A rapidly evolving MYB-related protein causes species isolation in Drosophila

Abstract

Matings among different species of animals or plants often result in sterile or lethal hybrids. Identifying the evolutionary forces that create hybrid incompatibility alleles is fundamental to understanding the process of speciation, but very few such alleles have been identified, particularly in model organisms that are amenable to experimental manipulation. We report here the cloning of the first, to our knowledge, Drosophila melanogaster gene involved in hybrid incompatibilities, Hybrid male rescue (Hmr). Hmr causes lethality and female sterility in hybrids among D. melanogaster and its sibling species. We have found that Hmr encodes a protein with homology to a family of MYB-related DNA-binding transcriptional regulators. The HMR protein has evolved both amino acid substitutions and insertions and deletions at an extraordinarily high rate between D. melanogaster and its sibling species, including in its predicted DNA-binding domain. Our results suggest that hybrid lethality may result from disruptions in gene regulation, and we also propose that rapid evolution may be a hallmark of speciation genes in general.

Figure 1

Physical and genetic maps of the Hmr region. A chromosome walk of cosmid (28) and P1 (29) clones with their cytological locations on the X chromosome is shown above a map of deficiency breakpoints. Rescue status refers to assays for high-temperature hybrid female lethality suppression. The vertical lines designate the region considered for further study. Below are shown predicted and known genes (19) in the Hmr region as well as the previously unannotated Rab9D (30) aligned with the deficiency map. The intron/exon structure is shown only for the two transcripts of the sprint gene (31), which are labeled above their respective 5′-most exons.

Figure 2

Identification of CG1619 as Hmr. (A) P element transgene constructs assayed for Hmr activity shown with the restriction sites used for cloning. Transgenes were classified as Hmr⁺ if they suppressed Hmr¹- and In(1)AB-dependent hybrid male rescue (Tables 1 and 2). (B) Northern blot analysis of CG1619 expression in wild-type (Oregon-R) and mutant D. melanogaster samples using the RE54143 cDNA as a probe. Reprobing with a RpL32 cDNA was used as a loading control. For mutant samples, RNA was from 24- to 48-h (lanes 6–8) or 48- to 72-h (lanes 9 and10) larvae. Df(1)EP307-1-2/Y animals were identified as the Gfp-negative progeny of a Df(1)EP307-1-2/FM7i, P{w^+mC = ActGFP}JMR3 stock. (C) cDNA structures with noncoding regions in white, coding regions in gray, and MADF domain-homologous regions in black. Putative translation start sites are shown as large arrows. Primers used for RT-PCR are shown as small arrows with the expected size of PCR products indicated between them. The location of the P element insertion in Hmr¹ is also shown. (D) PCRs using as templates the two CG1619 cDNAs and reverse transcribed RNAs from two different wild-type stocks. Location of the primers used is shown in Fig. 2C. (E) Alignment of the two MADF domains of HMR with regions of representative MADF domain (ADF1), MYB domain (human C-MYB), and SANT domain (yeast Swi3) proteins. The 65% consensus of the MADF domain (accession no. SM0595) in the Simple Modular Architecture Research Tool database (SMART; http://smart.embl-heidelberg.de) is shown at the top, with identical and similar residues in the alignment darkly and lightly shaded, respectively. a, aromatic; l, aliphatic; +, positively charged. Defining members of the SANT domain are the SWI3 and ADA2 components of the SWI-SNF and ADA transcription activation complexes, respectively, the transcriptional corepressor N-CoR and the TFIIIB B“ subunit of the RNA polymerase III initiation complex (21).

Figure 3

Hmr evolves rapidly in Drosophila. (A) Northern blot analysis of CG1619 expression in the sibling species. Note that the levels of CG1619 cannot be accurately compared between the species because the D. simulans probe used has incomplete homology to the other species, particularly D. melanogaster. rRNA was used as a loading control. (B) A 300-bp sliding window plot of average nucleotide divergence calculated by the method of Nei and Gojobori (32), aligned with the gene structure of Hmr as diagrammed in Fig. 2C. Mean divergence values are indicated. Of all possible nonoverlapping 50 codon windows (n = 999), a window centered over the end of the first DNA-binding domain had the highest mean replacement divergence between D. melanogaster and its siblings (0.183). Among 100,000 random shufflings of the order of codons, mean divergence values ≥0.183 occurred only 705 times, indicating that this high level of divergence is significant at P < 0.01. (C) Alignment of the MADF domains of Hmr from D. melanogaster and its sibling species. Amino acids of the sibling species proteins that differ from D. melanogaster are shown; identical residues are indicated as dots. The MADF consensus is shown as in Fig. 2E.