bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448721; this version posted June 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1 Evolution of Drosophila buzzatii wings: Modular genetic organization,
2 sex-biased integrative selection and intralocus sexual conflict
3 short running title: Evolution of Drosophila buzzatii wings
4 Iglesias PP1†*, Machado FA2†*, Llanes S3, Hasson E3, Soto EM3
5 1 Laboratorio de Genética Evolutiva, Universidad Nacional de Misiones – CONICET, Félix de
6 Azara 1552, N3300LQH, Misiones, Argentina.
7 2 Department of Biological Sciences, Virginia Polytechnic Institute and State University,
8 Blacksburg, United States.
9 3 Instituto de Ecología, Genética y Evolución de Buenos Aires (IEGEBA – CONICET), DEGE,
10 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires,
11 Argentina.
12 †These authors contributed equally to this work.
13 *Corresponding authors; e-mail: [email protected] , [email protected]
14
15 Abstract
16 The Drosophila wing is a structure shared by males and females with the
17 main function of flight. However, in males, wings are also used to produce songs, or
18 visual displays during courtship. Thus, observed changes in wing phenotype depend
19 on the interaction between sex-specific selective pressures and the genetic and
20 ontogenetic restrictions imposed by a common genetic architecture. Here, we
21 investigate these issues by studying how the wing has evolved in twelve populations
22 of Drosophila buzzatii raised in common-garden conditions and using an isofemale
1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448721; this version posted June 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
23 line design. The between-population divergence shows that sexual dimorphism is
24 greater when sex evolves in different directions. Multivariate Qst-Fst analyses
25 confirm that male wing shape is the target for multiple selective pressures, leading
26 males’ wings to diverge more than females’ wings. While the wing blade and the
27 wing base appear to be valid modules at the genetic (G matrix) and among-
28 population (D matrix) levels, the reconstruction of between-population adaptive
29 landscapes (Ω matrix) shows selection as an integrative force. Also, cross-sex
30 covariances reduced the predicted response to selection in the direction of the
31 extant sexual dimorphism, suggesting that selection had to be intensified in order to
32 circumvent the limitations imposed by G. However, such intensity of selection was
33 not able to break the modularity pattern of the wing. The results obtained here show
34 that the evolution of D. buzzatii wing shape is the product of a complex interplay
35 between ontogenetic constraints and conflicting sexual and natural selections.
36
37 Keywords
38 Adaptive landscapes, Genetic architecture, Intralocus sexual conflict, Morphological
39 integration/modularity, Multivariate selection.
40
41 Introduction
42 Understanding changes in morphological structures requires an integrative
43 approach that also considers constraints upon change. How is morphology
44 produced during development in the first place? Is selection in line with these
45 developmental rules? Does selection differ between the sexes? Morphological
46 integration, selection, and between-sex pleiotropy are key factors generating
47 association among traits at larger scales. An integrated developmental pattern or
2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448721; this version posted June 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
48 the occurrence of sex-specific selection on homologous structures controlled by
49 common genetic machinery (i.e. intralocus sexual conflict) may constrain the
50 evolutionary trajectories of such structures. It is only after evaluating the relative
51 effect of these factors that morphological changes can be understood in the light of
52 the interaction between constraints and their potential for functional adaptation.
53 The wing of Drosophila is a complex structure involved in different functions
54 such as flight and acoustic or visual communication (Wooton, 1992). For a long time,
55 it has been considered as a developmentally integrated structure that constrains
56 adaptive evolution (Houle, Bolstad, Van der Linde, & Hansen, 2017; Klingenberg,
57 2009; Klingenberg & Zaklan, 2000). While these conclusions are mostly based on
58 the hypothesis that the wing is divided into anterior and posterior (AP)
59 compartments (Klingenberg & Zaklan, 2000), recently Muñoz-Muñoz et al. (2016)
60 showed evidence supporting the compartmentalization of this structure along the
61 proximo-distal (PD) axis, forming two different modules, the wing base and the wing
62 blade. These modules were recognized not only on the phenotypic level but also at
63 the genetic and environmental levels, suggesting that it is possibly a consequence of
64 a modular developmental program.
65 From the wing primary task perspective (i.e. flight), the developmental
66 modules seem to match functional modules: the wing base transmits the forces
67 generated by the flight muscles and the wing blade generates the aerodynamic
68 forces necessary to lift the body (Dudley 2002). Although fly's ability is common to
69 both sexes, sex-biased dispersal has been documented in Drosophila (Begon, 1976;
70 Powell, Dobzhansky, Hook, & Wistrand, 1976; Fontdevila & Carson, 1978; Markow
71 & Castrezana, 2000; Mishra, Tung, Shree Sruti, Srivathsa, & Dey, 2020). An
3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448721; this version posted June 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
72 asymmetric dispersal of the sexes can exert different selective pressures on wing
73 morphology in each sex, leading to a sex-biased evolution of the modules.
74 Drosophila’s wings are also involved in premating behaviors that markedly
75 differentiate the wing’s function between sexes (Ewing, 1983; Dickson, 2008).
76 Except for a few species (within the Drosophila virilis species group; Satokangas,
77 Liimatainen, & Hoikkala, 1994), only males use wings for acoustic or visual
78 communication. Therefore, if wing morphology influences sound production or
79 visual display, only male wings will be subject to selection. This selection on males
80 can cause the displacement of females from their phenotypic optimum, reducing
81 their fitness. It is well documented that the direction and intensity of selection on
82 courtship song have been found to differ among populations and species according
83 to female preferences (Iglesias & Hasson, 2017; Iglesias et al., 2018a; Klappert,
84 Mazzi, Hoikkala, & Ritchie, 2007).
85 Here, we address these issues by investigating how the wing has evolved in
86 twelve populations of the cactophilic species Drosophila buzzatii raised in common-
87 garden conditions and using an isofemale line design. Only the males of the D.
88 buzzatii species use wings to produce a courtship song (Iglesias & Hasson, 2017;
89 Iglesias et al., 2018a; Iglesias, Soto, Soto, Colines, & Hasson, 2018b), and a previous
90 study has shown a rapid divergence of courtship song parameters among these
91 populations (Iglesias et al., 2018a). Thus, we first investigated whether the wings of
92 males and females are evolving differently among the sampled populations. At
93 present, wing modularity at the genetic level has only been tested in the model
94 species D. melanogaster (but see Soto, Carreira, Soto, & Hasson, 2008); however, D.
95 buzzatii is a cactophilic species distantly related to it. The effect of selection and drift
96 accumulate through time in each evolving lineage and can even change the modular
4
bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448721; this version posted June 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
97 pattern in an evolutionary context (Martín-Serra, Figueirido, & Palmqvist, 2020;
98 Melo & Marroig, 2015). Thus, we then tested whether the wing in each sex is
99 organized in two modules along the PD (proximo-distal) or the AP (antero-
100 posterior) axis in D. buzzatii. We test these hypotheses by estimating the posterior
101 distribution of the Among-Population (D) and Additive Genetic (G) within and
102 between sexes covariances from a landmark-based analysis. We also estimated the
103 covariance pattern among peaks on the realized adaptive landscape for these
104 populations (Ω) to evaluate the potential effect of selection in defining between-
105 populations patterns of phenotypic integration and modularity. We also applied a
106 multivariate FSTq –FST approach to determine the extent to which selection and
107 drift contributed to the evolution of the wing. Finally, we investigated how cross-sex
108 covariances constrain or facilitate the predicted responses to multivariate selection.
109 To do that, we combined the multivariate breeder’s equation with random and
110 empirical selection gradients and compared the predicted response to selection
111 when using a G with and without between sex covariances. If intralocus sexual
112 conflict is not fully resolved, we expect high and positive intersexual genetic
113 correlations resulting in an augmented response when cross-sex covariances are
114 setting to zeroes. Otherwise, if the genetic architecture of wings evolves to allow
115 independent adaptation in each sex, thus resolving intralocus conflict, we expect the
116 response to selection to be the same. However, if cross-sex covariances facilitate the
117 response to selection, we expect the magnitude of the response to be higher when
118 including them.
119
120 Materials and Methods
121
5
bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448721; this version posted June 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
122 Data collection and measurements
123 For this study, we analyzed 12 populations of D. buzzatii flies that have been
124 previously used to study variation in male courtship song (Iglesias et al., 2018a).
125 Each population was characterized by eight to 15 isofemale lines founded with wild-
126 collected gravid females. Flies were raised under common-garden and controlled-
127 density conditions (40 first-instar larvae per vial), and a photoperiod regimen of 12-
128 h light: 12-h dark cycle. First, they were raised on standard Drosophila medium for
129 four generations and then were moved one more generation to a ‘semi-natural’
130 medium prepared with fresh cladodes of the cactus Opuntia ficus indica (see Iglesias
131 et al., 2018a for more details). This cactus species represents the more widespread
132 host used by D. buzzatii in the study area.
133 We removed the right-wing of between three to five adult emerged flies per
134 sex and line, and mounted them on glass microscope slides for image acquisition.
135 Following Muñoz-Muñoz et al. (2016), a set of 15 landmarks was digitized in each
136 wing using the TPSdig software (Rohlf, 2001). Shape information was obtained from
137 the configurations of landmarks using standard geometric morphometrics methods
138 as implemented in the Geomorph package v3.0.7 (Adams, Collyer, Kaliontzopoulou,
139 & Sherratt, 2018). To examine shape variation, we performed a principal component
140 (PC) analysis based on the covariance matrix of Procrustes residuals. To evaluate
141 how many PCs should we retain as wing shape variables in subsequent analyses we
142 employed the broken-stick model criterion implemented in the package vegan
143 (Oksanen et al., 2019), which indicated that only the first 5 axes should be kept
144 (68.28% of total variation).
145
146 Genetic covariation, population divergence and selection
6
bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448721; this version posted June 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
147 We estimated the posterior distribution of the Among-Population (D) and
148 additive genetic (G) covariance matrices by using a Bayesian multivariate mixed
149 model implemented in the R package MCMCglmm (Hadfield, 2019). Population and
150 Line (nested within Population) were included as fixed and random factors,
151 respectively. The analysis was run for 105 generations with 50% burn-in, after
152 which we extracted 500 samples. Convergence was verified using trace plots for all
153 parameters.
154 The covariance matrices for the fixed effect Population and random effect
155 Line were considered as the Among-Population (D) and the genetic (G) covariance
156 matrices, respectively. Each sex-trait combination was treated as a different trait
157 (Sztepanacz & Houle, 2019) resulting in 10 traits for each level of comparison. Thus,
158 G and D are composed of four submatrices representing both patterns of integration
159 and divergence between and within sexes as follows