Introduction The majority of animal species reproduce sexually. Sexual reproduction is generally considered to be advantageous because it results in genetically variable progeny due to segregation and recombination events (Williams, 1975; Maynard Smith, 1978; Bell, 1982). The maintenance of variation in the population allows rapid evolutionary response to shifts in the environment through adaptation and speciation (Van Valen, 1973; Bell, 1982). Because asexual species lack mechanisms for recombination, they are generally considered to be genetically inflexible and therefore long term evolutionary dead ends. However, the advantage of sexual reproduction may not be universal, so that under certain conditions, asexual reproduction is advantageous (Vrijenhoek et al, 1989). There are about 70 vertebrate species that reproduce by various ameotic mechanisms which lack recombination, and therefore result in genetically identical (i.e. clonal) progeny (Vrijenhoek et al, 1989). Clonal reproduction transmits the entire genome intact to the next generation, thus ensuring that favorable gene combinations are maintained (Maynard Smith, 1975). Obligate self-fertilization is not ameotic, but once homozygosity is reached (following approximately10 generations of selfing), parent and progeny are genetically identical, and the reproduction system is effectively clonal (Bell 1982). Individuals of a self-fertilizing species are always assured of reproductive success, and these species avoid the costs and risks associated with sexual reproduction (Maynard Smith, 1975). This allows the rapid colonization of new habitats relative to a sexual species, by even a single individual. Self-fertilization spreads the genome over a wide range as individual clones migrate, so that the overall success of the clonal lineage may not be threatened by local habitat changes. A self-fertilizing clonal species may change environments to suit the genome rather than changing the genome to suit the environment. The marine killifish Rivulus marmoratus is an obligate self-fertilizing 117 hermaphrodite and is the only vertebrate known to reproduce by internal self-fertilization (Harrington, 1961, 1963). Natural populations are composed almost entirely of selfing hermaphrodites, so that the population structure has been described as arrays of homozygous clones. This killifish is one of the few organisms to exist in nature in a homozygous state (Harrington & Kallman, 1968, Turner, Elder, Laughlin, & Davis, 1992a). Populations sampled so far have had high clonal diversity, with low representation of each clone (Turner, Elder, Laughlin, and Davis. 1992b). Clonal composition at a particular locale appears highly variable over time, suggesting a very high rate of clonal turnover due to migration and/or local extinctions (Turner et al, 1992 b). Though most natural populations of R. marmoratus surveyed thus far consist almost entirely of selfing hermaphrodites, males have been collected at low frequencies in a few populations. In Floridian samples, males comprise less than 1% of individuals collected. In most other locations sampled, males have not been found at all, but there have been two conspicuous exceptions: males comprised up to 24% of the samples from several barrier islands off the coast of Belize in 1988-89 (Turner et al, 1992b) and high frequencies of males were also reported on Curacao in the Dutch Antilles during the 1960's (Kristensen, 1970). Males consistently appear in laboratory stocks, even among those descended from natural populations in which males were never recorded. R.W. Harrington (1967) discovered that in three Floridian clonal lines, males could be induced in the laboratory at very high frequencies (up to 100%) by incubation of embryos at low temperatures (19° C), and to a more variable extent, by rearing juveniles at high temperatures (30° C)(Harrington, 1967). He delineated a "phenocritical" period of embryonic development for sex phenotype (Harrington, 1968). To date, the adaptive significance of this phenomenon is unclear. Is temperature-dependent male induction part of an "environmental sex determination" (ESD) system in this species, perhaps one that facilitates outcrossing under certain conditions? Or is it simply a laboratory 2 phenomenon that is not relevant to most natural populations, so that these males induced in the laboratory may be viewed as developmental anomalies? Harrington detected some variation among the three clones he studied in the extent of male induction, especially at higher rearing temperatures. Do these differences stem from adaptive modifications of an ESD to particular local conditions, especially temperature, encountered by specific clones, or are these differences clone-specific effects indicating differing degrees of developmental stability between clones? The objective of this research is to detect differences between clonal lines in the low temperatures at which males are induced. If these differences correlate with the geographic location from which these clonal lines were originally collected, this may be evidence that low temperature induction of males in this species is part of an environmental sex determination system. If low temperature is important in the induction of males in nature, then the temperature at which males are induced may be related to the local temperature regime. Therefore, it was hypothesized that clonal lines originally collected from the extremes of the range (Florida and Brazil) will produce males at a lower threshold temperature than clonal lines originally collected from the more equatorial center of the range of this species.
Literature Review Research history: Rivulus marmoratus was originally described as a species from Cuba by Poey in 1880. Later, it was classified as a synonym of R. cylindraceus, a Cuban species to which it does not appear to be closely related, but it was revived as a distinct species by Rivas in 1945. The species was discovered in Florida by R.W. Harrington and Rivas in 1958. Harrington became the most prominent student of this fish, describing the self-fertilizing hermaphroditism unique to this species, as well as the male gender induced by incubation and rearing temperature manipulations (1967, 1968). Also, he and K.D. Kallman provided the first evidence for the homozygosity of the species through
39 tissue transplants among siblings (Kallman and Harrington 1968). R. marmoratus has been used as a subject for carcinogenicity studies (Koenig and Chasar 1984). Other studies have explored the relationship between homozygosity in this species and developmental stability through the examination of meristic characteristics (Swain and Lindsey 1985a, b, Harrington and Crossman 1976, Lindsey and Harrington, 1971). Embryological and developmental studies have included embryonic repair (Park and Yi 1989), skeletal development (Lee and Park 1989), and development of photoreceptors (Ali et al 1988). Current work has focused on the ecology of the species (Davis et al 1990, Taylor et al 1995), the comparisons of the fitness of different clones under different developmental and environmental conditions, and the role of rare males in natural populations (Turner et al 1991, Lubinski et al 1995). Also, phylogenetic studies using mtDNA are in progress by Thomas Dowling to determine the geographic origin of this widely dispersed species. This work may provide insight into the origins of self- fertilization by making comparisons with closely related sexual species possible. The species has also been used in toxicological studies (Davis 1986, 1988) Davis has proposed that the apparent dependence of this species upon mangrove swamps may make it useful indicator of the overall health of mangrove ecosystems (Davis et al 1995).
Geographic range: Rivulus marmoratus has been found in southeaster Brazil, near Rio de Janeiro, Venezuela (Taphorn 1980), Nicaragua, Guatemala , Belize, both mainland and on the barrier islands, Yucatan, and southern Florida, as far north as Vero Beach on the Atlantic coast. It seems likely that the species range is continuous between Brazil and Florida, so that gaps are the result of incomplete sampling rather than breaks in the range. R. marmoratus is also found throughout the Caribbean islands, including the Florida Keys, Tortugas, Cuba, the Bahamas, Isle of Pines, Sto. Domingo, Puerto Rico, St. Maarten, Grand Cayman, Jamaica, Curacao, Aruba, and Bonaire. This range is complementary to the range of other Rivulus species, although associations with these 4 species are rare to non-existent.
Habitat: Rivulus marmoratus is typically found in coastal mangrove swamps. Specimens have been collected across a wide range of salinities (0-68%), temperatures (7-30° C), and in water containing low dissolved oxygen and high levels of hydrogen sulfide from the decomposing mangral. Often, individuals are found emersed in detritus or leaf litter, likely stranded as temporal pools dried during low tides (Davis et al 1990). The species particularly favors the burrows of land crabs (Cardisoma or Ucides), though they have been recorded in shallow depression such as tires tracks, and have even been collected in numbers inside emergent rotting logs. There are also reports of individuals collected on mangrove leaves in the canopy and even flipping along the ground 100 m from the nearest water source (Huehner et al 1985).
General biology: The species is generally a marbled brown and gray with a distinct caudal peduncle ocellus (hence the synonym R. ocellatus). Maximum size is approximately 75 mm total length. Laboratory specimens of this size are typically 2-3 years old. Males have a characteristic orange wash which has proven to be a reliable indication of sex (Soto 1988). The possible role of males is discussed elsewhere. Rivulus marmoratus has been said to be rare in nature, but in fact it appears to be relatively common in certain micro-habitats, particularly the burrows of land crabs as mentioned above (Taylor 1988, Davis et al 1990). It is difficult to collect by conventional capture methods and seems to be resistant to rotenone. It is euryhaline and can tolerate abrupt changes in salinity, probably due to epidermal chloride cells. In the laboratory, it can be maintained in salinities ranging from fresh to seawater, though water of at least moderate salinity is best. Individuals readily emerge either to escape rising hydrogen sulfide levels and anoxia or in pursuit of food such as ant eggs. This habit of ready emersion may be used to escape poisons such as rotenone often used in sampling. Once emersed, 5 individuals moved by flipping, always in the direction in which the tail is pointing. Aerial respiration may be important as individuals captured in submerged traps often die. Aerial respiration is aided by epidermal capillaries (Grizzle and Thiyagarajah 1987). Frequent emergence, aerial respiration, and egg stranding are traits shared with several other Rivulus species, including R. chacunaque, R. hartii, R. isthmemsis, R. limonchocae, R. uroflammeus, and R. stagnatus (Davis et al 1990). In the laboratory, specimens have survived for up to 60 days in moist leaves (Davis 1990). Eggs are tolerant of drying substrate and are capable of entering diapause if stranded or buried (Ritchie and Davis 1986). The only ova collected in nature have been on leaves near the water (Taylor 1992). It is not known if this is a general strategy employed by this species to avoid predation on ova. Sexual maturity is reached in the laboratory in as little as 90 days, as marked by the onset of oviposition. It is not known if this correlates with the age of maturity in nature.
Gonadal morphology: The ovotestis is paired over much of its length. The testicular tissue is generally much less extensive than the ovarian tissue. The ovarian and testicular tissues are not separated by membranes, and mature sperm are found in ducts throughout the organ (Soto 1988). This proximity renders fertilization virtually simultaneous with ovulation. Fertilized ova can be held within the body for up to 77 hours (20%), thought 80% are oviposited within 24 hours of fertilization. Less than 1% are oviposited immediately upon fertilization (Harrington, 1963). Eggs are produced daily under laboratory conditions, with the number of eggs varying in accordance with diet.
Genetics: The Floridian fish studied by Kallman and Harrington (1964) proved to be homozygous and clonal by immunogenetic techniques. These clones proved indistinguishable by allozyme techniques, but different clonal lines are easily distinguished by DNA fingerprinting (Turner et al 1990). The population structure 6 appears to consist of many different homozygous clones, all at low frequency. The clonal composition of local populations changes rapidly, so that it is difficult to collect a member of a particular clonal line from year to year. It was proposed that this was due to an extraordinarily high mutation rate at the microsatellite loci used in DNA fingerprinting, but progeny tests over several generations proved that the species is clonally stable over at least three generations (Laughlin et al 1995). Lin and Dunson (1995) have reported fitness differences, measured by weight and survivorship, between clones under different incubation and rearing conditions. It seems that clonal lines may vary in fitness in different environments, though overall fitness across several environments is the same, so that changes in the local environment may lead to changes in the clonal lines represented in that particular area.
Environmental Sex Determination In some organisms, sex is determined through a combination of genotype and environment, rather than being entirely genetically determined (Charnov and Bull 1977). Environmental sex determination is found in many invertebrates (Charnov and Bull 1977), particularly those with a parasitic life history, and there are many cases among poikilothermic vertebrates, including turtles (Bull and Voight 1979), alligators (Ferguson and Joanen 1982), lizards (Bull 1980), and a few fishes (Conover and Kynard 1981, Middaugh and Hemmer 1987, Strussmann et al 1996). In the case of many species exhibiting environmental sex determination, incubation temperature during critical periods of embryological development appears to be the primary driver, and can result in highly skewed sex ratios. Charnov and Bull (1977) extended Ghiselin's size advantage (1969) model by proposing that if a change in the sex ratio in response to the environment is favored, then mechanisms would evolve whereby sex would be determined directly by the
7 environment. The criteria leading to the development of ESD (environmental sex determination) proposed by Charnov and Bull are: (1) the effect of the environment on fitness differs between sexes, and (2) the parents are unable to control the environment into which there progeny enter. Further, the environment must be 'patchy' so that environments favorable to both sexes occur in order that the overall sex ratio does not become overly skewed. It seems reasonable to divide discussion of the factors influencing environmental sex determination between the social factors which are involved in sex change in sequentially hermaphroditic species, usually dominance behaviors and/or hormones and pheromones, and other environmental factors which determine sex in non-hermaphroditic species, the most common being incubation temperature. Temperature mediated sex determination (TSD) has been recorded in a wide range of vertebrate species (Warner 1975). In lizards, alligators and fishes, low temperature incubation results in a higher percentage of females, while the pattern is reversed in turtles. Work by Conover (1984, 1985, 1990, Conover and Kynard 1981, 1984, Conover and Present 1990, Conover and Ross, 1982) with the Atlantic silverside, Menidia menidia, provide support for this model in fishes. This species is somewhat unique in that it completes its life cycle in one year, unlike most temperate fishes. It was demonstrated that in some populations, sex is determined largely by the incubation temperature an individual encounters during a critical phase of embryological development. Incubation temperatures fluctuating between 11-19° C produced significantly higher proportions of females, while temperatures between 17-15° C produced significantly more males. The lower temperatures correspond to those occurring during the earlier part of the breeding season, while the higher temperatures occur during the later part. This results in significant sexual dimorphism, as females hatch earlier in the season and therefore have longer to grow prior to mating. The growth rate of the sexes is the same. Because female fecundity is positively correlated with body 8 size, larger individuals are likely to reproduce more effectively as females, while smaller individuals basically make the best of a bad situation by reproducing as males. Mating involves aggregations of varying size, from as few as five to thousands of individuals of both sexes, mostly males (approximately 10:1). There are no courtship behaviors, breeding coloration or accessory structures involved with mating. It appears that gonad weight as a percent body weight increases much more rapidly in females than males, so while females gain reproductive advantage through larger size, this is much less true of males. The spawning patterns of male and females also differ. Spawning occurs at high tides corresponding to the new and full moon (approximately every two weeks) in intertidal marshes, so that there are 4-5 spawning periods during the breeding season (April through June). During these high tides over several days, females spawn only once a cycle, while males spawn several times (Conover 1985). These multiple spawnings are energetically costly for males, and reduce the time males are able to spend foraging. Conover concludes that TSD in this species allows those born early in the season, and have the greatest opportunity to become large, to become the sex in which large size has the greatest advantage, and also allows those born later in the season, with little chance of becoming large, to become the sex in which the penalty for small size is the least. This system also shows a cline in the degree of TSD between populations from Nova Scotia to South Carolina. Nova Scotian populations lack TSD, so that sex is entirely genetically determined. The degree of TSD increases as latitude decreases (Conover and Present 1990). Conover explains this cline in terms of the decrease in the length of the growing/spawning season from South Carolina to Nova Scotia. As the growing season shortens, the advantages of early birth in terms of future size are diminished, so that incubation temperature becomes an unreliable indication of expected size. Obviously, as the growing season lengthens, the advantages of early birth increase as well, and so does the adaptive value of TSD. Similar result were recently reported in another atherinid fish, Ondontesthes 9 argeninensis, the South American sea perjerry, again with females predominating among individuals incubated at lower temperatures (18 and 21° C), while males predominated among individuals incubated at 25° C (Strussmann et al 1996). However, in this species, the adaptive significance of TSD is less apparent, as these fish are not annual and can spawn for at least two years, so that the differences in reproductive success between individuals of different sizes are somewhat mitigated. It has been suggested that ESD may be maintained in longer lived species, as extreme sex ratios will tend to cancel out over time so that an overall balanced sex ratio is maintained (Bulmer and Bull 1989); in this species, ESD may be vestigial. Studies of TSD in the poecilleid Poeciliopsis lucida have provided evidence that the degree of lability of sex is under genetic control. Sullivan and Schultz (1985) demonstrated that there were differences in the degree of incubation temperature influenced lability of sex between inbred (homozygous) laboratory strains of this species. Whereas one strain produced a sex ratio of approximately 1:2 males to females at 27° C, a 1:1 ratio at 25.5° C, and 2:1 males to females at 24° C, another strain produced sex ratios of 1:1 at all three temperatures. The possibility that these differences could be due to differential mortality was eliminated, though there remained unexplained sources of variation between the sex ratios of individuals of the labile strain. It was suggested that seasonal fluctuations in the stream habitat of this species between the rainy and dry seasons might provide a basis under which one sex might have a greater advantage and provide an adaptive explanation for TSD. During the rainy season from July to November, the streams of northern Mexico to which this species is endemic are often flooded and nutrient rich, conditions under which individuals would have the opportunity to achieve greater size. Under the size advantage hypothesis, these individuals would reproduce more effectively as females. However, during the dry season, these streams often dry to small pools in which nutrients are scarce. Individuals born under these conditions have little opportunity to grow large before the breeding season, and therefore 1016 would achieve the greatest reproductive success as males. J.J. Bull (1981) has provided a theory for the evolution of ESD from purely genetical sex determination where one sex is heterogametic, as well as some constraints on this process. Assuming a situation where XX is female and XY is male, the process begins with the occurrence of some effect that overrides the genetic determination of sex so that occasional XX males appear. This might be an environmental factor, to which all members of the species are susceptible, but only a few encounter (e.g.. temperature extremes), or a segregating gene that causes sex reversal in its carriers under certain conditions. If the sex ratio changes as a result of this factor so that while most XX are female, some are male, and all XY are male, then the frequency of XY must change so that the sex ratio remains 1:1. The fitness of XX males must be equal to that of XY males. If there is a selection pressure which favors ESD, then selection will be for the loss of genetic sex determination.
Environmental Sex Determination, Sequential hermaphroditism, and Rivulus marmoratus
It is difficult to apply concepts in the general literature on ESD and sequential hermaphroditism to Rivulus marmoratus. R.W. Harrington has suggested that the low temperature induction of males in this species might be an environmental sex determination system (1968), possibly one designed to allow episodic outcrossing. Perhaps it is best to begin with what is known regarding male/hermaphrodite interactions, and then discuss other speculations. In the laboratory, Rivulus marmoratus males will readily court hermaphrodites using typical killifish courtship behavior. These courtship displays are often accepted, and it is not uncommon to witness spawning embraces between the two. Hermaphrodite/hermaphrodite spawning embraces have not been documented. The discovery of an outcrossed population on Twin Cays, Belize in the presence of high (10-25%) numbers of males clearly indicates that on that island at least, 113 some mechanism exist whereby hermaphrodites are able to mate with males. No females were found on the island. Successful matings have never been documented in the laboratory, though some attempts have been made in this direction. In our laboratory, male/hermaphrodite pairings were made in small (approximately 500 ml) containers, and often the male was killed by the hermaphrodite in less than two weeks. Less frequently, the two individuals would co-exist, but the hermaphrodite would cease to oviposit, and even more rarely, the male would kill the hermaphrodite, though only if he was larger (Personal observation). Beyond these facts and observations, almost nothing is known regarding mating in this species. Because the ovotestis is unrestricted, the presence of sperm in the lumen of the gonad virtually insures that ovulation leads to fertilization. For fertilization to routinely take place during mating, the hermaphrodite must have previously ceased or severely limited spermatogenesis and have depleted sperm stores. It has been speculated that males might fertilize the rare viable but unfertilized eggs that hermaphrodites emit. This cannot be discounted, but it is unlikely that this type of outcrossing would explain the phenomenon on Twin Cays, were every individual tested from that population was heterozygous. This means that either the entire population participated in outcrossing, or that the rare heterozygous individuals resulting from male fertilizations were so much more fit that they displaced their homozygous conspecifics within very few generations. Although the first hypothesis is more likely, there is as yet no explanation as to what could cause a hermaphrodite to cease spermatogenesis in order to outcross. The males captured on Twin Cays all appeared to be primary males, having no remnants of ovarian tissue indicating prior existence as a functional hermaphrodite. Shapiro (1987) warns against using indices such as these to determine the functional history of a gonad, but given that none of the male gonads sectioned exhibited ovarian tissue, it seems safe to conclude that these were primary males. It is likely that whatever factor resulted in these primary males, would also cause effective male sterility in the 12 hermaphrodites experiencing the same factor. Otherwise, males could not be certain that mates would be available upon their sexual maturation. It is difficult to envision such a factor. It does not seem that the mere presence of males is sufficient to induce male sterility in hermaphrodites. The general literature regarding gonochoristic species that exhibit TSD hypothesizes that temperature is a factor in sex determination because it predicts the type of environment into which an individual can expect to be born, and this in turn differentially affects the likely mating success of each sex. Therefore, an individual will become the sex in which it is likely to have the greatest mating success. If this hypothesis is applied to R. marmoratus, it implies that if there is a environmental sex determining factor, the males that result from this factor will be more reproductively successful than if they were hermaphrodites. Given that selfing hermaphrodites are guaranteed to reproduce, males must be quite successful at mating for this to be a viable reproductive strategy. The genetics involved with self-fertilization, in terms of the percent of one's genome that is transmitted to the succeeding generation, further complicate this. A homozygous selfing hermaphrodite transmits it's genome intact to it's progeny, but if it participates in sexual matings, it's progeny's genome will consist of only half of the parental hermaphrodite's genes. This is true for males as well, but it is the best that they can expect. The conditions under which it would be adaptive for a hermaphrodite to participate in sexual reproduction are yet to be determined.
Sequential Hermaphroditism Sequential hermaphroditism, in which individuals change sex at some point during their life, is a part of the life history of many species, particularly among invertebrates and some poikilothermic vertebrates, especially fishes (Atz 1964, Reinboth 1969). Species in which the initial sex is male are protandrous, and those in which the initial sex is female are protogynous.
13135 Ghiselin (1969) proposed three models with which to explain the adaptive significance of hermaphroditism in animals. Two models dealt with species in which it is difficult to locate a mate or with the importance of maintaining genetic variability in a population with low motility (e.g. marine invertebrates) or low population density. His size advantage model best applies to sequential hermaphrodites. Simply, this hypothesis proposes that sequential hermaphroditism is favored when an individual reproduces more efficiently as one sex when small, and more efficiently as the other sex when large. This idea is developed by Warner (1975), who concludes that sequential hermaphroditism is likely to arise in species with high population densities in which expected fecundity, survivorship, and mating characteristics combine to produce a situation where male and female size specific fecundities differ. It appears that there are varied combinations of expected survivorship and female fecundity under which pressure favoring sequential hermaphroditism could exist. Selection for protandry could exist under mating systems in which male reproductive success is not correlated with male size, but female reproductive success increases as size increases. This exists in some group spawning species in which mating is random so that small males are as likely to fertilize eggs as large males. Sperm production does not increase appreciably with size in males. However, egg production in females is closely correlated with size, so that large females, which are able to produce more eggs, produce more progeny than small females. In such a system, the greatest reproductive success would be achieved by being male when small and female when large. Protogyny could arise in mating systems were males monopolize female matings. Under this system, it is clearly disadvantageous to reproduce as a male when small. Therefore individuals increase their reproductive success by reproducing as females when small and then as males once a competitive size has been reached. Surveys of protogynous fishes conform to this prediction (Warner 1988). Virtually all protogynous species are ones in which males dominate female matings, either through controlling females themselves, or by controlling resources 144146 necessary for mating, usually nesting sites Selection for protygyny is likely in randomly mating species such as group spawners were male size is not an important factor in determining male fecundity, but female fecundity increases as a function of size (Warner 1988). The variety of theoretical mortality and fecundity schedules under which one sex has a greater expected fecundity than the other at some point in their life history raises the question as to why more species do not engage in sequentially hermaphroditic reproductive strategies. A likely explanation is the existence of strong genetic sex determining mechanisms in most species. Warner (1975) raises the question as to why sequential hermaphroditism is not generally found in higher vertebrates when it is relatively common among teleost fishes. Possibly, the costs associated with changing sex are prohibitively high for these organisms and the age/size specific fecundities are not as pronounced. Also, many higher vertebrates do not grow throughout there lives in the same manner as fishes, so there is little size differential between different age classes (Ohno 1967). The size advantage model allows the likelihood that a species will engage in sequential hermaphroditism as a reproductive strategy to be gauged. However, it does not provide information as to what environmental cues individual members of such a species might use to evaluate their reproductive potential and make decisions as to when it would be advantageous to change sex. These factors are likely to be species specific, and their description so far has been based on scrutiny of individual species. It is not sufficient to conclude that individuals change size at a specific age or size, because the reproductive success of an individual is affected also by the size and sex of the other individuals in the population, as well as on the size of the mating group as a whole. For example, in a large mating population, even a small male may be able to control enough females or resources to render mating as a small male successful, relative to the reproductive success of a similar sized female. 15157 In some species studied, sex change is under social control. The labrid fish Labroides dimidiatus, a reef cleaner fish, is a protogynous species, with the population consisting of mostly females (Robertson 1972). The social unit is a harem formed around one male and several females and immature individuals. The male dominates all of the females in the harem. Also, each female dominates all inferior females and immature individuals below them in the hierarchy. The male patrols the entire territory and engages in aggressive behavior with any males he might encounter attempting to invade the territory. Females control feeding territories within the larger territory of the male, with the dominant female holding the feeding territory at the center of the male's territory. Should an individual within the harem die or vacate a territory, the entire female hierarchy generally shifts up one territory, so that if the dominate female dies, her territory becomes the territory of the second most dominate female, who also abandons her previous territory. This is repeated throughout the hierarchy (Robertson 1972). In the event of the death of the male, two events can occur. Outside males may invade the territory and attempt to co-opt it en mass, by dominating all the females. Alternatively, the dominant female may undergo sex reversal and become the dominate male. This female begins characteristic male type dominant displays 1.5 to 2 hours after the death of the male. The entire process of sex reversal is complete within four days, and sperm can be released within two weeks. It appears that all females have testicular elements within the ovaries, so that any female in capable of undergoing sex reversal (Robertson 1972). The anemone fish Amphiprion bincintus and Amphiprion alkallopisos in the Red Sea are protandrous sequential hermaphrodites. The dynamics of the mating system are similar to that of L. dimidiatus, but with single large females dominating numbers of males, all inhabiting a single sea anemone. Only the largest male (the second largest fish on the anemone) participates in matings with the female. The hierarchy among the fishes is maintained by aggressive dominance, with most aggressive behavior directed at the individual directly below in the hierarchy. Each male is capable of changing into a 16168 female. Upon the death of a female, the dominant male changes sex and the next most dominant male is permitted to mate. This system appears designed to prevent the population from expanding beyond the point where it can be supported by the resources of the host anemone, while still ensuring a supply of potential mates of both sexes.
Sequential hermaphroditism and Rivulus marmoratus The well studied sequentially hermaphroditic mating systems have generally supported the size advantage model. While laboratory kept specimens of R. marmoratus will frequently change from selfing hermaphrodites to functional males, usually late in their life, it is difficult to explain this as adaptive under the size advantage model. Most importantly, males captured from natural populations are likely to have been primary males in which the ovarian tissue never matured, or matured only briefly. Most protogynous sequentially hermaphroditic species are those in which females choose mates based on size, or in which a males mating success is dictated by his ability to hold a mating territory, which is generally related to a males size relative to other males in the population. These types of behaviors are absent in Rivulus marmoratus. Also, it is difficult to theoretically envision how an individual of this species would increase its mating success by functioning only as a male, given the obligate self-fertilizing nature of hermaphrodites of this species.
Simultaneous hermaphroditism Simultaneous hermaphroditism, where individuals reproduce both as males and as females, is a much less common gender pattern than sequential hermaphroditism among teleost fishes. Excluding Rivulus marmoratus, all simultaneously hermaphroditic fishes participate in some type of reciprocal fertilization, egg trading being the most common form. Egg trading behavior has been described in Hyloplectus nigricans (Serranidae), a tropical reef fish. These fish are pair spawners. Individuals parcel their eggs rather than
17188 releasing then en mass. Pairs alternate egg release with fertilization and mating is discontinued if one partner ceases to offer eggs (Fischer 1980). Most simultaneous hermaphrodite fishes are members of the family Serranidae. Self-fertilization does not seem to be a strategy employed by these fishes. The gonadal structure of serranids differs from that of R. marmoratus in that the ovarian and testicular tissue are separated by membranes within the gonad capsule, and both have separate ducts to the outside (Reinboth 1962, Bortone 1977). The mating behavior of the tropical reef fish Serranus fasciatus has been described in detail (Hastings and Petersen 1986). This system differs from the egg trading seen in other species. Spawning typically involves two individuals, one acting as male and one as female. The individual acting in the female role contorts its body so that the abdomen is extended and it rises approximately a meter off the substrate. The second fish follows closely, and gametes are released at the peak of the rise. These fish spawn several times during the mating season both as males and as females. This mating system is complicated by the presence of pure males, who are always the largest individuals. These males control territories covering the feeding range of several hermaphrodites and mate with these hermaphrodites. Further, hermaphrodites will attempt to steal fertilizations by "streaking". These individuals invade a spawning display just as gametes are released, and release sperm as well. This strategy appears to be employed by hermaphrodites living in a male controlled territory who have already released their eggs. The presence of these large males appears to limit the success of smaller hermaphrodites attempting to mate as males. This mating system appears to be a basically polygynous system, with large males controlling harems of hermaphrodites, with these hermaphrodites utilizing "streaking" as an alternative mating strategy (Petersen 1987).
18 Materials and Methods Study Design The goal of this experiment was to detect variation between clonal lines in the production of males resulting from the incubation of embryos at low temperatures. The hypothesis to be tested is that the variation in the production of males at lower incubation temperatures will vary between clonal lines and that this variation will correspond to the geographic origin of these clonal lines. Therefore, this study used clonal lines originally collected from the northern and southern limits of the range (Florida and Brazil respectively) and clonal lines originally collected from several Belize cays and the Belize mainland, both close to the center of the range. Eggs were collected from three isogenic hermaphrodites (clones) from each clonal line and these eggs were incubated at one of three incubation temperatures. The resulting progeny of each clonal line were pooled in the final analysis. Three incubation temperatures were chosen. 26° C is a moderate temperature and one which the organism would likely to encounter in nature. It was expected that almost no males to appear among embryos incubated at this temperature, regardless of the geographic origin of the clonal line, reflecting what is observed in most natural population of this species. 22.5° C was chosen as an intermediate temperature. This temperature might occur in Floridian and Brazilian waters, but is not likely to be common in the Caribbean. Therefore, male production was expected to be higher at this temperature among clonal lines from the Belize cays and Belize mainland, relative to male production among the clonal lines originally from Florida and Brazil, where lower temperatures may have provided a selection pressure to depress the temperature at which males are produced. 19° C was chosen as an extreme low temperature, which would not likely be encountered regularly by any of these fish in nature. At this incubation temperature, up to 100% males were expected to be produced by all of the clonal lines in this study.
19 Specimens: Origins and Care Specimens used in this study were laboratory reared descendants of wild-caught fish. The original collection sites included Coco Plum Cay, Belize , Ragged Cay, Belize (two barrier islands off the coast of Belize), Charles Church Marsh, Florida, near Vero Beach , and Rio de Janeiro, Brazil. The Belize and Floridian fish were originally collected in 1991, and the Brazilian fish was originally collected in 1989. The hermaphrodites were maintained individually in glass bowls in 200 ml of dilute marine water (18 ppt), which was changed every two weeks. A plastic mesh false bottom was placed in each bowl as a means of preventing parental oophagy before the eggs could be collected. The entire colony was maintained in an environment chamber at 26° C (+/-0.5) with a 12 hour light/dark cycle. All adult hermaphrodites were fed brine shrimp daily, supplemented with minced earthworms twice weekly.
Egg Collection, Incubation, and Rearing Fertilized eggs were collected every two days and placed in plastic petri dishes in 10 ml of water of the same salinity as the adults. Though eggs are retained by the hermaphrodite for different periods before oviposition (Harrington 1963), the stage during which sex is labile is relatively late in development, so that no embryos will have developed beyond this point prior to oviposition or collection. These dishes were kept in total darkness in one of three environment chambers, set at 19° C, 22.5° C, and 26° C. Developing eggs were checked daily for either mortality or hatching. Eggs incubated at 26° C, were allowed to develop to hatching without intervention. However, embryos incubated at 22.5° C and 19° C frequently enter a state of diapause, and if they are not forced from the egg, these embryos die when their yolk sac is depleted (Ritchie and Davis, 1986). Therefore, embryos incubated at 19° C and 22.5° C required manual dechorionation in order to limit mortality. It is possible that males and hermaphrodites 20 suffer from differential mortality rates under these conditions. Manual dechorianation prevents this possibility from affecting the final sex ratios. Manual dechorionation involves puncturing the chorion with a needle and peeling it apart with forceps. Eggs were dechorionated when the embryo appeared to be fully developed and the yolk sac was almost depleted. In a pilot study, dechorionation itself had no effect on the eventual sex phenotype. Forty eggs were collected from a single hermaphrodite from a Floridian clonal line. These eggs were incubated at 26° C, the temperature at which males were not expected to be induced. Twenty of these eggs were manually dechorionated and twenty were allowed to hatched unassisted. No males appeared among the eggs allowed to hatch normally and one male appeared among the twenty dechorionated individuals. Once the fry were hatched, each was placed in the 26° C incubation chamber in 200 ml of water and maintained in isolation from birth. Fry were fed brine shrimp.
Data Collection and Analysis Each specimen was allowed to mature until phenotypic sex was expressed. Sex was determined by the coloration of each individual. The orange body wash particular to males has proven to be a reliable indicator of gender (Soto and Noakes, 1994). Sexual maturation, indicated by either orange coloration in males or the onset of egg-laying in hermaphrodites, typically occurs between 3 and 6 months post hatching. This broad time frame could result from several factors. Sexual maturation is correlated with body size. Individuals incubated at the lower temperatures are typically less vigorous and smaller than those incubated at higher temperatures and so, initially, at least, they are likely to have greater difficulty capturing food and their growth rate is therefore slower, likely delaying sexual maturity. This effect is compounded in individuals with significant deformities (discussed below). Because of this differential, phenotypic sex was scored on an individual basis, rather than at a set time post hatching. 21 While scoring animals for phenotypic sex, high numbers of physical deformities were noted, especially among individuals incubated at the lower temperatures. These deformities mainly included spinal kyphosis, pharyngeal hyperplasia, and belly sliding, a deformity of the swim bladder. These deformities were also noted and described by Harrington (1967), though they were largely attributed to factors other than temperature. The frequencies of these deformities were scored and analyzed in the same manner as the male induction data. The G-test for homogeneity of variance (Sokal and Rohlf, 1995) was used to determine the statistical significance of the variation of sex ratio both within and among clonal lineages. Within each clonal lineage, variation was examined both across all three temperatures, to detect an overall temperature effect, and between adjacent temperatures, so that male induction at 26° C was compared with male induction at 22.5° C, and male induction at 22.5° C was independently compared with the effect at 19° C. This same method was also used to analyze the percentages of deformities that appeared at each temperature treatment, both within and among clonal lineages. Significance was taken at the a = 0.05 level.
Results Variation in male production within clones/temperatures The overall variation in male production across the three incubation temperatures within each clonal lineage was highly significant in all seven clones (Gh = 14.95 - 147.46, df=2, p<0.0005 in each case). The pattern was similar among most clones, being lowest following incubation at 26° C, intermediate following 22.5° C incubation, and highest following incubation at 19° C (figure 1). The only exception was the clone from Charles Church Marsh, Florida, in which the highest numbers of males produced was at 22.5° C.
(53%), while only 38% males were produced at 19° C (Gh = 105.84, df=2 ,p<0.0005).
22 Difference in the percent males produced between incubation at 26° C and 22.5°
C was only significant in the line from Charles Church Marsh, Florida (Gh = 7.85, df=1, p<0.025). This was also the only line in which male production at 22.5° C was higher than that at 19° C. There was significant difference in male production following incubation at 22.5°
C and 19° C in the clones from Rio de Janiero, Brazil (Gh = 10.94, df=1, p<0.0005),
Ragged Cay, Belize (Gh = 8.26, df=1, p<0.01), and Dangriga, Belize (Gh = 9.62, df=1, p<0.0005). The remaining samples, except for the line from Charles Church Marsh, Florida, did not have statistically significant variation in male production between 19° C and 22.5° C, though male production was always higher at 19° C.
Variation in male production within temperatures among clones Incubation at 26° C produced no significant difference among clones in the percentages of males produced. The percentages ranged from 17 % (Tobacco Range, Belize) to 2% (Dangriga Belize). Following incubation at 22.5° C, male production ranged from 53 % (Charles Church Marsh, Florida), to 6% (Rio de Janiero, Brazil). The percentages of male produced divided into two statistically significant groups (Gh = 43.55, df=1, p<0.0005), one ranging from 53% to 22% males, and the other ranging from 25% to 6% males, with two lines (Coco Plum, Belize 1 and Tobacco Range, Belize) being common to each group. The group with higher male production included the lines from Florida and the four Belize Cay lines, while the lower male production group included lines from Dangriga, Belize, Rio de Janiero, Brazil, and three Belize islands (two lines from Coco Plum, and Tobacco Range) (figure 1). Male production following 19° C incubation also produced two statistically significant groups (Gh = 28.55, df=1, p<0.0005). The high male production group ranged from 74% to 47% males, and included lines from Tobacco Range, Coco Plum 2, and 23 Ragged Cay, all Belize islands, and Dangriga, Belize. The lower male production group ranged from 47% to 35% males and included lines from Dangriga, Belize (common to both groups), Coco Plum Cay, Belize 1, Florida, and Brazil. Higher levels of male production at 22.5° C were not always predictive of corresponding levels at 19° C, so that while the lines from Coco Plum, Belize 1 and Charles Church Marsh, Florida were high male producers at 22.5° C, they were both included in the low male production group at 19° C. The line from Dangriga, Belize was a high male producer at 22.5° C, but a low male producer at 19° C. The lines from Tobacco Range, Belize and Ragged Cay, Belize were included in both groups.
Variation in deformities produced within clones/temperatures There was significant difference in the percent deformed individuals produced
across the three incubation temperatures in five of the seven clones (Gh = 13.56 - 26.57, df=2, 0.025>p>0.0005). The clones in which the incidence of deformities across the three incubation temperatures were from Vero Beach, Fl., Ragged Cay, Belize, Tobacco Range, Belize, Dangriga, Belize, and Rio de Janiero, Brazil. The pattern of these results was similar as that of the male production data, being lowest following 26° C incubation and highest following 19° C incubation (figure 2). The Tobacco Range, Belize lineage produced deformities at all three temperatures, ranging from 17% deformed at 26° C to 36% deformed at 19° C. These results followed the same trend as the other samples, but the data were statistically indistinguishable. The results from the Ragged Cay, Belize lineage also followed the typical trend (0% to 20%), but were insignificant due to small sample size. There was significant variation in the percent deformities produced following 26°
C and 22.5° C variation in the clonal lineages from Charles Church Marsh, Florida (Gh =
4.79, df=1, p<0.05) and both lineages from Coco Plum, Belize (Gh = 9.90 and 13.58, df=1, p<0.0005). All the lineages showed a trend of higher percentages of deformed 24 individuals produced following 22.5° C incubation than following 26° C incubation, except for the Dangriga, Belize lineage (figure 2). There was no significant variation in the percent deformed individuals produced following incubation at 22.5° C and 19° C. The lineage from Rio de Janiero, Brazil showed the largest difference, but only approached significance.
Variation in deformities produced within temperatures among clones Following incubation at 26° C, there was no significant variation in the number of deformed individuals produced among the various clonal lineages. The percentage of deformed individuals ranged from 24% (Dangriga, Belize) to 0% (Ragged Cay, Belize). . Following incubation at 22.5° C, production of deformed individuals ranged from 66% to 6%, and the results divided into three distinct groups. The Coco Plum, Belize 1 produced 66% deformed individuals at this temperature, and was significantly different from all other lines (Gh = 9.57, df=1, p<0.0005). The lines from Tobacco Range, Belize, Charles Church Marsh, Florida, and Rio de Janiero, Brazil produced 22%, 20% and 19% deformed individuals respectively. This group was distinct from those with both higher and lower production of deformed individuals (Gh = 12.66, df=4, p<0.0005). The final group was composed of the lines from Dangriga, Belize and Coco Plum, Belize, which produced 6% and 2% deformed individuals respectively. Incubation at 19° C produced relatively high levels of deformed individuals (87% to 36%), but there was no statistically significant variation among the clonal lineages, possibly because of small sample sizes at this temperature treatment.
25 Table 1 : The number of hermaphrodites and males produced by several clonal lines at three different incubation temperatures
Clonal Line Incubation Herms Males Temperature (°C) ______26 38 7 Vero Beach 22.5 7 8 Fl. 19 5 3
26 12 1 Ragged 22.5 2 1 Cay 19 2 3
26 24 5 Tobacco 22.5 7 2 Range 19 5 17
26 33 2 Coco Plum 1 22.5 9 3 19 5 3
26 50 2 Coco Plum 2 22.5 9 2 19 3 5
26 40 1 Dangriga 22.5 35 3 19 8 7
26 30 1 Rio de 22.5 16 1 Janiero 19 7 9
26 Figure 1 : Percent males produced from several different clonal lines by incubation temperature
90 26 C 80 22.5 C 19 C 70
60
50
40
30
20
10
0 Vero Ragged Tobacco Coco Plum Coco Plum Dangriga Rio de Beach, Fl. Cay Range 1 2 Janiero
Geographic origin of clonal line
273 Table 2 : The number of normal and deformed individuals produced by several clonal lines at three different incubation temperatures
Clonal Line Incubation Normal Deformed Temperature (°C) ______26 44 1 Vero Beach 22.5 12 3 Fl. 19 4 4
26 13 0 Ragged 22.5 3 0 Cay 19 4 1
26 24 5 Tobacco 22.5 7 2 Range 19 14 8
26 29 6 Coco Plum 1 22.5 4 8 19 1 7
26 49 3 Coco Plum 2 22.5 5 6 19 3 5
26 31 10 Dangriga 22.5 34 4 19 9 6
26 29 2 Rio de 22.5 13 3 Janiero 19 7 9
28 Figure 2 : Percent deformed individuals produced from several clonal lines by incubation temperature
80 26 C 70 22.5 C 19 C 60
50
40
30
20
10
0 Vero Ragged Tobacco Coco Plum Coco Plum Dangriga Rio de Beach, Fl. Cay Range 1 2 Janiero
Geographic origin of clonal line
29 Discussion Low temperature induced male production among the clonal lineages included in this study does not appear to conform to any macro-geographic pattern. I had initially hypothesized that the production of males following low temperature incubation of embryos would show a gradient between clones derived from hermaphrodites collected at locations closer to the equator and those derived from individuals collected at the more temperate extremes of the range, with the highest percentages of males being produced by those lines derived from the individuals captured in the more equatorial (warmer) locations. There are two possible explainations for this hypothesis, which are indistinguishable in this study. The first is that the production of males, at least through low temperature incubation, may not be adaptive, as I found no evidence that males commonly participate in reproduction. If this is so, the production of males represents a decrease in fitness for the parental hermaphrodite, as males compete with hermaphrodites for resources, but do not pass any genes to subsequent generations, so that the overall reproductive success of a hermaphrodite that produces males is lowered. If this is the case, there would be a selective pressure to reduce the production of males by low temperature incubation. Sex theory dictates that the rate of population increase of a self- fertilizing species will be twice that of a sexually reproducing species, all else held equal. The intrinsic rate of increase of a selfing species is related to the number of individuals in the population (N), whereas the rate of increase of a sexual species is related to the number of females in the population (N/2), assuming a 1:1 sex ratio. This predicts that the only way in which a sexual population could compete with self-fertilizing conspecifics is if the progeny produced through sexual reproduction were on average twice as fit as those of the selfing population (Maynard Smith, 1975). If low temperature incubation of embryos is in fact the primary driver of male production in natural populations, then there would be a selective pressure to depress the temperature at which males are produced below temperatures at which embryos are likely to be incubated. 30 This threshold temperature would be lower for those individuals inhabiting the extremes of the range of R. marmoratus, where more extreme low temperatures occur. The second rationale is that male induction at low temperature is part of an ESD, so that male production and possibly outcrossing is mediated by below average temperatures. If this effect is part of an ESD system designed to allow low levels of outcrossing, then populations which encounter consistent lower temperatures should have a lower temperature threshold for male induction. If the temperature threshold were the same across all populations, then those inhabiting regions with lower temperature extremes would produce more males. This prediction is not supported by population samples. This study is an extension of similar work done by R.W. Harrington, in which only clonal lineages derived from hermaphrodites collected in Florida were used. Harrington noted that there was in fact a differential in the percentages of males produced by different clones following low temperature incubation, and he surmised that this difference might be the consequence of a differential in the average temperatures encountered by these clones in nature (Harrington, 1967). At that time, the full extent of the range of R. marmoratus was not known. Harrington believed it likely that males participated in the mating system to at least some extent, based on the bright orange coloration of mature males, and observations of mating behavior between hermaphrodites and males (Harrington, 1968). This behavior is noteworthy because hermaphrodites in this species are generally highly antagonistic toward conspecifics, rarely tolerating the presence of others, particularly when ovipositing. While the adaptive significance of low temperature induced males may be debated, it is clear that the deformities appearing among individuals incubated at the lower two temperatures are maladaptive. While these deformities have been noted previously, they have not been specifically linked to low temperature incubation. The numbers of deformed individuals produced at the only moderately low incubation temperature of 22.5° C suggest that even this is developmentally stressful for R. 31 marmoratus. Given that low temperature are clearly stressful, as evidenced by the level of deformed individuals, it seems unlikely that low temperature incubation would be a strategy employed by this species to induce males and facilitate outcrossing. These data presented here do not appear to support the hypothesis that male production is part of a temperature mediated ESD. The variation in male production between clonal lineages does not follow any obvious pattern. Rather than indicating clonal adaptation to local temperature conditions, as originally hypothesized, male production may be indicative of the developmental stability of individual clonal lineages, so that variation is clone specific rather than location specific. If different clones have different genetic mechanisms for maintaining developmental stability, then some may be more effective than others in canalizing development under low temperature stress. These differences may appear as differences in male production, resulting from differential success at buffering development of sex phenotype, or as differences in the percentage of deformed individuals produced. The contrast in rank order sequences between male production and deformity production within temperatures among clones may result from different buffering mechanisms for sex phenotype and other (somatic) characters, so that while a particular clone may buffer sex phenotype well, the genetic mechanism responsible for buffering other characters may be less effective. The same comparison between male and deformity production within clones among temperatures may also be indicative of differences in buffering mechanisms, so that a genotype that performs well at 22.5° C, relative to that of other clonal lineages, may perform poorly under the greater stress of 19° C. It may well be that what has been measured in this study are the relative effectiveness of different developmental buffering mechanisms among different clones. There does not seem to be any evidence supporting the theory that males are a regular part of the normal reproductive biology of R. marmoratus. Though males are functional, there is no apparent mechanism by which hermaphrodites could regularly emit eggs which males could fertilize. The gonad of the hermaphrodite is an ovotestis, in 32 which ovarian tissue is closely surrounded by testicular tissue. Mature eggs are fertilized well before oviposition. In the laboratory, at least, this process of internal fertilization is almost 100% efficient. It has been hypothesized that males are opportunistic in their reproductive efforts, fertilizing rare viable but infertile eggs emitted by hermaphrodites (Harrington, 1971). However, in a laboratory setting, the number of such eggs is generally less than 4% (Harrington, 1971) so that this mechanism for outcrossing is unlikely. This hypothesis is not likely to account for the extensive outcrossing described on Twin Cays. There has been speculation that the proximity of males, possibly through social or pheremonal interactions, could cause hermaphrodites to cease spermatogenesis, while continuing oogenesis, so that they would then emit viable but infertile eggs which the males could fertilize (Harrington, 1971). There are no current data which support this hypothesis. A possible explanation is that populations of R. marmoratus outcross episodically. Such episodes would likely require specific environmental cues to which most clones would respond. If such cues exist, it is likely that they do not include low temperature. It may be that these unknown conditions induce the production of large numbers of males, which then participate in matings. Population density could be the key condition which drives episodic outcrossing. In general, individual R. marmoratus inhabit high tidal waters which may be isolated for extended periods of time so that there is little chance for interaction between individuals. This would render sexual reproduction difficult, as individuals are not likely to come into contact and therefore the costs associated with finding a mate are high. This may change during periods of high population density. Clearly, the cost of finding a mate drops as population density rises. Also, periods of high population density are likely to correspond to times when environmental conditions are relatively benign. The tidal mangrove swamp habitat of R. marmoratus is a low energy, high stress environment. Conditions including extended anoxia, high hydrogen sulfide concentrations, high temperature, high salinity, and low food availability are 33 common (Davis, Taylor, and Turner, 1990; Abel, Koenig, & Davis, 1987, ). Under these conditions, phenotypes resistant to extreme stress are favored. This is particularly true of loci involved in developmental processes, as phenotypic variants resulting from failures of developmental canalization under stressed conditions are likely to result in lower fitness for the individual. The loci involved in canalizing developmental processes are thought to work in concert as a co-adapted gene complexes, so that a stress resistant phenotype is the result of several genes at different loci (Clarke, 1993). Outcrossing in a normally self-fertilizing species such as R. marmoratus may cause the disruption of these co-adapted gene complexes resulting in a phenotype less resistant to biotic stress factors during development. However, in periods of high population density, possibly corresponding to periods when stress is low and available energy is high, developmental canalization may not be as important, and a wider diversity of phenotypes may be possible (Parsons, 1993). When typical environmental constraints are relaxed, outcrossing might occur as the metabolic cost restraints associated with supporting variation are lowered. The questions remains as to whether or not it would ever be advantageous for an obligate self-fertilizing species such as R. marmoratus to outcross. This debate is rooted in the larger debate concerning heterosis and genomic co-adaptation and the relative importance of each in maintaining developmental stability. Co-adaptation is thought to result in developmental canalization, the ability to produce the desired phenotype over a variety of developmental conditions, and is assumed to be selectively advantageous (reviewed in Zahharov, 1989, 1992). The theory of heterozygote superiority claims that the most fit individuals or populations are those that are heterozygous at the most loci (Lerner, 1954; Soule, 1979). The level of heterozygosity is determined as the percentage of electrophoretically mobile enzyme systems that show polymorphism (Van Valen, 1962). This percentage is assumed to be a reliable measure of the overall heterozygosity within the genome. Studies of the effect of heterozygosity typically involved 34 measurements of Fluctuating Asymmetry (FA) between more and less heterozygous populations. The level of FA is determined as the difference in left/right morphometric characters (Palmer & Strobeck, 1986). In fishes, these include pectoral fin rays, scale counts, and numbers of gill rakers on paired pharyngeal arches. Variation in these characters is assumed to be a measure of the degree of developmental stability exhibited by an individual. Proponents of heterosis claim that heterozygosity imparts an increased degree of developmental stability in comparison to more homozygous individuals because development can be more effectively buffered against accidents of development as different alleles can complement each other. The most cited case in which increased heterozygosity was correlated with reduced FA is a series of studies conducted with Salmo gardnerei (Leary, Allendorf, and Knudson, 1983,1984,1992; Leary et al, 1985). However, studies of other organisms have failed to show any such correlation, or have even shown decreased heterozygosity correlated to reduced levels of FA (Zahkarov, 1981; Leary, Allendorf, & Knudson, 1985; Clarke & McKenzie, 1987, 1992), so that the role of heterozygosity in "buffering" development remains unclear. The theory of genomic co-adaptation claims that developmental stability arises not from heterozygosity, but from the actions of genes at different loci working in concert to canalize development (Dobzhansky, 1950, 1970; Thoday, 1955). Genes that perform well together tend to accumulate within the gene pool. The results of this process are co- adapted gene complexes (Maynard Smith 1975). There is some support for both the overdominance hypothesis and the genetic co-adaptation hypothesis and there may be no simple, definitive answer as to which is more important. The relationship may depend on the degree of outcrossing that normally occurs within a population (Clarke, 1993). These two theories provide opposite expectations regarding outcrossing in R. marmoratus, particularly as a response to stress. Obviously, outcrossing is related to the production of males in this species. Since the initial discovery of temperature dependent male induction in R. marmoratus, it has been assumed, explicitly (Harrington 1967) and 35 implicitly (Hughes 1989), that the phenomenon is part of an environmental sex determination system (ESD), and that males have some regular role in the reproductive biology of this species. However, it is not clear under what conditions, if any, outcrossing might be advantages for this species. In fact, it is by no means certain that the heterozygous progeny resulting from a sexual mating would enjoy an increase in fitness as compared to their homozygous conspecifics. It seems likely, given the homozygous nature of almost all R. marmoratus, that overdominance is not important in maintaining developmental stability in this species. If an individuals fitness is due to co-adaptation within the genome, then outcrossing may serve to disrupt these co-adaptated gene complexes, resulting in a lower fitness, because outcrossing increases the rate of linkage disequalibrium decay and could therefore disrupt co-adapted gene complexes. It is not unreasonable to suppose that individual clonal lines of R. marmoratus maintain different co-adapted gene complexes, and that these gene complexes are not necessarily compatible. The clonal differences in both male production and production of deformed individuals provide some support for this idea. If this hypothesis is correct, outcrossing may result in a decrease in the fitness of individuals resulting from sexual matings, due the disruption of co-adapted gene complexes. Given the lack of an obvious mechanism by which males could participate in matings, and the open question as to whether this would be selectively advantageous, it seems that males resulting from low temperature incubation are likely to be developmental anomalies rather than a normal part of the reproductive biology of the species. Development at temperatures below 22.5° C is clearly difficult for R. marmoratus, as seen in the relatively high percentages of deformed individuals resulting from incubation at these temperatures. Temperatures below 20° C, where male production and deformities are both at their highest levels, are likely too low to be physiologically reasonable for R. marmoratus, particularly given that all of these 362 embryos had to be manually dechorionated to achieve significant survival. Also, hermaphrodites typically cease egg production at temperatures below 22.5° C, and so seem to avoid the possibility of submitting their progeny to development under thermally stressful conditions, rather than laying eggs which could produce males, and then outcross as a response to this stress. Behavioral stress avoidance is seen in other aspects of the behavior of R. marmoratus, such as in the ability to emerge from poor quality water, and to travel long distances over land possibly in search of better habitat or food. The phenomenon of outcrossing on Twin Cays, a Belize barrier island, must be addressed. Progeny testing of individuals collected from this island in 1991 indicated that all of these individuals were heterozygous at mini- and microsatellite loci, and so clearly recent outcrossing had occurred (Lubinski, Davis, Taylor, and Turner, 1993). At the same time as these heterozygous individuals were collected, males comprised 24% of the population on Twin Cays. Also at this time, similar percentages of males were found on nearby barrier islands, but individuals collected from these islands proved to be homozygous when DNA fingerprinted. This finding makes interpretation of the phenomenon of outcrossing on Twin Cays difficult to explain. It seems unlikely that relatively high numbers of males in the population alone are not sufficient to trigger outcrossing. There was no obvious indication that environmental conditions on Twin Cays were significantly different from those on nearby islands. Also, temperatures on these islands were never low enough to produce the percentages of males that were documented, so that Twin Cays is certainly not an example of outcrossing in response to low temperature stress. This is not intended to trivialize the importance of the discovery of outcrossing on Twin Cays. However, future research must focus on factors other than temperature to explain the appearance of high numbers of males in natural populations of this species.
37 Literature Cited
Abel, D.C., C.C. Koenig, and W.P. Davis. 1987. Emersion in the mangrove fish Rivulus marmoratus: a unique response to hydrogen sulfide. Env. Biol. Fishes 18(1): 67-72.
Atz, J.W. 1964. Intersexuality in fishes, p. 145-232. In: Intersexuality in vertebrates including man. C.N. Armstrong and A.J. Marshall (eds.). Academic Press, London, England.
Bell, G. 1982. The masterpiece of nature: the evolution and genetics of sexuality. Univ. of California Press, Berkeley. pp. 336.
Bull, J.J. 1981. Evolution of environmental sex determination from genotypic sex determination. Heredity 47(2): 173-184.
Bull, J.J., and M.J. Bulmer. 1989. Longevity enhances selection of environmental sex determination. Heredity 63: 315-320.
Bull, J.J., and R.C. Vogt. 1979. Temperature dependent sex determination in turtles. Science 206: 1186-1188.
Bulmer, M.J. 1987. Sex determination in fish. Nature 362(2): 440-441.
Bulmer, M.G., and J.J. Bull. 1981. Models of polygenic sex determination and sex ratio control. Evolution 36: 13-26.
Charnov, E.L., and J.J. Bull. 1977. When is sex environmentally determined? Nature 266: 828-830.
Clarke, G.M. 1993. The genetic basis of developmental stability. I. Relationships between stability, heterozygosity, and genomic coadaptation. Genetica 89: 15-23.
Clarke, G.M. and J.A. McKenzie. 1992. Coadaptation, developmental stability and fitness of insecticide resistant genotypes in the Australian sheep blowfly, Lucilia cuprina: A review. Acta Zool. Fennica 191: 107-110.
Clarke, G.M. and J.A. McKenzie. 1987. Developmental stability of insecticide resistant phenotypes in blowfly; a result of canalizing natural selection. Nature 325: 345-346.
Conover, D.O. 1984. Adaptive significance of temperature-dependent sex determination in a fish. American Naturalist 123: 297-313.
Conover, D.O. 1985. Field and laboratory assessment of patterns of fecundity of a multiple spawning fish: the Atlantic silverside, Menidia menidia. Fishery Bulletin 83, 331-341.
38 Conover, D.O. 1990. The relation between capacity for growth and length of growing season: evidence for and implications of countergradient variation. Transactions of the American Fisheries Society 119: 416-430.
Conover, D.O., S.W. Heins. 1987. The environmental and genetic components of sex ration in Menidia menidia. Copeia 1987: 732-743.
Conover, D.O., and B.E. Kynard. 1981. Environmental sex determination: interaction of temperature and genotype in a fish. Science 213: 577-579.
Conover, D.O., and B.E. Kynard. 1984. Field and laboratory observations of spawning periodicity and behavior of a northern population of the Atlantic silverside, Menidia menidia (Pisces: Atherinidae). Environmental Biology of Fishes 11: 161-171.
Conover, D.O., and T.M. Present. 1990. Countergradient variation in growth rate: compensation for the length of the growing season among Atlantic silverside from different latitudes. Oecologica 83: 316-324.
Davis, W.P., D.S. Taylor, and B.J. Turner. 1990. Field observations on the ecology and habits of the mangrove rivulus (Rivulus marmoratus) in Belize and Florida (Teleostei: Cyprinodontiformes: Rivulidae). Icthyol. Explor. Freshwaters 1(2): 123-134.
Dobzhansky, T. 1950. Genetics of natural populations. XIX. Origin of heterosis through natural selection in populations of Drosophila pseudoobscura. Genetics 35: 288- 302.
Dobzhansky, T. 1970. Genetics of the Evolutionary Process. Columbia Univ. Press, NY. pp. 505.
Fishelson, L. 1970. Protogynous sex reversal in the fish Anthias squamipinnis (Teleostei, Anthiidae) regulated by the presence or absence of a male fish. Nature 227: 90-91.
Ferguson, M.W.J., and T. Joanen. 1982. Temperature of egg incubation determines sex in Alligator mississippiensis. Nature 296:850-853.
Fricke, H., and S. Fricke. 1977. Monogamy and sex change by aggressive dominance in coral reef fish. Nature 266: 830-832.
Ghislin, M.T. 1969. The evolution of hermaphroditism among animals. Quarterly Review of Biology 44: 189-208.
Harrington, R.W., Jr. 1961. Oviparous hermaphroditic fish with internal self- fertilization. Science 134: 1749-1750.
Harrington, R.W., Jr. 1963. Twenty-four hour rhythms of internal self-fertilization and of oviposition by hermaphrodites of Rivulus marmoratus. Physiol. Zool. 36: 325-341.
3940 Harrington, R.W., Jr. 1967. Environmentally controlled induction of primary male gonochorists from eggs of the self-fertilizing hermaphroditic fish Rivulus marmoratus. Biol. Bull. 132: 174-199
Harrington, R.W., Jr. 1968. Delimitation of the thermolabile phenocritical period of sex determination and differentiation in the ontogeny of the normally hermaphroditic fish Rivulus marmoratus. Physiol. Zool. 41: 447-460.
Harrington, R.W., Jr. 1971. How ecological and genetic factors interact to determine when self-fertilizing hermaphrodites of Rivulus marmoratus change into functional secondary males with a reappraisal of the modes of intersexuality among fishes. Copeia 3: 389-432.
Harrington, R.W., Jr. and K.D. Kallman. 1968. The homozygosity of clones of the self- fertilizing hermaphroditic fish Rivulus marmoratus (Cyprinodontidae Atheriniformes). Am. Nat. 102:337-343.
Kallman, K.D. and R.W. Harrington, Jr. 1964. Evidence for the existence of homozygous clones in the self-fertilizing hermaphroditic teleost Rivulus marmoratus (Poey). Biol. Bull. 126: 101-114.
Kristensen, I. 1970. Competition among three cyprinodont fish species in the Netherlands Antilles. Studies on the Fauna of Curacao and other Caribbean Islands 119: 82-101.
Leary, R.F., F.W. Allendorf, and K.L. Knudsen. 1983. Developmental stability and enzyme heterozygosity in rainbow trout. Nature 301: 71-72.
Leary, R.F., F.W. Allendorf, and K.L. Knudsen. 1984. Superior developmental stability of heterozygotes at enzye loci in salmonid fishes. Amer. Nat. 124: 540-551.
Leary, R.F., F.W. Allendorf, and K.L. Knudsen. 1985. Developmental instability and high meristic counts in interspecific hybrids of salmonid fishes. Evolution 39: 1318- 1326.
Leary, R.F., F.W. Allendorf, and K.L. Knudsen. 1992. Genetic, environmental and developmental causes of meristic variation in rainbow trout. Acta Zool. Fennica 191: 79- 96.
Lerner, I.M. 1954. Genetic Homeostasis. Wiley, NY. pp134.
Maynard Smith, J. 1978. The Evolution of Sex. Cambridge Univ. Press, Cambridge. pp. 222.
Middaugh, D.P., and M.J. Hemmer. 1992. Reproductive ecology of the inland
40 silverside, Menidia beryllina (Pisces: Atherinidae) from Blackwater Bay, Florida. Copeia 1992: 53-61.
Ohno, S. 1967. Sex chromosomes and sex-linked genes. Springer, Heidelberg. 192 pp.
Parsons, P.A. 1993. Developmental variability and the limits of adaptation: interactions with stress. Genetica 89: 245-253.
Reinboth, R. 1970. Intersexuality in fishes. Memorial Society of Endocrinology 18: 515- 543.
Robertson, D.R. 1972. Social control of sex reversal in a coral-reef fish. Science 177: 1007-1009.
Sadovy, Y., and D.Y. Shapiro. 1987. Criteria for the diagnosis of hermaphroditism in fishes. Copeia 1987(1): 136-156.
Shapiro, D.Y. 1987. Differentiation and evolution of sex change in fishes. BioScience 37 (7): 490-497.
Soule, M.E. 1979. Heterozygosity and developmental stability; another look. Evolution 33: 396-401.
Soto, C.G., and D.L.G. Noakes. 1994. Coloration and gender in the hermaphroditic fish Rivulus marmoratus Poey (Teleostei: Rivulinae). Icthyological Explorations of Freshwaters 5: 79-90.
Strussmann, C.A., J.C. Calsina Cota, G. Phonlor, H. Higuchi, and F. Tahkashima. 1996. Temperature effects on sex determination of two South American atherinids, Odontesthes argentinensis and Patatgonia hatcheri. Environmental Biology of Fishes 47: 143-154.
Sullivan, J.A., and R.J. Schultz. 1986. Genetic and environmental basis of variable sex ratios in laboratory strains of Poeciliopsis lucida. Evolution 40: 152-158.
Taylor, D.S. 1988. Observations on the ecology of the killifish Rivulus marmoratus (Cyprinodontidae) in an infrequently flooded mangrove swamp. Northeast Gulf Sci. 10(1): 63-68.
Thoday, J.M. 1955. Balance, heterozygosity and developmental stability. Cold Spring Harbor Symposium on Quantitative Biology 20: 318-326.
Turner, B.J., W.P. Davis, and D.S. Taylor. 1992. Abundant males in populations of a self fertilizing hermaphroditic fish, Rivulus marmoratus, from some Belize cays. J. Fish Biol. 40: 307-310.
Turner, B.J., J.F. Elder, Jr., T.F. Laughlin, and W.P. Davis. 1990. Genetic variation in
41 clonal vertebrates detected by simple-sequence DNA fingerprinting. Proc. Natl. Acad. Sci. USA 87: 5653-5657.
Turner, B.J., J.F. Elder, Jr., T.F. Laughlin, W.P. Davis, and D.S. Taylor. 1992. Extreme clonal diversity and divergence in populations of a selfing hermaphroditic fish. Proc. Natl. Acad. Sci. USA 89:10643-10647.
Van Valen, L. 1962. A study of fluctuating asymmetry. Evolution 16: 125-142.
Van Valen, L. 1973. A new evolutionary law. Evol. Theory 1:1-30.
Vrijenhoek, R.C. 1985. Homozygosity and interstrain variation in the self-fertilizing hermaphroditic fish, Rivulus marmoratus. J. Hered. 76: 82-84.
Vrijenhoek, R.C., R.M. Dawley, C.J. Cole and J.P. Bogart. 1989. A list of the known unisexual vertebrates. Pp. 19-32 in: Evolution and ecology of unisexual vertebrates, ed. by R.M. Dawley and J.P. Bogart. Bulletin 466, New York State Museum, Albany, NY.
Warner, R.R. 1975. The adaptive significance of sequential hermaphroditism in animals. American Naturalist 109: 61-82.
Warner, R.R. 1988. Sex change in fishes: hypothesis, evidence, and objections. Environmental biology of Fishes 22(2): 81-90.
Warner, R.R., and P. Lejeune. 1985. Sex change limited by paternal care: a test using four Mediterranean labrid fishes, genus Symphodus. Marine Biology 87: 89-99.
Williams, G.C. 1975. Sex and Evolution. Princeton Univ. Press, Princeton, NY. pp. 200.
Zahkarov, V.M. 1992. Population phenogenetics: Analysis of developmental stability in natural populations. Acta Zool. Fennica 191: 7-30.
42 CIRRICULUM VITAE
Michael Todd Fisher Department of Biology Virginia Polytechnic Institute and State University Blacksburg, Virginia 24061 (540) 953-1060 [email protected]
I. Personal Data
Born: 10 February 1969, Seattle, Washington. Citizenship: USA Marital Status: unmarried
II. Education
High School Diploma: June, 1987; Yorktown High School, Arlington, VA
Bachelor of Arts: December, 1994; Virginia Polytechnic Institute and State University, Blacksburg, VA. Major: History.
Currently enrolled: Master's of Science: January 1995 - present. Virginia Polytechnic Institute and State University, Blacksburg, VA. Major Advisor: B.J. Turner. Thesis: Low temperature induction of males and other developmental anomalies in a self-fertlizing hermaphroditic fish species.
III. Teaching Experience
Graduate teaching assistant, Virginia Polytechnic Institute and State University. Courses: General Biology Laboratory, Spring, 1996-Spring 1997 Principles of Biology Laboratory for Majors, Fall 1997.
IV. Research
1. Variation in induction of males through low temperature incubation of embryos of a self-fertilizing vertebrate, the marine killifish Rivulus marmoratus, January 1995 - December, 1997.
2. Characterization of the Major Histocompatibility Complex (Class I) in Rivulus marmoratus using PCR techniques, April, 1997 - present.
43 V. Papers presented at Professional Meetings
A possible explanation for the appearance of males in populations of self- fertilizing hermaphroditic fish. American Society of Icthyologists and Herpetologists Annual Meeting, 1997, Seattle, Washington.
VI. Military service
United States Marine Corps Reserve, Company B, 4th Combat Engineer Battalion, 4th Marine Division, Roanoke, Virginia. October, 1990 - April, 1997. Separation rank: Sergeant (E-5).
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