Group-living animals often experience within-group competition for resources like shelter and space, as well as for social status. Because of this conflict, residents may aggressively resist joining attempts by new members. Here we asked whether different forms of competition mediate this response, specifically competition over i) shelter abundance, ii) spatial position within groups, and iii) social or sexual roles. We performed experiments on wild groups of Neolamprologus multifasciatus cichlids in Lake Tanganyika, either increasing or decreasing the number of shelters (empty snail shells) within their territories. We predicted that increases in resource abundance would reduce conflict and lower the aggression of residents towards presented conspecifics, while decreases in resources would increase aggression. We explored the effects of social conflict and spatial arrangement by introducing same or opposite sex conspecifics, at greater or lesser distances from resident subterritories. We found that changing the abundance of shells had no detectable effect on the responses of residents to presented conspecifics. Rather, aggression was strongly sex-dependent, with male residents almost exclusively aggressing presented males, and female residents almost exclusively aggressing presented females. For females, this aggression was influenced by the spatial distances between the presented conspecific and the resident female sub-territory, with aggression scaling with proximity. In contrast, presentation distance did not influence resident males, which were aggressive to all presented males regardless of location. Overall, our results show that group residents respond to presented conspecifics differently depending on the type of competitive threat these potential joiners pose.

Field work and social group selection

Field work took place off the southern shore of Chikonde Village, Mutondwe Island, Zambia (8°42'49.0"S 31°07'22.9"E) in October and November 2018. This field site contains a large breeding population of N. multifasciatus, located on a shell bed at a depth of 9 – 11 m. Groups typically contain 1-3 males and 0-5 females, along with numerous juveniles (Jordan et al 2016), and while there is no pronounced sexual dimorphism in coloration, males are larger than females (males 24.5 mm median standard length; females 19.0 mm median standard length; Jordan et al in review), and males are typically more aggressive than females (Jordan et al 2016). Relatedness structure within and among groups is unclear, but it has been suggested that females are the dispersing sex and males may inherit their natal territories (Kohler 1998). Ten social groups were selected while SCUBA diving, each consisting of one adult male, two adult females, and several juveniles. Top-mounted video cameras (GoPro Hero 6) were installed 55 cm above each group (Figure 1 A). After cameras were set up, an observer (JG) remained motionless from a distance of approximately two meters away from the group and made a count of the number of visible gastropod shells in each group’s territory and the home shell of each individual (the shell into which it retreated when threatened). The sex composition of each group was also determined based on their social behaviour and relative body sizes, an approach that was confirmed by dissecting fish after similar field observations in a parallel study conducted concurrently in the same population (AB & AJ personal observations). The standard length (SL) of each resident fish and total territory area (cm²) were subsequently measured in Adobe Photoshop CC from still frames of the video recordings taken by the cameras in which a ruler was placed for reference.

Competition and resident response experiments

We applied three experimental treatments to the ten selected social groups, using a within-groups repeated-measures design. In the “shell addition” treatment, the number of shells in the focal territory was increased by as close to 20% as possible by taking empty, available shells from the wider shell bed environment (these shells were removed again immediately after the trial). In the “shell subtraction” treatment, ~20% of the shells in the territory were removed and temporarily placed 2 m away from the focal group (these shells were returned to their original locations in the territory immediately after the trial). In the control treatment, ~20% of the visible shells in the territory were taken away and then immediately returned to their original places. These 20% shell manipulations were spatially concentrated in areas of the males’ territories where i) there was no fish’s home shell, such that the home shell of a resident fish was never disturbed during the handling process, and ii) there was a sufficient number of shells present to be taken or supplemented. Previous studies (Jordan et al. 2016) suggest that manipulation of more than this ratio of shells increases risk of territory takeover by larger heterospecifics, so a ratio of ~20% was the maximum manipulation we considered reasonable for this study. The groups were all given 24 hours between each treatment and the following observation recording. Immediately after each observation, the groups were given their next treatment and again allowed 24 hours before their subsequent observation.

In each trial, a conspecific from a distant territory (at least 20 m away) was taken, along with its home shell, and placed in a transparent plexiglass cylinder (8 cm diameter). The cylinder was placed on the edge of the focal territory, within 2 cm of one of the peripheral shells, and interactions among the resident and presented fish were recorded (Figure 1 B). Both males and females were chosen to be presented, and each focal N. multifasciatus group received all three shell manipulation treatments in the presence of a presented female and also a presented male. The male presentations and the female presentations each took place in short succession to one another during the observation phases of each experimental treatment (counterbalancing for order). Thus, every social group experienced three shell manipulation treatments (one treatment per day, in randomized order), and the ensuing behavioural interactions between residents and presented fish were observed for each treatment. Trials took place between 9:00 and 14:00. The presented fish were returned to their home territories after completion of their trials and not used again in any further experimental trials.

Recording was started after the placement of the cylinder containing the presentation fish. The presented fish emerged from its shell while within the cylinder 85 ± 87 seconds (mean ± s.d., range = 15 - 423) after placing it on the territory edge. These presentations elicited appreciable levels of aggression towards the presented fish but also aggression amongst the resident fish themselves, which had previously showed little or no intra-group aggression. All aggressive interactions were scored for a 10-minute period. Behaviour was scored manually using the software BORIS (Friard and Gamba 2016). Because manipulations were visually apparent, the scorer (JG) could not be blind to treatment. Behaviours were scored using the ethogram presented in Table 1 and pooled into one count of aggression. Note that frontal displays were rare in our observations, and sometimes difficult to accurately assess from the top-down field video footage, and were therefore not included in our counts of aggression. Although we scored all aggressive acts occurring amongst the resident fish, aggression by resident females towards resident males was also exceptionally rare; across all of our 10-minute trials, resident females aggressed against their males a total of 19 times, a sample insufficient to draw statistical inferences from. Furthermore, aggression between resident females was also rare, occurring only 35 times and only in seven field videos. Our statistical comparisons of within-group aggression across experimental treatments therefore focus on resident male versus female aggression. Lastly, we measured the distances between each resident fish’s home shell and the presented cylinder for each trial using the Adobe Photoshop CC.

Statistical analysis

All statistical analyses were conducted in R (v. 3.6.2, R Core Team 2019). To test whether shell manipulations influenced the aggression by the resident fish towards the presented fish, we fit a generalized linear mixed effects model (GLMM) assuming a quasi-Poisson error distribution with a log link function (using the ‘nbinom1’ family from the glmmTMB R package, Brooks et al. 2017). We included the counts of aggressive acts by each resident fish towards the presented fish as the response variable, as well as treatment (3-level categorical variable: control, shell addition, shell subtraction), sex of the presented fish (2-level categorical variable: male, female), and sex of the resident fish as predictor variables, along with each of their pairwise interaction terms. In addition, we included the distance between the resident fish’s home shell and the presented fish (cm, but scaled so that mean = 0, s.d. = 1) as another predictor variable along with its interaction with sex of the resident fish. Finally, we also included the order in which the shell manipulation treatments were given to account for potential order effects. We included a random intercept of fish ID nested within territory ID to account for non-independence of responses (because multiple N. multifasciatus individuals per group were repeatedly tested across treatments). As a model offset term, we included the cumulative time durations over which both the resident fish and the presented fish were outside their shells and thus had the opportunity to interact (log-transformed). We tested whether inclusion of the interaction terms significantly improved model fit based on a likelihood ratio test (LRT), and if not, we omitted them. We used the ‘emmeans’ R package (Lenth 2020) to make further comparisons using the Tukey method.

Next, we tested whether resident male-to-resident female aggression varied with the sex of the presented fish. To do this, we fit a GLMM assuming a quasi-Poisson error distribution (‘nbinom1’ from glmmTMB). We included the counts of aggressive actions by the resident male towards resident females as the response variable. Treatment and sex of the presented fish were included as predictor variables, and we tested whether to include their interaction term based on a LRT (as above). We included a random intercept of female ID nested within male ID and also a model offset term to account for differing time windows when both the male and each resident female were out of their shells and thus had the opportunity to interact.

Finally, we focused only on the scenario when the presented fish was female, and we tested whether resident male aggression towards his resident females was disproportionately directed towards the resident females that were currently closer to the presented female. Here, we fit a binomial GLMM using the ‘logit’ link function. We included a binary response variable indicating whether or not the attacked resident female was the closer of the two females. We also included treatment as a predictor variable as well as a random intercept of female ID nested within male ID.