Rivulus marmoratus (Cyprinodont; Aplochelidae) is the only vertebrate known to reproduce by obligate internal self-fertilization. Selfing is the ultimate form of inbreeding and leads to virtual homozygosity in approximately ten generations, so that this mode of reproduction is effectively clonal. Populations of this species consist of arrays of homozygous clones, with high clonal diversity and low representation of each clone. Males occur at low frequency (<1%) in most populations, with a few notable exceptions. Females have never been recorded.High frequencies of males have been reported on two occasions; high numbers of males were reported on the island of Curacao in the 1970's, and males comprised up to 24% of the populations sampled on several Belize Cays during 1990-91. Hermaphrodites collected from one island, Twin Cays, proved to be heterozygous at mini-satellite loci when progeny tested and DNA fingerprinted. This was evidence that recent outcrossing had occurred.Earlier experiments with Floridian clonal lines had suggested that sex in this species was thermolabile; incubation of embryos at low temperatures (19° C)) produced up to 100% males. It was suggested that this might be part of an environmental sex determination (ESD) system in this species.If the induction of males is indeed part of and ESD in this species, then it may be that the temperature at which males are induced in a particular clone will be related to the temperature regime that clone encounters in nature. Therefore, it was hypothesized that the temperature at which males are induced would be related to the geographic origin of each clone, so that clones from the cooler extremes of the range would produce males at a lower threshold temperature than clones from the more equatorial center of the range. This study used laboratory reared descendants of clones from Vero Beach, Florida, Rio de Janiero, Brazil (the extreme north and south of the range), and from several Belize barrier islands and the Belize mainland (near the center of the range) in an effort to detect differences in the production of males between clonal lines based on their geographic origin. Eggs were collected from these hermaphrodites and incubated at 26° C)C, 22.5° C), or 19 ° C), representing normal, and below average temperatures for this species. These embryos were hatched and reared to sexual maturity at which time their sex was evaluated.Incubation at 26° C) resulted in 2-17% males, incubation at 22.5° C) resulted in 6-53% males, and incubation at 19° C) resulted in 47-74% males. However, the variation in male induction between clonal lines did not correspond to a geographic hypothesis.Also, significant numbers of deformed individuals were noted, particularly among individuals incubated at the lower two temperatures. Incubation at 26° C) resulted in 0-24% deformed, incubation at 22.5° C) resulted in 6-66% deformed, and incubation at 19° C) resulted in 36-87% deformed. It is clear that development at these lower temperatures is difficult for this species, and it may be that low temperature developmental stress is not a general challenge to this species. / Master of Science

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 109-117). Also available via the Internet.

The ability of natural selection to promote and preserve genetic variation at Major Histocompatibility Complex (MHC) class I loci was examined in two fish species known to have low genetic variation either as a consequence of their breeding system or population structure. The tempo and modes of molecular evolution acting on these loci has also been discussed. The marine killifish Rivulus marmoratus is the only vertebrate known to exist in nature in homozygous form. The findings of this study suggest that MHC class I genes at one locus in R. marmoratus have rapidly accumulated variation, particularly in region of the gene encoding functional important domains. This rapid accumulation of variation is likely due to the increased intensity of natural selection acting on these genes resulting from the homozygosity of the species. The contention that the variation characterized at one MHC locus is of recent origin is further supported by an analysis of the mitochondrial control region if R. marmoratus, which suggests that the individuals included in this study are recently diverged. The variation characterized here is the first evidence of genetic variation at coding loci in this species, and may be evidence of the cost of homozygosity for R. marmoratus. Variation at a single MHC class I locus was also characterized in several members of the Death Valley pupfish species complex. Two of these species, Cyprinodon diabolis and Cyprinodon salinus have undergone intense historical or ongoing population bottlenecks. Despite the action of random genetic drift acting on these species, both maintain multiple MHC class I alleles at one locus. The alleles characterized in C. diabolis and C. salinus were also recovered from other members of the Death Valley complex. This suggests that overdominant natural selection has acted to maintain ancestral genetic variation at this locus in both species. This result has important implications for the design of conservation programs for these endangered species, and may have broader implications for the design of captive breeding programs of species with highly reduced population sizes in general. / Ph. D.

The mangrove killifish, Kryptolebias marmoratus, has the potential of becoming a strong model organism for a range of biological disciplines thanks to its ability to self-fertilise, a process only known to occur in invertebrate animals until its discovery. Selfing, a natural occurrence in this species, has lead to the formation of clonal lineages composed of highly homozygous individuals. The aim of this thesis was to further establish K. marmoratus in the field of developmental biology by providing an information infrastructure to help advance research on this peculiar animal and further promote its place in the pantheon of model organisms. To do so, I first set out to standardise K. marmoratus embryology by providing defined developmental stages with clear visual representations of key embryonic structures. This staging series is an essential tool that will ensure repeatability and consistency within and across different laboratories. Secondly, I examined several techniques for embryonic manipulation and for imaging that can be used in an array of experimental designs. Using these techniques I demonstrated microinjection of embryos by monitoring the yolk syncytial layer and its nuclei, and time-lapse analyses of the yolk surface during embryonic development. Finally, I applied the knowledge gained from my first two studies and examined Bmp signalling in K. marmoratus embryos and its influence on body patterning. By inhibiting this pathway, I found a new phenotype characterised by an extremely short and split body axis. These data highlighted the importance of studying known signalling pathways in unknown organisms as species-specific differences may improve our understanding of fundamental developmental processes. This thesis demonstrates that with its easily obtainable and manipulated embryos, K. marmoratus can be used for embryological research in the same light as other model organisms such as zebrafish or medaka. The rising amount of information on mangrove killifish will help further take advantage of this unique and intriguing species, and supports the use of this hermaphroditic vertebrate as a strong comparative model in developmental biology.