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Copy number changes in co-expressed odorant receptor genes enable selection for sensory differences in drosophilid species

Nature Ecology & Evolution volume 6, pages 1343–1353 (2022)Cite this article

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Abstract

Despite numerous examples of chemoreceptor gene family expansions and contractions, how these relate to modifications in the sensory neuron populations in which they are expressed remains unclear. Drosophila melanogaster’s odorant receptor (Or) family is ideal for addressing this question because most Ors are expressed in distinct olfactory sensory neuron (OSN) types. Between-species changes in Or copy number may therefore indicate increases or reductions in the number of OSN populations. Here we investigated the Or67a subfamily, which exhibits copy number variation in D. melanogaster and its closest relatives: D. simulans, D. sechellia and D. mauritiana. These species’ common ancestor had three Or67a paralogues that had already diverged adaptively. Following speciation, two Or67a paralogues were lost independently in D. melanogaster and D. sechellia, with ongoing positive selection shaping the intact genes. Unexpectedly, the functionally diverged Or67a paralogues in D. simulans are co-expressed in a single neuron population, which projects to a glomerulus homologous to that innervated by Or67a neurons in D. melanogaster. Thus, while sensory pathway neuroanatomy is conserved, independent selection on co-expressed receptors has contributed to species-specific peripheral coding. This work reveals a type of adaptive change largely overlooked for olfactory evolution, raising the possibility that similar processes influence other cases of insect Or co-expression.

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Fig. 1: Molecular evolution of the Or67a subfamily in Drosophila.
Fig. 2: Evolutionary changes in Or67a orthologue and paralogue odour responses.
Fig. 3: Evolutionary analyses of Or67a expression and circuit neuroanatomy.
Fig. 4: Unique but overlapping contributions of Or67a paralogues to endogenous neuronal responses.

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Data availability

All raw data generated for this study are available in the supplementary files or in the GitLab repository at https://gitlab.com/roman.arguello/or67a_dsim_trio.

Code availability

The code for this study is available in the GitLab repository at https://gitlab.com/roman.arguello/or67a_dsim_trio.

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Acknowledgements

We thank M. Cardoso-Moreira, L.L. Prieto-Godino, J. A. Sánchez-Alcañiz, L. Keller, M. Long and members of the Arguello lab for comments on earlier versions of the manuscript; M. Khallaf, M. Knaden, A. Svatos and J. Weissflog at the Max Planck Institute for Chemical Ecology for the synthesized (R)-actinidine; and J. Carlson and D. Stern for sharing transgenic fly lines. T.O.A. was supported by a Human Frontier Science Program Long-Term Fellowship (no. LT000461/2015-L) and a Swiss National Science Foundation Ambizione Grant (no. PZ00P3 185743). Research in R.B.’s laboratory was supported by ERC Consolidator and Advanced Grants (nos 615094 and 833548, respectively) and the Swiss National Science Foundation. Research in J.R.A.’s lab is supported by the Swiss National Science Foundation grants no. PP00P3_176956 and no. 310030_201188.

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  1. These authors contributed equally: Thomas O. Auer, Raquel Álvarez-Ocaña.

Authors and Affiliations

  1. Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland

    Thomas O. Auer, Raquel Álvarez-Ocaña, Steeve Cruchet & Richard Benton

  2. Department of Ecology & Evolution, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland

    J. Roman Arguello

  3. Swiss Institute of Bioinformatics, Lausanne, Switzerland

    J. Roman Arguello

Authors
  1. Thomas O. Auer
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  2. Raquel Álvarez-Ocaña
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  3. Steeve Cruchet
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  4. Richard Benton
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  5. J. Roman Arguello
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Contributions

J.R.A. conceived the study. J.R.A., T.O.A. and R.A.-O. designed the experiments with input from R.B. J.R.A., T.O.A., R.A.-O. and S.C. carried out the experiments. J.R.A., T.O.A. and R.A.-O. analysed the data. J.R.A and T.O.A. prepared the original draft of the manuscript with revisions from R.A.-O. and R.B. All authors read and approved the final version of the manuscript.

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Correspondence to J. Roman Arguello.

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Nature Ecology & Evolution thanks Jean-Christophe Billeter, Carolyn McBride and Coral Warr for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Microsynteny for chromosomal regions containing Or67a.D and Or67a.P.

Alignment for the chromosome 3L interval containing Or67a.D and Or67a.P (and flanking genes, light blue) for six species. High sequence identity is indicated with black alignment blocks with low sequence identity indicated with light grey alignment blocks. Thin horizontal lines are alignment gaps. Red annotations indicate locations of transposable elements. Chromosome position on the horizontal axis are relative to the extracted interval. See Supplementary Files 1 for the alignment in a flat file.

Extended Data Fig. 2 Microsynteny for chromosomal region containing Or67a.3R.

Alignment for the chromosome 3R interval containing Or67a.3R (and flanking genes, light blue) for six species. High sequence identity is indicated with black alignment blocks with low sequence identity indicated with light grey alignment blocks. Thin horizontal lines are alignment gaps. Red annotations indicate locations of transposable elements. Chromosome position on the horizontal axis are relative to the extracted interval. See Supplementary Files 2 for the alignment in a flat file.

Extended Data Fig. 3 Genetic diversity in regions containing D. melanogaster’s Or67a.D and Or67a.3R deletions.

Nucleotide diversity (Π) and Tajima’s D over D. melanogaster’s chromosomal regions containing the intact Or67a.P gene and the deleted Or67a.D and Or67a.3R. The regions containing the deleted Or67a paralogs do not show differences in genetic diversity in comparison to the surrounding regions, as would be expected if the deletions were adaptive and swept in the population. The black line in each panel is the smoothed curve fit with LOESS, with the grey ribbon around it displaying the standard error. The sample size (number of genomes) = 84.

Extended Data Fig. 4 Evolution of odour sensitivity across Or67a orthologs.

The full set of dose-response experiments for the subset of odours that evoked high or intermediate responses in our initial screen of nine odours (Fig. 2b). For simplicity, the level of significance indicated above each concentration’s comparison is only for the single comparison with the largest difference (see Supplementary Table 4 for the full set of tests; *p < 0.05, **p < 0.01, ***p < 0.001). Colours correspond to those in Fig. 2d. p-values were calculated with a two-sided Dunn test; correction for multiple comparisons was done using the Holm method. For sample sizes and all test results see Supplementary Table 4.

Extended Data Fig. 5 Transgenic tools for Or67a expression analyses in D. simulans and D. melanogaster.

a, Schematics of the wild-type and knock-in Gal4 transcriptional reporter alleles at the DsimOr67a.3R (top) and DsimOr67a.D/P (bottom) loci. The first was created via a two-step process (CRISPR/Cas9 engineering + PhiC31 mediated integration) while the latter was resulting from a direct CRISPR/Cas9 mediated insertion. The red lines indicate sgRNA cutting sites; three and two sgRNAs were used to target the Or67a.3R and Or67a.P locus, respectively. Note that the intercalated sequence was removed upon donor vector integration. b, Antennal co-expression of the melOr67a.P-GFP transcriptional reporter and Or67a.P RNA in D. melanogaster. Scale bar = 25 μm. c, Antennal co-expression of the simOr67a.P-GFP and simOr67a.D-RFP transcriptional reporters (top) and the simOr67a.3R-GFP and simOr67a.D-RFP transcriptional reporters (bottom) in D. melanogaster. Scale bar = 25 μm. d, Pairing of the simOr67a.P-GFP promoter transcriptional reporter and melOr85f-Gal4, UAS-RFP expression in neighboring neurons in the antenna of D. melanogaster. Scale bar = 25 μm. Inset scale bar = 5 μm. (b-d) Experiments were repeated at least three times for each staining on independent days and pictures show representative examples for each condition.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. Codeml table of models tested and likelihoods. Supplementary Table 2. Codeml table of likelihood ratio tests. Supplementary Table 3. Relative effects of odours on receptor responses based on the npmv analysis. Supplementary Table 4. Dose–response test results and sample sizes. Supplementary Table 5. Pairwise identity between Or67a gene sequences. Supplementary Table 6. Pairwise identity between Or67a promoter sequences. Supplementary Table 7. Table with MEME results. Supplementary Table 8. Oligonucleotides used in this study.

Supplementary Data 1

Alignment for the 3L region containing Or67a.D and Or67a.P.

Supplementary Data 2

Alignment for the 3R region containing Or67a.3R.

Supplementary Data 3

D. simulans and D. melanogaster polymorphism summaries.

Supplementary Data 4

Odour response data from electrophysiology experiments: 10−2.

Supplementary Data 5

Odour response data from electrophysiology experiments: dose–responses.

Supplementary Data 6

Odour response data from electrophysiology experiments: fluorescence-guided single-sensillum recordings.

Supplementary Data 7

D. simulans Sanger sequences generated in this study.

Supplementary Data 8

D. simulans Sanger sequences generated in this study.

Supplementary Data 9

D. simulans Sanger sequences generated in this study.

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Auer, T.O., Álvarez-Ocaña, R., Cruchet, S. et al. Copy number changes in co-expressed odorant receptor genes enable selection for sensory differences in drosophilid species. Nat Ecol Evol 6, 1343–1353 (2022). https://doi.org/10.1038/s41559-022-01830-y

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