Birth, death, and replacement of karyopherins in Drosophila

Abstract

Nucleocytoplasmic transport is a broadly conserved process across eukaryotes. Despite its essential function and conserved mechanism, components of the nuclear transport apparatus have been implicated in genetic conflicts in Drosophila, especially in the male germ line. The best understood case is represented by a truncated RanGAP gene duplication that is part of the segregation distorter system in Drosophila melanogaster. Consistent with the hypothesis that the nuclear transport pathway is at the heart of mediating genetic conflicts, both nucleoporins and directionality imposing components of nuclear transport have previously been shown to evolve under positive selection. Here, we present a comprehensive phylogenomic analysis of importins (karyopherins) in Drosophila evolution. Importins are adaptor molecules that physically mediate the transport of cargo molecules and comprise the third component of the nuclear transport apparatus. We find that importins have been repeatedly gained and lost throughout various stages of Drosophila evolution, including two intriguing examples of an apparently coincident loss and gain of nonorthologous and noncanonical importin-α. Although there are a few signatures of episodic positive selection, genetic innovation in importin evolution is more evident in patterns of recurrent gene birth and loss specifically for function in Drosophila testes, which is consistent with their role in supporting host genomes defense against segregation distortion.

Fig. 1.

(A) Gene structure and intron–exon boundary of importin-αs from the Drosophila melanogaster genome. aKap4 is a partial retrogene, which has one intron in the same precise location as aKap3, suggesting its birth from aKap3. Note that introns are not shown to scale. (B) Expression patterns of aKap4 and aKap3 in D. melanogaster as revealed by reverse transcription polymerase chain reaction (RT-PCR). Whereas aKap3 is ubiquitously expressed in adult D. melanogaster and Drosophila yakuba tissues, aKap4 is only expressed in testes in both species. (C) Using PCR and primers flanking the syntenic location of aKap4 (which is found in an intron of the CG32406 gene in D. melanogaster), we amplified the genomic region containing aKap4 gene from a variety of species. Subsequent sequence analysis of the approximately 2.2 kb products revealed that all members of the melanogaster species subgroup possess aKap4, whereas all other Drosophila species assayed had a much smaller (650 bp) band that corresponded to the syntenic region in the intron of the CG32406 but lacked aKap4. We therefore presume that aKap4 was born in the ancestor of the melanogaster species subgroup (bold lines in schematic phylogeny).

Fig. 2.

(A) McDonald–Kreitman test for aKap4 from Drosophila melanogaster and Drosophila simulans. Using a Fisher's exact test to evaluate significance, we find that aKap4 has evolved under positive selection specifically in the lineage leading to D. simulans. (B) Free-ratio analysis for aKap4 in the melanogaster group of species reveals a single lineage leading to the sister species D. simulans, Drosophila mauritiana, and Drosophila sechellia with a dN/dS ratio exceeding 1. This elevated dN/dS value of 2.43 was statistically significant when compared with the neutral expectation of 1 for this branch (see fig. 2D). (C) NSsites analysis for aKap4 from the melanogaster group of species suggests that some residues in aKap4 have evolved recurrently under positive selection. (D) Test for statistical significance of lineage-specific positive selection for aKap4 in the branch highlighted in (B) using the branch-specific positive selection analysis.

Fig. 3.

(A) Gene structure of aKap5 showing the absence of the canonical IBB domain. (B) Expression pattern of aKap5 and aKap3 in Drosophila virilis and Drosophila ananassae as revealed by RT-PCR. Like in Drosophila melanogaster, aKap3 is expressed ubiquitously, whereas aKap5 is expressed only in testes in both D. virilis and D. ananassae. (C) PCR surveys using aKap5-syntenic primers in the melanogaster group of species reveal that aKap5 is ancestrally present in Drosophila species (bold lines on schematic phylogeny, larger band of 2.2 kb) but was lost in the melanogaster species subgroup (leading to the shorter band of ∼800 bp) coincident with the gain of aKap4 (fig. 1). Although the Drosophila yakuba, Drosophila santomea, and Drosophila teissieri species also yield a larger PCR product, sequencing of this product revealed no akap5 rather an independent genomic expansion. Further bioinformatic analyses of genome sequences in 12 Drosophila species further revealed not only the loss of akap5 in Drosophila grimshawi but also the birth of aKap2A in this lineage.

Fig. 4.

(A) Free-Ratio analysis for aKap5 in the ananassae species group reveals only one lineage with a dN/dS > 1; however, this branch did not meet the statistical threshold for significance using a branch-specific test (fig. 4C). (B) NSsites analysis for aKap5 in the ananassae species group reveals no evidence for recurrently positively selected codons in aKap5. (C) Statistical tests for significance of the finding of lineage-specific positive selection for aKap5 in (A) using the branch-specific test.

FIG. 5.

(A) Gene structure and tissue-specific expression of aKap2B compared with the parental gene aKap2 in Drosophila ananassae. Whereas parental aKap2 is expressed in testes and ovaries, we find aKap2B to be exclusively expressed in testes of D. ananassae adults. (B) Gene structure of aKap2C compared with the parental aKap2 gene in D. virilis. Genomic PCR analyses reveal that akap2C is a young Drosophila virilis–specific partial retrogene, whereas parental aKap2 is present in other members of the virilis group. RT-PCR analyses on adult tissues reveal that aKap2C is testes specific in D. virilis, whereas akap2 is expressed in both testes and ovaries. (C) Genomic region encompassing the aKap5 duplication in Drosophila pseudoobscura compared with the syntenic region in Drosophila persimilis. Tissue-specific RT-PCR analyses of aKap5 paralogs in D. pseudoobscura reveal testes-specific expression in contrast to the ubiquitously expressed akap3.

FIG. 6.

(A) Maximum-likelihood tree constructed using PhyML showing the phylogenetic relationship between all importins based on amino acid sequences. The five clades of aKap genes are highlighted aKap1 through aKap5. Lineage-specific new aKap genes are highlighted in bold. Bootstrap values refer to 100 trials on PhyML performed using the webserver at www.phylogeny.fr. (B) A summary of the 12 sequenced Drosophila genomes indicating the presence and absence of importins across the Drosophila phylogeny. All importin-αs are indicated, along with their location in associated Muller elements. Also indicated is the size of the encoded proteins as well as all noncanonical IBB-domain lacking importins (in gray).

FIG. 7.

(A) Model for importin evolution where pressure to escape from antagonism from transposable elements (or unknown viruses) that wish to access the germ line genome leads to amino acid sequence evolution in importins. (B) An alternate (favored) model for importin evolution where pressure for restoring transport to suppress segregation in male gametogenesis drives innovation in importins.