==== Front PLoS One PLoS One plos PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0287843 PONE-D-23-01131 Research Article Research and Analysis Methods Research Facilities Mesocosms Research and Analysis Methods Animal Studies Experimental Organism Systems Animal Models Xiphophorus Biology and Life Sciences Organisms Eukaryota Animals Vertebrates Fish Osteichthyes Xiphophorus Biology and Life Sciences Zoology Animals Vertebrates Fish Osteichthyes Xiphophorus Biology and Life Sciences Genetics Biology and Life Sciences Psychology Behavior Animal Behavior Animal Sexual Behavior Social Sciences Psychology Behavior Animal Behavior Animal Sexual Behavior Biology and Life Sciences Zoology Animal Behavior Animal Sexual Behavior Biology and Life Sciences Evolutionary Biology Population Genetics Genetic Polymorphism Biology and Life Sciences Genetics Population Genetics Genetic Polymorphism Biology and Life Sciences Population Biology Population Genetics Genetic Polymorphism Biology and Life Sciences Population Biology Population Metrics Death Rates Biology and Life Sciences Biochemistry Hormones Lipid Hormones Cortisol Biology and Life Sciences Biochemistry Hormones Steroid Hormones Cortisol Biology and Life Sciences Cell Biology Cellular Types Animal Cells Germ Cells Sperm Genotype and growth rate influence female mate preference in Xiphophorus multilineatus: Potential selection to optimize mortality-growth rate tradeoff Genotype and growth rate influence female mate preference in Xiphophorus multilineatus https://orcid.org/0000-0001-8043-9168 Fitschen-Brown Meredith Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing – original draft Writing – review & editing 1 2 * Morris Molly Conceptualization Data curation Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Writing – original draft Writing – review & editing 1 2 1 Department of Biological Sciences, Ohio University, Athens, OH, United States of America 2 The Ohio Center for Ecological and Evolutionary Studies, Ohio University, Athens OH, United States of America Tuckett Quenton M. Editor University of Florida, UNITED STATES Competing Interests: The authors have declared that no competing interests exist. * E-mail: mf854617@ohio.edu 29 6 2023 2023 18 6 e028784313 1 2023 13 6 2023 © 2023 Fitschen-Brown, Morris 2023 Fitschen-Brown, Morris https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The extent to which mate preferences are adaptive requires a better understanding of the factors that influence variation in mate preferences. Xiphophorus multilineatus is a live-bearing fish with males that exhibit alternative reproductive tactics (courter/sneaker). We examined the influence of a female’s genotype (courter vs sneaker lineage), growth rate, and social experience on mate preference for courter as compared to sneaker males. We found that females with a sneaker genotype and slower growth rates had stronger mate preferences for the faster growing courter males than females with a courter genotype, regardless of mating experience with one or both types of males. In addition, the relationship between strength of preference and growth rate depended on a females’ genotype; females with sneaker genotypes decreased their preference as their growth rates increased, a pattern that trended in the opposite direction for females from the courter genotypes. Disassortative mating preferences are predicted to evolve when heterozygous offspring benefit from increased fitness. Given male tactical dimorphism in growth rates and a mortality-growth rate tradeoff previously detected in this species, the variation in mating preferences for the male tactics we detected may be under selection to optimize the mortality-growth rate tradeoff for offspring. Ohio University Baker Fund Morris Molly This project was supported in part by the Ohio Center of Ecological and Evolutionary Studies graduate research fellowship program and a Baker Grant from Ohio University. The funders had no role in in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the paper and its Supporting Information files. Data Availability All relevant data are within the paper and its Supporting Information files. ==== Body pmcIntroduction Mate preference is an important evolutionary process that drives the exaggeration of phenotypic characters [1], influences the trajectories of population size, and accelerates speciation/extinction events [2,3]. Variation in female mate preferences has been suggested to dampen selection on individual male traits and maintain genetic variation [4] which can lead to maintaining multiple male traits or genetic polymorphisms [5]. Examining the different factors that create variation in female mate preferences provides insights into not only when and how mate preference will vary, but also the costs and benefits that drive the evolution of mate preferences. Teasing apart the different factors that influence mate preferences may require studies that consider how the environment, a female’s physiology, and genetics all interact to produce variation in mate preference. There is considerable evidence that both external and internal factors can alter female mate preferences [reviewed by 6,7]. For example, social interactions with available mates is an external factor that can also produce internal influences on mate preferences when females mate. Virgin females might be less choosy compared to experienced females because virgin females have not yet secured fertilization of the eggs [8], and depending on the available choices and a female’s mate choice strategy, fewer available males could alter mate preferences as the plasticity continues after mating [9–13]. And finally, genetic variation in mate preferences is a prerequisite for mate preferences to evolve [14–16] and yet we understand little about how female genotype influences mate preferences [17–19]. This is complicated by the fact that the heritable aspects of variation in preference can remain cryptic if genotypes are responding differently to environmental change [20]. We examined factors influencing variation in the strength of mate preference in a system where previous work provides us with extensive knowledge about the males that females choose and the factors influencing female mate preferences. The high-backed pygmy swordtail fish, Xiphophorus multilineatus is a freshwater live-bearing fish from the Pánuco River Basin in Mexico [21]. Males exhibit two alternative reproductive tactics (ARTs). Courter males are larger, deeper bodied [22], and faster growing [23] while reaching sexual maturity later. Sneaker males are smaller, more narrow bodied, and slower growing, reaching sexual maturity earlier [24,25]. The courter males are fixed in their use of courtship behavior, while the sneaker males are plastic depending on social context, using both sneak-chase and courtship mating behaviors (24). Females on average prefer to mate with courter males compared to sneaker males [26]. However, there is variation in this preference depending on both social and sexual experience [27], and variation in this preference may function to help maintain the two ARTs [26]. Variation in male size and age at sexual maturity in X. multilineatus, when males stop growing, is influenced by a sex-linked gene (mc4r) on the Y chromosome [28]. A tradeoff between survival to and size at sexual maturity helps to maintain the two genetically influenced ARTs via negative frequency dependent selection [29]. In addition, both laboratory [30] and field [25] studies have detected a mortality-growth rate tradeoff for males, where courter males optimize growing faster to increase liklihood of reaching sexual maturity and sneaker males grow slower to optimize living longer as adults. The genetic influences on growth rates have not been identified, however females with courter sires also have faster growth rates compared to females with sneaker sires [31]. In addition, the role of growth rate and ART lineage in relation to female mate preference in X. multilineatus is not known. Building off this body of work, we examined variation in female mate preference for males from the courter ART lineage as compared to males from the sneaker ART lineage, taking into consideration the interactions between female genotype in relation to the male ARTs, growth rates that are influenced by sire’s genotype, and social experience. In the first experiment, we manipulated both the composition of the female’s social environment and her genotype, where females either experienced 100% sneaker males and had a pure sneaker genotype (sneaker sire, dam with sneaker sire), experienced 100% courter males and had a pure courter genotype (courter sire, dam with courter sire) or experienced a 50/50 split of each tactic and had mixed genotype. In the second experiment, we tested the mate preferences of females from pure sneaker or pure courter genotype that had no sexual experience (virgins), followed by tests of their preferences after we provided them with sexual experience with both types of males. We wanted to examine the influence of sexual experience on strength of preference as we previously demonstrated that virgins are not as choosey, and that their experience with different frequencies of the male ARTs can influence their strength of preference [27]. In addition, given the mortality-growth rate tradeoff detected previously in this system, we wanted to examine the possibility of disassortative mating in relation to growth rates. While less common than assortative mating, disassortative mating is predicted in cases where traits experience strong balancing selection [32], and could be a mechanism for negative frequency dependent selection [33,34]. This hypothesis predicts that females from the slower growing genotype (sneaker) will have a stronger preference for the faster growing courter males than females from the courter genotype. Methods Animal care These experiments were approved by the IACUC of Ohio University (12-L-042). We minimized the welfare impacts on subjects, including not housing adult males together and environmental enrichment of artificial plants and gravel substrate in each individual tank. Every individual tank had an over the back carbon filter to ensure water movement, and remove any water transfer from tank to tank. Bi-weekly water changes occurred in all tanks to additionally support consistent water quality within and across different tanks. We fed all fish Ken’s Premium Flake ad libitum once daily seven days a week, and brine shrimp nauplii (Artemia species) ad libitum once daily five days a week. Fish were kept on a 12:12-hour light: dark cycle. After the study stimulus males in experiments were returned to their original breeding mesocosms. Female mate preference assay We tested females for their preference for courter males with a standard dichotomous choice test [26] using a 284 L tank dived into three sections partitioned by plexiglass (Fig 1). We marked two additional sections by dotted lines on the outside of the tank within the largest center section. Females were placed into an opaque tube in the center of the tank, and one courter male and once sneaker male were placed on opposite ends of the tank in the outermost sections. We gave all fish a 10-min acclimation period, after which the female was released and allowed to swim freely. If males/female were inactive during acclimation, the trial was ended and all individuals were returned to their respective “home” tank. Three trials were removed from the experiment and were not included in analyses. While the use of video animations completely controls for variation in male behaviors, there is also a reduction in the strength of female responses [35], which the use of live males allowed us to avoid. The time the female spent in the center zone adjacent to either male (choice zone) was recorded. We made all observations from behind a screen covered with one-way vision film. Each preference test consisted of two 10-minute trials with the side on which the males were placed switched between each trial to control for side bias. After trials were completed, we calculated a female’s strength of preference by subtracting the total time spent with the sneaker male from the total time spent with the courter male. Thus, positive preference scores were indicative of a courter male preference and negative values were indicative of a sneaker male preference. We calculated total association time with males as the time spent with the sneaker male plus the time spent with courter male. Higher positive values indicate more time spent overall with males and is an overall indicator of choosiness. 10.1371/journal.pone.0287843.g001 Fig 1 Schematic of experimental setup for dichotomous choice tank used in preference tests. Side view with a female located in center compartment and one sneaker and one courter stimulus male located on either the right or the left compartment. All fish were separated by plexiglass indicated by solid lines. Dotted-lines indicate female association zones for each male. Experiment one To examine the effect of a female’s genotype and the composition of male alternative reproductive tactics (ARTs) on mate preference we set up six breeding mesocosms as mock communities with an equal ratio of female and males: two mesocosms with equal ratio of both sneaker and courter males (mixed mesocosm), two mesocosms with only sneaker males (sneaker mesocosm), and two mesocosms only with courter males (courter mesocosm). Mesocosm were set up with virgin females and wild caught males and allowed to breed for 4–5 generations prior to testing. Given that the males in this species breed true to ART, and the mesocosms had been breeding for longer than the average female’s life span, females from the sneaker mesocosm will therefore be from a pure sneaker genotype (sneaker sire and dam with a sneaker sire), while females from the courter mesocosm will be from a pure courter genotype (courter sire and dam with a courter sire). In addition, females will have had experience with only one of the two types of males based on the community the female came from. We also tested females from mixed mesocosms where females would have more mixed genotypes and experience with both types of males. We tested females from replicate mesocosms for both the pure courter, pure sneaker, and mixed genotypes. Females were photographed and measured in ImageJ [36] 24 hours before mate preference tests to measure standard length. In total, we tested a total of 32 females from the two courter mesocosms (mean standard length = 33.03 mm; SE ± 0.64, standard length range: 26.58 mm—42.57 mm), 37 from the two sneaker mesocosms (mean standard length = 33.86 mm; SE ± 0.62, standard length range: 25.83 mm– 40.77 mm), and 18 females from the mixed mesocosm (9 early, 9 later, mean standard length = 28.82 mm; SE ± 0.82, standard length range: 24.95 mm– 38.37 mm) for a total of 87 females. Prior to testing, females were isolated in to 22.7-L aquaria for 7–14 days. None of the females dropped fry during this isolation period, suggesting similar stages of gestation [37]. We then tested their mate preferences as described above, using a randomly chosen unique pair of one sneaker stimulus male and one courter stimulus male for each female. We had a total of 20 stimulus males (10 courter, 10 sneaker) and we created 50 unique male pairs for the mate preference assay. Courter males had a mean standard length of 24.83 mm ± SE 0.34 (Range: 32.89 mm– 36.44 mm) and sneaker males had a mean standard length of 27.69 mm ± 0.22 (Range: 26.32 mm– 28.78 mm). The males in a stimulus pair differed in size by 7.09 mm on average (SE = 0.15). Experiment two We tested 20 females from the F2 generation of a paternal half-sibling lab-bred, pedigree population [38]. Each fish had been raised with at least one companion sibling until sexual maturity, at which point the fish were isolated into 18.9-L aquaria with refugia. None of the females in this study were siblings. Each female had a pure genotype (both dam and sire from the same ART lineage) of courter or sneaker ARTs (10 pure courter genotypes, 10 pure sneaker genotypes). We then preformed a female mate preference assay (as described above) using as stimuli male pairs of one sneaker stimulus male and one courter stimulus male. We had a total of 10 stimulus males (5 courter, 5 sneaker) and created 20 unique male pairs for the mate preference assay. Courter males had a mean standard length of 34.74 mm ± SE 0.61 (Range: 33.63 mm– 36.5 mm) and sneaker males had a mean standard length of 28.05 mm ± 0.24 (Range: 27.19 mm– 28.46 mm). The males in a stimulus pair differed in size by 6.84 mm on average (SE = 0.3). Once the females had been tested for their mate preference as virgins, they were given mate experience. The mate experience consisted of placing the female with a unique pair of males that were different that the stimulus males; one sneaker, and one courter male were randomly selected from one of the breeding mesocosm populations within laboratory. One companion female was also randomly selected and added to the tank to limit harassment of the focal female. The companion female’s caudal fin was clipped in order to identify her from the focal female. All four fish were placed in to 39-L aquaria with refugia and allowed to socialize freely for four weeks, after which we removed the males and companion female from the tanks. We then repeated the mate preference assay to assess a female’s preference for courter males following mate experience. Females were photographed and measured in ImageJ [36] 24 hours before each of the mate preference tests to measure standard length. The two measurements, 28 days apart, were used to calculate growth rates. Statistical analysis For experiment one, we used a non-parametric paired Wilcoxon rank t-test to compare the total time females from each genotype spent associating with the courter versus the sneaker male. We calculated effect size as the absolute of the test statistic divided by the square-root of the sample size. While time spent with courter males (K-S = 0.99 p = 0.77) was normally distributed, time spent with sneaker males (K-S = 0.97 p = 0.024) was not normally distributed. We examined the factors influencing the strength of female mate preference using a general linear mixed-effect model (GLMM), following a Gaussian distribution in package “nlme” v3.1–137 [39]. The model was checked for multi-collinearity using function vif in the “car” package [40] and normality using a Shapiro-Wilks normality test (p = 0.76). Our GLMM consisted of the following fixed effects that were of primary interest: female standard length (mm), mesocosm (mixed, courter, sneaker) and included replicate mesocosm as a nested factor in our analyses. The specific male pair combination used in the dichotomous choice tank was included as a random effect since the same male pair was repeated in multiple tests. The model was fit using restricted maximum likelihood. We used a Tukey’s post hoc test to assess mesocosm treatment relationships when a result was obtained with a p < 0.05. All statistical analysis was done in R software v3.4.1 [41]. We repeated the methods above with a GLMM with the same fixed effects to examine the factors influencing total association time with the males. For experiment two, we calculated a female’s strength of preference for each mate preference trial as the total time spent with the courter male minus the total time spent with the sneaker male. Change in strength of preference was calculated by subtracting a female’s virgin strength of preference from their experienced strength of preference. Standard length was measured in mm twice (measurements 28 days apart) and we calculated growth rate by subtracting the second measurement from the first and dividing by the number of days between each measurement. We tested for a difference in the strength of preference between inexperienced and experienced females using a Wilcoxon rank t-test. To test for differences in growth rates between females from the two genotypes we used a Welch two sample t-test. We calculated effect size as the absolute of the test statistic divided by the square-root of the sample size. In addition, three scores of female mate preference (strength of preference as virgin, strength of preference as experienced, and change in strength of preference) were examined using three general linear models (GLMs) to evaluate the effect of female genotype (courter vs sneaker), growth rate, and the interaction between female genotype and growth rate. We used the interaction between female genotype and growth rate within the model since we know for this species females with courter sires have a faster growth rate than females with sneaker sires [31]. Graphs presented throughout were created using the “ggplot2” package [42]. Datasets for experiment one (S1) and experiment two (S2) are available in supporting information. Results Experiment one Females from the sneaker community type (mesocosm) spent more time with courter males (median: 746s) compared to sneaker males (median: 269s; p <0.001, effect size = 0.95). There was no difference in the time the females from the courter mesocosm spent with courter males (median: 590s) compared to sneaker males (median: 496s; p = 0.49, effect size = 0.29). In addition, there was no difference in the time females from the mixed mesocosm spent with courter males (median: 594s) compared to sneaker males (median: 488s; p = 0.54, effect size = 0.14). Strength of mate preference for courter males was explained by mesocosm (GLMM: χ2 = 11.6 df = 48 p = 0.007), but not female’s standard length (GLMM: χ2 = 1.32 df = 48 p = 0.26). Strength of preference for females from the courter mesocosm was not different compared to strength of preference for the females from the mixed mesocosm (p = 0.83). However, both the courter mesocosm females (p = 0.017) and the mixed mesocosm females (p = 0.025) had a lower strength of preference for courter males compared to the sneaker mesocosm (Fig 2). 10.1371/journal.pone.0287843.g002 Fig 2 The strength of preference (s) for courter males from the different mesocosm treatments: Mixed (n = 18), courter (n = 32), and sneaker (n = 37). * = significance difference where p < 0.05. Total time spent associating with males was not explained by mesocosm (GLMM: χ2 = 0.64 df = 48 p = 0.61), and the influence of a female’s standard length (GLMM: χ2 = 4.07 df = 48 p = 0.054). Experiment two We did not detect a change in the strength of mate preference from prior to sexual experience (i.e., virgins) to after sexual experience for females from either genotype (sneaker virgins median = 568.5s, sneaker experienced median = 904.5s, p = 0.19, effect size = 0.41; courter virgins median = 454s; courter experience median = 495s, p = 0.31, effect size = 0.42). The courter females grew faster (0.027 mm/day SE = .0022) than the sneaker females (0.019 mm/day, SE = .002; t = 2.62, p = 0.017), similar to results from previous studies [31]. Strength of preference in virgin females was unrelated to female genotype (GLM: t = 1.22 df = 16 p = 0.24), growth rate (GLM: t = 0.85 df = 16 p = 0.41), or the interaction between female genotype and growth rate (GLM: t = -1.3 df = 16 p = 0.22). Strength of preference for experienced females was influenced by a female’s genotype (GLM: t = 2.53 df = 16 p = 0.02), an interaction between growth rate and genotype (GLM: t = -2.34 df = 16 p = 0.033) but unrelated to growth rate overall (GLM: t = 1.24 p = 0.23). Similar to the results for the females from the mesocosms, sneaker females had a stronger preference for courter males as compared to courter females after mating experience (Fig 3). There was an interaction between growth rate and genotype when explaining strength of preference (GLM: t = -2.34 p = 0.033, Fig 4), with faster growth rates correlated with a decrease in strength of preference in sneaker females and an increase in strength of preference in courter females. 10.1371/journal.pone.0287843.g003 Fig 3 Strength of preference (s) of females from two different genotypes (courter vs. sneaker). A.) Female’s strength of preference before mate experience B.) Female’s strength of preference after mate experience with each male tactic. * = significance difference where p < 0.05. 10.1371/journal.pone.0287843.g004 Fig 4 Interaction between growth rate (mm/day) and females genotype (courter vs. sneaker) on strength of preference (s). Green line and circles indicate courter genotype and red line and triangles indicate sneaker genotype. Change in preference when females were inexperienced as compared to experienced was unrelated to female genotype (GLM: t = -0.42 p = 0.68), growth rate (GLM: t = -0.02 p = 0.98), or the interaction between genotype and growth rate (GLM: t = 0.27 p = 0.79). Discussion A female’s genotype in relation to the male alternative reproductive tactic (ART) (courter lineage or sneaker lineage) affected the strength of her mate preference for the ARTs in Xiphophorus multilineatus. This pattern was detected in females from breeding mesocosms (experiment one) and females with known pedigrees (experiment two) after mating experience, regardless of whether experience was with only one tactic or both. Sneaker females that have slower growth rates had a stronger preference for the faster growing courter males than courter females. In addition, variation in strength of preference was influenced by a female’s growth rate, but not in the same direction for the two female genotypes. Within sneaker females, strength of preference for the larger courter males decreased with increased female growth rates, while within courter females strength of preference trended towards increase with increased female growth rates. Below we consider why mating experience may be necessary for detecting the differences between the genotypes in strength of preference, as well as potential mechanisms and hypotheses that could explain the variation in strength of preference based on genotype and growth rates. Given the known growth rate differences between the ARTs that are associated with a mortality-survival tradeoff in this system, we present a hypothesis suggesting that some of the variation in mate preferences in this system could be due to disassortative mate preferences in relation to growth rates. The differences in strength of preferences between the two genotypes of females was detected in females from the mesocosms that had mating experience, and in the pedigree females but only once they were given mating experience. This result concurs with previous findings in this species, that virgin females with no mating experience have weaker preferences for courter males than females with mating experience [27]. As experience interacting with males influenced mate preferences, one possible explanation for the differences found between the experienced courter and sneaker females could be differences in costs or benefits of socially learning a mate preference. Learning has been examined in this species, and females with bigger brains learned faster, as well as courter females learned faster than sneaker females [43]. However, we did not find that the change in preference after gaining mating experience was explained by genotype. Given that brains are energetically expensive, we expect that further study of the tradeoffs between brain size, growth rates, and fecundity may provide increased insights into the variation across the genotypes in response to mating experience that we detected. Finally, swordtail females store sperm [44] and aspects of sperm competition and longevity can differ among the ARTs [45]. Both are additional factors that could be examined in relation to the influence of sexual experience on mating preferences. The relationship we found between strength of preference and growth rate, which was different depending on a female’s genotype, has an interesting connection to previous work on female mate preferences and cortisol levels in this species. Courter females were found to have higher cortisol levels, and the strength of female mate preferences were associated with higher cortisol levels [46]. Cortisol is known to be associated with reduced growth, and stress responses in fish [47,48], and therefore cannot explain the faster growth rates we detected in the courter females. However, cortisol differences could explain the differences in strength of preference between the genotypes, as well as the reduction in mate preference in the sneaker females as their growth rates increased. Examining this possibility will require further study, but will be particularly important to consider for hypotheses that attempt to explain the variation in mate preferences in relation to growth rates in this species. Given that costs to mate preferences have been well documented [6,20] it is important to consider their potential impact on the relationships we detected between a female’s genotype, growth rate and strength of mate preference. Costs can range from energy invested while searching for mates, to tradeoffs with other life history traits like growth, to the sensory systems necessary to detect differences between mates. We assessed one of costs of being choosey by measuring the total time females associated with the males during testing (not the relative time spent with the two stimulus males), but detected no influence of a female’s genotype. However, if being choosey requires increased investment in sensory processing, such as larger brains, this could result in less energy available for growth and could explain why the genotype with slower growth (sneaker) had stronger preferences. Results from previous work in this system found the opposite relationship between brain size and growth, as the faster growing courter females were shown to learn faster and have larger brains than sneaker females [49]. Costs are likely to play an important role in the variation in mate preferences in most systems and further assessment of the costs in this system will be important in relation to testing hypotheses for evolution of these mate preferences [50]. Females can store sperm in these fishes [44], leading to additional arguments for examining both increased and decreased costs of mate preferences. Males provide no resources to females or offspring in swordtails, suggesting females may prefer larger males because male size is a proxy for some aspect of mate quality that provides indirect benefits—offspring inheritance of adaptive traits. Given that females showed variation in their preferences for the male ARTs and differences between the male ARTs in X. multilineatus have been studied extensively, this is an excellent system in which to further examine the adaptive benefits of female mate preference. Evolutionary hypotheses for the variation in mate preferences we detected will want to consider known differences between the male ARTs in this system. ARTs in X. multilineatus are dimorphic not only for body size and shape [22,51], but also plasticity in mating behaviors [22], age at sexual maturity when males stop growing [23], and sperm longevity [46]. Variation in the Mc4r gene on the Y-chromosome has been found to be associated with some these traits [24,28], but is unlikely to explain all the genetic variation and tactical dimorphism between the two ARTs. The two ARTs are maintained by frequency dependent selection and a tradeoff between the benefits of being larger and gaining more mating success (optimized by the courter males), and the benefits of a reaching sexual maturity sooner at a smaller size, which increases the probability of reaching sexual maturity (optimized by the sneaker males) [29]. Interestingly, the ARTs are also dimorphic for growth rate. Measured in the laboratory, the larger courter males grow faster than the smaller sneaker males [24]. This difference in growth rates leads to an additional tradeoff: juvenile growth rates and adult mortality (i.e. mortality-growth rate tradeoff, [52], which has been documented for X. multilineatus males in both the laboratory [30] and the field [25]. Assuming genetic variation is at least in part responsible for the differences in growth rates between female’s based on genotype (courter versus sneaker lineage), disassortative mating based on growth rates would provide offspring with benefits by optimizing the mortality/growth rate tradeoff. To further develop this hypothesis, it will be important to examine the heritability of growth rates, teasing apart the contribution of dam versus sire genotype to offspring growth rate. In addition, it would be interesting to examining the variation in female mate preference for male size within courter males, as there is still a relationship between male size and growth rates, but not the additional differences in other traits (e.g. behavior and mating success) found between the alternative reproductive tactics. Finally, two aspects of our results do not match with previous studies of mate preference for the courter male ART by wild-caught females from this species: we did not detect a significant preference in the females from the mixed or courter only mesocosms, and we did not detect an influence of female size on this preference. One possibility is that we need a better understanding the factors that influence variation in female size, in addition to the relationship between growth rates and female size in the laboratory as compared to wild-caught females which are not likely to be the same. We have recently increased our ability to assess the growth rates of wild-caught females using otoliths [25], and therefore future studies can examine the relationship between size, growth rates and preferences in wild-caught females to determine if the pattern we detected in lab reared females is the same for wild-caught females. Conclusion Variation in female mate preferences in Xiphophorus multilineatus have been implicated in the negative frequency dependent selection that maintains the male ARTs in this species [53], and found to correlate with female size [27] as well as levels of cortisol [46]. Here we document variation in relation to a female’s genotype and growth rate, suggesting that females with faster growth rates (courter lineage compared to sneaker lineage, and variation within sneaker genotype) have weaker preferences for the faster growing courter males. Neither the sensory bias hypothesis for larger males nor a hypothesis based on the costs to being choosey can adequately explain our results. Given what is known about the ARTs in this system, we suggest an additional hypothesis warrants further investigation. The growth rate optimization hypothesis suggests that when choosing between potential mates, females are selected to balance the mortality/growth rates tradeoff by providing their offspring with genotypes from their sires genotypes will optimally balance growing fast prior to sexual maturity (increased probability of reaching sexual maturity, larger adult size) with the costs of growing faster due to a shorter adult lifespan. While not the first hypothesis to suggest a relationship between behaviors and mortality-growth rate tradeoffs [54], our hypothesis is the first to suggest variation in female mate preferences have evolved in response to producing offspring that optimize these tradeoffs. In addition, recent work on the selection regimes and the genetic architecture favoring the emergence of disassortative mating [32] provides a theoretical framework in which to test this hypothesis. Studies of variation in female mate preferences, especially in systems with alternative reproductive tactics, can help disentangle the multiple influences on mate preferences and lead to a better understanding of their evolution. In this system, further investigation of the genetic influences on growth rates, selection on growth rates in females, and differences in the optimal growth rates between males and females are needed. Further study of the complex relationship between male size and growth rates [55] has the potential to provide additional support across taxa for the hypothesis that mate preference for male size may be under selection due to size being indicative of growth rate. Supporting information S1 Dataset Dataset for experiment one. (PDF) Click here for additional data file. S2 Dataset Dataset for experiment two. (PDF) Click here for additional data file. We would like to thank John Bobo for support in conducting behavioral trials in experiment two in this study, Hannah Griebling for providing us with the females to test both before and after mating experience, and for the students that helped maintain the fish in the Morris laboratory with safe animal care practices. 10.1371/journal.pone.0287843.r001 Decision Letter 0 Tuckett Quenton M. Academic Editor © 2023 Quenton M. Tuckett 2023 Quenton M. Tuckett https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version0 11 Apr 2023 PONE-D-23-01131Genotype and growth rate influence female mate preference in Xiphophorus multilineatus: potential selection to optimize mortality-growth rate tradeoffPLOS ONE Dear Dr. Fitschen-Brown, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by May 26 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Additional Editor Comments: Dear authors, First, let me apologize for the delay in getting this decision finalized; it was partly my doing and also an inability to located suitable reviewers. That said, we now have a decision based on two reviews of your manuscript "Genotype and growth rate influence female mate preference in Xiphophorus multilineatus: potential selection to optimize mortality-growth rate tradeoff". Both reviewers indicated the manuscript could be suitable for publication, but it will require major revisions. Please find the detailed comments from both reviewers below. In particular, consider how the results are explained, including consideration of alternate hypotheses; this was identified by both reviewers. Sincerely, Quenton M. Tuckett [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ********** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ********** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This MS deals with a study of a model system that has been detailed studied in the past for mate choice, the live-bearing fish Xiphophorus multilineatus. Previous studies have shown that males have genetically-based alternative reproductive tactics (ARTs): one male type is called COURTER (C type; having higher size and growth rate, later sexual maturity and displaying extreme courtship behaviour) while the other is called SNEAKER (S type; having smaller size, lower growth rate and earlier sexual maturity, with earlier courtship and coercive force copulation). Natural populations show a possible protected polymorphism in ARTs via presumably frequency dependent selection. Although much is known, the details of the mechanism acting as well as the different interactions between traits (size, growth rate, mate choice tactic) are basically unknown. Therefore, the study provides two new experiments trying to improve the knowledge on the system. The first experiment, a female choice design, try to test if the alternative reproductive genotypes (i.e. genotypes) show a pattern of assortative mating or not. The dependent variables were preference (female time expend with C – female time expend with S) and choosiness (female time expend with C + female time expend with S). The studied treatment were three genotypes (C; S and both included) in (non-virgin) females and two (C and S) in males. As the genotypes cannot be morphologically identified, they produced them by rearing specimens of each phenotype for 4-5 generations in mesocosms. The results show a trend consistent with negative assortative mating (a mechanism able to cause negative frequency-dependent sexual selection). The second experiment tested the mating preferences of 10 pure (F2) female genotypes with males of both genotypes as virgin females, then they got some mate experience (maintained 4 weeks in presence of another female plus two males) and them tested again for mating preference as before. As females were measured at both dates they could calculate female growth rate as well. This second experiment is important for understanding the results of the first experiment as well as identifying trait interaction contributing to mate choice. The preference showed a significant interaction between genotypes and growth rate (positive relationship in C and negative in S) a trend which do not support any of the former hypothesis used in this context. The authors discuss and speculate why they got these results as well as their possible interpretation. This study adds a useful piece of information to the study of mate choice in this system, it is reasonable well designed and analysed and so its merits publication. However, I found several potential drawbacks that should be discussed or even mentioned/discussed in the text as potential limitations of the study. I list them below 1. The analyses were done using several fishes repeated in different trials. For example, in experiment 1 they used 10 C and 10 S males, for an overall 87 trials. I understand that they tried to use different combinations of males in different trials (50 new, and so at least 40% of data was not independent). Actually, perhaps it is not bad to use the same males for different female genotypes, providing that they behaviour (of the males) do not change with experience (but see second experiment). I do not think that this invalidates the whole experiment but the potential consequences and limitations of this should be discussed. The same happened in experiment 2. 2. The authors claim that they got a results that supports negative assortative mating (lines 266-269). Actually I think that they got complex results. I will discuss results for both experiments. 2.1. Although in experiment 1 S female prefers to mate the other type, it did not happen with the C females. Notice that a pure negative assortative mating trend will require of both genotypes favouring the other or alternative that the four combinations of possible mates show a sexual isolation index that was negative, or similar. So the authors should be more caution with this result, I think. 2.2. Similarly, in the second experiment, S type female showed a trend consistent with growth rate favouring disassortative mating, as claimed in the text (lines 369-372). Again this was far from being so clear, I think, as the relationship is so in the S genotype but it is the opposite in the C one. Again the authors should be caution with the interpretation regarding assortative mating as they got complex results. 3. One interesting result has not achieved enough author attention, I think. They got that ART trait (their genotypes) show actually a plastic characteristic, as the trait needs of certain previous experience in order to produce certain aspects of the mate choice, as the preference for particular genotype needs of previous experience. I do not know if this has been observed before in other organism, and perhaps merits a particular discussion about the interaction between genetic determination and phenotypic plasticity, aspects considered two alternatives but which could also be two sides of the same coin. 4. The authors and too worried about to produce an evolutionary hypothesis/mechanism that explain their results, which can be understandable, but their new hypothesis seems to me too speculative, perhaps is too soon to advance new hypothesis and just more clear and compressive data is just needed. In my opinion, they should try to reduce the importance of the speculative ideas in discussion, and emphasize the new findings in relation to the previous ones, without any need to present a closed interpretation. 5. In addition, I have a few comments on the text . Lines 17-19. They only describe the results of one genotype, say that the other showed the opposite at least. . Lines 63-64. Too frequent repeat “females with courter sires” Once you explained how you got these females they are “courter females”. I think that some sentences could be simplified in the text. . Line 64-65. It should be explained the exact characteristic of the mate choice relationship in guppy . Line 81-82. Perhaps this explanation is obvious for the authors or specialized readers, but I think that general reader needs a better explanation about how such hypothesis works and produces its predictions. . Lines 85-87. Actually several theoreticians claim that disassortative mating may directly cause negative frequency-dependent sexual selection, and so disassortative mating would be the cause of the selection not the consequence (Pusey and wolf 1996. TREE, 11: 201-206; Hedrick several papers; for example 2016; Evolution 70: 757-766). Any fast search on the WEB should give a few more useful references to discuss this if interested. . Lines 132-134. I personally do not see clear the test group factor used, for me repeated measurements are typically analysed as a nested factor instead of a new factor, as you are not interested per se in such factor just to know if the main effects are true irrespective of such variation. . Legend of Figure 2. The P > 0.05, I assume that actually means < I enjoined the MS and so hope that authors may be able to produce an improved version. Reviewer #2: The authors of “Genotype and growth rate influence female mate preference in Xiphophorus multilineatus: potential selection to optimize mortality-growth rate tradeoff” report on two experiments used to identify the factors affecting female choice for males exhibiting alternative reproductive tactics. Overall, I enjoyed the manuscript; it wasn’t overly long and rarely strayed from the main objectives. There are some writing issues throughout, but always minor. I do note one issue that should be mentioned alongside their chosen hypotheses: the potential for sperm storage and quality to affect the results. See below for one specific issue (sperm storage) and some specific comments listed line by line. Specific Issues 1. Somewhere the authors should discuss sperm storage and competition might affect the results of this experiment and also how it might be his might be related to their hypothesis on 272-275. For example, there has been studies on X. nigrensis showing that male tactic is related to traits related to sperm competition (e.g., sneakers might have sperm with greater viability and longevity compared to courters; doi: 10.1098/rsbl.2011.0286). I also think a discussion of sperm storage (in general) would be warranted early on. Specific Comments L36: comma after physiology L93: “not hosing” L93-94: I am curious about how these sterile environments might affect mate choice experiments L95: any water quality testing? L104: remove “a” L102-: regarding the dichotomous choice tank; this is a bit unclear without a diagram. In doing an image search, these tanks will seemingly vary L124: “mesocosms” L141: lowercase females L141: 22.7-L aquaria L144: delete “number of” L146: more important than reporting the range for both males and females would be to examine if there are differences L156: delete “number of” L157: once again; differences between tested populations would be more informative, even if expected (e.g., sneaker and courter) L161: “experience males”? L208: how was effect size calculated? Was this presented in the methods? L218: sometimes test statistics and df are reported and at other times not L228: delete “from” L239: some variation in significant figures L257-260: that’s a mouthful L297: comma after “growth rates” L299-312: not quite convinced by this explanation and not quite sure why it was included. Some other explanations might be more fruitful. For example, why not mention sperm traits and alternative reproductive tactics? This seems like a missed opportunity, perhaps even something that should be discussed. L315-316: is this reference comparable to a swordtail that exhibits sperm storage? I’m not sure; I am also not sure how much or how little sperm storage affects the costs of assessing and choosing mates. Presumably, if females can store the sperm from multiple males, there could be arguments for both an increase and decrease in costs. L367: a possible place for sperm quality as well? ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Emilio Rolán-Alvarez (Universidade de Vigo, Spain) Reviewer #2: No ********** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0287843.r002 Author response to Decision Letter 0 Submission Version1 24 May 2023 Additional Editor Comments: In particular, consider how the results are explained, including consideration of alternate hypotheses; this was identified by both reviewers. Alternative hypotheses and additional explanation of results were addressed in the discussion section. Additional information can be found below in the responses to the reviewer’s comments. Reviewers' comments: Reviewer #1: 1. The analyses were done using several fishes repeated in different trials. For example, in experiment 1 they used 10 C and 10 S males, for an overall 87 trials. I understand that they tried to use different combinations of males in different trials (50 new, and so at least 40% of data was not independent). Actually, perhaps it is not bad to use the same males for different female genotypes, providing that they behaviour (of the males) do not change with experience (but see second experiment). I do not think that this invalidates the whole experiment but the potential consequences and limitations of this should be discussed. The same happened in experiment 2. For experiment 1, we attempted to control for the re-use of male pairs by including it as a random repeated effect in the model. Especially since male pairs in experiment 1 were repeated with different females. Within, the second experiment we only had unique pairs of males presented to the females, therefore we did not include it as a random effect in the model. Additionally, males that were inactive (sat on the bottom of the tank) or did not present courtship behavior (sigmodal displays) were not used for a trial and this was determined during the first acclimation period. This information has now been included in methods section (line 109). However, there is no way to insure exact behavioral consistency across trials without using video animations or mechanical models. While this is one of the drawbacks of using live males, previous work has found that using live males and video animations gave similar overall results, however there was a reduction in the strength of female responses using videos (which in the current study we chose to avoid). This point was included in the methodology section (line 115-117). 2. The authors claim that they got a results that supports negative assortative mating (lines 266-269). Actually I think that they got complex results. I will discuss results for both experiments. 2.1. Although in experiment 1 S female prefers to mate the other type, it did not happen with the C females. Notice that a pure negative assortative mating trend will require of both genotypes favouring the other or alternative that the four combinations of possible mates show a sexual isolation index that was negative, or similar. So the authors should be more caution with this result, I think. Yes, we agree that we did not detect what the reviewer is defining as “pure” negative assortative mating. While we detected significant differences in the strength of preference between the female genotypes, with a stronger preference for the faster growing males in the sneaker females that had slower growth, no preference was detected in the faster growing courter females. In addition, for experiment 2 where we had individual female growth rates and could examine the variation within the females of a genotype, the relationship was only found in one of the two genotypes (the females with the slower growth rate), but was different from the relationship for the courter genotype females. Therefore, we agree that the pattern is more complex than pure negative assortative mating, and have changed our language throughout the discussion to reflect this. 2.2. Similarly, in the second experiment, S type female showed a trend consistent with growth rate favouring disassortative mating, as claimed in the text (lines 369-372). Again this was far from being so clear, I think, as the relationship is so in the S genotype but it is the opposite in the C one. Again the authors should be caution with the interpretation regarding assortative mating as they got complex results. Discussion was redesigned to more accurately represent the complexity of the results, and not just focus on the disassortative mating hypotheses. 3. One interesting result has not achieved enough author attention, I think. They got that ART trait (their genotypes) show actually a plastic characteristic, as the trait needs of certain previous experience in order to produce certain aspects of the mate choice, as the preference for particular genotype needs of previous experience. I do not know if this has been observed before in other organism, and perhaps merits a particular discussion about the interaction between genetic determination and phenotypic plasticity, aspects considered two alternatives but which could also be two sides of the same coin. Yes, this is an interesting result, and one that is the focus of a recent meta-analysis (Richardson and Zuk 2023). While this review did not find that across studies virgin females were less choosey, most studies do not examine or at least report both as we do here. We included a paragraph specifically on the role of experience acting on genotypes and the role within mate preference and mate choice (see paragraph starting on line 340). Richardson, J., & Zuk, M. (2023). Unlike a virgin: a meta-analytical review of female mating status in studies of female mate choice. Behavioral Ecology, 34(2), 165-182. 4. The authors and too worried about to produce an evolutionary hypothesis/mechanism that explain their results, which can be understandable, but their new hypothesis seems to me too speculative, perhaps is too soon to advance new hypothesis and just more clear and compressive data is just needed. In my opinion, they should try to reduce the importance of the speculative ideas in discussion, and emphasize the new findings in relation to the previous ones, without any need to present a closed interpretation. Discussion was redesigned to focus more heavily on findings surrounding social experience and female’s genotype on strength of preference for courter males. We do still present discussion of hypotheses to potentially explain the influence of growth rates on preferences we detected. Judy Stamps (2007) presents clear reasoning/arguments for growth-mortality tradeoffs, and we have found support for these tradeoffs in our fish in both the laboratory and the field (line 602-603). We think that the influence this tradeoff may have on mate preferences is something that future scientists should investigate not only in our system, but in general. However, we have paired the discussion of the potential influence of this tradeoff on mate preferences to a paragraph within clear statements of need for future research. 5. In addition, I have a few comments on the text Lines 17-19. They only describe the results of one genotype, say that the other showed the opposite at least. Line 17-19 edited sentences to state: In addition, the relationship between strength of preference and growth rate depended on a females’ genotype; females with sneaker genotypes decreased their preference as their growth rates increased, a pattern that trended in the opposite direction for the females from the courter genotypes. Lines 63-64. Too frequent repeat “females with courter sires” Once you explained how you got these females they are “courter females”. I think that some sentences could be simplified in the text. After methods section changed females with courter sires to just courter females/sneaker females for the rest of the manuscript. Line 64-65. It should be explained the exact characteristic of the mate choice relationship in guppy The preference for male body size, as the study we cited specifically found preference for body size in that population of guppies (line 68-69). However, we ended up taking the citation out as we restructured the paragraphs to address other points. Line 81-82. Perhaps this explanation is obvious for the authors or specialized readers, but I think that general reader needs a better explanation about how such hypothesis works and produces its predictions. Thank you for noting that this prediction was not clear. Even preferences for genetic benefits can be plastic in relation to experience (reduced for example if costs are high on being choosey). We have restated our interest in examining virgin and experienced females (line 88). Lines 85-87. Actually several theoreticians claim that disassortative mating may directly cause negative frequency-dependent sexual selection, and so disassortative mating would be the cause of the selection not the consequence (Pusey and wolf 1996. TREE, 11: 201-206; Hedrick several papers; for example 2016; Evolution 70: 757-766). Any fast search on the WEB should give a few more useful references to discuss this if interested. Thank you for these references. We added some additional reverences here (see lines 94-96) and throughout the manuscript in relation to this hypothesis. Lines 132-134. I personally do not see clear the test group factor used, for me repeated measurements are typically analysed as a nested factor instead of a new factor, as you are not interested per se in such factor just to know if the main effects are true irrespective of such variation. We changed the model to using test group as a nested factor instead of a covariate/new factor. We agree with this interpretation of the test group factor since our focus was to understand if mesocosm or standard length influenced strength of female mate preference. We included this in both the methods and changed the results. However, the outcome did not change when using test group as a nested factor. We also changed the test group terminology to replicate to be more clear. Legend of Figure 2. The P > 0.05, I assume that actually means < Changed Figure 1 & 2 legend mistake. I enjoined the MS and so hope that authors may be able to produce an improved version. Thank you, and we really appreciate the comments and feedback provided as well as the time spent on improving our work. Reviewer #2: 1. Somewhere the authors should discuss sperm storage and competition might affect the results of this experiment and also how it might be his might be related to their hypothesis on 272-275. For example, there has been studies on X. nigrensis showing that male tactic is related to traits related to sperm competition (e.g., sneakers might have sperm with greater viability and longevity compared to courters; doi: 10.1098/rsbl.2011.0286). I also think a discussion of sperm storage (in general) would be warranted early on. We are aware of evidence for influence of sperm competition on male mate preferences in guppies (Dosen and Montgomerie 2003) but are unclear on how to evaluate the influence of sperm storage and/or competition in our case (female mate preferences). We did not quantify if females had actually mated with males. While they were in a social environment and exposed to males for a long period of time, we assume that courtship and mating (or at least attempted mating) occurred at some point. For this reason, we cannot disentangle in this study social dynamics from copulation. However, the differences in longevity of the sperm and sperm competition in relation to the ARTs are interesting aspects of this system that we have now included in the discussion of the influence of experience on mate preferences. Also, Line 166 we added a statement that we did not have any individuals drop fry during or after testing. Therefore, we assumed that females were more or less at the same gestational stage during experimentation, as this could potentially impact the results of the mate preference trials as well (Ramsey et al. 2011). Dosen, L. D., & Montgomerie, R. (2004). Female size influences mate preferences of male guppies. Ethology, 110(3), 245-255. Ramsey, M. E., Wong, R. Y., & Cummings, M. E. (2011). Estradiol, reproductive cycle and preference behavior in a northern swordtail. General and comparative endocrinology, 170(2), 381-390.) Specific Comments L36: comma after physiology Added comma L93: “not hosing” Fixed grammar error to not housing L93-94: I am curious about how these sterile environments might affect mate choice experiments Absolutely, we are too. While we have not identified the factors influencing the differences, we discuss how the environment of the lab as compared to the wild may influence mate preferences in the paragraph starting on line 421 L95: any water quality testing? Larger Tanks (50-100 gallons): Continuous monitoring of ammonia with Seachem patch, Weekly of nitrates and pH levels. Smaller tanks (5-10 gallons): ammonia, nitrates and pH levels tested once every 3 months. This information was added to the animal care section. L104: remove “a” Removed “a” L102-: regarding the dichotomous choice tank; this is a bit unclear without a diagram. In doing an image search, these tanks will seemingly vary Agreed, a schematic was included as a new figure to show both the set up and dimensions of the dichotomous choice thank that we use in the lab. L124: “mesocosms” Switched aquarium experiments to breeding mesocosm experiments L141: lowercase females Changed females to lowercase L141: 22.7-L aquaria Changed L144: delete “number of” Deleted L146: more important than reporting the range for both males and females would be to examine if there are differences In this line we were reporting the range in the sizes between courter and sneaker males that were used as stimuli in the mate preference trials. However, we agree that it would be useful to present the size differences of the pairs that were presented to the female. This information is now included. L156: delete “number of” Deleted L157: once again; differences between tested populations would be more informative, even if expected (e.g., sneaker and courter) Differences between the sizes of the males in the stimulus pairs is now presented (lines 172-173). We apologize that this was not clear. This range was referring to the courter stimulus males and sneaker stimulus males and was not actually describing any of the populations. L161: “experience males”? We want the readers to understand that these males were different males from the stimulus males used during the mate preference trails. Therefore, the females had never seen the stimulus males before being tested. We have clarified this point in the text. L208: how was effect size calculated? Was this presented in the methods? Effect size calculation was added to the statistical methods section. L218: sometimes test statistics and df are reported and at other times not We have standardize based on the statistical analysis we did both the test statistics and df being reported. L228: delete “from” Deleted L239: some variation in significant figures Added variation in individuals to boxplots via the jitter function in ggplot2. L257-260: that’s a mouthful Paired sentence down to make more straightforward. Got rid of preference for courter males because that was the same throughout the wholes study. L297: comma after “growth rates” Added comma L299-312: not quite convinced by this explanation and not quite sure why it was included. Some other explanations might be more fruitful. For example, why not mention sperm traits and alternative reproductive tactics? This seems like a missed opportunity, perhaps even something that should be discussed. We included the cortisol as a hypothesis for the relationship between growth rate differences and mate preferences, as we previously detected an association between cortisol and strength of mate preferences between the genotypes. In addition, as cortisol has been demonstrated to be associated with growth rates in addition to stress, it has the potential to be a mechanism influencing the variation we detected in both growth rates and preferences between the genotypes. We are still unclear how sperm traits in this system might play a role in female mate preference and genotype at this time. However, the differences in the ARTs in growth rates is one of the factors that drives the hypothesis for disassortative mating (females from sneaker lineage with slower growth rates having a stronger preference for courter males with faster growth rates). We reduced focus on the physiological and evolutionary future hypotheses in the discussion, and includ sperm trait differences in the ARTs in swordtails as an avenue of future research in relation to female mate preference. L315-316: is this reference comparable to a swordtail that exhibits sperm storage? I’m not sure; I am also not sure how much or how little sperm storage affects the costs of assessing and choosing mates. Presumably, if females can store the sperm from multiple males, there could be arguments for both an increase and decrease in costs. We have modified this paragraph on costs. Additional references have been included. Sperm storage is an interesting aspect of the system in relation to costs, however, as it suggests that females may not need to invest in more mate assessment after mating, in particular if mate choice is not adaptive. We have added a statement to make this point here (line 432). L367: a possible place for sperm quality as well? We moved discussion on sperm quality to the discussion paragraph with differences between experienced and non-experienced females and the discussion about difference in traits between the ARTs. Attachment Submitted filename: Response to Reviewers .docx Click here for additional data file. 10.1371/journal.pone.0287843.r003 Decision Letter 1 Tuckett Quenton M. Academic Editor © 2023 Quenton M. Tuckett 2023 Quenton M. Tuckett https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version1 13 Jun 2023 Genotype and growth rate influence female mate preference in Xiphophorus multilineatus: potential selection to optimize mortality-growth rate tradeoff PONE-D-23-01131R1 Dear Fitschen-Brown, Thank you for this thorough revision, which has now been reexamined by myself and one of the reviewers. We both found the manuscript to be be suitable for publication. We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact onepress@plos.org. Kind regards, Quenton M. Tuckett Academic Editor PLOS ONE 10.1371/journal.pone.0287843.r004 Acceptance letter Tuckett Quenton M. Academic Editor © 2023 Quenton M. Tuckett 2023 Quenton M. Tuckett https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 22 Jun 2023 PONE-D-23-01131R1 Genotype and growth rate influence female mate preference in Xiphophorus multilineatus: potential selection to optimize mortality-growth rate tradeoff Dear Dr. Fitschen-Brown: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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