Experimental evidence of resilience to size-selective harvesting in a protogynous hermaphroditic reef fish, Rhinogobiops nicholsii

Journal of Experimental Marine Biology and Ecology 525 (2020) 151320

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Experimental evidence of resilience to size-selective harvesting in a protogynous hermaphroditic reef fish, Rhinogobiops nicholsii

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Michael J. Schrama,b, , Mark A. Steelea a b

Department of Biology, 18111 Nordhoff Street, California State University Northridge, CA 91330-8303, United States of America College of Marine Science, 830 1st Street SE, University of South Florida, FL 33701, United States of America

A R T I C LE I N FO

A B S T R A C T

Keywords: Size-selective harvesting Field experiment Protogynous sex change Reproduction Growth Size structure

Most fishing methods are inherently size-selective, and many fisheries have minimum size limits, resulting in removal of the largest, most fecund size-classes of harvested species. Some modeling and correlative studies suggest protogynous (female-to-male sex changing) fishes may be particularly strongly impacted by removal of large individuals, but controlled experiments are necessary to test this expectation. By manipulating the density and size structure of populations of the blackeye goby (Rhinogobiops nicholsii) in the field via size-selective removals, this experiment aimed to identify the effects of size-selective harvesting on the (1) growth, (2) maturation, (3) sex change, and (4) reproductive output of a protogynous hermaphroditic reef fish that uses exogenous cues for sex change. Removals of ~ 25% of fish established in populations on standardized artificial reefs simulated a pulse of fishing mortality with minimum size limits, slot size limits, and maximum size limits, which are used in some fisheries. Size-selective removals had no statistically significant effects on the growth, maturation, sex change, or reproduction in blackeye gobies, indicating that this protogynous species is resilient to moderately intense size-selective removals. Some trends consistent with predicted effects of size-selective removals were present, but we had low power to detect some effects. Specifically, the rate of maturation was highest in the slot size limit treatment, and the rate of sex change was highest where large fish had been removed. Regardless of simulated fishing type, populations in all treatments exhibited similar size- and sexstructure by the end of the experiment, nearly two months after population structure had been manipulated. These results suggest that sequentially hermaphroditic fishes with exogenous cues for sex change may be able to quickly replace individuals with high reproductive value after experiencing moderately intense size-selective harvesting. However, experiments incorporating additional sex-changing species, particularly with different modes of reproduction and cues for sex change, as well as higher fishing intensities or longer durations of fishing are still necessary to reconcile differences in results reported herein and those from modeling and correlative studies.

1. Introduction Understanding how exploitation rates and management tactics interact to influence species, communities, and ecosystems is necessary for the development of effective management protocols (Dayton et al., 1995). Size-limits are commonly used to mitigate potential over-exploitation of fisheries species; however, implementation often produces disproportionate harvest pressure on specific size ranges within populations (Law, 2000; Molloy et al., 2007; Conover et al., 2009). Maximum size limits protect highly fecund adults in an attempt to maintain a large breeding stock, whereas slot size limits focus on intermediate sizes. Minimum size limits, the most commonly applied approach, ensure harvested individuals have had an opportunity to contribute to at ⁎

least one reproductive cycle, but they also promote removal of the largest, most fecund size-classes. Persistent size-selective harvesting is thought to cause populationlevel life history changes (Hamilton et al., 2007; Conover et al., 2009), which may cause dramatic changes in sustainable yield (Law, 2000; Conover et al., 2009) and local ecology (Selden et al., 2017). Observational studies have correlated persistent removal of large individuals, as the result of minimum size limits, with reductions in mean body size (e.g., Danylchuk and Fox, 1996; Brunton and Booth, 2003; Markert and Arnegard, 2007), which may also cause a directly proportional shift in size-at-maturity (Hutchings, 2005). Smaller adult body size and effective biomass ultimately reduces total reproductive potential (Conover et al., 2009), which has implications for resilience to

Corresponding author at: College of Marine Science, 830 1st Street SE, University of South Florida, FL 33701, United States of America E-mail address: [email protected] (M.J. Schram).

https://doi.org/10.1016/j.jembe.2020.151320 Received 14 July 2019; Received in revised form 5 November 2019; Accepted 14 January 2020 0022-0981/ © 2020 Elsevier B.V. All rights reserved.

Journal of Experimental Marine Biology and Ecology 525 (2020) 151320

M.J. Schram and M.A. Steele

history, and the mechanism controlling sex change (e.g., Armsworth, 2001; Alonzo and Mangel, 2004; Matthias et al., 2019). If fishing intensity is such that fertilization rates remain high, protogynous species may be less affected by fishing than gonochores (Robinson et al., 2017). However, if fishing significantly alters the sex-ratio of a target population, gamete limitation may occur (Alonzo and Mangel, 2004). The potential for stocks to significantly decline or crash is greatest if sex change is triggered by endogenous cues (e.g., fixed size or age); however, when age- or size-at-sex-change is flexible (due to use of exogenous cues for sex change), populations are able to maintain functional sex ratios at greater harvest intensities, partially mitigating losses (Hamilton et al., 2007; Mariani et al., 2013). In fact, fisheries models have demonstrated that protogynous populations may maintain higher effective biomass (Robinson et al., 2017) or population size (Waples et al., 2018) relative to gonochoric populations under similar model parameters. Experiments are necessary to establish the causal links between sizeselective removals and predicted long- or short-term changes in life history attributes and demographic rates of protogynous fishes. Such experiments are difficult with harvested species because they are mobile and slow growing, making experiments at spatially, temporally, and ecologically relevant scales difficult. This study experimentally tested the effects of size-selective harvesting on a protogynous hermaphroditic fish, the blackeye goby, Rhinogobiops nicholsii (Bean, 1882). This small, reef-associated fish has traits that make it a useful model study species. By selectively manipulating the density and size structure of blackeye goby populations, reflecting outcomes of typical fisheries management, the goals of this study were to identify how a pulse of size-selective harvesting influences the (1) growth, (2) maturation, (3) sex change, and (4) reproductive output of a protogynous hermaphrodite with exogenous cues for sex change.

further exploitation or stochastic environmental variability. Many commercially and recreationally important species are hermaphroditic (Alonzo and Mangel, 2004; Robinson et al., 2017; Matthias et al., 2019), and most of these species are sequential hermaphrodites, meaning individuals change from one sex to the other at some point in their life. The mechanism governing sex change may be endogenous, whereby individuals change sex at a specific size or age, or exogenous, whereby sex change is triggered by relative body size, potential fitness, and behavioral interactions (Ghiselin, 1969; Warner et al., 1975; Muñoz and Warner, 2003). Regardless of how sex change is controlled in sequential hermaphrodites, one sex achieves larger body size than the other, and typically the sex ratios are skewed. Thus, size-selective mortality can further bias already skewed sex ratios, possibly reducing the reproductive potential of the population. Many protogynous (female-first) hermaphroditic fishes have sizebased social dominance hierarchies, reinforced through behavioral interactions among conspecifics, which influence individual growth and reproductive potential (Ross, 1990; Munday et al., 2009). Males of protogynous species exhibiting polygyny maintain dominance over a harem through agonistic behaviors that ward off competitors and inhibit growth in subordinates that could otherwise grow and compete (Koebele, 1985; Munday et al., 2009). In species with exogenous cues for sex change, the loss of a harem's male typically triggers sex change in one of the largest remaining females (Cole and Shapiro, 1995; Munday et al., 2009), although smaller females sometimes change sex instead (Muñoz and Warner, 2003). During this period, female growth rates are expected to increase, due to lack of social suppression, allowing newly sex-changed males to more effectively dominate subordinates, defend harems, and ward off competitors (Munday et al., 2009). The system therefore favors individuals who reach competitively effective sizes quickly and change sex to male (Munday et al., 2009; Walker and McCormick, 2009). However, minimum size limits in fisheries create an artificial counter pressure against those same sizeclasses and further skews an already skewed sex ratio (Hamilton et al., 2007; Molloy et al., 2008; Matthias et al., 2019). The duration and intensity of size-selective harvesting (i.e., press vs. pulse fishing) may influence how sex-changing populations respond. Conover et al. (2009) showed that persistent size-based mortality over several generations in experimental populations of the gonochoric Atlantic silverside (Menidia menidia) resulted in irreversible, presumably genetically-based, life history changes. Conversely, Hamilton and Caselle (2014) observed that cessation of long-term size-selective harvesting in populations of the protogynous California sheephead (Semicossyphus pulcher) resulted in relatively quick reversal of fishinginduced life history changes. These findings suggest that persistent removals have the potential to induce evolutionary responses in harvested populations, but the extent of those effects is dependent upon species' ecology and life history. In contrast, pulse fishing, e.g., as a result of short seasons or rotational closures (e.g., 5-day Atlantic red snapper season; NOAA, 2018), often result in relatively high-intensity, short-duration harvesting. Coupled with size-based regulations, pulse fishing has the potential to induce significant changes to population demography and ecology by altering population size structure, sex ratio, and effective spawning stock in a relatively short period of time. The sudden removal of large numbers of large individuals in protogynous fishes would relieve socially derived growth repression (Koebele, 1985; Cole and Shapiro, 1995; Munday et al., 2009), which could allow for increased growth rates and potentially stimulate sex change in subordinate individuals. This compensatory response could result in the replacement of individuals with high reproductive value relatively quickly, partially mitigating predicted negative impacts of size-selective fisheries, provided harvest intensity is not so high that few adults remain. Fisheries models for sex-changing species generally agree that the effects of size-selective harvesting on populations of protogynous species are a complex function of fishing intensity and selectivity, life

2. Materials and methods 2.1. Study species The blackeye goby (Rhinogobiops nicholsii) is a small bottom dwelling fish that is found on rocky reefs from British Columbia, Canada to central Baja California, Mexico (Miller and Lea, 1972; Love, 2011). It is a protogynous hermaphrodite, which settles to rocky reefs at 15–25 mm standard length (SL) (Steele unpublished data) after a planktonic phase averaging ~ 60 days (Block, 2011). Fish mature two to three months after settlement at ~ 45 mm SL (Wiley, 1973), with most maturing as females, but some maturing as males that changed sex prior to maturity (Kroon, 1997). Sex change from mature female to male occurs across a broad range of sizes, documented from 58 to 85 mm SL (Wiley, 1973; Cole, 1983; Kroon, 1997). The primary cue for sex change in the blackeye goby is size relative to other females, and sex change occurs even when males are present (Cole, 1983; Kroon, 1997). Males attain larger sizes than females, and the largest size classes of populations (≥ 80 mm SL) are composed mostly of males (Wiley, 1973; Cole, 1983). Densities of adult blackeye gobies vary widely, but ~ 6 individuals per m2 are commonly found (Cole, 1984). Sex ratios tend to be female biased, with 1.7 to 4 females per male, though areas with abundant nesting sites can have unbiased sex ratios (Wiley, 1973; Breitburg, 1987). Peak spawning is speculated to occur between February and October in Southern California, where the present study was conducted (Wiley, 1973; Love, 2011), but spawning can occur year-round (Schram and Steele, 2016). Males establish nests within their territory by burrowing under a rock where females lay adhesive eggs on the rock ceiling (Ebert and Turner, 1962; Cole, 1984). Nesting males defend a brood of eggs laid by up to 6 females (Love, 2011) until they hatch. Incubation time is between 1 and 2 weeks (Schram and Steele, 2016). Both sexes defend territories. Nesting or not, most males (~ 80%) defend territories of up to 0.5 m2 in area, which are imbedded within 2

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home ranges of up to 1.2 m2 (Kroon et al., 2000). About one-third of mature females defend territories, too, and in both sexes, defense of territories occurs year round (Cole, 1984; Kroon et al., 2000). Territory size is positively correlated with body size, and larger individuals predictably win agonistic interactions, resulting in a stable social structure that is dominated by large individuals (Cole, 1984; Kroon et al., 2000).

2.3. Experimental methods From June to October 2013, we conducted a field experiment to evaluate the impacts of size-selective harvesting on populations of blackeye gobies on 20 rock rubble reefs constructed in Big Fisherman Cove, Santa Catalina Island, California (33°26′42”N, 118°29′8”W). Experimental reefs were arranged in two parallel lines of 10 reefs at ~ 10 and 13 m depth, with reefs separated by 10 m to limit migration among them. Reefs were 2.25 m2, built of ~ 60 L rocks ranging in size from 5 to 40 cm (see Schram and Steele, 2016 for additional details and photographs). To maximize habitable space, rocks were arranged in four interconnected piles, one in each corner of the 1.5 × 1.5 m area. Three artificial nest sites were placed on each reef, near the interior edges of the rock piles. These were made from inverted terracotta potting saucers (one of each of three sizes per reef: 20, 23, and 25 cm in diameter) with an opening cut along one side. Saucers were readily adopted as nests by blackeye gobies and were easily accessible by SCUBA divers, minimizing disturbance of the overall reef structure when checking for eggs. There was no evidence of eggs being laid anywhere else on the reefs during the study period. Piscivorous fishes were abundant at the study site, so exclosure cages (3.8 cm plastic mesh) were placed around each reef to mitigate unintended mortality. It took several weeks to establish similar populations of blackeye gobies on all 20 reefs. Once that was achieved, the populations were monitored for 3 weeks prior to imposing the experimental removal treatments. Reproductive output was measured for an additional 5 weeks, and somatic growth, maturation, and sex change were assessed 7.5 weeks after the removal treatments were imposed. Blackeye gobies placed on the experimental reefs were collected by SCUBA divers with hand nets from natural reefs 1–2 km away. In the laboratory, the sex and maturity of each fish was determined via genital papillae (Wiley, 1973; Cole, 1983) and standard length (mm SL) was measured. Individuals were injected with two subcutaneous, colored, elastomer tags (Northwest Marine Technology Inc.) indicating sex/maturity (one tag, green for juvenile, red for female, or blue for male) and size to the nearest mm SL (the other tag) based on color (4 colors) and position along the dorsal surface (10 possible positions for size, plus one for sex; see Schram, 2014 for a photograph). We began establishing resident populations on all the experimental reefs on 14 June, but based on regular surveys by divers, we did not consider them to be established until 21 July. Initially stocked densities were 11 ± 2 (mean ± 1 SD) blackeye gobies per 2.25 m2 reef, comprised of 2 ± 1 males (50–80+ mm SL), 6 ± 1 females (40–79 mm SL), and 4 ± 2 juveniles (10–49 mm SL). Many gobies, however, were lost to predators and several moved to different reefs. Such movements are not uncommon when blackeye gobies are initially stocked on reefs but seldom occur after they have established themselves on a reef. Additional gobies were collected and placed on the experimental reefs to achieve the desired densities and size, sex, and maturity distributions. Predator exclosure cages were placed on reefs on 19 July to limit predation on gobies. Populations were considered established on 21 July and we refer to this date as day 0 of the experiment. Three weeks later (12–14 August), the size-based removal treatments simulating fishing with different size limits were imposed. The populations were left undisturbed for 3 weeks prior to these removals to ensure that fish placed on the reefs had in fact established themselves as residents and had developed stable social hierarchies. At week 3, prior to imposing the treatments, average densities were 20 ± 4 (mean ± 1 SD) blackeye gobies per 2.25 m2 reef, comprised of 6 ± 2 males, 8 ± 3 females, and 6 ± 3 juveniles. This density and size-distribution reflected what was present on natural reefs at the collection sites based on surveys of them. It was higher than what was initially placed on reefs because additional gobies were added after the first round of reef stocking, and there was some immigration of larger individuals as well as recruitment of juveniles to the reefs. Size-based removal treatments were established by removing a

2.2. Experimental design Four treatments were used, a control to represent no fishing, and three size-based removal treatments simulating a pulse of fishing with different types of size limits. There were 5 replicates of each treatment. The removal treatments simulated fishing with a minimum size limit (only fish > 75 mm SL could be harvested), a maximum size limit (only fish < 40 mm SL could be harvested), and a slot size limit (only fish 40–75 mm SL could be harvested). Because protogynous species typically exhibit size-structured sexual dimorphism, removal of large individuals, as might occur in a fishery with minimum size limit, primarily reduced the density of males. Removal of medium sized individuals, as might occur in a fishery with a slot size limit, primarily reduced female densities. Removal of small individuals, as might occur in a fishery with a maximum size limit, primarily reduced densities of juveniles and small females. For simplicity, hereafter we refer to the removal treatments as Large Removal (removals of large fish, simulating a minimum size limit), Medium Removal (simulating a slot size limit), and Small Removal (simulating a maximum size limit). A typical target for managed fisheries is harvesting down to a density of 50% of carrying capacity, the theoretical point of maximum sustainable yield (Levinton, 2009), or lower. But due to the relatively small population sizes on the experimental reefs in this study (see next section), reductions of only ~25% were made to avoid having too few fish remaining per experimental reef to obtain estimates of demographic rates. If 25% reductions of populations were sufficiently large to affect demographic rates, we expected the following differences among treatments (summarized in Table 1). (1) For growth, we anticipated removal of large fish (“minimum size limit”) would increase average growth rates the most because large individuals socially dominate smaller ones, especially large females that might otherwise change sex (and grow rapidly), and because density-based removals result in a larger removal of biomass when large individuals are removed. (2) For maturation, we expected that removals of medium size individuals (“slot limit”) would increase maturation rates the most because mature females in this size-class socially repress the next size class down the most, and these are maturing juveniles. (3) For sex change, we expected the removal of large individuals would increase the rate of sex change the most because these large fish were mostly male, thus freeing up opportunities for females to assume male function. (4) We expected removal of large fish would cause the largest reduction in reproductive output because this size class was composed of both large, highly fecund and socially dominant females, and large males, perhaps leaving too few males to provide sufficient nests for females to deposit eggs in.

Table 1 Predicted differences among treatments for each response variable, assuming removal treatments were large enough to generate a response. Treatments were control (C), large removal (LR; “minimum size limit”), medium removal (MR; “slot size limit”), and small removal (SR; “maximum size limit”). Response variable

Predicted differences

Somatic growth rate Maturation rate Sex change rate Reproductive output

LR > MR > SR ≈ C MR > SR > LR > C LR > MR ≈ SR ≈ C C > SR > MR > LR

3

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protogynous species, the growth of different size-classes, sexes, or stages (e.g., juvenile vs. adult) might respond differently to the treatments. Therefore, a linear mixed effects model (LMM) was used to test whether manipulations affected the growth rates of males, females, or juveniles differentially. Average per capita growth rate was the response variable and predictor variables were treatment (fixed effect), initial sexual state (fixed effect), and reef (random effect). Growth data also met parametric assumptions of normality and homogeneity of variance. Differences among treatments in rate of maturation or sex change were tested with generalized linear mixed effects models (GLMM) with binomial error. Of the recovered fish, state change was recorded as a binary response (yes/no) as determined by genital papilla morphology at the end of the study period compared to their initial tagging as juvenile (for maturation) or female (for sex change). Maturation and sex change were tested in separate models with treatment (fixed effect) and reef (random effect) as predictor variables. Differences in final population structure of treatment groups were tested with multivariate two-way permutational MANOVA (PERMANOVA). Treatment group and size class (10-mm SL increments) were predictor variables and the abundance of males, females, and juveniles were the multivariate response variables. A non-significant interaction between treatment and size class would indicate that the different treatments had similar population structures, i.e., similar densities of males, females, and juveniles in each size class. Differences in egg production among size-selective removal treatments were tested with repeated-measures PERMANOVA (RM-PERMANOVA). Reefs were treated as replicates and the response variable was the number of new eggs produced per week with treatment as the categorical variable. Only data from the 5 weeks following treatment implementation were included in the analysis and those data met the assumption of homogeneity of multivariate dispersion for treatment but failed to meet this assumption for week, however, PERMANOVAs with a balanced design are robust to this violation (Anderson, 2017). Distribution-free permutational analyses were used because they alleviate the parametric assumption of normality, which is commonly not met by ecological data. Permutational P values were based on 10,000 permutations of the raw data without regard to treatment to generate a random distribution of test statistics to which the actual test statistic was compared. All analyses were conducted in MATLAB R2018a and using functions from the Fathom Toolbox (Jones, 2017).

predetermined number of individuals in the appropriate size class from each reef. To ensure all blackeye gobies, including those on control reefs, were subjected to the same handling stress, all gobies on each reef were captured by divers using hand nets. All fish were measured and sexed and those to be removed were retained and later released several km away from the artificial reefs. The blackeye gobies to remain on the reefs were re-tagged if they had grown, changed sex, or matured since being stocked. Juveniles that naturally recruited to the reefs and untagged immigrants that were desired on the reefs to achieve the intended density and size/sex/maturity structure were also tagged. This was all done underwater by SCUBA divers. To investigate population structure on each reef, population densities on the experimental reefs were visually estimated regularly. Following the implementation of treatments, reef surveys occurred every other day. Once it was determined reef populations were stable (~ 5 days post treatment implementation), survey frequency was reduced. Blackeye gobies are fairly cryptic and often hide in burrows and crevices on the reef. Therefore, time-averaged densities were used to estimate the number of individuals tagged as males, females, and juveniles present on each reef during the study period. Each experimental reef was visited independently by two divers during each survey, and presence/absence was recorded using the tags of fish, which were visible to divers. If an individual was seen during two consecutive observations, it was assumed to have been present on all days between the observation dates. If a fish was present during one observation and absent during the next, it was counted as present on half of the days between the two observations. If there were gaps between sightings of a fish, it was assumed to have been present during the gap but missed by the observers. Time-averaged densities were calculated as the sum of all days present divided by the duration of the experiment. Reproduction of blackeye gobies was measured weekly for 8 weeks, 3 weeks before and 5 weeks after removal treatments were imposed, following the methods detailed in Schram and Steele (2016). The three weeks pre-manipulation were used to ensure individuals were reproductively active, whereas the five weeks after were used to test the effects of the removal treatments on reproductive output. All 3 nesting saucers on each reef were checked weekly and all broods of eggs were photographed and the total number of new eggs produced on each reef was determined through digital analysis of photographs. Seven and a half weeks after the removal treatments were implemented (3–5 October), all remaining fish were collected and remeasured. Length measurements (mm SL) from the day removal treatments were imposed and the day remaining fish were collected at the end of the experiment were converted to mass using the following length-to-weight equation:

Mass (g) = 0.000019•SL (mm)

3. Results Size-selective removal treatments were successful in altering the structure of the blackeye goby populations as expected (Fig. 1). Throughout the experimental period, time-averaged total densities in the three size-selective removal treatments were ~ 25% lower than those in the control group (control = 19.90 ± 1.50, large removal = 15.05 ± 1.00, medium removal = 15.28 ± 1.12, and small removal = 13.40 ± 1.24, mean ± 1SE blackeye gobies per 2.25 m2 reef; Fig. 2) and the treatments were statistically distinct (MANOVA, F9,34 = 4.45, p = .001). Over the 7.5 weeks after treatments were implemented, time-averaged densities of fish initially tagged as male were significantly lower in the large removal treatment compared to all other treatments (F1,16 = 16.71, p = .001), as were densities of fish initially tagged as female in the medium removal treatment (F1,16 = 7.17, p = .02), as well as densities of fish initially tagged as juveniles in the small removal treatment (F1,16 = 6.82, p = .02). Despite effectively manipulating the size structure and density on the reefs in the size-selective removal treatments, there was little evidence that these differences affected the populations that remained on the reefs. Growth rates of individuals (g•day−1) did not differ among the four treatments (Fig. 3) or among sexual states (Fig. 4; LMM, Treatment x Sexual State: F6,185 = 0.77, p = .59, Treatment: F3,185 = 1.00, p = .39, Sexual State: F2,185 = 1.97, p = .14). As

2.9643

(n = 118, r2 = 0.97, p < .0001, Schram unpublished data). Growth was estimated as the difference between initial and final mass and expressed as daily per capita growth (g•day−1). Genital papillae were also re-inspected to determine which individuals had matured or changed sex. 2.4. Statistical analyses To determine whether the population manipulations were effective in establishing distinct treatments, multivariate analysis of variance (MANOVA) was used to compare the time-averaged densities of fish initially tagged as males, females, and juveniles present on each plot. Univariate a priori comparisons were also used to verify specific differences in the abundance of targeted sizes among the three removal treatments and control group (e.g., lower abundance of males in the large removal treatment relative to all other groups). These data met the assumptions of normality and homogeneity of variance. Because growth regulation is socially influenced in many 4

Journal of Experimental Marine Biology and Ecology 525 (2020) 151320

M.J. Schram and M.A. Steele

Fig. 1. Population size, sex, and maturity structures of blackeye gobies in each of three removal treatments and one control group, pooled across replicate reefs (n = 5 per group). Left panels show population structures after stocking experimental plots but prior to size-based removals. Middle panels show population structures 2–4 days after size-based removals. Right panels show population structures ~52 days after removals.

roughly half that present at the start of the experiment (20 ± 4). The average number of recovered individuals initially tagged as male (3 ± 2), female (5 ± 2), and juvenile (2 ± 1) were low relative to starting numbers (6 ± 2, 8 ± 3, and 6 ± 3, respectively). These low numbers of individuals at the end of the study limited the statistical power of our tests for differences among treatments for growth, maturation, and sex change. Control populations tended to have the highest reproductive output, about twice that of the three size-selective removal treatments, which were similar to one another, but this difference was not statistically significant (RM-PERMANOVA, Treatment: F3,16 = 0.57, p = .66). This trend was largely driven by reproduction in a single week (Fig. 7). Because of large variability of reproductive output among replicate reefs and among weeks, the power of this test was obviously low, with a ~30% chance to detect a 50% difference in reproductive output among

predicted by the disruption of the social structure, more juveniles matured in the medium removal treatment than in the other three treatments (Fig. 5); and similarly more females changed sex in the large removal treatment (Fig. 6), but neither of these trends were statistically significant (GLMM, Maturation: F3,31 = 1.10, p = .37; Sex Change: F3,95 = 0.82, p = .48). By the end of the experiment, the size and sex/maturity structure of the populations in the different size-removal treatments had converged on the control treatment (PERMANOVA: Treatment x Size Class: F15,96 = 0.94, p = .54, Fig. 1: “Final”). Although predator exclosure cages were used, population densities on the experimental reefs declined over the course of the experiment, presumably due to predation and migration, reducing the number of individuals that remained on reefs at the end of the study. The average number of individuals recovered per reef at the end of the study (10 ± 3, mean ± SD) was 5

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Fig. 4. Per capita growth of three classes of blackeye goby on replicate reefs in each of three removal treatments and one control group. Growth rates did not differ significantly among the three sex/maturity classes or among groups (see Results). Means ± 1SE are shown, based on n = 5 reefs for each group.

Fig. 2. Time-averaged densities of each sex/stage of blackeye goby in populations on replicate artificial reefs during the 7.5 weeks following removal treatments. Population structures differed among the density manipulations as expected. Specifically, male densities were significantly lower in the large removal (“minimum size limit”) group compared to all other treatments, as were female densities in the medium removal (“slot size limit”) group, and juvenile densities in the small removal (“maximum size limit”) group (see Results). Error bars represent ± 1SE based on n = reefs 5 for each bar.

selective removals on the growth, maturation, incidence of sex change, or reproductive output of the protogynous blackeye goby. This result indicates that this species may be resilient to pulse removals of moderate intensity (i.e., ~ 25%) that are size selective, or that our experiment lacked sufficient statistical power to detect biologically meaningful differences among treatments. Demographic rates of natural populations are often spatiotemporally variable, responding to changes in the biotic and abiotic conditions of their respective habitats (Gust et al., 2002; Figueira et al., 2008). Coupled with limited numbers of individuals on our experimental reefs (which reflected natural densities), our power to detect statistically significant changes in demographic rates was relatively low. For example, estimates of the rates of maturation and sex change on a reef were often based on only a few individuals, limiting the statistical power to detect differences in the binary response. Similarly, high variability in reproductive output of egg-spawning fishes is typical (Lowerre-Barbieri et al., 1996; Karjalainen et al., 2016), and cyclic patterns in some demersal spawners, caused by nesting preferences associated with egg presence (Sikkel, 1989; Knapp et al., 1995) could limit the power of tests of factors affecting reproductive output. Consequently, our experiment only had a ~30% chance to detect a 50% change in reproductive output among treatments. Limited statistical power may have compromised our ability to detect effects of size-selective removals on growth, maturation rate, and sex change rate, but even if so, the effect sizes were relatively small. Mean growth rates were nearly identical among treatments. For maturation rate, the general pattern, with highest maturation rate on reefs where medium size fish (predominately mature females) were removed was consistent with our expectations, but rate of maturation was not very different in control populations (~ 90% vs. 70% of juveniles matured, respectively). The pattern of the highest rate of sex change occurring in the large removal treatment was consistent with our predictions, but inconsistent with our predictions was that it was only slightly higher than in control populations. The lowest sex change rates were in the small and medium removals. If this non-significant pattern reflects an actual biologically meaningful difference, it may indicate that sex change in the study species is at least partly triggered by the presence of smaller individuals in populations – perhaps potential future mates. Future experiments with greater replication may be able to shed light on these possibilities, but they will be logistically very

Fig. 3. Per capita growth of blackeye gobies on replicate reefs in three removal treatments and one control group. Growth rates did not differ significantly among groups (see Results). Means ± 1SE are shown, based on n = 5 reefs for each group.

treatments. Reproductive output differed significantly among weeks (RM-PERMANOVA, Week: F4,64 = 4.15, p < .01). Permutational pairwise comparisons with a Bonferroni correction indicated that the reproductive output during week four (the first week following removals), was significantly lower than in weeks six, seven, and eight (all corrected p ≤ .03). There were no other significant pair-wise differences between weeks (all p > .42). 4. Discussion This study revealed no statistically significant effects of size6

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Fig. 5. Proportion of juvenile blackeye gobies that matured per replicate reef in each of three treatments and one control group during the 52-day-long period. Differences among groups were not statistically significant (see Results). Means ± 1SE are shown, based on n = 5 reefs for each group.

Fig. 7. Number of eggs produced per reef each week by blackeye gobies in each of three different size-selective removal treatments and one control group. The treatments were implemented after 3 weeks. Differences among groups were not statistically significant but were statistically significant among weeks (see Results). Means ± 1SE are shown, based on n = 5 reefs per group.

ecological effects of size-selective harvesting on protogynous fishes. Our findings of limited effects of size-selective removals on demographic rates of a protogynous fish are at odds with prior observational studies on protogynous hermaphrodites, which revealed patterns consistent with impacts of size-selective harvesting practices on size-atmaturity, size-at-sex-change, and altered growth rates (e.g., Hutchings, 2005; Hamilton et al., 2007; Caselle et al., 2011). The majority of those prior studies, however, focused on populations under persistent harvest pressure (i.e., press fishing), and possibly greater reductions in population density. Our study tested the short-term ecological effects of moderately intense, one-time size-selective removals. Some models predict that the demographic rates of sex-changing populations may not be affected much by size-selective removals if the sex ratios are not altered greatly (Alonzo and Mangel, 2004; Robinson et al., 2017), or if the altered sex ratio is within a range in which sufficient males still remain to fertilize eggs effectively (Easter and White, 2016). Sex-ratios may not be affected much by size-selective harvesting if individuals occupying the larger, terminal sex, escape harvest, which would be important for species with fixed age- or size-at-sex-change (endogenous control), or by flexibility in age- or size-at-sex-change (exogenous control). Results of our moderate intensity removals tend to agree with model predictions for scenarios in which sex ratios are within a range with sufficient males remaining to effectively fertilize eggs. However, a broader range of harvest intensities and durations (press instead of pulse) is still necessary to address the numerous outcomes proposed by fisheries models. Many fisheries aim to reduce population densities by 50% (Levinton, 2009), the point at which the theoretical maximum sustainable yield can be achieved, but often they reduce populations by much more than 50%, commonly by as much as 90% (Myers and Worm, 2003; Conover et al., 2009). Reductions of that magnitude, regardless of persistence, coupled with size-selective practices would be expected to have greater impacts on harvested populations than did the 25% removals imposed in the present experiment. Indeed, recent unpublished work on the blackeye goby using higher removal intensities (≥ 50%) has documented impacts on reproductive output, sex change, and growth (Adreani et al., unpublished data), possibly indicating a tipping point beyond which flexible age- and size-at-sex-change cannot

Fig. 6. Proportion of female blackeye gobies per replicate reef that changed sex to male in each of three treatments and one control group over the 52-day duration. Differences among groups were not statistically significant (see Results). Means ± 1SE are shown, based on n = 5 reefs for each group.

challenging to do. Our findings providing some insight into the magnitude of effects of size-selecting fishing on protogynous species. Much of what is known regarding fishery effects on populations of protogynous species are derived from observational studies, which typically rely on long-term before-after-control-impact type datasets, and fisheries models, which often include incomplete life history or demographic data. Our study, which took a controlled manipulative approach, was, to the best of our knowledge, one of the first attempts at addressing the causal mechanisms that govern how populations of sex-changing species respond to size-selective harvesting. Furthermore, our study illustrates an experimental approach that we hope will be expanded on to better resolve the 7

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control access to nest sites. It also is a demersal spawner, in contrast to fished protogynous species, which are broadcast spawners, many of which spawn in large aggregations (Aguilar-Perera and Aguilar-Dávila, 1996; Coleman et al., 1996). Additionally, cues for sex change in the blackeye goby are largely exogenous – specifically, being one of the largest females in a population cues sex change (Cole, 1983). In some fished species, the cue for sex change is largely or exclusively endogenous: body size (McGovern et al., 1998; Heppell et al., 2011). Models predict that species with endogenous cues for sex change will be more severely impacted by fishing than those using exogenous cues (Alonzo and Mangel, 2005; Easter and White, 2016). Overall, our study suggests that some protogynous hermaphroditic species may be fairly resilient to the impacts of a bout of size-selective harvesting; however, that resilience is probably dependent on a number of factors. Particularly for species that change sex based on exogenous cues, the loss of large individuals from populations may be quickly compensated for by sex change by large females, or by males already present but that had been socially suppressed from reproducing by larger, dominant males. Overall, the extent to which protogynous fishes can buffer impacts or recover upon cessation of fishing is likely a function of harvesting history, relative body size, life history, and population demography. Studies that explore the factors that cause a loss of such resilience to fishing, such as harvesting intensities and details of the reproductive ecology, may provide valuable insight into the most effective management strategies for protogynous species.

compensate. The apparent resilience of blackeye gobies to simulated size-selective harvesting may also be explained in part by their social structure or female mate choice. In the blackeye goby, it seems that a single male can maintain exclusive access to a harem of females. All of our treatments had at least two males per reef. Yet despite there being more than one male on all reefs, when eggs were present, they were typically only in a single nest, suggesting either that a single male can socially dominate an entire 2.25 m2 reef or that all females on a reef chose to mate with a single male. Cole (1984) reported that even the largest, presumably male, blackeye gobies have areas of activity that are < 1.0 m2, suggesting that a single dominant male should not be able to socially dominate an area of 2.25 m2. Indeed, on rare occasions, two or three nests with eggs were found on individual reefs and these nests appeared to be guarded by different males (authors' personal observations). Why more than one active nest per reef was not present more often is unclear. Averaged over the 5 weeks after the size-selective removals were made, reproductive output in our control group was nearly twice as high as that of the three size-selective removal treatments, but this difference was not statistically significant, and was largely driven by a single week's high reproductive output on control reefs. Reproduction was otherwise relatively similar and stable across treatments despite the alteration of densities and population structure by our treatments. Notably, reproductive output was very similar in the small fish removal treatment (which removed no adult males and only removed some smaller females, plus juveniles) and the medium and large removal treatments (in which only adults were removed), suggesting that the experimental populations had sufficient adults to buffer the loss of up to ~ 50% of males (large removal) or ~ 30% of females (medium removal). Even immediately after removals, all populations had at least one adult male and a few adult females. Nevertheless, reproductive output on all reefs was lowest during the week immediately following removals. This drop in reproduction may have more than one cause. One possibility is that the handling of fish induced a stress response that altered reproductive activity. Another possibility is that the sudden, albeit incomplete, disruption of the social hierarchy temporarily affected reproductive activity until functional social hierarchies were reestablished. This possibility seems unlikely because the drop in reproductive output was also seen in the control treatment, in which no individuals were removed. Another possibility is that physical disruption of the reefs to facilitate capture of the gobies caused the fish to reestablish territories and home ranges, which took time. Yet another possibility is that some environmental trigger caused reproduction to drop off. This possibility is supported by the generally low reproductive output on all reefs in the week prior to removals. The rapid recovery of our experimental populations to size structures and sex ratios similar to control populations may also explain, in part, why differences in growth rate among size-selective removal treatments were not detected in the present study. For many harem forming protogynous hermaphrodites, social structure heavily influences behavioral dynamics and resource availability and acquisition (Ross, 1990; Walker and McCormick, 2009). Although our removals were effective at altering population structure, as noted previously, the moderate intensity of our removals did not necessarily interrupt the entire social hierarchy. With only 25% of the fish being removed, and some remaining in all of the size classes, the general structure of the social hierarchy may have remained largely intact, maintaining some socially derived growth repression. It may require a larger fraction of the population be removed to affect socially controlled demographic rates more strongly. Although the blackeye goby is a useful model species because it is small, abundant, has a small home-range, and its reproduction is relatively easy to measure, thus facilitating field experiments, it differs from most harvested protogynous species in several potentially important ways. It is a pair-spawning species in which a dominant male can

Declaration of Competing Interest None. Acknowledgements We thank S. Ranson for field assistance. We appreciate the logistical support provided by the staff of the University of Southern California Wrigley Marine Science Center (WMSC) field station. M. Adreani, L. Allen, S. Hamilton, S. Pang, and J. White provided helpful discussion or comments on drafts of this manuscript. We appreciate the helpful comments of two anonymous reviewers. This research was supported by funding from CSU Northridge Association of Retired Faculty, CSUN Graduate Thesis Support, the CSUN Peter Bellinger student research award, CSU-COAST, Sigma XI, the USC Rose Hills Foundation Summer Fellowship, and the National Science Foundation (OCE-1437571). This is contribution number 298 of the CSUN Marine Biology program and 255 of the WMSC. References Aguilar-Perera, A., Aguilar-Dávila, W., 1996. A spawning aggregation of Nassau grouper Epinephelus striatus (Pisces: Serranidae) in the Mexican Caribbean. Environ. Biol. Fish 45, 351–361. https://doi.org/10.1007/BF00002527. Alonzo, S.H., Mangel, M., 2004. The effects of size-selective fisheries on the stock dynamics of and sperm limitation in sex-changing fish. Fish. Bull. 102, 1–13. Alonzo, S.H., Mangel, M., 2005. Sex-change rules, stock dynamics, and the performance of spawning-per-recruit measures in protogynous stocks. Fish. Bull. 103, 229–245. Anderson, M.J., 2017. Permutational multivariate analysis of variance (PERMANOVA), in: Balakrishnan, N., Colton, T., et al. (Eds.), Wiley StatsRef: statistics reference online. Am. Cancer Soc. 1–15. https://doi.org/10.1002/9781118445112.stat07841. Armsworth, P.R., 2001. Effects of fishing on a protogynous hermaphrodite. Can. J. Fish. Aquat. Sci. 58, 568–578. https://doi.org/10.1139/f01-015. Block, H.E., 2011. Selection on Larval Traits in Early Post-Settlement Temperate and Tropical Reef Fishes. Master of Science thesis. Breitburg, D.L., 1987. Interspecific competition and the abundance of nest sites: factors affecting sexual selection. Ecology 68, 1844–1855. Brunton, B.J., Booth, D.J., 2003. Density- and size-dependent mortality of a settling coralreef damselfish (Pomacentrus moluccensis Bleeker). Oecologia 137, 377–384. https:// doi.org/10.1007/s00442-003-1377-2. Caselle, J.E., Hamilton, S.L., et al., 2011. Geographic variation in density, demography, and life history traits of a harvested, sex-changing, temperate reef fish. Can. J. Fish. Aquat. Sci. 68, 288–303. https://doi.org/10.1139/F10-140. Cole, K.S., 1983. Protogynous hermaphroditism in a temperate zone territorial marine goby, Coryphopterus nicholsi. Copeia 1983, 809–812.

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