Sexual Selection, Fertilization Dynamics and the Use of Alternative Mating Tactics in the Hermaphroditic Seabass Serranus Subligarius Mia Susan Adreani

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2011 Sexual Selection, Fertilization Dynamics and the Use of Alternative Mating Tactics in the Hermaphroditic Seabass Serranus Subligarius Mia Susan Adreani

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SEXUAL SELECTION, FERTILIZATION DYNAMICS AND THE USE OF ALTERNATIVE

MATING TACTICS IN THE HERMAPHRODITIC SEABASS SERRANUS SUBLIGARIUS

MIA S. ADREANI

A dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Degree Awarded: Spring Semester, 2011

The members of the committee approve the dissertation of Mia Adreani defended on October 25, 2010.

______Don Levitan Professor Directing Dissertation

______Joseph Travis Professor Directing Dissertation

______Zuoxin Wang University Representative

______David Houle Committee Member

______Christopher Koenig Committee Member

Approved:

______P. Bryant Chase, Chair, Dept of Biological Science

The Graduate School has verified and approved the above-named committee members.

ii ACKNOWLEDGEMENTS

There are many people who have contributed, directly and indirectly, to this work, and without them, this would not have been possible. First, I thank my advisors, Joe Travis and Don

Levitan, whose intelligence, guidance and humor got me through these last several years. I thank my committee members: Chris Koenig, David Houle and Zuoxin Wang for their input and patience.

Many people have helped me in the field and I am forever grateful for their assistance:

Shannon Chaplin, Andy David, Mark Endries, Nikki Fogarty, Meghan Kirk, Clemens Lakner,

Megan Lowenberg, Katie McGhee, Chris Stallings, Brian Storz. In addition, I had a great deal of lab assistance: Shannon Chaplin, Dave Ferrell, Casey Grace, Nate Jue, Meagan Kirk, Alex

Marsh, Andres Plata-Stapper, Matt Schrader.

I also had a wonderful group of colleagues with whom I could discuss this research, both formally and informally: Denise Akob, Janna Fierst, Nikki Fogarty, Becca Hale, Phil Hastings,

Pierson Hill, Nate Jue, Katie Lotterhos, Megan Lowenberg, Katie McGhee, Andres Plata-

Stapper, Matt Schrader, Barb Shoplock, Anna Strimaitas, Casey terHorst, Bob Warner, as well as the many years of EERDG participants.

This work was partially funded a Brenda Weems Bennison grant, PADI grant, and NSF grants to both Joe Travis and Don Levitan. Site support was generously provided by∗ the NOAA marine fisheries lab in Panama City, FL, St. Andrews State Park, Panama City, FL and the FSU

Coastal and Marine Laboratory in St. Teresa, FL.

Finally, I want to thank my family for their continued love and support.

iii

TABLE OF CONTENTS

List of tables v

List of figures vi

Abstract viii

1. INTRODUCTION 1

2. DO SIZE STRUCTURE, HABITAT AND EARLY DEVELOPMENT 14 INFLUENCE THE USE OF ALTERNATIVE MATING TACTICS?

3. THE EFFECT OF POPULATION SIZE STRUCTURE AND ALTERNATIVE MATING TACTICS ON THE FERTILIZATION SUCCESS OF A HERMAPHRODITIC SEABASS 37

4. CONCLUSIONS 57

APPENDIX – ACUC PROTOCOL APPROVAL 61

REFERENCES 62

BIOGRAPHICAL SKETCH 77

iv LIST OF TABLES

Table 2.1: Summary of (a) ANOVA using the total number of spawns with season and site as factors and temperature as a covariate and (b) ANOVA using the proportion of streak spawns with season and site as factors and sea-surface temperature as a covariate. 26

Table 2.2: Percentage of histologically analyzed gonads of S. subligarius at each of five stages of development: immature, mature testis only, mature inactive, mature active, and mature postspawning. The samples analyzed histologically represent a subsample of the sample size used for GSI and age analysis. For each month, the range of gonadosomatic indices (GSI = 100*(gonad mass/body mass)) is shown in percent. 27

v LIST OF FIGURES

Figure 1.1: Comparisons of the Drosophila data of Bateman’s (1948) paper describing the conditions under which variation in mating may differ among males and females leading to sexual selection on traits that confer greater fitness (solid lines, modified from Bateman, 1948) to a simultaneous hermaphrodite in which selection is stronger on the male role (arbitrary data-dashed line). The gain curves are steeper for hermaphrodites and more similar for males and females than the gonochoristic flies. The gray dotted line indicates the conditions Charnov associated with hermaphroditism being favored (both male and female fitness curves are saturating). 6

Figure 1.2: Possible trade-offs between male and female function for hermaphroditism (convex line) and dioecy (concave line) as discussed by Charnov (1982). 8

Figure 2.1: Map of St. Andrews State Park in Panama City, Florida, highlighting the three locations of observation and their average fish densities; Outer Jetty, Lagoon, and Inner Jetty. 28

Figure 2.2: Number of spawns (clear diamonds) across the spawning season. Surface temperature (black squares) taken from a nearby NOAA buoy across spawning season. Water temperature correlates with spawning activity (Pearson’s correlation, r95 = 0.60, p = 0.032). 29

Figure 2.3: Fish densities taken along permanent 50-m band transects across two spawning seasons at each of the three spawning locations (inner jetty, lagoon, outer jetty). 30

Figure 2.4: Results of correlation between fish density and the proportion of streaking behaviors. Inner jetty (low density, diamonds): Pearson’s correlation, r15 = -0.147, p = 0.875; Lagoon (intermediate density, squares): Pearson’s correlation, r9 = 0.051, df = 1, p = 0.323; Outer jetty (high density, triangles): Pearson’s correlation, r13 = 0.079, df = 1, p = 0.192). 31

Figure 2.5: Incidence of pair spawning and streak spawning in S. subligarius from animals observed in the field, at each of three locations within the study site that vary naturally in density; inner jetty (low density), lagoon (intermediate density), outer jetty (high density). 32

Figure 2.6: Size structure data presented as a proportion of total density from observations along permanent density transects at each of three sites (a) Inner jetty, (b) Lagoon and (c) Outer jetty. Relative sizes were used and individuals were classified as small (<65 mm SL), medium (65-80 mm SL) or large (>80 mm SL). Black bars = small; dark gray bars = medium; light gray bars = large. 33

vi Figure 2.7: Frequency of different male mating behaviors (pair spawn and streak spawn) of each size class at the three different sites using total number of fish observed over two mating seasons. 34

Figure 2.8: Relationship between algal cover and incidence of streak spawning during two field seasons (a) 2006 and (b) 2007, where high algal cover at local spawning sites predicts a greater proportion of streak spawning than bare rock. Chi-sq; 5.20, df = 1, p = 0.023 (2006). Chi-sq; 5.97, df = 1, p = 0.015 (2007). 35

Figure 2.9: Cross section of a gonad of Serranus subligarius, taken from the area near the base (posterior) portion, indicating basic morphology and three of the gamete development stages; spermatozoa, primary oocytes and yolk globule oocytes. 36

Figure 3.1: Drawing showing typical pair spawning event, with courtship occurring over rocks and culminating in a short spawning rush into the water column, cupping behavior, and finally, the release of gametes. Black lines drawn onto to individual in cupping position indicate the body angle measurement taken at point of gamete release as a test of altered behavior using ImageJ software analysis. 47

Figure 3.2: Fertilization rates obtained from field-collected samples during the spawning seasons of 2006, 2007, 2008. Variation in fertilization rate increases with the spawning season. 48

Figure 3.3: Percent of eggs fertilized over the spawning season in 2006 and 2007. Spawns that included a streaker were removed from this analysis and a best fit regression line suggests a slightly negative, but nonsignificant result (2006 (solid line): n = 13, R2 = 0.0512; 2007 (dashed line): n = 19, R2 = 0.3016). 49

Figure 3.4: Truncated data showing the fertilization rates late in the spawning season (August-October). Pair spawning (dark circles, n = 23) and streak spawning (open circles, n = 16) fertilization rates are shown and participation of streakers results in lower overall fertilization success (t-test, df = 37, p = 0.001). 50

Figure 3.5: Total sperm output estimates (in millions) for pair spawns and pair spawns with at least one streaker participating (n = 22, t-test, p = 0.047). 51

Figure 3.6: The average number of eggs in a single parcel does not differ between spawns of a pair only and ones that include at least one streaker (n = 23, t-test, p = 0.67). 52

Figure 3.7: Proportion of spawns including a streaker from each of the four treatments of the field manipulation experiment. Streak spawning occurred at significantly higher rates in the high density, mixed size structure treatment and the presence of small fish drove the incidence of streak spawning (streak*density (hi/lo): Chi-sq=0.743, p=0.388; streak*structure (equal/mixed): Chi-sq: 3.930, p=0.047; streak*size (small/large): Chi-sq: 17.183, p<0.0001). 53

vii

Figure 3.8: Proportion of eggs fertilized during the manipulation experiment, with pair spawns that included at least one streaker (black bar) and those that were only a pair of fish (white bar). Pair spawning fish had a greater proportion of eggs fertilized than those with streak spawners (t-test: n = 27, t=3.14, p=0.012). 54

Figure 3.9: Body angle of female fish during gamete release, with pair spawning only (black bar) and pair spawners with streakers (white bar). Body angle was significantly greater for females when streak spawners were present (n = 32, Watson-Williams test, p = 0.041). 55

Figure 3.10: Body angle of male fish during gamete release, with pair spawning only (black bar) and pair spawners with streakers (white bar). Body angle was not significantly different for males in pairs or when streak spawners were present (n = 21, Watson-Williams test, p = 0.87). 56

viii ABSTRACT

The use of an alternative male mating tactic is frequently discussed as an option that makes the best of a bad situation for disadvantaged males and not as part of an adaptive system that can produce equal average levels of reproductive success for individuals using each tactic. It is easy to develop scenarios in which some combinations of tactics produce greater success than others; the difficulty of testing hypotheses about these scenarios and the optimal distribution of tactics revolves around the difficulty of understanding how tactics are expressed. To be specific, it is unknown whether alternative behaviors are conditional and if conditional, how an individual assesses and ultimately chooses which tactic to use in any given context. Under conditions of limited mate availability, variable resource allocation or altered physiological response (e.g. temperature or poor food quality), one might expect variation in mating patterns, but the direction and extent of these variations is relatively unknown.

In the simultaneously hermaphroditic marine fish, Serranus subligarius, male role individuals are known to pair spawn, group spawn and streak spawn. These mating strategies are common among marine reef fish and their behavior has been well studied. What is unclear is how each behavior translates into reproductive success and how these competing strategies are maintained within the same population. This research will focus on the use of alternative mating tactics among simultaneous hermaphrodites. The majority of simultaneous hermaphrodites mate in pairs, often cross-fertilizing each other. Alternative tactics among external fertilizers include group spawning, sneak spawning (males mimic females), or streak spawning, in which a male releases sperm over a spawning event already in progress. In this dissertation I examined the ecological and social contexts under which alternative tactics are utilized and their resulting outcomes with respect to mating behavior and fertilization success.

Chapter one investigates the role of simultaneous hermaphrodites in sexual selection and reviews several studies in which sexual selection is revealed in systems with equal allocation to sex. I

ix discuss ways in which sexual selection has been measured and suggest ways in which this may be modified in hermaphrodites. I also indicate ways in which males compete and females are chosen in these systems, despite their ability to mate with any other individual. Finally, I implicate some of the potential causes of sexual conflict in my study system; namely, that individuals vie for the male role and larger fish spawn more frequently in the male role.

In chapter two, I explored the ecological and physiological contexts under which streak spawning occurs and discuss their relative importance. Size structure was characterized at each of three sites within the field study site and was subsequently used as the impetus for the experiments performed in Chapter 3 to tease out its relevance. Size structure appears to play an important role in the occurrence of streak spawning, as more streaking appears in the sites, which have greater than 20% of individuals comprised of the small size class (measured by density transects at three sites). This chapter also aims to quantify the type of habitat over which a pair spawns and whether or not this differs when additional males participate. I found that at one of the sites, there was a preference for streak spawners when macroalgae was present than when the rocks were bare. In addition, this chapter demonstrates the peak spawning times to be July and August, which were confirmed by both field observations and investigation of the gonads of actively mating individuals.

In chapter three, I investigated the fertilization success of animals spawning with and without streakers participating, both in the field and in a controlled manipulation experiment. In the field I find that fertilization rates are lower in fish spawning with multiple males participating. I confirmed this finding under more controlled conditions in a field manipulation experiment. Rock rubble reef plots provided substrate on which I tested the role of density and size structure on the frequency of streak spawning and resulting fertilization success. Spawns with additional males resulted in lower fertilization success and the number of streak spawns was significantly greater when small size-class individuals

x were present. Thus, both field and experimental evidence suggest that small individuals predict the incidence of streak spawning and their participation lowers fertilization success. Reduced fertilization success means that both males and females incur a fitness cost. The mechanism for lowered success remains unclear but likely includes the physical disturbance created in the water column by the activity of additional males.

The results of this dissertation suggest that there may be a cost to certain mating systems and the contexts under which these different tactics are utilized are dependent on ecological, social and physiological factors. It points to the complexities of individual variation in behavior and the need for more rigorous experimental work to tease apart the factors associated with variation in mating tactic.

Interactions among environment, social group, and ontogeny are not often addressed simultaneously in study systems and more empirical work in these areas would help elucidate the mechanisms associated with the use of alternative mating tactics.

CHAPTER ONE

GENERAL INTRODUCTION

SEXUAL SELECTION IN SIMULTANEOUS HERMAPHRODITES

Background

Hermaphroditism is a widespread sexual allocation strategy among taxa of plants and invertebrates (Ji et al. 2004, Henter 2004, Scharer et al. 2004; Parachnowitsch and Elle 2004; Xhang and Jiang 2002), but is relatively rare among vertebrates, with the exception of the teleost fishes (Charnov 1979; Fischer 1981). Shifts in allocation to male and female function can lead to several outcomes with respect to operational gender, such as protogyny (female to male), protandry (male to female), self-compatible simultaneous hermaphroditism (both male and female gametes produced at the same time, selfing probable) and self-incompatible simultaneous hermaphroditism (both male and female gametes produced at the same time, selfing unlikely). In many cases, differences in environmental and/or social contexts can drive variation in sex allocation. When sex allocation is skewed toward one sex, selection may act to increase gamete output for that sex. If one sex role is favored, the strength of sexual selection may then be stronger for traits associated with that sex (ability to acquire mates, increased gamete number, color pattern). Sexual selection was originally discussed by Darwin (1859) as a struggle between males, which results not in the death of the losing competitor, but in fewer offspring. This definition, while somewhat vague, was inclusive of all taxa, including plants and simple invertebrate systems. In his later discussion of sexual selection, Darwin discounted hermaphroditic systems (including plants and simple invertebrates) as excepted from this phenomenon, as it was not thought that males in these systems competed (but instead have equal access among individuals) or that females were choosing mates based on attractive male traits. Male-male competition and female choice have been at the crux of most studies of sexual selection for decades and are thought to be key factors contributing to sexual selection. Some

1 researchers have created definitions based simply on competition for mates (Leonard 2006). One of the more widely used definitions today is that of Arnold (1994), which states that it is selection that arises from differences in mating success (mates or progeny). Thus, any traits that act to confer higher mating success for the male or female may be under sexual selection, including cues to attract a mate, parental care, or cryptic female choice. There is a wealth of theory on hermaphrodite mating systems and sex allocation as applied to both plants and animals (Charnov 1979, 1982; Fischer 1981; Warner 1984; Ghiselin 1969; Zhang and Jiang 2002). Much of the theory on sexual selection, however, deals with gonochoristic and sex changing species, who have more apparent differences in reproductive success based on sexual pattern (Marconato and Shapiro 1996; Taborsky 1998). Studies of sexual selection in simultaneously hermaphroditic groups, however, have lagged behind, largely due to the broad assumption that sexual selection doesn’t act on organisms that reproduce equally in both sexual roles (Darwin 1871; but see Zhang and Jiang 2002 for role of sexual selection in the plants). This assumption has been debunked over the past decade as mounting research has revealed clear differences in mating success between male and female role individuals, creating ample opportunity for sexual selection (Petersen 1991, 2006; Angeloni 2003; Leonard 2006). Measuring sexual selection in organisms is often done by measuring some proxy of reproductive success (such as number of mates or number of eggs produced), then extrapolating that measure to indicate which traits are being selected. The variance in success gives insight into the sex under selection and behavioral observations can determine whether they are due to some type of female choice or male competition (for example). Crow’s index is a commonly used method to measure the intensity of selection and considers the variance in progeny (Crow 1958). More recently, Shuster and Wade (2003) published a book, which utilizes the format

Imates as the measure of mating success. While this works well for gonochores, it is unclear how this can be adapted for use in a hermaphroditic system, since the measures are based on the fraction of male success and in hermaphrodites, there has to be some account for the success as both male and female (which may not have the same upper limit). For example, under conditions of sperm competition, sexual selection in hermaphrodites may act upon male traits that allow for greater output of sperm or closer access to the female (and thus, her eggs). When there is variation in success based on body size, a simultaneous

2 hermaphrodite is predicted to invest more in male function or utilize a different mating tactic (Charnov 1979) to overcome the lower success achieved by smaller individuals. Because sex allocation is generally labile in hermaphrodites and sperm is considered a low cost investment, it is often possible for males to adjust their allocation to the conditions of an individual mating event. Increasing the number of sperm per mating event may aid in swamping the competition with respect to fertilization of eggs and selection may act on investment in male gonad under conditions of sperm competition. Gonad allocation has often been used as a predictor of mating system as well as a predictor of the preferred role in hermaphroditic mating systems, whereby significant increases in testis size indicates a multiple male mating system and a preference for the male role. It has been argued, however, that prior experience in mating may be a more important predictor than sex allocation (Anthes and Michiels 2004). Accurate measures of sex allocation in simultaneous hermaphrodites is often difficult as the ovotestes is typically an intertwined structure and it has been argued that sex allocation has simply not been measured with the degree of accuracy required to make such predictions in most hermaphroditic systems (Scharer 2009). When allocation increases linearly with body size and does not appear to be adjusted on a per mating basis, the individual may employ an alternative tactic. These tactics may include sneak mating (Leonard 1990), whereby individuals mimic the female behavior/coloration to gain undetected access to the pair male or streak spawning (Oliver 1997; Warner 1984), which means that externally fertilizing animals release sperm over a mating event already in progress. While there have been many studies that have shown these types of tactics to be effective at gaining fertilizations (Petersen 1991; Wooninck et al., 2000; Lorenzi and Sella 2006), it is unclear to me whether one needs a separate measure of selection for individuals who fall outside the dominant mating system. Alternative tactics have been touted as a mechanism by which hermaphroditism is maintained, but alternative tactics are widely used in taxa that are not hermaphroditic, so this claim may not be well grounded in empirical data. Competition for mates is a key piece of evidence for sexual selection and measuring variance in reproductive success. Following these competitive matings can give insight into the level of sexual selection. The long-held assumption that the advantage of hermaphroditism was that one could mate with any other available mate is far too simplified. Data suggest that most often, there is clear male-male competition and/or female choice in these systems (Michiels

3 1998; Anthes 2008). Sperm competition can lead to increased female promiscuity and increased male sexual traits (Gage 1994; Snook 2005). Empirically, however, it can be hard to tease apart these components which often occur at the same time. Lorenzi and Sella (2008) have documented clear female mate choice and sperm competition in polychaete worms and suggest that females can discriminate sperm quality. In land slugs, love darts and their associated organs have apparently coevolved in a sexually antagonistic arms race matching that of single sex species under sperm competition. These conditions not only shape the mating system, but can offer insightful tests of selection theory that have previously been ignored.

Measures of fitness that are used in sexual selection

Crow’s index (Imates), measures the opportunity for selection and is the ratio of variance in progeny number to the squared mean number of progeny. This appears to work well when measuring potential for selection in gonochores, as it incorporates Bateman’s principles with respect to the variance in reproductive success among males and females (discussed in more detail later). This index might be appropriate for simultaneous hermaphrodites if the investment in male and female function were equivalent. This is commonly seen in pair mating organisms or spawners who are not in close proximity to one another. In these cases, more sperm may be needed to reach all of the eggs. Sometimes, however, this investment is not equal, particularly in reciprocal maters, who tend to invest more in female function, as males only need to invest in enough sperm to fertilize all eggs. This idea forms the basis for the local mate competition theory, as it was formulated by Hamilton (1967) and revised by Charnov (1982). In cases where there is variation in mate competition, selection would favor the ability to adjust allocation to increase a male’s ability to fertilize the most eggs. In addition, allocation to a particular sex can depend upon both social context and available resources. All of these components make the measurement of sexual selection in hermaphrodites very difficult. One study where these expectations under different conditions were tested compared monogamous and promiscuous populations of the polychaete worm, Ophryotrocha diadema (Lorenzi and Sella 2008). This worm is an out-crossing, pair-mating hermaphrodite who takes turns laying successive clutches of eggs. In low density, pairs are monogamous while in larger groups, they are promiscuous. In an elaborate experiment, which aims to separate the rearing environment from the functional sex role, they find that the potential for selection in these

4 outcrossers depends on the reproductive context. Under monogamy, selection is relaxed and reciprocity is a solution to sexual conflict (as has been suggested by Leonard and others). They also report that the opportunity for selection in many simultaneous hermaphrodites (across taxa) is generally low, when compared to mammals and other gonochores. In the promiscuous populations, the opportunity for selection was greater, even more so for female function.

Sexual selection versus fertility selection Fertility selection refers to the number of offspring produced by a mating and it may depend on the maternal and/or paternal genotype. A significant component of natural selection is fertility selection and ignoring it when measuring selection can often lead to very large differences in success, particularly in animals for whom parental care is a part of their mating system. It is important to consider fertility selection when discussing differences in reproductive variance between males and females because fitness gains are applied to a mating pair as opposed to separate sexes or roles (Shuster and Wade 2003). Interactions between males and females may affect an individuals’ fertility without necessarily affecting the variance in reproductive success. Mate number may not alter the variation if, for example, a male mates more frequently with females who have produced fewer eggs. This has lead some to use individual fitness as a measure in the case of hermaphroditic animals, but Shuster and Wade (2003) warn against using the individual fitness as a measure, particularly when there is a great degree of context dependence incorporated into the fitness. This can lead to incorrect evolutionary expectations and often assumes that all traits associated with a particular phenotype are adaptive. The use of individual quality traits, however, can give more information under variable conditions.

Bateman’s Principle: Bateman (1948) suggested that a female’s reproductive gain curve will be dependent upon available resources, whereas the male gain curve will be limited by the number of eggs available to him. This may be shifted when sperm becomes a limited resource. This also assumes that sperm production is not costly to a male. Because eggs are the limiting resource

5 for males, competition for access to a female’s eggs frequently occurs. This leads to a situation of male-male competition, a key factor in sexual selection. If, however, a female’s fitness increases with multiple mates, variation in reproductive success also increases and the male and female curves will look similar. This breaks down the predictions of Bateman’s principle

150 140 120 100 100 80 60 50 40 20 0 0 1 2 3 1 2 3 4 Number of offspring Number of offspring Number of mates Number of mates

Figure 1.1: Comparisons of the Drosophila data of Bateman’s (1948) paper describing the conditions under which variation in mating may differ among males and females leading to sexual selection on traits that confer greater fitness (solid lines, modified from Bateman, 1948) to a simultaneous hermaphrodite in which selection is stronger on the male role (arbitrary data- dashed line). The gain curves are steeper for hermaphrodites and more similar for males and females than the gonochoristic flies. The gray dotted line indicates the conditions Charnov associated with hermaphroditism being favored (both male and female fitness curves are saturating).

Does this apply to simultaneous hermaphrodites? Among studies of simultaneous hermaphrodites, mate choice has not been a prevalent topic, as it is widely held that the key advantage to this reproductive condition is the ability to mate with any other individual who is reproductively mature. In the few field studies that have investigated mate choice, however, hermaphroditic animals have been observed passing up matings with mature individuals to mate with another (Petersen 1990; Angeloni 2003). Many simultaneously hermaphroditic systems exhibit some form of reciprocity, whereby individuals trade eggs and often vie for a particular role (Oliver 1997, Leonard 2006). This can lead to sexual conflict among male and female role individuals. According to Bateman’s

6 principle, the female role would generally be the preferred role, but in many systems, this is not the case (Leonard 1990; Petersen 1991; Fischer and Petersen 1986). In order to apply Bateman’s principle to simultaneous hermaphrodites, one must consider male and female roles separately (Figure 1). Alternatively, hermaphroditism may be a strategy for reducing variance in reproductive success and this may fall more in line with Charnov’s predictions that hermaphroditism would evolve under conditions where Bateman’s principle did not apply (Figure 2). If there is a preference for a particular role, which is the case for many hermaphroditic species (Leonard 2006, Petersen 2006), then one would expect the curves to be steeper for one sex over the other (Figure 1). When matings are reciprocal (individuals assume both roles during mating bouts), variance in reproductive success can be restricted (Anthes et al, 2010). Charnov (1979) graphically described the fitness trade-offs of hermaphroditism and dioecy by suggesting that where fitness increases proportionately to investment in role, a concave fitness curve will result and where fitness of one sexual role does not interfere with fitness of the other, a convex fitness curve will result (Figure 2). If this is true, then what are the conditions under which each of these scenarios will occur? Low population density and low mobility have often been touted as the conditions favoring hermaphroditism (Ghiselin 1969). It has also been suggested by Leonard (1990) and others that dioecy produces greater variance in reproductive success and hermaphroditism may act to reduce this variance in offspring fitness. In addition, if the fitness curves of both males and females saturate over multiple matings, then hermaphroditism will be favored (Charnov 1979). These predictions would violate Bateman’s principle, as he predicted a linear increase for males. The saturating curve of male fitness is consistent with the conditions described above (low mobility, low population size) and could also apply to plants who have a limited number of pollinators.

Figure 1.2: Possible trade-offs between male and female function for hermaphroditism (convex line) and dioecy (concave line) as discussed by Charnov (1982).

Sexual conflict One of the complexities leading to deviations from Bateman’s principle in simultaneous hermaphrodites is that individuals often vie for one sexual role. This creates opportunity for sexual conflict and can lead to greater variation in reproductive success than that expected from Bateman’s principle. In the case where individuals vie for the male role and females can control the number of eggs released, one would get contradictory results as compared to the expected Bateman gain curves. This was first analyzed by Charnov (1979) who stated that where you have a preferred sexual role, sexual conflict may arise due to the motivation to give sperm away in order to gain access to eggs and this conflict may shape the mating system. Part of the conflict in externally fertilizing animals is the result of males controlling fertilization by virtue of releasing sperm over previously spawned eggs. The gamete-trading model suggests that the role that controls fertilization is the preferred role. For internally fertilizing animals, the preferred role can depend upon the latency between sperm deposition and fertilization. In some species, females may choose deposited sperm from multiple males and exert the control in fertilization, leading to much of the sperm being wasted (thus female role is preferred). When a partner leaves the pair mating before releasing or allowing access to eggs, they have failed to reciprocate and this is noted as direct evidence of sexual conflict in several hermaphroditic species (Fischer 1980, Axelrod and Hamilton 1981).

8 The Hermaphrodite’s Dilemma model (Leonard 1990) predicts that reciprocity will evolve in mating systems where there is a clear preference for one sexual role. In this model, conditional reciprocity results from partners assessing each other prior to mating and the choice to mate is based upon either the condition of the partner or the willingness of the partner to reciprocate. There are several examples of conditional reciprocity and measures of its evolutionary stability in polychaetes (Lorenzi and Sella 2008), terrestrial gastropods (Leonard 1990), and fish (Petersen 1991; Oliver 1997; Adreani, unpublished data). What is difficult to include in such a model, however, is the effect of animals that don’t reciprocate, or cheat. While game theory models (particularly Prisoners dilemma) have aided our understanding of how animals in mating games assure honesty, there is no clear way to explain defection by way of not forming a pair (as is the case with a streak spawner) or by leaving the pair. In addition, one major assumption of this model, that each partner is equally likely to act first, is violated in at least two species of Serranine fishes, S. tabacarius and S. subligarius (Petersen 2006). Because alternative tactics are common in this family of fishes (and others), it seems that models including this phenomenon must be utilized to make accurate predictions as to the success and stability of the mating system. Given that sexual conflict is typically evidenced by the occurrence of mating reciprocity (a potential resolution to strong sexual conflict), direct measurements of conflict are typically not given. One way to get better detail on the presence and/or strength of conflict is to have very detailed observations of matings in the field (i.e. natural environment). From these observations, one can get a better idea of the motivation for deserting a mating. In other words, if an animal deserts before reciprocating due to predator presence or other stimulus unrelated to mating, it should not be confused with a desertion motivated by an unwillingness to mate with a particular partner.

Serranus subligarius-a case study The belted sandfish, Serranus subligarius, is an egg-trading simultaneous hermaphrodite, whose typical mating system consists of pair spawning in bouts of 2-5 spawns, with at least one sexual role trade (Oliver 1997). Larger individuals (>70mm) defend territories during the mating season and small individuals frequently streak spawn. Individuals assess each other during a relatively long courtship period, during which each role is determined for the first

9 spawn. Mating is considered to be conditionally reciprocal, whereby mating is performed on the assumption of reciprocity. Individuals, however, vie for the male role (violating part of Bateman’s principle) and the larger individual in an asymmetrically matched pairing, will spawn more frequently in the male role (Oliver 1997). Overall fertilization rate of a pair spawn is high, owing in part to the pairs’ close proximity to one another upon release of gametes (Chapter 3). When streak spawners participate in the spawns, however, overall fertilization rate is lower than that of a pair spawn (Chapter 3), suggesting a cost to both male and female reproductive success. This is contrary to most studies that have found similar or higher fertilization with additional males participating (Taborsky 1998; Wooninck et al. 2000). One assumes that selection would favor traits that allow a male to gain closer access to a female (in the case of external fertilizers) as this has been shown to be the most significant factor contributing to the majority paternity share, even more so than contributing more sperm (Wooninck et al. 2000). This makes some intuitive sense if one considers the environment. In aquatic organisms and sessile terrestrial organisms such as plants, dilution and turbulence could have major effects on the distribution of sperm in a short amount of time. A competing male may release a greater concentration of sperm, but if it is distributed away from the release of eggs, it is a wasteful investment. This may be the case in S. subligarius, where streakers swim up and over the spawn in progress, release sperm, while at the same time disrupting both the behavior of the pair mates and the water surrounding the gametes. Given that these small males do not reciprocate as a pair spawner and if one includes them as part of a game, they may be considered defectors of reciprocity (Prisoner’s dilemma). Asymmetries in mating success, such as those associated with cheaters or where a clear size advantage exists would seemingly weaken the stability of egg trading. A large male phenotype could theoretically invade the population and break down the conditions of reciprocity if large males obtain more mates and larger mates, significantly increasing their reproductive success. Functional males happen rarely in this group of fishes and require certain environmental and social conditions to be maintained (Fischer and Petersen 1986; Petersen and Fischer 1996). In the two species that exhibit harem polygyny, S. baldwini and S. psitticinus, high density and spatial predictability appear to drive male monopolization of a number of hermaphrodites, resulting in loss of ovarian tissue for the large, dominant male (Fischer and

10 Petersen 1986). If this is true, then we can predict seeing a similar relationship between mating system, density and spatial distribution in other species within the Serranines, where such variation exists. For S. subligarius, however, these predictions do not appear to hold true. Egg trading has been demonstrated to be stable (Oliver 1997), despite variation in density and distribution, largely attributed to their requirement for reciprocal mating. While larger male role individuals mate more frequently with larger females and selection seems to favor traits that confer greater fitness for the male role, the large male phenotype has not invaded this system. One apparent difference between S. subligarius and the haremic species is the occurrence of alternative tactics and thus the level of sperm competition, which is fairly high in S. subligarius (up to 30% of all spawns) and very low (< 10% of all spawns)or absent in S. baldwini and S. psitticinus. Where streak spawning tactics are utilized with some frequency, it may be difficult for a large male to monopolize the matings of several hermaphroditic individuals. Perhaps defending a large territory, in addition to multiple mates is too energetically costly. The possible fitness advantage of a haremic system may therefore be dampened by the participation of multiple males. The other component lending to the stability of reciprocity is the parceling of eggs over the course of a spawning bout. In S. subligarius, batches of eggs are released over multiple matings throughout the day, while in the haremic species, large males mate multiply, but female role hermaphrodites release only a single parcel per day (Fischer and Petersen 1986). While recent studies (Hart 2010; Adreani, Chapter 3) on mating systems and behavioral ecology of the hermaphroditic seabasses lend greater insight into the traits associated with sexual selection and the strength of that selection, there is still much more data needed to ascertain lifetime fitness and how this changes across variation in mating system. In addition, the theoretical framework laid out by Charnov (1979), Fischer (1981) and others has been supported by many of the studies thus far, but there is a great need to properly include alternative tactics, asymmetries in reciprocation and preference for a particular sexual role to tease out the selective forces acting on both male and female role individuals under these circumstances. Sexual selection has often been discounted in hermaphroditic systems (including plants and simple invertebrates) as it was not thought that males in these systems competed (but instead have equal access among individuals) or that females were choosing mates based on

11 attractive male traits. Male-male competition and female choice have been at the crux of most studies of sexual selection for decades and are thought to be key factors contributing to sexual selection. Any traits, however, that act to confer higher mating success for the male or female may be under sexual selection, including cues to attract a mate, parental care, or cryptic female choice (Arnold, 1994). There is a wealth of theory on hermaphrodite mating systems and sex allocation as applied to both plants and animals (Charnov 1979, 1986; Fischer 1981; Warner 1984; Ghiselin 1969; Zhang and Jiang 2002). Much of the theory on sexual selection deals with gonochoristic and sex changing species, who have more apparent differences in reproductive success based on sexual pattern (Marconato and Shapiro 1996; Taborsky 1994). Studies of sexual selection in simultaneously hermaphroditic groups, however, have lagged behind, largely due to the broad assumption that sexual selection doesn’t act on organisms that reproduce equally in both sexual roles (Darwin 1871; but see Zhang and Jiang 2002 for role of sexual selection in the plants). This assumption has been debunked over the past decade as mounting research has revealed clear differences in mating success between male and female role individuals, creating ample opportunity for sexual selection (Leonard 2006; Petersen 1991, 2006; Angeloni 2003). Mating system theory predicts that differences in mating strategy within a population of simultaneous hermaphrodites are the result of sex allocation differences affected by body size (Charnov 1982, Angeloni 2003). Individuals should invest more in male function when small and more in female function when large. Small individuals who invest more in male function can skew paternity patterns by swamping matings with increased levels of sperm and this can often lead to alternative strategies, such as streaking and sneaking, to gain access to females that are paired with larger individuals, in populations that are typically pair spawners. These alternative tactics, however are not well understood in simultaneous hermaphrodites that do not show strong differences in sex allocation or differential mating based on body size (Oliver 1997, Hastings and Bortone 1980). This gap in our understanding of these mating systems leads to a central question of my research program, which is: when sex allocation does not differ strongly, what are the conditions associated with the use of alternative mating tactics? Variation in mating pattern may be expected when resources are variable, mate availability is limited or ontogenetic patterns alter development and behavior (Emlen and Oring, 1977). For example, passerine birds have

12 been shown to form long term pair bonds in habitats where highly productive resources are concentrated and thus, more easily defended by a territorial male (Verner and Wilson, 1966). In contrast, habitats with broad, sparse resources, appear to favor promiscuous mating systems (short-term pair bonds, multiple matings). Additionally, many animals utilize one mating system when young (e.g. promiscuous) and another once older, when they are able to acquire a territory (e.g. polygynous with long term pair bond). Behavioral phenotypes are often labile and the conditions under which we see variation in mating type may be heavily context-dependent. Here I describe a series of field observations, collections and field manipulations designed to investigate the following questions: (a) How do density and size structure affect the use of mating system? (chapter 2 and 3) (b) How does seasonality affect the use of alternative mating tactics? (chapter 2) (c) Under what environmental conditions are alternative mating tactics used? (chapter 2). (d) How can fertilization dynamics help to predict mating system and allocation to a given sex? (chapter 3).

13 CHAPTER TWO

DO SIZE STRUCTURE, HABITAT AND EARLY DEVELOPMENT INFLUENCE THE USE OF ALTERNATIVE MATING TACTICS?

ABSTRACT

While the use of alternative male mating tactics is common among marine reef fish, there are particular instances in which their employment is poorly understood. One of these instances is in simultaneous hermaphrodites in which some individuals attempt to mate only as males, acting surreptitiously, that is, as “streakers” invading a pair spawn. This situation creates a vastly different social context for the expression of mating preferences and sexual selection. I explored the ecological and physiological associations with streaking behavior in Serranus subligarius, a hermaphroditic seabass. In this species, individuals acting as males may pair spawn, group spawn or streak spawn. During the mating seasons of 2006-2008, I examined mating behaviors at three sites known to vary naturally in density. During the first field season, I recorded densities and size structure (small, medium, large) at each of the three sites. In the 2007 and 2008 spawning seasons, I also recorded the incidence of pair spawning and streak spawning and the type of local habitat over which each of those spawns occurred (bare rock or algal covered rock). In 2008 and 2009, samples of spawning fish were then collected and sacrificed to investigate the development of the gonads. The incidence of streaking was highest at sites with a greater number of individuals in small size classes (> 20% of the local population). Streaking rates increased late in the spawning season (late July-early September) when there is a mosaic of algae-covered and bare rock. More streak spawning occurred over algae-covered rock than over bare rock. While young fish (< 1 year) do pass through a male only phase early in their development, these fish have sexually mature gonads (male and female) during the time that streaking is at its highest frequency. The streaking behavior, then, is likely the result of an inability to obtain a territory (due to small size) and successfully court a female role individual. Streaking occurs at a higher frequency where there is an appropriate place for these fish to hide (dense macroalgae) and avoid direct interaction with larger, territorial fish. Small fish take advantage of spawning

14 opportunities, without reciprocation in many cases, in their first year, despite the cost of high levels of sperm competition.

15 INTRODUCTION

Several ecological factors can influence the expression of mating systems. Resource availability, habitat structure and the species in the surrounding communities may determine the densities, the distribution of females, as well as the suitability of territories or nesting sites (Sale 1990; Shuster and Wade 2003), all of which can play a role in shaping mating patterns. Preferences for habitat, spawning mode or recruitment patterns may induce trade-offs in allocation to resources (Sale 1990; Steele 1997; McDermott and Shima 2006) and have a strong influence on behavioral patterns and result in variation in the outcome of matings. The distribution of individuals across the local habitat can provide insight into the contexts under which animals use different mating tactics. Through these effects, these factors may also influence the adoption and success of alternative mating tactics (Taborsky 1994; Birkhead and Moller 1998; Shuster and Wade 2003; Angeloni 2006). The use of different mating tactics is most likely to occur when there are differences in mating success affected by body size and smaller-sized individuals can mate successfully by using tactics different from those used by larger individuals. Alternative mating tactics are usually context dependent but occur at a lower frequency than the standard mating system. These tactics have been described as a reproductive parasitism (Taborsky 1994) and are often associated with high levels of sperm competition (Birkhead and Moller 1998; Smith et al. 2003). They are also expected to occur when there is high variability in local environment that influences mating success within an individuals’ lifetime (Shuster and Wade 2003). Under the conditions typically associated with the use of alternative tactics such as limited mate availability, variable resource allocation, or altered physiological response (sometimes a result of ontogeny), one might expect variation in mating patterns and this is seen in a wide range of animals (Nicholls et al. 2001; Petersen and Warner 2002; Angeloni 2006) and plants (Parachnowitsch and Elle 2004; Zhang and Jiang 2002). Alternative tactics and the extent of variation in success, however, are not well understood in simultaneous hermaphrodites that do not show strong differences in sex allocation or differential mating based on body size (Hastings and Bortone 1980; Leonard 1990; Oliver 1998; Petersen 2006). One example of how ecological shifts can influence mating system is seen in Serranus psittacinus, a hermaphroditic marine reef fish. Under conditions of patchy habitat, there is specialization of the male role, such that an individual acts behaviorally as a male and mates with simultaneous

16 hermaphrodites in a harem style mating system (Fischer and Petersen, 1986). When resources are more homogeneous, individuals form pair bonds and alter male and female roles equally. Identifying similar patterns among behavior, physiology and life history allows the examination of processes that shape their evolution. This research aims to explain some of the mechanisms that influence the use of alternative mating tactics in a simultaneously hermaphroditic marine fish. I adapted the conditions discussed above (mate availability, resource allocation and physiology) into locally relevant factors for the fish being studied. Specifically, the factors explored were density, size structure, small-scale habitat differences and ontogenetic changes in gonad development. The following questions were asked for each factor: (a) do density and size structure vary among sites and does it influence the occurrence of streak spawning? (b) do small scale habitat differences alter the use of alternative mating tactics? and (c) do streak spawners have active, mature eggs and are they able to reciprocate during spawning events?

METHODS

Study species The seabasses are a diverse group of teleost fishes of the family Serranidae, whose mating systems are equally diverse and cover the spectrum of mating types, from monogamous pair spawning to very large group spawns and they also show great lability in their allocation to male and female function. The species used here to explore the mechanisms driving the use of alternative tactics is the belted sandfish, Serranus subligarius. They are commonly found in shallow waters of the northern Gulf of Mexico and on the Atlantic coast up to North Carolina (Clark, 1959). They tend to be associated with hard substrate and live up to about 4 years (Hastings and Bortone, 1980). They are simultaneous hermaphrodites who typically pair spawn (courtship and spawning with one other individual), but utilize streak spawning (swimming up and over a pair spawn already in progress and releasing sperm) and occasional group spawns (2- 3 male role fish engaged in active courtship with a female role fish; Oliver, 1997). Group spawns were rarely seen at the study site, so only pair and streak spawning will be discussed. Mating in S. subligarius consists of pelagic spawning rushes, over rocks, in bouts of 2-5 spawns, with at least one sexual role trade. Over the course of a 30-minute period, a set of 4 to 6

17 spawns may occur. They form temporary pair bonds but will also mate with neighbors and the larger territory holders are often in conflict with smaller fish for access to, and guarding of, mates (Oliver, 1997; Adreani, Chapter 3). Individuals mate multiple times throughout the day during the summer spawning season, late May through early October. Study site The study site used was St. Andrews State Park, near the channel opening to St. Andrews Bay, Panama City, Florida, along the northeast Gulf of Mexico (Figure 2.1). Field data were collected along a rock jetty on one edge of the park, which allowed for separated regions that differed in natural local population density and could easily be accessed by a short swim. Depth varied among the sites from 3-10m and also varied with tidal height and season. The separated regions were labeled inner jetty, lagoon, and outer jetty. The inner jetty site was characterized by open water, large shallow boulders and low density (approximately 0.03 fish/m3). The lagoon site consisted of shallow calm water, smaller boulders and intermediate density (approximately 0.11 fish/m3). The outer jetty, which is the slightly deeper site at approximately 8 m is the high- density site (approximately 0.23 fish/m3) and consisted of open water and large boulders. Spawning pattern During the months of May through September of 2006-2007, the number of individuals at each site was recorded weekly by swimming a permanent 50 m belt transect. Fish observed below the diver and 1 m to either side of the transect were noted on an underwater slate. In addition, the relative size of each individual was recorded. Small (S) corresponded to fish between 45-65 mm standard length, medium (M) were fish between 65-80 mm standard length and large (L) were those greater than 80 mm. At the beginning of each spawning season, 12-15 fish were observed underwater, sized, then captured and measured for verification of size estimates. Those same sites were used to record the number of mating events (spawns per day) witnessed. Each event was recorded as a pair, group or streak spawn (though group spawns were extremely rare) and the relative size of the pair spawning individuals was noted along with the presence of additional individuals, in the case of a streak spawn. At each of the three sites described above, spawning events were observed and the following information was collected: spawning rate (number of spawns per day), whether or not a streak spawner participated, the size class of the fish involved in each spawning bout. Divers would swim around a designated area (which differed during each sampling period) and stop

18 when any courtship or spawning was observed. For each observation, a focal individual was observed for approximately 15 minutes. When a complete spawning bout was witnessed, the number of times each focal fish was in the male or female sex role was noted. Each of the three sites was visited 1-2 days per week from May through October (or until spawning activity ceased). On days that density transects were done, spawning observations were made following the completion of the transects, at haphazardly selected sites in the surrounding area. In addition, local sea surface temperatures, timing of high tide and height of high tide were obtained from a nearby National Oceanic and Atmospheric Association (NOAA) buoy (< 1 km from field site). This allowed for the exploration of additional environmental correlates with respect to spawning pattern. Habitat For each spawning event witnessed at the outer jetty site, the habitat type directly below the courting and subsequently spawning individuals was recorded. More specifically, it was noted that the rocks over which they were courting were either algal covered (high relief macroalgae) or bare (largely the result of urchin grazing). I utilized the length of the permanent density transect as a reference point and the area approximately 2 m on either side of it in order to encompass a large number of patches, both with and without algae. This habitat appeared as a mosaic of bare/covered patches by the later part of the summer (August –September). Both habitats were usually present within a large fish’s territory (approximately 1m2). Gonad development During the 2008 and 2009 spawning seasons, approximately 20 individuals were collected from the high density outer jetty site per month (May through October) and approximately six individuals were collected from the low density inner jetty site per month, to avoid impacting the behaviors of the already low population size. Fish were captured using a baited hand net and brought to the surface, where they were euthanized by bathing them in an overdose solution of MS-222. They were then held on ice until back at the lab where they were dissected in order to remove gonadal tissue and otoliths. Otoliths were removed to verify that fish size was a good representation of approximate age. They were dried, sanded and read under a dissection microscope for evidence of annual rings indicating fish age. The readings were taken by two individuals on separate days and recorded independently, compared and read a third time where there were discrepancies.

19 Gonadal tissue was blotted, weighed and placed in a buffered formalin solution to preserve it for histological processing. A subset of samples, containing those gonads of each of the relative size class individuals were sent to a histology lab at Louisiana State University for sectioning and slide preparation. Tissue was processed using standard paraffin embedding, followed by staining in haemotoxylin-eosin. Both cross-sectional and longitudinal sections were taken, where possible. The slides were then observed under a compound microscope for evidence of egg and sperm development. Gonads were classified into five categories of maturity (modified from Sadovy and Shapiro, 1987; Erisman et al, 2010); immature; mature testis only; mature inactive; mature active; mature postspawning (discussed in detail in the results section). Gonads from each collection month were pooled across years.

RESULTS

Spawning pattern In 2006 and 2007, fish densities were recorded and compared to streak spawning activity across the spawning months (Figure 2.3). While there was high variation in density at the outer jetty site, there was no significant correlation between density and streaking at any of the sites

(Figure 2.4; (a) Inner jetty: Pearson’s correlation, r15 = -0.147, p = 0.875; (b) Lagoon: Pearson’s correlation, r9 = 0.051, df = 1 p = 0.323; (c) Outer jetty: Pearson’s correlation, r13 = 0.079, df = 1, p = 0.192). Recorded densities are clearly autocorrelated across these sampling events, which can present a statistical complication. The evidence presented here suggests that density is not an important factor and additional tests would tend to make these results less significant. During those same spawning seasons, a combined total of 1,092 spawns were observed. Of those observed spawns, only five represented group spawns. Pair spawns were observed most frequently, with 841 out of 1,092 spawns (77%). Streakers participated in approximately 22% of all spawns that occurred. During the 2006 reproductive season, spawning activity was recorded (# spawns observed) and plotted with sea surface temperatures, pooled across sites (Figure 2.2). Spawning activity was correlated with temperature (Pearson’s correlation, r88 = 0.56, p = 0.017) and tidal height (Pearson’s correlation, r88 = 0.88, p = 0.004), but not very strongly associated with the time of high tide (Pearson’s correlation, r89 = 0.08, p = 0.655).

20 In a spawning bout of 2-6 spawns, partners traded eggs by switching male and female sex roles. During the 2006 spawning season, when pairs were observed of relatively equal size (within 5 mm SL), they spawned in the female role with equal frequency (Wilcoxon signed rank test, Z = - 0.41, p = 0.38, n = 22 bouts). In spawning bouts where partners were of unequal size, the smaller fish was the female role more frequently than the larger one (Wilcoxon signed rank test, Z = - 3.35, p = 0.0001, n = 46 bouts). The daily spawning rate observed varied significantly across three seasonal levels (May+June, July+August, September+October) (Table 2.1a, ANOVA, df = 2, F = 6.021, p = 0.003). While temperature was a significant covariate, season was still highly significant and explains a much larger amount of the variance, even when adjusted for temperature. The May+June level appears to contribute most strongly to the seasonal differences seen. All other comparisons of streak vs. pair spawn reinforced the conclusion that pair spawning is the prevalent spawning mode (Figure 2.5). Very little to no streak spawning occurred in the lagoon, but pair spawning reached peaks in July and August (which did not differ from each other), as indicated by a comparison of monthly spawning activity at that site (ANOVA, df = 5, p = 0.832). In both the inner jetty and the outer jetty sites, streak spawning activity reached levels of well over 30% of the total number of spawns. The proportion of streak spawning that occurred across the three sites and three seasonal time periods were analyzed using ANOVA with temperature as a covariate (Table 2.1b). Site differed significantly and there was a positive interaction of season and site, indicating that a seasonal pattern affects streaking at the different sites. Post-hoc comparisons reveal significant differences between the early season lagoon and late season inner jetty as well as differences between the late season lagoon and late season outer jetty (Tukey test; p = 0.025 and p = 0.005, respectively). Streak spawning activity increased as the spawning season progressed, peaking between late July and early September, then dropping off in the early fall. The inner jetty site and the lagoon exhibit a peak in spawning in July and August, whereas the outer jetty site peaks in August and September (Figure 2.5). Both the inner jetty and the outer jetty had a high incidence of streak spawning, with very little streaking occurring at the lagoon site. Size structure

21 Size frequency data collected from my permanent density transects at each site indicate that medium (65-80 mm SL) and large (>80 mm SL) fish are abundant at all three sites, whereas small (< 65 mm SL) fish are occurring at higher frequency (> 20%)at the outer jetty and inner jetty sites (Figure 2.6). The lagoon had a low occurrence of small fish (< 7%) and this trend remained throughout the summer. The observed proportion of small individuals was significantly greater than a null expectation of evenly distributed size classes (small, medium, large) at the outer jetty site only (chi-square; df = 5, p = 0.037). The frequency of male pair spawning and streak spawning also differed among sites with respect to those size class distributions (Fig 2.7). Habitat Streak spawning behavior occurred more frequently over algal covered rock than over bare rock (Figure 2.8: Chi-sq; 5.20, df = 1, p = 0.023 (2006). Chi-sq; 5.97, df = 1, p = 0.015 (2007)) at the outer jetty site. The visible pattern of microhabitat changed to a more patchy distribution in the later summer months. These changes can be attributed to environmental changes, such as temperature and herbivore feeding activity, and these ideas will be explored in greater detail in the discussion. Pair spawning still occurred over all rocks, but small individuals were rarely seen swimming on or around bare rock. There is no observed evidence that territorial individuals will leave one area to preferentially spawn over one habitat or another. Gonad development Gonads of immature individuals were small, clear, thin and comprised of primary growth stage oocytes and spermatozoa, but lacked spermatids. Gonads of mature inactive individuals were also small and thin, but early stages of spermatocytes could be detected and developed sperm sinuses were visible in the testes. In the ovaries, primary growth oocytes were visible and the gonadal wall was thickened. Mature active gonads were larger, thicker and within the lamellae of the ovaries, all stages of oocyte were present, including the yolk globule stage, while testes were filled with spermatozoa in the testes, cortical alveoli with atretic eggs and gaps in the sinuses were visible from prior spawning (n = 68, Figure 2.9). All individuals were sexually mature when they reach sizes of 65 mm SL. All size and age classes observed spawning had the potential to release sperm and eggs during both pair and streak spawning events, which means that individuals breeding as male role streakers had the capacity to function as females, but may not be doing so. Individuals smaller than those classified as small were not observed spawning and thus were not collected. From

22 June-August, all individuals investigated were in mature spawning condition, with both active testes and ovaries, including the small size class individuals who were primarily streak spawning. Older (2-3 years) and larger (>65 mm) fish exhibited active oocytes at all stages described below and spermatocytes with flagellated sperm (Figure 2.9). Active, mature gonads were present during all months of the spawning season (May-October), but peaked in July and August with nearly all individuals inspected showing evidence of active spawning (Table 2.2). Gonadosomatic indices (ratio of gonad weight to somatic weight), which gave additional evidence for active spawning were also highest in July and August and did not vary dramatically among age or size classes.

DISCUSSION

There are four major findings of this research. First, streak spawning occurs at a higher frequency at both the inner jetty and outer jetty sites and very little streaking occurs at the lagoon site. Both the inner jetty and outer jetty sites have high numbers of small individuals (> 20% of total density), whereas the lagoon site had very few (< 7% of total density). One common explanation for the use of alternative mating tactics is density dependence, such that at high density, males are unable to monopolize matings with females (Petersen and Warner 1982; Oliver 1997; Smith et al. 2003). This work suggests that density is less tightly correlated with spawning type than size structure, thus the presence of small individuals is a better predictor for the incidence of streak spawning. While a previous study noted the difference in size structure between the intermediate-density site and high-density sites (Oliver, 1997), the low-density site (inner jetty) was not used. It is this lower-density site that is of particular importance to this study, as it highlights the differences in size structure and allows for the comparison of a four- fold difference in variation between the low-density and high-density sites. One plausible explanation for this discrepancy in size structure would be that newly recruited individuals are more likely to settle on the rocks closer to the outer edges of the jetty as the currents and tide would deposit them there first. They may not move into the shallow lagoon area until they are bigger and can actively compete for space along the more exposed rocks of the lagoon. There

23 may also be a preference for higher relief habitat structure that is provided by the large boulders of the inner and outer jetty and is less prevalent in the rocky and sandy lagoon site. Further experimental work in this area is needed to rule out other population differences between these sites. Second, the overall spawning pattern of S. subligarius is strongly influenced by both season and temperature, though season has a much stronger effect. This suggests a clear seasonal pattern with respect to spawning frequency and a strong influence from the early season factor (May+June) during which far fewer spawns were observed. The proportion of streak spawning that occurs appears to be most strongly influenced by site and these effects vary with season. Given what is known about the size structure and streaking patterns at each site, these data suggest that one plausible explanation is that more small individuals are recruiting to the outer and inner jetty sites than to the lagoon site. Third, I found that the proportion of streak spawns that occurred was greater when they spawned over algal covered rock by tracking the local micro-habitat during each spawn that occurred along my transect at the high-density site. This makes intuitive sense in that small individuals would need refuge and an abundant food source, both of which could be provided by the algae. Additionally, small fish would not be as detectable by the larger pair bonded fish or by predators when hiding in the macroalgae. (Steele 1997; Verweij et al. 2006). While no direct measurements of algal growth were taken, the bare rocks seen interspersed throughout the jetty were clearly created by the grazing of urchins and this activity increased as the summer progressed, creating a mosaic of covered and bare patches (Adreani, personal observation). This may be due to increased temperatures, increased productivity and thus increased activity levels of the urchins noted in other studies of similar habitats (Eklof et al. 2008). It could also suggest that territorial fish are moving to open space to avoid streakers, but observations of this behavior were not noted in this study. This mosaic pattern appears to create a patchy habitat and may allow an opportunity for additional mating types to be successful, without the shift in sex allocation seen in S. psittacinus (Fischer and Petersen 1986). Finally, ontogenetic changes that occur as the fish comes into maturity do not strongly affect the choice of mating tactic. While it is common for hermaphroditic fishes to go through a unisexual or bisexual phase during their early development (Hastings and Bortone 1980; Sadovy de Mitcheson and Liu 2008; Erisman et al. 2010), it does not appear to play a role in the mating

24 tactic used, as all fish are mature in both male and female role by the time the active spawning occurs. Fish that were born the previous year reach maturity during their first spawning season (usually by early June) and were fully functional as males and females. Given their small size and perhaps their lack of experience, however, they were unable to establish territories. By the middle of the spawning season, they were physically able to spawn in both roles, but may not have the physical space, dominance or experience to complete the courtship process, so they may streak spawn as a way to gain some reproductive success in their first year. It is also possible that many of the small fish do not spawn, even though the ones examined appeared to be able to do so. In summary, it appears that size structure may give us some insight into the occurrence of streak spawning, but the details of this prediction need to be more rigorously tested in order to tease out other environmental influences on the mating system. Microhabitat differences may make streaking a more viable option in certain areas along the reef than others and the success with which an individual mates in its first year may depend heavily on those environmental conditions. Finally, peak spawning occurs in July and August, evidenced by both field observation and investigation of the gonads. While gonads appear to mature in early May, some individuals are not actively spawning until late June or early July. All size and age classes studied appear to actively spawn and there is not the great variation in GSI often noted in mating systems under heavy sperm competition.

25 Tables and Figures

(a).

Source Type III SS df Mean Squares F-Ratio p-Value ______SEASON 2331.418 2 1,165.709 6.021 0.003 SITE 417.253 2 208.626 1.078 0.344 SEASON*SITE 275.874 4 68.969 0.356 0.839 TEMP 879.186 1 879.186 4.541 0.035 Error 108 193.602

(b).

Source Type III SS df Mean Sq F-Ratio p-Value ______SEASON 0.039 2 0.019 2.039 0.146 SITE 0.167 2 0.084 8.860 0.001 SEASON*SITE 0.121 4 0.030 3.214 0.025 TEMP 0.013 1 0.013 1.332 0.257 Error 33 0.009

26 Table 2.2: Percentage of histologically analyzed gonads of S. subligarius at each of five stages of development: immature, mature testis only, mature inactive, mature active, and mature postspawning. The samples analyzed histologically represent a subsample of the sample size used for GSI and age analysis. For each month, the range of gonadosomatic indices (GSI = 100*(gonad mass/body mass)) is shown in percent.

Size N Size Age GSI N % % % % % range Range range (histology) Immature Mature Mature Mature Mature (mm SL) (years) (%) testes inactive active postspawning only May 0.32- 9 3.03 S 10 47-62 <1 4 25 25 50 M 8 69-78 1-2 3 33 66 L 2 81-83 2-4 2 50 50 Jun 1.23- 14 3.33 S 8 46-60 <1-2 5 20 80 M 7 66-74 1-2 3 20 80 L 11 81-88 2-3 6 100 Jul 0.79- 10 3.67 S 9 48-59 <1-2 5 100 M 6 65-78 1-3 2 100 L 7 83-94 2-4 3 100 Aug 2.63- 14 3.86 S 8 44-62 1-2 4 100 M 12 66-76 2-3 5 100 L 10 80-86 3-5 5 80 20 Sep 0.96- 9 2.94 S 5 42-64 1-2 3 100 M 8 66-79 2-3 1 100 L 9 80-88 3-4 5 60 40 Oct 0.93- 10 1.63 S 4 47-53 <1-2 2 50 50 M 9 65-79 1-3 5 40 60 L 6 83-90 3-4 3 33 66

0.11 fish/m3

0.23 fish/m3

0.03 fish/m3

Figure 2.1: Map of St. Andrews State Park in Panama City, Florida, highlighting the three locations of observation and their average fish densities; Outer Jetty, Lagoon, and Inner Jetty.

28 75 32 70 31 65 30 60 55 29 50 28 45 27 40 26 35 # Spawns

30 25 Temp Water 25 24 20 23 15 22 10 5 21 0 20 5/5/06 6/4/06 7/4/06 8/3/06 9/2/06 10/2/06 11/1/06

Figure 2.2: Number of spawns per day (clear diamonds) across the spawning season. Surface temperature (black squares) taken from a nearby NOAA buoy across spawning season. Water temperature correlates with spawning activity (Pearson’s correlation, r95 = 0.60, p = 0.032).

29 (a) 2006 2007 0.4

0.3 3 0.2 Fish/m 0.1

0 14-May 3-Jul 22-Aug 11-Oct 30-Nov

(b) 0.4

0.3

3 0.2 Fish/m 0.1

0 14-May 3-Jul 22-Aug 11-Oct 30-Nov

0.3

3 0.2 Fish/m 0.1

0 14-May 3-Jul 22-Aug 11-Oct 30-Nov

Figure 2.3: Fish densities taken along permanent 50-m band transects across two spawning seasons at each of the three spawning locations (inner jetty, lagoon, outer jetty).

Inner jetty Lagoon Outer jetty 0.6 0.5 0.4 0.3 0.2

Proportion streaking 0.1 0 0 0.1 0.2 0.3 0.4

Density (fish/m3)

Figure 2.4: Results of correlation between fish density and the proportion of streaking behaviors. Inner jetty (low density, diamonds): Pearson’s correlation, r15 = -0.147, p = 0.875; Lagoon (intermediate density, squares): Pearson’s correlation, r9 = 0.051, df = 1 p = 0.323; Outer jetty (high density, triangles): Pearson’s correlation, r13 = 0.079, df = 1, p = 0.192).

31 Inner Jetty Pair only Inner Jetty Streak 60 50 40 30 20 10 0 Number of spawns

Lagoon Pair only Lagoon Streak 60 50 40 30 20 10 0 Number of spawns

Outer Jetty Pair only Outer Jetty Streak

60 50 40 30 20 10 0 Number of spawns

32 (a)

(b)

(c)

33 450 400 350 I-pair 300 I-streak 250 L-pair 200 # Fish L-streak 150 100 O-pair 50 O-streak 0 S M L

Figure 2.7: Frequency of different male mating behaviors (pair spawn and streak spawn) of each size class at the three different sites using total number of fish observed over two mating seasons. Bars indicate the site and spawning behavior: inner pair (solid green), inner streak (open green), lagoon pair (solid black), lagoon streak (open black), outer pair (solid blue), outer streak (open blue).

(a) (b)

120 Pair Streak 120 Pair Streak

100 100

80 80

60 60

40 40

20 20 Observed Behavior Observed Behavior 0 0 Bare Algae Bare Algae Habitat Type Habitat Type

36 CHAPTER THREE

THE EFFECT OF POPULATION SIZE STRUCTURE AND ALTERNATIVE MATING TACTICS ON THE FERTILIZATION SUCCESS OF A HERMAPHRODITIC SEABASS

ABSTRACT

In the simultaneously hermaphroditic marine fish, Serranus subligarius, male role individuals are known to pair spawn, group spawn and streak spawn. While these mating tactics, which are common among marine reef fish, have been well studied, it remains unclear whether these competing strategies affect fertilization success for the female role individuals. To investigate this issue, I observed mating behaviors and quantified fertilization success in natural and experimental setting during the summers of 2005-2008 at three sites with different local population densities. I observed focal individuals in 15-minute increments and recorded the total number of spawns, number of streak spawns, size of participating spawners and fertilization rate. The occurrence of small sized individuals in the local population is associated with higher frequencies of streaking behavior; these small fish are most often first year individuals reaching sexual maturity late in the spawning season (August/September). Spawns that included one or more streak spawners had a significantly lower average fertilization rate (89%) than pair spawns without a streak spawner (97%). This pattern was confirmed in a field manipulation experiment in which spawning events that included streakers again showed lower fertilization rates (93%) than spawning events that did not include streakers (98%). Spawns that included multiple males produced, on average, 20% more sperm than spawns involving only one male. These results indicate that females incur a significant fitness cost when streakers invade a spawning event, that streak spawning males fully participate in spawning and that sperm number is not a limiting factor responsible for the lower fertilization rates.

37 INTRODUCTION

The reproductive success of aquatic, externally fertilizing animals can be highly variable and the proportion of released gametes that are fertilized depends upon many factors including proximity of mate, current speed, and level of sperm competition (Levitan 1991; Petersen et al. 1992; Marconato et al. 1997). The potential difficulties in achieving high fertilization rates can be overcome in part by producing quantities of sperm in excess of the amount necessary to fertilize all eggs so that it can reach a greater distribution, and by changing the proximity of mating individuals to one another, thus increasing the chance of sperm and egg coming in contact. For example, many reef fish species spawn in pairs as well as groups; in a group spawn there is a greater average distance between males and females and males appear to compensate by increasing their output of sperm in a spawning burst and allow males in a more distant position to compete with multiple males (Petersen et al. 1992; Warner 1996; Levitan 2005; Yoshikawa 1992; Marconato et al. 1997). In situations like these, sexual selection may act upon male traits that promote closer access to the female; it may also act on female traits to ensure that fertilization rate is maximized. Sexual selection for access in males and fertilization rate in females becomes potentially more complicated and interesting when individuals in a population employ different mating tactics. Many marine reef fishes have labile mating systems and the mating tactics employed and allocation to a particular sex may depend upon a variety of local circumstances (Thresher 1984; Warner 1984; Taborsky 1998). In many cases, competing individuals employ different tactics; one male may attempt to pair spawn with a female, whereas another may attempt to spawn as a streaker, which means that he swims up and over a pair spawn already in progress and releases sperm. Such variation in tactics is predicted to emerge from sexual selection on males when differences in mating success are affected by body size and size-limited success can be overcome either by investing more in male function or by using different mating tactics (Charnov 1979). However, it is unclear if the existence of competing tactics like streaking and pair spawning cause relaxed or enhanced sexual selection on females for high fertilization rates. The increased sperm concentrations typically seen when streakers and pair spawners compete could relax the intensity of selection to ensure high fertilization rates; alternatively, if streaking creates turbulence in the water that disrupts the mixing of sperm and eggs, such selection could become more intense.

38 The situation is more interesting still in simultaneous hermaphrodites that do not show strong differences in sex allocation or differential mating based on body size (Oliver 1997, Hastings and Bortone 1980). In simultaneous hermaphrodites, individuals vie for the sex role that allows for greater reproductive output. In some cases, the choice of role is based on body size and relies on the animals’ assessment of the costs and benefits associated with mating in a given role. For example, in some systems, females attract males to them using visual cues of being gravid, then proceed to vie for the male mating role (Leonard 2006; Petersen 2006). This can increase the opportunity for sexual selection on traits associated with male roles and promote reciprocity (egg trading) (Leonard 1990). But just as in gonochores, streaking occurs in many hermaphrodite systems (Leonard 1993; Petersen 2006). The competition among individuals acting as sires is the same as in gonochores but the effects on fertilization rates and sexual selection to enhance those rates may be more complicated. Not only might streaking disrupt sperm and egg mixing, it adds an additional male role that can disrupt the fitness relationships that promote reciprocity between the pair spawners. All of these possibilities remain poorly understood and so whether these systems fit well with existing theory for sexual allocation remains to be seen. In this paper, I report the results of an investigation into the dynamics of fertilization in a simultaneous hermaphrodite in the presence and absence of streaking. I report the association of population size-structure with streaking, sperm output in pair and streak spawning, and the effects of streaking on fertilization rates. The results point to a clear fitness cost in female function associated with the presence of streaking and raises the question of how the reciprocity between pair spawners might be affected.

METHODS

Study Species The belted sandfish, Serranus subligarius (family Serranidae, the seabasses), is a simultaneously hermaphroditic marine fish that utilizes an interesting pattern of mating and sex allocation. They are classified as reciprocal egg traders (spawn alternately as male and female in pairs) and allocate equally to ovaries and testes, but do not spawn equally in male and female roles across their life history stages (Oliver 1997). There are three possible spawning tactics for male role individuals— pair spawn, group spawn, or streak spawn. Pair spawns involve one male courting and mating with a

39 female, group spawns involve multiple males attempting to court and mate with a single female and streak spawning is when an individual swims up and over a pair spawn already in progress and releases sperm. All mature fish will spawn as females, but the frequency of female spawning behavior decreases with increasing size. Thus, larger individuals tend to obtain a greater number of matings as a male. The incidence of streak spawning is skewed toward small individuals, but larger individuals will streak spawn at low levels (Oliver 1997). These fish commonly occur in the northern Gulf of Mexico and the Atlantic coast from Florida to North Carolina (Robins and Starck 1961). They primarily inhabit shallow reefs and jetties, but have been noted at depths of greater than 60 m (C. Koenig, personal communication). Spawning occurs in the summer months and developing larvae will remain in the water for approximately 30 days (Hastings and Bortone 1980). Settlement will occur on hard substrate (rock outcroppings, jetties) throughout the northern Gulf of Mexico and individuals will typically mature and reproduce within their first year (Hastings and Bortone 1980). Individuals form pair bonds for a spawning bout, but it is unclear how often or how long this pair remains together. Territories are acquired and defended early in the reproductive season (Oliver 1997), but site fidelity to a territory over multiple seasons is unknown. Territory acquisition and maintenance under conditions of high density may be more costly and play a role in the increased frequency of streak spawning seen at high density sites (Oliver 1997). In other words, territorial males may be swamped by subordinate, non-territorial males and chasing away all potential streakers may become impossible. The conflict created between small male role streak spawners and larger territorial male role spawners has been proposed as a mechanism to maintain simultaneous hermaphroditism in this species (Oliver 1997), thus preventing invasion by a protogynous strategy.

Study site Field data were collected along a jetty at St. Andrews State Park in Panama City, Florida, along the northeast Gulf of Mexico. The jetty site allows for separated regions that differ in natural local population density and can be accessed by a short swim. Site depth varied from 3-10 m and depended upon tidal height and season. The outer jetty site is the high density site (HD) with 0.96 fish/m2, the medium density site (MD) with 0.64 fish/m2 and the low density site (LD) with 0.25 fish/m2. These means remain relatively consistent from year to year and movement within each site during the mating season is minimal, largely due to their territoriality. The highest density site is

40 likely fed by new recruits at a higher rate than the other two sites. Given that streak spawning was previously described as a density-dependent tactic (Oliver 1997), streaking should increase with density, but the observed streaking frequency is highest at the low density (up to 20% of spawns including a streaker) and high density (up to 30% of spawns including a streaker) sites, with very little occurring at the intermediate density (<5%) site (see chapter 2).

Field data Behavioral data were collected from June through mid-October in 2006, 2007, and 2008. For each observation, a focal individual was observed for approximately 15 minutes while on SCUBA. If a spawn occurred, one diver continued to observe the fish and one collected the resulting gametes after 20 seconds to allow for fertilization to occur at a natural rate. Prior to the field observations, sperm from seven individuals were observed in the lab by gently squeezing fish to extract milt and verify the length of sperm activity. Sperm remained motile for approximately 20 seconds (with range of 18-36 seconds). The fish observer recorded the timing of the spawn, the size class of the focal individual and how many (if any) streak spawners participated in the spawn. Fish collected early in the season were used as a verification of our size class estimates underwater. Estimates of small, medium and large fish were recorded on slates and 10-15 of these fish were collected and measured at the surface. If this was done with less than 10% accuracy, this process was repeated. Gametes were collected in the field using a 48-liter plastic bag with a 450 ml plastic container inserted and glued into the bottom seam (modified from Marconato et al. 1997). Filled bags were taken to the surface to allow excess water to drain out of the Nytex mesh panels and flushed with clean seawater to ensure that eggs didn’t get stuck on the sides of the bag. Egg containers were then sealed with a screw top lid and brought back to the lab at the end of the dive. For any given dive, an average of five batches of eggs were collected. In the lab, eggs were observed for evidence of fertilization, namely the development of the chorion around the egg and multiple cell divisions, and counted. Counts were done approximately three hours after collection. In 2008, sperm was collected in addition to fertilized eggs to get an estimate of relative contribution of sperm when between a paired mating and a pair with at least one streaker involved. This was done using the same collection bag method, but without the Nitex panel. Full bags were taken to the shore, pored through a milipore filter, dried and oiled to clear the filter paper and make the sperm visible under a microscope to get sperm number estimates (modified from Marconato and

41 Shapiro 1996). Counts were taken approximately 24-48 hours after collection, allowing the filter paper to dry. Eggs were collected as described above and counted using a plexiglass maze, which aligns the eggs to allow for easier visualization under a dissection microscope. During about 50 % of observed spawns in 2008 and 2009, a digital video camera with underwater housing was used to capture images and short video clips of spawning bouts. A subset of these clips (those clear enough to analyze) were slowed down and observed frame by frame. Still images were captured of individuals in the “cupping” behavior, which is the position in which the vents of male role and female role individuals are in closest proximity and the point of release of gametes. These still images were then imported into to ImageJ (NIH) and angled lines were superimposed on their bodies from the point of bending to the caudal fin and to the head (Figure 3.1). This measurement gives an estimate for the overall body angle for both male role and female role during the cupping behavior.

Field experiment A field manipulation experiment was done to test the hypothesis that size structure and an individual’s size play a role in predicting the rate of streaking and the consequences of streaking to patterns of fertilization. Twelve mini-reefs were constructed using rocks collected from around the jetty and placed in a shallow (2-3m depth) lagoon adjacent to the jetty in a randomized block array. Each reef was approximately 1.5 m2 in diameter and 0.5 m2 in height and were placed 5 m apart from one another. They had a randomized combination of high density (6 fish), low density (3 fish) and either mixed sizes or same sizes (all large), where mixed size combinations included at least one small size-class individual. Each treatment was replicated three times. Observations in other locations have found higher density to be associated with increased incidence of streaking, although my field observations did not show this association. Size structure was manipulated after I gained evidence of high streak spawning at both the low- density site and high-density site and noted that at the intermediate-density site, there were very few small size-class individuals (chapter 2). The reefs were set up in July and monitored weekly throughout the late summer in 2007 for mating type frequency. During the first two weeks, a few fish left the plots and were immediately replaced to maintain the treatment. Once established, fish stayed on the mini reefs until the end of the spawning season. As in the field component, eggs were collected from observed spawns using the collection bag method previously described.

42 For each observed spawn, the reef number, treatment, focal fish size and number of participating spawners was noted.

RESULTS Field data Data from the outer jetty site indicate high fertilization success throughout the season, but a decrease in the percent of eggs fertilized late in the spawning season (Figure 3.2). Early in the season typical rates were 95% to 100% whereas late in the season rates ranged between 84% and 100%. A close examination of the individual data points revealed that lower rates of fertilization occurred in spawns that included at least one streak spawner. To rule out the possibility of sperm depletion late in the season, I removed the spawns that included a streaker and regressed fertilization rate on date (Figure 3.3). Though the slope is slightly negative, it is not significantly different from zero, suggesting that the reduction in fertilization rate late in the spawning season was not an issue of sperm depletion. When both types of spawning occurred, later in the season, spawns with streakers had lower average fertilization rates. I examined a truncated data set, using only spawns observed after the first eggs were collected from a streak spawn (Figure 3.4). Within this subset, the average fertilization rate for spawns with streaking was 89% whereas the average for spawns without any streaking activity was 98%. This difference is highly significant (t-test, p = 0.0001). There was greater overall sperm output when streakers participated in the spawn than with a pair spawn only (Figure 3.5, N = 22, t-test: p = 0.047). The total concentration of sperm was approximately 20% greater when streak spawners were present, suggesting that more than one male participates in the spawning activity. Without appropriate molecular markers, I cannot estimate the proportions of sperm released per male. Egg parcel number did not differ between spawns with and without streakers participating (Figure 3.6, N = 23, t-test, p = 0.67).

Experimental data The experiment revealed the importance of size structure in determining the incidence of streaking (Figure 3.7). While the incidence of streaking was highest in the high density/mixed size structure plots, there was no significant association of streaking rate with density variation (Chi-sq=0.743, p=0.388). There was a clear effect of size structure in that streaking occurred only in the mixed size treatments because it is the small fish that do the streaking

43 (streak*structure (equal/mixed): Chi-sq: 3.930, p=0.047; streak*size (small/large): Chi-sq: 17.183, p<0.0001). The pooled mean proportion of eggs fertilized when the spawn was just a pair or a pair with a streaker indicates a lower fertilization rate for males and females when a streaker is involved (Figure 3.8, t-test: t = 3.14, p = 0.012). Behavior Female body angle was wider when two or more individuals were acting as males in a streak spawning. Overall body angle was around 100 degrees for females mating with a single male and averaged 140 degrees when additional males participated (Figure 3.9, Watson- Williams test for circular data, p = 0.041). Male body angle did not differ significantly between pair matings and those with streakers (Figure 3.10, Watson-Williams test for circular data, p=0.87).

DISCUSSION

Four patterns emerge from these data. First, there appears to be a clear cost to both the male and female role pair spawners when a streaker participates. While several studies have shown a cost to individual participating males (Warner and Woonick 2003; Thresher 1984), a cost to the female in fertilization rates has never been shown before because of the presumption that all of her eggs will be fertilized, regardless of the number of males. Given that these data show that sperm is not a limiting resource late in the spawning season, lower fertilization rates must be the consequence of the streaking activity. One mechanism for the lowered rates could be advection of eggs and sperm away from each other due to the high velocity spawning rushes of all participating males in the water column. Another possibility is altered behavior by the pair male or female in reaction to the presence of additional male role individuals that may change his optimal position over the female during gamete release. Finally, polyspermy could cause lower fertilization success with multiple males and more sperm. Polyspermy is considered rare among teleost fishes because of their complex sperm blocking system. In fishes, the highly specialized micropyle is the only site where sperm can enter the egg (unlike many invertebrate eggs, which have multiple sites available to sperm). The diameter of the micropyle is only large enough for one sperm to enter, at which point, a plug is formed, which prevents additional sperm from

44 entering (Ginzberg 1972; Murata 2008). So, while polyspermy is unlikely, the data in hand do not allow us to distinguish the first two explanations. Second, the presence of more sperm when streaking supplements pair spawning and suggests that streaking could be an effective way for the small males to obtain some paternity. The data in hand cannot refute the hypothesis that all of that extra sperm was produced by the pair spawner in response to the presence of streakers, but this seems unlikely, given that streakers in other systems are known to shed sperm and often produce more sperm than the focal male. While it is unclear exactly how much sperm streak spawners release, it appears to supplement pair spawning concentrations, so sperm competition is likely to be intensified during streak spawning events. Third, there is an association between behavioral variation between spawning types and the different fertilization rates. The more open body angle of the female during gamete release in streak spawning may contribute to the lower fertilization rates. This altered body shape may allow eggs to escape contact with sperm as they are released. During a typical pair spawn, male and female role bodies are cupped tightly over one another. With a decrease in that body angle, more water flow may occur between the two bodies, thus deflecting more particles (i.e. sperm and egg). It is also possible that the individuals alter the timing of release of gametes in these situations, reducing the effectiveness of the tight cupping behavior with respect to fertilization rate. Given the small amounts of sperm that are released in most pair spawning fishes and the often murky water, direct field recordings of the movement of eggs and milt were not possible. More direct comparisons of video and gametes released in a laboratory setting would be necessary to test this hypothesis. Finally, local population size structure appears to be the strongest predictor of the use of alternative mating tactics in this population. While density is important in other systems, the presence of small individuals in the local population drive the incidence of streaking and in their absence (as in the intermediate density site) little to no streaking occurs. One possible mechanism by which this occurs at this specific site is through higher larval recruitment to the high and low-density sites, as they are more open to ocean input. Another possibility is that young individuals are just reaching sexual maturity at the end of their first year and are unable to obtain a territory for proper courtship activities. Physiologically, there is some evidence that the testicular tissue matures earlier than the ovarian tissue, thus limiting the young individuals to a

45 male-only tactic, thus bypassing any reciprocal mating (Hastings and Bortone 1980), but the pattern found in a further investigation did not support this idea (chapter 2). Several studies have shown a decrease in fertilization rate due to polyspermy (Franke et al., 2002; Levitan, 2004) but this does not appear to be occurring in this system. Additional empirical tests are needed here to tease apart the mechanisms contributing to the observed pattern. The absence of data on this topic is likely due to the difficulty of obtaining gametes in the field from highly mobile species. Additional lab studies investigating egg development under conditions of sperm competition are needed to test the effectiveness of the polyspermy blocking mechanisms in fishes. If more species follow this trend of low fertilization rate with male competition, it could suggest that many of the models surrounding mating system theory are lacking a key component in this phenomenon. More detailed behavioral studies coupled with manipulative field experiments could help to elucidate the subtleties of mating behavior and the resulting fertilization events. Molecular analyses to pinpoint the contribution of each participating individual would also aid in our understanding of sperm allocation sexual selection on traits associated with dominant male role behavior.

FIGURES

L. Allen

47 2006 2007 2008

100

80 % Fertilized eggs

70 0 20 May 10 20 Jun 2020 Jul 20 30Aug 20 Sep40 20 Oct 50

Figure 3.2: Fertilization rates obtained from field-collected samples during the spawning seasons of 2006, 2007, 2008. Variation in fertilization rate increases with the spawning season.

48 Fertilization 2006 Fertilization 2007 Linear(Fertilization 2006) Linear(Fertilization 2007)

2 100 R = 0.0512

R2 = 0.3016

85 % Eggs Fertilized

75 10-May 9-Jun 9-Jul 8-Aug 7-Sep 7-Oct

Figure 3.3: Percent of eggs fertilized over the spawning season in 2006 and 2007. Spawns that included a streaker were removed from this analysis and a best fit regression line suggests a slightly negative, but nonsignificant result (2006 (solid line): N = 13, R2 = 0.0512; 2007 (dashed line): N = 19, R2 = 0.3016).

49 pair streak

100

80 % Eggs fertilized 75

70 23-Jul 12-Aug 1-Sep 21-Sep 11-Oct

Figure 3.4: Truncated data showing the fertilization rates late in the spawning season (August- October). Pair spawning (dark circles, n = 23) and streak spawning (open circles, n = 16) fertilization rates are shown and participation of streakers results in lower overall fertilization success (t-test, df = 37, p = 0.001).

50 Pair Pair w/Streak 6

2 Sperm output (millions) 1

0 1 Spawn type

Figure 3.5: Total sperm output estimates (in millions) for pair spawns and pair spawns with at least one streaker participating (N = 22, t-test, p = 0.047).

51 Pair Pair w/streak 250

200

150

100

50 Number eggs per spawn 0 Pair Spawn type

Figure 3.6: The average number of eggs in a single parcel does not differ between spawns of a pair only and ones that include at least one streaker (N = 23, t-test, p = 0.67).

52 Streak Pair Only 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3

Proportion Spawns 0.2 0.1 0 Lo Equal Lo Mixed Hi Equal Hi Mixed

Pair Only Pair w/Streaker 1

0.95

0.9

0.85

Proportion Eggs Fertilized 0.8

0.75 Pair Only Pair w/Streaker Spawning Behavior

160 Pair Pair w/streak

140

120

100

60 Female body angle 40

0 Spawn type

55 Pair Pair w/ Streak 160

140

120

100

60 Male body angle 40

20 Pair 0 Pair Spawn type

56 CHAPTER FOUR

CONCLUSIONS

Understanding the role of mating systems and behavior with respect to male or female function as well as gaining a more comprehensive understanding of the utilization of alternative mating strategies as a possible way to maximize reproductive success was a primary goal of this research. Additionally, this work aims to expand our knowledge of the role of ecological context and social structure under which mating systems operate. This work has application across a broad array of taxa (plants, invertebrates, fishes) and disciplines (theoretical biology, molecular biology, physiology) and has the potential to provide a framework for the study of the selection and maintenance of specific traits associated with mating systems and reproductive behavior. One of the conflicts in externally fertilizing animals is the result of males controlling fertilization by virtue of releasing sperm over previously spawned eggs. The gamete-trading model suggests that the role that controls fertilization is the preferred role. For internally fertilizing animals, the preferred role can depend upon the latency between sperm deposition and fertilization. In some species, females may choose deposited sperm from multiple males and exert the control in fertilization, leading to much of the sperm being wasted (thus female role is preferred). When a partner leaves the pair mating before releasing or allowing access to eggs, they have failed to reciprocate and this is noted as direct evidence of sexual conflict in several hermaphroditic species (Fischer 1980, Axelrod and Hamilton 1981). The Hermaphrodite’s Dilemma model (Leonard 1990) predicts that reciprocity will evolve in mating systems where there is a clear preference for one sexual role. In this model, conditional reciprocity results from partners assessing each other prior to mating and the choice to mate is based upon either the condition of the partner or the willingness of the partner to reciprocate. There are several examples of conditional reciprocity and measures of its evolutionary stability in polychaetes (Lorenzi and Sella 2006), terrestrial gastropods (Leonard 1990), and fish (Petersen 1991; Oliver 1998; Adreani, unpublished data). What is difficult to include in such a model, however, is the effect of animals that don’t reciprocate, or cheat. While game theory models (particularly Prisoners dilemma) have aided our understanding of how animals in mating games assure honesty, there is no clear way to explain defection by way of not

57 forming a pair (as is the case with a streak spawner) or by leaving the pair. In addition, one major assumption of this model, that each partner is equally likely to act first, is violated in at least two species of Serranine fishes, S. tabacarius and S. subligarius (Petersen 2006). Because alternative tactics are common in this family of fishes (and others), it seems that models including this phenomenon must be utilized to make accurate predictions as to the success and stability of the mating system. Given that sexual conflict is typically evidenced by the occurrence of mating reciprocity (a potential resolution to strong sexual conflict), direct measurements of conflict are typically not given. One way to get better detail on the presence and/or strength of conflict is to have very detailed observations of matings in the field (i.e. natural environment). From these observations, one can get a better idea of the motivation for deserting a mating or utilizing an alternative. When streak spawners participate in the spawns, however, overall fertilization rate is lower than that of a pair spawn, suggesting a cost to both male and female reproductive success (Chapter 2). This is contrary to most studies that have found similar or higher fertilization with additional males participating (Taborsky 1994; Wooninck et al. 2003). One assumes that selection would favor traits that allow a male to gain closer access to a female (in the case of external fertilizers) as this has been shown to be the most significant factor contributing to the majority paternity share, even more so than contributing more sperm (Wooninck et al. 2003). This makes some intuitive sense if one considers the environment. In aquatic organisms, dilution and turbulence could have major effects on the distribution of sperm in a short amount of time. A competing male may release a greater concentration of sperm, but if it is distributed away from the release of eggs, that investment may be wasted. This appears to be the case in S. subligarius, where streakers swim up and over the spawn in progress, release sperm, while at the same time disrupting both the behavior of the pair mates and the water surrounding the gametes. Given that these small males do not reciprocate, they may be considered defectors of the reciprocity game (e.g., Prisoner’s dilemma). Asymmetries in mating success, such as those associated with cheaters or where a clear size advantage exists would seemingly weaken the stability of egg trading. A large male phenotype could theoretically invade the population and break down the conditions of reciprocity if large males obtain more mates and larger mates, significantly increasing their reproductive success. Functional males happen rarely in this group of fishes and require certain environmental

58 and social conditions to be maintained (Petersen and Fischer 1986; Petersen 1987). For S. subligarius, however, these predictions do not appear to hold true and egg trading has been demonstrated to be stable (Oliver 1997), despite variation in density and distribution. While larger male role individuals mate more frequently with larger females and selection seems to favor traits that confer greater fitness for the male role, the large male phenotype has not invaded this system. One apparent difference between S. subligarius and the haremic species is the occurrence of alternative tactics and thus the level of sperm competition, which is fairly high in S. subligarius (up to 30%) and very low (< 10%) or absent in S. baldwini and S. psitticinus. Where streak spawning tactics are utilized with some frequency, it may be difficult for a large male to monopolize the matings of several hermaphroditic individuals. Perhaps defending a large territory, in addition to multiple mates is too energetically costly. The possible fitness advantage of a haremic system may therefore be dampened by the participation of multiple males. The other component lending to the stability of reciprocity is the parceling of eggs over the course of a spawning bout. In S. subligarius, batches of eggs are released over multiple matings throughout the day, while in the haremic species, large males mate multiply, but female role hermaphrodites release only a single parcel per day (Petersen and Fischer 1986). In chapter two, the results suggest that overall mating activity peaks between July and August (based on observations of spawning and histological gonad sampling). During August and September, streak spawning peaks and occurs at significantly higher rates at the Inner Jetty (low density) and Outer Jetty (high density) sites than the intermediate density Lagoon site. A closer look at the habitat type over which pairs spawn reveals that high algal covered rocks are preferred by streak spawners and this may be due to the shelter and food provided by such microhabitat. Finally, gonad development via histological analysis reveals that all size/age classes are mature by June; both ovaries and testes show active oocytes and spermatocytes. Sperm and egg regression begins in early September. In chapter three, I demonstrate the importance of size structure and fertilization dynamics on the incidence of streak spawning. The high level of sperm in the water at the time of spawning suggests that small streakers are actively participating in the spawning, yet I repeatedly saw lower fertilization success when additional males participated (<90%). This may be the result of disruption of the water column, causing sperm and egg to deflect away from each other,

59 the altered behavior seen by females in opening up their body angles or the possibility of polyspermy, which has been shown to affect other externally fertilizing marine invertebrates under high density, resulting in embryo death (Levitan, 1995). While recent studies (Hart 2010; Adreani, Chapter 2 and 3) on mating systems and behavioral ecology of the hermaphroditic seabasses lend greater insight into the traits associated with sexual selection, there is still much more data needed to ascertain lifetime fitness and how this changes across variation in mating system. In addition, the theoretical framework laid out by Charnov (1979), Fischer (1980) and others has been supported by many of the studies thus far, but there is a great need to properly include alternative tactics, asymmetries in reciprocation and preference for a particular sexual role to tease out the selective forces acting on both male and female role individuals under these circumstances.

60 APPENDIX Approval letter from the Animal Care and Use Committee

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66 BIOGRAPHICAL SKETCH

Curriculum vitae

MIA ADREANI ______

ACADEMIC POSITIONS • Postdoctoral researcher. California State University, Northridge, CA. June 2010-present. Project title: Reproductive output, growth, and food-chain support of fishes on the Wheeler J. North Artificial Reef Supervisor: Mark Steele

EDUCATION Graduate: Florida State University, Tallahassee, FL, PhD, Ecology and Evolution. 2003-2010. California State University, Northridge, CA, M.S., Biology. 2000-2003. Northeastern University, Boston, MA. East/West Marine Biology Program 1997

Undergraduate: University of California, Santa Barbara, CA. B.S., Aquatic Biology. 1992-1996

TEACHING EXPERIENCE • Seminar in Ecology. California State University, Northridge, CA. Spring 2011. • Tallahassee Community College, Tallahassee, FL, 2009-2010. Adjunct Faculty in the Science and Mathematics Division.

• Florida State University and Leon County School’s GK-12 program. Science teaching at Belle Vue Middle School and Hartsfield Elementary, Tallahassee, FL. 2006 -National Science Foundation Graduate Students in K-12 Education grant (GK-12) allows graduate students in the sciences to integrate science activities in local elementary and middle school classroom around Leon County

• Florida State University, Tallahassee, FL, 2003-2010. Teaching assistant -Ecology of Fishes: Co-teach course with lecture/lab/field components designed to introduce ichthyology and ecology, physiology of fishes to advanced undergrads/grads. 2008-2009. - Animal Diversity: Biology lab for majors which includes a lecture and lab portion. Responsible for teaching multiple labs and assisting with large scale practical exams. - Intro Biological Science: Biology lab for non-majors which includes learning cell processes, enzyme function, molecular techniques. - Experimental Marine Ecology: Teaching assistant for course in designing and implementing a scientific experiment in the marine environment

• Saturday at the Sea: outreach program taking 5-7th graders to the marine lab for field/lab experience. 2004- 2010.

• Florida State University, Athletic Academic Support Tutor. Fall 2003-Spring 2005. -Tutor in all disciplines of biological and environmental science for athletes

• University of Southern California, Los Angeles, CA. August 2003. Instructor for college-level marine ecology course for high school students at Wrigley Marine Science Center, Santa Catalina Island, CA. Lectures, field experiments and individual research projects

• California State University, Northridge, 2000-2003. Teaching assistant for introductory biology labs, marine biology (field and lab components), physiological ecology (lab), fish ecology (field and lab), and algal ecology (field and lab)

• Shoals Marine Lab, Appledore Island, ME, Summers 1998-1999. Teaching assistant for college- level marine ecology course for high school students. Students had lectures, lab excercises and field experiments in an intensive short course in marine biology and oceanography

AWARDS AND GRANTS • Animal Behavior Society, Travel Grant, Pirenopolis, Brasil, June 2009. $1000 • NSF GK-12 Fellowship, Florida State University, January 2005-January 2006, $30,000 • Outstanding Teaching Assistant Award nominee. 2005. • Bennison Memorial Scholarship, Florida State University. April 2005. $1000 • Sigma Xi Grants-in-Aid of Research. January 2003. $175 • E.E. Stoye Award for Ecology and Ethology, Amer Soc of Ichthyology and Herpetology, July 2002 • PADI Foundation Research Grant. March 2002. $1450 • Graduate Research and International Programs, CSU Northridge. November 2001. $1000 INVITED LECTURES/SCIENTIFIC PRESENTATIONS • Animal Behavior Society, Allee Competition Seminar, June 2009 • Florida State University Coastal and Marine Laboratory, Research lecture, May 2009 • Washington University, ecology seminar series, July 2007 • Society for the Study of Evolution, annual meeting, June 2006, 2008 • University of Southern California, Wrigley Marine Science Center. Summer 2001, 2002 • Cornell University, Shoals Marine Lab, Summer 1999, 2000. Lectures on fish biology, invert zoo • California State University, Student Research Seminar. November 2001, 2002 • Western Society of Naturalists annual meeting. November 2001, 2002. 2003, 2006 • American Society of Ichthyologists and Herpetologists meeting July 2002, 2003, 2006, 2007, 2009 • Benthic Ecology Meeting. March 2003, 2009 PROFESSIONAL MEMBERSHIPS • Society for the Study of Evolution. 2002-present • American Society of Ichthyologists and Herpetologists. 2000-present. • American Society of Naturalists, 2004-2005 • Animal Behavior Society. 2002-present.

68 • Western Society of Naturalists. 2000-present. ORGANIZATION OFFICES HELD • FSU Ecology Evolution Research Discussion Group- President. 2005-2006. • FSU Ecology Evolution Research Discussion Group- Treasurer. 2004-2005. • CSU Northridge Biology Program- Vice President. 2002-2003.

PUBLICATIONS • Adreani, MS. 2010. Fertilization dynamics and the effect of local population size structure on the use of alternative mating tactics in Serranus subligarius. Evolutionary Ecology.

• Adreani, MS, BE Erisman, RR Warner 2004. Observations of Courtship and Spawning Behavior in the California Sheephead, Semicossyphus pulcher. Env Biology of Fishes 71: 13-19.

• Adreani, MS and LG Allen. 2008. Reproductive Behavior and Mating System of the Temperate Wrasse, Halichoeres semicinctus. Copeia 2: 467-475.

• Adreani, MS and MA Steele. Life history traits of the temperate wrasse, Oxyjulis californica. In prep.

• Adreani, MS. Spatial, temporal and behavioral patterns associated with streak spawning in the hermaphroditic seabass, Serranus subligarius. In Review, MEPS.

• Schrader, M, Adreani, MS and Travis, J. Local population size and the level of multiple paternity in the least killifish, Heterandria Formosa. In prep.