The deep sea is the largest and least known ecosystem on Earth. Recent exploration has revealed a surprisingly varied and dynamic environment, and quite unexpectedly high biodiversity at both community and landscape levels. While a picture of ecological patterns is emerging, the evolutionary origin of this rich and highly endemic fauna is unknown. This represents a huge gap in our understanding of basic evolutionary phenomena and presents a number of major theoretical challenges. The primary evidence of evolutionary divergence in other environments is genetic population structure. Our first grant was devoted to developing molecular methods to extract mitochondrial DNA from preserved deep sea molluscs. The success of these new methodological advances enabled us to perform an analysis of genetic differentiation along a complete depth gradient (119 5000 m) within the North American Basin. Results provide the first clear insight into how and where genetic variation accumulates and differentiates to produce the enormously high biodiversity observed in the deep ocean. Genetic patterns confirmed the only explicit model of population differentiation in deep sea populations (Etter and Rex 1990). The ability to sequence DNA in preserved deep sea specimens makes it possible to explore, for the first time, many facets of the historical evolutionary development of this unique fauna by using the vast archived collections of deep sea material. We propose to continue this research by expanding in 2 directions. First, the unexpectedly large number of mutations characterizing 16S mtDNA haplotypes sampled in several species, while exciting, can confound interpretations of population level processes because it cannot be ascribed to intraspecific versus interspecific divergence (e.g., cryptic morphospecies). We propose to develop techniques to assay single copy nuclear genes from formalin fixed tissues. Phylogenies based on nuclear DNA can be compared to those from the mtDNA enabling us to distinguish between the hypotheses of large scale intraspecific polymorphism versus the alternative of co existing 'clades' of deep sea taxa with limited contemporary gene flow. We will target the internally transcribed spacer (M) regions of the rRNA cluster as well as introns within protein coding regions because of their relatively rapid rate of evolution. Working with the nuclear DNA from formalin fixed tissues is more challenging than our successes with mtDNA loci, primarily due to differences in genome copy number/cell. However, our preliminary success with two loci would indicate that nuclear gene assays from those individuals previously characterized for mtDNA are feasible. The proposed research is essential to the confident interpretation of our within basin results, allows for the development of molecular tools necessary for interpreting patterns at larger scales, and provides the strength of an analysis of several independent loci for elucidating concordant evolutionary processes in the deep sea. Second, we propose to extend the analysis of genetic population structure to ocean wide scales in five species that we have successfully analyzed on a regional scale and to test whether specific potential isolating barriers influence population structure. Like many deep sea organisms, these species are very widely distributed. A complete understanding of population differentiation, species formation and adaptive radiation of higher taxa clearly requires analysis of geographic variation on very large scales including those on which geographic isolating barriers may operate. Potential barriers include distance, depth, major topographic features and ocean current patterns. We will investigate the importance of isolating barriers on large scale population structure by comparing the genetic structure of two gastropods with different modes of dispersal, which should respond to the isolating effects of currents and bottom topography in very different ways. One species develops planktotrophically in the surface currents and the other has lecithotrophic development in abyssal currents. Statistical models will be used to determine whether genetic distance can be predicted by differences in depth and distance within and among basins, and corresponds to potential isolating barriers. Large scale genetic population structure in bivalves will contribute significantly to our understanding of speciation in the deep sea, and test the generality of the trend discovered in our current research that interpopulation genetic distance decreases with increasing depth. This research is the first oceanwide controlled comparison of species that has the potential to reveal the basic causes of evolutionary divergence in the deep sea ecosystems on scales that are appropriate to the processes involved. Expanding our analysis of regional differentiation to include the effects of isolating barriers on very large scales would be a major advance in our understanding of evolutionary dynamics in the deep sea and will provide essential information to formulate future questions about the origin of deep sea biodiversity. A 1.
