Nikolaj Sørensen:
Marine picoeukaryotes in cold water: diversity and ecology

Date: 15-08-2014    Supervisor: Niels Daugbjerg & Katherine Richardson

Picoeukaryotes (protists ≤3 μm) form an important part of marine ecosystems, both as primary producers, bacterial grazers and parasites. The Arctic is experiencing accelerated global warming. Thus, the organisms living there are already being impacted by climate change. This PhD thesis investigates picoeukaryotes in the Arctic. The objective of this thesis is to investigate the diversity, ecology, population dynamics and biogeography of Arctic picoeukaryotes.

The picoeukaryotic community is very diverse in size, ecology and taxonomy and a bulk-approach will not be able to identify population dynamics within the community. Methods allowing taxonomic resolution therefore form an essential part of this thesis. A secondarily objective of this thesis is to improve and expand upon these methods.

In manuscript I, the hypothesis that ciliate and dinoflagellate phylotypes from picoplanktonic samples stem from extracellular DNA is tested. A meta-analysis of picoeukaryotic molecular surveys and a real-time qPCR experiment with environmental samples finds the relative abundance of ciliate and dinoflagellate DNA in the picoplanktonic size fraction (<3 μm) to be negatively correlated with the pore size of the end filter: filters with a pore size of 0.8 μm retain 84-89% less ciliate and dinoflagellate DNA than 0.2 μm filters. This would either imply that most pico-sized ciliates and dinoflagellates are smaller than 0.8 μm, and not 0.8-3 μm, or that extracellular DNA from larger organisms is attached to particles <3 μm that are more readily retained on end filters with smaller pore sizes. Considering that the smallest known eukaryote, Ostreococcus tauri, measures 0.8 μm and that sediment is known to harbour large amounts of extracellular DNA, the latter is the more probable explanation.

In manuscript II, the succession of picophytoplankton during the spring bloom is investigated in an Arctic Bay using HPLC-CHEMTAX. Although picoplanktonic chlorophyll a is relatively stable over time when compared to larger algae, rapid species succession takes place within the picophytoplanktonic community, with groups of picoeukaryotes showing the same degree of variation in their contribution to total chlorophyll a as larger algae. Interestingly, haptophytes and pelagophytes are found to be the dominant picophytoplankton, whereas molecular studies typically find Micomonas to dominate in the Arctic. Molecular underrepresentation of haptophytes has previously been observed in other oceanic regions and a high GC content of haptophytes, leading to decreased amplification efficiency during PCR, has been suggested as the cause of this bias. However, a real-time qPCR experiment conducted as part of the study reported in manuscript II did not detect decreased amplification of relatively GC rich haptophyte 18S rDNA compared to GC poor mamiellophyte 18S rDNA. Instead, the larger size of picohaptophytes compared to picomamiellophytes, and their comparable 18S rDNA copy number, may be the source of the molecular underrepresentation of haptophytes.

In manuscript III, an in-depth study of picoeukaryotes along the east coast of Greenland during late summer is presented. Specific groups of picophytoplankton are found to spatially vary in their chlorophyll a contribution to a degree exceeding that of larger algae, similar to what was found in manuscript II. Despite dinoflagellate sequences being abundant in the study, key pigments for this group are practically absent, indicating that their molecular abundance may be an artefact as suggested in manuscript I. Several bipolar phylotypes are identified which have not been found in any oceans in the Southern Hemisphere, except for the Antarctic Ocean, suggesting that advection may not always be responsible for long-range dispersal events. The relative abundance of MAST-1 is found to have a strong negative correlation with temperature (suggesting that it may be a primarily Arctic clade) and the abundance of non-chlorophyte picophytoplankton is found to correlate positively with both the influx of Atlantic water and nutrient availability. Thus, both increasing temperature and changes in ocean currents and nutrient availability are likely to influence the Arctic picoeukaryotic community. As all three factors are likely to be influenced by global warming, it is possible that the Arctic picoeukaryotic community is facing substantial changes in the near future.

In manuscript IV, the first sequencing study of sympagic (living in sea ice) picoeukaryotes is presented, thus providing the first in-depth description of picoeukaryotic diversity in sea ice. While the functional groups identified are similar to those that have been found for pelagic picoeukaryotic communities (phototrophs, bacterivores and parasites), the taxonomic compositions of these groups are completely different from those reported for pelagic communities. A significant proportion of the sympagic phylotypes reported have never been recovered from a pelagic setting, despite also being found in sea ice in the Baltic Sea or Southern Ocean. This implies that the entire sympagic community is unlikely to be recruited from the pelagic community but other, as of yet unknown, pathways of dispersal and sea ice colonization may exist. This echoes the finding from manuscript III, where advection by ocean currents is found to be unable to account for the global distribution of some picoeukaryotic phylotypes.

In manuscript V, the recruitment of rare pelagic pcioeukaryotes into sea ice is investigated. Although the vast majority of microbial species in all communities are found at low abundances, the ecological role, if any, of such locally rare species is poorly understood. One hypothesis is that the rare microbial biosphere may function as a seed bank by maintaining species that can exploit, and become abundant under, changed environmental conditions. Here, using an example of microbe recruitment to sea ice, we present the first empirical evidence for the rare biosphere serving as a seed bank in nature. Sea-ice formation represents a dramatic and long-lived (compared to microbe generation times) environmental change and the sea-ice microbial community is primarily recruited from the surrounding waters. If the rare pelagic biosphere serves as a seed bank for the sea ice community, the seed bank hypothesis predicts that rare pelagic species should be recruited more often to and be relatively more abundant in sea ice than abundant pelagic species. By comparing the species diversity of picoeukaryotes collected from Arctic water and sea ice, this study provides empirical evidence for both predictions and illustrates that rare species in the pelagic picoplankton community serve as a seed bank for the sea ice community.

The findings presented in this thesis expand our knowledge of picoeukaryote diversity and distribution and by going beyond a bulk-approach new insights into picoeukaryote population dynamics are obtained. The thesis also presents a protocol for 18S rDNA sequencing on the Ion Torrent PGM protocol which should be useful for molecular surveys of all protists and highlights the influence of extracellular DNA in molecular studies.