Factors affecting egg production in the selfing mangrove rivulus (Kryptolebias marmoratus)

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Factors affecting egg production in the selfing mangrove rivulus (Kryptolebias marmoratus) Justin L. Lomax 1 , Rachel E. Carlson 1 , Judson W. Wells, Patrice M. Crawford, Ryan L. Earley ∗ Department of Biological Sciences, University of Alabama, 300 Hackberry Lane, Box 870344, Tuscaloosa, AL 35487, USA

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Article history: Received 30 November 2016 Received in revised form 13 February 2017 Accepted 15 February 2017 Available online xxx Keywords: Mangrove rivulus Hermaphrodite Outcrossing Self-fertilization

a b s t r a c t The mangrove rivulus, Kryptolebias marmoratus, is one of two known vertebrate species with preferentially self-fertilizing hermaphrodites. Males also exist, and can outcross with hermaphrodites. Outcrossing events vary across wild populations and occur infrequently in laboratory settings. This study sought to add dimension to our understanding of mangrove rivulus reproductive habits by probing the effects of male presence on hermaphroditic unfertilized egg production. Specifically, we quantified egg production of solitary hermaphrodites compared to hermaphrodites exposed to males and exposed to other hermaphrodites. Hermaphrodites tended to produce more fertilized eggs in the presence of males but unfertilized eggs were produced relatively rarely and did not vary significantly among treatments. The probability that hermaphrodites would produce eggs changed as a function of genetic dissimilarity with their partner and in a season-dependent manner. In the fall, the probability of laying eggs decreased as a function of increased genetic dissimilarity, regardless of the sex of the partner. In the winter/spring, however, the probability of laying eggs increased markedly with increased genetic dissimilarity, regardless of the sex of the partner. Our findings indicate that reproductive decisions are modulated by factors beyond male presence, and we discuss a number of alternative hypotheses that should be tested in future studies. © 2017 Elsevier GmbH. All rights reserved.

1. Introduction Androdioecious mating systems are characterized by the presence of both hermaphrodites and males, and have evolved independently in the plant and animal kingdoms. Pannell (2002) defines functional androdioecy as a mating system in which the hermaphrodites make a significant genetic contribution through both male and female functions. Within the animal kingdom specifically, androdioecy is observed among various invertebrate groups including shrimp, barnacles, and nematodes (Stewart and Phillips, 2001; Hollenbeck et al., 2002; Zardus et al., 2013). Interestingly, among vertebrates, only the mangrove rivulus (Kryptolebias marmoratus) and its sister species (K. hermaphroditus; Costa, 2011), killifish native to sub-tropical and tropical climates, exhibit an androdioecious mating system. Two types of males exist in K. marmoratus − those that develop from embryos as primary males and, more commonly, males that result from

∗ Corresponding author. E-mail address: [email protected] (R.L. Earley). 1 These authors contributed equally.

hermaphrodites undergoing sex change (Harrington, 1971). Proportions of males and hermaphrodites are determined largely by environmental influences. For example, Harrington (1967, 1968) showed that the propensity of embryos to develop as either males or hermaphrodites was a function of the temperature the eggs developed in, which was recently substantiated by Ellison et al. (2015). Furthermore, hermaphrodites exposed to warm temperatures during early adulthood and subsequently to a short-day photoperiod tend to undergo sex change (Harrington, 1971, 1975), but the likelihood of responding to these environmental factors appears to vary among genotypes (Turner et al., 2006). Although every population is unique in its sex ratio, most populations of K. marmoratus maintain fewer than 5% males (Tatarenkov et al., 2015). K. marmoratus hermaphrodites simultaneously maintain functional ovarian and testicular tissue, and possess the ability to fertilize their own eggs. If hermaphrodites exclusively self-fertilize, heterozygosity will decrease by, on average, one half in each successive generation, which can, over many generations, lead to individuals with completely homozygous genotypes (Mesak et al., 2014; Tatarenkov et al., 2015). However, K. marmoratus hermaphrodites exhibit a mixed-mating system and possess the ability to outcross by means of laying an unfertilized egg. Because

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Please cite this article in press as: Lomax, J.L., et al., Factors affecting egg production in the selfing mangrove rivulus (Kryptolebias marmoratus). Zoology (2017), http://dx.doi.org/10.1016/j.zool.2017.02.004

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hermaphrodites are unable to fertilize each other’s eggs, it is essential that males are present for an outcrossing event to occur (Furness et al., 2015). Once an outcrossing event does occur, heterozygosity can potentially be restored across all loci in the filial generation, depending on genetic differences between the parents. Therefore, the level of heterozygosity is directly related to the frequency of outcrossing events. Wild populations have varying levels of heterozygosity and thus it can be inferred that these populations vary in the frequency of outcrossing events. Analyses of 36 microsatellites in field-caught populations throughout south Florida and the Bahamas show rates of heterozygosity that range from 0 to 16%, while populations in Belize averaged over 46% heterozygous loci (Mackiewicz et al., 2006a). This raises questions regarding the mechanisms that trigger outcrossing events between hermaphrodites and males. The reproductive flexibility of K. marmoratus, specifically their ability to either self-fertilize or outcross, has raised questions about its evolutionary history. Both self-fertilization and laying an unfertilized egg for a male to fertilize carry inherent benefits and costs. Selfing offers reproductive assurance and preservation of favorable phenotypes; i.e. favorable genes related to high fitness will not be diluted by an outside genetic contribution of a potentially less fit male (Baker, 1955; Clegg and Allard, 1972). Outcrossing, however, introduces genetic diversity and guards against inbreeding depression (Charlesworth et al., 1993). Current hypotheses suggest that a mixed-mating system may allow K. marmoratus to cope with dynamic selection pressures, such as continually evolving parasites (Ellison et al., 2011). Though there are many hypotheses surrounding the evolutionary costs and benefits of a mixed-mating system, little is known about how outcrossing is triggered. A primary objective of the present research is to determine factors that induce hermaphroditic mangrove rivulus to forego selffertilization, which ultimately enables outcrossing with males in a population. Outcrossing is known to occur in the wild as evidenced by the prevalence of heterozygosity in field-caught individuals (Tatarenkov et al., 2007); however, the proximate cause behind outcrossing remains unknown. We investigated the potential effects of visual and chemical cues from a male on the propensity of hermaphrodites to self-fertilize versus lay unfertilized eggs. We hypothesized that hermaphrodites would exhibit decreased selffertilization (i.e. they would produce more unfertilized eggs) while in the presence of a male possessing a different genotype from their own. This hypothesis is rooted in the notion that hermaphrodites should only lay unfertilized eggs if there is a means for the eggs to be fertilized, and if the genetic contribution is distinct from that of the hermaphrodites. Based on this hypothesis, we expected to see a marked decrease in the number of self-fertilized eggs from hermaphrodites exposed to males in comparison to the eggs produced by hermaphrodites that were not exposed to males. To test our hypothesis, we constructed an experiment that isolated the presence, or lack thereof, of males as our independent variable across several treatments, and examined the status of eggs laid by hermaphrodites.

2. Materials and methods 2.1. Experimental setup To test the effects of male presence on the production of unfertilized eggs in hermaphroditic mangrove rivulus, we developed three treatments. In treatment HM, we exposed hermaphrodites to males across a mesh partition. In treatment HH, the objective was to account for the presence of another fish by exposing hermaphrodites to other hermaphrodites. Treatment H0 served as our control with hermaphrodites housed on opposite sides of a

tank with no opportunity for visual or chemical interaction. Details about the treatments, sample sizes, and ages of the animals used can be found in Table 1. The experiment was conducted from 17 November 2014 to 11 December 2014 (fall; 25 days), and again from 5 February 2015 to 2 April 2015 (winter/spring; 57 days). Conducting the experiment in two phases was motivated by historical data and by our own data indicating that rates of egg-laying in K. marmoratus follow a seasonal pattern in the laboratory. Harrington (1968) showed that peak reproduction occurs between mid-March and early August with notable nadirs in September and December. Harrington’s (1968) study was conducted in the laboratory and the fish were exposed to natural photoperiod through a window. Our laboratory has demonstrated that similar seasonal patterns of egg-laying persist even when photoperiod is controlled, light is provided artificially, and all other parameters (e.g., temperature, feeding) are strictly regulated (Marson and Earley, unpublished data); a formal analysis of these data coupled with field-based quantification of reproductive seasonality is forthcoming. While the mechanisms underlying the retention of seasonal patterns of egg-laying in the laboratory remain unknown, we refer to the two phases of the experiment cautiously as ‘seasons’. 2.2. Housing conditions Sterilite FlipTop containers (clear polypropylene; Sterilite Corp., Townsend, MA, USA) with the dimensions 19.4 cm x 11.4 cm x 16.5 cm were used as tanks. Each pre-manufactured tank was modified with the addition of a partition, yielding two compartments with the dimensions 9.7 cm x 11.4 cm x 16.5 cm. The tanks of treatments HM and HH were partitioned using clear mesh, with the experimental hermaphrodites on one side, and the males or control hermaphrodites on the other (Fig. 1A and B). The clear mesh allowed for visual contact and water exchange between the two sides. In treatment H0, the tanks were partitioned with black corrugated plastic rather than mesh. The plastic was siliconed with Marineland aquarium sealant (Marineland Spectrum Brands, Blacksburg, VA, USA) to create a watertight and opaque division, simulating isolation while keeping the volume of water in each treatment standard (Fig. 1C). In each setup, the tanks were filled with 25 ppt synthetic seawater (Instant Ocean sea salt; Instant Ocean Spectrum Brands, Blacksburg, VA, USA). For each treatment, every experimental hermaphrodite was kept in the side of the tanks opposite the hinge. The fish were held in a room with minimal foot traffic and a temperature of 26 ± 1 ◦ C on a 12 h light: 12 h dark photoperiod provided by overhead incandescent lights. The fish were fed a daily diet of 0.07 g of brine shrimp (Artemia) nauplii suspended in 4 ml water of 25 ppt salinity. 2.3. Data collection Poly-Fil (100% polyester fiber) was placed in the corner farthest from the partition in the experimental hermaphrodites’ side of the tank (Fig. 1), acting as a substrate for egg-laying when placed partially in the water. Placing the egg-laying substrate away from the partition minimized the likelihood of eggs being fertilized by sperm from the males on the opposite side of the mesh partition. Hermaphrodites do not fertilize one another’s eggs (Furness et al., 2015). The egg-laying substrate was checked for eggs twice weekly from 17 November 2014 to 11 December 2014 (fall), and again with the same experimental hermaphrodites but different partners from 5 February 2015 to 2 April 2015 (winter/spring). To check for eggs, the fibrous material was removed from the tank, and the lid was closed to ensure that the fish did not escape. Because the eggs were clear and camouflaged by the fibrous material, the fuzz was carefully rolled through the collector’s fingers and checked for hard, spherical objects. Once the eggs were located, each egg was gen-

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Table 1 Summary of the treatments, sample sizes, and ages of mangrove rivulus involved in the present study. H = hermaphrodite; M = male; 0 = no partner; N/A = not applicable. Averages are shown ± standard error of the mean (SEM). Ages are shown in days. Treatment

# Fish H/M

Age experimental hermaphrodite

Age paired fish

Age range experimental hermaphrodites

Age range paired fish

HH HM H0

16/16 42/42 24/–

185.57 ± 8.65 193.7 ± 5.09 198.61 ± 7.79

272.68 ± 22.23 377.39 ± 30.06 N/A

141–264 138–260 134–299

114–585 164–656 N/A

Fig. 1. Experimental setup. Experimental mangrove rivulus hermaphrodites received one of three treatments: (A) paired with a male (orange coloration), (B) paired with a hermaphrodite, or (C) isolated/unpaired. The tanks were plastic containers and were modified with a divider. Tanks A and B had a mesh divider that did not obstruct visual interaction and permitted water exchange between the two sides. Tank C had an opaque, waterproof divider. Experimental hermaphrodites were provided with a fibrous material that acted as a surface on which to lay eggs.

Fig. 2. Classification of eggs. Eggs were classified into three categories: (A) fertilized, (B) unfertilized, and (C) inviable. Fertilized eggs were identified by the presence of a perivitelline space (ps, marked by white arrows). Unfertilized eggs lack the perivitelline space. Inviable eggs were identified by their marked discoloration and opaque appearance (indicated by a white arrow in C).

tly removed from the fuzz and placed in a cup filled with water of 25 ppt salinity to prevent dehydration of the egg until it was analyzed. Each cup was labeled with the tank number, the number of eggs collected, and the identity of the fish that laid them. The polyester fiber was then dipped slightly back in the water and placed in its original position. After collecting eggs from every experimental hermaphrodite, each egg was examined under a Zeiss Stemi 2000-C dissection microscope (Carl Zeiss Microscopy, Jena, Germany) to determine whether it was fertilized, unfertilized, or inviable. Eggs were determined to be fertilized or unfertilized based on the presence (or absence) of a perivitelline space (Fig. 2A and B). The perivitelline space is present in fertilized eggs and absent in unfertilized eggs. Eggs that were completely opaque were classified as inviable (Fig. 2C). The lineage, treatment, and number of fertilized, unfertilized, and inviable eggs were recorded, and the total number of eggs (across all categories) was calculated for each individual. Pictures were taken with an iPhone through the eye piece of the dissecting microscope, and each egg was preserved in formalin. A summary of the egg yield in each treatment and season (fall vs. winter/spring) is provided in the supplementary material (Table S1 in the online Appendix). Because only 10 inviable eggs were recorded during the study, and never in the HH treatment, no further analysis was conducted on this category.

2.4. Fish selection Fish in the various treatments were progeny from multiple isogenic or near-isogenic lineages (see Tables S1–S3 in the online Appendix for details on the lineages). All focal animals were between one and eight generations removed from a wild-caught progenitor that was homozygous at a minimum of 30 out of 32 microsatellite loci; the vast majority of animals were F1 or F2 generation. Each progenitor had been previously genotyped at 32 microsatellite loci using the protocol of Mackiewicz et al. (2006b). Fish were selected based on three main factors: egg yield, age, and genetic diversity. Hermaphrodites were selected from lineages that were determined to be “high yielding” in terms of egg production, which meant that representatives of the lineage reliably produced at least one egg on a weekly basis. Once the most appropriate lineages were determined, experimental hermaphrodites that were at peak egg-laying ages, which is between 100 days post hatch (dph) and 700 dph in our laboratory, were selected (Table 1). After all of the experimental hermaphrodites were selected, they were paired with either a control hermaphrodite, a male, or placed in a solitary tank. The complementary hermaphrodites and males were chosen based on their genetic diversity compared to the experimental hermaphrodites. For our experiment, a threshold of 20 microsatellite allele differences (out of 64 possible) was

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chosen as the minimum that had to exist between the experimental hermaphrodite and its pair mate (see Tables S1 and S2 for details). Here, the goal was to determine whether experimental hermaphrodites would be more likely to produce unfertilized eggs when exposed to individuals with varying degrees of relatedness. 2.5. Data analysis Before the data were processed, all of the eggs were qualitatively assessed for a second time by two independent contributors. In order to avoid any biases, the contributors were not aware of which eggs came from which treatments. Following the second round of analysis, the results from the two contributors were reconciled against each other, and then against the original data and original photos. Any discrepancies were analyzed for a third time by the authors until a unanimous decision was reached for each egg. Following this process, the results were analyzed using JMP Pro version 12.0.1 statistical software (SAS Institute, Cary, NC, USA). To test the hypothesis that the probability of laying eggs (unfertilized, fertilized or total) would vary among treatments, we conducted logistic analyses with season (fall, winter/spring), treatment (H0, HH, HM), and the season × treatment interaction as predictors and the binary egg-laying score for each animal (yes or no) as the response. Analyses for each egg type were run separately. To test the hypothesis that the number of eggs laid per day in each category (unfertilized, fertilized or total) would vary among treatments, we conducted a generalized regression with a zero-inflated gamma distribution, log link function, and maximum likelihood estimation of the parameters; the gamma distribution accommodates continuous positive data that show a strong right skew. Season, treatment, and the season × treatment interaction were included as predictors and the number of eggs laid by each animal per day (rate of egg-laying) was the response. Rates of egg-laying were used because the fall and winter/spring trials were conducted for different lengths of time (25 versus 57 days). For purposes of analysis, rates of egg-laying were multiplied by 100 (but true means are reported in the text and figures). These two types of analysis (logistic and generalized regression) were repeated for the data set excluding the H0 treatment to determine whether the number of allelic differences between the partners (focal hermaphrodite vs. hermaphrodite partner in HH; focal hermaphrodite vs. male partner in HM) influences the probability of egg-laying and the number of eggs laid per day. In these analyses, the model included season, treatment, and allelic differences plus all two-way interactions as the predictors, and either the binary condition (yes or no for egglaying) or the number of eggs laid per day as the response. Because some animals failed to lay eggs, we conducted the same analyses as described above on only animals that laid eggs (except with standard least squares analysis for rates of fertilized and total egg production), and the results were identical. True means ± standard error of the mean are reported in the text and figures. 3. Results Unfertilized egg production was relatively rare, accounting for a maximum of 17% of all eggs laid across all treatments (see Table S4 in the online Appendix). The probability of laying eggs of any type did not vary with treatment, season, or the treatment × season interaction (Table 2A). Rates of daily egg-laying also did not vary significantly with treatment, season, or the treatment × season interaction (Table 2B). There was a qualitative but not statistically significant trend towards higher rates of fertilized and total egg production when hermaphrodites were paired with males (Fig. 3). Overall, individuals in the HM treatment laid 3–5 times as many eggs as solitary hermaphrodites and 5–7 times as many eggs as

Fig. 3. Seasonal differences in rates of fertilized egg production. Rates of fertilized egg production in the fall and winter/spring in each of the three treatments (HO, HH, HM). There were no significant differences but there was a qualitative trend towards higher production in the HM treatment.

hermaphrodites paired with other hermaphrodites (Table S4) but this was due largely to a couple of animals with very high rates of egg production (see supplementary Figs. S1 and S2 in the online Appendix). To examine the effects of genetic differences between the experimental hermaphrodite and its partner (hermaphrodite or male) on the probability of egg-laying and on rates of eggs production, models were constructed including only the HH and HM treatments. Only the season × allele differences interaction affected the probability of laying fertilized eggs (Table 3). In both treatments (HH and HM), the probability of laying fertilized eggs decreased as a function of increasing genetic differentiation between the focal hermaphrodite and its partner in the fall. However, the probability of laying fertilized eggs increased as a function of increasing genetic differentiation between the focal hermaphrodite and its partner in the winter/spring (see supplementary Fig. S3 in the online Appendix). The same trend was evident when examining the probability of laying eggs of any type (total; Table 3 and Fig. S4). There was an interesting but non-significant treatment × allele differences interaction for the probability of laying unfertilized eggs; in HH, hermaphrodites were more likely to lay unfertilized eggs with more genetically dissimilar partners but in HM, the probability of laying unfertilized eggs decreased with increasing genetic dissimilarity (Fig. S5). There were no significant effects of allele differences alone on rates of egg-laying (Table 4). Analyses accounting for genetic dissimilarity revealed higher rates of fertilized egg and total egg production in the fall than in winter/spring (Table 4 and Fig. 3). Two significant effects were found in the model for rates of unfertilized egg production (Table 4). The treatment × season interaction was due primarily to a significant decrease from fall (0.04 ± 0.04 eggs/day) to winter/spring (0.007 ± 0.005 eggs/day) in the HH treatment (Tukey’s HSD, P = 0.009). There was a weak seasonal effect on the relationship between allele differences and rates of unfertilized egg production. This was due to there being a slight negative relationship in the fall and a slight positive relationship in the winter/spring for the HM treatment (Fig. S6). Given the small sample sizes, especially for the HH treatment in the fall, and the fact that relatively few unfertilized eggs were laid, these results should be interpreted with caution.

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Table 2 (A) The effects of treatment, season, and the treatment × season interaction on the probability of mangrove rivulus laying eggs of any type (total) and of laying fertilized or unfertilized eggs. L-R 2 is the likelihood-ratio chi-square derived from the logistic analyses. (B) The effects of treatment, season, and the treatment × season interaction on the rate of laying eggs of any type (total) and the rates of laying fertilized or unfertilized eggs. Wald 2 is the test statistic derived from the zero inflated model; df = degrees of freedom. (A) Probability

Overall model Treatment Season Treatment × season

Fertilized 2

L-R 

P-value

L-R 

P-value

L-R 2

P-value

5 2 1 2

3.38 0.10 0.82 2.93

0.64 0.95 0.37 0.23

1.96 0.54 0.11 0.76

0.85 0.76 0.74 0.68

3.19 0.13 0.23 2.73

0.67 0.94 0.63 0.26

Fertilized

2

Total

df

(B) Rates

Treatment Season Treatment × season

Unfertilized

Unfertilized

Total

df

Wald 2

P-value

Wald 2

P-value

Wald 2

P-value

2 1 2

0.33 0.03 1.26

0.85 0.87 0.53

0.93 0.04 4.43

0.63 0.83 0.11

0.10 0.21 1.92

0.95 0.64 0.38

Table 3 The effects of treatment, season, allele differences, and all two-way interactions on the probability of mangrove rivulus laying eggs of any type (total) and of laying fertilized or unfertilized eggs. L-R 2 is the likelihood-ratio chi-square derived from the logistic analyses; df = degrees of freedom. Statistically significant effects are denoted with bold type and an asterisk; marginally non-significant effects are denoted with italic type and an asterisk. Fertilized

Overall model Treatment Season Treatment × season Allele difference Treatment × allele diff. Season × allele diff.

2

Unfertilized 2

Total

df

L-R 

P-value

L-R 

P-value

L-R 2

P-value

6 1 1 1 1 1 1

9.49 0.12 0.12 0.85 0.00 1.11 6.33

0.14 0.73 0.73 0.36 0.99 0.29 0.01*

5.11 1.12 0.19 0.03 1.20 2.61 0.06

0.53 0.29 0.66 0.86 0.27 0.11 0.80

6.91 0.00 0.00 0.74 0.02 0.03 3.09

0.33 0.95 0.99 0.39 0.89 0.87 0.08*

Table 4 The effects of treatment, season, allele differences and all two-way interactions on the rates of mangrove rivulus laying any type of egg (total) and rates of laying fertilized or unfertilized eggs. Wald 2 is the test statistic derived from the zero inflated model; df = degrees of freedom. Statistically significant effects are denoted with bold type and an asterisk; marginally non-significant effects are denoted with italic type and an asterisk. Fertilized

Treatment Season Treatment × season Allele difference Treatment × allele diff. Season × allele diff.

Unfertilized

Total

df

Wald 2

P-value

Wald 2

P-value

Wald 2

P-value

1 1 1 1 1 1

0.20 3.51 0.06 0.03 0.27 0.00

0.65 0.06* 0.81 0.86 0.60 0.98

0.24 2.86 6.00 1.22 0.36 4.18

0.62 0.10 0.01* 0.27 0.55 0.04*

0.06 8.30 0.01 0.25 0.34 0.14

0.81 0.004* 0.91 0.62 0.56 0.71

4. Discussion This study investigated the effects of the chemical and visual presence of males on egg production in mangrove rivulus hermaphrodites. This fish is becoming an important model organism for biological research (Lee et al., 2008; Earley et al., 2012), but much remains unknown about its reproductive behaviors and the factors that mediate outcrossing between hermaphrodites and males. We hypothesized that unfertilized egg production, which is a pre-requisite for external fertilization and outcrossing, would be induced by the presence of males. We predicted an increase in unfertilized egg production in treatments where hermaphrodites could interact with males both visually and chemically relative to treatments in which only hermaphrodites could interact or in which hermaphrodites were housed individually. Our hypothesis and predictions were not supported by the data. There was no significant variation among treatments in the production of unfertilized eggs, and individuals rarely laid unfertilized eggs. While the presence of males did not significantly affect the probability of producing unfertilized eggs, our data indicate that males do play

some role in reproductive patterns because some hermaphrodites appeared to lay consistently more fertilized eggs in the HM treatment than in the HH or H0 treatment (Fig. 3). It remains possible that some of the eggs were fertilized by the males, although this is highly unlikely considering the distance between the males and the egg-laying substrate. As noted by Harrington (1968), egg production is seasonal in mangrove rivulus held under laboratory conditions with exposure to natural photoperiod. Our own data corroborate seasonal patterns of egg production even in the absence of natural photoperiod (Marson and Earley, unpublished data). Our current results show some seasonality in egg production but, surprisingly, rates of egg-laying were higher in the fall (November–December) than in the winter/spring (February–March). This might have resulted from a slight increase in egg production during the month of November, and from the winter/spring phase being conducted just as the animals are expected to resume higher rates of egg production (Harrington, 1968). Future studies should be conducted longitudinally with the same animals throughout the year or, to control for age-related effects, with replicate individuals from a given lineage begin-

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ning treatment at different times of year at the same age. There also appeared to be seasonal differences in how hermaphrodites responded to partners of varying genetic similarity. The probability of laying fertilized eggs decreased with increased genetic variation between the focal hermaphrodite and its partner, regardless of the partner’s sex, in the fall (Fig. S4). Conversely, the probability of laying fertilized eggs increased with increased genetic variation in the winter/spring (Fig. S4). Mangrove rivulus has the ability to distinguish between adults that are genetically similar and those that are dissimilar (Edenbrow and Croft, 2012; Ellison et al., 2013). Wells and Wright (unpublished data) recently observed in mangrove rivulus that kin discrimination plays an important role in mediating cannibalism. When hermaphrodites encountered both their own embryos and embryos of a different genetic lineage, they were more likely to exhibit cannibalism towards non-kin. The risk of cannibalism may be especially costly in the fall, as eggs might be more valuable, in terms of energy expenditure, when animals are preparing for the reproductive offseason. Although rates of egg production were higher in the fall than in winter/spring in the present study, this species shows a consistent and considerable reproductive nadir between late fall and early spring. This could explain the decrease in the production of fertilized eggs in the fall when the focal hermaphrodites were paired with genetically dissimilar partners. In contrast, fertilized egg production in the winter/spring increased as a function of increasing genetic dissimilarity between the focal hermaphrodite and its partner. This may be due to greater perceived competition between genetically different animals, making it beneficial to lay more eggs to provide a greater chance for offspring survival. While the fish typically lay eggs year round, there might be a distinct benefit associated with having flexible patterns of egglaying based on the threat of cannibalism and levels of perceived competition. Olofsson et al. (2009) suggest that bet-hedging might explain how animals optimize their long-term fitness in variable environments at the expense of lowering mean fitness between years. In the case of mangrove rivulus, variability in the level of cannibalism and competition, perhaps cued by genetic similarity of other individuals in the population, may drive fluctuations in egglaying. Bet-hedging can result in the production of fewer, larger eggs (conservative approach) or more eggs of variable sizes (diversified approach) (Seger and Brockmann, 1987; Philippi and Seger, 1989). When entering the low reproductive period (late fall and winter), it might be beneficial to reduce egg production in the presence of non-kin, perhaps using the energy that would have been committed to eggs that have a high probability of being eaten for other fitness-related activities. However, when entering the high reproductive period (spring and early summer), it might be most beneficial to increase egg production in the presence of non-kin so as to account for cannibalism. If this were the case, we would expect to see decreased egg mass during periods of high egg production and larger eggs during periods of low egg production. While our experiment did not examine the possible tradeoff between egg number and egg size, it may be useful to consider this relationship in future studies with regard to the treatments that we employed. Our hypothesis aimed to understand the effects of the presence of males on the production of unfertilized eggs. Our findings indicate that the visual and chemical presence of males does not convincingly modulate whether hermaphrodites opt to lay unfertilized eggs. Male presence is just one of many factors that could influence the reproductive output of hermaphrodites. Park et al. (2017) recently demonstrated that when hermaphrodites were held for 60 days at elevated temperatures (30 ◦ C), production of unfertilized eggs increased markedly. This leads to an alternative hypothesis − that environmental stressors (e.g., variation in abiotic conditions such as temperature, water availability and salinity, food availability, and changes in predation pressure or parasite

encounter rates) trigger the production of unfertilized eggs and promote outcrossing. Selection might favor this strategy because outcrossing would produce genetic variants that might be more likely to survive such challenges. Variable selection pressures might promote the maintenance of mixed-mating strategies in mangrove rivulus such that long periods of self-fertilization are punctuated by episodes of outcrossing. A recent study by Vergara et al. (2014) explored the potential effect of parasites in sustaining both sexually and asexually reproducing lineages within a snail population in an attempt to understand how environmental factors control reproductive behaviors. This study demonstrated a negative relationship between parasite loads and the proportion of asexual snails; moreover, it implicated sexual reproduction as the strategy with higher fitness for four of the five years, while asexual reproduction had higher fitness for the remaining year. This fluctuating advantage (between asexual and sexual strategies) across an evolutionary timeframe could be responsible for the development and maintenance of variable reproductive strategies in snail populations. In a broader sense, Lively and Morran (2014) contrast the favorability of sexual reproduction in novel environments, across a variety of experiments and model organisms, with the favorability of asexual reproduction in stable environments with fewer stressors. Similarly, in the mangrove rivulus’ androdioecious mating system, varied biotic and abiotic stressors could potentially explain the disproportionate occurrence of outcrossing among wild populations. Future investigations could probe the occurrence of outcrossing events when the animals are challenged by different stressors. Such investigations would be conducted under the hypothesis that selection has favored reproductive flexibility, as this allows mangrove rivulus populations to selectively introduce genetic variation (via outcrossing) as a means of coping with novel environments and novel stressors. Before these other factors are explored, it will be important to further investigate the degree to which male presence affects reproductive habits. Our experimental design prevented direct contact between the fish in an effort to avoid the uncertainty of paternity. This eliminated the need to genotype all of the fertilized eggs; however, a consequence was that the males could not physically interact with the hermaphrodites. Courting behaviors between males and females have been documented in other killifish, and male behavior has been shown to be the key factor (above color and size) in determining mate choice and spawning success (McGhee et al., 2007). Ellison et al. (2013) noted the tendency of mangrove rivulus hermaphrodites to closely associate with males over other hermaphrodites, but did not describe any notable courtship behavior. Furthermore, their study assessed the role of genetic dissimilarity (specifically MHC dissimilarity) in mate choice. Hermaphrodites did not exhibit preference based on MHC dissimilarity; however, the less common males (2–25% of natural populations) did exhibit strong preference for hermaphrodites with great MHC dissimilarity. These findings suggest the potential role of male preference in initiating outcrossing events. In another study, Luke and Bechler (2010) identified 23 unique behaviors between rivulus pairings, and reported a marked increase in courting behaviors in male–hermaphrodite pairings as compared to hermaphrodite–hermaphrodite and male–male pairings. Despite the increase in courting behavior, there was no observed spawning between male–hermaphrodite pairs. Finally, Martin (2007) found that hermaphrodites preferentially associate with males in tight, close-proximity burrows. Although this study did not record any spawning events between males and hermaphrodites, it did identify the existence of a social prerequisite (male–hermaphrodite association) for the hypothesized male-mediated outcrossing events. These studies illustrate an element of the hermaphrodite-male dyad − close physical association − that was not considered in our present design. Future experi-

Please cite this article in press as: Lomax, J.L., et al., Factors affecting egg production in the selfing mangrove rivulus (Kryptolebias marmoratus). Zoology (2017), http://dx.doi.org/10.1016/j.zool.2017.02.004

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mental designs should permit direct contact, but will also have to include a protocol for genotyping every fertilized egg. The mangrove rivulus is increasingly being used as a model organism in scientific research. With this vertebrate model, we can probe the effects of different developmental environments using isogenic individuals, but we can also use genetically distinct individuals under common developmental environments (Earley et al., 2012). Application of the rivulus as a model organism is especially promising in the areas of genomics, oncology, endocrinology, and aquatic toxicology (Lee et al., 2008). As rivulus’ popularity grows, it becomes increasingly imperative that we better understand the factors that modulate its reproductive behavior. Selectively crossing distinct isogenic lineages with divergent phenotypes will allow us to produce a large number of recombinant inbred lines, and thus will permit identification of quantitative trait loci (QTL). Mackay (2001) enumerates the power of generating recombinant lines, including the ability to map pleiotropic genes as well as genes that exert subtle, additive effects. Additionally, Mackay (2001) details the dependence of high-resolution QTL on the ability to generate recombination through extensive (and selective) crosses. Before any outcrossing events can occur between lineages, an unfertilized egg ultimately must be produced by, or harvested from, a hermaphrodite. Currently, two protocols have successfully produced outcrossed offspring in a laboratory setting but both have their limitations. The first involves directly pairing males and hermaphrodites and allowing them to reproduce. Though both selfing and outcrossing events will occur, they will happen extremely disproportionately: Mackiewicz et al. (2006b) produced 32 offspring through hermaphrodite–male pairings, and only 2 (6.25%) of these offspring were identified to be progeny of an outcrossing event. Additionally, this technique requires an intensive process to determine which progeny are products of self-fertilization and which have been produced by outcrossing. The offspring must all be raised and individually genotyped to determine their paternity. This method is inefficient in terms of cost, the number of outcrossed progeny produced, and the effort required to identify them as heterozygous individuals. A second technique eliminates the uncertainty of paternity and need for genetic analysis, but carries its own inherent disadvantages. Hermaphrodites must be culled, dissected, and their eggs must be harvested (Nakamura et al., 2008). These eggs can then be exposed to sperm harvested from male testis, and an outcrossing event can be induced (Harrington and Kallman, 1968). Though paternity is selectively controlled, this technique requires the sacrifice of two parent organisms and still only produces a small number of outcrossed progeny per parent. A strategy that consistently stimulates outcrossing without the aforementioned limitations would be ideal. This system would have to rely on two major events: (1) hermaphrodites would need to lay an unfertilized egg and (2) a male must externally fertilize the egg. Additionally, an ideal system would provide certainty that any eggs that are fertilized have indeed been fertilized by the male. Establishing a reliable outcrossing protocol will open the door to further understanding the biology of the mangrove rivulus, and will facilitate the generation of large amounts of genetic variation that can then be quantitatively assessed, allowing researchers to map traits with high resolution and explore the genetic basis for virtually any phenotype. The mangrove rivulus has great promise as a model organism, but its full potential will not be realized until we can readily cross genetically distinct isogenic lineages. We hypothesize, based on the field evidence of high rates of outcrossing, that some combination of male presence, physical interaction with males, and a constellation of environmental cues might trigger unfertilized egg production and outcrossing. We are currently at a stage where many more controlled laboratory studies are needed to identify the most salient proximate cues.

7

Acknowledgements We would like to thank Sydney Sudderth and Mingyuan Li for their assistance in qualitatively assessing the status of each egg collected. We thank Evan Harrison for his contribution to data collection, and we thank previous researchers, Genevieve Miller and Ricky Seeber, for their insight and previous contributions to the project. Two anonymous reviewers provided excellent suggestions on a previous draft, which greatly improved the manuscript. The research described herein was approved by the Institutional Animal Care and Use Committee at the University of Alabama (Protocol #13-10-0048). This work was supported by the University of Alabama Undergraduate Research & Creative Activities grant awarded to J.L.L. and R.E.C.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.zool.2017.02.004.

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Please cite this article in press as: Lomax, J.L., et al., Factors affecting egg production in the selfing mangrove rivulus (Kryptolebias marmoratus). Zoology (2017), http://dx.doi.org/10.1016/j.zool.2017.02.004