The molecular characterization of fixed inversions breakpoints unveils the ancestral character of the Drosophila guanche chromosomal arrangements

Abstract

Cytological studies revealed that the number of chromosomes and their organization varies across species. The increasing availability of whole genome sequences of multiple species across specific phylogenies has confirmed and greatly extended these cytological observations. In the Drosophila genus, the ancestral karyotype consists of five rod-like acrocentric chromosomes (Muller elements A to E) and one dot-like chromosome (element F), each exhibiting a generally conserved gene content. Chromosomal fusions and paracentric inversions are thus the major contributors, respectively, to chromosome number variation among species and to gene order variation within chromosomal element. The subobscura cluster of Drosophila consists in three species that retain the genus ancestral karyotype and differ by a reduced number of fixed inversions. Here, we have used cytological information and the D. guanche genome sequence to identify and molecularly characterize the breakpoints of inversions that became fixed since the D. guanche-D. subobscura split. Our results have led us to propose a modified version of the D. guanche cytological map of its X chromosome, and to establish that (i) most inversions became fixed in the D. subobscura lineage and (ii) the order in which the four X chromosome overlapping inversions occurred and became fixed.

Figure 1

Drosophila guanche and D. subobscura chromosomes affected by fixed inversions. The localization of inversion breakpoints in D. subobscura chromosomes J, E, O and A (Muller elements in parentheses) through their flanking markers is given on a schematic representation of the Kunze-Mühl and Müller map (not at scale). Localization of each marker sequence in the D. guanche genome sequence is represented above on a horizontal line (in Mb units). Fragments delimited by differently colored lines include inversion breakpoints, with the D. guanche fragments spanning both breakpoints of a given inversion labeled with the same low-case letter (e.g., a, proximal breakpoint; a*, distal breakpoint). The O chromosome of D. guanche differs from the O_st arrangement of D. subobscura by two overlapping inversions, the O_f inversion and the D. subobscura polymorphic inversion O₃. Although both inversions are shown, only markers flanking the O_f inversion —fixed since the two species divergence— are given. For the four overlapping inversions of the A chromosome (#), fragments labeled a, b, c and d correspond to the breakpoints of inversions A_f1, A_f2, A_f3 and A_f4, respectively. Markers delimiting inversion breakpoints are labeled 1 to 39.

Figure 2

Newly proposed cytological map of the D. guanche A chromosome. Colored boxes represent conserved blocks between D. guanche and D. subobscura based on the localization of breakpoints in the recently assembled D. guanche genome (Puerma et al.). Sections delimiting each conserved block, given in their upper part, were inferred from the localization in the Kunze-Mühl and Müller map of D. subobscura of the fragments spanning the six fixed inversions breakpoints in D. guanche (Puerma et al.). Differently colored lines stemming from a given breakpoint in the D. guanche map indicate each of its flanking regions and their localization in the Kunze-Mühl and Müller map of D. subobscura, as revealed by in situ hybridization.

Figure 3

Functional annotation of autosomal inversion breakpoints. Schematic representation of the sequenced and annotated breakpoint regions corresponding to the four autosomal inversions fixed since the D. subobscura-D. guanche split. D. guanche, above; D. subobscura, below. Dashed lines between chromosomal arrangements indicate the limits and orientation of homologous regions. Arrowed bars represent annotated coding regions whereas rhombuses represent annotated transposable elements and other repetitive sequences. Thick dark red lines above or below a particular breakpoint region indicate its collinearity relative to D. pseudoobscura.

Figure 4

Functional annotation of A chromosome inversion breakpoints. Schematic representation of the annotated breakpoint regions corresponding to X chromosome inversions A_f2 to A_f5 that originated and became fixed since the D. subobscura-D. guanche split. D. guanche, above; D. subobscura, below. Dashed lines between chromosomal arrangements indicate the limits and orientation of homologous regions. Arrowed bars represent annotated coding regions whereas rhombuses represent annotated transposable elements and other repetitive sequences. Thick dark red lines above a particular breakpoint region indicate its collinearity relative to D. pseudoobscura.?, missing information.

Figure 5

Distribution across the subobscura cluster phylogeny of the ten fixed inversions since the D. subobscura-D. guanche split. The ancestral arrangement of chromosomes J, E, O and A (X), as inferred in the present study, is given in the cluster ancestor. Inversions that became fixed in these chromosomes are presented in the different branches of the phylogeny. The arrangement for each chromosome upon the fixation of the 10 inversions is given for each lineage, with numbers in parentheses referring to presently polymorphic arrangements as a result of inversions that originated thereafter in the four affected chromosomes. *Inversion O₃ is currently polymorphic in D. subobscura and always associated with inversion O₄.

Figure 6

Sequential order of occurrence and fixation in the D. subobscura lineage of the four A chromosome overlapping inversions. Schematic representation of the order established in the present study (I) and its discarded alternative (II) for the sequential occurrence and fixation of inversions A_f1 to A_f4 of the A chromosome in the D. subobscura lineage after its split from D. guanche (see text). Horizontal bars with differently colored boxes represent the different chromosomal arrangements, with the ancestral order (now only present in D. guanche) in the upmost part of the figure and the D. subobscura A_st order in its lowest part. Boxes colored as in Fig. 2 reflect conserved blocks relative to D. subobscura. Pairs of crossed lines between arrangements represent the regions affected by each of the four inversions. Arrows connecting the different chromosomal arrangements represent the sequential accumulation of inversions from the ancestral D. guanche arrangement (see text).