Post-copulatory sexual selection (PSS) is an important selective force that determines fitness in polyandrous species. PSS can be intense in some cases and can drive the evolution of remarkable ejaculate properties. In males, investment in ejaculate plays an important role in the outcome of PSS. Thus, males are expected to adaptively tailor their ejaculate according to the perceived competition in their vicinity. Plastic responses in ejaculate investment to variation in intra-sexual competition are disparate and widespread in males.

We investigated the evolution of plasticity in reproductive traits using Drosophila melanogaster populations evolving for more than 150 generations under male- or female-biased sex ratios. When exposed to different numbers of competitors early in their life, males from these two regimes responded differently in terms of their copulation duration and sperm competitive ability. In addition, the effect of this early life experience wore off at different rates in males of male-biased and female-biased regimes with increasing time from the removal of competitive cues. Furthermore, our study finds that males change their reproductive strategies depending upon the identity of rival males. Together, our results provide evidence of the evolution of male reproductive investment that depends on socio-sexual cues experienced early in life.

METHODOLOGICAL INFORMATION

Maintenance of populations 

Baseline population – LHst is a large laboratory-adapted population of D. melanogaster that was derived from the LH population by introgressing a benign, autosomal recessive scarlet eye colour mutation (Nandy et al. 2013a). The maintenance protocol of both LH and LHst populations is described elsewhere (Nandy et al. 2012). Briefly, both LH and LHst are maintained on a 14-day discrete generation cycle, at 25⁰C, on standard cornmeal-molasses-yeast food and on a 12 hours: 12hours light-dark cycle. Maintenance of LHst and LH population is similar to M, and F populations (described below) except that (1) Flies are not collected as virgins for LHst and LH populations, (2) Equal sex ratios are maintained in LHst and LH populations, i.e. 16 males: 16 females per vial, hence a total of 32 flies/vial. 
Selection Regimes – Experimental evolution lines with two different selection regimes –M or male biased and F or female-biased were used for all the experiments. First, three replicate populations (C1-3) were derived from LHst and maintained under equal sex ratio (16 males:16 females) (Nandy et al. 2013a). After 5 generations, M and F populations (M1-M3 and F1-F3) were derived from each of the C populations (C1-C3). Replicate populations bearing the same numerical subscript were derived from same ancestral populations. For example, M1 and F1 were derived from C1 population, M2 and F2 from C2 population and similarly, M3 and F3 were derived from C3 population. Hence populations carrying the same numerical subscript (e.g., M1 and F1) are more closely related to each other than to any other population with a different numerical subscript. Also, populations related by ancestry (e.g. M1 and F1) are handled on the same day every generation during regular maintenance as well as during the assays performed in this study. Thus, M1 and F1 populations correspond to block-1, M2 and F2 to block-2 and M3 and F3 to block-3, making a total of 3 statistical “blocks” for our analyses. 
Hence, a total of six populations from the two selection regimes (the male-biased or M regime and the female-biased or F regime), each having 3 replicates:   (M1, M2, M3 and F1, F2, F3) were used for this study. Populations belonging to both regimes are maintained in an identical manner except that they have different adult sex ratios - male: female ratio is 3:1 for the M populations and 1:3 for the F populations. A discrete 14- day generation cycle is followed for population maintenance at 250C and ~60% relative humidity (RH) and 12:12 hours light/dark cycle. Flies are provided with standard cornmeal–molasses–yeast food in standard vials (90-mm length × 25-mm diameter). They are reared at a controlled larval density of 140– 160 per vial. Virgin flies are collected each generation and held in single-sex vials (eight individuals per vial) till the 12th day after egg collection. Therefore, during regular maintenance, the early-life adult densities are identical for both M and F selection regimes. On the 12th day, flies are then combined in fresh food vials following the respective sex ratio regimes i.e. 8 males: 24 females for F regime and 24 males: 8 females for M regime, making a total of 32 flies per vial. Every generation, a total of 20 such vials are maintained for each population from both male-biased and female-biased regimes (hence, making adult population size as 20×32=640 flies for both M and F regimes). A fixed quantity of live yeast per female, i.e. 0.47 mg is provided to flies in food vials. After 2 days, flies are transferred to new vials for oviposition. The eggs laid during the next 18 hours are used to start the next generation.

Standardization and generation of experimental flies

Before generating experimental flies, we followed one generation of standardization of populations (Rose 1984). This was done to eliminate any potential non-genetic parental effects between the two regimes. During standardization, populations were maintained just like the ancestral LHst population, i.e. they were not subjected to virgin collection and the sex ratio was not altered. Experimental flies were generated from these standardized populations.
For generating experimental flies, we collected eggs for our test males from M and F regimes, for females from LHst and for the second male for the sperm defense assay from LH, all on the same day. All the experimental flies were reared under controlled larval density and standard culture conditions (25⁰C, 60–80% RH, 12 hours–12 hours light / dark cycle). Eggs were cultured at a density of 150 eggs / vial in 8–10 mL of cornmeal-molasses-yeast food for each of the population. On the 9-10th day post egg collection, M and F males were collected as virgins by isolating them within 6 hours of eclosion, using light CO2 anaesthesia. Soon after eclosion, focal males from M and F regimes were assigned to experimental treatments. LHst females were collected as virgins and held in single-sex vials at a density of 8 females / vial. LH males were collected as adults from culture vials on 12th day using CO2 anaesthesia.

Experimental design

Experiment (1) Effect of evolutionary history and number of competitors on reproductive traits (Data in Tab 1 "Response to coevolved rivals" of "Raw_data.xlsx")
Mating latency and Copulation duration assay - Virgin males were collected from the M and F populations and common virgin females were collected from the ancestral population LHst. Soon after virgin collection, both M and F males were assigned randomly to one of three experimental treatments- (a) 1 male/vial (b) 8 males/vial (c) 32 males / vial. For each treatment, 30 replicates were sampled. Males were kept undisturbed in these treatments for 2 days. After 2 days, one conditioned male from each treatment was pulled out at random, 2 hours before the mating trials and kept singly in individual vials. Individual virgin LHst females were introduced into these vials containing single males. Each pair was observed individually for recording mating latency and copulation duration. In all the experiments, flies that did not mate within 2 hours were discarded.
Sperm defense ability assay (Proportion of progeny sired by the 1st male or P1) - After mating observations, males from the M and F selection regimes were discarded using light CO2 anaesthesia and females were retained. Following this, after about 1 hour, LHst females previously mated with M and F males were paired with control red-eyed LH males in new vials to remate. These vials were left undisturbed for the next 48 hours. Since the second mating of females with LH (red-eyed) males was not observed, our measure of P1 incorporates a composite measure of actual P1 i.e. direct sperm competition between the males in the FRT as well as the first male’s ability to prevent the female from remating. After 48 hours, LH males were discarded and females were transferred individually to test tubes (12 mm × 75 mm) containing cornmeal-molasses-yeast food. Females were allowed to oviposit in these test tubes for 18 hours after which they were discarded. After 12 days, when all the flies had eclosed, progeny from each test tube were scored for eye colour to determine paternity.

Experiment (2) Consequences of changing the identity of rival males (Data in Tab 2 "LH rivals" of "Raw_data.xlsx")
Virgin males were collected from M, F and LH populations, whereas common virgin females were collected from the ancestral population LHst. After virgin collection, single M/F males were assigned to one of three treatments: 
(a) 1 M/F male/vial
(b) 1 M/F male + 7 LH males = 8 males/vial
(c) 1 M/F male + 31 LH males = 32 males/vial
Males were kept undisturbed in these treatments for 2 days. After 2 days, LH males were discarded and individual conditioned M/F males were pulled out using light CO2 anaesthesia before the mating assay. Then common virgin LHst females were presented to conditioned M/F males for recording mating latency and copulation duration. Flies that did not mate within 2 hours of observation were discarded. Sperm defense ability (P1) was measured in the same way as described previously in experiment (1).

Experiment (3) Effect of increase in time lag between removal of competitive cues and assessment of reproductive traits (Data in Tab 3 "Assayed after 3 days" and Tab 4 "Assayed after 5 days" of "Raw_data.xlsx")

For this experiment, we collected 2 different sets of males to observe changes in the pattern of reproductive investment with an increase in the time between the removal of competitive cues and assessment of the reproductive traits. These 2 sets of males were assayed at 2 different time periods after conditioning them with rival males for 2 days (same as experiment 1). Virgin males were collected from the M and F populations and common young virgin females were collected from the ancestral population LHst for each mating assay. Virgin males from each of the 2 sets were assigned randomly to previously described 3 kinds of treatments (different number of competitors- 1, 8, 32) in early life similar to experiment (1). These two sets of males from both M and F regimes were kept undisturbed for 2 days. After the conditioning period of 2 days, males from both the sets were removed from the treatments and were held singly in individual vials.
Following this, these 2 sets from both M and F regime were assayed at 2 different time periods i.e; 
(a) after 3 days of removal of cues 
(b) after 5 days of the removal of cues. 
Mating assays were executed in the same way as described above (experiment 1) for assaying mating latency, copulation duration and sperm defense ability (P1).

This file ("Raw_data.xlsx") was generated in 2016 and 2017 by Komal Maggu.

GENERAL INFORMATION

1. Title of Dataset: "Divergence of responses to variable socio-sexual environments in laboratory populations of Drosophila melanogaster evolving under altered operational sex-ratios."

2. Author Information
    A. Principal Investigator Contact Information
        Name: Prof. N. G. Prasad
        Institution: Indian Institute of Science Education and Research, Mohali
        Address: IISER Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India.
        Email: prasad@iisermohali.ac.in

    B. Associate or Co-investigator Contact Information
        Name: Komal Maggu
        Institution: Indian Institute of Science Education and Research, Mohali
        Address: IISER Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India.
        Email: komal024maggu@gmail.com

3. Duration of data collection: 2016-2017

4. Geographic location of data collection: Mohali, Punjab, India 

5. Information about funding sources that supported the collection of the data: IISER Mohali, University Grants Commission, Govt. of India.

#######SHARING/ACCESS INFORMATION
 
Links to publications that cite or use the data: 

Manuscript id: 20-0494.R1
Manuscript title: Divergence of responses to variable socio-sexual environments in laboratory populations of Drosophila melanogaster evolving under altered operational sex –ratios.

DATA & FILE OVERVIEW

1. File List:"Raw_data.xlsx"

Note: This file contains four separate data sheets (please see below for methodological details) in the following four tabs:

Tab 1. "Response to coevolved rivals" (Data for Experiment 1)
Tab 2. "LH rivals" (Data for Experiment 2)
Tab 3. "Assayed after 3 days" (Data for Experiment 3)
Tab 4. "Assayed after 5 days" (Data for Experiment 3)

DATA-SPECIFIC INFORMATION FOR "Raw_data.xlsx"

This file has four separate tabs. Below, we provide details separately for each tab.

Tab 1. "Response to coevolved rivals" (Experiment 1)

1. Number of variables: 6

2. Number of cases/rows: 360

3. Variable List: 

Selection (Selection regime; male-biased "M" or female-biased "F")
Block (3 independent replicates of each selection regime; "1", "2" or "3")
Treatment (Number of early-life competitors; "1", "8" or "32")
Mating Latency (Time taken by focal males to begin copulating with a virgin female in minutes)
Copulation Duration (Time spent by focal males in copula with virgin females in minutes)
Sperm Defense Ability (P1) (Proportion of progeny sired by focal males)

Tab 2. "LH rivals" (Experiment 2)

1. Number of variables: 6

2. Number of cases/rows: 540

3. Variable List: 

Selection (Selection regime; male-biased "M" or female-biased "F")
Block (3 independent replicates of each selection regime; "1", "2" or "3")
Treatment (Number of early-life competitors; "1", "8" or "32")
Mating Latency (Time taken by focal males to begin copulating with a virgin female in minutes)
Copulation Duration (Time spent by focal males in copula with virgin females in minutes)
Sperm Defense Ability (P1) (Proportion of progeny sired by focal males)

Tab 3. "Assayed after 3 days" (Experiment 3)

1. Number of variables: 6

2. Number of cases/rows: 359

3. Variable List: 

Selection (Selection regime; male-biased "M" or female-biased "F")
Block (3 independent replicates of each selection regime; "1", "2" or "3")
Treatment (Number of early-life competitors; "1", "8" or "32")
Mating Latency (Time taken by focal males to begin copulating with a virgin female in minutes)
Copulation Duration (Time spent by focal males in copula with virgin females in minutes)
Sperm Defense Ability (P1) (Proportion of progeny sired by focal males)

Tab 4. "Assayed after 5 days" (Experiment 4)

1. Number of variables: 6

2. Number of cases/rows: 359

3. Variable List: 

Selection (Selection regime; male-biased "M" or female-biased "F")
Block (3 independent replicates of each selection regime; "1", "2" or "3")
Treatment (Number of early-life competitors; "1", "8" or "32")
Mating Latency (Time taken by focal males to begin copulating with a virgin female in minutes)
Copulation Duration (Time spent by focal males in copula with virgin females in minutes)
Sperm Defense Ability (P1) (Proportion of progeny sired by focal males)