A fast-evolving X-linked duplicate of importin-α2 is overexpressed in sex-ratio drive in Drosophila neotestacea

Abstract

Selfish genetic elements that manipulate gametogenesis to achieve a transmission advantage are known as meiotic drivers. Sex-ratio X chromosomes (SR) are meiotic drivers that prevent the maturation of Y-bearing sperm in male carriers to result in the production of mainly female progeny. The spread of an SR chromosome can affect host genetic diversity and genome evolution, and can even cause host extinction if it reaches sufficiently high prevalence. Meiotic drivers have evolved independently many times, though only in a few cases is the underlying genetic mechanism known. In this study we use a combination of transcriptomics and population genetics to identify widespread expression differences between the standard (ST) and sex-ratio (SR) X chromosomes of the fly Drosophila neotestacea. We found the X chromosome is enriched for differentially expressed transcripts and that many of these X-linked differentially expressed transcripts had elevated K_a /K_s values between ST and SR, indicative of potential functional differences. We identified a set of candidate transcripts, including a testis-specific, X-linked duplicate of the nuclear transport gene importin-α2 that is overexpressed in SR. We find suggestions of positive selection in the lineage leading to the duplicate and that its molecular evolutionary patterns are consistent with relaxed purifying selection in ST. As these patterns are consistent with involvement in the mechanism of drive in this species, this duplicate is a strong candidate worthy of further functional investigation. Nuclear transport may be a common target for genetic conflict, as the mechanism of the autosomal Segregation Distorter drive system in D. melanogaster involves the same pathway.

Keywords: genetic conflict; meiotic drive; nuclear transport; spermatogenesis; testes.

Figure 1.

Differentially expressed (DE) transcripts are enriched on the X-chromosome. The total number of transcripts that mapped to each chromosome is listed beneath the bars. The large Muller elements are X, B, C, D, and E. F is the small, non-recombining dot chromosome, mito is the mitochondria, Y is the Y-chromosome, and unk stands for unknown location. Dark and light grey indicate the proportion of ST and SR biased transcripts, respectively, given the total number of transcripts that mapped to that chromosome. The number of DE transcripts in each category is printed within each bar.

Figure 2.

Sequence differences between ST and SR in transcripts. A) There is weak relationship between differential expression between ST and SR (shown as the absolute value of log₂ fold change) and sequence differences between ST and SR (Pearson’s correlation, r = 0.125). Percent sequence difference is calculated as the total number of differences divided by the length of the transcript times 100. Transcripts with significant differential expression are marked in red. Only transcripts meeting the minimum coverage criteria for detecting sequence differences and had at least one difference are included. The mean percent different of transcripts with nucleotide differences was 0.53%. B) Transcripts with the highest number of differences are not the same as those with the highest K_a/K_s values. Only transcripts with more than three differences and at least one synonymous difference are included in the figure. Transcripts with more than three differences but no synonymous differences were also included in the K_a/K_s > 1 set. The identified candidates are marked in red; one of these had no synonymous differences and is not pictured. The inset Venn diagram shows the criteria used to identify K_a/K_s candidates and the number of transcripts in each category. The total number of transcripts with K_a/K_s > 1 or more than three synonymous differences was 46, the total number of ST-SR DE transcripts was 729, and the total number of testes-specific transcripts was 14,392. K_a/K_s was calculated between ST and SR.

Figure 3.

X-importin-α2 is a fast-evolving X-linked duplicate of the autosomal gene importin-α2. Branch lengths were estimated with a neighbor-joining tree of 1,000 bootstraps. All nodes had bootstrap support > 99. Each of the three importin-α clades is labeled. X-importin-α2 includes TR10603, TR2814, TR37105. Estimated d_N/d_S values larger than 0.1 are labeled, and branches with d_N/d_S values > 0.60 are marked in red.

Figure 4.

Molecular evolutionary patterns of top candidates are consistent with positive selection on SR and relaxed purifying selection on ST. A) Top candidates (top) on SR have significantly lower silent polymorphism than on ST. Silent sites include synonymous sites as well as non-coding sites. The star denotes statistical significance at p < 0.05, two-tailed Mann-Whitney U-test. B) The top candidates have higher non-synonymous polymorphism on ST than the non-candidates, though not significantly so (p = 0.056, two-tailed Mann-Whitney U-test). C) Tajima’s D is elevated in the top candidates on SR compared to the non-candidates, but not significant (p = 0.071, two-tailed Mann-Whitney U-test). In panels A through C, SR is represented by white boxes and ST by grey boxes. All data points are shown; the edges of the boxes are the first and third quartiles, and the middle line is the median. D) Haplotype structure of X-importin-α2. Each row is a chromosome, with ST phenotype males above the solid black line and SR phenotype males below. Each column is a single segregating site. Dark grey represents the individual carries the major allele, and light grey is the minor allele. Some sites have a third segregating allele, which is represented by white. Sites with a gap are marked with an X. Non-synonymous sites are denoted with an N; deletion polymorphisms are denoted with a G. Unlabeled sites are either synonymous or non-coding. Fixed differences between ST and SR are marked with a star. Sites in the intron and UTR are labeled. Sites located in specific protein domains are also labeled by grey blocks: the importin-ß binding (IBB) domain, the cargo binding domains (Armadillo [ARM] repeats 1 through 9), and the nuclear export factor (CAS) binding domain (ARM repeat 10) (Goldfarb et al., 2004).

Figure 5.

Developing bundle of 64 spermatids in the testes of ST males (left) and SR males (right) at 650× magnification. The DNA is stained with DAPI, revealing the heads of the spermatids. In SR, roughly half of the sperm do not develop properly, which are presumably Y-bearing spermatids. Arrows point out the heads of sperm that are not maturing properly.