Chromosomal Inversions between Human and Chimpanzee Lineages Caused by Retrotransposons

The long interspersed element-1 (LINE-1 or L1) and Alu elements are the most abundant mobile elements comprising 21% and 11% of the human genome, respectively. Since the divergence of human and chimpanzee lineages, these elements have vigorously created chromosomal rearrangements causing genomic difference between humans and chimpanzees by either increasing or decreasing the size of genome. Here, we report an exotic mechanism, retrotransposon recombination-mediated inversion (RRMI), that usually does not alter the amount of genomic material present. Through the comparison of the human and chimpanzee draft genome sequences, we identified 252 inversions whose respective inversion junctions can clearly be characterized. Our results suggest that L1 and Alu elements cause chromosomal inversions by either forming a secondary structure or providing a fragile site for double-strand breaks. The detailed analysis of the inversion breakpoints showed that L1 and Alu elements are responsible for at least 44% of the 252 inversion loci between human and chimpanzee lineages, including 49 RRMI loci. Among them, three RRMI loci inverted exonic regions in known genes, which implicates this mechanism in generating the genomic and phenotypic differences between human and chimpanzee lineages. This study is the first comprehensive analysis of mobile element bases inversion breakpoints between human and chimpanzee lineages, and highlights their role in primate genome evolution.     

Molecular Definition of Pericentric Inversion Breakpoints Occurring during the Evolution of Humans and Chimpanzees

High-resolution G-banding analysis has demonstrated remarkable morphological conservation of the chromosomes of the Hominidae family members (humans, chimpanzees, gorillas, and orangutans), with the most notable differences between the genomes appearing as changes in heterochromatin distribution and pericentric inversions. Pericentric inversions may have been important for the establishment of reproductive isolation and speciation of the hominoids as they diverged from a common ancestor. Here the previously published primate karyotype comparisons, coupled with the resources of the Human Genome Project, have been used to identify pericentric inversion breakpoints seen when comparing the human karyotype to that of chimpanzee. Yeast artificial chromosome (YAC) clones were used to detect, by fluorescencein situhybridization, five evolutionary pericentric inversion breakpoints present on the chimpanzee chromosome equivalents of human chromosomes 4, 9, and 12. In addition, two YACs from human 12p that detect a breakpoint in chimpanzee detect a similar rearrangement in gorilla.     

Characterization of the human lineage-specific pericentric inversion that distinguishes human chromosome 1 from the homologous chromosomes of the great apes

The human and chimpanzee genomes are distinguishable in terms of ten gross karyotypic differences including nine pericentric inversions and a chromosomal fusion. Seven of these large pericentric inversions are chimpanzee-specific whereas two of them, involving human chromosomes 1 and 18, were fixed in the human lineage after the divergence of humans and chimpanzees. We have performed detailed molecular and computational characterization of the breakpoint regions of the human-specific inversion of chromosome 1. FISH analysis and sequence comparisons together revealed that the pericentromeric region of HSA 1 contains numerous segmental duplications that display a high degree of sequence similarity between both chromosomal arms. Detailed analysis of these regions has allowed us to refine the p-arm breakpoint region to a 154.2 kb interval at 1p11.2 and the q-arm breakpoint region to a 562.6 kb interval at 1q21.1. Both breakpoint regions contain human-specific segmental duplications arranged in inverted orientation. We therefore propose that the pericentric inversion of HSA 1 was mediated by intra-chromosomal non-homologous recombination between these highly homologous segmental duplications that had themselves arisen only recently in the human lineage by duplicative transposition.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Justyna M. Szamalek and Violaine Goidts are contributed equally to the paper.     

Discovery of human inversion polymorphisms by comparative analysis of human and chimpanzee DNA sequence assemblies

With a draft genome-sequence assembly for the chimpanzee available, it is now possible to perform genome-wide analyses to identify, at a submicroscopic level, structural rearrangements that have occurred between chimpanzees and humans. The goal of this study was to investigate chromosomal regions that are inverted between the chimpanzee and human genomes. Using the net alignments for the builds of the human and chimpanzee genome assemblies, we identified a total of 1,576 putative regions of inverted orientation, covering more than 154 mega-bases of DNA. The DNA segments are distributed throughout the genome and range from 23 base pairs to 62 mega-bases in length. For the 66 inversions more than 25 kilobases (kb) in length, 75% were flanked on one or both sides by (often unrelated) segmental duplications. Using PCR and fluorescence in situ hybridization we experimentally validated 23 of 27 (85%) semi-randomly chosen regions; the largest novel inversion confirmed was 4.3 mega-bases at human Chromosome 7p14. Gorilla was used as an out-group to assign ancestral status to the variants. All experimentally validated inversion regions were then assayed against a panel of human samples and three of the 23 (13%) regions were found to be polymorphic in the human genome. These polymorphic inversions include 730 kb (at 7p22), 13 kb (at 7q11), and 1 kb (at 16q24) fragments with a 5%, 30%, and 48% minor allele frequency, respectively. Our results suggest that inversions are an important source of variation in primate genome evolution. The finding of at least three novel inversion polymorphisms in humans indicates this type of structural variation may be a more common feature of our genome than previously realized.     

CHROMOSOMAL INVERSIONS AND SPECIES DIFFERENCES: WHEN ARE GENES AFFECTING ADAPTIVE DIVERGENCE AND REPRODUCTIVE ISOLATION EXPECTED TO RESIDE WITHIN INVERSIONS?

Many factors can promote speciation, and one which has received much attention is chromosomal inversions. A number of models propose that the recombination suppressing effects of inversions facilitate the maintenance of differences between interbreeding populations in genes affecting adaptive divergence and reproductive isolation. These models predict that such genes will disproportionately reside within inversions, rather than in collinear regions. This hypothesis has received some support, but exceptions exist. Additionally, the effects of known low levels of recombination within inversions on these models are uninvestigated. Here, simulations are used to compare the maintenance of genetic differences between populations following secondary contact and hybridization in different inversion models. We compare regions with no recombination within them to regions with low recombination and to collinear regions with free recombination. Our most general finding is that the low levels of recombination within an inversion often result in the loss of accentuated divergence in inverted regions compared to collinear ones. We conclude that inversions can facilitate the maintenance of species differences under some conditions, but that large or qualitative differences between inverted and collinear regions need not occur. We also find that strong selection facilitates maintenance of divergence in a manner analogous to inversions.     

Polymorphic micro-inversions contribute to the genomic variability of humans and chimpanzees

A combination of inter- and intra-species genome comparisons is required to identify and classify the full spectrum of genetic changes, both subtle and gross, that have accompanied the evolutionary divergence of humans and other primates. In this study, gene order comparisons of 11,518 human and chimpanzee orthologous gene pairs were performed to detect regions of inverted gene order that are potentially indicative of small-scale rearrangements such as inversions. By these means, a total of 71 potential micro-rearrangements were detected, nine of which were considered to represent micro-inversions encompassing more than three genes. These putative inversions were then investigated by FISH and/or PCR analyses and the authenticity of five of the nine inversions, ranging in size from approximately 800 kb to approximately 4.4 Mb, was confirmed. These inversions mapped to 1p13.2-13.3, 7p22.1, 7p13-14.1, 18p11.21-11.22 and 19q13.12 and encompass 50, 14, 16, 7 and 16 known genes, respectively. Intriguingly, four of the confirmed inversions turned out to be polymorphic: three were polymorphic in the chimpanzee and one in humans. It is concluded that micro-inversions make a significant contribution to genomic variability in both humans and chimpanzees and inversion polymorphisms may be more frequent than previously realized.     

The chimpanzee-specific pericentric inversions that distinguish humans and chimpanzees have identical breakpoints in Pan troglodytes and Pan paniscus

Seven of nine pericentric inversions that distinguish human (HSA) and chimpanzee karyotypes are chimpanzee-specific. In this study we investigated whether the two extant chimpanzee species, Pan troglodytes (common chimpanzee) and Pan paniscus (bonobo), share exactly the same pericentric inversions. The methods applied were FISH with breakpoint-spanning BAC/PAC clones and PCR analyses of the breakpoint junction sequences. Our findings for the homologues to HSA 4, 5, 9, 12, 16, and 17 confirm for the first time at the sequence level that these pericentric inversions have identical breakpoints in the common chimpanzee and the bonobo. Therefore, these inversions predate the separation of the two chimpanzee species 0.86-2 Mya. Further, the inversions distinguishing human and chimpanzee karyotypes may be regarded as early acquisitions, such that they are likely to have been present at the time of human/chimpanzee divergence. According to the chromosomal speciation theory the inversions themselves could have promoted human speciation.     

The Genomics of Speciation in Drosophila: Diversity, Divergence, and Introgression Estimated Using Low-Coverage Genome Sequencing

In nature, closely related species may hybridize while still retaining their distinctive identities. Chromosomal regions that experience reduced recombination in hybrids, such as within inversions, have been hypothesized to contribute to the maintenance of species integrity. Here, we examine genomic sequences from closely related fruit fly taxa of the Drosophila pseudoobscura subgroup to reconstruct their evolutionary histories and past patterns of genic exchange. Partial genomic assemblies were generated from two subspecies of Drosophila pseudoobscura (D. ps.) and an outgroup species, D. miranda. These new assemblies were compared to available assemblies of D. ps. pseudoobscura and D. persimilis, two species with overlapping ranges in western North America. Within inverted regions, nucleotide divergence among each pair of the three species is comparable, whereas divergence between D. ps. pseudoobscura and D. persimilis in non-inverted regions is much lower and closer to levels of intraspecific variation. Using molecular markers flanking each of the major chromosomal inversions, we identify strong crossover suppression in F₁ hybrids extending over 2 megabase pairs (Mbp) beyond the inversion breakpoints. These regions of crossover suppression also exhibit the high nucleotide divergence associated with inverted regions. Finally, by comparison to a geographically isolated subspecies, D. ps. bogotana, our results suggest that autosomal gene exchange between the North American species, D. ps. pseudoobscura and D. persimilis, occurred since the split of the subspecies, likely within the last 200,000 years. We conclude that chromosomal rearrangements have been vital to the ongoing persistence of these species despite recent hybridization. Our study serves as a proof-of-principle on how whole genome sequencing can be applied to formulate and test hypotheses about species formation in lesser-known non-model systems.     

Coalescent patterns for chromosomal inversions in divergent populations

Chromosomal inversions allow genetic divergence of locally adapted populations by reducing recombination between chromosomes with different arrangements. Divergence between populations (or hybridization between species) is expected to leave signatures in the neutral genetic diversity of the inverted region. Quantitative expectations for these patterns, however, have not been obtained. Here, we develop coalescent models of neutral sites linked to an inversion polymorphism in two locally adapted populations. We consider two scenarios of local adaptation: selection on the inversion breakpoints and selection on alleles inside the inversion. We find that ancient inversion polymorphisms cause genetic diversity to depart dramatically from neutral expectations. Other situations, however, lead to patterns that may be difficult to detect; important determinants are the age of the inversion and the rate of gene flux between arrangements. We also study inversions under genetic drift, finding that they produce patterns similar to locally adapted inversions of intermediate age. Our results are consistent with empirical observations, and provide the foundation for quantitative analyses of the roles that inversions have played in speciation.     

Evaluation of the genomic extent of effects of fixed inversion differences on intraspecific variation and interspecific gene flow in Drosophila pseudoobscura and D. persimilis

There is increasing evidence that chromosomal inversions may facilitate the formation or persistence of new species by allowing genetic factors conferring species-specific adaptations or reproductive isolation to be inherited together and by reducing or eliminating introgression. However, the genomic domain of influence of the inverted regions on introgression has not been carefully studied. Here, we present a detailed study on the consequences that distance from inversion breakpoints has had on the inferred level of gene flow and divergence between Drosophila pseudoobscura and D. persimilis. We identified the locations of the inversion breakpoints distinguishing D. pseudoobscura and D. persimilis in chromosomes 2, XR, and XL. Population genetic data were collected at specific distances from the inversion breakpoints of the second chromosome and at two loci inside the XR and XL inverted regions. For loci outside the inverted regions, we found that distance from the nearest inversion breakpoint had a significant effect on several measures of divergence and gene flow between D. pseudoobscura and D. persimilis. The data fitted a logarithmic relationship, showing that the suppression of crossovers in inversion heterozygotes also extends to loci located outside the inversion but close to it (within 1-2 Mb). Further, we detected a significant reduction in nucleotide variation inside the inverted second chromosome region of D. persimilis and near one breakpoint, consistent with a scenario in which this inversion arose and was fixed in this species by natural selection.     

Divergence between the Drosophila pseudoobscura and D. persimilis genome sequences in relation to chromosomal inversions

As whole-genome sequence assemblies accumulate, a challenge is to determine how these can be used to address fundamental evolutionary questions, such as inferring the process of speciation. Here, we use the sequence assemblies of Drosophila pseudoobscura and D. persimilis to test hypotheses regarding divergence with gene flow. We observe low differentiation between the two genome sequences in pericentromeric and peritelomeric regions. We interpret this result as primarily a remnant of the correlation between levels of variation and local recombination rate observed within populations. However, we also observe lower differentiation far from the fixed chromosomal inversions distinguishing these species and greater differentiation within and near these inversions. This finding is consistent with models suggesting that chromosomal inversions facilitate species divergence despite interspecies gene flow. We also document heterogeneity among the inverted regions in their degree of differentiation, suggesting temporal differences in the origin of each inverted region consistent with the inversions arising during a process of divergence with gene flow. While this study provides insights into the speciation process using two single-genome sequences, it was informed by lower throughput but more rigorous examinations of polymorphism and divergence. This reliance highlights the need for complementary genomic and population genetic approaches for tackling fundamental evolutionary questions such as speciation.     

Pervasive gene conversion in chromosomal inversion heterozygotes

Chromosomal inversions shape recombination landscapes, and species differing by inversions may exhibit reduced gene flow in these regions of the genome. Though single crossovers within inversions are not usually recovered from inversion heterozygotes, the recombination barrier imposed by inversions is nuanced by noncrossover gene conversion. Here, we provide a genomewide empirical analysis of gene conversion rates both within species and in species hybrids. We estimate that gene conversion occurs at a rate of 1 × 10^–5 to 2.5 × 10^–5 converted sites per bp per generation in experimental crosses within Drosophila pseudoobscura and between D. pseudoobscura and its naturally hybridizing sister species D. persimilis. This analysis is the first direct empirical assessment of gene conversion rates within inversions of a species hybrid. Our data show that gene conversion rates in interspecies hybrids are at least as high as within‐species estimates of gene conversion rates, and gene conversion occurs regularly within and around inverted regions of species hybrids, even near inversion breakpoints. We also found that several gene conversion events appeared to be mitotic rather than meiotic in origin. Finally, we observed that gene conversion rates are higher in regions of lower local sequence divergence, yet our observed gene conversion rates in more divergent inverted regions were at least as high as in less divergent collinear regions. Given our observed high rates of gene conversion despite the sequence differentiation between species, especially in inverted regions, gene conversion has the potential to reduce the efficacy of inversions as barriers to recombination over evolutionary time.     

Speciation and inversions: chimps and humans

A new set of models has resurrected a role for chromosomal inversions in the formation of new species. Traditional models, which are generally considered to be unlikely in most cases, had imagined that inversions might aid speciation by directly causing low hybrid fitness. In contrast, the newer models focus on the effect that inversions have on local recombination rates. A test of these models found a strikingly high rate of amino-acid substitution within regions where humans and chimpanzees differ by inversions, suggesting perhaps that our ancestral species underwent a divergence process in which gene flow and inversions played a key role. However, it remains uncertain whether this interesting finding is actually consistent with the proposed model.     

Molecular characterisation of the pericentric inversion that distinguishes human chromosome 5 from the homologous chimpanzee chromosome

Human and chimpanzee karyotypes differ by virtue of nine pericentric inversions that serve to distinguish human chromosomes 1, 4, 5, 9, 12, 15, 16, 17, and 18 from their chimpanzee orthologues. In this study, we have analysed the breakpoints of the pericentric inversion characteristic of chimpanzee chromosome 4, the homologue of human chromosome 5. Breakpoint-spanning BAC clones were identified from both the human and chimpanzee genomes by fluorescence in situ hybridisation, and the precise locations of the breakpoints were determined by sequence comparisons. In stark contrast to some other characterised evolutionary rearrangements in primates, this chimpanzee-specific inversion appears not to have been mediated by either gross segmental duplications or low-copy repeats, although micro-duplications were found adjacent to the breakpoints. However, alternating purine–pyrimidine (RY) tracts were detected at the breakpoints, and such sequences are known to adopt non-B DNA conformations that are capable of triggering DNA breakage and genomic rearrangements. Comparison of the breakpoint region of human chromosome 5q15 with the orthologous regions of the chicken, mouse, and rat genomes, revealed similar but non-identical syntenic disruptions in all three species. The clustering of evolutionary breakpoints within this chromosomal region, together with the presence of multiple pathological breakpoints in the vicinity of both 5p15 and 5q15, is consistent with the non-random model of chromosomal evolution and suggests that these regions may well possess intrinsic features that have served to mediate a variety of genomic rearrangements, including the pericentric inversion in chimpanzee chromosome 4.     

Adaptive chromosomal divergence driven by mixed geographic mode of evolution

Chromosomal inversions are ubiquitous in nature and of great significance for understanding adaptation and speciation. Inversions were the first markers used to investigate the genetic structure of natural populations, leading to the concept of coadapted gene complexes and theories concerning founder effects and genetic drift in small populations. However, we still lack elements of a general theory accounting for the origins and distribution of inversions in nature. Here, we use computer simulations to show that a "mixed geographic mode" of evolution involving allopatric separation of populations followed by secondary contact and gene flow generates chromosomal divergence by natural selection under wider conditions than previous hypotheses. This occurs because inversions arising in allopatry contain a full complement of locally adapted genes. Once gene flow ensues, reduced recombination within inversions keeps these favorable genotypic combinations intact, resulting in inverted genomic regions being favored over collinear regions. This process allows inversions to establish to high frequencies. Our model can account for several classic patterns in the geographic distribution of inversions and highlights how selection on standing genetic variation allows rapid chromosomal evolution without the waiting time for new mutations. As inversion differences often separate closely related taxa, mixed modes of divergence could be common.     

Structural divergence between the human and chimpanzee genomes

The structural microheterogeneity evident between the human and chimpanzee genomes is quite considerable and includes inversions and duplications as well as deletions, ranging in size from a few base-pairs up to several megabases (Mb). Insertions and deletions have together given rise to at least 150 Mb of genomic DNA sequence that is either present or absent in humans as compared to chimpanzees. Such regions often contain paralogous sequences and members of multigene families thereby ensuring that the human and chimpanzee genomes differ by a significant fraction of their gene content. There is as yet no evidence to suggest that the large chromosomal rearrangements which serve to distinguish the human and chimpanzee karyotypes have influenced either speciation or the evolution of lineage-specific traits. However, the myriad submicroscopic rearrangements in both genomes, particularly those involving copy number variation, are unlikely to represent exclusively neutral changes and hence promise to facilitate the identification of genes that have been important for human-specific evolution.     

Sequence signatures of a recent chromosomal rearrangement in <Emphasis Type="Italic">Drosophila mojavensis</Emphasis>

The X-chromosome inversion, Xe, distinguishes Drosophila mojavensis and D. arizonae. Earlier work mapped the breakpoints of this inversion to large intervals and provided hypotheses for the locations of the breakpoints within 3000-bp intergenic regions on the D. mojavensis genome sequence assembly. Here, we sequenced these regions directly in the putatively ancestral D. arizonae X-chromosome. We find that the two inversion breakpoints are near an inverted gene duplication and a common repetitive element, respectively, and these features were likely present in the non-inverted ancestral chromosome on the D. mojavensis lineage. Contrary to an earlier hypothesis, the inverted gene duplication appears to predate the inversion. We find no sequence similarity between the breakpoint regions in the D. mojavensis ancestor, excluding an ectopic-exchange model of chromosome rearrangements. We also found no evidence that staggered single-strand breaks caused the inversion. We suggest these features may have contributed to the chromosomal breakages resulting in this inversion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reexamination of the African hominoid trichotomy with additional sequences from the primate beta-globin gene cluster.

Additional DNA sequence information from a range of primates, including 13.7 kb from pygmy chimpanzee (Pan paniscus), was added to data sets of beta-globin gene cluster sequence alignments that span the gamma 1, gamma 2, and psi eta loci and their flanking and intergenic regions. This enlarged body of data was used to address the issue of whether the ancestral separations of gorilla, chimpanzee, and human lineages resulted from only one trichotomous branching or from two dichotomous branching events. The degree of divergence, corrected for superimposed substitutions, seen in the beta-globin gene cluster between human alleles is about a third to a half that observed between two species of chimpanzee and about a fourth that between human and chimpanzee. The divergence either between chimpanzee and gorilla or between human and gorilla is slightly greater than that between human and chimpanzee, suggesting that the ancestral separations resulted from two closely spaced dichotomous branchings. Maximum parsimony analysis further strengthened the evidence that humans and chimpanzees share the longest common ancestry. Support for this human-chimpanzee clade is statistically significant at P = 0.002 over a human-gorilla clade or a chimpanzee-gorilla clade. An analysis of expected and observed homoplasy revealed that the number of sequence changes uniquely shared by human and chimpanzee lineages is too large to be attributed to homoplasy. Molecular clock calculations that accommodated lineage variations in rates of molecular evolution yielded hominoid branching times that ranged from 17-19 million years ago (MYA) for the separation of gibbon from the other hominoids to 5-7 MYA for the separation of chimpanzees from humans. Based on the relatively late dates and mounting corroborative evidence from unlinked nuclear genes and mitochondrial DNA for the close sister grouping of humans and chimpanzees, a cladistic classification would place all apes and humans in the same family. Within this family, gibbons would be placed in one subfamily and all other extant hominoids in another subfamily. The later subfamily would be divided into a tribe for orangutans and another tribe for gorillas, chimpanzees, and humans. Finally, gorillas would be placed in one subtribe with chimpanzees and humans in another, although this last division is not as strongly supported as the other divisions.     

Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17

A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.