DOI: 10.5061/dryad.xksn02vq2
title: Can developmental plasticity shape sexual competition and promote reproductive isolation?

description: Environmental factors such as dietary nutrients can shape the expression
 of developmentally plastic sexual traits in many species. However, while
 there has been extensive research into the developmental plasticity of
 sexual traits at the individual level, the broader consequences of this
 variation at the population scale remain poorly understood. Here, we asked
 whether plastic responses to the developmental environment can shape
 sexual competition and initiate reproductive isolation between
 populations. We reared neriid flies, Telostylinus angusticollis, on
 nutrient-rich and nutrient-poor larval diets, generating adult flies that
 differed in body size and secondary sexual trait expression. We then
 investigated sexual competition in experimental populations from each
 developmental environment, and tested for reproductive isolation between
 flies from mismatched environments. We found that, compared with poor-diet
 populations, rich-diet populations exhibited more frequent and escalated
 male-male combat and more frequent mating and mate-guarding. However, we
 found no evidence that sexual selection was affected by the developmental
 environment. Mismatched female-male pairs tended to take longer to mate
 and rich-diet females often rejected poor-diet males, but mismatched pairs
 were not less likely to mate within 1 hour or produce viable offspring.
 Our findings suggest that developmental plasticity could generate dramatic
 differences in sexual competition between populations, and could
 contribute to reproductive isolation.
description: <em>Rearing and Culturing of Flies</em>
 The individuals used in the experiments described below were
 second-generation individuals reared from<em> T.
 angusticollis</em> collected from Fred Hollows Reserve in Randwick,
 New South Wales, Australia (33˚54’44.04”S, 151˚14’52.14”E). The flies were
 housed in population cages with moist cocopeat and were given three
 separate petri dishes containing brown sugar, yeast, and oviposition
 medium. The oviposition medium consisted of a nutrient-rich diet (as
 described below) which had been left to mould for approximately 4 days and
 then mixed to encourage oviposition. The cages were sprayed with reverse
 osmosis (RO) water every second day. To generate focal flies for
 experiments, eggs were collected from oviposition medium  in stock cages
 and transferred to 500 mL jars containing 200 mL of either a nutrient-rich
 diet (“rich diet”) or a nutrient-poor diet (“poor diet”) to represent
 developmental environments differing in availability of macronutrients.
 Approximately 20 eggs were transferred to each container, with 19
 containers for each diet treatment (N = 760 eggs in total). The rich diet
 consisted of 32.9 g soy protein (Nature’s Way Instant Natural Protein) and
 41.3 g of brown sugar (Coles Brown Sugar), whereas the poor diet consisted
 of 5.5 g soy protein and 6.9 g brown sugar, mixed with 1 L dry cocopeat
 (coconut husk shavings), and approximately 800 mL RO water (Sentinella et
 al., 2013). Virgin adults were separated within 24 hours after emergence
 into 12 L plastic tanks by sex and diet, with approximately 20 males or 25
 females per tank (N = 18 tanks in total). Tanks contained adult flies of
 similar age, with flies from multiple larval containers combined randomly
 in adult tanks. Each tank contained a layer of moist cocopeat for
 humidity, and petri dishes containing an excess of brown sugar, yeast, and
 oviposition medium that were replaced approximately every 7 days. The
 tanks were sprayed with RO water every second day. Flies, larval medium
 containers, and experiments were all kept and conducted in a controlled
 lab environment with a temperature of 25 °C (± 2 °C). The focal flies were
 used in two separate experiments, as described below.   
 <em>Sexual Competition Experiment</em>
 We observed a total of 13 experimental replicate populations
 (i.e., unique groups of 5 males and 5 females combined and observed inside
 an experimental arena) from each larval diet (N = 26 replicate populations
 in total). Each replicate population was created by combining five virgin
 females and five virgin males (all of similar age and reared on the same
 larval diet, and each male marked with a different colour as described
 below) together in a transparent plexiglass arena (26 x 36 x 20 cm) with a
 mesh sleeve (Fig. 2). Replicate populations were comprised of focal
 individuals drawn randomly from the same adult tank (when possible), and
 focal individuals were not re-used in this experiment. Focal flies were
 between 14 and 45 days old when used in experiments. Flies reared on the
 poor larval diet emerged ~ 3 days later on average than flies reared on
 the rich larval diet, as typically observed in this species (Bonduriansky,
 2007; Hooper et al., 2017). However, since <em>T.
 angusticollis</em> can live for &gt;130 days in the laboratory
 (Hooper et al., 2017), all focal flies were relatively young when used in
 experiments and the small average difference in adult age between rich-
 and poor-diet focal flies is unlikely to have substantially influenced our
 results. The arena was designed to simulate natural mating aggregations on
 rotting tree bark, where females oviposit and feed and males compete for
 matings (Bonduriansky, 2006, 2007). The bottom of the arena contained a
 layer of moist cocopeat, and a petri dish (5cm diameter) filled with
 oviposition medium was placed in the centre. The arena was illuminated
 with a broad-spectrum light source placed above the oviposition medium to
 encourage interaction. In order to identify individual
 males in replicate populations, the focal males were anaesthetised with
 CO<sub>2</sub> using a Flystuff Benchtop Flow Buddy System
 (59-122BCU, Genesee Scientific, USA) with Ultimate Fly Pad and Gun, and a
 spot of enamel paint (Tamiya Color Enamel Paint, Japan) was applied to the
 thorax, with each of the five males in each replicate population marked
 with a different colour. The males were placed in individual vials to
 allow the paint to dry and for recovery from the anaesthesia, and then
 kept in tanks with other males (as described above) for at least 24 hours
 before being allocated to replicate populations for the
 experiment. During each day of the experiment, we
 collected data on one rich-diet replicate population and one poor-diet
 replicate population using the same dish of oviposition medium. Each
 replicate population was left in the arena for 30 minutes to acclimatise,
 and was then observed for one hour. For each individual male, we recorded
 the duration and number of matings and mating attempts, number and
 duration of guarding bouts after mating, number of male wing flicking
 bouts, number and duration of combat interactions, and number of times the
 male was rejected by a female. Mating was recorded when a male positioned
 himself above or behind a female and mounted the female for at least 20 s
 (Bath et al., 2012; Wylde et al., 2019b). Mating duration was quantified
 as the time in seconds between a male mounting a female and removing his
 genitalia from the female’s oviscape (Wylde et al., 2019b) (Fig. 1a). A
 mounting interaction that lasted less than 20 s was counted as a mating
 attempt. Female rejection was recorded when a female kicked a male or flew
 and/or ran away when a male attempted to mate with her (Bath et al.,
 2012). Guarding was identified as a male standing above a female after
 mating and using the span of his legs to enclose the female (Bonduriansky,
 2006) (Fig. 1b). Male-male combat interactions were recorded when two
 males used their forelegs and/or body to strike each other (Bath et al.,
 2012) (Fig. 1c). Wing flicking involved males flicking their wings at
 another individual (Wylde et al., 2019b).
 <em>Reproductive Isolation Experiment</em>
 To determine whether individuals reared on different larval diets
 can mate and produce offspring, males and females were paired in all
 possible combinations (poor male with rich female, poor male with poor
 female, rich male with poor female, and rich male with rich female), with
 23 replicates of each combination (Fig. 3). To ensure that the males were
 sexually experienced, males from the sexual competition experiment were
 re-used in the reproductive isolation experiment. However, all females
 were virgins. Each pair was placed in a 500 mL container with moist
 cocopeat on the bottom and a small petri dish of oviposition medium. After
 the female and male flies were combined, they were left to adjust for 10
 s, and then observed for one hour. We recorded latency to mate, duration
 and number of matings, number of mating attempts, and female rejection
 behaviours. Here, female rejection was evidenced by a female wing-flicking
 or kicking a male as he tried to mate, or when a female ran/flew away from
 a male during a mating attempt. On each day of the experiment, we set up
 one replicate of each of the four larval diet combinations. A
 broad-spectrum light source was positioned above the containers throughout
 the experiment. After one hour, the male was removed
 from the container and the female was left to lay eggs. Each oviposition
 dish was checked for eggs every day for a maximum of six days after the
 pairing was conducted. After six days, the female was removed and frozen
 for body size measurement. For each pair, 20 eggs (where possible) were
 transferred to a container with 200 mL of standard larval diet consisting
 of 11 g soy protein and 13.8 g brown sugar per 1 L of dry cocopeat and
 800mL of water (Sentinella et al., 2013). After the first adult emergence
 in each container, the container was left for 10 days and then the emerged
 flies were counted. The larval containers with eggs but with no emerging
 flies were left for 40 days before they were discarded.
 <em>Body Size Measurement </em>
 The focal males used in the sexual competition experiment and
 reproductive isolation experiment, and females used in the reproductive
 isolation experiment, were frozen at -20°C and then used to quantify body
 size. One wing from each individual was removed, mounted on a microscope
 slide using double-sided tape, and then covered with cling-wrap. The wings
 were then photographed at a magnification of 6.3 x using a Leica MC170 HD
 camera mounted on a Leica MS5 stereo-microscope (Wetzlar, Germany). The
 linear distance from the intersection of the R<sub>2+3</sub>
 wing vein with the wing margin to its intersection with the
 R<sub>s</sub> wing vein was measured using ImageJ software
 (Schneider et al., 2012).  <em>Statistical
 Analysis</em> All statistical tests were carried
 out using R 4.0.3 (R_Core_Team, 2020). To verify that our larval diet
 manipulation resulted in the expected effects on adult phenotype, we
 modelled wing length using a linear model with larval diet, sex and their
 interaction as fixed effects. Although we did not keep track of family
 identity or larval container, the flies were derived from eggs laid in
 stock tanks containing numerous flies and eggs were distributed among 38
 larval containers. Effects of genotype and shared (container-specific)
 environments are therefore unlikely to have biased the results of this
 analysis. In the sexual competition experiment, some
 males did not fight or interact with females, such that the individual
 data set was zero-inflated. Thus, analysis was carried out on means (for
 duration of mating, combat interactions, guarding) or total counts (number
 of matings, guarding bouts, female rejections, combat interactions, and
 wing flicks) for replicate populations. General and generalised linear
 mixed models (GLMMs) were fitted to the replicate population means or sums
 using the R package lme4 (Bates et al., 2015), with each response variable
 modelled separately. Models included larval diet as a fixed effect, and
 day as a random block effect (but the random effect of day yielded
 extremely small variance components and was removed from two models to
 facilitate model fit: see Table 1). Combat duration was log-transformed to
 improve model diagnostics. For over-dispersed count data, an
 observation-level random effect (unique code for each replicate
 population) was included. For Gaussian models, effects were tested using
 <em>t</em>-tests with Satterthwaite’s degrees of freedom using
 the package LmerTest (Kuznetsova et al., 2017). For Poisson models,
 effects were tested using <em>z</em>-tests. For rejection
 behaviours by females, the model failed to converge and a Wilcoxon
 matched-pairs test was used instead to compare data from rich- vs.
 poor-diet replicate populations within days. Mating
 success skew represents the opportunity for sexual selection (Cattelan et
 al., 2020; Jones, 2009). To estimate mating success skew, we calculated
 coefficients of variation of the number of matings by individual males
 within replicate populations. The coefficients of variation were then
 compared using a Gaussian mixed-effects model with larval diet as the
 fixed effect and day as a random block effect. To test for and compare
 sexual selection on male body size in rich-diet vs. poor-diet replicate
 populations, we used a Poisson model of individual male mating success
 (number of matings) as a function of male body size, with larval diet and
 body size as fixed effects and day as a random block effect. For this
 analysis, individual male mating success and body size (wing length) were
 both standardized (z-transformed) within replicate populations. In this
 model, a main effect of male body size would indicate overall sexual
 selection on male body size, whereas a larval diet × body size interaction
 would indicate a difference between larval diet treatment groups in sexual
 selection on male body size. Note that the main effect of larval diet is
 not meaningful in this model because standardization within replicate
 populations brings the effect estimate to ~ 0. To test
 for reproductive isolation, models were fitted with male larval diet,
 female larval diet, and their interaction as fixed effects and day as a
 random block effect. An environmentally induced reproductive barrier would
 be indicated by reduced propensity to mate or produce viable offspring by
 flies reared on mismatched diets, detectable statistically as a male
 larval diet x female larval diet cross-over interaction. Mating outcome
 and female rejection behaviour were modelled as binomial (0 or 1) response
 variables. A binomial model was also used to investigate treatment effects
 on egg-to-adult viability, represented for each replicate pair as a matrix
 of successes (number of eggs that resulted in adult flies) and failures
 (number of eggs that did not result in adult flies). For over-dispersed
 binomial data, an observation-level random effect (unique code for each
 pair) was included in the model. Latency to mate was modelled with
 Gaussian error.
description: # Can developmental plasticity shape sexual competition and promote
 reproductive isolation?
 [https://doi.org/10.5061/dryad.xksn02vq2](https://doi.org/10.5061/dryad.xksn02vq2) #### Data files: (1) **BodySize**: Individual body size (wing length) measurements for the focal males and females used in the study. (2) **SexCompData**: Individual behavioural data for males and females in the Sexual Competition Experiment, grouped by replicate population, day (block), and larval diet treatment. (3) **SexCompSummary**: Summary statistics (means or counts) of behavioural data from the Sexual Competition Experiment, grouped by day and larval diet treatment. Most analyses for this experiment were carried out on means or counts for replicate populations using this data file. (4) **Rejections_Paired**: Rejection behaviours by females (from the Sexual Competition Experiment) in wide form for a Wilcoxon matched-pairs test, with day as the grouping factor. (5) **RIData**: Data from the Reproductive Isolation Experiment, grouped by day, male larval diet and female larval diet. ## Description of the data and file structure (1) **BodySize**: Individual body size (wing length) measurements for the focal males and females used in the study. Variables: DIET: Larval diet treatment (RICH or POOR) SEX: Female or Male WL: Wing length (mm), used as an index of body size (2) **SexCompData**: Individual behavioural data for males and females in the Sexual Competition Experiment, grouped by replicate population, day (block), and larval diet treatment. Variables: Day: Temporal block, with one Rich and one Poor trial (= "replicate population") assayed each day. Trial: An assay (1 hour of behavioural observations) conducted on a replicate population Diet: Larval diet treatment (Rich or Poor) Male: The colour of paint used to mark each individual male Matings: The number of matings achieved by each individual male Matinglength: The duration of each mating in seconds Matingattempt: The number of mating attempts observed for each individual male Guardings: The number of guarding bouts observed for each individual male; note that there are empty cells for males that did not mate, and therefore did not guard Guardinglengthtotal: The total duration (in seconds) of guarding observed for each individual male; note that there are empty cells for males that did not mate, and therefore did not guard  GuardingsperMating: Number of guarding bouts divided by the number of matings observed for individual males; note that there are empty cells for males that did not mate, and therefore did not guard Wingflicking: The number of wing flicks observed for each individual male Femalesresisting: Number of times that each male encountered resistance from a female Shortfight: Number of short (~ 1 s) combat bouts for each individual male Longfight: Number of prolonged (&gt; 1 s) combat bouts for each individual male Fightlength: Duration (s) of each combat bout for each individual male WL: Wing length (mm) of each individual male STDWL: Individual male wing length standardized (converted to z-scores) within replicate populations STDMatings: Individual male mating success (number of matings) standardized (converted to z-scores) within replicate populations Individual: Unique code for each individual male (3) **SexCompSummary**: Summary statistics (means or counts) of behavioural data from the Sexual Competition Experiment, grouped by day and larval diet treatment. Most analyses for this experiment were carried out on means or counts for replicate populations using this data file. Variables: Trial: An assay (1 hour of behavioural observations) conducted on a replicate population Day: Temporal block, with one Rich and one Poor trial (= "replicate population") assayed each day LarvalDiet: Larval diet treatment (Rich or Poor) MatingsPerMale: Total number of matings observed in the trial divided by 5 MatingsTotal: Total number of matings obsreved in the trial CVMatings: Coefficient of variation of individual male mating success within trial; note that there are empty cells for trials where no matings occurred and the CV therefore couldn't be calculated MatingLengthMean: Mean mating duration (s) for each trial; note that there are empty cells for trials where no matings occurred and the mean duration of mating therefore couldn't be calculated MatingAttemptsTotal: Total number of mating attempts observed in the trial GuardingsTotal: Total number of guarding bouts observed in the trial; note that there are empty cells for trials where no matings occurred and the number of guarding bouts therefore wasn't relevant (since guarding occurs after mating)  GuardingLengthMean: Mean duration of guarding bouts in the trial; note that there are empty cells for trials where no matings occurred and the mean duration of guarding bouts therefore couldn't be calculated (since guarding occurs after mating)  GuardingsPerMating: Number of guarding bouts divided by number of matings observed; ; note that there are empty cells for trials where no matings occurred and the number of guarding bouts per mating therefore couldn't be caulculated WingFlicksTotal: Total number of wing flicks observed in the trial FemaleresistancesTotal: Total number of resistance behaviours by females observed in the trial ShortFightsTotal: Total number of short combat bouts (~ 1 s) observed in the trial LongFightsTotal: Total number of long combat bouts (&gt; 1 s) observed in the trial Fightlength: Total duration (s) of all combat bouts observed in the trial (4) **Rejections_Paired**: Rejection behaviours by females (from the Sexual Competition Experiment) in wide form for a Wilcoxon matched-pairs test, with day as the grouping factor. Variables: RejectRICH: Number of rejection behaviours by females observed in the Rich-diet replicate population assayed on a specific day (row) RejectPOOR:  Number of rejection behaviours by females observed in the Poor-diet replicate population assayed on a specific day (row) (5) **RIData**: Data from the Reproductive Isolation Experiment, grouped by day, male larval diet and female larval diet. Variables: Day: Temporal block, with one replicate of each treatment combination assayed each day Individual: Unique code for each replicate Malediet: Larval diet treatment of the male in the replicate Femalediet: Larval diet treatment of the female in the replicate Mated: Binomial variable indicating whether mating occurred during the assay (1 = yes, 0 = no) MatingLatency: Time (s) from the start of the assay until the start of mating; if no mating occurred, a value of 3600 was assigned Resistingbinomial: Binomial variable indicating whether resistance by the female occurred during the assay (1 = yes, 0 = no) EggsCollected: Number of eggs collected from the female for the egg-to-adult viability assay Hatched: Number of eggs that resulted in adult flies; note that there are empty cells for replicate pairs that produced no eggs and for which the number of eggs hatched was therefore not meaningful Unhatched: Number of eggs that did not result in adult flies; note that there are empty cells for replicate pairs that produced no eggs and for which the number of eggs unhatched was therefore not meaningful  ## Code/Software An R script is provided with code to carry out the analyses reported in the paper.