Graduation Year

2016

Document Type

Thesis

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Marine Science

Major Professor

Pamela Hallock Muller, Ph.D.

Committee Member

Mya Breitbart, Ph.D.

Committee Member

James Garey, Ph.D.

Committee Member

Dale Griffin, Ph.D.

Committee Member

John Paul, Ph.D.

Keywords

DNA Extraction, Sequencing, Denaturing Gradient Gel Electrophoresis, Symbiosis, Phylogenetics, Bioinformatics

Abstract

Diatoms associated with foraminifers of the genus Amphistegina were assessed using a combination of morphological and molecular techniques. These included: 1) microscopic identification of diatoms cultured from the host, 2) sequencing of portions of the small subunit of the ribosomal RNA gene (18S) and the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase [i.e., RubisCO] gene (rbcL) from DNA extracted directly from the Amphistegina hosts and also from diatoms cultured from these hosts, and 3) denaturing gradient gel electrophoresis (DGGE) profiles of rbcL and internal transcribed spacer 1 (ITS1) PCR amplicons from DNA extracted directly from hosts and from cultures.

Consistent with previous culture studies, multiple species of pennate diatoms of the genera Nitzschia, Fragilaria (including Nanofrustulum), Amphora, and Navicula, were cultured from >900 host specimens collected from >20 sites in the western Atlantic and four sites in the Pacific. Diatoms of the genus Nitzschia grew in about half of all successful cultures. The genetic identities of selected cultures were consistent with those based on morphological taxonomy.

Diatom sequences from DNA extracted directly from the cytoplasm of the Amphistegina hosts were species specific and distinct from sequences obtained from cultured diatoms and from sequences in GenBank of diatom taxa previously reported as endosymbionts. Multiple phylogenetic analyses revealed that the 18S and rbcL diatom sequences from specimens of A. gibbosa collected from the Atlantic sites and of Amphistegina spp. from Hawai’i were most similar to the 18S and rbcL sequences of an unnamed Fragilariaceae diatom in GenBank (Accession # JX413542.1 for 18S and JX413559.1 for rbcL) and other closely related diatoms in that family.

Of diatom taxa previously reported as endosymbionts of larger foraminifers, Nanofrustulum shiloi was the most similar, but not identical, to the sequences from hosts collected from the Atlantic and Hawai’i. The 18S and rbcL diatom sequences from the Atlantic host species, A. gibbosa, were all nearly identical, but small intra-species differences (subclades) were observed from specimens collected from the deepest (75 m) site in the Florida Keys and also from the eastern-most site, Young Island near St. Vincent. The 18S and rbcL diatom sequences from the two host species from Hawai’i, A. lobifera and A. lessonii, were more variable but still within the family Fragilariaceae.

The diatom sequences from A. radiata collected from two sites in Papua New Guinea (PNG) were most similar to diatoms of the family Plagiogrammaceae and therefore distinct from sequences obtained from other Amphistegina species in this study, as well as from all diatoms previously reported as endosymbionts. A small difference was observed between the diatom sequences from host specimens collected from a Pacific site as compared to a Bismarck Sea site.

The ITS1 DGGE profiles of DNA extracted directly from A. gibbosa specimens at different depths, locations, and seasons in the western Atlantic were nearly identical. Differences were seen between rbcL DGGE profiles of DNA extracted directly from the different Amphistegina host species. The rbcL DGGE profiles directly from all hosts were clearly different from those extracted from diatoms cultured from the same host specimens, as well as from Nitzschia laevis, a commonly reported diatom endosymbiont in past culture-based studies.

My findings are consistent with ultrastructural studies of endosymbionts of Amphistegina published in the early 1980s and congruent with recent molecular studies of endosymbionts in other diatom-bearing foraminifers, all of which indicate specificity. Nevertheless, the consistency with which several diatom taxa have been reported in culture studies from all oceans indicates the possibility of some relationship with Amphistegina spp., either as important food items, epiphytes, or minor opportunistic symbionts that can thrive in culture media.

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