Date of Award

8-2006

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology

Advisor

Paul D. Rawson

Second Committee Member

Irving L Kornfield III

Third Committee Member

Michael T. Kinnison

Abstract

Extreme environmental heterogeneity on spatial and temporal scales in the rocky intertidal zone has been shown to be a strong selective force maintaining genetic polymorphisms for many enzymes (e.g. Levinton and Fundiller 1975; Koehn et al. 1980; Gosling and McGrath 1990; Riginos and Cunningham 2005). In particular, the mannose- 6-phosphate isomerase (MPI; EC 5.3.1.8) locus is highly polymorphic, and is thought to be under selection based on a combination of factors including temperature, dessication, exposure, and variation in dietary carbohydrates (Schmidt 2001). While previous studies have documented significant shifts in MPI allozyme allele frequencies as a function of tidal height andlor wave exposure (McDonald 1991 ;Rand et al. 2002), a more rigorous sequence-based analysis of selection has not been undertaken in any marine species. In order to better understand the potential role of the MPI locus in relation to adaptation to heterogeneous intertidal habitats, full-length MPI cDNAs were isolated from the blue mussel, Mytilus edulis, which were sampled along intertidal (high intertidal, tidepool, subtidal) and wave exposed (sheltered, protected) gradients. Surprisingly, paralogous MPI loci (MPI-A and MPI-B) were discovered in the isolation process, which represents the first known instance of gene duplication in a molluscan central metabolic enzyme. To test the hypothesis that MPI is a target of selection in M. edulis, I used three separate approaches: 1. A frequency-based approach using RFLP variation at MPI-A and two putatively neutral markers (LAP and COIII); 2. SYBR-green-based real-time quantitative RT-PCR assays to measure differential expression patterns of MPI-A and MPI-B across tissue types; and 3. Intraspecific and interspecific comparative sequence analyses of MPI-A and MPI-B from alternate tidal heights and locations. Significant shifts in MPI-A RFLP variation were documented between habitat types in central and Downeast Maine populations, while no significant variation was found for either the LAP or COIII markers. Expression assays revealed significant changes in transcript levels between tissue types, with MPI-A showing strongest expression in mantle tissue while MPI-A was preferentially expressed in gill and hepatopancreas tissues. Finally, the comparative sequence analyses revealed substantial changes in dN/dS ratios between tidal heights and locations and significant departures from neutral sequence evolution for both loci. The data suggest that: 1) the maintenance of genetic polymorphism through microgeographic selection may be an important evolutionary force in intertidal environments where steep gradients in environmental heterogeneity exist; and 2) the retention of duplicate MPI loci in M. edulis may an effective evolutionary strategy for coping with physiological stress.

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