Glue Genes Are Subjected to Diverse Selective Forces during Drosophila Development

GBE Glue Genes Are Subjected to Diverse Selective Forces during Drosophila Development Flora Borne 1 , Rob J. Kulathinal1,2, and Virginie Courtier-Orgogozo 1, * 1 CNRS, Institut Jacques Monod, Universite de Paris, France 2 Department of Biology, Temple University, USA Accepted: 30 October 2021 Abstract Molecular evolutionary studies usually focus on genes with clear roles in adult fitness or on developmental genes expressed at multiple time points during the life of the organism. Here, we examine the evolutionary dynamics of Drosophila glue genes, a set of eight genes tasked with a singular primary function during a specific developmental stage: the production of glue that allows animal pupa to attach to a substrate for several days during metamorphosis. Using phenotypic assays and available data from transcriptomics, PacBio genomes, and sequence variation from global populations, we explore the selective forces acting on glue genes within the cosmopolitan Drosophila melanogaster species and its five closely related species, D. simulans, D. sechellia, D. mauritiana, D. yakuba, and D. teissieri. We observe a three-fold difference in glue adhesion between the least and the most adhesive D. melanogaster strain, indicating a strong genetic component to phenotypic variation. These eight glue genes are among the most highly expressed genes in salivary glands yet they display no notable codon bias. New copies of Sgs3 and Sgs7 are found in D. yakuba and D. teissieri with the Sgs3 coding sequence evolving rapidly after duplication in the D. yakuba branch. Multiple sites along the various glue genes appear to be constrained. Our population genetics analysis in D. melanogaster suggests signals of local adaptive evolution for Sgs3, Sgs5, and Sgs5bis and traces of selective sweeps for Sgs1, Sgs3, Sgs7, and Sgs8. Our work shows that stage-specific genes can be subjected to various dynamic evolutionary forces. Key words: Drosophila, glue genes, adaptation, pupal development, Sgs, bioadhesive. Significance Drosophila larvae produce a glue to stick themselves to a substrate for several days during metamorphosis. Here, we observe wide variation in stickiness among Drosophila melanogaster strains and we analyze the molecular evolution of eight glue genes. We find several recent gene duplications and heterogenous rates of evolution among these genes. Introduction Our understanding of evolutionary patterns and processes in multicellular eukaryotes derives primarily from observations and analyses of adult stages. For example, in Gephebase, a database that compiles genes that have been found to contribute to evolutionary changes in animals and plants (Courtier-Orgogozo et al. 2020), about 95% of the phenotypic traits refers to the adult stage, whereas only 5% corresponds to earlier developmental stages. Yet, a large component of an individual’s relative fitness may occur before the adult stage. Here we use a simple model system to study the influence of evolutionary forces on genes whose function appears to be restricted to a specific developmental stage, the genes encoding for the glue that attaches the animal to external substrates during the pupal stage in Drosophila. In Diptera, development transitions through several larval stages followed by a pupal stage during which metamorphosis occurs. During the pupal stage, insects are particularly ß The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Genome Biol. Evol. 13(12) https://doi.org/10.1093/gbe/evab248 Advance Access publication 12 November 2021 1 *Corresponding author: E-mail: . GBE Borne et al. 2 D. melanogaster genes and, so far, they have only been found within the Drosophila genus, likely due to the rapid divergence of nucleotide sequences (Da Lage et al. 2019). In Drosophila, glue genes have undergone multiple gains and losses of copies as well as extensive genetic changes in their coding sequences (Farkas 2016; Da Lage et al. 2019). In particular, repeat regions of Sgs1, Sgs3, Sgs4, and Eig71Ee have evolved rapidly in terms of the number of repeats as well as the motif sequence of these repeats (Da Lage et al. 2019). A first attempt was made to study the evolutionary rate of the glue genes but was primarily limited to glue genes without repeats due to the limited quality of the available genome assemblies at the time (Da Lage et al. 2019). Recent advances in long read sequencing (e.g., PacBio, Oxford Nanopore) now make it possible to utilize higher quality genomes with reliable sequences spanning across multiple repeats. High quality assemblies have been recently generated in D. melanogaster and its closely related species (e.g., Chakraborty et al. 2019; Kim et al. 2021) and can be applied for the study of repeat-laden genes such as glue genes. Glue genes seem to be expressed exclusively in the salivary glands over a relatively short period of time during the larval stage (Andres et al. 1993; Li and White 2003; Duan et al. 2020), with the exception of Eig71Ee which is also expressed in hemocytes and in the gut where it is probably involved in immunity and clotting (Korayem et al. 2004). Their function thus appears to be limited to glue properties where they may play an important and very specific role in the fly’s ultimate survival. This set of tissue- and developmental stage-specific genes together with our recently developed phenotypic assay to quantify pupal adhesion (Borne et al. 2020) provides a promising model to understand how patterns of genetic variation are related to phenotypic variation in adhesion as well as the role of adaptation in early metamorphic stages. In this study, we investigate the phenotypic variation in pupal adhesion and the genetic variation of glue genes in Drosophila. We apply a force assay (Borne et al. 2020) on individual pupae from a set of 12 inbred D. melanogaster lines originating from different geographic locations (Chakraborty et al. 2019) as well as three sister species to survey differences in pupal adhesion. Using 15 high-quality PacBio genomes that correspond to these phenotyped lines and three additional lines from D. melanogaster, as well as high-quality PacBio genomes of five sister species, we then investigated the evolutionary dynamics of these glue genes. We observed low levels of codon bias on these highly expressed genes, discovered several gene duplications, and identified rapidly evolving lineages. Putative sites with signals of negative and positive selection were found in these glue genes among D. melanogaster lines as well as global populations from the D (...truncated)