DOI: 10.5061/dryad.5hqbzkhhd
title: Data from: Reproductive transitions and sperm utilisation in a facultatively parthenogenetic stick insect

description: Facultative parthenogenesis enables females to switch from asexual
 (parthenogenetic) to sexual reproduction after mating, but the process of
 fertilisation is poorly understood in such animals. In particular, it is
 not known whether switching reproductive modes requires changes in the
 eggs themselves, delaying the transition from laying unfertilised to
 fertilised eggs. Likewise, very little is known about patterns of sperm
 precedence in facultatively parthenogenetic females that mate with
 multiple males. In this study, we manipulated reproductive mode in females
 of the facultatively parthenogenetic stick insect Megacrania batesii. We
 used offspring sex ratio, fertilisation rate, and paternity analysis to
 investigate how females descended from distinct natural populations switch
 between reproductive modes and utilise sperm from different males. In the
 switch treatment group, females were first allowed to lay unfertilised
 eggs, and then paired with a male. In the non-switch treatment group,
 females were instead paired successively with two different males. We
 collected eggs laid over two successive 10-day periods after male
 introduction (switch treatment) or substitution (non-switch treatment). We
 found little difference between the treatment groups in fertilisation rate
 or in the number of sons produced during the first and second 10-day
 egg-collection. We also observed similar reproductive performance between
 switch and non-switch treatment groups, but females’ population of origin
 influenced fertilisation rate and offspring sex ratio. In the non-switch
 group, we found near-equal fertilisation rates by the first and second
 male. Our results show that M. batesii females can quickly switch from
 producing parthenogenetic to fertilised eggs, suggesting that this
 transition does not require production of distinct types of eggs. Our
 results also show that M. batesii females can rapidly utilise sperm from a
 new mate, and exhibit near-complete sperm mixing which suggests that
 paternity may be evenly distributed in this species.
description: We used lab-reared females that were collected as eggs from
 natural Northern genotype mixed-sex (BK, CO, MB, MK) and Southern genotype
 all-female (B1, CB, TB) populations (Fig. 1) of <em>M.
 batesii</em> in far-north Queensland, Australia (population codes
 correspond to Wilner et al., 2025a). Adult females were divided into two
 treatment groups: “switch” (<em>N</em> = 17) and “non-switch”
 (<em>N</em> = 18). In the switch group, females were first
 housed alone and, after the onset of oviposition, allowed to lay
 unfertilised eggs for 20 days. In the non-switch group, females were
 paired with a male (first male) soon after their adult moult (mean = 1.78
 ± 1.06 days) and, after the onset of oviposition, allowed to mate and lay
 eggs for 20 days. The eggs laid over the first 20 days were then removed.
 The number of eggs laid during this 20-day period did not differ between
 unpaired (switch treatment) females and male-paired (non-switch treatment)
 females (see Fig. 4A in Wilner et al., 2025a). Switch treatment females
 were then paired with a male for 20 days, while non-switch treatment
 females were paired with a new male (second male) for 20 days (Fig. 2).
 Following male-pairing (switch treatment) or replacement (non-switch
 treatment), we collected eggs laid by each female over two successive
 10-day periods.
description: # Data from: Reproductive transitions and sperm utilisation in a
 facultatively parthenogenetic stick insect Dataset DOI:
 [10.5061/dryad.5hqbzkhhd](10.5061/dryad.5hqbzkhhd) ## Description of the
 data and file structure: There are five main csv files (listed below). ###
 Files and variables #### File: all_seq_daughters.csv
 **Description:** Sequenced daughters  ##### Variables: 9 *
 origin_mom_type: Whether the population is Mixed-sex (reported as NMS) or
 All-female (reported as SAF). * dna_extracted_date: DNA extracted date *
 treat_id: Treatment group ID * treat_grp: Treatment group names: the
 reproductive switch mode, represented as switch for those females paired
 with males throughout the experiment while non-switch for those
 females' initial reproductive mode was asexual. * timing_of_eggs: E1
 represents first 10-day period while E2 represents second 10-day period
 egg collections * hatched_date: Hatched date for each hatchling *
 sex_offspring: The sex of the hatchling, represented as F for female *
 adult_hatchling: Whether extracted individuals were hatchlings or adults *
 percentage_hetero: Heterozygosity level #### File: hatchling_dev_time.csv
 **Description:** Hatchling developmental time ##### Variables: 8 *
 treat_id: Treatment group ID * treat_grp: Treatment group names: the
 reproductive switch mode, represented as switch for those females paired
 with males throughout the experiment while non-switch for those
 females' initial reproductive mode was asexual. * timing_of_eggs: E1
 represents first 10-day period while E2 represents second 10-day period
 egg collections * sex_hatchling: The sex of the hatchling *
 origin_mom_type: Whether the population is Mixed-sex (reported as NMS) or
 All-female (reported as SAF). * initial_mode: Whether females'
 initial reproductive modes were asexual (Parthenogenetic) or sexual
 (Mating) * male_id: The unique individual ID of each male individual *
 dev_time: Development time (days) for each hatchling #### File:
 hatchling_sex_ratio.csv **Description:** Hatchling sex ratio  #####
 Variables: 14 * origin_mom_id: The unique individual ID of each female
 individual * origin_mom_type: Whether the population is Mixed-sex
 (reported as NMS) or All-female (reported as SAF). * initial_mode: Whether
 females' initial reproductive modes were asexual (Parthenogenetic) or
 sexual (Mating) * male_id: The unique individual ID of each male
 individual * treat_grp: Treatment group names: the reproductive switch
 mode, represented as switch for those females paired with males throughout
 the experiment while non-switch for those females' initial
 reproductive mode was asexual. * treat_id: Treatment group ID *
 timing_of_eggs: E1 represents first 10-day period while E2 represents
 second 10-day period egg collections * num_females: Number of females
 hatched * num_males: Number of males hatched * ratio_females: Proportion
 of female hatchlings * ratio_males: Proportion of male hatchlings *
 total_emerged_hatchlings: Total emerged hatchlings *
 unemerged_hatchlings: Total unemerged hatchlings * total_eggs_laid: Total
 eggs laid #### File: heterozygosity_all_offspring.csv
 **Description:** Heterozygosity level for all offspring  ##### Variables:
 9 * dna_extracted_date: DNA extracted date * treat_id: Treatment group ID
 * treat_grp: Treatment group names: the reproductive switch mode,
 represented as switch for those females paired with males throughout the
 experiment while non-switch for those females' initial reproductive
 mode was asexual. * initial_mode: Whether females' initial
 reproductive modes were asexual (Parthenogenetic) or sexual (Mating) *
 timing_of_eggs: E1 represents first 10-day period while E2 represents
 second 10-day period egg collections * hatched_date: Hatched date for each
 hatchling * sex_offspring: The sex of the hatchling, represented as F for
 female and M for male. * adult_hatchling: Whether extracted individuals
 were hatchlings or adults * percentage_hetero: Heterozygosity level ####
 File: paternity_full_active.csv **Description:** Sperm precedence
 analysis  ##### Variables: 13 * origin_mom_type: Whether the population is
 Mixed-sex (reported as NMS) or All-female (reported as SAF). *
 treat_id: Treatment group ID * timing_of_eggs: E1 represents first 10-day
 period while E2 represents second 10-day period egg collections *
 mother_id: The unique individual ID of each female individual *
 offspring_id: The unique individual ID of each hatchling *
 offspring_sex: The sex of the hatchling, represented as F for female and M
 for male. * sire_a: First male mated with a female * sire_b: Second male
 mated with a female * heterozygosity_offspring: Heterozygosity level *
 count_sire_a: The number of mismatched loci from first male *
 count_sire_b: The number of mismatched loci from second male *
 which_father: Potential father for each hatchling whether it was first or
 second male * full_partial: Full parents data represented as full when all
 parents DNA sequence was done, as partial for either of the parents DNA
 sequence is missing ## Code/software We carried out all statistical
 analyses in R version 4.4.3 (R Core Team, 2025). We fitted GLMMs with the
 function *glmmTMB* from the package *glmmTMB* (Brooks et al., 2017), and
 the LMM with the *lmer* function from the package *lme4* (Bates et al.,
 2007). GLMM effects were tested using Wald z-tests. We made *ad-hoc*
 pairwise comparisons using the function *emmeans* from the package
 *emmeans* (Lenth, 2025). We also examined whether paternity success
 differed overall between the first and second male using a Wilcoxon
 matched-pairs test, with female identity as the grouping factor. Model
 assumptions were verified using the package DHARMa (Hartig, 2024). We did
 not detect overdispersion in any of the models according to the function
 *check_overdispersion* from the package *performance* (Lüdecke et al.,
 2021).