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Patterns of molecular evolution of the germ line specification gene oskar suggest that a novel domain may contribute to functional divergence in Drosophila

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Abstract

In several metazoans including flies of the genus Drosophila, germ line specification occurs through the inheritance of maternally deposited cytoplasmic determinants, collectively called germ plasm. The novel insect gene oskar is at the top of the Drosophila germ line specification pathway, and also plays an important role in posterior patterning. A novel N-terminal domain of oskar (the Long Oskar domain) evolved in Drosophilids, but the role of this domain in oskar functional evolution is unknown. Trans-species transgenesis experiments have shown that oskar orthologs from different Drosophila species have functionally diverged, but the underlying selective pressures and molecular changes have not been investigated. As a first step toward understanding how Oskar function could have evolved, we applied molecular evolution analysis to oskar sequences from the completely sequenced genomes of 16 Drosophila species from the Sophophora subgenus, Drosophila virilis and Drosophila immigrans. We show that overall, this gene is subject to purifying selection, but that individual predicted structural and functional domains are subject to heterogeneous selection pressures. Specifically, two domains, the Drosophila-specific Long Osk domain and the region that interacts with the germ plasm protein Lasp, are evolving at a faster rate than other regions of oskar. Further, we provide evidence that positive selection may have acted on specific sites within these two domains on the D. virilis branch. Our domain-based analysis suggests that changes in the Long Osk and Lasp-binding domains are strong candidates for the molecular basis of functional divergence between the Oskar proteins of D. melanogaster and D. virilis. This molecular evolutionary analysis thus represents an important step towards understanding the role of an evolutionarily and developmentally critical gene in germ plasm evolution and assembly.

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Acknowledgments

Thanks to Paul Macdonald for the plasmid containing the D. virilis oskar cDNA, to John Srouji for Sanger sequencing and discussion of the results, to Victor Zeng and Amit Indap for assistance with preliminary analyses, and to Extavour lab members for discussion of the manuscript. This work was partly supported by NSF grant IOS-0817678 to CGE and funds from Harvard University.

Competing interests

The authors declare that they have no competing interests.

Author contributions

AA conceived of the study, created alignments, and performed evolutionary rate analyses. CGE assisted with study design and performed analyses of amino acid physicochemical properties and phylogenetic distribution of germ plasm morphology. Both authors wrote and approved the final manuscript.

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  1. Abha Ahuja

    Present address: Curriculum Fellows Program, Department of Cell Biology, Harvard Medical School, Boston, MA, USA

Authors and Affiliations

  1. Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA, 02138, USA

    Abha Ahuja & Cassandra G. Extavour

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  1. Abha Ahuja
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Correspondence to Abha Ahuja or Cassandra G. Extavour.

Additional information

Communicated by: Claude Desplan

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1

Nucleotide sequences and accessions of sequences of oskar orthologs used for analyses. (PDF 99.1 KB)

Online Resource 2

Oskar amino acid alignment generated with sequences from 18 Drosophilids using the MUSCLE MSA, and the results of multiple PAML analyses of this alignment. (PDF 296 kb)

Online Resource 3

Oskar amino acid alignment generated with sequences from 18 Drosophilids using the PRANK MSA, and the results of multiple PAML analyses of this alignment. (PDF 306 kb)

Online Resource 4

Amino acid alignments of conserved sequence blocks from specific predicted structural and interaction Oskar domains from 18 Drosophilids using the PRANK MSA, and the results of the PAML analysis of these alignments. (PDF 170 kb)

Online Resource 5

Amino acid alignments of Oskar from the five melanogaster subgroup Drosophila species using the MUSCLE MSA, and the results of multiple PAML analyses of this alignment. (PDF 192 kb)

Online Resource 6

Amino acid alignments of specific predicted structural and interaction Oskar domains from seven Drosophilids (melanogaster subgroup members plus D. virilis and D. immigrans) using the PRANK MSA, and the results of multiple PAML analyses of these alignments. (PDF 173 kb)

Online Resource 7

Clustal alignment of the amino acid translation of the three D. virilis alleles used in this study: (1) annotated from genomic sequence; NCBI accession XM_002053233.1; (2) cDNA sequence reported by Webster et al. (1994) (“Macdonald allele”); NCBI accession L22556.1; and (3) cDNA sequence obtained from Macdonald lab and verified by Sanger sequencing. Residues that are different between the different alleles and/or under positive selection are indicated. (PDF 101 kb)

Online Resource 8

Amino acid alignments of the Long Osk domain from 18 Drosophilids including the D. virilis allele sequence from Webster et al. (1994) using the MUSCLE MSA, and the results of multiple PAML analyses of these alignments. (PDF 134 kb)

Online Resource 9

Amino acid alignments of the Long Osk and Lasp-binding domains from 18 Drosophilids including the D. virilis allele sequence from Webster et al. (1994) using the PRANK MSA, and the results of multiple PAML analyses of these alignments. (PDF 148 kb)

Online Resource 10

Phylogenetic distribution of germ plasm morphology in Drosophila species for which data are available, as determined by histology and electron microscopy (Mahowald 1962, 1968; Counce 1963). In the unipolar morphology all or most germ granules are aggregated into a single, large perinuclear body. In the perinuclear morphology, germ granules are aggregated into multiple perinuclear bodies distributed over the cytoplasmic face of nuclear membrane. In the dispersed morphology, observed only in D. melanogaster, some germ granules are perinuclear and others are distributed throughout the cytoplasm. Species included in the present molecular evolution analysis are indicated in bold face; colored text (red/blue) indicates functional conservation of oskar with respect to germ plasm assembly in D. melanogaster (Webster et al. 1994; Jones and Macdonald 2007). No sequence data are currently publicly available for the remaining species shown, with the exception of D. willistoni, whose oskar sequence was incomplete and excluded from the present analysis (see Methods for details). Phylogenetic relationships from (Oliveira et al. 2012; Da Lage et al. 2006; van der Linde et al. 2010; Robe et al. 2005). (PDF 359 kb)

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Ahuja, A., Extavour, C.G. Patterns of molecular evolution of the germ line specification gene oskar suggest that a novel domain may contribute to functional divergence in Drosophila . Dev Genes Evol 224, 65–77 (2014). https://doi.org/10.1007/s00427-013-0463-7

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