Can sexual conflict drive transitions to asexuality? Female resistance to fertilization in a facultatively parthenogenetic insect

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.

Methods
STUDY SYSTEM

The peppermint stick-insect Megacrania batesii (Figure 1A) is a facultative parthenogen endemic to far-north Queensland, Australia. Parthenogenesis occurs via automixis (Miller 2024), and fertilized M. batesii 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 M. batesii 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 M. batesii, suggesting an XX/XY sex-determination system.  

Two discrete types of M. batesii 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 Fst 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 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.  

EXPERIMENT 1: ARE FEMALES FROM LONG-ESTABLISHED ALL-FEMALE POPULATIONS RESISTANT TO FERTILIZATION?

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 Pandanus tectorius leaves ad libitum. 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 M. batesii 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 M. batesii females are rarely found on the same small host plant or tree branch (R.B., unpublished data); by contrast, most adult M. batesii 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 M. batesii males eat much less than females (Boldbaatar 2022), and food was provided ad libitum. 

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.

EXPERIMENT 2: IS LOW FERTILIZATION RATE EXPLAINED BY GENETIC INCOMPATIBILITY?

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”) versus 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.

EXPERIMENT 3: IS RESISTANCE DRIVEN BY GENOTYPIC OR MATERNAL EFFECTS? AND ARE MALE TRAITS DECAYING IN ALL-FEMALE POPULATIONS?

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.