Chromosomal Elements Evolve at Different Rates in the Drosophila Genome

Corresponding author: Alfredo Ruiz, Facultat de Ciències-Edifici C, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain., Alfredo.Ruiz{at}uab.es (E-mail)

Communicating editor: D. CHARLESWORTH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Recent results indicate that the rate of chromosomal rearrangement in the genus Drosophila is the highest found so far in any eukaryote. This conclusion is based chiefly on the comparative mapping analysis of a single chromosomal element (Muller's element E) in two species, D. melanogaster and D. repleta, representing the two farthest lineages within the genus (the Sophophora and Drosophila subgenera, respectively). We have extended the analysis to two other chromosomal elements (Muller's elements A and D) and tested for differences in rate of evolution among chromosomes. With this purpose, detailed physical maps of chromosomes X and 4 of D. repleta were constructed by in situ hybridization of 145 DNA probes (gene clones, cosmids, and P1 phages) and their gene arrangements compared with those of the homologous chromosomes X and 3L of D. melanogaster. Both chromosomal elements have been extensively reshuffled over their entire length. The number of paracentric inversions fixed has been estimated as 118 ± 17 for element A and 56 ± 8 for element D. Comparison with previous data for elements E and B shows that there are fourfold differences in evolution rate among chromosomal elements, with chromosome X exhibiting the highest rate of rearrangement. Combining all results, we estimated that 393 paracentric inversions have been fixed in the whole genome since the divergence between D. repleta and D. melanogaster. This amounts to an average rate of 0.053 disruptions/Mb/myr, corroborating the high rate of rearrangement in the genus Drosophila.

CHROMOSOME repatterning is commonly thought to be of universal occurrence during the evolution of the eukaryotic genomes, even though only a few precise comparative analyses have been performed (GALE and DEVOS 1998 ; O'BRIEN et al. 1999 ; SEOIGHE et al. 2000 ; RANZ et al. 2001 ). Comparative mapping allows us to describe and estimate the amount of chromosomal evolution that has occurred during the divergence of species from their common ancestor, that is, the patterns and rates of genome reshaping (NADEAU and TAYLOR 1984 ; NADEAU and SANKOFF 1998A ; O'BRIEN et al. 1999 ). The amount of chromosomal evolution between two species can be expressed as the number of chromosomal rearrangements separating their genomes. Furthermore, by comparing the physical maps, segments in which the linear order of contiguous markers has been conserved can be identified. Closely related species are expected to have accumulated fewer rearrangements, and thus to share longer conserved segments, than distantly related species. Whether the particular gene combinations found in the conserved segments are preserved by natural selection, by structural DNA features that promote or restrict chromosome breakage, or simply by random processes is a matter of discussion (HARTL and LOZOVSKAYA 1994 ; NADEAU and SANKOFF 1998A ; HUYNEN et al. 2001 ).

In the genus Drosophila, there is a remarkable synteny conservation; that is, the gene content of the five major chromosomal elements usually is preserved during the evolution of most lineages (MULLER 1940 ; STURTEVANT and NOVITSKI 1941 ). This has allowed the establishment of homologies between the chromosomes of different species (POWELL 1997 ). Per contra, the order of genes within each chromosomal arm is scrambled from species to species via the fixation of paracentric inversions (HARTL and LOZOVSKAYA 1994 ; SEGARRA et al. 1995 , SEGARRA et al. 1996 ; VIEIRA et al. 1997A , VIEIRA et al. 1997B ; RANZ et al. 1997 , RANZ et al. 1999 , RANZ et al. 2001 ; GONZALEZ et al. 2000 ), which are by far the most frequent chromosomal rearrangements in this genus (KRIMBAS and POWELL 1992 ; POWELL 1997 ). Exceptionally, a few cases of gene transposition have also been reported (BROCK and ROBERTS 1983 ; NEUFELD et al. 1991 ; YI and CHARLESWORTH 2000 ).

Remarkable differences in the rate of chromosomal evolution between phylogenetic lineages have been reported. In vertebrates, for instance, rates of synteny disruption vary >15-fold among lineages (NADEAU and SANKOFF 1998A ; MURPHY et al. 2001 ). Some vertebrate lineages (e.g., human, carnivores, and common shrew) show remarkable conservation while others (e.g., mice and the lesser apes) show extensive chromosomal rearrangement (EHRLICH et al. 1997 ; BURT et al. 1999 ). However, the highest rate recorded so far in eukaryotes is that of Drosophila. RANZ et al. 2001 carried out a detailed comparative study of the largest chromosomal element (Muller's element E) between the species Drosophila repleta and D. melanogaster, representative of the two main lineages in the genus Drosophila. Their results revealed an extensive reshuffling of gene order from centromere to telomere and a rate of disruptions per megabase per million years two orders of magnitude higher than that of mammals and 5-fold higher than that of the most dynamic plant genomes. Only yeast genomes seem to exhibit rates of chromosomal rearrangement comparable (or perhaps greater) to those of Drosophila (LLORENTE et al. 2000 ; SEOIGHE et al. 2000 ). The between-lineages variation in evolution rate can be explained in terms of differential mutation rate, fluctuations of population size, variation in generation time, or differential fertility effects of chromosome rearrangements (BURT et al. 1999 ; RANZ et al. 2001 ).

Besides the variation among lineages, different chromosomes or chromosomal elements may also show unequal evolution rates. RICE 1984 pointed out that X-linked mutations with antagonistic effects in the two sexes should increase when rare under a much wider range of conditions compared to autosomal mutations. Moreover, CHARLESWORTH et al. 1987 showed that the fixation rate of underdominant and advantageous partially recessive mutations should be higher for the X chromosome (due to the hemizygosity of males) than for the autosomes. For these reasons, the X chromosome has probably played a main role during the genetic differentiation associated with speciation. For example, in Drosophila, many hybrid sterility genes are X-linked (ORR 1997 ) and, in mice, the X chromosome harbors an unexpectedly large number of genes involved in sperm formation (HURST 2001 ; WANG et al. 2001 ). Whether this functional specialization is related to the particular chromosomal dynamics of the X chromosome and the autosomes is unknown. In addition, X-linked genes undergo dosage compensation and the X chromosome must contain dispersed nucleotide sequences that act as a target for specific proteins and noncoding RNAs involved in this process (KELLEY and KURODA 1995 ; STUCKENHOLZ et al. 1999 ). The autosomes may also exhibit variable evolution rates. Within several Drosophila species groups, such as the Hawaiian Drosophila or the repleta species group, the number of polymorphic and fixed paracentric inversions recorded in each chromosomal element is very unequal (KRIMBAS and POWELL 1992 ). Also, using the comparative mapping approach, VIEIRA et al. 1997A , VIEIRA et al. 1997B found different evolution rates between chromosomes within the virilis species group. So far, however, only relatively short-range phylogenetic comparisons have been carried out in the genus Drosophila.

We have investigated whether the chromosomal elements of Drosophila show nonhomogeneous evolution rates over long phylogenetic distances. Physical maps of the D. repleta chromosomes X and 4 have been constructed by in situ hybridization of 145 DNA clones (gene clones, cosmids, and P1 phages) and their gene arrangements compared with those of the homologous chromosomes X and 3L of D. melanogaster (Muller's elements A and D; see Fig 1). D. repleta belongs to the repleta species group of the Drosophila subgenus (WASSERMAN 1992 ) whereas D. melanogaster belongs to the melanogaster species group in the Sophophora subgenus (POWELL 1997 ). They are representative species of the farthest lineages within the genus Drosophila, separated by 80–124 million years (myr; Fig 1). The aims of this study are (i) to estimate the number of inversions fixed in chromosomal elements A and D between D. melanogaster and D. repleta; (ii) to compare the evolutionary rates of these two chromosomes with those previously reported for Muller's elements E (RANZ et al. 2001 ) and B (GONZALEZ et al. 2000 ); and (iii) to shed light on the molecular organization of Drosophila chromosomes, find out conserved chromosomal segments, and test for functional constraints on the evolution of the Drosophila genome.

View larger version (15K): In this window In a new window Download PPT slide   Figure 1. Phylogenetic relationships and chromosomal homologies of the Drosophila species cited in the text. Estimates of the divergence time taken from BEVERLY and WILSON 1984 , SPICER 1988 , and RUSSO et al. 1995 .

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Fly stocks:
One stock of D. melanogaster (Canton-S), one stock of D. repleta (no. 1611.6 from the National Drosophila Species Resource Center, Bowling Green, OH), and one stock of D. buzzatii (39.13st) were used. The three stocks are homokaryotypic for the standard arrangement in all chromosomes (LEMEUNIER and AULARD 1992 ; WASSERMAN 1992 ; RUIZ and WASSERMAN 1993 ).

Probes:
A total of 198 clones (46 gene clones, 64 cosmids, and 88 P1 phages) were used as probes. All these markers were previously known to map on chromosome X (111) or chromosomal arm 3L (87) of D. melanogaster (Table 1). Of the 46 gene clones, 14 are cDNAs from the Drosophila melanogaster Gene Collection (RUBIN et al. 2000 ); 29 gene clones also come from D. melanogaster and were provided by different authors (Table 2); the remaining three gene clones (Sod, sina, and Rh4) were isolated by PCR in our laboratory and the PCR products cloned into a PGEM-T vector (Promega, Madison, WI) and partially sequenced to confirm their identity. The Sod fragment was amplified from D. buzzatii DNA according to the conditions and primers (N and O) reported for D. melanogaster (HUDSON et al. 1994 ). The sina clone was produced from D. buzzatii DNA using primers (5'-GGAATTCCAGCTCTTCACTGTCGT-3' and 5'-GGAATTCCCCAGTCGATAGACAAA-3') designed to match conserved sina nucleotide sequences between D. melanogaster and D. virilis (NEUFELD et al. 1991 ). Finally, the Rh4 clone was isolated from D. virilis DNA using primers (5'-GCCAAGTTGCTGTGCATT-3' and 5'-ATCAGGCGGAGTTCGATT-3') designed according to the Rh4 nucleotide sequence of D. virilis (NEUFELD et al. 1991 ). Cosmid clones come from the European Drosophila Genome Project cosmid library (MADUENO et al. 1995 ) and P1 phages from the Berkeley Drosophila Genome Project P1 library (HARTL et al. 1994 ; KIMMERLY et al. 1996 ). DNA from all these clones was extracted following standard procedures (SAMBROOK et al. 1989 ). Isopropyl thiogalactoside (0.1 M) was added to the overnight cultures of P1 phage clones (HARTL and LOZOVSKAYA 1995 ). Cosmid clone 28C2 was digested with BamHI and subcloned into pBluescript; four subclones were used as probes.

In situ hybridization and chromosome maps:
All clones were hybridized to the chromosomes of D. repleta to determine their physical localization in this species and to those of D. melanogaster as control. All hybridizations to the chromosomes of D. melanogaster gave positive results. In most cases, the hybridization signal was localized at the expected chromosomal site (TABLE A11 1). However, two cosmids (156H1 and 13F10) and two P1 phages (DS08585 and DS00004) mapped to distant sites from those previously reported. We take this as an indication that these clones were probably mislabeled during the distribution process. Nevertheless, this does not diminish the utility of these clones as physical markers and they have been included accordingly in our marker set. In a few cases (see TABLE A11 1) the map position of a marker in D. repleta was inferred from its localization in D. buzzatii, another species of the repleta species group (WASSERMAN 1992 ; RUIZ and WASSERMAN 1993 ). This can be safely done with markers mapping to homosequential chromosomal regions, i.e., those regions not rearranged by paracentric inversions and thus with the same sequence of bands in the two species. Only female larvae were used for the hybridization of the X chromosome probes because the efficiency of hybridization on the female X is equivalent to that on the autosomes whereas the single X of the male shows a somewhat reduced level of hybridization (PARDUE et al. 1987 ). Polytene chromosome squashes, hybridization, and detection were carried out as in RANZ et al. 1997 . Probes were labeled with biotin-16-dUTP by nick translation. Hybridization signals were localized using the photographic maps of D. melanogaster polytene chromosomes (LEFEVRE 1976 ) and the cytological maps of D. repleta (WHARTON 1942 ) and D. buzzatii (RUIZ and WASSERMAN 1993 ). Hybridization results were recorded as photographs taken with a phase contrast Nikon Optiphot-2 microscope at x600 magnification. Examples of the hybridization results have been pictured in previous publications of our laboratory (RANZ et al. 1997 , RANZ et al. 1999 , RANZ et al. 2001 ; GONZALEZ et al. 2000 ).

Data analysis:
Most of the genomic clones (cosmids and P1 phages) hybridized in this study have terminal sequence tagged sites (STSs; HARTL et al. 1994 ; MADUENO et al. 1995 ; KIMMERLY et al. 1996 ) that allowed us to localize them precisely on the genome sequence of D. melanogaster (ADAMS et al. 2000 ). When only one terminal STS was available, both the average size of each clone type [80 kb for P1 phages (HARTL et al. 1994 ) and 40 kb for cosmids (MADUENO et al. 1995 )] and its physical orientation were taken into account to anchor the clone in the genome sequence. The positions in the sequence of a few genomic clones with no STSs available were inferred from their cytological site (HARTL et al. 1994 ). Each pair of contiguous markers in the D. melanogaster (the reference species) map delimits a chromosomal segment of known size. All these chromosomal segments were checked for conservation in D. repleta. Those segments in which the relative order of contiguous markers is equivalent in both species were considered as conserved segments. Likewise, all clones yielding a single hybridization signal were considered as conserved segments ("singletons"). Otherwise, the segments were classed as nonconserved and assumed to bear at least one fixed inversion breakpoint. The maximum-likelihood method described in RANZ et al. 1997 was used to estimate the number of inversions fixed between the two species in each chromosomal element. This method does not require a particular distribution of markers along the chromosomes although it does assume random distribution of breakpoints in the reference species. The upper limit of the divergence time (Fig 1) was used to estimate the rates of evolution. Our evolution rate estimates are therefore conservative.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Positive hybridizations to the chromosomes of D. repleta:
Nearly three-quarters of the assayed DNA clones (145/198 = 73.2%) yielded one or more hybridization signals on the chromosomes of D. repleta (or D. buzzatii; see MATERIALS AND METHODS). The distribution of successful hybridizations by clone type and chromosome is shown in Table 1, which also includes our previous results for clones from D. melanogaster chromosomal arms 2L (GONZALEZ et al. 2000 ), 3L (RANZ et al. 1997 ), and 3R (RANZ et al. 2001 ) for comparison. Overall, the rate of success is remarkably high (328/414 = 79.2%) given the long divergence time between D. melanogaster and D. repleta (Fig 1). There seem to be no differences between clone types (G = 3.63, d.f. = 2, P > 0.05) but there are highly significant differences between chromosomal elements (G = 23.31, d.f. = 3, P < 0.001). Chromosome X shows the smallest proportion of successful hybridizations (66.7%), significantly lower than that of the autosomes (83.8%; G = 13.59, d.f. = 1, P < 0.001). This difference seems to be due chiefly to cosmids (G = 9.86; d.f. = 1, P < 0.01) and P1 phages (G = 17.05, d.f. = 1, P < 0.001) rather than to gene clones, which show a similar hybridization rate (G = 0.42, d.f. = 1, P > 0.05).

All gene clones hybridizing to the chromosomes of D. repleta but one (CG1716; see below) gave a single signal (TABLE A11 1). Likewise, most cosmid clones and P1 phages (98 out of 113) also gave a single hybridization signal (TABLE A11 1). Nevertheless, 15 cosmid clones or P1 phages gave two or more (up to four) hybridization signals in D. repleta chromosomes. These 15 genomic clones were considered to contain one or more (up to three) rearrangement breakpoints fixed during the divergence of D. melanogaster and D. repleta. This interpretation is supported by our previous results. Several cosmids and P1 phages giving multiple hybridization signals have been subcloned and the signals physically separated when the subclones were independently hybridized (RANZ et al. 1999 ; GONZALEZ et al. 2000 ). The present hybridization of two genes (CG3585 and Ubi-p5E) included in cosmid clone 143G11 provides further evidence in favor of this interpretation. This cosmid gave two hybridization signals while each gene produced only one of them. Likewise, cosmid clone 28C2, giving three hybridization signals, was subcloned and hybridization of two of the subclones allowed us to physically separate two of the three signals. These results provide a firm basis for our interpretation of multiple signals as the result of the presence of fixed breakpoints in these genomic clones.

Thirteen of the gene clones are included in 10 of the genomic clones hybridized in this study (TABLE A11 1). As expected, each genomic clone and the gene (or genes) included within it hybridized to the same chromosomal site in most cases. However, in four exceptions (sd, Hsp22-26, Hsp23-27, and tra) a different localization was observed. These apparent inconsistencies can be resolved by taking into account that in each case the genes are localized at one end of the genomic clone and by assuming that the genomic clone contains a fixed inversion breakpoint. In this case, it seems reasonable to expect a single signal caused by the hybridization of the major portion of the genomic clone instead of the two signals usually seen when a breakpoint is present.

Physical map of the D. repleta X chromosome:
The localization of the 74 clones from the D. melanogaster X chromosome mapped in D. repleta is given in the TABLE A11 and shown in Fig 2 The euchromatic portion of the D. melanogaster X chromosome is 21.8 Mb long (ADAMS et al. 2000 ) and was divided by Bridges into sections 1–20 (LEFEVRE 1976 ). Our markers come from all sections of the D. melanogaster X chromosome (2–6 markers per section with an average of 3.7 markers) with an average density of 1 marker/295 kb.

View larger version (54K): In this window In a new window Download PPT slide   Figure 2. Comparison of the molecular organization of Muller's element A (chromosome X) between D. melanogaster and D. repleta. Connecting lines match the cytological position of orthologous markers. Shaded rectangles show conserved segments with two or more consecutive markers. The estimated size of each conserved segment is given on the leftmost column. The asterisk (*) indicates those clones yielding more than one hybridization signal. The names of those clones in parentheses are incorrect (these clones have probably been mislabeled during the clone distribution process).

All the clones but two hybridized to the X chromosome of D. repleta, as expected according to the accepted chromosomal homologies (Fig 1). The two exceptional clones (Lsp1alpha and 174F6) likely represent transposition events, which are discussed below. In addition, one gene clone, CG1716, gave two hybridization signals in two different D. repleta chromosomes: X(F3g) and 4(C3c-d). This gene shows significant sequence homology with two other D. melanogaster genes (BERKELEY DROSOPHILA GENOME PROJECT 2001): ash1 localized in 3L(76B9) and CG4976 localized in 3R(98B2). We interpret the signal in the D. repleta X chromosome as pointing to the orthologous gene of CG1716 in this species whereas the signal in the D. repleta chromosome 4 can be tentatively attributed to the orthologous gene of ash1.

The physical map of the D. repleta X chromosome contains 81 markers (TABLE A11 and Fig 2). This includes the 72 markers mapped in this study and a few additional markers mapped previously by our group (RANZ et al. 1997 , RANZ et al. 1999 ) or other authors (NAVEIRA et al. 1986 ; KOKOZA et al. 1992 ; H. NAVEIRA, personal communication). The genome size of the repleta group species is 220 Mb with 69% (150 Mb) of single-copy DNA (SCHULZE and LEE 1986 ). Thus, the euchromatic portion of the D. repleta X chromosome, which represents 18% of the total (WASSERMAN 1992 ), must contain 27 Mb of DNA and the average marker density is 1 marker per 333 kb. Inspection of Fig 2, however, reveals that the markers are far from being distributed in a uniform manner along this chromosome. If the chromosome is divided in four equal-length quarters, the number of markers in each quarter differs significantly from the random expectation (G = 21.20; d.f. = 3, P < 0.001) and suggests that gene density varies up to six times between the most distal and most proximal quarters.

Physical map of the D. repleta chromosome 4:
The localization of the 71 clones from chromosomal arm 3L of D. melanogaster successfully hybridized to the chromosomes of D. repleta is given in the TABLE A11 and shown in Fig 3. The euchromatic portion of chromosomal arm 3L is 24.4 Mb long in D. melanogaster (ADAMS et al. 2000 ) and is composed of sections 60–80 of the cytological map drawn by Bridges (LEFEVRE 1976 ). We have mapped 1–6 markers per section (average 3.5 markers) with an average density of 1 marker per 344 kb.

View larger version (58K): In this window In a new window Download PPT slide   Figure 3. Comparison of the molecular organization of Muller's element D between D. melanogaster (chromosomal arm 3L) and D. repleta (chromosome 4). For meaning of the symbols, see legend of Fig 2.

All 71 clones hybridized to chromosome 4 of D. repleta (TABLE A11), which is homologous to chromosomal arm 3L of D. melanogaster (Fig 1). Thus, after including 8 markers from our previous work (RANZ et al. 1997 ) and those of other authors (NAVEIRA et al. 1986 ; LAAYOUNI et al. 2000 ), the physical map of D. repleta chromosome 4 bears 79 markers (TABLE A11 and Fig 3). The size of the euchromatic portion of chromosome 4 is 27 Mb and the average density is 1 marker per 342 kb, both values similar to those for chromosome X. In contrast to the previous results of the X chromosome, however, the markers are distributed uniformly along chromosome 4 with similar numbers in the four quarters (G = 1.72, d.f. = 3, P > 0.05).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Exceptions to the chromosomal homologies and rate of gene transposition:
The ancestral karyotype of the genus Drosophila consisted of five acrocentric chromosomes and a dot (Muller's elements A–F). Our results are in good agreement with an extensive conserved synteny of Muller's elements A and D during the evolution of the D. melanogaster and D. repleta lineages. Therefore, and with the exception of 2 out of 145 clones, the established chromosomal homologies between chromosome X of D. melanogaster and D. repleta and between chromosomal arm 3L of D. melanogaster and chromosome 4 of D. repleta (Fig 1) are firmly corroborated. Our results also indicate that no exchange of information occurred via pericentric inversions after the centric fusion between Muller's elements D and E that gave rise to the metacentric chromosome 3 in the D. melanogaster lineage. Mammals also show an extensive conserved synteny of chromosome X, even though translocations have often rearranged the genome of mammalian species (OHNO 1967 ; LANDER et al. 2001 ). However, the conservation of the X chromosome in mammals and in the Drosophila genus likely results from different mechanisms. In the case of mammals, there is a need to keep the level of expression of the X-linked genes adjusted to one single copy due to the dosage compensation mechanism (HARTL and LOZOVSKAYA 1994 ; GRAVES 1996 ). In Drosophila, without discarding adjustments on gene dosage, the main reason is probably the reported lack of interchromosomal rearrangements, which holds for both the X chromosome and the autosomes (POWELL 1997 ).

Because of this absence of interchromosomal rearrangements in Drosophila, the two exceptional clones that fail to obey the synteny conservation likely indicate transposition events.

1. The gene Lsp1alpha is located on the X chromosome (element A) of D. melanogaster but maps to chromosome 2 (element E) of D. repleta. This gene is also localized in element E in eight different species belonging to the Sophophora and Drosophila subgenera (BROCK and ROBERTS 1983 ) and it has been suggested that it recently transposed onto the X chromosome in the species belonging to the melanogaster subgroup (SMITH et al. 1981 ). The D. buzzatii Lsp1 genes are being cloned and sequenced in our laboratory to test this hypothesis (J. GONZÁLEZ, F. CASALS and A. RUIZ, unpublished results).

2. Cosmid clone 174F6 maps to the euchromatin- heterochromatin boundary of the D. melanogaster X chromosome (20A-C). However, it hybridized near the centromere (polytene band G5d) of chromosome 4 in D. repleta. This cosmid clone contains the suppressor of forked [su(f)] gene (MADUENO et al. 1995 ). Analysis of a 33-kb chromosomal walk around the su(f) locus in D. melanogaster revealed that most of this interval consists of repetitive sequences. In fact the su(f) gene is flanked by a 1.5-kb direct repeat sequence (TUDOR et al. 1996 ). Sequences homologous to the 1.5-kb repeats are found in the euchromatin-heterochromatin boundary of chromosome arms 2L, 2R, and 3L of D. melanogaster. Therefore, ectopic exchange events involving some homology between donor and target site, which are a possible mechanism of gene transposition in the Drosophila genome (YI and CHARLESWORTH 2000 ), could explain the case of cosmid clone 174F6.

A crude estimate of the rate of gene transposition in the Drosophila genus can be produced by combining the results of the present work with results previously obtained for chromosomal elements B (GONZALEZ et al. 2000 ) and E (RANZ et al. 2001 ). In these latter works no cases of gene transposition were detected. Therefore we have observed two possible transposition events out of a total of 328 clones hybridized to the D. repleta chromosomes (Table 1). This amounts to a rate of 4.9 x 10^-5 transpositions/gene/myr, which is quite low. This rate, however, does not include tandemly repeated genes such as histone or rRNA genes, which often show transposition (ALONSO and BERENDES 1975 ; FITCH et al. 1990 ). It also does not include intrachromosomal transpositions. Given the differentiation undergone by the banding pattern and morphology of the polytene chromosomes of so distantly related species as D. repleta and D. melanogaster, the only transpositions that we can safely detect with our mapping procedure are those taking place between different chromosomal elements. Transpositions within the same chromosomal element are probably overlooked, although they do exist. For instance, the seven in absentia (sina) gene is nested within an intron of the Rh4 opsin gene in chromosomal arm 3L of D. melanogaster (MONTELL et al. 1987 ). However, in D. virilis (NEUFELD et al. 1991 ) and D. repleta (this work) the two genes are located at distant sites of the homologous element (chromosome 3 in D. virilis and chromosome 4 in D. repleta). To explain the different structural arrangement of these two genes between D. melanogaster and D. virilis, NEUFELD et al. 1991 proposed a retrotransposition event of the Rh4 gene in the lineage leading to D. virilis. Our results support their interpretation because two P1 phages (DS00383 and DS00052) from the sina/Rh4 region in D. melanogaster map near sina but far from Rh4 in D. repleta. In addition, if a single transposition event was involved, our result indicates that it took place after the separation of the D. melanogaster and D. virilis ancestral lineages but before the divergence between the D. virilis and D. repleta lineages (30 myr ago). Further work with phylogenetically closer species, whose polytene chromosomes are more easily compared, is required to obtain more accurate estimates of the rate of gene transposition.

Comparative mapping and rates of fixation of paracentric inversions:
Our fairly dense physical maps of D. repleta chromosomes X and 4 allow a detailed comparison of their gene arrangements with those of the homologous D. melanogaster elements X and 3L (Fig 2 and Fig 3). In both elements, a considerable reshuffling of gene order extends from telomere to centromere. The rank order correlation is in both cases nonsignificant (for chromosome X, Spearman's R = 0.062, P > 0.05, six ties; for chromosome 4, Spearman's R = 0.184, P > 0.05, two ties), indicating that gene order is effectively randomized. This profound rearrangement found for Muller's elements A and D can be attributed mainly to the fixation of paracentric inversions if we consider that transposition rates are low (see above) and that paracentric inversions are the prevailing chromosomal rearrangement in Drosophila both as intraspecific polymorphisms and as interspecific fixed differences (HARTL and LOZOVSKAYA 1994 ; POWELL 1997 ; RANZ et al. 2001 ). Using the maximum-likelihood method devised by RANZ et al. 1997 we have estimated the number of inversions fixed between D. repleta and D. melanogaster since their most recent common ancestor (Fig 1). An estimate (±SD) of 118 ± 17 for Muller's element A and 56 ± 8 for Muller's element D were obtained (Table 3). The coefficient of variation (CV) of both estimates is reasonably low (14%) and comparable to the most detailed comparative maps carried out in Drosophila (RANZ et al. 2001 ). Obviously, the accuracy with which the rates of chromosomal evolution are estimated increases with the number of markers used in comparative mapping (SCHOEN 2000 ). However, computer simulations made with the method of RANZ et al. 1997 show that the CV is not a lineal function of the number of markers but follows a negative exponential function (D. SCHOEN, personal communication). This implies that a decrease of the CV below the actual values would require a disproportionate increase in the number of markers.

The evolution rates for chromosomal elements A and D can be compared with those for elements B and E, which have been previously estimated using the same pair of species (GONZALEZ et al. 2000 ; RANZ et al. 2001 ). Because the different chromosomal elements vary in size, to make the data comparable, we have calculated the density of breakpoints per megabase by dividing the number of breakpoints by the size of each element in megabases in D. melanogaster (Table 3). Breakpoint density varies up to four times among chromosomal elements and the differences are statistically significant. The breakpoint density for element A (X chromosome) is the highest (10.83) but there are differences between the three analyzed autosomes as well. Element E exhibits the highest density (8.14) whereas element B shows the lowest (2.57) and element D is intermediate (4.63). The weighted average for the whole genome (the euchromatic portion of the six chromosomal elements A–F) is 6.54 breakpoints/Mb, which allows us to infer that 393 paracentric inversions have become fixed in the whole genome between D. melanogaster and D. repleta (Table 3). Taking 62 myr as the divergence time between the two subgenera, we obtain conservative estimates for the rate of disruptions per megabase per million years. These estimates range from 0.021 for element B up to 0.087 for element A with a weighted average of 0.053 for the whole genome (Table 3).

These results agree fairly well with the scarce reliable estimates previously reported in other Drosophila species. SEGARRA et al. 1995 compared the X chromosome between D. melanogaster and D. pseudoobscura and estimated that 0.086 disruptions/Mb/myr have occurred since the divergence of these two lineages. Likewise, VIEIRA et al. 1997A , VIEIRA et al. 1997B compared the gene order of three different chromosomes among D. virilis, D. montana, and D. novamexicana, three species of the virilis group of subgenus Drosophila. Their rates (taking D. melanogaster as the reference species for chromosome sizes as before) were 0.036–0.056 disruptions/Mb/myr for chromosome X, 0.032 for chromosome 2 (Muller's element E), and 0.009–0.014 for chromosome 3 (Muller's element D). It is remarkable that the ranking order between chromosomal elements in these studies (A > E > D) is the same that we have observed (Table 3), which suggests a genus-wide pattern regardless of the phylogenetic distance of the species compared.

In addition, our results support the previous finding that the rate of genome rearrangement in Drosophila is about two orders of magnitude higher than that in mammals and several times higher than that in the most dynamic plant lineages (RANZ et al. 2001 ). This conclusion was drawn from the comparative analysis of the Muller's element E between D. melanogaster and D. repleta, which represents the 23% of the euchromatic fraction of the D. melanogaster genome. Now, with comparative data in D. repleta of >60% of the D. melanogaster genome, the same conclusion still holds. Most current comparative maps of mammals (and also plants) have a relatively poor resolution. Consequently, as the number of orthologous markers mapped increase, it is likely that more rearrangements (e.g., paracentric inversions) will be discovered in some lineages and the evolution rates in these lineages rise accordingly (SUN et al. 1999 ; MULLER et al. 2000 ; PUTTAGUNTA et al. 2000 ; FRONICKE and WIENBERG 2001 ). In our view, however, such increase will not equalize the disparate evolution rates that we have observed. The plausible reasons for the faster chromosomal evolution in the genus Drosophila have been discussed elsewhere (RANZ et al. 2001 ).

What factors can account for the remarkable variation in evolution rate observed between the chromosomal elements of Drosophila? Factors affecting the rate of chromosomal evolution can be classed in two groups: mutational and selective. A higher rate of inversion fixation would be expected if mutation rate were higher (other things being equal). In Drosophila, transposable elements (TEs) have been implicated in the origin of natural inversions, which can originate through ectopic recombination between TE copies located in opposite orientation in different sites of the same chromosome (MONTGOMERY et al. 1991 ; LIM and SIMMONS 1994 ; ANDOLFATTO et al. 1999 ; CACERES et al. 1999A , CACERES et al. 2001 ; MATHIOPOULOS et al. 1999). Thus, a higher mutation rate could be due to a higher proportion of repetitive DNA or to a higher recombination rate. If we consider the recently sequenced genome of D. melanogaster (Table 4), it would appear that the X chromosome, which in our study showed the highest breakpoint density, does not show a TE density higher than that of the autosomes (RIZZON et al. 2002 ). On the other hand, chromosome X does possess a microsatellite density that is at least twice that of any of the autosomes (KATTI et al. 2001 ), a fact that likely reflects the presence of several repeated sequences that are exclusive of the X chromosome euchromatin or more abundant in the X chromosome than in the autosomes (HUIJSER et al. 1987 ; PARDUE et al. 1987 ; WARING and POLLACK 1987 ; LOWENHAUPT et al. 1989 ; DIBARTOLOMEIS et al. 1992 ; BACHTROG et al. 1999 ). Moreover, the microsatellite density of the autosomes (Table 4) parallels their evolution rates (Table 3). This is an intriguing observation because microsatellite sequences can generate unstable secondary structures (MITAS 1997 ; MOORE et al. 1999 ) that could be involved in the origin of chromosome rearrangements (PLETCHER et al. 2000 ; PUTTAGUNTA et al. 2000 ). So far, however, no evidence for the implication of microsatellites in the origin of Drosophila inversions has been found. Obviously, more data are needed on the distribution of TEs and repeated sequences among chromosomal elements in other Drosophila species apart from D. melanogaster.

The second factor that might be affecting inversion production is recombination rate. If we assume that ectopic recombination is correlated with regular meiotic recombination (MONTGOMERY et al. 1991 ), then meiotic recombination rates in D. melanogaster can be considered in search of a pattern. Chromosome X exhibits a higher average recombination rate (3.35 cM/Mb) than the autosomes (Table 4) in good agreement with its faster chromosomal evolution. However, the average recombination rate of chromosomal arm 2L, which shows the slowest evolution rate, is comparable (or superior) to that of both arms of chromosome 3 (Table 4). Therefore, no consistent effect of recombination on evolution rate is apparent. On the other hand, it is clear that recombination rates vary between Drosophila species (TRUE et al. 1996 ; CACERES et al. 1999B ) and the D. melanogaster rates may not have a genus-wide validity.

Another group of factors comprises those selective causes affecting the probability of fixation of inversions. For instance, CHARLESWORTH et al. 1987 showed that the X chromosome should evolve faster than the autosomes due to a higher fixation probability of underdominant and favorable partial or fully recessive rearrangements. Also, when the chromosomal rearrangements have an antagonistic effect in the two sexes, they will invade the population under a wider range of conditions if they are X linked than if they occur in the autosomes (RICE 1984 ). These predictions might help to explain the fast evolution rate of the X chromosome but would not explain the rate variation between the autosomes. A higher evolution rate could also be due to less functional constraints, as would be expected in regions with low gene density (LANDER et al. 2001 ). Fixed inversions are more likely to have their breakpoints between genes (as found by CIRERA et al. 1995 and CACERES et al. 1999A ) than within transcriptions units (as in SCHNEUWLY et al. 1987 ). In the latter case, an inversion would probably have a strong deleterious effect and would be quickly eliminated by natural selection before fixation. The average density in Drosophila is one gene per 9 kb but there is substantial variation in gene density throughout the genome (ADAMS et al. 2000 ). Nevertheless, the average gene density (as inferred from release 2 of the D. melanogaster genome sequence; ADAMS et al. 2000 ) seems to be comparable for elements X, 2L, and 3L and only slightly higher for elements 2R and 3R (Table 4). Thus, no systematic correlation is apparent between gene density and evolution rate in Drosophila. However, we have to take into account that there can be local differences in gene density within chromosomal elements. JABBARI and BERNARDI 2000 pointed out that the gene concentration in GC-rich regions is sevenfold higher than that in GC-poor regions in the Drosophila genome and our results suggest comparable density differences within the D. repleta X chromosome. Chromosomal arms rich in gene-poor intervals might have more fixed breakpoints than arms with little or no variation in gene density.

Chromosomal inversions may have diverse effects at the genetic and phenotypic level, which will affect their probability of fixation in a complex manner. For instance, in heterokaryotypes, inversions reduce recombination rate in the inverted chromosomal segment (NAVARRO et al. 1997 ) but may increase it in the nonhomologous chromosomes (LUCCHESI and SUZUKI 1968 ). Accordingly, the fate of an inversion is considered to depend strongly on the epistatic combinations of alleles caught by the inversion at the moment of its appearance (CHARLESWORTH and CHARLESWORTH 1973 ; CHARLESWORTH 1974 ) and the species recombination rate (CACERES et al. 1999B ). Finally, in some cases (e.g., the sex ratio arrangement of chromosome X), inversions may be associated with meiotic drive alleles and be preferentially transmitted to the offspring (ASHBURNER 1989 ). Given these manifold effects of inversions, it seems improbable that a single factor explains the variation in evolution rate among Drosophila chromosomal elements and we must cautiously conclude that several causes, some of them discussed above, contribute to this variation.

Conserved chromosomal segments and functional constraints:
The chromosomes of D. repleta can be regarded as a mosaic of relatively small segments homologous to those in D. melanogaster chromosomes. Our estimates of the number of inversions fixed in elements A and D allow us to predict that the average size of such segments is 92 kb for chromosome X and 214 kb for chromosome 4 (Table 3). The comparison of the physical maps of D. melanogaster and D. repleta led us to the identification of 9 and 13 conserved segments with two or more consecutive markers in elements A and D, respectively (see Fig 2 and Fig 3). There were also 39 and 29 singletons, segments that contained only a single marker. In chromosome X the size of the 9 segments with two or more consecutive markers ranged from 4.4 to 576 kb with an average length of 170.7 kb. Likewise, in chromosome 4 the size of the 13 conserved segments ranged from 152 to 939 kb with an average length of 386.9 kb. In both cases, the average length of the observed conserved segments is bigger than the predicted average size (Table 3). This is expected because there is a discovery bias that favors big conserved segments at the initial stages of comparative mapping and also because only conserved segments delimited by two or more markers have been considered to estimate the average length of observed segments (NADEAU and SANKOFF 1998B ).

Blocks of genes that are conserved during long periods of time may represent gene combinations that interact functionally and are therefore maintained together by natural selection, the so-called "functional constraints" hypothesis (MAIER et al. 1993 ; RANDAZZO et al. 1993 ; WRIGHT 1996 ). However, because all genomes are phylogenetically related, colinear groups of genes may also reflect the fixation of a limited number of genomic rearrangements with random breakpoints since both species diverged, the "random breakage" (RB) hypothesis (NADEAU and TAYLOR 1984 ; NADEAU and SANKOFF 1998A ). Previous comparative mapping results in Drosophila (GONZALEZ et al. 2000 ; RANZ et al. 2001 ) and other organisms (NADEAU and SANKOFF 1998A ; HUYNEN et al. 2001 ; LANDER et al. 2001 ) have found little evidence for functional constraints. The RB hypothesis can be tested comparing the observed length distribution of conserved segments with that predicted under the RB hypothesis, which will approximate a negative exponential distribution (Fig 4). It can be seen that, in both chromosomal elements, the empirical distribution fits in general inside the theoretical distribution as expected because only a subset of all conserved segments has been detected. Only one segment in each chromosome seems to depart significantly from the expectations (Fig 4). The size and gene content of these segments should be further investigated.

View larger version (21K): In this window In a new window Download PPT slide   Figure 4. Expected () and observed () distribution of the length of conserved segments under the random breakage hypothesis (NADEAU and TAYLOR 1984 ; NADEAU and SANKOFF 1998B ). The empirical distribution fits in general inside the theoretical distribution as expected because only a subset of all conserved segments has been detected (see text for details).

We can look at the functional constraints hypothesis from a different perspective. There are a few examples of gene complexes in D. melanogaster chromosomes X and 3L whose members show a coregulated expression. We can ask whether or not such complexes are conserved in D. repleta. The achaete-scute complex (AS-C) has been studied extensively (reviewed in MODOLELL and CAMPUZANO 1998 ). It spans 90 kb of the X chromosome where only six transcription units are separated by very large stretches of nontranscribed DNA. This DNA contains many cis-regulatory sequences that coregulate the achaete and scute (but not the lethal-of-scute and asense) genes of the complex. The molecular organization of the 210-kb D. melanogaster segment delimited by the markers 125H10-65F1 has been studied in D. repleta. This 210-kb segment contains, among others, the genes of the AS-C (see TABLE A11 1). When the physical maps of this region are compared, only a segment of 130 kb, which includes the genes achaete and scute, has been conserved. This gene complex is also conserved in D. virilis (BEAMONTE 1990 ). Thus, the molecular organization of this gene complex seems to have been preserved during the 80–124 myr of divergence of these species. Two gene complexes of chromosome 3L also appear to show coregulated expression. The genes araucan (ara) and caupolican (caup), two members of the Iroquois complex, have similar patterns of expression and apparently share cis-regulatory sequences (GOMEZ-SKARMETA et al. 1996 ). They are closely linked in D. melanogaster (comprising a genomic segment of 40 kb) and also in D. repleta (as shown by the coincident hybridization sites of caup, ara, and DS08512). Finally, knirps (kni) and knirps-related (knrl) are two neighboring and functionally equivalent genes mapping to a region of 100 kb (LUNDE et al. 1998 ). They are affected by a cis-acting regulatory sequence (ri) lying immediately upstream of the kni transcription unit. A chromosomal segment of 245 kb around the kni locus (comprising kni, DS01369, and DS00239) is conserved in D. repleta, suggesting that both genes and their regulatory sequences have not been disrupted. In summary, the conservation of these three small gene complexes suggest that natural selection may play a role in some (perhaps exceptional) cases to keep together functionally related Drosophila genes.

Overall our results are in agreement with a modular organization of the Drosophila genome (RANZ et al. 2001 ). Thus, the genome of Drosophila can be seen as a mosaic of independent modules that can change their localization within the euchromatin without loss of function. Usually these modules change their localization within the chromosomal arm and only occasionally between chromosomal arms. Each module may consist of a gene plus its regulatory sequences (as proposed by RANZ et al. 2001 ) or perhaps a small group of nearby genes. If Drosophila euchromatin possess expression domains organized by insulators or boundary elements (GERASIMOVA et al. 2000 ; MONGELARD and CORCES 2001 ), this undoubtedly will influence the molecular consequences of inversion breakpoints. We can speculate that breaks taking place within trancriptionally independent domains will have more disturbing consequences than those occurring between domains. The rough agreement between the size of these expression domains (GERASIMOVA et al. 2000 ) and the average size of the conserved segments (Table 3) is certainly intriguing and deserves more work. Recent comparisons of yeast genomes show the prevalence of small inversions in gene order evolution between Saccharomyces and some Candida species (LLORENTE et al. 2000 ; SEOIGHE et al. 2000 ). A similar result was found when comparing zebrafish and human genomes (POSTLETHWAIT et al. 2000 ), supporting the hypothesis that inversions have been a more frequent force in the shaping of vertebrate karyotypes than translocations. Altogether these results suggest a major role for inversions in the genome shuffling process (HUYNEN et al. 2001 ).

	FOOTNOTES

¹ Present address: Department of Organismic and Evolutionary Biology, Harvard University, D. L. Hartl Laboratory, Cambridge, MA 02138.

	ACKNOWLEDGMENTS

We thank all the authors who sent us clones and/or information: A. Bidwai (West Virginia University); H. Biessman (University of California, Irvine); O. Cabré (Universitat Autònoma de Barcelona); A. Chovnik (University of Connecticut, Storrs); C. Ferraz (Centre National de la Recherche Scientifique); J. W. Fristrom (University of California, Berkeley); R. de Frutos (Universidad de Valencia); C. Kraemer (Universität Mainz); E. Madueño, J. Modolell, and S. Campuzano (Centro de Biología Molecular Severo Ochoa, CSIC, Madrid); D. G. McEwen (University of North Carolina); H. Naveira (Universidade da Coruña); K. O'Hare (Imperial College Science Technology, London); N. Perrimon (Harvard Medical School, HHMI, Boston); and L. Sánchez (Centro de Investigaciones Biológicas, CSIC, Madrid). We are grateful to F. Casals for his help with the cloning of sina and Rh, to S. Misra (Berkeley Drosophila Genome Project) for the analysis of the GadFly database, and to D. Schoen for his computer simulations of the inversion fixation process. The helpful comments of E. Betrán, J. Modolell, C. Segarra, D. Shoen, A. Villasante, and two anonymous referees contributed to improve previous versions of the manuscript. Work was supported by grant PB98-0900-C02-01 from the Dirección General de Enseñanza Superior e Investigación Científica (Ministerio de Educación y Cultura; Spain) awarded to A.R. and a doctoral FI fellowship from the Universitat Autònoma de Barcelona awarded to J.G.

Manuscript received November 12, 2001; Accepted for publication March 21, 2002.

	APPENDIX

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

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