Supplemental Information. Guarding Males Protect Females. from Predation in a Wild Insect. Rolando Rodríguez-Muñoz, Amanda Bretman, and Tom Tregenza

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1 Current Biology, Volume 21 Supplemental Information Guarding Males Protect Females from Predation in a Wild Insect Rolando Rodríguez-Muñoz, Amanda Bretman, and Tom Tregenza Figure S1. Spatial Use of Burrow Surroundings in Relation to Sex and Sharing Status Distances from individual crickets to the entrance of their burrow, recorded at 15min intervals over a two hour period. Squares, median; box, interquartile range; whiskers, non-outlier range (values within 1 times the interquartile range outside the closest quartile); circles outliers. Paired vs. isolated females n = 11. Paired vs. isolated males n = 15.

2 Figure S2. Spatial Use of Burrow Surroundings within Pairs Distance from individual males and females to the entrance of the burrow when they are paired. Squares, median; box, interquartile range; whiskers, non-outlier range (values within 1 times the interquartile range outside the closest quartile); circles outliers; stars, extremes (n = 17). Figure S3. Contribution of Different Predators to Cricket Mortality through Predation Number of attacks of different types of predators on crickets observed around burrows.

3 Supplemental Experimental Procedures Life History Gryllus campestris is a field cricket living in and around burrows excavated among the grass. Females lay eggs in the early summer, eggs hatch shortly afterwards and nymphs of both sexes dig burrows in which they overwinter. By late summer all adults are dead and the following generation emerges from their burrows in spring and undergoes a final moult to adulthood immediately outside the burrow. Adults are very territorial, and burrows are usually occupied by isolated crickets or opposite sex pairs. Triads composed of one male and two females are exceptional. Burrows are narrow with a constant diameter that prevents the crickets from turning around or mating when they are inside. Each cricket has a territory with a radius of 10-15cm around the burrow, which it rarely strays outside. If it does leave this territory (for instance to move to a different burrow), then when disturbed it will not attempt to reach the burrow. Study Species During the reproductive seasons of , we studied postinsemination associations in a wild population of Gryllus campestris in a meadow in Northern Spain. Both sexes are highly territorial and burrow sharing is almost exclusively restricted to opposite sex pairs. Although they frequently switch burrows, their dependency on burrow occupancy allowed us to observe the vast majority of their adult lives using infra-red video cameras that allow us to record cricket activity 24h a day. Individual Identification All crickets and burrows in the population were marked to allow individual identification. Acetate tags with a 2-symbol code were glued onto the thorax with cyanoacrylate adhesive. Tags were 6-7mm in length and 5 mm in width. Each tag had a 2-symbol (capital letter or digit) code, avoiding symbols that are similar to one another (e.g. S and 5). Most crickets were tagged shortly after they emerged and the remainder, as soon as we could catch them after they were seen for the first time (range = 0-6.3d, mean = 1.72, SD = 1.09, n = 135). Overall, only 3.2% of video footage included untagged crickets. After tagging, crickets were returned to their burrow in all cases they resumed normal activity within a few minutes of release, and departures from burrows were no more common following release than at any other time. Our tagging system worked well, no crickets had to be re-tagged in 2006 and 2008, and only 5 of them in Before the start of the study, we ran a laboratory test on a closely related species, G. bimaculatus, comparing lifespan of control vs. tagged and leg cut specimens in a crowded enclosure in the lab, which showed no difference among both groups (ANOVA, F 1,27 = 1.46, P = 0.247). All crickets were genotyped for a set of 11 microsatellite loci, and parents from the previous generation were assigned to each offspring using the program COLONY2 [26, 27]. DNA was collected from a 2mm piece of the tip of a hind leg sampled when crickets were captured for tagging. Detailed laboratory procedures and statistical methodology for parentage assignment can be found in Rodríguez-Muñoz et al. [19]. Monitoring Cricket Behavior with CCTV To monitor burrows, we installed a video system using closed circuit television (CCTV) technology. We used infra-red (IR) analogue cameras that allow us to record cricket activity 24h

4 a day. Each camera monitored an area of approximately 150cm 2 around the burrow, which covers all cricket activity in nearly all cases, except for the time spent moving among burrows. We installed a maximum of 80 cameras provided with 32 IR light emitting diodes, a wide angle lens and a photocell to automatically switch between daylight and IR illumination, depending on lighting conditions (Pak-NV30 night vision camera Pakatak.co.uk). Cameras were connected to independent wires 5-30 m in length. The ends of these wires were plugged into a connection box located at the centre of the meadow. This box was connected to six computers located in a building 30m away through four 50-core cables. Each computer recorded the signal from 16 cameras through a commercial DVR board, using software designed for security video surveillance (Diginet, Kodicom Co., Ltd This software provides control over recording speed, video resolution, quality and colour vs. black and white (B/W) recording. Recorded video can be played back in groups of 1, 4, 9 or 16 cameras at different speeds. The system started with 64 cameras in 2006 and was updated to 95 in However, cabling and connection malfunctions meant we consistently had 61 cameras in 2006 and 80 in 2007 (the number of cameras exceeded the number of burrows in 2008). Monitoring extended from the beginning of the adult emergence period to the date of the last adult observation in late June or early July. Overall, we recorded over 200,000 hours of video, with over 70,000 hours featuring cricket activity. A single observer (RRM) extracted data from the video. Target burrows were selected at random among those being used by the crickets, and cameras covering burrows where no activity was observed for more than 2-3 consecutive days were moved to an occupied burrow. We marked each burrow by placing a flag with a small plastic number on its base adjacent to the entrance. Monitoring extended from the beginning of the adult emergence period to the date of the last adult observation in late June or early July. Given the large number of cameras distributed across the meadow, and the fact that crickets move regularly among burrows (and hence between monitored and unmonitored burrows) the first and last observations we made are likely to be good surrogates of the start of reproductive behavior and death dates. Video watching was carried out in two consecutive steps on a per-day basis. A first viewing was done to separate recordings from occupied and unoccupied burrows. The second viewing allowed us to extract information on individual cricket behavior. After the viewing was finished, data were imported into a database (MSAccess) to facilitate subsequent processing. Evidence of Mate Guarding Crickets always run into their burrow very quickly when disturbed by vibration. We searched for evidence of mate guarding, by analysing male spatial and temporal use of the burrow territory in relation to both the presence/absence of a female and her mating receptivity towards the guarding male. To assess how far away from the burrow individuals positioned themselves, we selected a single video frame every 15mins over 2h of recording. For each frame, we measured the distance (in arbitrary Screen Units, hereafter SU; one SU = 1.1mm at the entrance of the burrow) of each individual cricket to the entrance of the burrow. We then calculated the mean of those distances and assigned it to the individual cricket being monitored. We use these SU s rather than converting distances to mm measured on the screen, because the cameras are not perpendicular to the ground and hence measurements would be underestimated when the cricket is displaced towards the camera relative to when they move perpendicular to the camera. However these errors will be unbiased in relation to sex or burrow sharing status (there is no

5 relationship between these factors and camera angle or distance). To standardise measurements across burrows, we calibrated them using the size of the tags on the cricket as a reference. To assess the effect of female receptivity on how long males spent at a burrow, we compared the duration of associations between a male and a female ( paired time ) between mated and unmated pairs. We selected only those associations where a male arrived at a burrow occupied by a female and subsequently left the burrow before the female (so that the paired time is male dependent). Additionally, using all pairs, we compared the proportion of occasions when the male left the burrow before the female in relation to whether they had mated or not. For males, one potential benefit of mate guarding is that it reduces the chance that their partner will mate to a different male following their own mating. We tested whether mating had any effect on the chances that a resident male wins a contest against an approaching rival male and whether females tend to mate with the winners of the contest. None of the measured variables were normally distributed, and some of them had a positive skew. We ran analyses using non-parametric tests based on median values. We used Mann- Whitney U tests for independent data, Sign tests of difference for paired skewed data, and Wilcoxon matched-pairs tests only for paired data that were not skewed (a requirement of this test that is frequently ignored [28].To test for differences between dichotomous variables we used 2 x 2 Contingency tables. In all analyses, each sample comes from the mean value obtained for a different individual, so sample sizes reflect the actual number of different individuals that have been tested. Sexual Conflict or Mutual Benefit? We analysed benefits and costs of mate guarding for both sexes to assess whether the balance supports the existence of sexual conflict or mutual benefits. We tested whether guarding increases male contribution to female sperm stores (as estimated from the contribution to female matings) and whether that increases the proportion of offspring sired by the guarding male. We estimated potential costs to males in terms of increased predation risk and reduced number of mates. For females, we looked for direct observational evidence of male coercion and tested the hypothesis that mate guarding reduces female predation risk at the cost of reduced level of polyandry. We tested the existence of a correlation between time spent guarding and number of copulations, using only pairs where the target male arrived at the burrow where they met later than the female and left earlier. This ensures that the time spent together depends on decisions by the target male. To assess the effect of repeated copulations on paternity we correlated the proportion of matings males achieved with particular females with the proportion of that female s offspring they sired. To carry out this correlation avoiding pseudo replication, we calculated a mean value for each of male of the proportion of the lifetime matings of each of his mates that the male s matings with her represented (including only females that left offspring in the next generation) and correlated this with the mean proportion of their offspring that the male sired. To analyse the effects of mate guarding on the acquisition of extra-pair copulations, we tested for correlations between time spent per association and the frequency of extra-pair associations that resulted in matings or not. We calculated the frequency of extra-pair associations as the total number of partners observed for the target cricket divided by the total amount of time that it was monitored. We independently analysed correlations for the total

6 number of partners and for those that mated. We used Pearson correlations for parametric data and Spearman rank correlations for data where these assumptions were not met. To determine the effects of mate guarding on predation risk, we first examined potential predator preferences in relation to sex (the rate of predator attacks on lone males vs. lone females), and pairing status (rate of predator attacks on crickets on their own vs. paired crickets). We then tested for differences in the probability of being predated for males vs. females when isolated or paired. Every day during the breeding season, we calculated the portion of the day each burrow was observed within each of three categories: burrows occupied by a single male, a single female, or a male-female pair. We used the sum of the values for all the burrows during the breeding season (burrow-days) within each category, as an estimate of the availability of that category for predators, and tested predator preferences for males vs. females and isolated vs. paired crickets, with a 2 x 2 contingency chi-squared test including the total number of burrowdays and the total number of attacks for each of the two categories. To estimate the daily probability that a cricket suffers a predator attack within each category, we divided the observed number of attacks by the observed number of burrow-days for that category. Likewise, we estimated the probability of being killed in an attack as the number of successful attacks divided by the total number of attacks within each category. Supplemental References 26. Wang, J. (2004). Sibship reconstruction from genetic data with typing errors. Genetics 166, Wang, J., and Santure, A.W. (2009). Parentage and sibship inference from multilocus genotype data under polygamy. Genetics 181, Whitlock, M.C., and Schluter, D. (2009). The Analysis of Biological Data (Greenwood Village, CO: Roberts and Company).