DOI: 10.5061/dryad.4tmpg4fcc
title: Strength and direction of sexual direction and sex roles vary between social groups in a coral reef cardinalfish

description: The strength and direction of sexual selection can vary among
 geographically distinct populations, and within populations across time.
 However, spatial and temporal variability at the level of the social unit
 is rarely explored. Here we investigate sexual selection and sex roles in
 the paternally mouthbrooding and site-attached pajama
 cardinalfish, Sphaeramia nematoptera, where social groups can vary in size
 and adult sex ratio. We found that reproductive success scaled with mating
 success for both sexes, consistent with positive sexual selection at
 population level. However, within social groups, we show that adult
 sex-ratio and group size can influence the strength and direction of
 sexual selection. Our results show the Bateman gradient is steeper for
 females in groups with male-biased adult sex ratios and sex roles are
 reversed, but steeper for males in female-biased groups, and equally steep
 when the sex ratio is even. In medium sized groups, reproductive skew was
 highest for both sexes and males had a steeper Bateman gradient than
 females, whereas in small and large groups the gradients were equal. We
 conclude that the strength and direction of sexual selection and sex roles
 can be masked by social dynamics in group-living species when considering
 only population and demographic processes.
description: Study system and tagging  This study was conducted over 9 months, spanning
 two years, over five different periods; October - November 2012, February
 - March 2013, July - August 2013, March - April 2014 and September 2014,
 in Kimbe Bay, Papua New Guinea(5°30’S, 150°05’E). To reflect approximately
 the same fieldwork timespan data were grouped by year. Social units or
 groups of the pajama cardinalfish, Sphaeramia nematoptera, were defined as
 occupying the same distinct patch of the stony coral Porites cylindrica at
 a minimum distance of 2m from another group (Fig. 1a). A total of 379
 individuals from 18 groups at 5-17 m depth on five reefs were included in
 the study (see map in Rueger et al. 2019). All fish were caught using
 handnets and diluted clove oil solution (Munday and Wilson, 1997), and
 individually marked using Visible Implant Elastomer (VIE) tags (Northwest
 Marine Technology). For each individual, we recorded a unique identifier,
 their life-stage and sex (see Group size and structure), behaviour (see
 Behavioural observations), collected fin clips (see Measuring mating and
 reproductive success) and morphological measures (see Sexual dimorphism). 
 Group size and structure: Spatial and temporal variation The number of
 individual fish in 18 spatially different groups were counted and adult
 and juvenile status were determined based on standard length (Rueger et
 al. 2016b). Maturity was determined according to standard length (Rueger
 et al. 2016b). Sex was determined by observing the distended buccal cavity
 during brooding (male) and bulging abdomen shortly before brooding
 (female) as well as pairing behaviour (Rueger et al. 2016b, 2018). Sex
 change is not known to occur in cardinalfish. A total of 168 individuals
 in 18 groups were identified as adults based on standard length with the
 remaining 211 being juveniles or subadults. Detailed demographics,
 including adult sex ratio and adult group size (number of adults per
 group), were gathered for 18 groups (Fig. 1b & c). For 10 groups
 three years of observations were available, for four groups two years of
 observations were available and for four groups only one year of
 observations was available. Reproduction was observed for 121 adult
 individuals in 16 groups; in one group reproduction was observed in all
 three years, in nine groups reproduction was observed in two years and in
 six groups reproduction was observed in only one year. To determine
 whether adult sex ratio and group size varied significantly across years
 and within groups, we conducted a linear mixed model (LMM) analysis using
 the lme4 package (Bates et al. 2015). We used adult sex ratio and group
 size as response variables in two separate models, year and group ID and
 their interaction as fixed factors and group ID as random intercept.
 Significance tests for LMMs were performed by likelihood ratio tests of
 the full model with the effect in question tested against the model
 without the effect. No obvious deviations from homoscedasticity were
 detected by visually inspecting the residual plots. No outliers or high
 variance inflation factors (VIF) were detected in any of the best-fit
 models, using performance (Luedecke et al., 2020). Conditional and
 marginal R2 were calculated using Nakagawa’s R2 in performance. All
 statistical analyses in this study were done in R version 4.0.3 (R Core
 team, 2020). Figure 1. a) Photo of a group of Sphaeramia
 nematoptera in Porites cylindrica in Kimbe Bay, Papua New Guinea (credit:
 TR). Frequency of b) mean adult sex ratio and c) mean group size for
 groups of S. nematoptera (N = 18 groups). Dashed green lines indicate
 categories used for analysis. d) Schematic of S. nematoptera, including
 location of the dorsal fin filament. Measuring mating and reproductive
 success: Genetic parentage analysis  Relative mating and reproductive
 success were measured by analyzing the parentage of embryos and matching
 them to known adult individuals in the population. A DNA sample was taken
 from each individual (N = 379) in the 18 groups by clipping their caudal
 fin using surgical scissors. The resulting fin-clips were stored in high
 grade 95% ethanol. Within the study periods, all broods were collected by
 catching brooding males (see Rueger et al. 2019 for detailed methods).
 Clutches were then subsampled for genetic analysis and individual embryos
 were stored in high grade 95% ethanol. To take into account the
 possibility of multiple mothers and fathers in each clutch, approx. 10
 eggs were sampled from different parts of each egg mass, including several
 points on the surface and the center of the congealed egg mass. A total of
 1056 embryos from 105 broods carried by 64 males were assayed.  All
 individuals were genotyped at 23 microsatellite loci with a range of 3 to
 34 alleles observed per locus. Four markers that showed high genotyping
 error (≥ 6%) were excluded from the analyses. The remaining 19 loci had an
 average genotyping error of 2.2% ± 0.4 SE. Parentage assignments were
 conducted with the software COLONY v2.0 (Jones & Wang 2010) to
 identify the most likely mother or mothers, and father or fathers, of a
 selection of eggs carried by male cardinalfish. We identified the number
 of genetic mates (mating success) and number of offspring (reproductive
 success) for each reproductive individual. Detailed methods of genotyping
 and parentage analysis as well as marker specific statistics can be found
 in Rueger et al. (2015) and Rueger et al. (2019).  Bateman gradient  We
 investigated the Bateman gradient by estimating the linear regression
 slope between mating success (number of genetic mates) and reproductive
 success (number of offspring assigned through parentage) (Lande and
 Arnold, 1983; Arnold and Wade, 1984). We compared the slopes between males
 and females collating all data for each individual for the five
 observational periods and using linear mixed model analysis (lme4 package,
 Bates et al. 2015). To explore the influence of group size and structure
 we calculated the mean number of adults for each group (Fig. 1b), and the
 mean adult sex ratio for each group (Fig. 1c) over all observational
 periods. We fit separate models for groups with female-biased (&gt;1,
 n = 6), equal (1, n = 7) and male-biased (&gt;1, n = 5) sex ratio and
 separate models for number of adults per group (group sizes were
 categorized into small (&lt;5, n = 9), medium (5-10, n =7), and large
 (&gt;10, n = 2). To account for different numbers of observations
 between individuals, we weighted the number of sampling periods that we
 recorded for each individual in the model. To account for non-independence
 of samples we included group ID as random intercept. Models were checked
 for fit and violations of assumptions as above. Reproductive variance  We
 quantified reproductive variance (variance in reproductive success as
 number of embryos assigned to each parent) for each sex per year of
 observation for the 16 groups in which reproduction was observed. We then
 used linear mixed effect models, to test for differences between males and
 females (fixed factor), using year as a random effect and weighting
 residuals by the number of reproductive success observations (number of
 clutches sampled) within a given year and sex. To explore the influence of
 group size and sex structure on reproductive variance, for each of the 16
 groups for each of the three years where reproduction was observed (number
 of groups reproducing n2012= 6; n2013= 11; n2014= 13), we assigned a
 categorical variable representing group size (small: n2012 = 2, n2013= 5,
 n2014 = 4; medium: n2012 = 2, n2013= 3, n2014 = 8; large: n2012 = 2,
 n2013 = 3, n2014 = 1), and a category for adult sex ratio (female-biased
 (&lt;1: n2012 = 1, n2013 = 4, n2014 = 3), equal (1: n2012 = 1, n2013 =
 3, n2014 = 6), male-biased (&gt;1: n2012 = 4, n2013 = 4, n2014 = 4)).
 We calculated reproductive variance for each category, sex and year and in
 separate models, we added number of adults and adult sex ratio as fixed
 factors, as well as their interaction with sex. In these models we kept
 year as random effect and weighted residuals by the number of observations
 as above. Models were checked for fit and violations of assumptions as
 above. Reproductive skew  To calculate skew in reproductive success (RS),
 we used the multinomial index implemented in the SkewCalc package (Ross et
 al. 2020). The multinomial index (M) is related to Nonacs’ binomial
 index B (Nonacs and Hager, 2011), and accounts for heterogeneity in the
 number of observational periods in which an individual was observed (Ross
 et al. 2020). We calculated M for males, females and the population as a
 whole, as well as for the different adult sex ratio and group size
 categories for each year (see Reproductive variance). Sexual dimorphism 
 Forty-seven adult S. nematoptera were caught and euthanized to extract
 their gonads and determine sex (see Rueger et al. 2018 for detailed
 methods). There were 20 females and 27 males in the sample. Their standard
 length (SL; fish length from the tip of the snout to the posterior end of
 the last vertebra excluding the length of the caudal (tail) fin) and the
 length of the dorsal filament (Fig. 1d) were measured to the nearest
 millimeter using calipers and wet weight (mass) was measured using a
 digital scale to the nearest 0.1g.  We conducted a directed comparison
 between three morphological traits, SL, mass and dorsal fin filament, that
 are: 1) highly distinguishable in this species (dorsal fin filament
 length) (Fig. 1d), and 2) commonly sexually dimorphic among other species
 (body size). While dorsal fin filament length may simply be represented by
 a univariate measurement (mm), body size, in contrast, is better
 represented as a multivariate morphological trait (Freeman and Jackson
 1990). Therefore, we first constructed a multivariate metric of body size
 by loading both SL (mm) and mass (g) into a principle component analysis
 (PCA) and extracting the first component from the PCA (the allometric size
 variable) for each individual (henceforth defined as ‘Body PC1’;
 explaining 97% of variance in SL and mass measurements). To aid in visual
 interpretation, PC1 values were normalized between 0 and 100. PCA was
 conducted in R using the function ‘prcomp’, and mass was not log
 transformed before loading into the PCA because both mass and dorsal
 filament scaled linearly (Linear Model: β = 2.96, t = 10.10, df =
 45, p &lt; 0.001, adjusted R2 = 0.687). Sexual dimorphism in body size
 was tested using a linear mixed-effects model with body size (Body PC1) as
 the Gaussian distributed response variable and sex (binomial) as the sole
 fixed effect predictor.  Sexual dimorphism in dorsal filament was tested
 using a similar linear mixed-effects model, however, body size was
 replaced with dorsal filament (mm) as the response variable (Gaussian
 distributed). To account for an influence of body size on dorsal filament
 alone, and to test for differences in allometric scaling between body size
 and dorsal filament between sexes, we included sex (binomial), body size
 (numeric), and an interaction between sex and body size as fixed effect
 predictors.  Behavioural observations During each of the five
 observational periods, all individuals from 18 groups were located every
 two to three days via visual census on SCUBA. On each day we noted whether
 males were brooding, which individuals were in pairs (see Rueger et al.
 2018 for details) and we recorded the subject and object of any aggressive
 behaviours (chases or bites) over 15-20 min. Overall, 500 hours of
 observational data on behaviours and social pair formation was collected.
 We compared the number of aggressive behaviours recorded per individual
 between males and females using a Fisher’s exact test. To determine
 whether adult sex ratio or the number of adults per group had an influence
 on the number of observed aggressions, we fitted a generalized linear
 mixed model (lme4 package, Bates et al. 2015) using a Poisson error
 distribution and fitting an observation level random effect term to
 account for overdispersion. 
description: Datafiles for “Strength and direction of sexual direction and sex roles
 vary between social groups in a coral reef cardinalfish”.
 ‘Parentage_Ruegeretal_2022.csv’ Data from molecular parentage analysis
 on Sphaeramia nematoptera embryos matched to adults in the population.
 ‘Bateman_Ruegeretal_2022.xlsx’ Number of offspring and number of mates per
 year for Sphaeramia nematoptera in different social groups.
 ‘Aggression_Ruegeretal_2022.xlsx’ Number of aggressions directed at same
 sex and opposite sex individuals. ‘Trait_Ruegeretal_2022.xlsx’ Size,
 weight and dorsal filament length data on a subset of the Sphaeramia
 nematoptera population.