Ancestral Populations

LH – It is a large laboratory adapted population of Drosophila melanogaster, established by, and named after Lawrence G. Harshman. The population is maintained on a 14 day discrete generation cycle, under 25 °C, 60–80% relative humidity, 12 hours light/12 hours dark cycle and on standard cornmeal – molasses – yeast food. The flies are grown under moderate larval density of 140–160 per 8-dram vial (25 mm diameter × 90 mm height) containing 8–10 ml food. On the 12th day post egg collection, flies from different vials are mixed and redistributed across fresh food vials containing limiting amount of live yeast grains with 16 males and 16 females per vial. On the 14th day, flies are transferred to fresh vials and are allowed a window of 18 hours to lay eggs which (after discarding the adults and controlling density) start the next generation39.

LH st – This population was derived by introducing the scarlet eye colour (recessive, autosomal and benign) gene into the LH population, hence the subscript. LH st is maintained under the same conditions as LH with N e > 2500. The genetic backgrounds of these two populations are homogenised by periodic back crossing.

Selection Regimes

The study was done on six populations of Drosophila melanogaster – M 1–3 and F 1–3 representing male biased and female biased operational sex ratio respectively. All these populations were created from the LH st population.

We derived the male biased (M 1–3 ) and female biased (F 1–3 ) regimes, each having three independent replicates, from LH st by varying the operational sex ratio to male: female:: 3:1 and 1:3 respectively. The maintenance of these populations differs from that of LH and LH st in the following ways:

a. In these populations adult flies are collected as virgins 9–10 days after egg collection, during the peak eclosion period and held in vials (containing 8 flies of one sex) for two days. b. The sexes are combined on the 12th day in fresh food vials seeded with measured amount of live yeast (0.47 mg per female) following the selection regime – 24 males + 8 females in each vial for M and 8 males + 24 females in each vial for F.

The effective population sizes of all the populations are maintained at >450 or >350 depending on the method used to calculate them4. For more details on the evolutionary history and detailed maintenance protocol, see 23.

Standardisation and Generation of Experimental Flies

In order to equalise the potential non-genetic parental effects across different regimes, we maintained all populations under ancestral condition which does not include virgin collection and sex ratio alteration- essentially following the same life cycle as LH st populations- for one generation before obtaining individuals for the experiment. This process is called standardisation40.

Eggs laid by the standardised flies were collected at a density of 150(±2) per vial (containing 8–10 ml of cornmeal food) to obtain the experimental flies. On the 9th−10th day after egg collection, males and females were collected as virgins during the peak of their eclosion and held as a single individual per vial.

Ancestral flies (LH), whenever they were used in this study, were raised in similar conditions. LH males were sorted on the 12th day post eclosion and held as single individuals. Eggs for LH flies were collected on the same day as that of the selection lines. Thus, the age of the experimental flies of all the populations was the same during the experiment.

General Experimental Design

For all our assays, we compared reproductive behaviour and/or fitness related traits between two types of individuals within a regime:

a. Within replicate (WR): These are individuals from the same replicate number of a given selection regime i.e., M i ♂ and M i ♀ are WR with respect to each other where i denotes the replicate number (e.g., M 1 ♂ and ♀) and similarly for F. b. Between replicate (BR): These are individuals from different replicate numbers of a given selection regime, i.e., M i ♂ and M j ♀ are BR with respect to each other –where i, j denote replicate numbers and (i, j) ∈ {(1,2), (2,3), (3,1)} (e.g. M 1 ♂ and M 2 ♀) and similarly for F. We took BR individuals in a round robin manner to avoid the problem of pseudo-replication16.

Assay for Assortative Mating

We combined a virgin female with two virgin males from the same selection regime –one WR and one BR – in vials containing fresh food. That is, a female from a given replicate number was combined with a male from the same replicate number and another from a different replicate number (all within the same selection regime), e.g., one M 1 female + one M 1 male + one M 2 male and so on. Thus, we had three combinations within each selection regime, denoted by female replicate number. Males were marked by pink or green Day-Glo dust for identification. Previous studies using the same dust found no effect on individuals in terms of mating behaviour or female preference41. However, to account for any mating bias brought about solely by green and/or pink colouration, we had reverse colouration treatments for all combinations. Thus, each combination had two treatments, e.g., one M 1 female + one green M 1 male + one pink M 2 male; one M 1 female + one pink M 1 male + one green M 2 male and so on. We had 30 replicate vials per combination per colour treatment (Table 1). In some vials we observed no mating till one hour after combining the flies. Those vials were discarded and excluded from the analysis (Number of discarded vials: 4, 4, and 2 out of 60 trials each from the three replicates in the F regime; 4, 7, and 6 out of 60 trials each from the three replicates in the M regime). Data are provided in Supplementary Dataset 1.

Assay for Mating Latency and Copulation Duration

For this assay we combined one virgin male and one virgin female according to treatment (WR or BR, see results) in a vial containing fresh food. After combining a male and a female, the pair was observed till they finished mating. Time taken for a pair to start mating after they were combined was recorded as mating latency and the time they spent in-copula was recorded as copulation duration. If a pair failed to mate after one hour, they were discarded. However, the number of failed mating in all treatments was very low (6, 3, 0 and 3 failures out of 60 trials in M-WR, M-BR, F-WR and F-BR respectively). Mating latency and copulation duration values for each vial was used as the unit of replication. Data are provided in Supplementary Dataset 2.

Assay for Competitive fertilisation success

As a measure of competitive fertilisation success, we measured sperm defense ability of males, the rationale for which is provided in the results section. For assaying sperm defense ability, we set up crosses following the same method as mentioned above and the vials were observed for mating for one hour. The females that did not mate with the first male were discarded. After the first mating, we sorted the females using light CO 2 -anaesthesia and held them back into the vials and discarded the males. After allowing a recovery time (from anesthesia) of half an hour, we introduced a second male (red eyed, LH) in each vial and kept the vials undisturbed for 24 hours, during which they could mate with the females. After this exposure window, the second males were discarded and the females were transferred singly (under light anesthesia) to test tubes (dimensions: 12 mm diameter × 75 mm length) provisioned with food. There they were allowed an oviposition window of 18 hours. The adult progeny emerging from the eggs laid during this window were scored for their eye colour marker after 12 days. The proportion of scarlet progeny was taken as an estimate of P1 of the male. 90 males from each of the crosses were assayed for P1. Since we did not observe the second mating, instances where all progeny was sired only by the first male (P1 = 1) could arise due to second male failing to mate. Such instances were excluded from the analysis. Final sample size for P1 analysis was n = 83–87 and 70–73 per cross type (WR/BR) in F and M populations respectively. P1 value from a single vial was used as the unit of replication. Data are provided in Supplementary Dataset 3.

Statistical Analysis

To test for assortative mating, we used logistic regression using a mixed model in a nested structure. In the model, successful mating by a WR male was used as the response variable, selection regime was used as a fixed effect, and replicate population (to which the female belonged) nested within selection regime was used as a random effect using the following model:

WR_Success ~ Selection + (Selection|Block), family = binomial(logtit)

The ‘glmer’ function in the ‘lme4’ package42 in R43 with binomial (logit) family was used for the analysis.

For the rest of the assays, we performed a two-way ANOVA with selection regime and treatment (type of individuals involved in a cross: BR/WR) as fixed factors and male and female replicate nested within selection regime as random effects using the following model:

response ~ Sel. Reg*Mating. Type + (1|Female.Replicate) + (1|Male.Replicate)

In case of significant Sel.Reg*Mating.Type interaction, we performed Tukey’s HSD (with α = 0.05) for post-hoc analysis. Linear model fitting, ANOVA and post-hoc tests were performed in R using packages ‘lme4’, ‘lmerTest’44 and ‘lsmeans’45 respectively.