Could adult or juvenile dispersal shape geographical parthenogenesis? Evidence from the facultatively parthenogenetic phasmid Megacrania batesii

Despite their individual reproductive flexibility, populations of many facultatively parthenogenetic animals vary in sex ratio and reproductive mode. Sex-specific dispersal could contribute to such spatial variation. We asked if sex-specific dispersal by adults or nymphs occurs in the facultatively parthenogenetic phasmid Megacrania batesii, which forms a geographical mosaic of mixed-sex (mostly sexually reproducing) and all-female (parthenogenetic) populations. If sex-specific dispersal contributes to sex-ratio variation in this species, we would expect to observe greater dispersal by females than by males. We carried out a mark-resighting field study over three years to investigate adult dispersal in mixed-sex and all-female populations. To better understand how males affect female behaviour, we also investigated pairing and mate-guarding. In addition, we investigated dispersal by hatchling nymphs in a semi-natural enclosure. Mean nightly movement distances did not differ between unpaired (single) females and males in the mixed-sex population. However, unpaired females moved further in mixed-sex than in all-female populations. Many adult females in the mixed-sex population continually carried guarding males on their dorsum. Pairs often remained together for multiple days, and few females or males were observed pairing with multiple partners. Paired females moved shorter distances than unpaired females, and such females’ movement increased following experimental removal of males. Hatchling nymphs rarely moved between plants. Our findings suggest that guarding males affect females’ movement patterns, but nymph and adult dispersal is unlikely to shape spatial variation in sex ratio in Megacrania batesii.

Methods
Mark-resighting methodology

Within each population, we used a mark-resighting method to track movements of Megacrania batesii adults (and a few final-instar female nymphs). Individuals were marked on the first day of the study each year, and additional unmarked individuals were marked on subsequent days. Resighting data were collected either daily or every other day, starting on the 2nd or 3rd day of the study. Newly sighted individuals were marked with unique codes on their thorax and/or wings. using black permanent marker (Artline xylene-free permanent marker, Shachihata, Japan) in situ on their host plants, without handling or restraining them. Host plants on which Megacrania batesii individuals were marked or resighted were tagged with numbered plastic tags. Both males and females can spray a defensive fluid when disturbed (Cermak and Hasenpusch 2000; Jones and Bulbert 2020), which can complicate marking by depositing moisture on the thorax. If a spraying response occurred, we waited a few minutes and then attempted to write the same code on the forewings. When an individual was found at the base of a leaf, we gently tapped the tip of its abdomen to trigger it to crawl upwards to become accessible for marking. When marking male-female pairs, the male’s antennae sometimes blocked the female’s thorax. In such cases, the antennae were pushed to the side or the male was moved gently to the side or backwards to expose the female’s thorax.

In 2020, we conducted a mark-resighting study from 2nd to 14th February at CO and CB (only within the Benstonea monticola patch). At CO, we marked individuals on 2nd February, and then collected resighting data daily until 4th of February, and every second day thereafter until 14th of February. At population CB, we marked individuals on 3rd February, and then collected resighting data daily from 4th February, and every second day thereafter until 14th February. In addition to adult females, we also marked three final-instar female nymphs at CB. Almost no rain occurred during our mark-resighting study, and average maximum temperature was ~34.6ºC (Table S1-S3). In 2021, we conducted a mark-resighting study from 25th February to 8th March at CO and KB. At CO, we marked and resighted Megacrania batesii individuals in the same patch of P. tectorius plants as in 2020, as well as an adjacent patch (see Results). We marked individuals on 26th February, and then collected resighting data every other day until 8th March. At KB, we marked individuals on 26th February, and then collected resighting data every other day until 8th March. There was considerable rainfall during our mark-resighting study, and the average maximum temperature was ~30.5ºC (Table S1-S3). In 2022, we conducted a mark-resighting study from 22nd February to 4th March at CO and CB (only within the Pandanus tectorius patch). At CO, we marked individuals in the same two patches as in 2021 on 22nd February, and then collected resighting data every other day until 4th March. At CB, we marked individuals on 23rd February, and then collected resighting data every other day until 5th March. There was little rainfall during our field-study, and average maximum temperature was ~34.7 ºC (Table S1-S3). 

Dispersal

To estimate dispersal rates, we quantified how far individuals moved between successive sightings. We checked for previously marked individuals on each plant (absence or presence) on each resighting day, measured the distance between the previous sighting location and new location, and recorded the identity and pairing state (unpaired or paired) of each marked individual. We collected the Global Positioning System (GPS) coordinates of each individual at its point of initial sighting or resighting using a Garmin eTrex 20x GPS. Because GPS coordinates are imprecise over small distances, we also measured the linear distance between plants using a tape-measure and use these measurements in analyses of dispersal distance. The “nightly dispersal” distance was calculated for each individual each time it was resighted as the distance between the previous and current location divided by the number of nights between the current sighting and the previous sighting (since Megacrania batesii are mostly active at night). The mean nightly dispersal distance for each individual was calculated as the sum of the nightly dispersal distances divided by the number of nights between the initial sighting/marking and the final resighting.

Male removal experiment

To further investigate the effect of male guarding on female dispersal, we removed males from a patch of host plants at CO on the 3rd of March 2021, thoroughly checking both during the day and at night and removing any males that we could find. Locations of all females that had been separated from guarding males were recorded daily until the 7th of March, and their nightly dispersals were measured. The removed males were kept in individual mesh cages in our field-laboratory, misted with water and provided fresh host plant leaves daily. The males were released in the same patch on 7th of March.

Dispersal of hatchlings

We observed dispersal of Megacrania batesii hatchling nymphs from 25th July to 28th August 2020 in a semi-natural enclosure set up inside a controlled-temperature room at UNSW Sydney. Hatchlings were obtained from eggs collected in the field between Cow Bay and Cape Tribulation, Queensland, in February 2020. Each day, newly hatched nymphs were sexed based on the morphology of the terminal abdominal sternites (see Miller et al. 2024a), marked individually with distinctive patterns of coloured dots using permanent marker (Sharpie, Australia), and released on Pandanus tectorius plants distributed on the floor of a controlled-temperature room (maintained at 25 ± 2°C and 60 ± 20% relative humidity; 12 hrs of cycle day/night with white and red lights). Six immature Pandanus plants (40 – 60 cm in height) were placed in the room, with one plant in each corner and two plants in the centre. Each morning, we recorded the locations of all hatchlings. Each nymph was monitored until it underwent its first molt, after which we were not able to track individuals because their individual markings were shed along with their exoskeletons. In total, we marked and released 55 hatchlings (39 females, 16 males), and observed each hatchling over 10 ± 2 days.