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