PROTISTAN SPECIES AND THEIR DIVERSITY FROM A MOLECULAR PERSPECTIVE A dissertation presented by Angela Schena to The Department of Biology In partial fulfillment of the requirements for the degree of Doctor of Philosophy in the field of Biology Northeastern University Boston, Massachusetts February 2012 1 PROTISTAN SPECIES AND THEIR DIVERSITY FROM A MOLECULAR PERSPECTIVE by Angela Schena ABSTRACT OF DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology in the Graduate School of Science of Northeastern University, February 2012 2 ABSTRACT Traditionally protists have been described based on their morphology using what is often referred to as alpha taxonomy approaches. However, today it is all but certain that these approaches have not revealed the real scale of protistan diversity. The two main reasons are the current uncultivability of most of microbial eukaryotes, which often makes direct observations impossible, and the lack of a comprehensive concept of species. Today, the molecular based (beta) taxonomy of comparisons of DNA sequences is increasingly used to bypass the limitations of the alpha taxonomy. The challenge is that the two approaches are typically used separately, and with different units of diversity. For example, it is not known to what extent the genetic distance between two taxa corresponds to morphological differences between them, and it is not clear if morphologically defined species do or do not cluster as phylogenetically distinct genetic groups of rRNA gene sequences (Operational Taxonomic Units, or OTUs). In the end, there is little understanding whether traditionally defined species vs OTUs combine identical, similar, or entirely different populations. Since traditional morphology and molecular taxonomy will likely both be used for the foreseeable future, it is important to understand what a morphologically defined species means in terms of gene sequence variability among cells composing this species. Here we use marine ciliates as model representatives of protists to investigate the level of intra- and interspecies heterogeneity in the most widely used genetic marker, the 18S rRNA gene. Using single-cell analysis and molecular cloning, we show that OTUs comprising 18S rRNA gene sequences that share ≥99% homology correspond well to species as defined by alpha taxonomy. Therefore, at least in ciliates, there appears to be a level of genetic variability in 18S 3 rRNA gene sequences that could be used as a proxy for morphologically defined species. Merging alpha and beta taxonomy is very convenient for protistan diversity studies as this opens a way to assess this diversity faster and more objectively. Capitalizing on this, we surveyed ciliate diversity in several marine habitats, and statistically estimated the total ciliate richness in these habitats. The resulting throughput compares favorably to, and at times exceeds, what would have been achieved by more traditional alpha taxonomy approaches. 4 ACKNOWLEDGEMENTS I would like to acknowledge and thank the people who helped me throughout my project and made my work possible. I thank my husband, my family, especially my father, and my friends, who have always supported and motivated me with any mean necessary. I would not have completed my studies without them. I am especially grateful to my advisor, Slava Epstein, a true mentor who gave me the guidance and the confidence I needed and stimulated me with his thought-provoking conversations. I would like to thank my committee members for their support and helpful suggestions: Virginia Edgcomb, Veronica Godoy-Carter, Edward Jarroll, Steven Vollmer. I thank all the members of the Epstein Lab, past and present, for the many insightful discussions about science and life. I especially thank Sun Hee Hong for her friendship and her teachings, Bill Orsi, Tine Hohmann and Annette Bollmann for their help during my field trips. I am indebted to Thorsten Stoeck for his secondary structure model and his words of molecular wisdom. I thank Chesley Leslin and Nathan Cahoon for navigating me into the unfamiliar territory of bioinformatics and John Bunge for providing the CatchAll software. I would also like to mention the people of the Estación de Investigationes Marinas de Margarita (Margarita Island, Venezuela) for their kindness and help during my field work. 5 DEDICATION This dissertation is dedicated to my mother Maria and my son Luca, my past and my future. 6 TABLE OF CONTENTS Abstract 3 Acknowledgments 5 Dedication 6 Table of Contents 7 List of Figures 8 List of Tables 10 Chapter 1. Introduction 11 Chapter 2. Material and Methods 20 Chapter 3. The rRNA gene sequence variability and the species molecular 27 signature Chapter 4. Species diversity and richness predictions on small local scale 44 Chapter 5. Conclusions 66 References 69 7 LIST OF FIGURES Figure 1. Geleid species used as model organisms in this study. A: G. simplex. 16 B: G. fossata. C: G. swedmarki (courtesy of R. Droste and E. Murphy). Figure 2. Maximum likelihood (ML) tree of 18S rRNA gene sequences showing 17 the phylogenetic position of the geleids within the class Karyorelictea. The first number at the nodes represents bootstrap values (percentage out of 1000 replicates) for ML and the second the posterior probability values of the Bayesian analysis. Bootstrap values over 50% are shown. Geleia simplex is strain 0.2Nah. The class Heterotrichea was used as outgroup. Figure 3. Phylogenetic conservation map and the nucleotide exchange 30 superimposed onto the G. simplex consensus SSU rRNA secondary structure. Number of sequences = 1960. Nucleotides categories: ACGU -98+% conserved; acgu -90-98% conserved; acgu -<90% conserved. Letters in bold with green arrows show a position where at least one of the 20 sequences has a nucleotide different from the consensus. Numbers in brackets: numbers of sequences which show a nucleotide different from the consensus. Letters in brackets: type of nucleotide exchange. Figure 4. Phylogenetic novelty of the OTUs retrieved from Nahant. (A) 46 Distribution of the similarity to the CEM and CIM. (B) Novelty pattern of the environmental OTUs. Red circles represent the similarity to the CEM and the CIM for each OTU. Figure 5. Phylogenetic novelty of the OTUs retrieved from Canada. (A) 47 Distribution of the similarity to the CEM and CIM. (B) Novelty pattern of the environmental OTUs. Red circles represent the similarity with the CEM and CIM for each OTU. Figure 6. Phylogenetic novelty of the OTUs retrieved from Greenland. (A) 48 Distribution of the similarity to the CEM and CIM. (B) Novelty pattern of the environmental OTUs. Red circles represent the similarity with the CEM and CIM for each OTU. Figure 7. Phylogenetic novelty of the OTUs retrieved from Venezuela. (A) 48 Distribution of the similarity to the CEM and CIM. (B) Novelty pattern of the environmental OTUs. Red circles represent the similarity with the CEM and CIM for each OTU. 8 Figure 8. (Page 50) ML phylogenetic tree of 18S rDNA clones retrieved from 50 Nahant. The first number at the nodes represents the bootstrap value (percentage out of 1000 replicates) for ML and the second the posterior probability value of the Bayesian analysis. Black dots indicate the nodes with 100% bootstrap/1 Bayesian probability values. Nahant OTUs appear in red. Phy = Phyllopharingea. Olig = Oligohymenophorea. Figure 9. (Page 52) ML phylogenetic tree of 18S rDNA clones retrieved from 52 Canada. The first number at each node represents the bootstrap value (percentage out of 1000 replicates) for ML and the second the posterior probability value of the Bayesian analysis. Black dots indicate nodes with 100% bootstrap/1 Bayesian probability values. Canada OTUs appear in red. Figure 10. (Page 54) ML phylogenetic tree of 18S rDNA clones retrieved from 54 Venezuela. The first number at each node represents the bootstrap value (percentage out of 1000 replicates) for ML and the second the posterior probability value of the Bayesian analysis. Black dots indicate nodes with 100% bootstrap/1 Bayesian probability values. Venezuela OTUs appear in red. Figure 11. (Page 56) ML phylogenetic tree of 18S rDNA clones retrieved from 56 Greenland. The first number at each node represents the bootstrap value (percentage out of 1000 replicates) for ML and the second the posterior probability value of the Bayesian analysis. Black dots indicate nodes with 100% bootstrap/1 Bayesian probability values. Greenland OTUs appear in red. Figure 12. Taxonomic affiliation of the OTUs from the four studied locations. 58 Figure 13. The number of OTUs (at 99% sequence similarity) shared among the 59 four clone libraries Figure 14. Frequency counts distributions of OTUs in three clone libraries 62 (Nahant, Canada, Venezuela), with the best-fitted parametric curve model (red) compared to other three competing parametric models. 9 LIST OF TABLES Table 1. Classification of the phylum Ciliophora. 15 Table 2. 18S rRNA gene sequence similarities among different Geleia species 18 (direct sequencing). Table 3. 18S rRNA gene sequence similarities between several populations of 19 G. simplex. The sequences from Nah 0.0m, 0.2m, 0.6m, 200m are from samples collected in 2002, spatially separated by 0.0, 0.2, 0.6, and 200 m respectively. Data from Droste 2003. (Nah= Nahant, MA; WH=Woods Hole, MA) Table 4. Primers used in the PCR amplifications of ciliate 18S rRNA gene. In 22 the primer names (F) indicates the forward direction and (R) indicates the reverse direction; in the primer sequences R* is an A or a G. Table 5. Variability of G. simplex 18S rRNA gene sequences within a population 27 and single cells. SD, Standard deviation. Table 6. Nucleotides composition of the G. simplex consensus sequence. 31 Table 7. Nucleotides composition of 18S rRNA in the different nucleotide 33 categories and the number of mismatches between cells in a 20-cells collection and within single cells. Table 8. Variability of G. simplex 18S rRNA gene sequences within a population 36 and single cells.