Reproductive capacity evolves in response to ecology through common developmental mechanisms in Hawai’ian Drosophila

Lifetime reproductive capacity, or the total number of offspring that an individual can give rise to in its lifetime, is a fitness component critical to the evolutionary process. In insects, female reproductive capacity is largely determined by the number of ovarioles, the egg-producing subunits of the ovary. Recent work has provided insights into the genetic and environmental control of ovariole number in Drosophila melanogaster. However, whether regulatory mechanisms discovered under laboratory conditions also explain evolutionary variation in natural populations is an outstanding question. Here we report, for the first time, insights into the mechanisms regulating ovariole number and its evolution among Hawai’ian Drosophila, a large adaptive radiation of fruit flies in which the highest and lowest ovariole numbers of the genus have evolved within 25 million years. Using phylogenetic comparative methods, we show that ovariole number variation among Hawai’ian Drosophila is best explained by adaptation to specific oviposition substrates. Further, we show that evolution of oviposition on ephemeral egg-laying substrates is linked to changes the allometric relationship between body size and ovariole number. Finally, we provide evidence that the developmental mechanism principally responsible for controlling ovariole number in D. melanogaster also regulates ovariole number in natural populations of Hawai’ian drosophilids. By integrating ecology, organismal growth, and cell behavior during development to understand the evolution of ovariole number, this work connects the ultimate and proximate mechanisms of evolutionary change in reproductive capacity.


Abstract 30
Lifetime reproductive capacity, or the total number of offspring that an individual can give rise 31 to in its lifetime, is a fitness component critical to the evolutionary process. In insects, female 32 reproductive capacity is largely determined by the number of ovarioles, the egg-producing 33 subunits of the ovary. Recent work has provided insights into the genetic and environmental 34 control of ovariole number in Drosophila melanogaster. However, whether regulatory 35 mechanisms discovered under laboratory conditions also explain evolutionary variation in 36 natural populations is an outstanding question. Here we report, for the first time, insights into the 37 mechanisms regulating ovariole number and its evolution among Hawai'ian Drosophila, a large 38 adaptive radiation of fruit flies in which the highest and lowest ovariole numbers of the genus 39 have evolved within 25 million years. Using phylogenetic comparative methods, we show that 40 ovariole number variation among Hawai'ian Drosophila is best explained by adaptation to 41 specific oviposition substrates. Further, we show that evolution of oviposition on ephemeral egg-42 laying substrates is linked to changes the allometric relationship between body size and ovariole 43 number. Finally, we provide evidence that the developmental mechanism principally responsible 44 for controlling ovariole number in D. melanogaster also regulates ovariole number in natural 45 populations of Hawai'ian drosophilids. By integrating ecology, organismal growth, and cell 46 behavior during development to understand the evolution of ovariole number, this work connects 47

Introduction 51
Reproductive capacity is an important life history trait that directly influences fitness by 52 determining how many offspring an individual can leave behind. There is a wide range in 53 potential fecundity across species (1, 2), which is often interpreted as trade-offs with presumed 54 ecological and developmental constraints . Trade-offs have been invoked to explain patterns of 55 egg-laying in animals, where total fecundity can correlate negatively with egg mass, clutch size 56 or lifespan (3-10), and positively with body size (11)(12)(13). In addition to these hypothesized 57 physical or growth-related constraints, life history parameters including predation risk, 58 environmental variability, host specialization and levels of parental care have been proposed to 59 influence evolutionary change in fecundity (1,(14)(15)(16)(17), suggesting that this trait could represent a 60 complex intersection between ecology and physiology. However, few studies have addressed 61 how female reproductive capacity evolves in response to ecology, and how these pressures 62 manifest as different phenotypes through changes in development. 63 In insects, female reproductive capacity is strongly influenced by the number of egg-64 producing structures called ovarioles (1,(18)(19)(20)(21)(22)(23). Ovariole number is species-specific and 65 genetically determined (24,25). Most insects have limited intraspecific variation in ovariole 66 number, and the effect of ovariole number on fecundity has been observed by comparing mean 67 ovariole numbers within or between species. In many insects, including beetles, fruit flies, and 68 aphids, ovariole number is positively correlated with fecundity between and within species (1, 69 21-23). For example, Drosophila melanogaster strains with naturally occurring or genetically 70 manipulated higher ovariole numbers both show increased fecundity (18,26). While 71 physiological traits like egg production rate may also play an important role in determining 72 reproductive capacity (27), these can be difficult to assess in laboratory settings where egg-laying conditions may not be suitable for some insects. In contrast, ovariole number has served 74 as a proxy for reproductive capacity for decades (18) ovariole number of the group (1). This reduction in ovariole number has been hypothesized to be 100 the result of increased egg size as an adaptation to feeding on the toxic Morinda (40), or to be 101 due to lower insulin signaling levels evolved in response to the relatively constant nutritional 102 input provided by substrate specialization (35). Reviewing data on oviposition behavior in 103 melanogaster subgroup species, Lachaise (37) proposed that the high ovariole number observed 104 in the generalists D. melanogaster and D. simulans may be driven by the frequent oviposition 105 opportunities available to these species, as they oviposit on most decaying fruit. However, the 106 melanogaster subgroup is not well-suited for a broader understanding of ovariole number 107 evolution, as most species share similar oviposition substrates (i.e. rotting fruit) and there are few 108 independent instances of evolution of specialists. 109 In contrast, Hawai'ian Drosophila have evolved to specialize on a variety of oviposition 110 substrates, including decaying flowers, leaves, fungi, sap fluxes, and bark of native plants, and 111 eggs of native spiders (41). Moreover, these flies exhibit the most extreme interspecies range of 112 ovariole number reported in the genus, ranging from two to 101 per ovary (42). Hawai'ian 113 Drosophila have undergone rapid island radiation from a common ancestor in the last 25 million 114 years, leading to over 1000 extant species (43-45). The high species diversity of Hawai'ian 115 Drosophila is spread across five monophyletic species groups that share genetic, morphological 116 and ecological similarities, and rely on different oviposition substrates (44,(46)(47)(48), as follows 117 breeding (44). 126 Ovariole number is highest in the PW species (up to 202 per female), and lowest in 127 Scaptomyza and AMC species (as few as 2 per female) (42). Dramatic differences in ovariole 128 number between species have been hypothesized to be a result of shifts between their varied 129 oviposition substrates (42, 51). Other studies have posited that the divergent ovariole number 130 observed in Hawai'ian Drosophila may be a result of r-K evolution (42), given the surface area 131 of decaying trees, and the predictability of this substrate in the field (36), is greater than that of 132 other oviposition substrates (51, 52). However, the studies supporting these hypotheses primarily 133 sampled PW species, and used phylogenies that have since been substantially improved upon in 134 more recent studies that include expanded taxon sampling and additional loci (44,46,48,53). 135 To investigate the linked effects of ecology and development underlying ovariole number 136 evolution in Hawai'ian Drosophila, we conducted phylogenetic comparative analyses of life 137 history traits from 60 species, and comparative development analyses from ten species using 138 both wild-caught flies and laboratory strains. Our results identify potential mechanisms of 139 evolutionary change in ovariole number operating at three levels of biological organization. First, 140 we found that evolutionary shifts in ecological niche could predict the dramatic differences in ovariole number or egg volume differed between species groups with different oviposition 143 substrates, suggesting that the allometric growth relationships between these traits evolves 144 dynamically. Finally, we found that changes in ovariole number from two to 60 per individual 145 can be explained by changes in total TFC number, suggesting that ovariole number diversity 146 evolves through the same developmental mechanism, regardless of the specific ecological 147 constraints or selective pressures. 148 149

Adult reproductive traits of Hawai'ian Drosophila 151
We measured three major adult traits relevant to reproductive capacity (body size, 152 ovariole number and egg volume), from field-collected females, lab-reared F1 offspring of field-153 collected females, and females from laboratory strains ( Figure 1; Table S1). Species identities of 154 field-collected females were assigned based on morphological keys or DNA barcoding (Tables 155 S2, S3). All traits ranged over an order of magnitude within Hawai'ian Drosophila: body size 156 ranged from 0.71mm for S. devexa to 3.12mm for D. melanocephala, ovariole number per 157 female ranged from 2 for S. caliginosa to 88.5 for D. melanocephala, and egg volume ranged 158 from 0.01 um 3 for Bunostoma spp. group (S. palmae/ S. anomala) to 0.2um 3 for D. adunca, 159 highlighting the diversity of life history traits in Hawai'ian Drosophila. 160 Within the melanogaster subgroup species, species-specific differences in ovariole 161 number are largely heritable (25,54,55). To test whether this is also the case in Hawai'ian 162 Drosophila, we compared ovariole number of wild-caught females and their lab-reared F1 163 offspring, across five species with different egg-laying substrates. We observed no significant 164 differences between the ovariole numbers of these two generations regardless of natural substrate ( Figure S1), indicating that species-specific differences in ovariole number are also strongly 166 genetically determined in Hawai'ian Drosophila. 167 168

Larval ecology influences ovariole number evolution 169
A previous study based almost exclusively on picture wing species proposed that 170 evolutionary shifts in larval ecology had driven ovariole number diversification in these flies 171 (51). To test this hypothesis across the major groups of Hawai'ian Drosophila, we compared the 172 fit of evolutionary models of ovariole number that accounted for ecologically driven evolution, 173 to those that did not. Our dataset included both specialist species that oviposit on one of bark, sap 174 flux, leaf, fungus, fruit, flower or spider-eggs, as well as generalist species that oviposit on 175 multiple decaying substrates ( Figure S2). We compared the fit of five models to our data, two of 176 which ((i) Brownian Motion, BM, and (ii) an Ornstein Uhlenbeck model with a shared optimum 177 for all species, OU1) do not take into account the oviposition substrate, and three of which were 178 nested ecological models based on alternative methods of substrate classification: (iii) OU2 179 assumed two states, bark breeders and all other species, to test previous suggestions that bark-180 breeding may drive evolution of ovariole number (51, 52); (iv) OU3 assumed three states, 181 Scaptomyza specialists on spider eggs and flowers, bark-breeders, and species using any other 182 substrate, to test proposals that substrates influence ovariole number evolution because of their 183 differences in carrying capacity and field predictability (36, 42); and (v) OU8 categorized each 184 oviposition substrate separately. These five models were fit over 100 trees sampled from the 185 posterior distribution of a Bayesian phylogenetic analysis to account for phylogenetic 186 uncertainty.
We found that models accounting for larval ecology explained the ovariole number 188 diversification in Hawai'ian Drosophila (Table 1) better than those that did not. Comparing the 189 three ecological models, we found that the three-state model (OU3), which accounted for both 190 bark breeders and Scaptomyza specialists, was supported as the best-fit model across a majority 191 of trees for ovariole number (ΔAICc > 2 as compared to OU2 and OU8 models; Table S4). 192 Estimated theta values for the OU3 model showed that bark breeders have more ovarioles than 193 species that oviposit on other substrates, suggesting that evolution of higher ovariole numbers 194 accompanied the transition to bark breeding from likely non-bark breeding ancestors ( Fig Figure S1). We therefore speculate that specific 211 substrate components may not only allow females to distinguish between hosts for oviposition, 212 but also may contribute to species-and substrate-specific egg laying behavior in Hawai'ian 213 Drosophila. 214 215

Evolution of specialist habitats changes allometry of reproductive traits 216
The range of Hawai'ian Drosophila body sizes is greater than that of other members of 217 the genus, spanning an order of magnitude (Table S1). To determine whether changes in 218 allometric growth might underlie reproductive trait evolution, we analyzed the allometric ratio of 219 such traits using a phylogenetic least squares (PGLS) analysis and thorax volume (thorax 220 length 3 ) as a proxy for body size. We found that across all Hawai'ian Drosophila, thorax volume 221 was significantly positively correlated with both ovariole number ( Figure 3A; Table 2; Table S6) 222 and egg volume ( Figure 3B; Table 2; Table S6) Hawai'ian Drosophila species groups (41). In sum, while a positive correlation between body 241 size and fecundity is commonly posited in egg-laying animals (11, 13), we did not find universal 242 support for this trend across Hawai'ian Drosophila. This is, however, consistent with previous 243 studies on Diptera, wherein trends toward higher fecundity or ovariole number in larger animals 244 observed within species (11) contrast with between-species differences in ovariole number that 245 do not always correlate with body size (22,37,61). showed that even over a range of ovariole numbers spanning an order of magnitude (Figure 4; 255 Table S7), larval TF number essentially corresponded to adult ovariole number (Table S8).
Although TFC number per TF varied somewhat between species ( Figure 4A; Table S7), PGLS 257 analysis showed no correlation between TFC number per TF and total TF number (Table 3). In 258 contrast, average total TFC number was strongly positively correlated with TF number (Table 3; 259 Figure 4B; Table S7), suggesting that, as in laboratory populations of D. melanogaster, changes 260 in TFC number underlie ovariole number evolution in Hawai'ian Drosophila. 261 The developmental mechanism underlying ovariole number evolution is particularly 262 interesting in light of the allometric changes in Hawai'ian Drosophila species groups. There has 263 been some debate as to whether allometry constrains or facilitates adaptive evolution (62-64). In 264 Hawai'ian Drosophila, the allometric relationship between two important female reproductive 265 traits, ovariole number and egg size, was coupled to body size in different groups in different 266 ways: when ovariole number was coupled with body size, egg size was not, and vice versa 267 hand, on ovarian development during larval and pupal stages. For example, we speculate that on 278 certain substrates, the larval ovary may become less sensitive to nutritionally-mediated growth factors by evolving lower expression levels of growth factor receptors, and relying more on 280 tissue-specific growth factors, which could include local insulin release or cell proliferation 281 pathways such as Hippo signaling. 282 Taken together, we found that highly divergent ovariole number, and by proxy female 283 reproductive capacity, have evolved together with changes in egg-laying substrate across 284 Hawai'ian Drosophila. Moreover, this remarkable adaptive radiation is linked to evolutionary 285 changes in a key reproductive trait that is regulated by variation in the same developmental 286 mechanisms operating in the model species D. melanogaster. 287 288

Materials and Methods 289
Hawai'ian Drosophila were collected (69)   We combined sequence data for 18 genes reported in four previous studies (44,46,48,298 53) from GenBank with additional newly identified mitochondrial sequences (Table S9)

Figure 3. Allometric relationship between life history traits in Hawai'ian Drosophila. 512
Scatter plots of log transformed adult measurements with phylogenetically transformed trend 513 lines generated by averaging runs from PGLS analysis across 100 posterior distribution BEAST 514 trees, performed with the R package nlme v.3.1-121 (76). Trend line of the consensus tree is 515 denoted in red when there was a significant relationship between the two traits, and black when 516 PGLS analysis did not support a significant relationship (Table 2)   analysis of relationships between ovariole number and thorax volume (mm 3 ), egg volume (µm 3 ) and thorax volume, and ovariole 555 number and proportional egg volume (µm 3 /mm 3 ) are listed. Regression analyses were performed with the R package nlme v.3.1-121 556 (76) on 100 trees from a BEAST posterior distribution using nuclear and mitochondrial genes, and the minimum, average, and 557 maximum slope and p-value for the analysis is included in the table. P-values below 0.01 are indicated in bold.