Chromosome Inversions,Genomic Differentiation and Speciation in the African Malaria Mosquito Anopheles gambiae

The African malaria vector, Anopheles gambiae, is characterized by multiple polymorphic chromosomal inversions and has become widely studied as a system for exploring models of speciation. Near complete reproductive isolation between different inversion types, known as chromosomal forms, has led to the suggestion that A. gambiae is in early stages of speciation, with divergence evolving in the face of considerable gene flow. We compared the standard chromosomal arrangement (Savanna form) with genomes homozygous for j, b, c, and u inversions (Bamako form) in order to identify regions of genomic divergence with respect to inversion polymorphism. We found levels of divergence between the two sub-taxa within some of these inversions (2Rj and 2Rb), but at a level lower than expected and confined near the inversion breakpoints, consistent with a gene flux model. Unexpectedly, we found that the majority of diverged regions were located on the X chromosome, which contained half of all significantly diverged regions, with much of this divergence located within exons. This is surprising given that the Bamako and Savanna chromosomal forms are both within the S molecular form that is defined by a locus near centromere of X chromosome. Two X-linked genes (a heat shock protein and P450 encoding genes) involved in reproductive isolation between the M and S molecular forms of A. gambiae were also significantly diverged between the two chromosomal forms. These results suggest that genes mediating reproductive isolation are likely located on the X chromosome, as is thought to be the case for the M and S molecular forms. We conclude that genes located on the sex chromosome may be the major force driving speciation between these chromosomal forms of A. gambiae.     

Chromosome Differentiated Populations of Cruzii: Evidence for a Third Sibling Species

Anopheles cruziiis the most common species of mosquito in Southeast Brazil and a vector of human and monkey malaria. The banding pattern of the ovarian polytene chromosomes and the frequencies of paracentric inversions of individuals from two populations were studied. A new sequence of bands on the sex chromosome, defined as form C, was disclosed. In both populations where forms A (considered as standard) and C are sympatric no heterozygotes were detected. A sequence of events that could account for the observed changes in the banding sequences of the X chromosome forms was proposed. The frequencies of 22 paracentric inversions were used to assess panmixia and the results indicated the presence of two distinct genetic pools in each population. We consider these results as evidence of another sibling species in the taxon cruzii, characterized by a distinctive form of the X chromosome and provisionally designated Anopheles cruziispecies C.     

Evidence for subdivision within the M molecular form of Anopheles gambiae

The principal vector of malaria in sub-Saharan Africa, Anopheles gambiae is subdivided into two molecular forms M and S. Additionally, several chromosomal forms, characterized by the presence of various inversion polymorphisms, have been described. The molecular forms M and S each contain several chromosomal forms, including the Savanna, Mopti and Forest forms. The M and S molecular forms are now considered to be the reproductive units within A. gambiae and it has recently been argued that a low recombination rate in the centromeric region of the X chromosome has facilitated isolation between these forms. The status of the chromosomal forms remains unclear however. Therefore, we studied genetic differentiation between Savanna S, Forest S, Forest M and Mopti M populations using microsatellites. Genetic differentiation between Savanna S and Forest S populations is very low (F(ST) = 0.0053 +/- 0.0049), even across large distances. In comparison, the Mopti M and Forest M populations show a relatively high degree of genetic differentiation (F(ST) = 0.0406 +/- 0.0054) indicating that the M molecular form may not be a single entity, but could be subdivided into at least two distinct chromosomal forms. Previously it was proposed that inversions have played a role in the origin of species within the A. gambiae complex. We argue that a possible subdivision within the M molecular form could be understood through this process, with the acquisition of inversions leading to the expansion of the M molecular form into new habitat, dividing it into two distinct chromosomal forms.     

Divergence with gene flow in Anopheles funestus from the Sudan Savanna of Burkina Faso, West Africa

Anopheles funestus is a major vector of malaria across Africa. Understanding its complex and nonequilibrium population genetic structure is an important challenge that must be overcome before vector populations can be successfully perturbed for malaria control. Here we examine the role of chromosomal inversions in structuring genetic variation and facilitating divergence in Burkina Faso, West Africa, where two incipient species (chromosomal forms) of A. funestus, defined principally by rearrangements of chromosome 3R, have been hypothesized. Sampling across an approximately 300-km east-west transect largely contained within the Sudan-Savanna ecoclimatic zone, we analyzed chromosomal inversions, 16 microsatellite loci distributed genomewide, and 834 bp of the mtDNA ND5 gene. Both molecular markers revealed high genetic diversity, nearly all of which was accounted for by within-population differences among individuals, owing to recent population expansion. Across the study area there was no correlation between genetic and geographic distance. Significant genetic differentiation found between chromosomal forms on the basis of microsatellites was not genomewide but could be explained by chromosome 3R alone on the basis of loci inside and near inversions. These data are not compatible with complete reproductive isolation but are consistent with differential introgression and sympatric divergence between the chromosomal forms, facilitated by chromosome 3R inversions.     

Selective Introgression of Paracentric Inversions between Two Sibling Species of the Anopheles Gambiae Complex

The Anopheles gambiae complex includes the major vectors of malaria in sub-Saharan Africa where >80% of all world-wide cases occur. These mosquitoes are characterized by chromosomal inversions associated to the speciation process and to intraspecific ecological and behavioral flexibility. It has been postulated that introgressive hybridization has selectively transferred inversions on the second chromosome between A. gambiae and A. arabiensis, the two most important vectors of malaria. Here we directly test this hypothesis with laboratory experiments in which hybrid populations were established and the fate of chromosomal inversions were followed. Consistent with the hypothesis, ``foreign' X chromosomes were eliminated within two generations, while some ``foreign' second chromosomes persisted for the duration of the experiments and, judging from the excess of heterozygotes, established stable heterotic polymorphisms. Only those second chromosome inversions found naturally in the species could be introgressed.     

Positive selection near an inversion breakpoint on the neo-X chromosome of Drosophila americana

Unique features of heteromorphic sex chromosomes are produced as a consequence of sex-linked transmission. Alternative models concerning the evolution of sex chromosomes can be classified in terms of genetic drift or positive selection being the primary mechanism of divergence between this chromosomal pair. This study examines early changes on a newly acquired chromosomal arm of the X in Drosophila americana, which was derived from a centromeric fusion between the ancestral X and previously autosomal chromosome 4 (element B). Breakpoints of a chromosomal inversion In(4)a, which is restricted to the neo-X, are identified and used to guide a sequence analysis along chromosome 4. Loci flanking the distal breakpoint exhibit patterns of sequence diversity consistent with neutral evolution, yet loci near the proximal breakpoint reveal distinct imprints of positive selection within the neo-X chromosomal class containing In(4)a. Data from six separate positions examined throughout the proximal region reveal a pattern of recent turnover driven by two independent sweeps among chromosomes with the inverted gene arrangement. Selection-mediated establishment of an extended haplotype associated with recombination-suppressing inversions on the neo-X indicates a pattern of active coadaptation apparently initiated by X-linked transmission and potentially sustained by intralocus sexual conflict.     

Construction of Chromosomal Rearrangements in Salmonella by Transduction: Inversions of Non-Permissive Segments Are Not Lethal

Homologous sequences placed in inverse order at particular separated sites in the bacterial chromosome (termed ``permissive') can recombine to form an inversion of the intervening chromosome segment. When the same repeated sequences flank other chromosome segments (``non-permissive'), recombination occurs but the expected inversion rearrangement is not found among the products. The failure to recover inversions of non-permissive chromosomal segments could be due to lethal effects of the final rearrangement. Alternatively, local chromosomal features might pose barriers to reciprocal exchanges between sequences at particular sites and could thereby prevent formation of inversions of the region between such sites. To distinguish between these two possibilities, we have constructed inversions of two non-permissive intervals by means of phage P22-mediated transduction crosses. These crosses generate inversions by simultaneous incorporation of two transduced fragments, each with a sequence that forms one join-point of the final inversion. We constructed inversions of the non-permissive intervals trp ('34) to his ('42) and his ('42) to cysA ('50). Strains with the constructed inversions are viable and grow normally. These results show that our previous failure to detect formation of these inversions by recombination between chromosomal sequences was not due to lethal effects of the final rearrangement. We infer that the ``non-permissive' character of some chromosomal segments reflects the inability of the recombination system to perform the needed exchanges between inverse order sequences at particular sites. Apparently these mechanistic problems were circumvented by the transductional method used here to direct inversion formation.     

A standard cytogenetic map for Anopheles sundaicus (Diptera: Culicidae) and evidence for chromosomal differentiation in populations from Thailand and Indonesia.

A standard photographic map of polytene chromosomes of Anopheles sundaicus was constructed from ovarian nurse cells and is described herein. Polytene chromosomes of wild specimens collected from 9 different geographical areas in Thailand and Indonesia have been analyzed. Specimens from these populations appear to share banding patterns with standard gene arrangements, except for some specimens from Purworejo, in Central Java, and South Tapanuli and Asahan, both of North Sumatra, which exhibited distinct banding patterns at the tip of chromosome X (Xb) compared with the standard sequence (Xa). Moreover, some specimens collected from Asahan, North Sumatra, consistently showed distinct loosely diffuse bands in zone 19 of chromosome arm 2R (2Rb) compared with the standard banding patterns (2Ra). The existence of the 2Rb pattern correlates perfectly with the presence of an extra block of centromeric heterochromatin in autosome 2 as revealed by mitotic karyotype analysis (2n = 6). These cytological differences have led to the recognition of 3 distinct forms, viz., A, B, and C, within the taxon An. sundaicus. In addition, forms A and C show a normal size for chromosome Y, (Y1), while form B has a relatively larger type of chromosome Y, (Y2). Form A is widely distributed in Thailand and Indonesia, while form B has been found in North Sumatra and Central Java. Form C, however, has been found only in Asahan, North Sumatra. Key words : Anopheles sundaicus, polytene chromosome map, mitotic karyotype, chromosomal differentiation.     

Genetic differentiation in theAedes atropalpus complex. II. Chromosomal divergence betweenAe. atropalpus andAe. epactius

Analysis of meiotic chromosomes from hybrids betweenAedes atropalpus andAe. epactius has revealed that the two species are fixed for alternate arrangements of four inversions: a paracentric inversion of chromosome 1, two paracentric inversions of chromosome 2, and a pericentric inversion of chromosome 3. This chromosomal heterozygosity in the interspecific hybrids has resulted in extensive meiolic chromosomal asynapsis. Dicentric bridges, acentric fragments, and chromosomal breakage were also associated with the heterozygous inversions. This disruption of meiosis was sufficient to account for the partial sterility observed in interspecific hybrids. No chromosomal polymorphisms, aberrations, or reduction in fertility was observed in parental strains of intraspecific hybrids of the two species.     

Multilevel analyses of genetic differentiation in Anopheles gambiae s.s. reveal patterns of gene flow important for malaria-fighting mosquito projects

Malaria control projects based on the introduction and spread of transgenes into mosquito populations depend on the extent of isolation between those populations. On the basis of the distribution of paracentric inversions, Anopheles gambiae has been subdivided into five subspecific chromosomal forms. Estimating gene flow between and within these forms of An. gambiae presents a number of challenges. We compared patterns of genetic divergence (F(ST)) between sympatric populations of the Bamako and Mopti forms at five sites. We used microsatellite loci within the j inversion on chromosome 2, which is fixed in the Bamako form but absent in the Mopti form, and microsatellites on chromosome 3, a region void of inversions. Estimates of genetic diversity and F(ST)'s suggest genetic exchanges between forms for the third chromosome but little for the j inversion. These results suggest a role for the inversion in speciation. Extensive gene flow within forms among sites resulted in populations clustering according to form despite substantial gene flow between forms. These patterns underscore the low levels of current gene flow between chromosomal forms in this area of sympatry. Introducing refractoriness genes in areas of the genome void of inversions may facilitate their spread within forms but their passage between forms may prove more difficult than previously thought.     

Chromosomal elements evolve at different rates in the Drosophila genome

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.     

Molecular Characterization of Hobo-Mediated Inversions in Drosophila Melanogaster

The structure of chromosomal inversions mediated by hobo transposable elements in the Uc-1 X chromosome was investigated using cytogenetic and molecular methods. Uc-1 contains a phenotypically silent hobo element inserted in an intron of the Notch locus. Cytological screening identified six independent Notch mutations resulting from chromosomal inversions with one breakpoint at cytological position 3C7, the location of Notch. In situ hybridization to salivary gland polytene chromosomes determined that both ends of each inversion contained hobo and Notch sequences. Southern blot analyses showed that both breakpoints in each inversion had hobo-Notch junction fragments indistinguishable in structure from those present in the Uc-1 X chromosome prior to the rearrangements. Polymerase chain reaction amplification of the 12 hobo-Notch junction fragments in the six inversions, followed by DNA sequence analysis, determined that each was identical to one of the two hobo-Notch junctions present in Uc-1. These results are consistent with a model in which hobo-mediated inversions result from homologous pairing and recombination between a pair of hobo elements in reverse orientation.     

Polytene chromosome relationships of five species of the Anopheles dirus complex in Thailand.

Photographic maps and rearrangements of each salivary gland polytene chromosome arm of Anopheles nemophilous (species F) and of An. dirus species A, B, C, and D of the Dirus group from natural populations in Thailand are presented. Structural conformation of heterokaryotypes and comparison of chromosome banding sequences reveal 10 paracentric inversions. The data on fixed inversion of 3Rb and inversion polymorphism of the X chromosome shared by these species were used to construct a phylogeny of the five members of the An. dirus complex, thereby outlining their patterns of speciation through chromosomal rearrangements.     

Molecular Cytogenetic Characterization of Multiple Intrachromosomal Rearrangements in Two Representatives of the Genus Turdus (Turdidae,Passeriformes)

Turdus rufiventris and Turdus albicollis, two songbirds belonging to the family Turdidae (Aves, Passeriformes) were studied by C-banding, 18S rDNA, as well as the use of whole chromosome probes derived from Gallus gallus (GGA) and Leucopternis albicollis (LAL). They showed very similar karyotypes, with 2n = 78 and the same pattern of distribution of heterochromatic blocks and hybridization patterns. However, the analysis of 18/28S rDNA has shown differences in the number of NOR-bearing chromosomes and ribosomal clusters. The hybridization pattern of GGA macrochromosomes was similar to the one found in songbirds studied by Fluorescent in situ hybridization, with fission of GGA 1 and GGA 4 chromosomes. In contrast, LAL chromosome paintings revealed a complex pattern of intrachromosomal rearrangements (paracentric and pericentric inversions) on chromosome 2, which corresponds to GGA1q. The first inversion changed the chromosomal morphology and the second and third inversions changed the order of chromosome segments. Karyotype analysis in Turdus revealed that this genus has derived characteristics in relation to the putative avian ancestral karyotype, highlighting the importance of using new tools for analysis of chromosomal evolution in birds, such as the probes derived from L. albicollis, which make it possible to identify intrachromosomal rearrangements not visible with the use of GGA chromosome painting solely.     

Karyotype evolution in South American subterranean rodents Ctenomys magellanicus (Rodentia: Octodontidae): chromosome rearrangements and (TTAGGG)n telomeric sequence localization in 2n=34 and 2n=36 chromosomal forms

Ctenomys is the most numerous genus of South American subterranean rodents and one of the most karyotypically diverse clades of mammals known. Ctenomys magellanicus is the southernmost species of the group and the only one living in Isla Grande de Tierra del Fuego (Argentina). This species presents two chromosomal forms, i.e. 2n=34, and 2n=36 (FN=68). Recent studies suggest that genetic divergence between both karyotypic forms resulted from a chromosomal speciation process. In order to identify the chromosomal rearrangement involved in the process of karyotype evolution in this species, we used chromosome banding techniques and fluorescence in situ hybridization with a telomeric probe to metaphase chromosomes of the two chromosomal forms of Ctenomys magellanicus. Chromosome analysis of Giemsa-stained and G-banding preparations showed that Cm34 and Cm36 karyotypes differ in one rearrangement involving chromosomes A9 from Cm34 and B12 and B17 from Cm36. In addition FISH analysis showed that all of the chromosomes from both chromosomal forms exhibit a telomeric-only distribution pattern of the (TTAGGG)n sequence, indicating that none of the chromosomal forms of Ctenomys magellanicus has true telocentric chromosomes. Our results suggest that a chromosome fission event would have occurred during the process of karyotype evolution in this species.     

Polytene chromosome map and inversion polymorphism in Drosophila mediopunctata

Drosophila mediopunctata belongs to the tripunctata group, and is one of the commonest Drosophila species collected in some places in Brazil, especially in the winter. A standard map of the polytene chromosomes is presented. The breakpoints of the naturally occurring chromosomal rearrangements are marked on the map. The distribution of breaking points through the chromosomes of D. mediopunctata is apparently non-random. Chromosomes X, II and IV show inversion polymorphisms. Chromosome II is the most polymorphic, with 17 inversions, 8 inversions in the distal region and 9 in the proximal region. Chromosome X has four different gene arrangements, while chromosome IV has only two.     

比较染色体涂色显示小熊猫(Ailurus fulgens)具有高度保守的核型

以狗的整条染色体特异探针,通过比较染色体涂色（Comparative Chromosome Painting）,建立了小熊猫和狗的比较染色体图谱。狗的38条常染色体探针在小熊猫染色体上共检出71个同源片段。其中狗的18条常染色体每一条在小熊猫染色全上各有1个同源片段,其余的20条常染色体每一条在小熊猫染色体上各有2至5个同源片段。广泛的染色体结构重排造成了小熊猫与狗的核型差异：至少需要经过28次断裂,49次融合,4次倒位才能将狗的核型（2n=78）“转变”为小熊猫的核型（2n=36）。结合已发表的狗与家猫的比较染色体图谱,我们推测：小熊猫与家猫之间共存在26个同源片段,二者的核型之间显示了较高的同源性。通过比较分析狗的染色体同源片段在小熊猫与家猫染色体上的分布和排列,可以看出：4次染色体易拉,2次倒位造成了小熊猫与家猫的核型差异。我们的工作进一步证实了利用基因组高度重排的物种（如：狗）的染色体特异探针与核型保守的物种（如：家猫、水貂、小熊猫）进行比较染色体涂色研究,不但可以准确快速地鉴别物种进化过程中所发生的染色体间的结构重排,而且还可揭示染色体内的结构重组。     

Photographic polytene chromosome maps for Glossina morsitans submorsitans (Diptera: Glossinidae): cytogenetic analysis of a colony with sex-ratio distortion.

Photographic polytene chromosome maps from trichogen cells of pharate adult Glossina morsitans submorsitans were constructed. Using the standard system employed to map polytene chromosomes of Drosophila, the characteristic landmarks were described for the X chromosome and the two autosomes (L1 and L2). Sex-ratio distortion, which is expressed in male G. m. submorsitans, was found to be associated with an X chromosome (X8) that contains three inversions in each arm. Preliminary data indicate no differences in the fecundity of X(A)X(A) and X(A)X(B) females, but there are indications that G. m. submorsitans in colonies originating from Burkina Faso and Nigeria have genes on the autosomes and (or) the Y chromosome that suppress expression of sex-ratio distortion.     

Chromosomal evolution within the family Estrildidae (Aves) II. The Lonchurae

Thirteen species of estrildid finches belonging to the Lonchurae were examined cytogenetically by G- and C-banding. The major forms of karyotypic change, both within and between species, were pericentric inversions and changes in the amount of heterochromatin. It appears that the direction of chromosome change in this lineage is towards an entirely telocentric karyotype because inversions converting a biarmed chromosome into a telocentric one only occur when all the macrochromosomes of smaller size are also telocentric. A comparison of hybrid fertility data and karyotypic differences indicates that genic factors affecting gonadal development, and not chromosomal rearrangements, are the primary influence in determining hybrid fertility. The chromosomal data was also used to clarify systematic relationships within the Lonchurae and demonstrate that the genus Lonchura as presently construed is polyphyletic.     

Evolutionary conserved chromosomal segments in the human karyotype are bounded by unstable chromosome bands

In this paper an ancestral karyotype for primates, defining for the first time the ancestral chromosome morphology and the banding patterns, is proposed, and the ancestral syntenic chromosomal segments are identified in the human karyotype. The chromosomal bands that are boundaries of ancestral segments are identified. We have analyzed from data published in the literature 35 different primate species from 19 genera, using the order Scandentia, as well as other published mammalian species as out-groups, and propose an ancestral chromosome number of 2n = 54 for primates, which includes the following chromosomal forms: 1(a+c(1)), 1(b+c(2)), 2a, 2b, 3/21, 4, 5, 6, 7a, 7b, 8, 9, 10a, 10b, 11, 12a/22a, 12b/22b, 13, 14/15, 16a, 16b, 17, 18, 19a, 19b, 20 and X and Y. From this analysis, we have been able to point out the human chromosome bands more "prone" to breakage during the evolutionary pathways and/or pathology processes. We have observed that 89.09% of the human chromosome bands, which are boundaries for ancestral chromosome segments, contain common fragile sites and/or intrachromosomal telomeric-like sequences. A more in depth analysis of twelve different human chromosomes has allowed us to determine that 62.16% of the chromosomal bands implicated in inversions and 100% involved in fusions/fissions correspond to fragile sites, intrachromosomal telomeric-like sequences and/or bands significantly affected by X irradiation. In addition, 73% of the bands affected in pathological processes are co-localized in bands where fragile sites, intrachromosomal telomeric-like sequences, bands significantly affected by X irradiation and/or evolutionary chromosomal bands have been described. Our data also support the hypothesis that chromosomal breakages detected in pathological processes are not randomly distributed along the chromosomes, but rather concentrate in those important evolutionary chromosome bands which correspond to fragile sites and/or intrachromosomal telomeric-like sequences.