Diaz et al. BMC Evolutionary Biology (2018) 18:144 https://doi.org/10.1186/s12862-018-1250-x RESEARCHARTICLE Open Access Positive selection at sites of chemosensory genes is associated with the recent divergence and local ecological adaptation in cactophilic Drosophila Fernando Diaz1, Carson W. Allan1 and Luciano M. Matzkin1,2,3* Abstract Background: Adaptation to new hosts in phytophagous insects often involves mechanisms of host recognition by genes of sensory pathways. Most often the molecular evolution of sensory genes has been explained in the context of the birth-and-death model. The role of positive selection is less understood, especially associated with host adaptation and specialization. Here we aim to contribute evidence for this latter hypothesis by considering the case of Drosophila mojavensis, a species with an evolutionary history shaped by multiple host shifts in a relatively short time scale, and its generalist sister species, D. arizonae. Results: We used a phylogenetic and population genetic analysis framework to test for positive selection in a subset of four chemoreceptor genes, one gustatory receptor (Gr) and three odorant receptors (Or), for which their expression has been previously associated with host shifts. We found strong evidence of positive selection at several amino acid sites in all genes investigated, most of which exhibited changes predicted to cause functional effects in these transmembrane proteins. A significant portion of the sites identified as evolving positively were largely found in the cytoplasmic region, although a few were also present in the extracellular domains. Conclusions: The pattern of substitution observed suggests that some of these changes likely had an effect on signal transduction as well as odorant recognition and protein-protein interactions. These findings support the role of positive selection in shaping the pattern of variation at chemosensory receptors, both during the specialization onto one or a few related hosts, but as well as during the evolution and adaptation of generalist species into utilizing several hosts. Keywords: Odorant receptor, Gustatory receptor, Population genetics, Molecular evolution, Adaptation, Cactophilic Drosophila Background shifting to a new host can therefore affect survival of Studying host adaptation is critical to understanding larva and adult stages, fecundity of feeding females as the ecological and evolutionary history of phytopha- well as behavioral patterns of oviposition and mating, gous insects [1–5]. Host shifts represent a distinct causing ultimately substantial impact on fitness. This and sometimes novel set of complex challenges since has led to the hypothesis that in addition to genes in- new host plants can differ in nutritional content, volved in performance relative to a new environment, chemical composition, microorganisms and defensive adaptation to new hosts often involves mechanisms of quality [6–8]. The use of novel resources when host preference [8–10]. Given their role in odorant and gustatory sensory and ultimately in the discrimin- * Correspondence: [email protected] ation of suitable hosts, chemoreceptors have been the 1 Department of Entomology, University of Arizona, Tucson, AZ 85721, USA focus of several studies examining the evolutionary 2Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA mechanisms underlying host adaptation [11–17]. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Diaz et al. BMC Evolutionary Biology (2018) 18:144 Page 2 of 13 Chemical stimuli recognition occurs through odorant preference, presumably by detecting volatiles associated (Or) and gustatory receptor (Gr) proteins, converting vola- with either the presence of nutrients or toxins produced tile and soluble chemicals from the environment into elec- in rotting cacti [35–37]. Or67c and Gr63a are known to trical outputs through nerve impulses [18, 19]. In insects, play a role in detecting the by-products of alcoholic fer- these proteins have seven trans-membrane domains mentation as well as CO2 [38, 39], while Or83c1 and located on the dendritic membranes of sensory neurons Or83c2 have been suggested to play role in food detec- housed in sensilla of receptor organs, with a reverse tion [38, 40]. orientation when compared to mammalian topology Drosophila mojavensis offers a unique case to study (COOH-tail extracellular) [20, 21]. While the number of host adaptation since its biogeographical history has genes in these families seems to be largely stable across been shaped by multiple host shifts in a relatively short Drosophila evolution, there have been constant gene loss period of time [41]. Since its divergence from its sister and gain events through the birth-and-death model in species D. arizonae, the D. mojavensis lineage has ex- insects [22–28] where divergence arises from the selection panded through a rapid specialization process (< 0.5 of beneficial mutations between paralogous genes [29]. Mya; [42, 43]) producing four host populations adapted This mode of evolution, as well as the clustered to different cactus species (Fig. 1): agria (Stenocereus organization of these genes has provided strong support gummosus) in Baja California; coastal prickly pear to the hypothesis that these loci play a critical role in (Opuntia littoralis) in Santa Catalina Island; red barrel ecological shifts and specialization [9, 11–17, 22, 23]. In (Ferocactus cylindraceus) in the Mojave Desert and particular, the ecological specialization observed within organpipe (S. thurberi) in the Sonora Desert population Drosophila has been suggested to have contributed to the [33, 34]. We investigated patterns of molecular evolution higher pseudogenization levels observed in sensory genes in four chemoreceptor genes across the four host popu- [14, 15]. However, the analysis of 12 Drosophila species lations of D. mojavensis and the generalist D. arizonae genomes [30], has shown that genome size or endemism to test for positive selection using a multiple approach could also predict pseudogenization rates, but the few of gene-wide, polymorphic-based, codon-based and specialist species compared and the lack of evolutionary functional tests. Then, we analyzed the distribution of data over short time scales has prevented efforts to sites showing evidence of positive selection on the struc- disentangle these causes [31]. ture of these trans-membrane proteins in order to test The dramatic heterogeneity in family size and diver- whether selection has been biased towards specific do- gence among chemosensory gene paralogs suggest that mains and discussed the implication of these results in these gene families have evolved rapidly. However, the terms of the role of positive selection in host preference pattern of variation and divergence at a majority of adaptation. genes are more consistent with purifying selection and only a small portion of genes have shown evidence of Results positive selection, some of which have been linked to Genetic diversity, neutrality tests and divergence specialization [14, 15, 29, 31]. Thus, specialist species For each locus examined, between 7 and 15 sequences such as D. sechellia and D. erecta have shown higher Ka/ were generated for each of the four D. mojavensis popu- Ks ratios when compared with their closest generalists lations (Sonora Desert, SON; Santa Catalina Island, species [15]. The majority of amino acid sites showing CAT; Mojave Desert, MOJ and Baja California, BAJ), as signatures of positive selection in these families have well as between 8 and 12 sequences of D. arizonae. After been found in the cytoplasmic domain, suggesting that excluding introns, these amplicons had between 1170 bp selection changes are associated with signal transduction for Or83c2 to 1479 bp for Gr63a (Table 1). Given the rather than the detection of odorant molecules [32]. To population structure in D. mojavensis (Additional file 1: date most comparisons have involved a few highly diver- Table S2) [44, 45], we sampled multiple individuals per gent species; here we examine the case of the cactophilic population. Here we report average values per popula- local specialist D. mojavensis and its generalist sister tion of D. mojavensis to make them comparable with species D. arizonae to investigate the role of positive se- those in D. arizonae (Table 1). Between 12 and 35 poly- lection in four candidate chemoreceptor genes (one Gr morphic sites and 6 and 12 different haplotypes were de- and three Or). These species have adapted to develop tected across the loci and species examined (Table 1). and feed on necrotic cacti, showing partially overlapping Diversity estimates in D. arizonae often fall between the distributions in xeric regions of southwestern United estimates for individual population in D. mojavensis States and northwestern Mexico [33, 34]. The four can- (Additional