive DNA [117] might reflect selection against rearrangement events (e.g. gain of repetitive DNA) near certain genes. Alternatively, repeat-rich regions might arise because the invasion of a chromosomal region by repetitive DNA tends to lead to a cascade, as recently activated transposable elements continually jump into older transposable elements [55]. That is, the formation of non-random concentrations of repetitive DNA might leave other genomic regions `gene-rich' by default rather than by selection. A related observation in plants is that quantitative trait loci (QTLs) are frequently found concentrated in common genomic regions that affect different traits [118]. Computer simulations have suggested that domestication can confer a selective advantage to the de novo evolution of tightly linked combinations of genes or `supergenes' [119]. Rearrangement by mechanisms such as transposition and illegitimate recombination might increase the opportunity for such supergenes to form. However, there are several plausible alternatives to the hypothesis of de novo evolution of tightly linked combinations of domestication-related supergenes, such as ancient evolution of QTL clusters and non-random distribution of allelic variation [118]. Concluding remarks Despite 80 years of research, many gaps remain in our understanding of the mechanisms of both small-scale and large-scale chromosomal rearrangements and the role that they have in evolution. Although there are many similarities among the eukaryotic kingdoms with respect to the characteristics of chromosomal rearrangements and their tendency to colocalize with repetitive DNA and duplicated regions, there are also significant differences. Polyploidization, a dominant force in the evolution of plants and fungi, occurs far less frequently in invertebrates, and is a rare event in most vertebrate lineages. Chromosomal rearrangements can vary by more than an order of magnitude both within and between kingdoms, and the rate of rearrangement within a lineage appears to be a variable that can change over time. What evolutionary pressures are responsible for these dramatic rate changes? Over the next few years, our understanding of these phenomena will be greatly aided by sequencing deeply into the evolutionary tree. The National Human Genome Research Institute (http://www.genome.gov/10002154) and the US Department of Energy (http://www.jgi.doe. gov/sequencing) together list w20 fungal species, w40 invertebrates and w25 vertebrates that are in various phases of preparation for or execution of whole-genome sequencing. In plants, sequencing is under way for the maize (http://www.jgi.doe.gov/sequencing/) and Medicago trunculata (legume) genomes. This collection of sequenced genomes, together with those that are already published, will provide us with a tremendous range of variation in genome size and rearrangement rate both within and between species. For example, among the 12 species of the genus Drosophila scheduled for sequencing are those with numerous fixed and polymorphic inversions (e.g. D. willistoni), those with few (D. simulans) and some that have chromosomes with disparate numbers of inversions TRENDS in Genetics Chromosome 1 Chromosome2 Figure 3. The Australian grasshopper Keyacris scurra carries two inversion polymorphisms. Individuals carrying the nine possible genotypes of these polymorphisms differ in body size and viability [13]. This is one of the most striking examples known of an inversion polymorphism with a visible phenotype. Review TRENDS in Genetics Vol.21 No.12 December 2005 679 www.sciencedirect.com on different chromosomes (e.g. D. grimshawi [13]). Analysis of these genomes might reveal why inversions proliferate in some species and often cluster along dipteran chromosomes [13,120]. The Drosophila sequences should also shed light on the evolution of genome size, which varies from w150 Mb (D. erecta) to w330 Mb (D. virilis) among the twelve species. In plants, it will be of great interest to compare the genome of rice, a 450-Mb diploid, with that of maize, an ancient tetraploid with a repeat-rich 2.2-Gb genome. Another project that is likely to help elucidate the biology of genome organization is the Oryza Map Alignment Project (http://www.omap. org), an effort to develop genome maps of 11 wild relatives of domestic rice. The collection of species to be sequenced is evenly split between tetraploids (e.g. O. minuta), and their diploid relatives (e.g. O. punctata), thereby enabling multiple independent polyploidization events to be studied. Acknowledgements The following funding sources supported this work: A.C is supported by the Wellcome Trust; E.E.E. holds a NIH grant (GM58815); A.H.P holds grants from the National Science Foundation (DBI-0115903 and DBI- 0211700) and USDA-NRI (02­01412); S.G.O holds grants from the BBSRC and the Wellcome Trust; L.S holds grants from NSF (DBI- 0321685), USDA (CSREES-00­52100­9622) and NIH (HG02639). A.C. thanks Richard Durbin and Des Higgins for generously allowing her to complete this work in their laboratories. References 1 Darwin, C. (1859) On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life, John Murray 2 Powell, J.R. (1987) In the air ­ Theodosius Dobzhansky's `Genetics and the Origin of Species'. Genetics 117, 363­366 3 Sturtevant, A.H. (1921) A case of rearrangement of genes in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 7, 235­237 4 Dobzhansky, T. 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