DOI: 10.5061/dryad.k98sf7mhf
title: Can sexual conflict drive transitions to asexuality? Female resistance to fertilization in a facultatively parthenogenetic insect

description: Facultatively parthenogenetic animals could help reveal the role of sexual
 conflict in the evolution of sex. Although each female can reproduce both
 sexually (producing sons and daughters from fertilized eggs) and asexually
 (typically producing only daughters from unfertilized eggs), these animals
 often form distinct sexual and asexual populations. We hypothesized that
 asexual populations are maintained through female resistance as well as
 the decay of male traits. We tested this via experimental crosses between
 individuals descended from multiple natural sexual and asexual populations
 of the facultatively parthenogenic stick-insect Megacrania batesii. We
 found that male-paired females descended from asexual populations produced
 strongly female-biased offspring sex-ratios resulting from reduced
 fertilization rates. This effect was not driven by incompatibility between
 diverged genotypes but, rather, by both genotypic and maternal effects on
 fertilization rate. Furthermore, when females from asexual populations
 mated and produced sons, those sons had poor fertilization success when
 paired with resistant females, consistent with male trait decay. Our
 results suggest that resistance to fertilization resulting from both
 maternal and genotypic effects, along with male sexual trait decay, can
 hinder the invasion of asexual populations by males. Sexual conflict could
 thus play a role in the establishment and maintenance of asexual
 populations.
description: <strong>STUDY SYSTEM</strong> The
 peppermint stick-insect <em>Megacrania batesii</em> (Figure
 1A) is a facultative parthenogen endemic to far-north Queensland,
 Australia. Parthenogenesis occurs via automixis (Miller 2024), and
 fertilized <em>M. batesii </em>eggs yield approximately equal
 proportions of male and female hatchlings; whereas unfertilized eggs yield
 only daughters (Miller et al. 2024b). Therefore, any consistent
 female-bias in offspring sex ratios suggests that some daughters were
 produced parthenogenetically, with higher proportions of daughters
 indicating a higher rate of parthenogenesis and lower rate of
 fertilization. A single generation of parthenogenetic reproduction
 typically results in complete or near-complete homozygosity in
 <em>M. batesii </em>females, making it possible to use the
 level of heterozygosity to differentiate impaternate females from
 paternate females and males (Miller 2024; Miller et al. 2024b).
 Spontaneous (parthenogenetically produced) males can occur in systems with
 XX/XO and ZZ/ZW sex determination (Pijnacker 1966, 1969; Lampert 2009;
 Morgan-Richards et al. 2010; Schwander et al. 2013; van der Kooi and
 Schwander 2014; Boyer et al. 2023). However, spontaneous males have never
 been observed in <em>M. batesii</em>, suggesting an XX/XY
 sex-determination system.   Two discrete types of
 <em>M. batesii </em>populations—all-female and mixed-sex—occur
 in a geographical mosaic (Figure 1B), sometimes in close proximity and
 with no obvious barriers to dispersal (Cermak and Hasenpusch 2000; Miller
 et al. 2024b). The all-female populations could have arisen via dispersal
 of unmated females, or via extinction of males (Miller et al. 2024b;
 Figure 1C). The known species range spans only 1.8º in latitude (with most
 populations occurring within 0.24º latitude), and there are no appreciable
 differences in climate, habitat or density between mixed-sex and
 all-female populations (Miller et al. 2024b). Annual field-surveys since
 2019 have shown that all-female populations contain only females, and all
 eggs collected from such populations have hatched into females (see Miller
 et al. 2024b for a summary of the first 4 years of field-data). Mixed-sex
 populations typically have approximately even or slightly female-biased
 sex ratios and reproduction is usually sexual, although approximately 10%
 of females in natural mixed-sex populations were found to have been
 produced parthenogenetically (Miller et al. 2024b). Males develop more
 quickly and mature several weeks before females (DW, pers. obs.), and
 females in mixed-sex populations tend to be almost constantly guarded by a
 male (Boldbaatar et al. 2025). Populations located
 north of Noah Creek form one genetic cluster (“Northern genotype” or
 simply “Northern”); while populations south of Noah Creek form another
 genetic cluster (“Southern genotype” or simply “Southern”). The two
 genetic clusters are clearly differentiated, with high inter-cluster
 <em>F</em>st values (Miller et al. 2024b). Both population
 types (all-female and mixed-sex) occur in each genetic cluster, but most
 known Southern populations are all-female while most known Northern
 populations are mixed-sex. Although the ages of these populations are not
 known, the Southern all-female populations appear to be relatively
 long-established and exhibit high<em> </em>genetic
 differentiation (Miller 2024; Miller et al. 2024b). We therefore expected
 the Southern all-female populations to exhibit evolved resistance to
 fertilization. By contrast, some Northern all-female populations (such as
 NS; Figure 1B) are both geographically and genetically very close to
 Northern mixed-sex populations (Miller 2024; Miller et al. 2024b), and
 might therefore lack evolved resistance to fertilization.  
 <strong>EXPERIMENT 1: ARE FEMALES FROM
 LONG-ESTABLISHED ALL-FEMALE POPULATIONS RESISTANT TO
 FERTILIZATION?</strong> To determine
 whether females descended from all-female populations are resistant to
 fertilization, we paired females from 4 all-female populations (Southern
 genotype) and 4 mixed-sex populations (Northern genotype) with males (all
 from the Northern mixed-sex populations), and we compared the resulting
 offspring sex ratios from the two types of mothers as an index of
 fertilization rate (Experiment 1, Figure 2; See Table 1 for sample sizes).
 We validated the use of offspring sex ratio as an index of fertilization
 rate by sequencing the DNA of a subset of daughters and using
 heterozygosity at 260 polymorphic (SNP) loci to differentiate paternate
 vs. impaternate daughters (Supporting Information, Heterozygosity, Figures
 S1-S2, Tables S1-S3). Additionally, we quantified
 fecundity and egg hatching success of these male-paired females and of
 control (unpaired) females (see Table 1 for sample sizes), to investigate
 whether sex ratios were biased by deaths of male embryos, and to compare
 reproductive outcomes (Supplemental Material, Fecundity and Viability). If
 female-biased offspring sex ratios are caused by the death of male embryos
 (e.g., due to male-killing bacteria, Engelstädter and Hurst 2009), then
 mated females with female-biased offspring sex ratios should also have
 reduced fecundity (if male death occurs before eggs are laid) or reduced
 hatching success (if male death occurs after eggs are laid).
 We first collected eggs from 4 Northern mixed-sex populations
 (BK, MB, MK, CO; Figure 1B), and 4 Southern all-female populations (B1,
 CB, KR, TB; Figure 1B) in Far-North Queensland, Australia, in early
 February 2020. We reared the hatchlings inside clear plastic cylindrical
 containers (200 mm diameter x 400 mm height) with mesh lids, in controlled
 temperature rooms (~27º C; 12-hour light cycle) at UNSW Sydney. They were
 sprayed daily with de-ionized water (for drinking and to maintain high
 humidity) and fed <em>Pandanus tectorius</em> leaves
 <em>ad libitum</em>. Hatchlings were first placed on small
 potted host plants inside the plastic containers, in groups of 2-4
 same-sex full-sib nymphs (juveniles). As they grew and started to
 defoliate their plant, larger nymphs were transferred to containers
 without plants but with a cloth to retain moisture, and provided fresh-cut
 leaves every 2-3 days. Once insects underwent their final moult to the
 adult stage, they were separated into individual containers. Some of the
 experimental females were sisters (from 12 mothers from Southern
 all-female populations, and 10 male-guarded mothers from Northern
 mixed-sex populations), and some of the experimental males were brothers
 (from 16 male-guarded mothers from Northern mixed-sex populations). We
 distributed siblings randomly across treatments, using each individual
 only once. We paired 34 newly moulted adult females (20
 Southern all-female-population females, and 14 Northern
 mixed-sex-population females) with 34 adult males from Northern mixed-sex
 populations. Most females were paired with a non-sibling male from their
 same population (but 3 mixed-sex population females were paired with males
 from a different population, and one was paired with her brother). On
 average, females were paired 1.5 days after their final moult (SD = 1.1
 d), and males were paired 28.6 days after their final moult (SD = 8.3 d).
 Because <em>M. batesii</em> males develop more quickly than
 females, these age ranges probably mimic natural conditions. We collected
 eggs from each pair 20 days after the female started laying, keeping the
 pair together throughout this time (three males died during this time, but
 all females had access to a male for at least 18 days). We later also
 paired 8 females from an isolated all-female population at the southern
 edge of the species range (population BL, Southern genotype; Miller et al.
 2024b) with Northern mixed-sex-population males to test whether this
 distant population is capable of sexual reproduction (Supporting
 Information, BL Crosses, Figure S3). As a control, we
 kept 35 females (23 from Southern all-female-populations; 12 From Northern
 mixed-sex populations) in individual containers to allow parthenogenetic
 reproduction, and collected their eggs 20 days after they started laying.
 Our experimental vs. control housing was designed to mimic natural
 conditions: in all-female populations, two adult <em>M.
 batesii</em> females are rarely found on the same small host plant
 or tree branch (R.B., unpublished data); by contrast, most adult
 <em>M. batesii</em> females in mixed-sex populations are
 constantly guarded by a male (Boldbaatar et al. 2025). These conditions
 are also unlikely to have substantially affected food availability because
 adult <em>M. batesii</em> males eat much less than females
 (Boldbaatar 2022), and food was provided <em>ad
 libitum</em>.  Eggs collected from paired (n =
 937; mean = 27.6; S.E. = 0.9) and unpaired (n = 925; mean = 26.4; S.E. =
 0.8) females were checked daily for hatching until 20 weeks after the last
 female had started laying eggs. Hatchlings (n=600; mean = 17.6; S.E. = 1.3
 from paired females; and n = 358; mean = 10.2; S.E.= 1.1 from unpaired
 females) were sexed based on the morphology of the 8th and 9th abdominal
 sternites (Miller et al. 2024b).  We also quantified the number of eggs,
 hatching success, and number of hatchlings (Supplemental Material,
 Fecundity and Viability). All statistical analyses were
 done in R 4.2.1  (R Core Team 2023). We used the the MuMIn package (Bartoń
 2023) to compare models with and without our predictor of interest, using
 “corrected” AIC (AICc) in all cases to avoid bias associated with small
 sample sizes (Johnson and Omland 2004). To test the effect of female
 population type (Southern all-female, or Northern mixed-sex) on offspring
 sex ratio, we compared a model with female population type as the only
 fixed effect to a null (intercept-only) model. Both models were
 generalized linear mixed models (package glmmTMB, Brooks et al. 2017),
 using the binomial family and logit link, and including an
 observation-level random effect (female ID) to account for overdispersion.
 We did not include population of origin in our models because of small
 sample sizes from some populations; however, the individual populations
 within each population type showed similar trends to each other (Table S4,
 Figures S4-S5). We used a similar approach to investigate the effects of
 female population type and pairing treatment on female fitness measures
 (Supplemental Material, Fecundity and Viability). The large language model
 ChatGPT (OpenAI 2024) was used as an aid in writing the R code for this
 study. <strong>EXPERIMENT 2: IS LOW
 FERTILIZATION RATE EXPLAINED BY GENETIC
 INCOMPATIBILITY?</strong> Reduced
 fertilization rates can be a consequence of incompatibility between male
 and female genotypes (Dobzhansky 1937; Mayr 1963; Howard 2003; Matute and
 Cooper 2021). This could potentially explain the results of Experiment 1
 because females from all-female populations were of the Southern genotype
 whereas all males were of the Northern genotype (i.e., population type was
 confounded with genotype). To address this, we performed 3 additional
 crosses using males and females from the same genetic cluster (“matching
 genotype”) <em>versus</em> differing genetic clusters (see
 Table 2 for sample sizes). These crosses (Experiment 2, Figure 2) were:
 “Southern Pairs” cross (matching genotypes: Southern all-female population
 female x Southern mixed-sex population male), “Northern Pairs” cross
 (matching genotypes: Northern all-female population female x Northern
 mixed-sex population male), and “Northern female x Southern male” cross
 (non-matching genotypes: Northern all-female population female x Southern
 mixed-sex population male). If female resistance caused the low
 fertilization success we observed in crosses between Southern
 all-female-population females and Northern mixed-sex-population males in
 Experiment 1, then the Southern Pairs cross in Experiment 2 should also
 produce more female-biased offspring sex ratios (i.e., we should see a
 female genotype effect). But if the low fertilization success was due to
 genetic incompatibility, then the Northern female x Southern male cross
 should produce more female-biased offspring sex ratios (i.e., we should
 see a genotype-matching effect). We used lab-reared
 insects that had been collected from natural populations as eggs or
 hatchlings in August 2022 (except for one lab-colony male). All females
 were collected from all-female populations (and were therefore
 impaternate), and all males were descended purely from Northern or
 Southern mixed-sex populations. Southern all-female-population females
 were collected as first-instar hatchlings from population CB (Figure 1B).
 The Northern all-female-population females (from population NS) and
 Southern mixed-sex-population males (from population VS) were collected as
 eggs. Three of the Northern mixed-sex-population males were collected as
 hatchlings from population CO, and the fourth was lab-bred (from Northern
 mixed-sex population stock). Females were housed and paired as described
 above. However, average age at pairing was 17.4 days (SD = 10.1 d) for
 females, and 61.5 days (SD = 26.1 d) for males. Four of the Southern
 mixed-sex-population males were used twice (paired once with a Southern
 all-female-population female and once with a Northern
 all-female-population female, in alternating order, with at least 4 days
 of rest between pairings to minimize sperm depletion effects). We
 collected eggs (n=437 total; mean = 27.3; S.E. = 1.3) from each pair after
 20 days of laying (but 21 days for one female). Four females started
 laying before pairing (two from the Southern Pairs cross treatment and two
 from the Northern female x Southern male cross treatment); for these four
 females, any pre-pairing eggs were removed, and eggs were collected 20
 days after pairing; the offspring sex ratios produced from these four
 females were similar to those produced by other females in their
 respective treatments. We again quantified the number of eggs, hatching
 success, and number of hatchlings (Supplemental Material, Fecundity and
 Viability), and the hatchlings (n=257 total; mean = 16.1 per female; S.E.
 = 1.5) were sexed as described above. Based on the data
 from these three crosses, we used AICc to compare two models of offspring
 sex-ratio, each containing a single fixed effect, as well as a null
 (intercept-only) model. In the first model, the fixed-effect was female
 genotype (Southern vs Northern); whereas in the second model, it was
 genotype matching (matching or non-matching). All three were generalized
 linear mixed models with a binomial family error structure with logit
 link, and they included an observation-level random effect to account for
 overdispersion. <strong>EXPERIMENT 3: IS
 RESISTANCE DRIVEN BY GENOTYPIC OR MATERNAL EFFECTS? AND ARE MALE TRAITS
 DECAYING IN ALL-FEMALE POPULATIONS?</strong>
 In Experiment 1, as in nature, impaternity was confounded with
 female population type and genotype, since females from all-female
 populations are always impaternate, and females from mixed-sex populations
 are usually paternate. To disentangle these effects, we performed another
 set of crosses using our second generation of lab-reared insects
 (Experiment 3, Figure 2). These crosses included impaternate females
 (i.e., females produced by unmated mothers in Experiment 1) descended from
 both Northern mixed-sex and Southern all-female populations. If resistance
 to fertilization is due to a trait in Southern all-female populations
 (i.e., a genotypic effect), both paternate and impaternate females
 descended from Northern mixed-sex-populations should lay more fertilized
 eggs and produce more sons than Southern all-female-population females
 when paired with males. If impaternity results in female resistance, then
 impaternate females descended from Northern mixed-sex-populations should
 produce more female-biased offspring sex ratios than paternate Northern
 females. To test the male trait decay hypothesis, we
 also included two types of males in these crosses (Experiment 3, Figure
 2): fully Northern males (descended from Northern mixed-sex-populations
 only, from Experiment 1) and North-South intraspecific hybrid males
 (produced by Southern all-female population mothers and Northern mixed-sex
 population fathers in Experiment 1). If male traits have decayed in the
 Southern all-female populations, intraspecific hybrid males should have
 poor fertilization success. If there is no such decay, intraspecific
 hybrid males should have similar success to fully Northern males (or
 greater success than them with Southern females, in case of
 incompatibilities between Northern and Southern genotypes). For
 comparison, we also included North-South intraspecific hybrid females in
 our crosses. We thus paired four types of females (11
 impaternate Southern all-female-population females, 11 impaternate
 Northern mixed-sex-population females, 19 paternate Northern
 mixed-sex-population females, and 12 North-South intraspecific hybrid
 females) with the two types of males (26 fully Northern males and 27
 North-South intraspecific hybrid males), in a full-factorial design (Table
 3).  Paternate females from Northern mixed-sex populations were descended
 from primarily sexual lineages; impaternate Southern all-female population
 females were descended from entirely parthenogenetic lineages; and
 impaternate Northern mixed-sex population females were descended from
 primarily sexual lineages via a single generation of
 parthenogenesis. Subsequent heterozygosity analysis
 showed that 3 of the putatively paternate North-South intraspecific hybrid
 females used in this experiment were actually impaternate (see
 Supplemental Material, Heterozygosity). We did not remove these from our
 analyses because we were not able to sequence DNA from all focal females.
 Our estimates of the differences between paternate and impaternate females
 are therefore conservative. We reared and paired the
 insects and collected their eggs as in Experiment 1, except that average
 pairing age (days since final moult) was 10 days (SD = 4.6) for females
 and 34.7 days (SD = 11.6) for males, and we collected eggs laid over 15
 days instead of 20 days. We also kept 63 additional females laying
 parthenogenetically (Table 3) for comparison. The eggs (n=1811; mean =
 15.6; S.E. = 0.4) were checked daily for hatching until 21 weeks after the
 last egg was collected, and hatchlings (n=1281; mean = 11; S.E. = 0.5)
 were sexed as described above. Additionally, we propagated 20
 intraspecific hybrid females parthenogenetically for an additional
 generation to check for infertility (Supplemental Material, Performance of
 second-generation intraspecific hybrid females). We again collected
 fecundity and viability data (Supporting Information, Fecundity and
 Viability), and sequenced DNA from a subset of daughters to quantify their
 heterozygosity (Supplemental Material, Heterozygosity). We used AICc model
 selection to investigate effects on offspring sex ratio. We tested three
 main effects: focal female impaternity (paternate vs impaternate), focal
 females’ maternal genotype (Southern vs. Northern), and male (mate)
 genotype (fully Northern vs North-South intraspecific hybrid). We used
 only the female’s mother’s genotype (ignoring that of her father) because
 all paternate females necessarily had Northern mixed-sex-population
 fathers and all impaternate females had none. We also tested interactions
 among these factors. We therefore compared 18 models (see Table S19 for
 the full list of models and the predictions they test). All were
 generalized linear mixed models, and all used a binomial distribution and
 included an observation-level random effect to account for
 overdispersion.
description: # Can sexual conflict drive transitions to asexuality? Female resistance
 to fertilization in a facultatively parthenogenetic insect
 [https://doi.org/10.5061/dryad.k98sf7mhf](https://doi.org/10.5061/dryad.k98sf7mhf) ## Description of the data and file structure **Data file: Experiment 1_main** This data file contains data from Experiment 1, in which impaternate (parthenogenetically produced) females descended from Southern genotype all-female populations and putatively paternate (sexually produced) females descended from Northern genotype mixed-sex populations were either paired with Northern genotype males or housed alone and allowed to reproduce parthenogenetically. **Data file: Experiment 2** This data file contains data from Experiment 2, in which impaternate (parthenogenetically produced) females descended from a Southern genotype all-female population and impaternate (parthenogenetically produced) females descended from a Northern genotype all-female population were paired with males of either the Northern or Southern genotype. **Data file: Experiment 3_main** This data file contains data from Experiment 3, in which impaternate (parthenogenetically produced) females descended from Southern genotype all-female populations, putatively paternate (sexually produced) females descended from Northern genotype mixed-sex populations, impaternate (parthenogenetically produced) females descended from Northern genotype mixed-sex populations, or putative intraspecific hybrid females produced by an impaternate Southern genotype mother and Northern genotype father were either paired with Northern genotype males or intraspecific hybrid males produced by an impaternate Southern genotype mother and Northern genotype father (“Hybrid Male”), or housed alone to reproduce parthenogenetically. **Data file: BL_crosses** This data file contains data from an additional set of crosses in which impaternate (parthenogenetically produced) females descended from the Southern genotype all-female population BL were paired with Northern genotype males. **Data file: Experiment 1_Heterozygosity_17mothers** This data file contains data on mean heterozygosity of each of the 17 focal females from Experiment 1 whose DNA was sequenced (reduced representation sequencing by Diversity Arrays Technology using the DArTag process) **Data file: Experiment 1_Heterozygosity_84daughters** This data file contains data on mean heterozygosity of each of the 84 daughters from Experiment 1 whose DNA was sequenced (reduced representation sequencing by Diversity Arrays Technology using the DArTag process) **Data file: Experiment 3_Heterozygosity_25mothers** This data file contains data on mean heterozygosity of each of the 24 focal females from Experiment 3 whose DNA was sequenced (reduced representation sequencing by Diversity Arrays Technology using the DArTag process), and lists one additional focal female that was not sequenced but had one daughter sequenced **Data file: Experiment 3_Heterozygosity_124daughters** This data file contains data on mean heterozygosity of each of the 124 daughters from Experiment 3 whose DNA was sequenced (reduced representation sequencing by Diversity Arrays Technology using the DArTag process) ### Files and variables #### **Data file: Experiment 1\_main** This data file contains data from Experiment 1, in which impaternate (parthenogenetically produced) females descended from Southern genotype all-female populations and putatively paternate (sexually produced) females descended from Northern genotype mixed-sex populations were either paired with Northern genotype males or housed alone and allowed to reproduce parthenogenetically. FemaleID: Identity of focal female Female_popn_type: Population type (mixed-sex or all-female) that the focal female is descended from Treatment: Experimental treatment applied to the focal female: paired with a male (“Sexual”) or housed alone (“Asexual”) Male_ID: Identity of male paired with the focal female (“NA” for females housed alone) FemalePopulation: The population from which the focal female was descended (codes based on Miller et al. 2024 American Naturalist 203: 727511) MalePopulation: The population from which the male partner was descended (codes based on Miller et al. 2024 American Naturalist) NumberEggs_20d: The number of eggs laid by the focal female over 20 days, starting from the day of first oviposition total_hatched: Total number of eggs hatched, out of the number of eggs laid by the focal female Total_sons: Total number of male hatchlings obtained from the eggs laid by the focal female Total_daughters: Total number of daughters obtained from the eggs laid by the focal female first_egg_dmy: Date of onset of oviposition (i.e., first egg laid) by the focal female first_hatchling_dmy: Date of first egg hatching EggDeveTime_days: Number of days from the onset of oviposition to the first egg hatching PairedOrTransfered_mdy: Date when the focal female was assigned to experimental treatment (either paired with a male or housed alone) FirstMaleDeath_Note: Note indicating death of the male partner (“NA” if no death occurred during the experiment) Substitute_male: Identity of the substitute male used to replace a male that had died (“NA” if no substitute male was used) MaleDEAD_mdy: Date when the male partner died (“NA” if no death occurred during the experiment) **Data file: Experiment 2** This data file contains data from Experiment 2, in which impaternate (parthenogenetically produced) females descended from a Southern genotype all-female population and impaternate (parthenogenetically produced) females descended from a Northern genotype all-female population were paired with males of either the Northern or Southern genotype. FemaleID: Identity of focal female FemaleGenotype: Whether the focal female was descended from the Southern or Northern genetic cluster (based on Miller et al. 2024 American Naturalist 203: 727511) MaleGenotype: Whether the male partner was descended from the Southern or Northern genetic cluster (based on Miller et al. 2024 American Naturalist 203: 727511) SameGenotype: Whether the focal female and the male partner were descended from the same genetic cluster (“Same”) or from different genetic clusters (“Different”) FemalePopulation: The population from which the focal female was descended (codes based on Miller et al. 2024 American Naturalist 203: 727511) MalePopulation: The population from which the male partner was descended (codes based on Miller et al. 2024 American Naturalist) Treatment: The inter-population cross (female’s population x male’s population) PrevMatedMale: Whether the male had been paired with a different female prior to this pairing FOCAL_EGGS_num: The number of eggs laid by the focal female over 20 days, starting from the day of first oviposition (or from the date of pairing, if the female had started laying before pairing –see Extra_eggs_note and Focal_eggs_comments) total_hatched: Total number of eggs hatched, out of the number of eggs laid by the focal female Total_daughters: Total number of daughters obtained from the eggs laid by the focal female Total_sons_withzeroes: Total number of male hatchlings obtained from the eggs laid by the focal female Datepaired: Date when the focal female was paired with a male FIRST_EGGS: Date of onset of oviposition (i.e., first egg laid) by the focal female FOCAL_EGGS_date: Date when the eggs laid by the focal female after pairing were collected Extra_eggs_note: Note indicating whether the focal female had started laying eggs prior to being paired with the male (“parth eggs before pairing”), or not (“NA”) Focal_eggs_comments: Note indicating when eggs were collected (“NA” indicates that oviposition started after the pairing and eggs were collected 20 days following onset of oviposition) Female_JuvContainerID: Code for the juvenile container from which the focal female was obtained Femaledateadult: Date when the focal female underwent her adult moult FemaleAgeAtPairing: Age (days) of the focal female when paired with a male Paired_who: Identity of the male paired with a focal female Male_JuvContainerID: Code for the juvenile container from which the male was obtained Maledateadult: Date when the male underwent his adult moult MaleAgeAtPairing: Age (days) of the male when paired with the focal female **Data file: Experiment 3_main** This data file contains data from Experiment 3, in which impaternate (parthenogenetically produced) females descended from Southern genotype all-female populations, putatively paternate (sexually produced) females descended from Northern genotype mixed-sex populations, impaternate (parthenogenetically produced) females descended from Northern genotype mixed-sex populations, or putative intraspecific hybrid females (produced by an impaternate Southern genotype mother and Northern genotype father) were either: paired with either Northern genotype males or intraspecific hybrid males (“Hybrid Male”, produced by an impaternate Southern genotype mother and Northern genotype father), or housed allone to reproduce parthenogenetically. Female_ID:  Identity of focal female treatment_pairedYN: Experimental treatment applied to the focal female: paired with a male (“Paired”) or housed alone (“Unpaired”) FemaleType: Whether the focal female was descended from the Northern genetic cluster and produced sexually (“sexually-produced North”), descended from the Northern genetic cluster and produced asexually (“impaternate North”), descended from the Southern genetic cluster and asexually produced (“impaternate South”), or produced by an impaternate Southern genotype mother and Northern genotype father (“Hybrid female”) MaleType: Whether the male was descended purely from a Northern mixed-sex population (“Full-north Male”), or produced by an impaternate Southern genotype mother and Northern genotype father (“Hybrid Male”) FemaleImpaternity: Whether the focal female was produced putatively sexually (“Paternate”) or asexually (“Impaternate”) FemalesMomGenotype: Whether the focal female’s mother was descended from a Southern-genotype all-female population (“South_AFP”) or a Northern-genotype mixed-sex population (“North_MSP”) FemalesMomID: Identity of the focal female’s mother FemaleMomPopulation: Population from which the focal female’s mother was descended (codes based on Miller et al. 2024 American Naturalist 203: 727511) EggsAfter15Days: Number of eggs collected from the focal female over 15 days following the onset of oviposition total_hatched: Number of eggs that hatched unhatched: Number of eggs that did not hatch Total_females: Total number of female nymphs obtained from the eggs that hatched Total_males_withzeroes: Total number of male nymphs obtained from the eggs that hatched FemaleAdultDate_mdy: Date when the focal female underwent her adult moult PairDate_mdy: Date when the focal female was paired with a male (“NA” for females that were housed alone) FirstEggs_mdy: Date of onset of oviposition (i.e., first egg laid) by the focal female ymd_15daysofeggs: Date when the eggs were collected from the focal female first_hatch_mdy: Date when the first egg hatched MaleAdultDate_mdy: Date when the male mate underwent his adult moult (“NA” for females that were housed alone) Female_AgeAtPairing: Age (days) of the focal female when paired with a male (“NA” for females that were housed alone) Male_AgeAtPairing: Age (days) of the male when paired with the focal female (“NA” for females that were housed alone) MaleData_Mom: Identity of the male mate’s mother (“NA” for females that were housed alone) **Data file: BL_crosses** This data file contains data from an additional set of crosses in which impaternate (parthenogenetically produced) females descended from the Southern genotype all-female population BL were paired with Northern genotype males. FemaleID: Identity of focal female FemaleType: Population of origin of the focal female (all from Bingil, code BL Miller et al. 2024 American Naturalist 203: 727511) FOCAL_EGGS_num: The number of eggs laid by the focal female over 20 days, starting from the day of first oviposition total_hatched: Total number of eggs hatched, out of the number of eggs laid by the focal female HatchingSuccess: Proportion of eggs hatched, out of the number of eggs laid by the focal female Total_daughters : Total number of daughters obtained from the eggs laid by the focal female Total_sons_withzeroes: Total number of male hatchlings obtained from the eggs laid by the focal female SR: Sex ratio of offspring (proportion hatchlings that were male) datepaired_ymd: Date when the focal female was paired with a male Paired_who: Identity of the male that was paired with the focal female female_dateadult_ymd: Date when the focal female underwent her adult moult male_dateadult_ymd: Date when the male mate underwent his adult moult female_ageAtPairing: Age (days) of the focal female when paired with a male male_ageAtPairing: Age (days) of the male when paired with the focal female MalePopulation: The population from which the male mate was descended (codes based on Miller et al. 2024 American Naturalist 203: 727511); three of the males were descended from Northern genotype mixed-sex populations but their exact population or origin was not known (“LabColony_Ngenotype”) PrevMatedMale: Whether the male had been paired previously with a different female (“Mated”) or not (“No”) **Data file: Experiment 1_Heterozygosity_17mothers** This data file contains data on mean heterozygosity of each of the 17 focal females from Experiment 1 whose DNA was sequenced (reduced representation sequencing by Diversity Arrays Technology using the DArTag process) FemaleID: Identity of focal female DNAsampleID: Code of the DNA sample (focal female) Ho: Mean heterozygosity of the DNA sample based on genotypes at 260 SNP loci Female_popn_type: Population type (All-female or Mixed-sex) that the focal female was descended from **Data file: Experiment 1_Heterozygosity_84daughters** This data file contains data on mean heterozygosity of each of the 84 daughters from Experiment 1 whose DNA was sequenced (reduced representation sequencing by Diversity Arrays Technology using the DArTag process) MomID: Identity of focal female (mother of the genotyped offspring) Female_popn_type: Population type (mixed-sex or all-female) that the focal female (mother of the genotyped offspring) is descended from Treatment: Experimental treatment applied to the focal female: all genotyped daughters were produced by focal females that had been paired with a male (“Sexual”) DNAsampleID: Code of the DNA sample (individual daughter) Ho: Mean heterozygosity of the DNA sample based on genotypes at 260 SNP loci **Data file: Experiment 3_Heterozygosity_25mothers** This data file contains data on mean heterozygosity of each of the 24 focal females from Experiment 3 whose DNA was sequenced (reduced representation sequencing by Diversity Arrays Technology using the DArTag process), and one additional focal female that was not sequenced but had one daughter sequenced Female_ID: Identity of focal female FemaleType: Whether the focal female was descended from the Northern genetic cluster and produced sexually (“sexually-produced North”), descended from the Northern genetic cluster and produced asexually (“impaternate North”), descended from the Southern genetic cluster and asexually produced (“impaternate South”), or produced by an impaternate Southern genotype mother and Northern genotype father (“Hybrid female”) MaleType: Whether the male was descended purely from a Northern mixed-sex population (“Full-north Male”), or produced by an impaternate Southern genotype mother and Northern genotype father (“Hybrid Male”) DNAsampleID: Code of the DNA sample (focal female) Ho: Mean heterozygosity of the DNA sample based on genotypes at 260 SNP loci Ho_suggests: Inferred sexual (“fert”) or parthenogenetic (“parth”) origin of the focal female, based on her estimated genome-wide heterozygosity: mean heterozygosity values &gt; 0.05 were assumed to indicate sexual origin while heterozygosity values &lt;= 0.05 were assumed to indicate parthenogenetic origin (based on Miller 2024 Thesis, UNSW) n_daughters_sequenced: Number of the focal female’s daughters whose DNA was sequenced **Data file: Experiment 1_Heterozygosity_124daughters** This data file contains data on mean heterozygosity of each of the 124 daughters from Experiment 3 whose DNA was sequenced (reduced representation sequencing by Diversity Arrays Technology using the DArTag process) Mom_ID: Identity of focal female (mother of the genotyped offspring) MotherType: Population type (mixed-sex or all-female) that the focal female (mother of the genotyped offspring) is descended from treatment_pairedYN: Experimental treatment applied to the focal female: all sequenced daughters were produced by focal females that had been paired with a male (“Paired”) MaleType: Whether the male (sire of the daughter that was sequenced) was descended purely from a Northern mixed-sex population (“Full-North Male”), or produced by an impaternate Southern genotype mother and Northern genotype father (“Hybrid Male”) DNAsampleID: Code of the DNA sample (individual daughter) Ho: Mean heterozygosity of the DNA sample based on genotypes at 260 SNP loci Ho_suggests: Inferred sexual (“fert”) or parthenogenetic (“parth”) origin of the sequenced daughter, based on her estimated genome-wide heterozygosity: mean heterozygosity values &gt; 0.05 were assumed to indicate sexual origin while heterozygosity values &lt;= 0.05 were assumed to indicate parthenogenetic origin (based on Miller 2024 Thesis, UNSW) ## Code/software R