Genome reduction and evolution in an obligate luminous symbiont. by Tory A. Hendry A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ecology and Evolutionary Biology) in the University of Michigan 2012 Doctoral Committee: Professor Paul V. Dunlap, Chair Associate Professor Yin-Long Qiu Assistant Professor Gregory James Dick Assistant Professor Patrick D. Schloss Illustration by Patricia Beals Copyright Tory A. Hendry 2012 To my grandfather for giving me a love of nature, my mother for giving me the courage to try, my husband for giving constant encouragement, and my son for being the best thing I have done. ii ACKNOWLEDGEMENTS Support and advice from many people have guided me through my graduate career and made this work possible. First and foremost I thank my committee members Paul Dunlap, Greg Dick, Pat Schloss and Yin-Long Qiu for their invaluable assistance and constructive comments. I would especially like to thank my advisor, Paul, for his time, advice, and support. I have been privileged to learn how to be a careful and thoughtful researcher under his guidance, while enjoying the freedom to follow the research directions that inspired me. I am also grateful to lab members past and present, particularly Jennifer Ast, Henryk Urbanczyk, Alison Gould, and Beth Pringle, for their thoughts and assistance. A special thank you goes to Katherine Dougan for all of her meticulous help. Numerous people went out of their way in helping me obtain samples. I thank Jay Hemdal from the Toledo Zoo and Seth Wolters from the Steinhart Aquarium for allowing me access to their flashlight fish exhibits. I am grateful to Richard Rosenblatt and the Scripps Institution of Oceanography for the loan of flashlight fish specimens. I greatly appreciate the help of Quality Marine Suppliers, Grant Norton from Sustainable Reef Suppliers, Brad Remmer from Sea Dwelling Creatures, and Fish Doctors Aquarium in finding, collecting, and transporting fish. And I would especially like to thank Margo Haygood for the generous gift of DNA. iii My graduate career has been greatly enhanced by discussions with, and support from, my amazing cohort and other graduate students in the department. Thanks to the best big sib of all, Wendy Grus, for taking me under her wing. I can not thank Michael Sheehan enough for reading nearly every word I’ve written and hearing every theory I’ve come up with. There is also no way to sufficiently thank Susanna Messinger and Rachel Vannette for the gift of sanity, which they gave to me over many cups of coffee. I greatly appreciate the sympathetic ear of Abby Woodroffe, who was there through many rocky starts. And an enormous thank you to my pseudosister, Flynn Boonstra, whose love and support were even more meaningful for having been given long distance. I have received unlimited help from many people in EEB, especially members of the office staff, Julia Eussen, Jane Sullivan, Cindy Carl, Sonja Botes, and Amber Stadler. I have been funded personally by the departmental Edward’s fellowship and this work was funded by departmental Block Grant awards and Rackham graduate student research grants. iv TABLE OF CONTENTS Dedication………………………………………………………………………….. ii Acknowledgements………………………………………………………………… iii List of Figures……………………………………………………………………… vi List of Tables………………………………………………………………………. viii List of Appendices…………………………………………………………………. ix Abstract…………………………………………………………………………….. x Chapter I. Introduction………………………………………………………………. 1 II. The uncultured luminous symbiont of Anomalops katoptron represents a new bacterial genus……………………………………………………... 12 III. Genome reduction and host dependence in a luminous symbiont……... 44 IV. The obligate luminous symbionts of flashlight fish are specific to host genera…………………………………………………………………... 67 V. Extremely low genetic diversity and population dynamics in an obligate luminous symbiont.……………………………………………………….. 84 VI. Genomic divergence between obligate luminous symbiont species…… 117 VII. Conclusion…………………………………………………………….. 138 Appendices…………………………………………………………………………. 147 v LIST OF FIGURES Figure 2.1. Maximum likelihood trees from housekeeping genes (16S rRNA gene, atpA, gapA, gyrB, pyrH, recA, rpoA, and topA)…………………………………… 31 Figure 2.2. Maximum likelihood trees from the lux operon………………..…….... 32 Figure 2.3. A. The structure of the lux operon and flanking regions in the A. katoptron symbionts and representative members of Vibrionaceae. B. Alignment of the regulatory region upstream of the lux operon in the A. katopron symbiont and Vibrio harveyi………………………………………………………………….. 33 Figure 2.4. Maximum likelihood tree of ‘Ca. Photodesmus katoptron’ luxR and Vibrionaceae homologs……………………………………………………………. 34 Fig. 3.1. Graph of genome size versus number of genes…………………………... 57 Fig. 3.2. Chromosome comparisons between ‘Ca. Photodesmus katoptron’ and A. fischeri (ES114) and V. campbellii (ATCC BAA-1116)…………………………... 58 Fig. 3.3. Phylogenetic tree with genome sizes of ‘Ca. Photodesmus katoptron’ and relatives…………………………………………………………………………….. 59 Fig. 3.4. Genes in TIGRfam functional categories for ‘Ca. Photodesmus katoptron’ and A. fischeri (ES114) and V. campbellii (ATCC BAA-1116)……….. 60 Fig. 3.5.Amino acid synthesis pathways showing gene loss in ‘Ca. Photodesmus katoptron.’………………………………………………………………………….. 61 Fig. 3.6. Genomic hierarchical clustering dendogram showing that ‘Ca. Photodesmus katoptron’ is more similar in gene content to distantly related obligate symbionts than it is to free-living close relatives…………………………. 62 Fig. 4.1. Maximum likelihood trees from housekeeping genes (16S rRNA gene, atpA, gapA, gyrB, pyrH, recA, rpoA, and topA)…………………………………… 78 Fig. 4.2. Maximum likelihood trees from lux operon (luxCDABEG)……………… 79 vi Fig. 4.3. Divergence between strains from different host genera for the symbionts Aliivibrio fischeri and ‘Ca. Photodesmus spp.’……………………………………. 80 Fig. 4.4. Cladograms comparing anomalopid host phylogeny and anomalopid symbiont phylogeny………………………………………………………………... 81 Fig. 5.1. Diversity estimates for the anomalopid symbionts and various facultatively host-associated relatives……………………………………………… 105 Fig. 5.2. Diversity estimates for anomalopid symbionts, free-living luminous symbionts, and genetically monomorphic obligately host-associated bacteria……. 106 Fig. 5.3. Percentage of sites polymorphic for a given polymorphism index (number polymorphic reads at a site) in ‘Ca. Photodesmus katoptron.’…………... 107 Fig. 5.4. Percentage of sites polymorphic for a given polymorphism index (number polymorphic reads at a site) in ‘Ca. Photodesmus blepharus’ Ppalp…….. 108 Fig. 5.5. ‘Candidatus Photodesmus katoptron’ host distribution, sample sites and genetic divergence…………………………………………………………………. 109 Fig. 6.1. A. Alignments of syntenic contigs from ‘Ca. Photodesmus katoptron’ (Pk) and ‘Ca. Photodesmus blepharus’ Ppalp (Pp) and B. Alignments of chromosome I and chromosome II from Vibrio cholerae N16961, Vibrio parahaemolyticus RIMD 2210633, and Vibrio vulnificus CMCP6………………... 133 Fig. 6.2. Genes shared by ‘Ca. Photodesmus katoptron’ and ‘Ca. Photodesmus blepharus’ Ppalp arranged by percent identity between the anomalopid symbionts compared to percent identity to relatives…………………………………………... 134 vii LIST OF TABLES Table 2.1. GenBank accession numbers and strain designations for all taxa in the housekeeping gene analysis………………………………………………………... 35 Table 2.2. GenBank accession numbers and strain designations for taxa in the lux gene analysis……………………………………………………………………….. 38 Table 3.1. Genome characteristics that vary with lifestyle compared to ‘Ca. Photodesmus katoptron’……………………………………………………………. 63 Table 3.2. Contig and assembly information showing size (bp) and read coverage depth of contigs by category……………………………………………………….. 63 Table 5.1. Polymorphism values for anomalopid symbionts. Single nucleotide polymorphisms per kilobase are shown for chromosome I, chromosome II, and the plasmid for each species………………………………………………………. 110 Table 5.2. Polymorphism values for free-living, host-associated Vibrionaceae species……………………………………………………………………………… 111 Table 5.3. Rates of nonsynonymous and synonymous substitutions for anomalopid symbionts, facultatively host-associated relatives and B. aphidicola… 112 Table 5.4. Genetic divergence between symbiont populations for anomalopid symbionts and free-living luminous symbionts……………………………………. 112 Table 5.5. Genes used to investigate divergence between populations of ‘Ca. Photodesmus katoptron.’ Numbers of polymorphisms in each population are shown………………………………………………………………………………. 113 Table 6.1. Genome content information for the ‘Ca. Photodesmus katoptron’ and ‘Ca. Photodesmus blepharus’ Ppalp genomes……………………………………... 135 Table 6.2. Comparison of how genes unique to one symbiont species were lost in the other species. Absolute numbers and percentages are shown………………….. 135 viii LIST OF APPENDICES Appendix 1. List of conserved genes present in the ‘Ca. Photodesmus katoptron’ genome……………………………………………………………………………... 147 Appendix 2. List of genes in selected TIGRfam categories found in the ‘Ca. Photodesmus katoptron’ genome…………………………………………………... 151 Appendix 3. Vibrionaceae strains used in diversity estimates…………………….. 172 Appendix 4. List of unique genes found in the ‘Ca. Photodesmus katoptron’ and ‘Ca. Photodesmus blepharus’ Ppalp genomes……………………………………... 176 ix ABSTRACT Genome reduction and evolution in an obligate luminous symbiont. by Tory A. Hendry Chair: Paul V. Dunlap The luminous bacteria symbiotic with