UC Santa Cruz UC Santa Cruz Electronic Theses and Dissertations Title Quantitative biogeography of distinct clades of picoplanktonic marine green algae establishes that Bathycoccus ecotypes coexist more frequently than Ostreococcus ecotypes Permalink https://escholarship.org/uc/item/09c977pb Author Limardo, Alexander Joseph Publication Date 2016 Supplemental Material https://escholarship.org/uc/item/09c977pb#supplemental Copyright Information This work is made available under the terms of a Creative Commons Attribution- NonCommercial-ShareAlike License, availalbe at https://creativecommons.org/licenses/by- nc-sa/4.0/ Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA SANTA CRUZ Quantitative biogeography of distinct clades of picoplanktonic marine green algae establishes that Bathycoccus ecotypes coexist more frequently than Ostreococcus ecotypes A thesis submitted in partial satisfaction of the requirements for the degree of MASTER OF SCIENCE in OCEAN SCIENCES by Alexander Joseph Limardo December 2016 The Thesis of Alexander Joseph Limardo is approved: Professor Marilou Sison-Mangus Professor Alexandra Z. Worden Professor Jonathan Zehr Tyrus Miller Vice Provost and Dean of Graduate Studies Copyright © by Alexander Joseph Limardo 2016 Table of Contents Title Page i Copyright ii Table of Contents iii List of Tables and Figures iv Acknowledgements vi Abstract vii Chapter 1: Background and Introduction 1 Chapter 2: Quantitative biogeography of distinct clades of picoplanktonic marine green algae establishes that Bathycoccus ecotypes coexist more frequently than Ostreococcus ecotypes 14 Summary 14 Introduction 17 Materials and Methods 22 Results 32 Discussion 55 Chapter 3: Conclusions and Future Directions 73 iii List of Figures & Tables Figure 1: Alignment of partial ITS1 region showing regions targeted by Bathycoccus BI and BII qPCR primers and probes. 28 Figure 2: Bathycoccus BI and BII qPCR assays testing different mixtures of BI and BII genomic DNA. 29 Figure 3: Map showing locations of environmental samples and phylogenetic tree of Mamiellophyceae ITS sequences. 34 Figure 4: Results from Station M1 of Monterey Bay Time-Series (MBTS). 40 Figure 5: Results from Station ALOHA of the Hawaii Ocean Time-series Study (HOT). 41 Figure 6: Results from GOC12 transect. 47 Figure 7: Results from WFAD09 transect. 48 Figure 8: Clade ratios (BI/BII or OI/OII) from GOC12 and WFAD09 cruises plotted against depth. 49 Figure 9: QPCR abundance for all samples where Bathycoccus or Ostreococcus were detected plotted against temperature and salinity. 53 Figure 10: Non-metric multidimensional scaling with k-means clustering analysis. 54 Table 1: Metadata for cruises and time-series sampled in this study. 23 Table 2: Nucleotide sequences for Bathycoccus BI and BII qPCR assays. 29 Table 3: Cross reactivity of Bathycoccus BI and BII qPCR assays. 30 Table 4: Results from environmental clone libraries targeting Mamiellophyceae. 33 iv Table 5: Environmental variables at Station M1 (MBTS) where BII or OII were detected and where absent. 42 Table 6: Environmental variables at Station ALOHA (HOT) where BII and OII were detected and where absent. 42 Table 7: Spearman’s rank correlation coefficient (ρ) between ecotype qPCR abundances and environmental variables. 52 v Acknowledgements First, I want to thank my parents, Lynn and Ken Limardo. Obviously I wouldn’t exist without them, but they also helped instill in me the value of education, and as a child they nurtured my scientific curiosity. Also, none of this would have been possible without the love and support of my significant other, Amy Klebesadel, who has stood by my side throughout the good times as well as the bad. I wanted to say a big THANK YOU to all Worden Lab members (past and present) who have helped me tremendously in so many ways these last 4 years (5 years including my time in the lab prior to becoming a grad student) as well as many MBARI staff members. A special thanks to those of you that sacrificed your time to give constructive feedback on my thesis presentation and on the thesis text itself. I feel fortunate to be a part of such a talented lab group with people who genuinely care about each other, and who are willing to help fellow lab-mates succeed in achieving their goals. Additional thanks to the amazing UCSC Ocean Sciences Department, my thesis reading committee (Marilou Sison-Mangus, Alex Worden and Jon Zehr), and all of my Santa Cruz and MBARI friends. Last, but certainly not least, I wanted to give a special thanks to my advisor Alex Worden for all the support and guidance she has provided these last few years. During my time with Alex, she has provided me with invaluable experiences, both in my professional as well as personal life, and these experiences will continue to influence my life for many years into the future. vi Abstract Quantitative biogeography of distinct clades of picoplanktonic marine green algae establishes that Bathycoccus ecotypes coexist more frequently than Ostreococcus ecotypes by Alexander Joseph Limardo We describe the biogeography of two clades of Bathycoccus, a picoplanktonic group of marine green algae, that are phylogenetically distinct based on analysis of the ITS1/5.8S/ITS2 region of the nuclear rRNA operon, but identical across the 18S rRNA gene. QPCR assays were developed against the ITS1 to allow quantification of these taxa (clades BI and BII). These were used alongside prior 18S rRNA primer- probes for two Ostreococcus clades that have previously been classified as different ecotypes (clades OI and OII) to analyze 266 photic zone samples from the tropical Atlantic and North Pacific Oceans. Clades BI and OI were observed in mesotrophic locations, whereas BII and OII were found in oligotrophic regions. Bathycoccus ecotypes co-occurred more often than Ostreococcus ecotypes. In contrast to Ostreococcus ecotypes, the adaptive differences between Bathycoccus clades have yet to be resolved and both Bathycoccus ecotypes appear to be more flexible in their niche(s) than Ostreococcus. vii CHAPTER 1: Background & Introduction Marine phytoplankton are responsible for approximately half of global net primary production, and comprise the base of the marine food web (Field et al 1998). By utilizing energy from sunlight to fix CO into organic carbon molecules, the growth 2 of phytoplankton biomass has a strong impact on the entire marine ecosystem as well as the physico-chemical conditions of the surrounding environment (Redfield 1958). Phytoplankton growth and mortality rates are controlled by numerous physical, chemical and biological factors. Understanding how these complex environmental parameters interact to determine phytoplankton community structure and biomass production is crucial to improving models of biogeochemical cycling and trophic interactions, and thus our ability to predict future changes to those systems. Oceanographers conventionally classify plankton based on size fractions retrieved from sequential filtration through different pore sizes. The smallest size class, the picophytoplankton (<3 µm cell diameter) are widely distributed in all aquatic habitats, and comprise a diverse group of organisms, including cyanobacteria and the ‘picoeukaryotes’ (Johnson and Sieburth 1982, Waterbury et al 1979). Due to their small cell size which has a high surface area to volume ratio, picophytoplankton are effective competitors for nutrients, therefore this group dominates primary production in marine habitats where nutrients are scarce (Chisholm 1992, Raven 1998). However, the small size of picophytoplankton and lack of distinguishable morphological features presents major obstacles for researchers attempting to study 1 these organisms, therefore they were largely overlooked until advances such as flow cytometry, epifluorescence microcopy (combined with in-situ hybridization methods) and nucleic acid sequencing enabled more detailed investigations. Flow cytometry, a tool utilized frequently to study picophytoplankton, allows oceanographers to discriminate, sort and quantify cyanobacteria (Synechococcus and Prochlorococcus) and photosynthetic picoeukaryotes (PPE) based on optical properties such as fluorescence of photosynthetic pigments and cell size-dependent light scattering (Olson et al 1991). Using flow cytometry to measure cell abundances, researchers began to recognize that picophytoplankton account for a significant proportion of photosynthetic biomass in many regions of the world’s oceans (Campbell et al 1994, Li 1994). While cyanobacteria were often numerically more abundant than PPE, studies incorporating biomass and growth estimates found that PPE can dominate primary productivity due to their larger cell volumes and relatively high growth rates (Li 1994, Worden et al 2004). However, a significant drawback of flow cytometry is limited taxonomic resolution of picoeukaryotes, where cell abundances of many different species are often summed together and reported as a single group. When DNA sequencing techniques were applied to ocean samples, the immense genetic diversity and complexity of the PPE component was revealed, and several novel branches were added to the eukaryotic tree of life (Diez et al 2001, Lopez-Garcia et al 2001, Moon-van der Staay et al 2001). Efforts to understand the ecological roles played by different PPE taxa were confounded by massive biodiversity and a lack of quantitative observations, although certain PPE taxa 2